Identification of a potent and safe vitamin D receptor
agonist for the treatment of inflammatory bowel disease
Doctoral Dissertation
To be presented by permission of the Faculty of Natural and Environmental Sciences
of the University of Kuopio for public examination in Auditorium L21,Snellmania Building,
University of Kuopio,on Thursday 10th December, at 3 p.m.
Department of Biosciences
University of Kuopio
Kuopio University Library
P.O. Box 1627
Tel. +358 207 87 2200
Fax +358 17 163 410
Series Editors:
Professor Pertti Pasanen, Ph.D.
Department of Environmental Science
Author’s address:
Department of Biosciences
University of Kuopio
P.O. Box 1627
Tel. +358 40 355 3084
E-mail: [email protected]
Professor Carsten Carlberg, Ph.D.
Department of Biosciences
University of Kuopio
Luciano Adorini, M.D.
Intercept Pharmaceuticals
Corciano (Perugia), Italy
Professor Annemieke Verstuyf, Ph.D.
Laboratorium voor Experimentele Geneeskunde en
endocrinologie (Legendo)
Leuven Belgium
Professor Alberto Muñoz, Ph.D.
Instituto de Investigaciones Biomédicas "Alberto Sols"
Madrid, Spain
Professor Hans van Leeuwen, Ph.D.
Department of Internal Medicine
Erasmus MC
Rotterdam, Netherlands
ISBN 978-951-27-1402-5
ISBN 978-951-27-1297-7 (PDF)
ISSN 1235-0486
Kuopio 2009
Laverny, Gilles. Identification of a potent and safe VDR agonist for the treatment of IBD.
Kuopio university publications C. natural and environmental sciences 264. 2009. 135 p.
ISBN 978-951-27-1402-5
ISBN 978-951-27-1297-7 (PDF)
ISSN 1235-0486
The bioactive form of vitamin D, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), is a secosteroid
hormone that binds to the vitamin D receptor (VDR), a member of the nuclear receptor superfamily
expressed in many cell types, and modulates a variety of biological functions. 1α,25(OH)2D3 is
essential for bone and mineral homeostasis but also regulates growth and differentiation of multiple
cell types, and displays immunoregulatory and anti-inflammatory activities. The anti-proliferative,
pro-differentiative, anti-bacterial, immunomodulatory and anti-inflammatory properties of synthetic
VDR agonists could be exploited to treat a variety of chronic inflammatory and autoimmune diseases,
such as benign prostatic hyperplasia (BPH) and inflammatory bowel diseases (IBDs).
We have analyzed the capacity of VDR agonists to treat BPH, a complex syndrome characterized by a
static component related to prostate overgrowth, a dynamic component responsible for urinary
irritative symptoms, and an inflammatory component. Data in this thesis demonstrate that VDR
agonists, and notably elocalcitol, reduce the static component of BPH by inhibiting the activity of
intraprostatic growth factors downstream of the androgen receptor, the dynamic component by
targeting the RhoA/ROCKpathway in prostate and bladder cells, and the inflammatory component by
targeting the NF-κB pathway.
Inflammatory bowel diseases (IBDs) comprise Crohn’s disease (CD) and ulcerative colitis (UC), both
characterized by relapsing inflammation of the gastrointestinal tract due to unbalanced activation of
the mucosal immune system in genetically predisposed individuals. In addition to genetic factors,
including VDR gene polymorphisms, environmental factors are also implicated in IBD development
and notably vitamin D deficiency, suggesting VDR agonists as potential therapeutic agents.
1α,25(OH)2D3 efficacy has been shown in different models of experimental colitis; but its calcemic
liability could pose safety issues in patients and limit its clinical use. Inhibition of TLR4-mediated
TNF-α secretion in PBMCs from healthy donors was used to identify the less calcemic 1α,25(OH)216-ene-20-cyclopropyl-vitamin-D3 (BXL-62) as a VDR agonist with superior TNF-α inhibitory
activity (IC50:1.5x10-15M), compared to 1α,25(OH)2D3 (IC50:8x10-9M). This higher anti-inflammatory
potency from BXL-62 compared to 1α,25(OH)2D3 was confirmed in PBMCs from IBD patients. In
addition, PBMCs from CD and UC patients activated by different TLR agonists are equally sensitive
to the anti-inflammatory properties of BXL-62. Other pro-inflammatory cytokines, such as IL12/23p40 and IL-6, were also inhibited both at mRNA and protein levels. BXL-62 induced VDR
primary response genes, like CYP24 and CAMP, at lower concentrations than 1α,25(OH)2D3,
indicating VDR-mediated effects. The strong potency of BXL-62 can be explained by a different
metabolism resulting in the accumulation of its stable 24-oxo metabolite, which shows antiinflammatory properties superimposable to the parent compound. The efficacy of BXL-62 in
experimental IBD was shown in DSS-induced colitis. Intrarectal administration of BXL-62 induced
faster recovery of clinical symptoms of colitis at normocalcemic doses, and its beneficial effects were
significantly superior to 1α,25(OH)2D3.
The results obtained in this thesis confirm the beneficial effects of VDR agonists in chronic
inflammatory and autoimmune disorders like IBD and BPH and suggest BXL-62 as a potentially
promising compound for IBD treatment.
Medical subject headings: autoimmunity, benign prostate hyperplasia, toll like receptor, antigenpresenting cell, colitis, inflammatory response, elocalcitol, proinflammatory cytokines, metabolite,
lamina propria mononuclear cells, peripheral blood mononuclear cells.
I would like to express my sincere thanks to all people who supported me professionally and
privately during the last years to achieve this milestone in my life and create this thesis.
I acknowledge the department of Biosciences from the University of Kuopio, BioXell S.p.a.
and Intercept Pharmaceuticals from Milan for the friendly and inspiring environment where it
is pleasure to study and to do research.
I am grateful to the European Union Marie Curie Research Trainings Network “NucSys”
which supported this work.
I would like to express my deepest gratitude to my principal supervisors Prof. Carsten
Carlberg and Luciano Adorini, M.D. for the possibility to do my doctoral studies under their
supervision. Thank you for your support, advices and time that lead to the success of this
I acknowledge Giuseppe Penna for his technical and scientific guidance as well as his
Prof. Alberto Muñoz and Prof. Annemieke Verstuyf, the official pre-examiners of this thesis,
are acknowledged for their valuable comments and advices, which helped to improve this
I would like to thank Dr. Arja Hirvonen and Taru Nylund who helped me a lot for all the
administrative tasks to get the thesis accepted.
I would like to thank all my co-authors because without them, none of this work would have
been achieved. Dr. Milan Uskokovic, Dr. Hubert Maehr and Prof. Satya Reddy for sharing
with me their knowledge in chemistry and metabolism of VDR agonists, Elisa Corsiero,
Laura Giudici and Thomas Lemeur for their help in performing the experiments and their
daily good mood and Silvio Danese, M.D. and its group, who introduced me to the
gastrointestinal field and shared their knowledge.
I would like to acknowledge all the members of NucSys for enjoyable and productive
meetings, lots of helpful discussions and all the support, especially Fabio, Carole, Pedro(s),
Marcin, Claudia and Thomas. A special acknowledgement to Tatjana who hosted me in
Kuopio and always support me during these three years, and to Tom for our lovely dinners
and discussions.
I would like to thank people in the Laboratory of Computational Biology from the University
of Luxembourg, specially Aleksandra, Anna and Janine for the kind atmosphere in the lab.
A special thanks to the personnel from BioXell and the Humanitas Institute from Milan for
creating a friendly and productive working environment.
To all my friends scattered around the world who were always present despite the distance,
especially Julien, Bertrand, Manu, Pierre and Béatrice.
I dedicate my doctoral thesis to my parents and grandmother, for their eternal support during
the totality of my studies and this even in times of trouble and difficulties. To my grandfather,
who is certainly taking care of me.
Finally to you, that shared my life, for your love and unlimited support.
1α,25-dihydroxy-vitamin D3
25-hydroxy-vitamin D3
activation function
antigen presenting cell
B cell receptor
benign prostatic hyperplasia
body weight
1α,25(OH)2-16-ene-20-cyclopropyl-24-oxo-vitamin D3
1α,25(OH)2-16-ene-20-cyclopropyl-vitamin D3
cathelicidin anti-microbial peptide
caspase recruitement domain
Crohn's disease
cluster of differentiation
cyclin dependent kinase
5' cytosine-phospho-guanine
cytotoxic T lymphocyte
cytochrome P450
disease activity index
DNA-binding domain
dendritic cell
desoxyribonucleic acid
dextran sodium sulfate
experimental autoimmune prostatitis
fetal bovine serum
fetal clone 1
fragment crystalisable receptor
fibroblast growth factor
granulocyte macrophage colony stimulating factor
genome wide association
human leukocytes antigen
inflammatory bowel disease
immature dendritic cell
immunoglobulin-like transcripts
inducible nitric oxide synthase
immunoreceptor tyrosine-based activation motifs
jun N-terminal Kinase
ligand-binding domain
lamina propria mononuclear cell
leucine-reach repeat domain
lower urinary tract symptoms
mitogen activated protein
monocyte chemotactic protein
mature dendritic cell
myeloid dendritic cell
muramyl dipeptide
major histocompatibility complex
mixed lymphocyte reaction
maximal tolerated dose
molecular weight
nuclear factor of activated T cells
nuclear factor κB
nucleotide binding site (NBS)–leucine-rich repeats
nuclear localisation signal
nitric oxyde
non obese diabetic
nucleotide oligomerisation domain
nuclear receptor
pathogen-associated molecular pattern
peripheral blood mononuclear cell
phosphate-buffered saline
plasmacytoid dendritic cell
prostaglandin E2
polymorphonuclear leukocytes
pathogen recognition pattern
parathyroid hormone
ribonucleic acid
RAR-related orphan receptor
reverse transcription
retinoid X receptor
signal transducer and activator of transcription
type 1 diabetes
T cell receptor
transforming growth factor
T helper cell
toll like receptor
2,4,6-trinitrobenzene sulfonic acid
tumor necrosis factor
regulatory T cell
transient receptor potential vanilloid
ulcerative colitis
vitamin D receptor
vitamin D response element
List of original publications
This thesis is based on the following publications referred to in the text by roman numerals (IV):
Penna G, Fibbi B, Amuchastegui S, Cossetti C, Aquilano F, Laverny G, Gacci M,
Crescioli C, Maggi M, Adorini L Human Benign Prostatic Hyperplasia Stromal Cells
as Inducers and Targets of Chronic Immuno-mediated Inflammation. (2009) Journal
of Immunology 182(7):4056-64
Penna G, Fibbi B, Amuchastegui S, Corsiero E, Laverny G, Silvestrini E,
Chavalmane A, Morelli A, Sarchielli E, Vannelli GB, Gacci M, Colli E, Maggi M,
Adorini L. The vitamin D receptor agonist elocalcitol inhibits IL-8-dependent benign
prostatic hyperplasia stromal cell proliferation and inflammatory response by targeting
the RhoA/Rho kinase and NF-kB pathways. (2009) Prostate 69(5):480-93
Laverny G, Penna G, Uskokovic M, Marczak S, Maehr H, Jankowski P, Ceailles
C, Vouros P, Smith B, Robinson M, Reddy GS, Adorini L. Synthesis and Antiinflammatory Properties of 1alpha,25-Dihydroxy-16-ene-20-cyclopropyl-24-oxovitamin D(3), a Hypocalcemic, Stable Metabolite of 1alpha,25-Dihydroxy-16-ene-20cyclopropyl-vitamin D(3). (2009) Journal of Medicinal Chemistry. 52(8):2204-13
Laverny G, Penna G, Vetrano S, Correale C, Danese S, Adorini L Identification of
a potent and safe VDR agonist for the treatment of inflammatory bowel disease.
(2009) submitted
Laverny G, Penna G, Vetrano S, Correale C, Danese S, Adorini L Toll like
receptor4-dependent selective defect in IL-10 production by blood leukocytes from
inflammatory bowel disease patients. (2009)
Table of content
ABSTRACT ........................................................................................................................................................... 3 ACKNOWLEDGEMENTS .................................................................................................................................. 5 ABBREVIATIONS ............................................................................................................................................... 7 LIST OF ORIGINAL PUBLICATIONS........................................................................................................... 11 TABLE OF CONTENT ...................................................................................................................................... 13 1. INTRODUCTION ........................................................................................................................................... 17 1.1 Overview of the immune system ................................................................................................................ 19 1.1.1 Components of the innate immunity .................................................................................................... 19 Toll like receptors..........................................................................................................................................19 Nucleotide oligomerisation domain-Like Receptors (NLR) ..........................................................................22 Complement ..................................................................................................................................................23 Fragment crystallizable Receptor (FcR) ........................................................................................................24 1.1.2 Cells of the innate immunity................................................................................................................ 25 Mucosal epithelia ..........................................................................................................................................25 Phagocytes.....................................................................................................................................................25 1.1.3 Components of the adaptive immunity ................................................................................................ 27 Major histocompatibility complex.................................................................................................................27 Major histocompatibility complex class I molecules .....................................................................................27 Major histocompatibility complex class II molecules ...................................................................................28 B cell receptor ...............................................................................................................................................28 T cell receptor................................................................................................................................................29 1.1.4 Cells of the adaptive immunity, the lymphocytes ................................................................................ 29 B cells ............................................................................................................................................................30 Effector T cells ..............................................................................................................................................31 Regulatory T cells .........................................................................................................................................35 1.1.5 Dendritic cells, a key role in innate and adaptive immunity ............................................................... 36 Inflammatory dendritic cells..........................................................................................................................37 Myeloid dendritic cells ..................................................................................................................................37 Plasmacytoid dendritic cells ..........................................................................................................................38 Tolerogenic dendritic cells ............................................................................................................................38 1.2 VDR and 1α,25(OH)2D3 ............................................................................................................................. 40 1.2.1 A brief history ..................................................................................................................................... 40 1.2.2 1α,25(OH)2D3 ..................................................................................................................................... 41 Synthesis of vitamin D ..................................................................................................................................41 Catabolism of vitamin D ...............................................................................................................................41 Vitamin D analogs .........................................................................................................................................43 1.2.3 VDR..................................................................................................................................................... 45 NR superfamily .............................................................................................................................................45 Classification .................................................................................................................................................45 Structural features..........................................................................................................................................46 Structure and functions of VDR ....................................................................................................................47 1.2.4 Target genes and biological role ........................................................................................................ 50 Calcium and phosphate homeostasis .............................................................................................................50 1α,25(OH)2D3 metabolism and catabolism.................................................................................................... 51 Vitamin D deficiency ....................................................................................................................................51 1.2.5 VDR-mediated non-calcemic activities ............................................................................................... 53 Regulation of cell proliferation and tumorigenesis ........................................................................................53 Regulation of the immune system .................................................................................................................54 1.2.6 Anti-inflammatory properties of VDR agonists ................................................................................... 55 Dendritic cells ...............................................................................................................................................55 T cells ............................................................................................................................................................58 Regulatory T cells .........................................................................................................................................60 Treatment of autoimmune diseases ...............................................................................................................62 1.3 Begnin prostate hyperplasia ........................................................................................................................ 64 1.3.1 Definition ............................................................................................................................................ 64 1.3.2 VDR agonists in BPH treatment ......................................................................................................... 64 Elocalcitol ameliorates experimental autoimmune prostatitis .......................................................................65 VDR agonists treat BPH-associated LUTS ...................................................................................................66 1.4 Inflammatory bowel disease ....................................................................................................................... 68 1.4.1 Diagnosis and clinical features........................................................................................................... 69 Epidemiology ................................................................................................................................................70 1.4.2 Etiology ............................................................................................................................................... 71 Environmental factors ...................................................................................................................................71 Genetic factors...............................................................................................................................................71 Immunological factors ...................................................................................................................................72 1.4.3 Current treatments .............................................................................................................................. 75 1.5 Vitamin D and inflammatory bowel disease ............................................................................................... 77 1.5.1 Vitamin D deficiency and VDR polymorphisms in IBD patients ......................................................... 77 1.5.2 VDR agonists in IBD treatment .......................................................................................................... 77 In vitro activity ..............................................................................................................................................77 In vivo activity ..............................................................................................................................................78 2. AIMS OF THE STUDY .................................................................................................................................. 80 3. MATERIALS AND METHODS .................................................................................................................... 81 3.1 VDR agonists .............................................................................................................................................. 81 3.2 Cell cultures ................................................................................................................................................ 83 3.2.1 Primary prostate cell lines .................................................................................................................. 83 3.2.2 Immortal cell lines .............................................................................................................................. 83 3.2.3 Peripheral blood mononuclear cells ................................................................................................... 84 3.2.4 Lamina propria mononuclear cells ..................................................................................................... 84 3.3 In vitro experiments .................................................................................................................................... 85 3.3.1 Mixed lymphocyte reaction ................................................................................................................. 85 3.3.2 TLR-activated PBMCs or LPMCs....................................................................................................... 85 3.3.3 BPH cell activation ............................................................................................................................. 86 3.3.4 Enzyme-linked immunosorbent assay (ELISA) ................................................................................... 86 3.3.5 Total RNA purification ........................................................................................................................ 87 3.3.6 cDNA synthesis ................................................................................................................................... 87 3.3.7 Real time PCR ..................................................................................................................................... 87 3.4 In vivo experiments .................................................................................................................................... 88 3.4.1 Mice .................................................................................................................................................... 88 3.4.2 Assessment of the MTD ....................................................................................................................... 88 3.4.3 Induction of experimental colitis......................................................................................................... 89 3.4.4 Administration of VDR agonists ......................................................................................................... 89 3.4.5 Assessment of inflammation ................................................................................................................ 89 3.4.6 Histology ............................................................................................................................................. 90 3.5 Statistical analysis....................................................................................................................................... 91 4. RESULTS ......................................................................................................................................................... 92 4.1 BPH cells can act as non-professional APCs to induce chronic prostate inflammation ............................. 92 4.2 VDR agonist elocalcitol inhibits IL-8-dependent BPH cell proliferation and inflammatory response ....... 93 4.3 Potent anti-inflammatory properties of 1α,25(OH)2-16-ene-20-cyclopropyl-vitamin D3 (BXL-62) in
inflammatory bowel disease models ................................................................................................................. 95 4.4 24-oxo BXL-62 metabolite exerts biological activities similar to its parent compound ............................. 98 4.5 Specific IL-10 production deficiency in inflammatory bowel disease patients compared to healthy controls
.......................................................................................................................................................................... 99 5. DISCUSSION................................................................................................................................................. 102 5.1 Prostatic cells as inducers and targets of chronic inflammation ............................................................... 102 5.2 VDR agonists inhibit intraprostatic inflammatory responses ................................................................... 103 5.3 TLR specific deficiency for IL-10 production .......................................................................................... 104 5.4 BXL-62 ameliorates symptoms in experimental model of colitis............................................................. 106 5.5 24-oxo metabolite accumulation, a key event for BXL-62 potency ......................................................... 108 6. SUMMARY AND CONCLUSIONS ............................................................................................................ 110 7. FUTURE ASPECTS...................................................................................................................................... 112 8. REFERENCES .............................................................................................................................................. 113 APPENDIX: ORIGINAL PUBLICATIONS .................................................................................................. 136 1. Introduction
The Roman numerals (I-V) refer to the manuscripts included in the thesis, as classified in the
list of original publications.
Despite all the efforts for the improvement of sanitary conditions and vector control,
infections remain the leading cause of morbidity and mortality worldwide and represent a
major challenge for the biomedical sciences. The development of vaccines and therapeutics is
absolutely required, and these implicate a deeper understanding of the host immune system.
The mammalian immune system is divided between innate immunity and adaptive immunity,
which cooperate to protect the host against microbial and viral infections. The innate immune
system is the phylogenetically older system to control microbe invasion and represents an
immediate and direct immune response, induced after recognition of specific composite of
bacteria, called pathogen-associated molecular pattern (PAMP). Conversely, the adaptive
immune system, evolutionary more recent, is a later addition to the immune system, mediated
by antibodies (humoral immunity) or by T and B lymphocytes (cell-mediated immunity).
Such host-pathogen discrimination is essential for the host capacity to eliminate the pathogen
without excessive damage to its own tissues. This avoidance of destruction of self-tissues is
referred to as self-tolerance. Environmental factors, in genetic predisposed individuals could
lead to failure of this control system, leading to autoimmune diseases.
Vitamin D is produced in the skin by enzymatic modifications of cholesterol after
exposure to ultraviolet B (UVB) radiation. 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), the
active form of vitamin D, is produced in the kidney by hydroxylation of its precursor, 25hydroxyvitamin D3 (25(OH)D3), and plays a central role in calcium homeostasis and bone
remodelling. 1α,25(OH)2D3 effects are mediated by its receptor, the vitamin D receptor
(VDR), a member of the superfamily of nuclear receptors (NRs). Since the discovery of VDR
expression in cells regulating the immune response, 1α,25(OH)2D3 was shown to have
benefits in various models of autoimmune and chronic inflammatory diseases. In addition,
recent epidemiological studies correlate auto-immune disorders with low 25(OH)D3 serum
levels. Since the supra-physiologic doses of 1α,25(OH)2D3 required to show robust antiinflammatory effects induce hypercalcemia, vitamin D analogues were synthesized in order to
potentiate anti-inflammatory properties of VDR agonists without inducing hypercalcemic side
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD
Inflammatory bowel diseases (IBD) are chronic, relapsing inflammatory disorders of
the gastrointestinal tract, most commonly the terminal ileum and colon, with two major forms
recognized: Crohn’s disease (CD) and ulcerative colitis (UC). Whether or not these IBD
subtypes fully share pathogenic mechanisms, the underlying factors are similar. Their current
pathogenesis is focussed on deregulated mucosal immune response against intestinal bacterial
flora in genetically predisposed individuals. In addition to genetic factors, including VDR
gene polymorphisms, many environmental factors are also implicated in IBD development,
and in this context vitamin D deficiency, especially observed in Northern latitudes, is now
well documented as a high-risk factor for IBD pathogenesis. VDR expression is required to
control inflammation of spontaneous and induced colitis models, as demonstrated by
exacerbation of symptoms in Vdr-deficient mice. In addition, 1α,25(OH)2D3 has been shown
to ameliorate spontaneous colitis in mice fed with a low calcium diet.
This thesis extends the research on the potential used of VDR agonists in autoimmune
disorder by identifying a potent anti-inflammatory VDR agonist as potential treatment for
IBD and in addition, through the understanding of the mechanisms of action of this analog,
contributes to the identification of events involved in the pathogenesis of experimental colitis.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
1.1 Overview of the immune system
1.1.1 Components of the innate immunity
The innate immune system includes defence mechanisms that are encoded in the host
germline. These include the epithelial barriers and the mucociliary blanket that sweeps away
inhaled or ingested particles. They also include soluble proteins and bioactive small molecules
that are either constitutively present in biologic fluids (for example, the complement proteins
and defensins) or that are released from cells as they are activated (including cytokines that
regulate the function of other cells, chemokines that attract inflammatory leukocytes, lipid
mediators of inflammation, and bioactive amines and enzymes). Lastly, the innate immune
system includes pattern recognition receptors (PRR) that bind PAMP expressed on the
surfaces of invading microbes or viruses. Toll like receptors
The Toll like receptor (TLR) family has been extremely conserved during evolution. This
family is the homolog of the Drosophila protein called Toll that was identified as a maternaleffect gene (Hashimoto, Hudson et al. 1988). TLRs are type I transmembrane proteins
characterized by an extracellular leucine-rich repeat domain (LRR) coupled to an intracellular
Toll/IL-1 receptor (TIR) with homology to the cytoplasmic domain of the IL-1 receptor
(Hashimoto, Hudson et al. 1988; Medzhitov, Preston-Hurlburt et al. 1997; Medzhitov 2001;
Athman and Philpott 2004). So far, 13 members have been identified in mammals, 11 in
humans (TLR1-11) and 12 in mice (TLR1-9 and TLR11-13). For most of them, a specific
ligand has been identified (Athman and Philpott 2004). These ligands are lipid, carbohydrate,
peptide and nucleic acid structures representing common structural features of
microorganisms, known as PAMP (Table 1).
Subcellular localisation of the TLR is associated to its type of ligand. TLR1, 2, 4, 5
and 6, which recognize microbial specific components, are expressed on the cell surface,
while TLR3, 7, 8 and 9, which recognize amino acids, are localized in endosomes or
lysosomes (Athman and Philpott 2004).
The primary function of TLRs is to signal that microbes have breached the body’s
barrier defenses. TLRs are highly expressed on macrophages and dendritic cells (DCs) but are
also expressed on neutrophils, eosinophils, epithelial cells and keratinocytes. Activation of
most TLR induces cellular responses associated with acute and chronic inflammation
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD
(Medzhitov 2001). When TLR ligands interact with their specific TLRs, intracellular adaptor
proteins transduce signals that lead to enhanced expression of genes encoding proinflammatory cytokines and other inflammatory mediators. For example, tissue-resident
macrophages stimulated by TLR agonists produce pro-inflammatory cytokines, including
tumor-necrosis factor α (TNF-α), interleukin-1β (IL-1β), IL-12 and IL-6, which coordinate
local and systemic inflammatory responses (Medzhitov, Preston-Hurlburt et al. 1997; Athman
and Philpott 2004). In addition, TLR activation is the main signal for dendritic cell (DC)
maturation (Steinman, Hawiger et al. 2003). Molecular mechanisms of TLR activation
involve numbers of proteins enrolled in two main pathways depending on the adaptor protein
Myd88 implication. For example, binding of bacterial lipopolysaccharide (LPS) to TLR4 in
association with the MD2 and CD14 coreceptors elicits signaling through both the MyD88
pathway (using the adaptor TIR-domain-containing adaptor protein (TIRAP), also known as
MyD88-adaptor-like protein, MAL) to activate nuclear factor κB (NF-κB) and proinflammatory responses, and also through an MyD88-independent pathway, including the
adaptors TRIF (TIR domain–containing adaptor inducing IFN-β), TRAM (TRIF-related
adaptor molecule) and TBK1 (TRAF family member-associated NF-κB activator-binding
kinase) leading to the phosphorylation and nuclear translocation of IFN-regulatory factor 3
(IRF3) and expression of IFN-β and induction of anti-inflammatory responses (Athman and
Philpott 2004).
TLRs, in addition to their role in driving the innate immune system, are able to shape
the adaptive immune system and especially the Th1/Th2 balance. Activation of nearly all
TLRs programs T helper cell type 1 (Th1) by TLR-induced IL-12 production, as shown by the
defect of Th1 response in Myd88-deficient mice upon immunization with ovalbumin in
Freund’s complete adjuvant. However, a specific TLR2 ligand suppresses IL-12 and enhances
IL-10 production, favoring a T helper cell type 2 (Th2) response via an ERK/MAPKdependent mechanism (Medzhitov, Preston-Hurlburt et al. 1997). Furthermore, TLRs that
induce strong production of TGF-β, IL-6 and IL-23 promote IL-17 producing T helper cell
type 17 (Th17) (Manicassamy and Pulendran 2009).
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
Table 1. Human TLRs and their respective ligands. Eleven TLR were identified in the human
genome and only for nine of them the ligands have been characterized. The ligand is representated of
the subcellular localization, since the cellular TLR1, 2, 4, 5 and 6 are expressed on the cell membrane
and recognize specific surface microbial components, while TLR3, 7, 8 and 9 are localized in
endosomes or lysosomes and recognize amino acids (Akira and Takeda 2004).
Triacyl lipopeptides
Soluble factors
Lipoteichoic acid
Phenol-soluble modulin
Atypical lipopolysaccharide
Atypical lipopolysaccharide
Double-stranded RNA
Fusion protein
Envelope protein
Heat-shock protein 60
Diacyl lipopeptides
Lipoteichoic acid
Single-stranded RNA
Single-stranded RNA
CpG-containing DNA
Origin of ligand
Bacteria and mycobacteria
Neisseria meningitidis
Various pathogens
Gram-positive bacteria
Gram-positive bacteria
Staphylococcus epidermidis
Trypanosoma cruzi
Treponema maltophilum
Leptospira interrogans
Porphyromonas gingivalis
Gram-negative bacteria
Respiratory syncytial virus
Mouse mammary-tumor virus
Chlamydia pneumoniae
Gram-positive bacteria
Synthetic compounds
Synthetic compounds
Synthetic compounds
Synthetic compounds
Bacteria and viruses
Uropathogenic bacteria
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Nucleotide oligomerisation domain-Like Receptors (NLR)
As discussed in the previous section, detection of invaders by the host is mediated by the
recognition of PAMPs by specific PRRs. While TLR recognition involves mainly membrane
receptors, in the cytoplasm are present PRR, termed nucleotide binding site (NBS)–leucinerich repeats (NLR), able to sense cytosolic invasion. NLRs homologues are found in plants
and are involved in the hypersensitive response against virulent plant pathogens (Athman and
Philpott 2004). Bioinformatics approaches revealed the presence in the human genome of 23
NLRs genes, whereas about 34 genes are found in the mouse genome. General domain
organisation of NLRs include a variable N‑terminal effector binding domain, a central
nucleotide-binding domain (NBD) and C‑terminal leucine-rich repeats detecting PAMPs
(Kanneganti, Lamkanfi et al. 2007).
Based on the structure of the N terminal domains, NLRs contain 3 sub-families:, the
caspase recruitement domain (CARD)-containing nucleotide oligomerization domain (Nods),
the pyrin (PYD) and baculovirus inhibitor repeat (BIR). Nod1 and Nod2 (CARD15) sense
bacterial molecules resulting from the synthesis and/or degradation of peptidoglycan (PGN).
Nod1 recognizes the dipeptide-γ-D-glutamyl-meso-diaminopimelic produced by Gramnegative bacteria and specific Gram-positive bacteria. Nod2 is mainly expressed in cells of
the myeloid lineage and is sensing the muramyl dipeptide (MDP), present on all types of
PGN. Upon ligand binding, conformation changes lead to the recruitment of the serinethreonine kinase RICK (also called RIP2) through the CARD domain. This dimerization
results in the degradation of NF-κB inhibitor IκBα, allowing the nuclear translocation of
active NF-κB. In addition, Nod1 and Nod2 activation could also activate mitogen activated
protein (MAP) kinases such as p38 or JNK (Jun N-terminal kinase). Finally, NF-κB and MAP
kinases activation induce production of pro-inflammatory cytokines and promote the
recruitment of neutrophils to the site of infection (Inohara, Chamaillard et al. 2005).
Furthermore, Nod2 is essential in the production of anti-microbial peptides, such as defensins
in Paneth cells (Lala, Ogura et al. 2003).
The importance of NLRs is highlighted by their genetic variation, which correlates
with disease susceptibility. Mutations in Nod2 were recently correlated with an increased
susceptibility to the chronic intestinal inflammatory disease, Crohn’s disease. One of the most
common mutation associated with this disease leads to the inability of the mutant protein to
respond to MDP and to its incapacity to activate NF-κB. However, it is still not understood,
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how the inability to sense bacterial products might lead to the aberrant inflammation that
characterizes Crohn’s disease (Cho and Abraham 2007). Complement
The complement system is part of the innate immune response and underlies one of the main
effector mechanisms of antibody-mediated immunity. The main role of complement is to
defend the organism against bacterial infections, bridging innate and adaptive immunity and
disposing of immune complexes and the products of inflammatory injury (Walport 2001). The
complement system is composed of more than 30 plasma and cell surface proteins and 3
different pathways have been described, the classical pathway, the alternative pathway and
the mannose-binding lectin pathway (Walport 2001; Chaplin 2003; Chaplin 2006).
The first pathway discovered, and then called classical pathway, is initiated by the
binding of the C1 complex (which consists of a C1q molecule, two molecules of C1r, and two
molecules of C1s) to antibodies bound to an antigen on the surface of a bacterial cell via
fragment crystalisable receptor (FcR). C1s first cleaves C4, which binds covalently to the
bacterial surface, and then cleaves C2, leading to the formation of a C4b2a enzyme complex,
the C3 convertase of the classical pathway (Walport 2001; Walport 2001; Chaplin 2003;
Chaplin 2006).
The mannose-binding lectin pathway is triggered by microbial cell wall components
containing mannans and is called the lectin pathway of complement activation. Interaction
between mannan-containing microbes and mannose-binding lectin–associated proteases 1 and
2 (MASP1 and MASP2, respectively) result to the lise of the mannose groups on the surface
of a bacterial cell. These form a protease analogous to the activated C1 of the classic pathway
that then goes on to activate C4, C2, and the remainder of the pathway (Walport 2001;
Walport 2001; Chaplin 2003; Chaplin 2006).
The alternative pathway is antibody-independent and is initiated by the covalent
binding of a small amount of C3b to hydroxyl groups on cell-surface carbohydrates and
proteins and is activated by low-grade cleavage of C3 in plasma. This C3b binds factor B, a
protein homolog to C2, to form a C3bB complex. Factor D cleaves factor B bound to C3b to
form the alternative pathway C3 complex C3bBb (Walport 2001; Walport 2001; Chaplin
2003; Chaplin 2006).
The C3 convertase enzyme is really efficient to cleave C3 in C3b. Then C3b binds
covalently around the site of complement activation. Some C3b binds to the C4b and C3b in
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the classical and alternative pathways, respectively, forming C5 convertase enzymes. This
C3b acts as an acceptor site for C5, which is cleaved to form C5a and C5b, and initiates the
formation of the membrane-attack complex, a complex of the complement proteins C6, C7,
C8, and C9, which are assembled into a membrane pore that causes lysis of the cells where
the complement is activated (Chaplin 2003; Chaplin 2006). Fragment crystallizable Receptor (FcR)
In contrast to B Cell Receptors (BCRs) and T Cell Receptors (TCRs), receptors for the
fragment crystallizable (Fc) domain of the immunoglobulins (Ig) do not recognize antigens
but the Fc portion of antibodies. FcR play an important role in immune defense. FcRs for IgG
and IgE are present on the surface of several cell types of the immune system. These
receptors, designated FcγRs for those that bind IgG, and FcεRs for those that bind IgE,
interact with antibody-antigen complexes to activate various biological responses. The
biological responses elicited include antibody-dependent cell-mediated cytotoxicity,
phagocytosis, release of inflammatory mediators and regulation of lymphocyte proliferation
and differentiation (Daeron 1997).
Affinities and genes encoding FcR present heterogenity within the family. FcγR is
divided in three major classes (FcγRI, FcγRII, FcγRIII) and FcεRs in two (FcεRI and FcεRII).
The form I recognizes with high affinity the Fc, while forms II and III bind Fc with a lower
affinity. The Ig-binding portions of FcγRI, FcγRII, FcγRIII and FcγRI (the γ chains) are
members of the Ig gene superfamily, all type I transmembrane proteins containing an
extracellular region with two or more Ig-like domains and a polypeptide or lipid anchor in the
membrane. The extracellular regions of the FcεR and FcγRI receptors show significant
sequence similarity to each other: 70-98% sequence identity within the FcγRs and about 40%
sequence identity between FcεRs and FcγRI (Raghavan and Bjorkman 1996).
FcRs capable of triggering cell activation possess one or several intracytoplasmic
activation motifs designated immunoreceptor tyrosine-based activation motifs (ITAMs),
which resemble to those of the BCR and TCR signal transduction subunits. This signal
activates sequentially src family tyrosine kinases and syk family tyrosine kinases that connect
transduced signals to common activation pathways shared with other receptors (Ravetch and
Bolland 2001).
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1.1.2 Cells of the innate immunity
Cells involved in innate immune response are essentially phagocytes differentiated from the
same myeloid progenitor. These cells do not need a somatic rearrangement, because most of
the genes involved in the innate immune response are encoded in the genome. The old
concept that cells of the innate immune system have as only role the engulfement of
pathogens and then its destruction by apoptosis is incorrect. A wealth of evidence shows that
engagement of innate immune mechanisms is required for shaping the adaptive immune
response, such as the maturation of DCs, enhancing major histocompatibility complex (MHC)
class II expression on their surface and promoting antigen presentation to T cells. Mucosal epithelia
The mucosal epithelia is one of the most ancient and universal modules of innate immunity.
The mucosal epithelia from the skin, airways, reproductive tract and intestine are the main
interface between the host and the microbial world (including both pathogenic and symbiotic
microorganisms) and therefore are susceptible to colonization and invasion of pathogens, such
as viruses, bacteria, fungi or parasites. Maintaining homeostasis with symbiotic bacteria while
protecting the host from pathogen invasion represents a hard challenge for the mucosal
epithelia (Artis 2008).
During evolution, mammals have developed a mucosa associated lymphoid tissues,
such as the Peyer’s patches in lamina propria in the gut, which are rich in cells involved in
innate or adaptive immune responses, in order to lighten the mucosal challenge. Epithelia
present the complete array of PRRs and in addition to the soluble factors produced after
activation; they are able to produce anti-microbial peptides, like defensins or cathelicidin antimicrobial peptide (CAMP), which are potent immuno-regulators. In addition, epithelial cells
at the mucosal surface can produce mucins inhibiting the attachment and entry of pathogens
(Medzhitov 2007; Artis 2008). Phagocytes
Phagocytes are white blood cells, deriving from stem cell and having a common myeloid
precursor. The primary function of phagocytes is to identify and engulf microbes.
Phagocytosis is a very complex process having as main role the production of molecules
required for efficient antigen presentation to the adaptive immune system after pathogen
recognition. This is accompanied by intracellular signals that trigger cellular processes as
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diverse as cytoskeletal rearrangement, alterations in membrane trafficking, activation of
microbial killing mechanisms, production of pro- and anti-inflammatory cytokines and
chemokines and activation of apoptosis (Underhill and Ozinsky 2002).
The two most important members of this family are macrophages and neutrophils that
share the same initial stage, the monocyte. Circulating monocytes represent 5-10% of
circulating leukocytes, leave the bone marrow incompletely differentiated and then give rise
to a variety of tissue-resident neutrophils and macrophages throughout the body, as well as to
specialized cells, such as DCs (Volkman and Gowans 1965).
In order to discriminate between infectious agents and self components, phagocytes
have evolved a restricted number of phagocytic receptors, such as mannose and lectin
receptors, PRR, FcR and the broad class of complement proteins. In addition, they express
MHC class I and II molecules, but they represent poorly efficient antigen-presenting cells
(APC) (Jensen 2007).
In addition to their crucial role in the embryogenesis, the main functions of
macrophages in the immune system are recognition and phagocytosis of pathogens, antigen
presentation, production of superoxide, cytokines and chemokines for the recruitement of
effector cells to the site of inflammation (Artis 2008). Macrophages are pleiomorphic in
different tissues, defined as Langerhans cells in the epidermis, Kuppfer cells in the liver,
microglia in the central nervous system, osteoclast in the bone and alveolar macrophages in
the lung. In the gut, macrophages resides in the lamina propria but are not specifically named.
The important step in the functional maturation and inflammation of macrophages is the
conversion from a resting to an activated macrophage, meaning that they have increased their
capacity to kill microbes (Medzhitov 2007).
Polymorphonuclear leukocytes (PMNs or neutrophils) are the most important
population of leukocytes. Neutrophils play an essential role in the human innate immune
system because they are usually the first cell type recruited to sites of infection or areas of
inflammation. Then, neutrophils are able to orchestrate the inflammatory response by
recruiting, activating and programming APCs. As macrophages, neutrophils destroy
microorganisms by phagocytosis. Interestingly, neutrophils present a short half-life, a
mechanism of resolution of the inflammatory response (Theilgaard-Monch, Porse et al. 2006).
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1.1.3 Components of the adaptive immunity
In contrast with innate immunity, mechanisms for generating receptors in the adaptive
immune system involve great variability and rearrangement of receptor gene segments. The
adaptive immune system can provide specific recognition of foreign antigens, immunological
memory of infection and pathogen-specific adaptor proteins. However, the adaptive immune
response is also responsible for allergy, autoimmunity and the rejection of tissue grafts
(Janeway and Medzhitov 2002).
Two main pathways encompass the adaptive immune system, humoral and cellmediated immunity where Ig secreted by B cells, and the TCR are the proteins presenting the
somatic rearrangements. Cell-mediated immunity, orchestrated by T cells, serves as a defense
mechanism against microbes that survive within phagocytes or infect nonphagocytic cells,
while humoral immunity, mediated by secreted antibodies, protects mostly against
extracellular microbes and microbial toxins (Janeway and Medzhitov 2002). Major histocompatibility complex
T cell-mediated immune response recognize antigen present only on infected cells, but not
free antigen in solution. This mechanism is possible because T cells, in addition to the
microbial antigen, have to recognize self structures. These self structures are the antigenic
peptide-binding major histocompatibility complex (MHC) molecules, also called human
leukocytes antigen (HLA) in humans. Major histocompatibility complex class I molecules
MHC class I molecules are cell surface heterodimers, consisting of a polymorphic
transmembrane 44 kDa α-chain (named also class I heavy chain) and a 12 kDa nonpolymorphic β2 microglobulin protein, both non covalently linked. Three distinct classes of
MHC class I molecules have been defined in humans, called HLA-A, HLA-B and HLA-C
determined by α-chain genes encoded within the same chromosome. The fully assembled
class I molecule is a heterotrimer consisting of a β2-microglobulin chain, a bound antigenic
peptide, and the α chain stably expressed on cell surfaces (Chaplin 2003).
Binding of antigen to the MHC class I pocket results in a structure that is the
molecular target for TCR. Antigens presented by MHC class I molecules are described as
“endogenous peptides” or self antigens due to the fact that they derive from proteins produced
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within the cells and processed by the proteasome and the endoplasmic reticulum. Antigen can
be recognized by the TCR only in combination with the MHC molecule and this phenomenon
is known as “MHC restriction”. The simultaneous recognition of antigenic peptide bound to
MHC molecules by the TCR ensures efficacy and specificity of T cell responses, allowing T
cells to ignore free extracellular antigen and to focus on infected cells. MHC class I
extracellular domains interact only with CD8+ cytotoxic T lymphocytes (CTLs) resulting in
the lysis of the target cell expressing the appropriate peptide-MHC combination (Jensen
2007). Major histocompatibility complex class II molecules
As the MHC class I molecules, MHC class II molecules are formed by two polypeptide
chains, but both are MHC-encoded transmembrane proteins, called α and β chains. The three
major human MHC class II molecules are designated as HLA-DR, HLA-DP and HLA-DQ.
Each MHC class II chain contains a cytoplasmic anchor, a transmembrane domain and two
extracellular domains. The α2 and β2 domains provide a unique support for CD4 binding
(Chaplin 2003).
In contrast to MHC class I molecules, which are expressed on all cell types, MHC
class II molecules are expressed only on particular cells called APCs. Professional APCs are
DCs and macrophages. As non-professional APCs are considered neutrophils, basophils and
B cells (Jensen 2007). This list could be extended, because many cell types are able to present
MHC class II molecules on their surface. Antigenic peptides presented by MHC class II
molecules result from the lysosomal and endosomal degradation of phagosized products,
before transport into a specialized MHC class II loading compartment. Thus, MHC class II
molecules present exogenous antigens to CD4+ T cells, alerting them about the presence of
intracellular invaders (Watts 2004). B cell receptor
As previously mentioned, the BCR is a member of the multichain immune recognition
receptor family that includes the TCR and FcεR1. The BCR, expressed only in mature B cells,
is composed of a membrane-anchored specific immunoglobulin associated with a 32kDa
phosphoprotein Ig-α and Ig-β. The latter, also defined as CD79α and CD79β, form a dimer
through a disulfide bridge and show homology with CD3α and β from the TCR. They present
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a 26 aa intra-cytoplasmic region called immunoreceptor tyrosine-based activation motif
(ITAM) (Chaplin 2003).
Once activated, ITAM is phosphorylated by the Src family kinase Lyn, providing a
binding site for the SH2-domain-containing kinase Syk, triggering the signalling cascades.
These phosphorylations events require co-stimulatory molecule such CD19 and CD45 that
once activated, induce the dephosphorylation of the negative regulatory site of src family
kinases. Antigen recogntion result in its internalisation and its processing in the MHC-II
complex, where the peptide takes place (Kurosaki 1997). T cell receptor
The TCR, expressed on the surface of T lymphocytes, is a transmembrane protein consisting
of a heterodimer formed by α/β chains or by γ/δ chains, resulting in the subclassification of T
cells in α/β or γ/δ T cells. As in antibodies, each chain of the TCR heterodimer contains a
variable and a constant region, where the variable region possesses 3 complementarity
determining regions allowing the recognition of peptides presented by MHC molecules.
TCRs, as BCRs, are associated with transmembrane molecules that allow signal transduction.
For the TCR, these proteins form the CD3 complex, constituted by the transmembrane
accessory molecules CD3γ, CD3δ, CD3ε, and a CD3ζ intracytoplasmic homodimer
(Zidovetzki, Rost et al. 1998).
As discussed for B cells, the cytoplasmic domain of CD3 contains an ITAM domain,
that once phosphorylated by receptor-associated kinases Lck and Fyn, initiates an activation
cascade involving the proteins ZAP-70, LAT, and SLP-76. These phosphorylation events
result in the stimulation of phospholipase C, activation of the G proteins Ras and Rac and
both protein kinase C and the MAP kinases. Activation of this pathway controls T cell
activation and proliferation (Smith-Garvin, Koretzky et al. 2009).
1.1.4 Cells of the adaptive immunity, the lymphocytes
Phagocytes and lymphocytes share the same precursors, the hematopoietic stem cells.
However, this common precursor gives rise to two main subclasses, myeloid stem cells, from
which phagocytes originate and lymphoid stem cells generating T and B cells and
plasmacytoid DCs (Fig. 1).
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Fig. 1. Major haematopoietic stem cell maturation pathways. Within the haematopoietic
system, the haematopoietic stem cell (HSC) has self-renewal potential and multipotent differentiation
potential. Its progeny include multipotent common lymphoid progenitors (CLPs) and common myeloid
progenitors (CMPs). In turn, these progenitors give rise to progenitors that have more limited
differentiation potential, Pro-B and Pro-T cells, megakaryocyte erythroid progenitors (MEPs) and
granulocyte monocyte progenitors (GMPs). In the myeloid lineage, MEPs give rise to mature
erythrocytes and platelets in the peripheral blood. GMPs give rise to monocytes and the various
granulocyte lineages. In the lymphoid lineages Pro-B and Pro-T cells give rise to mature (naive) B and
T lymphocytes and then stimulated B and T cells, respectively, following exposure to antigen. Adapted
from (Huntly and Gilliland 2005). B cells
B cells are lymphocytes that play a large role in the humoral immune response, as opposed to
the cell-mediated immune response, which is governed by T cells. Their definition comes
from the bursa of Fabricius in birds, where they mature. In mammals, immature B cells are
formed in the bone marrow (Raff 1973).
B cells differentiate from hematopoetic stem cells in the bone marrow under the
control of IL-7 produced by stromal cells (Burrows and Cooper 1997). Their maturation takes
subsequently place in lymph node follicles. B cells represent about 15% of the total
leukocytes (Chaplin 2003). On their surface they express co-stimulatory molecules like
CD19, CD81 or CD21, but also MHC-II molecules (Burrows and Cooper 1997). The main
function of B cells is the production of Igs, antigen-binding proteins also known as antibodies
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(LeBien and Tedder 2008). Antibodies are polypeptides formed by two heavy and two light
chains linked by disulfide bonds. Antigen specificity is provided by the diversity of Nterminal regions (called variable regions), while the conserved C-terminal part is defined Fc
(Chaplin 2003). An antibody is composed of two identical light (L) and two identical heavy
(H) chains, and the genes specifying them are found in the 'V' (Variable) region and the 'C'
(Constant) region. In the heavy chain 'V' region there are three segments; V, D and J, which
recombine randomly, in a process called VDJ recombination, to produce a unique variable
domain in the immunoglobulin of each individual B cell (Chaplin 2003). Similar
rearrangements occur for light-chain 'V' region except there are only two segments involved;
V and J. The amino terminal portion of each heavy chain is created by somatic joining of
genes encoding a variable (VH), diversity (DH), and joining (JH) region (Chaplin 2003). The
VH-JH and VL-JL light chain junctions formed by this recombination make up the third
hypervariable region that contributes to the antigen-binding site. B cell development occurs
through several stages, each stage representing a change in the genome content at the antibody
loci (Schatz, Oettinger et al. 1992).
B cells are the key cell of the humoral immunity, representing an essential component
of the adaptive immune system, but can also secrete a number of cytokines and chemokines.
In addition, B cells express MHC class II molecules and can act as non-professional APCs,
thus playing a role also in immunoregulation. They eventually develop into memory B cells
after activation by antigen interaction B cells. A critical difference between B cells and T cells
is based on their mode of antigen recognition. B cells recognize their cognate antigen in its
native form (Batista and Harwood 2009). They recognize free (soluble) antigen in the blood
or lymph using their BCR or membrane-bound Ig. In contrast, T cells recognize their cognate
antigen in a processed form, as a peptide fragment presented to the TCR by MHC molecules
of APCs (Smith-Garvin, Koretzky et al. 2009). Effector T cells
All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic
progenitors derived from hematopoietic stem cells populate the thymus and expand by cell
division to generate a large population of immature thymocytes (Schwarz and Bhandoola
2006). The earliest thymocytes express neither CD4 nor CD8, and are therefore classified as
double-negative (CD4-CD8-) cells. As they progress through their development they become
double-positive thymocytes (CD4+CD8+), and finally mature to single-positive (CD4+CD8- or
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CD4-CD8+) thymocytes that are then released from the thymus to peripheral tissues (Ellmeier,
Sawada et al. 1999). About 98% of thymocytes die during the development processes in the
thymus, which involves positive and negative selection (Delves and Roitt 2000).
Positive selection "selects for" T-cells capable of interacting with MHC molecules
(Delves and Roitt 2000). Double-positive thymocytes (CD4+/CD8+) move deep into the
thymic cortex where they are presented with self-antigens (i.e., antigens that are derived from
molecules belonging to the host of the T cell) complexed with MHC molecules on the surface
of cortical epithelial cells (Robey and Fowlkes 1994). Only those thymocytes that bind the
MHC/antigen complex with adequate affinity will receive a "survival signal". Developing
thymocytes that do not have adequate affinity cannot serve useful functions in the body (i.e.
the cells must be able to interact with MHC molecules and peptide complexes in order to
affect immune responses) (Marrack and Kappler 2004). Because of this, the thymocytes with
low affinity die by apoptosis and are engulfed by macrophages (Cohen, Duke et al. 1992) .
A thymocyte's fate is also determined during positive selection (Starr, Jameson et al.
2003). Double-positive cells (CD4+/CD8+) that are positively selected on MHC-II molecules
will eventually become CD4+ cells, while cells positively selected on MHC-I molecules
mature into CD8+ ells (Ellmeier, Sawada et al. 1999). A T cell becomes a CD4+ cell by downregulating expression of its CD8 cell surface receptors (Delves and Roitt 2000). If the cell
does not lose its signal through the ITAM pathway, it will continue down-regulating CD8 and
become a CD4+, single positive cell. But if there is signal drop, the cell stops down-regulating
CD8 and switches over to down-regulating CD4 molecules instead, eventually becoming a
CD8+, single positive cell (Starr, Jameson et al. 2003).
Negative selection removes thymocytes that are capable of strongly binding with
"self" peptides presented by the MHC complex (Starr, Jameson et al. 2003). Thymocytes that
survive positive selection migrate towards the boundary of the thymic cortex and thymic
medulla. While in the medulla, they are again presented with self-antigen in complex with
MHC molecules on APCs, such as DCs and macrophages (Anderson, Moore et al. 1996).
Thymocytes that interact too strongly with the antigen receive an apoptotic signal that leads to
cell death. The vast majority of all thymocytes end up dying during this process. The
remaining cells exit the thymus as mature naive T cells. This process is an important
component of immunological tolerance and serves to prevent the formation of self-reactive T
cells that are capable of generating autoimmune diseases in the host (Starr, Jameson et al.
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Although the specific mechanisms of activation vary slightly between different types
of T cells, the "two-signal model" in CD4+ T cells holds true for most. Activation of CD4+ T
cells occurs through the engagement of both the TCR and CD28 on the T cell by the MHCpeptide complex and B7 family members on the APC, respectively. Both are required for
production of an effective immune response; in the absence of CD28 co-stimulation, TCR
signaling alone results in anergy (Schwartz 1997). The signaling pathways downstream from
both CD28 and the TCR involve many proteins (Smith-Garvin, Koretzky et al. 2009).
The first signal is provided by binding of the TCR to a short peptide presented by the
MHC molecule. This ensures that only a T cell with a TCR specific to that peptide is
activated. The partner cell is usually a professional APC, a dendritic cell in the case of naïve
responses, although B cells and macrophages can be important APCs (Itano and Jenkins
2003). The peptides presented to CD8+ T cells by MHC-I molecules are 8-9 amino acids in
length; the peptides presented to CD4+ cells by MHC-II molecules are longer, as the ends of
the binding cleft of the MHC-II molecule are open (Jensen 2007).
The second signal comes from co-stimulation, in which surface receptors on the APC
are induced by a relatively small number of stimuli, usually products of pathogens, but
sometimes breakdown products of cells, such as necrotic-bodies or heat-shock proteins
(Akira, Takeda et al. 2001). The only co-stimulatory receptor expressed constitutively by
naïve T cells is CD28, so co-stimulation for these cells comes from the CD80 and CD86
proteins on the APC (Adorini, Penna et al. 2004). Other receptors are expressed upon
activation of the T cell, such as OX40 and ICOS, but these largely depend upon CD28 for
their expression (Smith-Garvin, Koretzky et al. 2009). The second signal licenses the T cell to
respond to an antigen. Without it, the T cell becomes anergic, and it becomes more difficult
for it to activate in future. This mechanism prevents inappropriate responses to self, as selfpeptides will not usually be presented with suitable co-stimulation (Schwartz, Mueller et al.
As the Th cells continue to respond to the activating signal, they progress toward polar
extremes of differentiation designated Th1 and Th2, depending on the nature of the cytokines
present at the site of activation (Zenewicz, Antov et al. 2009) (Fig. 2).
Naïve T cells can be differentiated in vitro into Th1 cells by culturing with IL-12, an
innate-system-derived cytokine that is highly expressed by activated macrophages and DCs
(Hsieh, Macatonia et al. 1993). IL-12 activates signal transducer and activator of transcription
4 (STAT4) signaling pathways, resulting in activation of the genes encoding the cytokine
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IFN-γ and the T-box family transcription factor T-bet essential for Th1 development (TBX21)
(Kaplan, Sun et al. 1996). Additionally, expression of the IL-12 receptor is up-regulated by Tcells and thus makes Th1 cells even more sensitive to this polarizing signal, thereby enabling
their expansion (Mullen, High et al. 2001). In combating infectious diseases, Th1 cells are
especially useful at eliminating intracellular pathogens, including viruses and intracellular
bacteria. These cells secrete high levels of IFN-γ, which is important for macrophage
activation, and IL-2, which is important for directing cytotoxic CD8+ T cell responses. In
contrast, aberrant Th1 responses are thought to be important for driving autoimmune diseases
and chronic inflammation (O'Garra and Arai 2000).
IL-4 is the signature molecule of Th2 cells; the role of this cytokine is not only to
promote auto-stimulation, but also to trigger isotype switching towards IgE, an antibody
isotype necessary for combating extracellular parasites, in B cells. IL-4 stimulation leads to
activation of STAT6 pathways, which are necessary for GATA-3 expression, a master
transcription factor for the Th2 regulation. In addition, GATA-3 drives expression of IL-4,
creating a positive feedback loop and inducing expression of other cytokines (O'Garra and
Arai 2000). Th2 cells direct the immune response against extracellular parasites, including
helminths. However, in some circumstances, they cause asthma and allergies. Th2 cells
produce a myriad of cytokines with distinct functions such as IL-4, IL-5 for eosinophil
recruitment, IL-9 important for mast cells and T cells and mucin production by epithelial cells
during allergies or the potent anti-inflammatory cytokine IL-10 (Zenewicz, Antov et al. 2009).
Recently, the Th1/Th2 paradigm has been expanded; following the discovery of a third
subset of effector Th cells that produce IL-17 and exhibits effector functions distinct from
Th1 and Th2 cells. Development of Th17 cells can be divided into three stages: differentiation
(driven by TGFβ and IL-6), amplification (triggered by IL-21) and, lastly, stabilization
(maintained by IL-23). Naïve T cells can be differentiated into Th17 cells in vitro by
activation in the presence of TGF-β, which drives Smad signaling, and a secondary
inflammatory stimulus, driven by signaling molecules such as IL-6 or IL-21 that activates the
transcription factor STAT3 (Zenewicz, Antov et al. 2009). Activated STAT3 drives
expression of two transcription factors essential for shaping Th17, retinoid-acid-receptorrelated orphan receptor (ROR) γ and RORα. IL-21 is induced by IL-6 and leads to activation
of RORγ, driving expression of IL-17A and other cytokines. Thus, this IL-21 loop represents
an important autocrine factor for amplification of Th17 cells (Nurieva, Yang et al. 2007). IL23 was originally thought to be the cytokine driving Th17 differentiation. However, as shown
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by the unresponsiveness of naïve T cells for IL-23, this cytokine is not needed for Th17
differentiation, but is instead important for maintenance of these cells. At the initial stage of
Th17 differentiation, TGF-β-mediated activation leads to the increased expression of the
receptor IL-23R, enabling the cells to become responsive to IL-23 (Mangan, Harrington et al.
2006; Zhou, Lopes et al. 2008). Signature cytokines secreted by Th17 include IL-17A, IL17F, IL-21 and IL-22 (Korn, Bettelli et al. 2009).
Th17 cells have been implicated in the progression of inflammatory diseases, but it
seems that they could act also as anti-inflammatory and protect against certain conditions,
mainly through production of IL-22, that activates anti-apoptotic and proliferative responses
(Zenewicz, Antov et al. 2009). Regulatory T cells
Among the various populations of regulatory and suppressor T cells described, naturally
occurring thymic and peripheral CD4+ T cells that co-express CD25 are currently most
actively investigated (Shevach, DiPaolo et al. 2006). CD4+CD25+ Treg cells prevent the
activation and proliferation of potentially autoreactive T cells that have escaped thymic
deletion. They fail to proliferate and secrete cytokines in response to polyclonal or antigenspecific stimulation, and are not only anergic but also inhibit the activation of responsive T
cells. Although CD25, CD152, and glucocorticoid-induced TNF-related protein (GITR) are
markers of CD4+ CD25+ Treg cells, they are also expressed by activated T cells (Shevach and
Stephens 2006). A more faithful marker distinguishing CD4+CD25+ Treg cells from recently
activated CD4+ T cells is Foxp3, a member forkhead family of transcription factors that is
required for CD25+ Treg cell development and is sufficient for their suppressive function
(Sakaguchi, Wing et al. 2007) (Fig. 2).
Foxp3+ CD4+ CD25+ Treg cells play an important role in preventing the induction of
several autoimmune diseases, such as the autoimmune syndrome induced by day 3
thymectomy in genetically susceptible mice, IBD, type 1 diabetes (T1D) in thymectomized
rats and in non obese diabetic (NOD) mice. A defect in peripheral regulatory cells affecting
both CD25+ Treg cells and natural killer cells has been described also in T1D patients, and
autoreactive T cells in diabetics are skewed to a pro-inflammatory Th1 phenotype lacking the
IL-10-secreting T cells found in non-diabetic, HLA-matched controls. The clinical relevance
of CD4+ CD25+ Treg cells has also been shown in patients affected by rheumatoid arthritis and
multiple sclerosis (Baecher-Allan and Hafler 2006).
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CD4- Naïve
CD8- T cell
Fig. 2. T cell differentiation. T-cell differentiation is tightly controlled by cytokines and
transcription factors that determine the type of inflammatory response. CD4+ T cells differentiate in T
helper cells (Th) or T regulatory (Treg), while CD8+ differentiates in cytotoxic T lymphocytes (CTL).
Adapted from (Korzenik and Podolsky 2006).
1.1.5 Dendritic cells, a key role in innate and adaptive immunity
DCs derive, as phagocytes, from monocytes, and after stimulation with IL-4 and granulocyte
macrophage colony stimulating factor (GM-CSF) differentiate into immature DCs (iDCs).
iDCs are continuously produced from hematopoietic stem cells in the bone marrow and are
widely distributed in lymphoid and nonlymphoid tissues.
Mature DCs are professional APCs, which are strategically positioned at the
boundaries between the inner and the outside world, thus bridging innate and adaptive
immunity. DCs, including epidermal Langerhan’s cells, splenic marginal zone DCs and
interstitial DCs within nonlymphoid tissues, continuously sample self-antigen to maintain T
cell self-tolerance (Banchereau and Steinman 1998).
At the immature stage, iDCs express PRRs and cytokine receptors, allowing them to
sense pathogens and contribute to the innate immune response and induced DCs maturation.
DCs are known as the most efficient APC to activate naïve T cells. However, they are able to
do more than just efficiently present antigen to T cells. They are key modulators of the
immune response that can influence Th cell differentiation by preferentially inducing Th1 or
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Th2 cell responses, and the differential polarization of CD4+ T cells appears to be mediated
by discrete dendritic cell subsets (Steinman, Hawiger et al. 2003). Inflammatory dendritic cells
In most tissues, DCs are present in an immature stage, lacking the requisite accessory signal
for T cells activation. In contrast, they express the complete panel of receptors involved in
sensing pathogen invasion such as TLRs or NLRs and many antigen-capturing Fcγ and Fcε
receptors. Antigen uptake by phagocytosis or recognition of PAMPs by its receptor transform
immature DCs into mature DCs, showing now a reduced capacity for antigen uptake but an
exceptional capacity for T cell stimulation. Thus, mature DCs express T cells co-stimulatory
molecules, such as CD40, CD86, CD80 and MHC-II. This maturation process induces DC
migration from the periphery to lymphoid organs, where antigen presentation to T cells could
occur (Itano and Jenkins 2003).
DCs are heterogeneous not only in terms of maturation stage, but also of origin,
morphology, phenotype and function. Two distinct DC subpopulations were originally
defined in the human blood based on the expression of CD11c, and they have been
subsequently characterized as belonging to the myeloid or lymphoid lineage, and defined as
myeloid (M-DCs) and plasmacytoid (P-DCs) (Colonna, Trinchieri et al. 2004). Myeloid dendritic cells
M-DCs are characterized by a monocytic morphology; express myeloid markers like CD13
and CD33, the β2 integrin CD11c, the activatory receptor Ig-like transcripts 1 (ILT1), and low
levels of the IL-3 receptor α chain CD123 and at a high level the complete TLRs familly
(Steinman and Banchereau 2007).
M-DCs are the most efficient APCs directly able to prime naïve T cells and can
become, under different conditions, immunogenic or tolerogenic (Steinman and Banchereau
2007). As already mentioned, MHC present on the M-DCs surface is recognized by TCR and
with engagement of co-receptors, such as CD86, CD80 or CD40-CD40L. This lead to the
production of IL-12 by M-DCs driving the CD4+ naïve T cells to a Th1 phenotype
(Banchereau, Briere et al. 2000).
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Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD Plasmacytoid dendritic cells
Conversely, P-DCs have a morphology resembling plasma cells, are devoid of myeloid
markers, express high levels of CD4, CD62L and CD123 and ILT3. Oppositely to M-DCs, PDCs express high level of TLRs involved in virus recognition, such as TLR3, 7 and 9, but not
those TLRs that are involved in bacteria component sensing (Colonna, Trinchieri et al. 2004).
P-DCs produce high levels of IFN-α (Colonna, Trinchieri et al. 2004), cytokines with clearly
distinct effects on T cell activation and differentiation due to its IL-12 inhibiting properties. PDCs are poor APCs due to its low MHC-II surface expression. However even if P-DCs are
less efficient than M-DCs as APCs, P-DCs are able to drive Th1-mediated response after
virus infection and to activate CTLs (Banchereau, Briere et al. 2000).
Interestingly, P-DCs primed CTLs present a poor proliferation capacity due to
substantial production of IL-10. In addition, a very important aspect of P-DC-mediated
regulation of adaptive immunity is the ability, through the production of both type I
interferons and IL-6, to induce human B cells to differentiate into plasma cells and produce
immunoglobulin. These observations, coupled to the high expression of ILT3 suggest a role
for P-DCs, under steady-state conditions, in the maintainance of peripheral immune tolerance
as naturally occurring tolerogenic DCs (Penna, Roncari et al. 2005). Tolerogenic dendritic cells
It is now clear that DCs can be not only immunogenic but also tolerogenic, both
intrathymically and in the periphery, and they can modulate T cell development (Steinman
and Banchereau 2007). Tolerogenic DCs are characterized by reduced expression of costimulatory molecules, in particular CD40, CD80, CD86, although this is not an absolute
requirement. In addition, they usually show reduced IL-12 and increased IL-10 production,
and often an early stage of maturation (Steinman, Hawiger et al. 2003).
While these well-established phenotypic and functional properties of tolerogenic DCs
can easily explain their propensity to induce regulatory rather than effector T cells, several
other mechanisms may play a role in favoring Treg cell induction by tolerogenic DCs.
However, the simplistic concept that iDCs are intrinsically and uniquely able to induce
regulatory/suppressor T cells has been dispelled by the observation that mature DCs can also
be very efficient inducers of Treg cells (Yamazaki, Iyoda et al. 2003), a property already noted
for semi-mature DCs (Lutz and Schuler 2002).
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Fig. 3. Dendritic cell subsets. DCs are heterogeneous not only in terms of maturation stage,
but also of origin, morphology, phenotype and function. Two distinct DC subpopulations were originally
defined in the human blood based on the expression of CD11c, and they have been subsequently
characterized as belonging to the myeloid or lymphoid lineage, and defined as myeloid (M-DCs) and
plasmacytoid (P-DCs). M-DCs could differentitated in tolerogenic or inflammatory DCs, resulting in a
phenotype and cytokine production different, whileP-DCs differentitate and express BDCA2, TLR3-7-9
and produce high amont of IFN-α (Colonna, Trinchieri et al. 2004).
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1.2 VDR and 1α,25(OH)2D3
1.2.1 A brief history
The first scientific description of a vitamin D deficiency, namely rickets, was provided in the
17th century by both Dr. Daniel Whistler (1645) and Professor Francis Glisson (1650). In
1824, German scientists found that cod-liver oil have excellent anti-rickets properties, and in
1861, Trousseau in France proposed that rickets was induced by lack of sun exposure and a
faulty diet, where a cure was as simple as cod-liver oil ingestion. Before the scientific
agreement to define the biologically active form of vitamin D as a secosteroid hormone, it
was accidentally classified as vitamin (vital-amine). Around 1920, Sir Edward Mellanby was
working with dogs raised exclusively in the absence of UVB. He developed a diet that
allowed him to unequivocally establish that the bone disease, rickets, was caused by a
deficiency of a trace component present in the diet (Mellanby 1976). In 1921 he wrote "The
action of fats in rickets is due to a vitamin or accessory food factor which they contain,
probably identical with the fat-soluble vitamin." Furthermore, he established that cod-liver oil
was an excellent anti-rachitic agent. Shortly thereafter, McCollum and associates observed
that oxidized cod-liver oil still retained its calcium-depositing properties. Based on this, they
concluded that the anti-rachitic substance found in certain fats was distinct from fat-soluble
vitamin A and its “specific property was to regulate the metabolism of the bones.”
In the sequence of discovery of vitamins, the newly discovered anti-rachitic substance
was the fourth; hence it was called vitamin D (McCollum, Simmonds et al. 1995). The
chemical structures of the vitamins D were determined in the 1930s in the laboratory of
Professor A. Windaus at the University of Göttingen in Germany. Vitamin D3 was not
chemically characterized until 1936, when it was shown to result from the UVB irradiation of
7-dehydrocholesterol (Windaus A 1936). Virtually simultaneously, the elusive anti-rachitic
component of cod-liver oil was shown to be identical to the newly characterized vitamin D3.
These results clearly established that the anti-rachitic substance vitamin D was chemically a
steroid, more specifically a secosteroid.
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1.2.2 1α,25(OH)2D3
Fig. 4. Structure of vitamin D3
(cholecalciferol) and its numbering system.
2 Synthesis of vitamin D
Vitamin D is composed of 3 rings named A, C and D, of a seco-B ring and 1 side chain for a
total of 27 carbons (Fig. 4). 1α,25(OH)2D3, the bioactive hormone, is synthesized from
vitamin D3 in a highly regulated multistep process. The initial step is the transformation by
UVB light of the 7-dehydrocholesterol circulating in the skin, in an unstable precursor, the
pre-vitamin D3 (Fig. 5). This precursor is transformed in vitamin D3 in a heat dependent
process. In the liver, vitamin D3 is hydroxylated to 25(OH)D3, by a mitochondrial cytochrome
P450 (CYP450) enzyme, the 25-hydroxylase (encoded by the gene CYP27A1).
Next, in the proximal renal tubule, another enzyme belonging to the CYP450 family,
the 1α-hydroxylase (encoded by the gene CYP27B1), transforms the precursor in the
bioactive form, the 1α,25(OH)2D3 (Bell 1998; Deeb, Trump et al. 2007). CYP27B1 is not
expressed exclusively in the proximal renal tubule, but extra renal sites of 1α,25(OH)2D3
synthesis have been found in cells of the immune system, as well as in breast, prostate and gut
cells (Bell 1998). The mechanisms leading to less active or completely inactive metabolites
are now fully characterized. Catabolism of vitamin D
The limiting rate from the bioactive hormone turnover is the product of its hydroxylation by
24-hydoxylase-1α,25(OH)2D3 (CYP24A1) to form 1α,24,25(OH)3D3 (Haussler, Whitfield et
al. 1998). Interestingly, CYP24A1 is directly up-regulated by 1α,25(OH)2D3 via a VDRdependent manner, confirming a negative feedback induced by the hormone to control its
concentration (Ohyama, Ozono et al. 1994). The principal pathway leading to the elimination
of 1α,25(OH)2D3 is mediated by the hydroxylation on carbon 24 (C24) (1α,24,25(OH)2D3),
then this hydroxyl group is reduced in a keto group leading to 24-oxo-1α,25(OH)2D3.
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Interestingly, the oxydation of C24-hydroxy group in C24-keto group is also catalyzed by
CYP24A1 (Uskokovic, Norman et al. 2001). After a second hydroxylation step, always
1α,23,25(OH)3D3) and finally oxidized into calcitroic acid, the final water soluble metabolite
excreted by the kidneys (Reddy and Tserng 1989) (Fig. 5). In some tissues, a secondary
pathway was described using successively an hydroxylation at carbons C23 and C26 resulting
in the calcitriol lactone (Reddy and Tserng 1989). More recently, an alternative pathway was
discovered in some tissues or malignant cell lines (Uskokovic, Norman et al. 2001), This
alternative pathway, named C3 epimerization pathway, induces a stereochemical modification
on the A ring resulting to the 3-epi-1α,25(OH)2D3 (Siu-Caldera, Sekimoto et al. 1999). Next,
elimination of 3-epi-1α,25(OH)2D3 is following the classical pathway by CYP24A1
hydroxylation (Kamao, Tatematsu et al. 2004) (Fig. 5). This metabolite has been shown to be
an inactive metabolite, while C24 and C23 metabolites were able to show, in some aspects,
activities similar to the parent compound (Lemire, Archer et al. 1994; Siu-Caldera, Sekimoto
et al. 1999).
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Fig. 5. Metabolism and catabolism of 1α,25(OH)2D3. After UV exposure, the 7-dehydrocholesterol is
transformed into vitamin D3, that after two successive hydroxylations in the liver (CYP27A1) and the
kidney (CYP27B1) is converted into the active metabolite, 1α,25(OH)2D3. The hormone is catabolized
into calcitroic acid, the final water soluble metabolite excreted by the kidneys. All these elimination
steps involve the same enzyme CYP24A1 and could occur in different tissues (Haussler, Whitfield et
al. 1998). Vitamin D analogs
The discovery of the immunomodulatory properties of VDR prompted the study of
1α,25(OH)2D3 as a therapeutic agent for immuno-mediated diseases (DeLuca 2004).
Unfortunately, the dominant role of 1α,25(OH)2D3 is to adjust serum calcium and phosphorus
concentrations, and its in vivo immunomodulatory properties are mostly achieved at
hypercalcemic doses. These observations have opened up a new research area, where the
design of 1α,25(OH)2D3 analogs with stronger anti-inflammatory properties but lower
calcium-increasing capacity has generated interesting compounds (Adorini 2002). Two
classes of 1α,25(OH)2D3 analogs containing 16-ene or 20-cyclopropyl moieties were
intensively studied because of their unique biological activity (Uskokovic, Manchand et al.
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In the early 90s, the first report of 16-ene VDR agonist demonstrated potent leukemic
cell growth inhibition without modification of intestinal calcium absorption (Norman, Zhou et
al. 1990). Since then, many different analogs were described containing this 16-ene
modification and their peculiar metabolism was shown to be responsible for their increased
potency (IV) (Lemire, Archer et al. 1994). As described previously, CYP24A1 is the main
enzyme catalysing 1α,25(OH)2D3 degradation, leading by various steps to calcitroic acid.
While 24-oxo-1α,25(OH)2D3 is rapidly converted in 24-oxo-1α,23,25(OH)3D3, it appears that
analogs containing 16-ene are protected from the C23-hydroxylation step, leading to an
accumulation of C24-oxo. This metabolite is a non-calcemic metabolite, which presents
similar activity than its parent compound, both in vitro and in vivo (Lemire, Archer et al.
1994; Uskokovic, Norman et al. 2001). Elocalcitol (BXL-628, 1α-fluoro-25-hydroxy-16,23Ediene-26,27-bishomo-20-epi-vitamin D3) (Fig. 12 and table 4) is an example of a 16-ene
modified compound, and it was proposed as a treatment for benign prostatic hyperplasia
(BPH) (II) (Maggi, Crescioli et al. 2006).
The second well-described family includes the 20-epi analogs. The 20-epi
modification leads to a conformational change, where the hydrogen at C20 is converted from
R to S (Binderup, Latini et al. 1991; Sicinska and Rotkiewicz 2009). This modification leads
to a stabilization of the VDR-retinoid X receptor (RXR) complex and a modification of the
coactivator (CoA) or corepressor (CoR) recruitment (Schwinn and DeLuca 2007). An
example of a potent 20-epi compound is KH1060 (1α,25(OH)2-20-epi-22-oxa-24,26,27trishomo-vitamin D3) that was shown to have enhanced anti-proliferative and antiinflammatory properties in many in vitro and in vivo models, such as T1D or IBD (Mathieu,
Waer et al. 1995; Stio, Treves et al. 2002; Penna, Amuchastegui et al. 2006). Molecular
mechanisms induced by this compound were extensively studied, and the crystal structure of
KH1060-VDR-RXR complex was solved, showing higher stability and longer half-life
compared to the natural hormone (Tocchini-Valentini, Rochel et al. 2001). A similar group of
analogs, based on similar stereochemistry, was synthesized later on, the 20-cyclopropyl.
These family of compounds show higher potency than the natural hormone in inhibiting the
proliferation of cancer cell lines, in inhibiting production of pro-inflammatory cytokines, such
as IFN-γ or TNF-α, and a stronger potency in primary VDR target gene induction with a
controlled calcemic activity (III) (Uskokovic, Manchand et al. 2006). In addition, introduction
of 16-ene moiety in 20-cyclopropyl analogs has been shown to increase anti-proliferative and
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anti-inflammatory properties of VDR agonists without enhancing calcemic activity (III)
(Uskokovic, Manchand et al. 2006).
1.2.3 VDR
The biological effects of 1α,25(OH)2D3 are mediated by the VDR, a member of the
superfamily of NRs. The VDR was discovered as a NR in 1975, but it was cloned a decade
later (Baker, McDonnell et al. 1988); two years after the first genomic identification of a
steroid receptor (glucocorticoid receptor) (Weinberger, Hollenberg et al. 1985). The
sequencing of other receptors highlighted the presence of many more NRs genes than
previously expected (Chawla, Repa et al. 2001).
The VDR is now classified as member of the endocrine receptor subfamily together
with the glucocorticoid receptor. Its natural ligand, 1α,25(OH)2D3, presents an affinity
constant for the VDR in the nanomolar range (Haussler, Whitfield et al. 1998). Recently, a
novel classification based on mouse tissue expression and function, considered the VDR as a
NRs also involved in bile acid and xenobiotic metabolism based on its high expression in
gastroenteric tissues (Bookout, Jeong et al. 2006). However, the main physiological process
regulated by the VDR is the calcium and the phosphate homeostasis. NR superfamily Classification
NRs belong to a large superfamily of transcription factors comprising 48 members in the
human genome. These transcription factors regulate the expression of target genes to affect
processes as diverse and important as reproduction, development and metabolism (Chawla,
Repa et al. 2001; Novac and Heinzel 2004). They are classified based on ligand-binding
affinity, but more recently a new classification based on the interpretation of physiological
role from tissue-specific expression patterns has been proposed (Chawla, Repa et al. 2001;
Bookout, Jeong et al. 2006). Based on the ligand sensitivity, NRs could be divided in three
subgroups. The first class called “endocrine receptors” presents a high affinity for hormonal
lipids (at the nanomolar range). The second group is called “sensors” and presents a lower
affinity in the micromolar range. This class senses xenobiotic or nutritional components, such
as cholesterol, lipids or fatty acids. The third group contains NR, for which no ligand has yet
been identified. More recently, a novel classification, based on expression levels and tissue
distribution of NRs in mice, classified NRs by their physiological shared functions. This
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Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD
classification highlights the important role of NRs in the regulation of inflammation, cell
growth and differentiation, development and reproduction and nutrient use and storage
(Bookout, Jeong et al. 2006).
Fig. 6. Circular dendrogram representing the relationship between NR expression and
their physiological functions. The relationship between NR expression, function and physiology is
depicted as a circular dendrogram using the hierarchical, unsupervised clustering of NR tissue
expression distribution. The analysis reveals the existence of a higher order network tying NR function
to reproduction, development, central, and basal metabolic functions, dietary-lipid metabolism and
energy homeostasis. Adapted from (Bookout, Jeong et al. 2006). Structural features
The structural and functional organisation within the NRs is highly conserved. NRs genes
present five distinct domains, named A to E. The N-terminal region, containing domains A/B,
is highly variable and contains at least one constitutively active transactivation function-1
(AF-1). This domain is implicated in transactivation and acts in a ligand-independent manner
outside of the receptor context. In addition, the C-terminal part of the A/B domain, due to its
proximity with C domain (DNA-binding domain) may play a role in the interaction with DNA
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by regulating the ability of the receptor to interact with other members of the family or by
altering the choice of the DNA target sequence (Robinson-Rechavi, Escriva Garcia et al.
The DNA-binding domain is the most conserved region and contains the P-box,
responsible for the DNA-binding specificity and two zinc-binding motifs that maintain the
domain architecture. The variability of this domain correlates with the DNA-binding
preferences of the NRs. Two NRs, the small heterodimerizing partner 1 (SHP1) and dosagesensitive sex reversal congenital adrenal hypoplasia critical region on the X chromosome
(DAX1) lack a DBD and function principally as dominant negative repressors for other NRs
(Nagy and Schwabe 2004).
Between the DNA-binding and the ligand-binding domain (E domain) is a less
conserved region (D domain) that behaves as a flexible hinge between the C and E domains,
and contains the nuclear localization signal (NLS) (Robinson-Rechavi, Escriva Garcia et al.
Finally, the large E domain, ligand-binding domain, is moderately conserved as
sequence but well conserved as 3D structure. The ligand-binding domain is responsible for
many functions, mostly ligand induced, notably the AF-2 (helix 12) transactivation function, a
strong dimerization interface, another NLS, and often a repression function (RobinsonRechavi, Escriva Garcia et al. 2003).
Fig. 7. Schematic representation of NR domains. Most of the NR exhibit five specific
domains, the N terminal containing AF-1 involved in transactivation, followed by the DNA-binding
domain (DBD). The hinge is a poorly conserved region presenting the nuclear localisation signal
(NLS). The C terminal portion contains the ligand-binding domain (LBD) and the helix 12 (AF-2),
crucial for the ligand-induced activity (Robinson-Rechavi, Escriva Garcia et al. 2003). Structure and functions of VDR
The human VDR gene, located on chromosome 12q, is composed of a promoter and a
regulatory region and 8 exons encoding the 427 amino acid of the VDR protein (MW 48 kDa)
(Fig. 8) (Deeb, Trump et al. 2007).
The VDR is composed of domains that allow translocation to the nucleus, ligandbinding, heterodimerization with its partner RXR, DNA-binding and the co-factor interactions
(Fig. 8) (Carlberg 2003). In the absence of ligand, VDR is partitioned between the cytoplasm
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and the nucleus. The ligand induces interaction of VDR with importin β, via its nuclear
localization signal regions, increasing nuclear translocation of VDR, as well as the
translocation of the VDR-RXR heterodimer (Yasmin, Williams et al. 2005). The DNA
binding domain (DBD) recognizes a specific DNA sequence present in regulatory regions of
primary 1α,25(OH)2D3 responding genes, which is referred to as a vitamin D response
element (VDRE). VDREs are hexameric DNA sequence composed of the consensus sequence
RGKTSA (R=A or G, K=G or T, S=C or G) separated by three or four spacing nucleotides
(Carlberg and Seuter 2009).
VDR-RXR heterodimers bind to VDREs form by a direct repeat (DR) of two
hexameric core binding motifs with 3 intervening nucleotides (DR3-type) (Carlberg, Bendik
et al. 1993), but also to DR4-type REs along with other members of the nuclear receptor
superfamily (Quack and Carlberg 2000). It should be noted that effective VDR binding has
also been observed on everted repeat (ER)-type REs with 6 to 9 spacing nucleotides (ER6,
ER7, ER8, ER9) (Schrader, Muller et al. 1994; Schrader, Nayeri et al. 1995)
The ligand-binding domain contains the 1α,25(OH)2D3-binding pocket and the
transactivation domain called AF-2 (Carlberg 2003). This last domain is essential for the
ability of NRs to activate gene transcription, as the change of positioning of helix 12, upon
ligand binding, creates a binding surface that favors the interaction with CoAs instead of
CoRs (Nagy and Schwabe 2004). CoRs suppress the expression of responsive genes, while
CoAs favor transcription and act as a bridge between the VDR-RXR heterodimer and the
basal transcription machinery (Nagpal, Na et al. 2005). CoRs recruit histone deacetylase
involved in chromatin condensation that wrap VDREs, which silences gene expression. CoAs
recruit histone acetyltransferases, destabilize the nucleosome core and unravel DNA for
transcription (Nagpal, Na et al. 2005).
Passive diffusion across cell membrane allows 1α,25(OH)2D3 to bind to its receptor
and induce VDR phosphorylation at serines 51 and 208. Phosphorylations have been
proposed to induce conformational changes in ligand- and DNA-binding domains that allow
heterodimerization of VDR with RXR (Arriagada, Paredes et al. 2007). As a consequence,
CoRs (such as NR co-repressors and the silencing mediator for retinoid and thyroid hormone
receptors) are released. Next, the CoA complexes (steroid receptor co-activators, nuclear coactivator 62 kDa–SKI-interacting protein, chromatin modifiers CREB binding protein–p300,
polybromo and SWI‑2 related gene 1 associated factor) are recruited to VDRE region to
initiate the transcription with the help of transcription factor 2B and RNA polymerase II
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(Haussler, Whitfield et al. 1998). VDR binding does not necessarily induce transcript upregulation, but it can result in direct or indirect inhibition of gene expression (White 2004;
Carlberg and Seuter 2009).
VDR expression is regulated by positive feedback, whereby VDR activation increases
its mRNA synthesis, while increased expression of PTH inhibits VDR synthesis (Brown,
Zhong et al. 1995). As observed in various cell line, VDR can be down-regulated due to the
inhibitory effect of SNAIL 1 or 2 (zinc-finger transcription factors involved in cell
movement) but it could be up-regulated in colon and breast cancer cells after estradiol
treatment in a ERK dependent manner (Palmer, Larriba et al. 2004; Gilad, Bresler et al. 2005;
Larriba, Martin-Villar et al. 2009). Interestingly, in inflammatory conditions, VDR expression
could be up-regulated as shown in IL-8 stimulated BPH cells (II).
Fig. 8. Chromosomic and protein domains of the Vitamin D receptor. The human VDR
gene located on chromosome 12q, is composed of non translated exons (1a–1f) and exons 2–9,
which encode 6 domains (A–F) of the full-length VDR protein. VDR nuclear localization signals (blue)
direct the receptor into the nucleus. VDR associates with RXR through the dimerization domains
(yellow). The 1α,25(OH)2D3–VDR–RXR complex binds to VDREs through the DNA-binding domain in
the regulatory region of target genes. Conformational changes in the VDR result in the dissociation of
the CoR, silencing mediator for retinoid and thyroid hormone receptors (SMRT), and allows interaction
of the transactivation domain AF-2 (light grey) with stimulatory CoAs (Haussler, Whitfield et al. 1998)
that mediate transcriptional activation. Non-synonymous (FokI) and synonymous (BsmI, ApaI, TaqI
and Tru9I) single-nucleotide polymorphisms (SNPs) have been identified in VDR (defined by
restriction enzymes, polymorphisms are indicated in parentheses). FokI polymorphism at translation
initiation codon results in a smaller VDR that interacts with transcription factor 2B (TF2B) more
efficiently and has greater transcriptional activity than the full length VDR. Adapted from (Deeb, Trump
et al. 2007)
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1.2.4 Target genes and biological role
About 1000 different genes are believed to be under the direct control of 1α,25(OH)2D3. As
revealed by rickets development caused by VDR loss of function, most of the VDR-regulated
genes are involved in the calcium/phosphate homeostasis and in the maintenance of bone
content. However, VDR expression in cells not involved in these processes, especially actors
of the immune system, led to the recognition of non-calcemic action of 1α,25(OH)2D3
(Nagpal, Na et al. 2005). Calcium and phosphate homeostasis
Biological functions of 1α,25(OH)2D3 are the regulation of calcium and phosphate
homeostasis and maintenance of bone integrity. These activities are achieved through direct
actions of the hormone on the intestine, kidney or bone and through feedback inhibition of
parathyroid hormone (PTH) production at the parathyroid glands. However, bone deficiency
in children’s severe rickets could be rescued by intra-venous calcium administration,
indicating an important role of vitamin D-dependent calcium regulation in the intestine rather
than in bone, kidney or parathyroid gland (Bouillon, Van Cromphaut et al. 2003).
Calcium ion channel transient receptor potential vanilloid type 5 and 6 (TRPV5-6 also
known as epithelial calcium channel 1 and 2) present VDREs in their regulatory regions and
are up-regulated after 1α,25(OH)2D3 treatment. TRPV5 and 6 are respectively involved in
transepithelial uptake of calcium by the kidney and in the absorption of calcium from the
intestinal lumen, and were considered as the “gatekeepers” of epithelial calcium transport
(Pike, Zella et al. 2007). Interestingly, Trpv6 and CalbindinD9K deficient mice present a
physiological intestinal calcium absorption and 1α,25(OH)2D3 treatment in Trpv6 deficient
mice respond equally well for the intestinal calcium regulation compared to the wild type
(Benn, Ajibade et al. 2008; Kutuzova, Sundersingh et al. 2008). Thus, the mechanisms
underlying the 1α,25(OH)2D3 intestinal calcium regulation appears to be still incompletely
defined. Moreover, up-regulation after 1α,25(OH)2D3 treatment and substantial reduction in
both Cyp27b1-null and VDR-null mice can explain the important role of 1α,25(OH)2D3 in
calcium homeostasis (Van Cromphaut, Dewerchin et al. 2001; van Abel, Hoenderop et al.
Phosphate homeostasis involves a phosphaturic hormone, fibroblast growth factor 23
(FGF23), which is secreted by osteoblasts and functions as a suppressor of phosphate
reabsorption from the kidney filtrate and represses 1α,25(OH)2D3 synthesis, closing this
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
endocrine regulatory loop (Haussler, Whitfield et al. 1998). In addition, 1α,25(OH)2D3 upregulates also receptor activator of NF-κB ligand (RANKL), an osteoblast cell surface protein
that stimulates osteoclastogenesis and bone resorption (Boyce and Xing 2008). Interestingly,
1α,25(OH)2D3 acts as a direct repressor of PTH in contrast with its positive effect on calcium
regulation. 1α,25(OH)2D3 metabolism and catabolism
1α,25(OH)2D3 has a direct regulatory effect on its metabolic and catabolic enzymes. First,
CYP27B1 is up-regulated by PTH and down-regulated by 1α,25(OH)2D3, highlighting a direct
negative control of the bioactive hormone on its actual synthesis (Brenza and DeLuca 2000).
Similarly, CYP24A1, encoding for the principal enzyme involved in 1α,25(OH)2D3
catabolism, is the most important positively regulated gene by 1α,25(OH)2D3 (Ohyama,
Ozono et al. 1994). Vitamin D deficiency
As previously discussed, vitamin D discovery was directly linked to the rickets, a disease
caused by vitamin D deficiency. The main source of vitamin D precursor is the UVBirradiated skin, while limited quantities are contained in the diary nutriments, except oily fish
and fish liver oil (Hollis 2005). This implicates sun exposure as a critical step in vitamin D
synthesis, since the main catalyzer of its synthesis is UVB.
Then, it became obvious that living in higher latitudes, lack of sunlight represents as
an important environmental factor for vitamin D deficiency (Cantorna and Mahon 2004;
Holick 2007). Vitamin D deficiency was also shown to be correlated with the incidence and
the severity of osteoporosis, a bone disorder (Lips 1996). The discovery of the non-calcemic
effects of 1α,25(OH)2D3 and the symptoms amelioration in autoimmune animal models after
1α,25(OH)2D3 treatment confirmed vitamin D deficiency as a potential environmental factor
in autoimmune diseases (Cantorna and Mahon 2004).
Nowadays, it is evident that low 25(OH)D3 serum levels are linked to the development
and the severity of many disorders from autoimmune disease to cancer (Brenza and DeLuca
2000; Jurutka, Bartik et al. 2007). Lack of sun exposure does not represent the sole cause of
vitamin D deficiency, but skin absorption and loss of function of VDR or enzymes
responsible for its synthesis are equally important (Holick 2007).
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Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD
Fig. 9. Physiological functions of 1〈,25(OH)2D3. Vitamin D3 made in the skin or ingested in the
diet can be stored in and then released from fat cells. Vitamin D in the circulation is bound to the vitamin
D–binding protein, which transports it to the liver, where vitamin D is converted by CYP27A1. This is the
major circulating form of vitamin D that is used by clinicians to determine vitamin D status. This form of
vitamin D is biologically inactive and must be converted in the kidneys by CYP27B1 to the biologically
active form 1α,25(OH)2D3. Serum phosphorus, calcium, FGF-23, and other factors can either increase
(+) or decrease (–) the renal production of 1α,25(OH)2D3 that decreases its own synthesis through
negative feedback and decreases the synthesis and secretion of PTH by the parathyroid glands.
1α,25(OH)2D3 increases the expression of CYP24A1 to catabolize 1α,25(OH)2D3 to the water-soluble,
biologically inactive calcitroic acid, which is excreted in the bile. 1α,25(OH)2D3 enhances intestinal
calcium absorption in the small intestine by interacting with the VDR-RXR to enhance the expression of
the epithelial calcium channel TRPV6 and calbindin D9K, a calcium-binding protein (CaBP).
1α,25(OH)2D3 is recognized by its receptor in osteoblasts, causing an increase in the expression of
RANKL, which induces preosteoclasts to become mature osteoclasts. Mature osteoclasts remove
calcium and phosphorus from the bone, maintaining calcium and phosphorus levels in the blood.
Adapted from (Deeb, Trump et al. 2007) and (Holick 2007).
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1.2.5 VDR-mediated non-calcemic activities
Epidemiological data on increased susceptibility to various diseases in people suffering of
25(OH)D3 deficiency, and the observation that VDR is also present in cells other than those of
the intestine, bone, kidney and parathyroid gland led to the recognition of non-calcemic
actions of VDR ligands. Discovery of VDR expression in lymphocytes and its antiproliferative properties were the first evidence for the potential role of 1α,25(OH)2D3 as antiproliferative and anti-inflammatory agent (Abe, Miyaura et al. 1981; Colston, Colston et al.
1982; Provvedini, Tsoukas et al. 1983). The immunomodulatory and anti-inflammatory
properties of VDR agonists will be described in detail below. Regulation of cell proliferation and tumorigenesis
The important role of 1α,25(OH)2D3 in the regulation of cell proliferation and tumorigenesis
is reflected by the phenotype observed in Vdr deficient mice. These mice present numerous
tumors after exposure to oncogene or carcinogen, compared to wild-type mice, and colorectal
hyper-proliferation. However, they do not present spontaneous tumors despite precancerous
lesions in mammary glands (Kallay, Pietschmann et al. 2001; Bouillon, Eelen et al. 2006). As
previously mentioned, vitamin D deficiency is directly correlated with cancer, but also genes
coding the metabolic enzymes, CYP24A1 and CYP27B1, are found to be down-regulated in
many cancers, such as in breast or prostate tumors (Palmer, Gonzalez-Sancho et al. 2001).
Mechanisms underlying 1α,25(OH)2D3 anti-proliferative effects are principally mediated by
its capacity to perturbate the cell cycle (Deeb, Trump et al. 2007).
E-cadherin, a transmembrane linker of the intercellular adherens junctions, is a
membrane protein classified as tumor suppressor gene. E-cadherin binds to β-catenin,
inhibiting its nuclear translocation. In the nucleus, β-catenin binds to T cell transcription
factor/lymphoid enhancer-binding factor 1 (TCF/LEF1), a transcription factor involved in the
cell proliferation control. 1α,25(OH)2D3 treatment increases the level of E-cadherin, resulting
in a sequestration of β-catenin in the cytoplasm and blocking the TCF/LEF1 gene regulation
(Palmer, Gonzalez-Sancho et al. 2001). Transition from G0/G1 to S phase is directed by cyclin
dependent kinases (CDKs) or CDK inhibitors (CDKIs) causing phosphorylation
/dephosphorylation events of the tumor suppressor retinoblastoma protein. VDREs were
found in regulatory regions of some CDK and CDKI, such as GADD45A or p21waf1,
demonstrating the direct regulatory process of 1α,25(OH)2D3 by arrest of G1 cycle (Deeb,
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Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD
Trump et al. 2007). An alternative anti-tumor mechanism of 1α,25(OH)2D3 is to promote cell
apoptosis. Key mediators of apoptosis such the anti-apoptotic, pro-survival proteins BCL2
and BCL-XL are repressed by 1α,25(OH)2D3, while it induces expression of pro-apoptotic
proteins, such as BAX, BAK and BAD (Ylikomi, Laaksi et al. 2002). Recent evidence
involves 1α,25(OH)2D3 also in angiogenesis inhibition, as demonstrated by inhibition of
vascular endothelial growth factor (VEGF) and up-regulation of the potent anti-angiogenic
factor thrombospondin 1 (THBS1) in SW480-ADH human colon tumor cells (FernandezGarcia, Palmer et al. 2005).
However, tumors exhibit many mechanisms to escape the VDR-dependent antiproliferative activity. As previously mentioned, VDR expression in late tumor phases is
down-regulated. In addition, in colorectal cancer, SNAIL1 and 2 repress VDR gene promoter,
inducing the inhibition of 1α,25(OH)2D3 dependent E-cadherin up-regulation, resulting in the
abolition of its anti-proliferative activity (Palmer, Gonzalez-Sancho et al. 2001; Palmer,
Larriba et al. 2004; Larriba, Martin-Villar et al. 2009). Regulation of the immune system
VDR is expressed in most cell types of the immune system, in particular in APCs, such as
macrophages and DCs, as well as in both CD4+ and CD8+ T cells (Veldman, Cantorna et al.
2000). Moreover, macrophages and DCs express, under the control of pro-inflammatory
signals, such as IFN-γ or NF-κB, the functional enzymatic machinery to synthesize and
metabolize the active hormone (van Etten and Mathieu 2005). From these observations, it is
conceivable that 1α,25(OH)2D3 could contribute to physiological regulation of the innate and
adaptive immune responses; thus VDR agonists could represent valuable anti-inflammatory
agents (Adorini and Penna 2008). Data accumulated in the last few years clearly demonstrate
that, in addition to exert direct effects on T-cell activation, VDR agonists markedly modulate
the phenotype and functions of APCs, in particular DCs. It is also possible that 1α,25(OH)2D3
may contribute to the physiological control of immune responses, and possibly be also
involved in maintaining tolerance to self antigens, as suggested by the enlarged lymph nodes
containing a higher frequency of mature DCs in Vdr-deficient mice (Griffin, Lutz et al. 2001).
Recently, novel regulatory roles of 1α,25(OH)2D3 were highlighted in wound repair
enabling keratinocytes to recognize and respond to microbes and to protect wounds against
infection, as well as its role as key component of innate immunity in microbial recognition
and anti-microbial response during injury (Liu, Stenger et al. 2006). This appealing concept
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
has emerged by the observation that vitamin D3 induced by sunlight in the skin is
hydroxylated by local DCs into the active hormone, which in turn up-regulates on activated T
cells expression of the epidermiotropic chemokine receptor CCR10, a primary VDRresponsive gene, enabling them to migrate in response to the epidermal chemokine CCL27
(Sigmundsdottir, Pan et al. 2007). Thus, the autocrine production of 1α,25(OH)2D3 by DCs
can program the homing of skin-associated T cells, which could include Treg cells able to
counteract the pro-inflammatory effects induced in the skin by sun exposure. In addition, high
CYP27B1 expression was found in wounds that were induced in keratinocytes in response to
TGF-1ß, triggering production of 1α,25(OH)2D3 by keratinocytes, which in turn increased
expression of CAMP and induced TLR2 and CD14 expression (Schauber, Dorschner et al.
2007). Thus, VDR agonists possess not only anti-inflammatory but also anti-infective
properties, which could provide additional clinical benefits in different inflammatory
1.2.6 Anti-inflammatory properties of VDR agonists Dendritic cells
Earlier indications for the capacity of VDR agonists to target APCs were corroborated by
their ability to inhibit the production of IL-12. More recent work has demonstrated that
1α,25(OH)2D3 and its analogs have profound effects on the phenotype and function of mDCs.
VDR agonists arrest the differentiation and maturation of DCs, maintaining them in an
immature state, as shown by decreased expression of maturation markers and increased
antigen uptake (Penna and Adorini 2000; Ferreira, van Etten et al. 2009). Studies performed
either on monocyte-derived DCs from human peripheral blood or on bone marrow-derived
mouse DCs have consistently shown that in vitro treatment of DCs with VDR agonists leads
to down-regulated expression of the co-stimulatory molecules CD40, CD80, CD86, to
markedly decreased IL-12, to enhance IL-10 production, and the modulation of chemokine
production; resulting in inhibition of T-cell activation. The near abrogation of IL-12
production and the strongly enhanced secretion of IL-10 highlight the important functional
effects of 1α,25(OH)2D3 and its analogs on DCs and are, at least in part, responsible for the
induction of DCs with tolerogenic properties (Penna and Adorini 2000). In addition, DCs
treated with VDR agonists up-regulate the expression of ILT3, an inhibitory molecule
associated with tolerance induction, although ILT3 expression is dispensable for the capacity
of 1α,25(OH)2D3-treated DCs to induce regulatory T cells (Penna, Roncari et al. 2005).
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Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD
1α,25(OH)2D3 utilizes different mechanisms to regulate cytokine production by DCs.
IL-12 secretion is inhibited by targeting the NF-κB pathway, via NF-κB proteins, such as Vrel reticuloendotheliosis viral oncogene homolog B (RelB) and c-Rel. RelB is essential in
differentiation and maturation of DCs, and exhibits an active VDRE that could explain is
strongly down-regulation in mice splenocytes after 1α,25(OH)2D3 intra-peritoneal (IP)
administration (Griffin, Dong et al. 2007). RelB inhibition is directly mediated by binding of
liganded-VDR-RXR complex in VDREs present in its promoter. This induces recruitment of
CoR complexes containing HDAC1 and 3 and SMRT. Direct RelB inhibition seems to occur
selectively in APCs, confirming predominant immunomodulation of 1α,25(OH)2D3 on this
subtypes (Griffin, Dong et al. 2007). Interestingly, antigen-exposed DCs, in which RelB
function is inhibited induce a population of antigen-specific CD4+ cells that regulate immune
responses in an IL-10-dependent manner (Griffin, Lutz et al. 2001). Suppression of the
monocyte recruited GM-CSF is achieved by interaction of ligand-VDR monomers with
functional repressive complexes in the promoter region of the cytokine (Towers and
Freedman 1998). In this case, the ligand-VDR complex acts selectively on the two
components required for activation of this promoter/enhancer: it competes with the
transcription factor NFAT1 for binding to the composite site and positioning itself adjacent to
Jun–Fos on the DNA. Co-occupancy apparently leads to an inhibitory effect on c-Jun
transactivation function. These two events mediated by VDR effectively block the NFAT1AP-1 activation complex, resulting in an attenuation of GM-CSF transcription (Towers and
Freedman 1998; Towers, Staeva et al. 1999).
The prevention of DC differentiation and maturation, as well as the modulation of
their activation and survival, which leads to DCs with tolerogenic phenotype and function that
result in T-cell hyporesponsiveness, are not limited to in vitro activity. 1α,25(OH)2D3 and its
analogs can also induce DCs with tolerogenic properties in vivo, as demonstrated in models of
allograft rejection by oral administration directly to the recipient or by adoptive transfer of in
vitro-treated DCs (Griffin, Lutz et al. 2001). Tolerogenic DCs induced by a short treatment
with 1α,25(OH)2D3 are probably responsible for the capacity of this hormone to induce
CD4+CD25+ Treg cells that are able to mediate transplantation tolerance (Adorini, Penna et al.
2003) (detailed in Regulatory T cells). Moreover, in autoimmune diabetic NOD-treated mice
that exhibit a defect in Treg cells, VDR agonists restored the Treg cell population and arrest the
development of autoimmune diabetes (Mathieu, Waer et al. 1995).
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Although the immunomodulatory effects of VDR agonists on DCs are well
established, the capacity of this hormone to modulate DCs subsets has been addressed only
recently. Two DC subsets, M-DCs and P-DCs have been identified. These subsets are
characterized by a distinct expression of PRRs and co-stimulatory molecules, and by the
selective production of immunomodulatory cytokines. Analysis of immunomodulatory effects
exerted by 1α,25(OH)2D3 on human blood M-DCs and P-DCs demonstrates a differential
capacity of this hormone to modulate cytokines and chemokines production in DC subsets,
showing marked effects in M-DCs and negligible ones in P-DCs (Penna, Amuchastegui et al.
2007). In addition, inhibition of Th1 development and enhancement of CD4+ suppressor Tcell activity are selectively induced by 1α,25(OH)2D3 in M-DCs but not P-DCs. This
differential capacity of DC subsets to respond to 1α,25(OH)2D3 is not due to a diverse VDR
expression or VDR-dependent signal transduction, but is associated with different effects of
this hormone on NF-κB p65 phosphorylation and nuclear translocation in DC subsets (Penna,
Amuchastegui et al. 2007). Thus, 1α,25(OH)2D3 appears to up-regulate tolerogenic properties
in mDCs, down-regulating IL-12 and Th1 cell development, while promoting CD4+
suppressor T cell activity and enhancing the production of CCL22, a chemokine able to
recruit Treg cells. By contrast, no immunomodulatory effects appear to be induced by
1α,25(OH)2D3 in P-DCs (Liu 2005). P-DCs, characterized by an intrinsic ability to prime
naïve CD4+ T cells to differentiate into IL-10-producing T cells and CD4+CD25+ Treg cells,
and to suppress immune responses, may represent naturally occurring regulatory DCs, and the
lack of P-DCs modulation by 1α,25(OH)2D3 would thus leave this tolerogenic potential
unmodified (Liu 2005).
Innate immune regulation by 1α,25(OH)2D3 was recently emphasized by the discovery
of a “nonclassical” mechanism mediated by the enhancement of anti-microbial peptide.
Discovery of VDREs in CAMP gene encoding, a potent anti microbial peptide, proposes new
functions for 1α,25(OH)2D3 in immunomodulation of innate immunity (Wang, Nestel et al.
2004). Protection by 1α,25(OH)2D3-induced CAMP expression was demonstrated in human
PBMCs infected by tuberculosis, decreasing pro-inflammatory cytokines production and
down-regulating HLA-DR expression (Schauber, Dorschner et al. 2007). Moreover, a clinical
correlate of this important link is provided by the observation that sera from AfricanAmerican individuals, known to have increased susceptibility to Mycobacterium tuberculosis,
have reduced levels of 25(OH)D3, the 1α,25(OH)2D3 precursor, and are inefficient in CAMP
mRNA induction, suggesting that 1α,25(OH)2D3 sufficiency contributes to decreased
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Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD
susceptibility to microbial infections (Schauber, Dorschner et al. 2007). Interestingly, in
autoimmune disorders, especially psoriasis, CAMP overexpression in wound healing has been
proposed as a pro-inflammatory signal for P-DCs (Lande, Gregorio et al. 2007). However,
clear evidences for anti-inflammatory properties, such as arrest of DCs maturation, are also
described. DCs maturation after TLR activation is inhibited by CAMP, as show by inhibition
of maturation markers, such as CD86, CD80, CD83 and HLA-DR, as well as by inhibition of
pro-inflammatory cytokines, via inhibition of NF-κB nuclear translocation (Kandler,
Shaykhiev et al. 2006). Morevover, the immunomodulatory properties of CAMP could also
affect T cells, as shown by the inhibition of IFN-γ and IL-2 production in DCs co-cultured
with naive T cells in the presence of CAMP (Kang, Azad et al. 2005; Bandholtz, Ekman et al.
2006; Yu, Mookherjee et al. 2007). In vivo data on the relationship between 1α,25(OH)2D3
and CAMP, especially in mice models, are difficult due to the absence of VDREs in the
promoter of the murine homolog, cramp (Gombart, Borregaard et al. 2005). However, cramptreated mice present amelioration of symptoms in chemically induced colitis or allergic
contact sensitization, as also observed with 1α,25(OH)2D3 treatment (Di Nardo, Braff et al.
2007; Tai, Wu et al. 2007). T cells
Soon after the discovery of VDR expression in T cells, 1α,25(OH)2D3 was shown to inhibit
antigen-induced T-cell proliferation and cytokine production. Later studies demonstrated
selective inhibition of Th1 cell development, although it was not clarified how much of this
effect could be accounted for by modulation of DC functions. Indeed, several key cytokines
are direct targets for VDR agonists in T lymphocytes, in particular Th1-type cytokines, such
as IL-2 and IFN-γ (Nagpal, Na et al. 2005). In activated T cells, inhibition of IL-2
transcription is mediated by antagonism of the ligand-VDR-RXR complex for the formation
of the NFAT/AP-1 complex, resulting to a stable association of VDR to the NF-AT/AP-1
binding site on the IL-2 promoter (Bemiss, Mahon et al. 2002). On the other hand, IFN-γ
transcription inhibition is directly mediated by interaction of the ligand–VDR–RXR complex
via a negative VDRE in the promoter region of the gene (Cippitelli and Santoni 1998).
1α,25(OH)2D3 has been also shown to enhance the development of Th2 cells via a
direct effect on naïve CD4+ cells. This could contribute to account for the beneficial effect of
VDR agonists in the treatment of inflammatory conditions (Boonstra, Barrat et al. 2001). The
capacity of 1α,25(OH)2D3 to skew T cells towards the Th2 pathway had been suggested
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previously, but could not be confirmed by other studies. Th2 cells can be targets of VDR
agonists but this depends on their activation and differentiation status (Mahon, Wittke et al.
2003). Thus, 1α,25(OH)2D3 can apparently up-regulate, down-regulate or have no effect on
IL-4 production and, consequently, on Th2 cell development, illustrating the complexity of
immunoregulatory pathways set in motion by 1α,25(OH)2D3.
Treatment with VDR agonists also inhibits IL-17 production (Tang, Zhou et al. 2009),
a proinflammatory cytokine shown recently to be produced by pathogenic T cells in various
models of chronic inflammation and immune-mediated tissue injury, including organ-specific
autoimmunity in the brain, heart, synovium and intestines, allergic disorders of the lung and
skin, and microbial infections of the intestines and the nervous system (Steinman 2007).
Interestingly, IL-17 production is induced by IL-23 where p40 chain is strongly inhibited by
VDR agonists. As expected, 1α,25(OH)2D3 inhibits IL-17 produced by T cells, showing an
amelioration of symptoms in different autoimmune diseases (Adorini and Penna 2008).
Recent work on experimental autoimmune uveitis, a Th17 autoimmune visual disorder, show
1α,25(OH)2D3 potency in the inhibition of IL-17 leading to a suppression of Th17-mediated
inflammation. This suppression involves the direct inhibition of IL-17 production by CD4+ T
cells and indirect inhibition of IL-17 lineage commitment by down-regulation of the ability of
DC to support priming of T cells toward the Th17 effectors pathway (Tang, Zhou et al. 2009).
Thus, in addition to controlling Th1 and Th2 cells, 1α,25(OH)2D3 also modulates the Th17
lineage. Interestingly, 1α,25(OH)2D3 has also been shown to prevent and treat TNBS-induced
colitis, by reducing Th1 and Th17 cells while up-regulating Foxp3+ Treg cells, associated
with significant reduction of IL-12p75, IL-23p19, and IL-6 production by DCs (Daniel,
Sartory et al. 2008).
In conclusion, 1α,25(OH)2D3 in vivo appears primarily to inhibit pro-inflammatory,
pathogenic T cells, such as Th1 and Th17, and, under appropriate conditions, may favor a
deviation to the Th2 pathway. These effects could be, in part, a consequence of direct T-cell
targeting by 1α,25(OH)2D3 and its analogs, but modulation of DC function by VDR agonists
certainly plays an important role in shaping the development of T-cell responses. Thus, VDR
agonists can target T cells both directly and indirectly, selectively inhibiting T-cell subsets
able to mediate inflammation and tissue damage.
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Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD
T cell
Fig. 10. Immunomodulatory effects of VDR agonists on myeloid DCs and CD4+ T cells.
VDR agonists inhibit in M-DCs, but not in P-DCs, expression of surface co-stimulatory molecules, for
example, CD40, CD80 and CD86, as well as MHC class II and CD54 molecules. Production of
cytokines affecting T-cell differentiation into Th1 and Th17, IL-12 and IL-23, respectively, are also
inhibited in M-DCs. Conversely, expression of surface inhibitory molecules, like ILT3, and of secreted
inhibitory cytokines, such as IL-10, are up-regulated markedly. Chemokines potentially able to recruit
CCR4+ regulatory T cells, such as CCL22 are also up-regulated, whereas the CCR4 ligand CCL17 is
down-regulated. Upon interaction with M-DCs, CD4+ T cells up-regulate expression of the inhibitory
molecule CD152 (CTLA-4). DCs expressing low levels of co-stimulatory molecules, secreting IL-10,
and expressing high levels of inhibitory molecules (for example, ILT3) favor the induction and/or the
enhancement of regulatory/suppressor T cells (Adorini 2002; Adorini and Penna 2008). Regulatory T cells
As discussed previously, induction of DCs with tolerogenic phenotype and function plays an
important role in the immunoregulatory activity of VDR agonists. Tolerogenic DCs induced
by a short treatment with 1α,25(OH)2D3 or its analogs are probably responsible for the
capacity of the hormone to induce CD4+CD25+ suppressor T cells that are able to mediate
transplantation tolerance (Adorini, Penna et al. 2003) and to arrest the development of
autoimmune diabetes (Gregori, Giarratana et al. 2002). VDR agonists enhance CD4+CD25+
Treg cells and promote tolerance induction in transplantation and autoimmune disease models.
A short treatment with 1α,25(OH)2D3 and mycophenolate mofetil, a selective inhibitor of T
and B cell proliferation that also modulates APCs, induces tolerance to islet allografts
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associated with an increased frequency of CD4+CD25+ Treg cells able to adoptively transfer
transplantation tolerance (Gregori, Casorati et al. 2001).
The induction of tolerogenic DCs could indeed represent a therapeutic strategy
promoting tolerance to allografts (Adorini 2002) and the observation that immature mDCs can
induce T cell tolerance to specific antigens in human volunteers represents an important proof
of concept for this approach (Dhodapkar, Steinman et al. 2001). CD4+CD25+ Treg cells able to
inhibit the T cell response to a pancreatic autoantigen and to significantly delay disease
transfer by pathogenic CD4+CD25- T cells are also induced by treatment of adult NOD mice
with the VDR agonist BXL-219 (Gregori, Giarratana et al. 2002). This treatment arrests
insulitis, blocks the progression of Th1 cell infiltration into the pancreatic islets and inhibits
T1D development at non-hypercalcemic doses (Gregori, Giarratana et al. 2002). Although the
T1D and islet transplantation models are quite different, in both cases administration of VDR
agonists doubles the number of CD4+CD25+ Treg cells, in the spleen and pancreatic lymph
However, tolerogenic DCs may not always be necessarily involved in the generation
of Treg cells by VDR agonists. A combination of 1α,25(OH)2D3 and dexamethasone has been
shown to induce human and mouse naïve CD4+ T cells to differentiate in vitro into Treg cells,
even in the absence of APCs. Upon transfer, these IL-10 producing T cells could prevent CNS
inflammation, indicating their capacity to exert a suppressive function in vivo (Barrat, Cua et
al. 2002).
VDR agonists not only favour induction of CD4+CD25+ Treg cells, but can also
enhance their recruitment to inflammatory sites. Blood-borne M-DCs, in contrast to P-DCs,
constitutively produce high levels of CCL17 and CCL22 ex vivo, which are enhanced further
by CD40 stimulation (Penna, Amuchastegui et al. 2007). CCL22 and CCL27 are chemokines
able to recruit activated T cells and, in particular, Th2 cells via CCR4. In addition, these
chemokines can recruit CD4+CD25+ Treg cells (D'Ambrosio, Sinigaglia et al. 2003). CCL22, a
chemokine mostly produced by DCs, has been found to selectively recruit, in ovarian
carcinoma patients, Foxp3+ Treg cells able to suppress anti-tumor responses, leading to
reduced patient survival (D'Ambrosio, Sinigaglia et al. 2003). Similarly, CCL22 secreted by
lymphoma B cells attracts Foxp3+CCR4+CD4+CD25+ Treg cells able to suppress proliferation
and cytokine production by tumor-infiltrating CD4+CD25- T cells (Yang, Novak et al. 2006).
Thus, the high constitutive and inducible production of CCR4 agonists by immature mDCs
could lead to the preferential attraction of CD4+CD25+ Treg cells. Intriguingly, the production
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Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD
CCL22 is markedly enhanced by 1α,25(OH)2D3 in blood M-DCs (Penna, Amuchastegui et al.
2007), indicating that VDR agonists may favor the recruitment of Treg cells by this DC subset,
a novel finding consistent with previous in vivo data (Gregori, Casorati et al. 2001; Gregori,
Giarratana et al. 2002; Penna, Amuchastegui et al. 2007). Treatment of autoimmune diseases
A common pattern shared by most autoimmune diseases is the Th1-mediated inflammatory
profile. Epidemiological analysis shows reasonably strong ecological and case-control
evidence that vitamin D reduces the risk of autoimmune disease, including multiple sclerosis
and T1D, and weaker evidence for rheumathoid arthritis (Grant 2006). The high prevalence of
vitamin D inadequacy in the general population could indeed favor chronic inflammatory
diseases, in addition to bone diseases and cancer (Holick 2007).
The anti-inflammatory and immunoregulatory properties of VDR agonists have been
studied in different models of autoimmune diseases. Notably, 1α,25(OH)2D3 and its analogs
can prevent systemic lupus erythematosus in MRLlpr/lpr mice, experimental allergic
encephalomyelitis (EAE), collagen-induced arthritis, Lyme arthritis, IBD, and autoimmune
diabetes in NOD mice. VDR agonists are able not only to prevent but also to treat ongoing
autoimmune disease, as demonstrated by their ability to inhibit T1D development in adult
NOD mice, and the recurrence of autoimmune disease after islet transplantation in the NOD
mouse, or to ameliorate significantly the chronic-relapsing EAE induced in Biozzi mice by
spinal cord homogenate (Mattner, Smiroldo et al. 2000; Penna, Amuchastegui et al. 2006).
As discussed previously, an important property of VDR agonists is their capacity to
modulate both APCs and T cells. Distinct regulatory mechanism may predominate in different
autoimmune disease models but a common pattern, characterized by inhibition of Th1 cell
development, has been frequently observed. The induction of tolerogenic DCs, which leads to
an enhanced number of CD4+CD25+ Treg cells renders them appealing for clinical use,
especially for the prevention and treatment of autoimmune disease (Adorini, Giarratana et al.
2004; Hackstein and Thomson 2004). However, topical treatment of psoriasis is the only
clinical application so far established for VDR agonists in the therapy of autoimmune
diseases. The calcemic liability of systemically applied VDR agonists still hampers progress
towards clinical applications, a situation that may be resolved by the ongoing development of
more potent and less calcemic analogs. In addition, additive and even synergistic effects have
been observed between VDR agonists and immunosuppressive agents, such as cyclosporine A
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and sirolimus, in autoimmune diabetes and EAE models (van Etten and Mathieu 2005). In the
following chapters, BPH and principally IBD will be extended.
Peripheral lymphoid organs
Target tissue
Target cell
Fig. 11. Immunomodulatory effects of VDR agonists in autoimmune diseases. VDR
agonists can modulate the inflammatory response via several mechanisms in secondary lymphoid
organs and in target tissues. In secondary lymphoid organs, VDR agonists inhibit IL-12 and IL-23
production and ILT3 expression. M-DC modulation by VDR agonists inhibit development of Th1 and
Th17 cells, while inducing CD4+CD25+Foxp3+ regulatory T cells and, under certain conditions, Th2
cells. VDR agonists can also inhibit the migration of Th1 cells, and they up-regulate CCL22 production
by M-DC, enhancing the recruitment of CD4+CD25+ regulatory T cells and of Th2 cells. In addition,
VDR agonists exert direct effects on T cells by inhibiting IL-2 and IFN-γ production. In target tissue,
pathogenic Th1 cells, that can damage target cells via induction of cytotoxic T cells and activated
macrophages, are reduced in number and their activity is further inhibited by CD4+CD25+ Treg cells and
by Th2 cells induced by VDR agonists. IL-17 production by Th17 cells is also inhibited. In
macrophage, important inflammatory molecules like COX-2 and iNOS, are inhibited by VDR agonists,
leading to decreased production of NO and prostaglandin E2 (PGE2). Macrophages, as well as DCs
and T cells, can synthesize 1α,25(OH)2D3 and this may also contribute to the regulation of the local
immune response (Adorini 2002; Adorini and Penna 2008).
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1.3 Begnin prostate hyperplasia
1.3.1 Definition
BPH is the most frequent benign neoplasm in aging men and one of the most common chronic
conditions in the male population, with a histological prevalence at autopsy of 50% in men
aged 50 to 60 years and of 90% over 80 years (McVary 2006). Clinical BPH refers to the
lower urinary tract symptoms (LUTS), associated with benign prostatic enlargement leading
to bladder outlet obstruction. BPH is defined histologically by hyperproliferation of stromal
and epithelial cells of the prostate, caused by complex cellular alterations including changes
in proliferation, differentiation, apoptosis and senescence (Lee and Peehl 2004).
Compared to normal prostate tissue, hyperplastic nodules are characterized by reduced
epithelium-to-stroma ratio, determined by an imbalance between growth and death programs
of stromal cells (Ishigooka, Hayami et al. 1996; Claus, Berges et al. 1997; Lin, Wang et al.
2000), leading to increased final stromal volume. Histological micronodular alterations appear
early in young men, characterized by an immature mesenchyme displaying features of
embryonic mesenchyme, able to differentiate into myofibroblasts and smooth muscle cells to
generate a “reactive stroma” (Peehl and Sellers 1997; Rumpold, Untergasser et al. 2002; Lee
and Peehl 2004; Untergasser, Madersbacher et al. 2005). These changes in stromal
architecture and homeostasis, and in the microenvironment of prostatic stromal-epithelial cell
interactions, induce subsequent epithelial rearrangements and BPH progression (McNeal
1990; Donjacour and Cunha 1991; Bierhoff, Walljasper et al. 1997).
1.3.2 VDR agonists in BPH treatment
A link between the vitamin D system and the prostate was first established by epidemiological
correlations of increased prostate cancer incidence and mortality rates in patients with vitamin
D insufficiency(Schwartz 2005). The prostate was then recognized as an extrarenal site of
vitamin D synthesis and action through the expression of 1α-hydroxylase and VDR,
respectively (Ali and Vaidya 2007). VDR expression in tissues derived from the urogenital
sinus, as prostate and bladder, is quantitatively similar to classic target organs for calcitriol, as
liver, kidney, and bone, although lower than in the bowel and in malignant prostate or bladder
cell lines (Maggi, Crescioli et al. 2006). Moreover, a growth-regulating role of calcitriol
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synthetized by the prostate is suggested by the demonstration of in vitro and in vivo antiproliferative, prodifferentiative, proapoptotic and anti-invasive properties in several models of
prostate cancer (Ali and Vaidya 2007), and by a marked decrease of 1α-hydroxylase activity
in prostate cancer cell lines (Maggi, Crescioli et al. 2006). Based on these preclinical results,
the chemotherapic efficacy of calcitriol, alone or in association with classical anti-tumoral
drugs (dexamethasone, docetaxel, carboplatin, estramustine), was tested by several clinical
trials in prostate cancer patients (Harzstark and Ryan 2008).
Pharmacological management of BPH is a novel application of vitamin D analogs,
prompted by the detection of VDR expression in cultured prostatic and bladder stromal cells
derived from BPH patients (Maggi, Crescioli et al. 2006). Reducing prostate overgrowth by
decreasing intra-prostatic androgen signalling, without directly interfering with systemic
androgen action, would obviate the adverse systemic side effects of anti-androgens, such as
5α-reductase inhibitors. In addition, VDR agonists display marked anti-inflammatory
properties and this class of agents could therefore represent an interesting therapeutic option
for the pharmacological treatment of BPH (Maggi, Crescioli et al. 2006). Elocalcitol ameliorates experimental autoimmune prostatitis
Based on marked inhibitory activity of the VDR agonist elocalcitol on basal and growth factor
induced proliferation of human prostate cells (Crescioli, Ferruzzi et al. 2004), this compound
was tested in experimental autoimmune prostatitis induced by injection of prostate
homogenate-complete Freund’s adjuvant in NOD mice (Penna, Amuchastegui et al. 2006).
Administration of elocalcitol, in normocalcemic doses for 2 weeks in already established
experimental autoimmune prostatitis (EAP) inhibits significantly the intraprostatic cell
infiltrate, leading to a profound reduction in the number of CD4+ and CD8+ T cells, B cells,
macrophages, DCs and I-Ag7-positive cells. Immunohistological analysis demonstrates
reduced cell proliferation and increased apoptosis of resident and infiltrating cells.
Therapeutic administration of elocalcitol in NOD mice with established EAP
decreases IFN-γ production by anti-TCR-stimulated lymph node cells, indicating inhibition of
Th1 cell responses in prostate-draining periaortic lymph nodes. Treatment with elocalcitol
also inhibits ex vivo production of IL-17, that could be relevant to the therapy of chronic
prostate inflammation because this cytokine has been found elevated in situ in prostate
specimens from patients affected by BPH, a condition characterized by prostate cell growth
associated with an important inflammatory component (Hackstein and Thomson 2004). In
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addition, a significantly decreased expression of inducible nitric oxide synthase (iNOS), a key
enzyme required for the synthesis of the inflammatory agent nitric oxide (NO), and a
markedly decreased production of NO itself is observed in peritoneal macrophages from
elocalcitol-treated NOD mice with established EAP (Penna, Amuchastegui et al. 2006). A
number of studies have associated excessive NO production with acute and chronic
inflammation in model systems, and have also demonstrated that administration of NO
synthase inhibitors can induce beneficial anti-inflammatory properties (Tinker and Wallace
2006). The capacity of elocalcitol to inhibit iNOS expression and NO production may thus
represent an additional mechanism explaining its anti-inflammatory properties. VDR agonists treat BPH-associated LUTS
Inhibition of prostatic inflammation and proliferation has been also observed in human BPH
cells. Elocalcitol treated human BPH cells inhibits significantly the inflammatory cytokine
(IFN-γ, IL-17 and TNF-α)-stimulated production of IL-8 responsible for their proliferation via
autocrine/paracrine mechanisms, but also inhibits the IL8-induced RhoA/ROCK pathway,
known to be involved in contractile signaling in many tissues, accompanied by inhibition of
cyclooxygenase 2 (COX-2) transcripts, prostaglandin E2 production and arrest of NF-κB p65
nuclear translocation (I and II). The capacity of elocalcitol to inhibit prostate inflammation
could complement the anti-proliferative effects of this VDR agonist on prostate growth,
providing a novel mechanism of action accounting for the arrest of prostate growth observed
in BPH patients treated with elocalcitol (Penna, Amuchastegui et al. 2006). Interestingly,
inhibition of prostatic inflammation by VDR agonists which is mediated by several different
mechanisms including inhibition of COX-2 and prostaglandin E2, could contribute to the
potential of these agents in prevention and treatment of prostate cancer (Krishnan, Moreno et
al. 2007).
Preclinical studies have shown reduced testosterone-induced BPH cell proliferation to
a similar extent than finasteride and cyproterone acetate, prompting apoptosis even in
presence of intraprostatic growth factors and completely antagonizing the effect of androgenic
stimulation at subpicomolar concentrations in cells treated with VDR agonist elocalcitol,
proposing this agonist as a potente regulator of the growth and survival of primary BPH
stromal cells (Maggi, Crescioli et al. 2006).
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Elocalcitol significantly decreased prostate growth both in unmanipulated and in
castrated T-replaced rats, with an effect comparable to finasteride but without affecting testis
androgenic secretion or pituitary function (Crescioli, Ferruzzi et al. 2004). Interestingly, both
prostatic epithelial and stromal cells from elocalcitol-treated rats showed increased apoptotic
rate and clusterin expression, confirming in vitro observations (Crescioli, Ferruzzi et al.
2004). Since rats do not develop BPH spontaneously, the anti-hyperplasic potential of
elocalcitol was tested in male beagle dogs chronically treated for 9 months with 5μg/kg/die. In
this model, reduction of prostate weight was observed, more evident after a 2-month recovery,
suggesting a prolonged pharmacological activity of this compound, in the absence of side
effects (Adorini, Penna et al. 2007).
These preclinical data prompted a 12-week phase IIa, multicenter, double-blind,
randomized, placebo-controlled clinical trial aimed at evaluating the efficacy and safety of
elocalcitol administration (150 μg/die) in BPH patients (Colli, Rigatti et al. 2006). Elocalcitol
exhibited a 7.2% reduction in prostate volume, measured by pelvic MRI, compared to
placebo. Importantly, 92% of elocalcitol-treated patients did not experience a clinically
significant growth in prostate volume compared with 48% in the placebo group. Thus, the
reduction of prostate volume in elocalcitol-treated group against its marked increase in the
placebo group indicates the ability of this VDR agonist to block the ongoing BPH process.
During the trial, no difference was observed in symptom score or urodynamic parameters,
probably because of the short duration of this proof-of-concept study and because patients
were not screened for symptoms but only for prostatic volume (Colli, Rigatti et al. 2006).
To elucidate this point, a 6-months phase IIb study was performed to measure
maximum urinary flow rate and symptom severity as secondary end-points in patients with at
least moderate symptomatology. Elocalcitol was effective in improving maximum urinary
flow rate (Qmax) and ameliorating LUTS, as well as arresting prostate growth and preventing
the risk of AUR and need for surgery (Fibbi, Penna et al. 2009), all key parameters of BPH
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1.4 Inflammatory bowel disease
The major forms of idiopathic IBD, UC and CD have been empirically defined by clinical,
pathological, endoscopic and radiological features (Podolsky 2002). IBD are chronic
inflammatory disorders of the gastrointestinal tract due to unbalanced activation of the
mucosal immune system in response to luminal antigens in genetically predisposed
individuals (Cho 2008). The onset of IBD typically occurs in the second and third decades of
life and a majority of affected individuals progress to relapsing and chronic disease.
Family aggregation has long been recognized. First-degree relatives of patients have a
relative risk of five-fold or greater and the inheritable component seems stronger in CD than
in UC (Tysk, Lindberg et al. 1988; Orholm, Munkholm et al. 1991). The remarkable increase
in the incidence of IBD during the last half century implicates changes in the environment as
a major cause for this evolution, since genetic variations are negligible in such a short period
of time, while the ‘‘hygiene hypothesis’’ of allergic and autoimmune diseases has been
invoked to explain the world-wide spreading of IBD (Danese, Sans et al. 2004).
Whilst CD and UC both fall under the collective term IBD, these conditions can be
quite distinct, with different pathogenesis, underlying inflammatory profiles, symptoms and
treatment strategies. UC is a relapsing non-transmural inflammatory disease that is restricted
to the colon, with characteristic histological findings such as acute and chronic inflammation
of the mucosa by polymorphonuclear leukocytes and mononuclear cells, crypt abscesses,
distortion of the mucosal glands and goblet cell depletion. In comparison, CD is a transmural
disorder affecting the entire gastrointestinal tract from the mouth to the anus, including at the
histological level small superficial ulcerations over a Peyer’s patch and focal chronic
inflammation extending to the submucosa, sometimes accompanied by non-caseating
granuloma formation (Baumgart and Carding 2007; Baumgart and Sandborn 2007; Xavier
and Podolsky 2007).
Both types of patients typically suffer from frequent and chronically relapsing flares,
resulting in diarrhea, abdominal pain, rectal bleeding and malnutrition. UC and CD are
manifestations are usually related to intestinal disease activity and may precede or develop
concurrently with intestinal symptoms (Danese, Semeraro et al. 2005).
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1.4.1 Diagnosis and clinical features
UC and CD are generally diagnosed using clinical, endoscopic and histologic criteria.
However, no single finding permits an absolute differential diagnosis. About 10-20% of
patients have a clinical picture that falls between CD and UC; and they are diagnosed as
affected by indeterminate colitis (Baumgart and Sandborn 2007).
The most consistent feature of UC is the presence of blood and mucus in the stool with
lower abdominal cramping, which is most intense during the passage of bowel movements.
The location of abdominal pain depends on the extent of colonic involvement. Pain is present
in the left lower quadrant with distal disease and extends to the entire abdomen with
pancolitis. Pediatric patients have a higher frequency of pancolonic involvement, a higher
likelihood of proximal extension of disease over time, and a higher risk of colectomy
compared to adult patients (Cho 2008).
In contrast to UC, the symptoms in CD could be subtle, leading to a delay in
diagnosis. Gastrointestinal symptoms depend on the location, extent, and severity of
involvement. In patients with ileocolonic involvement, abdominal pain is usually
postprandial, usually in the periumbilical area, especially in children. Gastroduodenal CD
presents with early satiety, nausea, emesis, epigastric pain or dysphagia. Due to postprandial
pain and delay in gastric emptying, patients with gastroduodenal CD often limit their caloric
intake to diminish their discomfort. Extensive small bowel disease causes diffuse abdominal
pain, anorexia, diarrhea and weight loss and may result in lactose malabsorption (Cho 2008).
While IBD can limit quality of life, due to pain, vomiting, diarrhea and rectal bleeding,
it is rarely fatal on its own. In part because of recurrences after surgery performed, quality of
life is lower in patients with CD than with UC. In case of IBD-dependent death, the most
common causes are septic peritonitis, malignancy and surgery complications (Baumgart and
Sandborn 2007).
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Table 2. Differential diagnosis of UC and CD. UC and CD are associated with both intestinal
and extraintestinal manifestations. Extraintestinal manifestations are usually related to intestinal
disease activity and may precede or develop with intestinal symptoms. While UC and CD have a
number of similarities in their clinical presentations, characteristic features highlight a number of
diversities. UC is a relapsing non-transmural inflammatory disease that is restricted to the colon. CD is
a relapsing, transmural inflammatory disease of the gastrointestinal mucosa that can affect the entire
gastrointestinal tract from the mouth to the anus. Typical presentations include the discontinuous
involvement of various portions of the gastrointestinal tract and the development of complications
including strictures, abscesses, or fistulas (Cho 2008). Adapted from (Baumgart and Sandborn 2007).
Clinical features
Passage of mucus or pus
Small-bowel disease
Can affect upper-gastrointestinal tract
Abdominal mass
Extraintestinal manifestations
Small-bowel obstruction
Colonic obstruction
Fistulas and perianal disease
Biochemical features
Anti-neutrophil cytoplasmic antibodies Common
Anti-saccharomyces cerevisia
Pathologival features
Transmural mucosal inflammation
Distorted crypt atchitecture
Cryptitis and crypt abscesses
Fissures and skip lesions
Common Epidemiology
IBD occurs most frequently in people in their late teens and twenties but also occurs in
children. There have been cases in children as young as two years old and in older adults in
their seventies and eighties. IBD is a disease of industrialized countries; the highest incidence
rates and prevalence have been reported from Northern Europe, the UK and North America.
Recently, it has been estimated to affect approximately four million people worldwide. In
North America, prevalence rates of CD for Hispanic (4:1 per 100000) and Asian people (5:6
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per 100000) is much lower than those for white individuals (43:6 per 100000) and AfricanAmerican people (29:8 per 100000) (Baumgart and Carding 2007).
Etiology Environmental factors
IBD‘s higher prevalence in more developed, or “Westernized” countries, could be explained
by an increased sanitation and the lifestyles within these regions. This hypothesis is supported
by large epidemiological studies in North America and Europe that shown a higher incidence
in urban compared with rural communities. It has been proposed that the exposure to
unhygienic conditions during development can prime the intestinal environment leading to
optimal mucosal immune development and regulation, thus preventing a future inflammatory
response. There are many environmental modifications that can be ascribed to the hygiene
hypothesis, including better housing, safer food and water, improved hygiene and sanitation,
vaccines, the widespread use of antibiotics, lack of parasites, fewer infections and better but
selective nutrition (Danese, Sans et al. 2004; Geier, Butler et al. 2007).
Some distinct environmental factors are equally considered as risk factors for IBD,
such as smoking, diet, drugs, social status, stress, the enteric flora, altered intestinal
permeability and appendectomy. Remarkably, smoking has a completely opposite effect on
CD compared to UC, further indicating that distinct pathogenic mechanisms underlie each
form of IBD (Danese, Sans et al. 2004). Genetic factors
A positive family history is still the largest independent risk factor for the disease,
highlighting the transmission of genetic information as a key feature in disease onset. The
frequency of familial occurrence among unselected individuals with IBD has been reported to
be as high as 20-30% in referral-based studies, but has ranged between 5 and 10% in
population-based surveys. Epidemiological studies have also shown that 75–80% of families
with members affected by the disease present concordance for disease type. All family
member affected by IBDs will be or CD or UCs but inside a family not both (Cho and
Abraham 2007; Cho 2008).
The strongest evidence of genetic factors contributing to susceptibility to IBD comes
from concordance studies in twins. Monozygotic-twin concordance for CD has ranged
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between 42 and 58% and between 6 and 17% for UC. This indicates that genetic factors are
more significant in CD than in UC (Baumgart and Carding 2007).
UC and CD are polygenic diseases: twelve genome-wide association (GWA) studies
have identified susceptibility regions on chromosomes 16, 12, 6, 14, 5, 19, 1, 16, and 3. These
regions have been renamed as IBD1–9, respectively (Baumgart and Carding 2007).
Nod2 was the first specific gene on chromosome 16 associated with IBD (and so is
also known as IBD1) and has been established as a susceptibility locus for CD but not UC.
Because genetic factors are more significant in CD, most of the first studies were performed
only on CD. However, recent studies extend analysis to UC and discover specific candidate
genes like IL-10. Extensive GWA results open a window into the complex biology of IBD,
revealing common candidate genes implicate in adaptive and innate immune system
pathways, such as IL-23R, STAT3 or IL-12B (encoding IL12/23p40). In addition, these studies
permit the identification of unexpected pathways, such as autophagy (especially ATG16L1 in
CD) (Cho 2008; Budarf, Labbe et al. 2009). Immunological factors
The dominant hypothesis in IBD pathogenesis is the abnormal dynamic balance between
microbes, particularly commensal flora, and host defensive responses at the mucosal frontier.
As observed in conventional animal facilities, the Il-10 KO mice develop enterocolitis within
5–8 weeks of life. This is caused by uncontrolled immune responses to conventional
microflora, since germ-free Il-10 KO mice do not develop the disease. In addition, mice raised
in specific pathogen-free facilities develop a milder disease, which does not result in death
(Kuhn, Lohler et al. 1993). Confirmation of the important role of the bacterial flora is also
provided by treatment with antibiotics, exterting beneficial effects in both CD and UC
patients (Baumgart and Sandborn 2007). However, precisely how commensal bacteria in the
intestine interface locally with cells of the immune system to initiate and perpetuate intestinal
inflammation remains unclear.
The first line of defence of the mucosal immune system is the epithelial barrier. Per
consequence, disturbances in epithelial barrier permeability represent the first signal required
for an abnormal response to luminal antigens. The intestinal epithelium, which is considered
to be a part of the innate immune system, plays an active role in the maintenance of mucosal
homeostasis (Artis 2008). Epithelial cells form a tight, highly selective barrier between the
body and the intraluminal microenvironment. Failure of this barrier may result in intestinal
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inflammation, most likely through exposure to fecal antigens leading to inappropriate
activation of the mucosal immune system. IBD patients present lowered epithelial resistance
and increased permeability of the inflamed and non-inflamed mucosa (Soderholm, Olaison et
al. 2002). Similar defect were shown to be involved in the pathogenesis in mice models. The
classical model used to study experimental colitis is a dextran sodium sulphate (DSS)-induced
colitis model. This chemical, once ingested, provokes severe bloody stool and a wasting
disease by disruption of the intestine epithelial barrier. Genetically modified mice that express
a dominant-negative N-cadherin transgene in epithelial cells present also a disruption of interepithelial cell adhesion resulting in modest inflammation, particularly in areas of the intestine
that lie beneath areas of barrier disruption (Elson, Cong et al. 2005).
Discovery of Nod2 mutations, a PRR recognizing the muramyl dipeptide, associated
with a group of CD patients (those with small bowel and stricturing disease) suggests an
important role of the innate immune system in disease pathogenesis (Danese and Fiocchi
2006). In the last few years, mutations in both TLRs and NLRs have been found to be
associated with IBD, confirming their required dysfunctions for the pathology onset. Even if
Nod2 mutations are the most important mutations observed in IBD patients, their exact role is
still undefined. Nod2 silencing does not induce spontaneous colitis symptoms, but mice are
more susceptible to intra-oral Listeria infection correlated to decreased expression of antimicrobial peptides (Kobayashi, Chamaillard et al. 2005). A recent study has highlighted a
dysfunction in IL-10 production after TLR activation of PBMCs from IBD patients carrying a
Nod2 mutation, by inhibiting MAP kinase phosphorylation (Noguchi, Homma et al. 2009).
Thus, Nod2 seems to play a role in the regulation of the balance between pro- and antiinflammatory cytokine production.
Nod2 is not the only PRR member involved in IBD. Most of the TLRs, especially
TLR4, have been proposed in some GWA studies as possible hot spots, and hyporesponsiveness of certain TLR4 polymorphisms have been observed for CD patients in
Northern Europe. In addition, colitis was exacerbated in mice treated with a TLR4 antagonist
or in tlr4 KO mice (Fort, Mozaffarian et al. 2005; Fukata, Michelsen et al. 2005). Still, GWA
studies could not confirm Nod2 mutations in all IBD patients.
An important role for the adaptive immune system is clearly demonstrated by GWA
studies showing IL-23R and IL-12B as hotspot mutations (Cho 2008).
The two IBD subsets were thought to involve two different T helper cell-mediated
responses. CD, characterized by elevated levels of IL-2 and IFN-γ, was considered as a
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predominant Th1-mediated disease. Conversely UC, showing higher amount of IL-5 and IL13 was considered as Th2-mediated. Recently, this view was abandoned with the emergence
of a new Th subset, the Th17 (Korzenik and Podolsky 2006). Mechanistic studies have
highlighted the role of IL-23 in the Th17 lineage in IBD patients and in experimental models.
Colitis initiation was originally correlated with IL-12 (Neurath, Fuss et al. 1995).
However, recent findings on the shared p40 chain in both IL-12 and IL-23 highlight IL-23 as
a required candidate for colitis development. Il-10 KO mice that were made Il-23 deficient do
not develop colitis (Noguchi, Homma et al. 2009). These observations, confirmed by a GWA
study, have highlighted the impact of the IL23/Th17 axis in IBD development.
IL-23-dependent colitis does not require IL-17, but it seems to inhibit the frequency of
Treg cells in the colon (Izcue, Hue et al. 2008). Although IL-23 has a pronounced
inflammatory role in IBD, the individual Th17-associated cytokines have more complex roles.
The role of IL-17 in colitis is controversial, because there are data suggesting that it is proinflammatory, protective, or that it has no observable role. IL-17 stimulation of colonic
epithelial cells induces pro-inflammatory cytokines, such as IL-6 and IL-8, and chemokines
like monocyte chemotactic protein-1 (MCP-1) (Andoh, Takaya et al. 2001). Accordingly, IL17 deficiency ameliorates colitis in mice (Zhang, Zheng et al. 2006). By contrast,
neutralization of IL-17 using antibodies during acute colitis leads to more severe disease,
suggesting a protective role of this cytokine (Ogawa, Andoh et al. 2004), while in a T-cellmediated model of colitis, IL-17 has been shown to be non-essential in disease development
(Izcue, Hue et al. 2008). Each of these studies was performed using different colitis models
and distinct ways of neutralizing IL-17 (anti-IL-17 antibody administration versus cytokine
KO versus receptor KO). This might explain why differing roles for IL-17 have been
described. Lastly, it is important to note that IL-17 is expressed by T cells in the healthy
gastrointestinal tract and that this expression is dependent on the presence of commensal flora
(Niess, Leithauser et al. 2008).
IL-21 is also upregulated in lesions of IBD patients and seems to regulate the balance
between Th17 and Treg cells in the gastrointestinal tract. Actually, IL-21 is required, in
combination with TGF-β, to drive the Th17 lineage. In its absence, TGF-β stimulation leads
to preferential induction of Treg cells (Fantini, Rizzo et al. 2007). In mice models, IL-21
deficiency leads to a reduced severity of chemically-induced colitis, and wild type mice
present a significant increase of IL-21 levels (Fina, Sarra et al. 2008). Thus, IL-21-driven
Th17 differentiation seems to actively contribute to the development of colitis.
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Unlike other Th17 cytokines, IL-22 is protective in IBD. Mice deficient in Il-22, either
genetically or through antibody depletion, display a more severe disease in models of acute
and chronic colitis. In addition, gene delivery approaches have shown that IL-22 can be
therapeutic, when directed to pre-existing inflammation (Sugimoto, Ogawa et al. 2008). The
protective effects of IL-22 in the gastrointestinal tract are not limited to IBD, but IL-22 has
also been shown to be protective to infection with the gastrointestinal pathogen Citrobacter
rodentium (a cause of murine colonic mucosal hyperplasia) (Zheng, Valdez et al. 2008). IL-22
induces pro-survival and anti-apoptotic signaling pathways in the cells of the intestinal
epithelium. IL-22 secretion by Th17 cell might provide protection through maintaining the
integrity of the epithelial barrier and protect mucin secreting goblet cells (Sugimoto, Ogawa et
al. 2008).
Th17 pathways could represent novel candidates for the regulation of colitis, but their
precise role in disease onset is still unresolved. Th1 pathways remain interesting targets, as
shown by IL-12p40 antibody treatment in CD patients (Baumgart and Sandborn 2007).
1.4.3 Current treatments
Glucocorticosteroids have been used in the treatment of active IBD for many decades and are
effective in inducing clinical remission of CD and UC. However, corticosteroids are not
effective for maintenance of remission and their long-term use is associated with sometimes
severe and irreversible side effects (Faubion, Loftus et al. 2001). Within one year from the
start of steroid therapy, most patients relapse or develop corticosteroid dependency. The
introduction in 1998 of biologics, such as infliximab (Remicade; Centocor), a chimeric
monoclonal antibody directed against TNF-α, for the treatment of CD, has changed the
treatment of refractory IBD dramatically (Hanauer, Feagan et al. 2002).
However, ideal therapeutic strategies for all IBD patients, inducing and maintaining
long-term remission without steroid exposure or surgery, have yet to be developed.
Epidemiological studies show that vitamin D deficiency correlates with IBD severity,
leading to a growing interest on the role and action of VDR agonists in this pathology
(Cantorna and Mahon 2004). VDR agonists show indeed potential therapeutic effects in IBD
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD
Table 3. Novel agent currently under investigation for treatment of IBD. Recent advances
in the understanding of UC and CD pathogenesis provide new biological candidates for therapeutic
approach targeting diverse cellular pathways. Adapted from (Baumgart and Sandborn 2007).
T-cell differentiation
Selective adhesion
Innate immune
Intestinal repair
Certolizumab (CDP-870)
Growth hormone
Trichuris suis ova
Balance of
gut flora
Autologous bone marrow
Drug class
Disease Phase
T cells?
Leukocytes Device
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
1.5 Vitamin D and inflammatory bowel disease
Immunomodulatory effects of VDR agonists are primarily observed on the inhibition of Th1mediated responses, but they can also inhibit Th17 responses (Penna and Adorini 2000; Tang,
Zhou et al. 2009). In addition to these direct immunomodulatory effects, VDR agonists induce
anti-microbial peptide expression, which shows benefits in colitis models (Tai, Wu et al.
1.5.1 Vitamin D deficiency and VDR polymorphisms in IBD patients
IBD is more common in higher latitudes in Europe and North America, described as the
‘‘North–South’’ gradient in IBD incidence. A possible explanation for this higher prevalence
could be vitamin D deficiency, since the Northern hemisphere receives less sunlight,
especially during winter. Vitamin D deficiency has been observed as a common feature in
IBD patients, and notably among pediatric one and in the Iranian population (Pappa, Grand et
al. 2006; Naderi, Farnood et al. 2008). Furthermore, two studies have shown that fish oil,
which is a rich source of vitamin D, decreases IBD severity (Cantorna and Mahon 2004),.
Recently, a pilot clinical study was performed on IBD patients, resulting in short-term
beneficial effect on bone metabolism and on disease activity after a one year administration of
calcitriol (Miheller, Muzes et al. 2009). This study underlines the potential role of vitamin D
in IBD treatment. In addition, the VDR maps to the IBD2 locus on chromosome 12, and VDR
gene polymorphisms have been described in CD patients (Simmons, Mullighan et al. 2000;
Gaya, Russell et al. 2006).
These genetic observations coupled to the environmental hypothesis support the use of
vitamin D analogs as therapeutic agents.
1.5.2 VDR agonists in IBD treatment In vitro activity
Lymphocytes represent key cells in IBD pathogenesis, and their recruitment in the gut
promotes the inflammatory cascade (Korzenik and Podolsky 2006). Phytohaemagglutininactivated peripheral blood T lymphocytes from UC patients or healthy controls cultured in the
presence of 1α,25(OH)2D3 or the VDR agonists EB1089 and KH1060 show significant and
dose dependent inhibition of proliferation from day 3 of culture (Stio, Bonanomi et al. 2001).
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
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This anti-proliferative effect of VDR agonists is induced by increased apoptosis, as observed
by higher protein levels of pro-apoptotic signals, such as Poly(ADP-ribose)-polymerase
(PARP) cleavage or caspase-3 (Martinesi, Treves et al. 2008). In addition, VDR agonisttreated PBMCs activated by TNF-α or LPS and co-cultured with human umbilical vein
endothelial cells (HUVEC) present an inhibition of ICAM-1, an adhesion molecule upregulated in IBD patients. This demonstrated the capacity of VDR agonists to inhibit cell–cell
contacts as well as cell–cell interactions between endothelial cells and lymphocytes
(Martinesi, Treves et al. 2008).
Based on the key role of DCs in driving T cell responses, DC modulation would
represent an important feature for VDR agonist activity in the context of IBD. Co-treatment
with dexamethasone and 1α,25(OH)2D3 arrests the differentiation and the maturation of DCs
activated with enteroantigen, as shown by decreased expression of maturation markers, such
as CD40, CD80 and CD86 as well as MHC-II (Pedersen, Schmidt et al. 2009). These cotreated DCs are impaired in T-cell activation, as demonstrated by inhibition of T-cell
dependent cytokine production, such as IL-4, IFN-γ, IL-2 or IL-17 (Pedersen, Schmidt et al.
TNF-α represents a validated target in IBD, since this cytokine plays an important role
in the initiation and perpetuation of intestinal inflammation in IBD and anti-TNF-α antibodies
are approved therapies also for this indication. The VDR agonist TX-527 [19-nor-14,20bisepi-23-yne-1α,25(OH)2D3] inhibits proliferation and TNF-α production by PBMCs from
CD patients (Stio, Martinesi et al. 2007). TNF-α inhibition in LPS-activated PBMCs is not
restricted to CD, but also PBMCs from UC patient are responsive to VDR agonists (IV).
Moreover, VDR agonists inhibit pro-inflammatory cytokines production such as IFN-γ, IL12p40, IL-6 or IL-1β in PBMCs from IBD patients activated by bacterial components or
cytokine stimuli (IV, V) (Ardizzone, Cassinotti et al. 2009). After treatement with the VDR
agonist TX-527, PBMC purified from CD patients stimulated by TNF-α present an inhibition
of NF-κB nuclear translocation together with an inhibition of IκBα degradation (Stio,
Martinesi et al. 2007). In vivo activity
Il-10 KO mice fed with a low calcemic diet develop diarrhea and a severe wasting disease
earlier than mice fed with a normocalcemic diet or supplemented with vitamin D (Cantorna,
Munsick et al. 2000). Moreover, administration of 1α,25(OH)2D3 significantly ameliorates
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
IBD symptoms in Il-10 KO mice and treatment for as little as 2 weeks blocks the progression
and ameliorates symptoms in mice with already established IBD (Cantorna, Munsick et al.
2000). As expected, VDR expression is required to control inflammation in the IL-10 KO
mouse, since colitis is exacerbated in Il-10/Vdr double-deficient mice, associated with high
local expression of IL-2, IFN-γ, IL-1β, TNF-α and IL-12 (Froicu, Weaver et al. 2003). Vdrdeficient mice are also extremely sensitive to DSS-induced colitis and then both dietary
calcium and intrarectal administration of 1α,25(OH)2D3 directly and indirectly inhibits the
TNF-α pathway, decreasing the severity and extent of DSS-induced colitis in wild-type mice.
Moreover, treatment of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis with the
VDR agonist 22-ene-25-oxa-1α,25(OH)2D3 (ZK156979) inhibits disease at normocalcemic
doses, accompanied by down-regulation of myeloperoxidase activity, TNF-α, IFN-γ, and Tbet expression, whereas in local tissue IL-10 and IL-4 protein levels increased (Daniel,
Radeke et al. 2006). The same group has recently proposed that 1α,25(OH)2D3, administered
together with dexamethasone, inhibits IL-12, IL-23p19 and IL-17 linked with increased
FoxP3 and TGF-β expression, hypothesizing a switch from Th1 and Th17 cells to Treg cells
(Daniel, Sartory et al. 2008).
Vdr-deficient mice undergoing DSS present impaired wound healing preceded by
increased transepithelial electric resistance (Kong, Zhang et al. 2008). These observations
suggest that VDR plays a critical role in mucosal barrier homeostasis by preserving the
integrity of junction complexes and the healing capacity of the colonic epithelium. Therefore,
vitamin D deficiency may compromise the mucosal barrier, leading to increased susceptibility
to mucosal damage and increased risk of IBD. Vdr-deficient mice present mortality after
intravenous LPS administration, likely due to a loss of negative control in the TLR activation
pathway in absence of VDR (Froicu and Cantorna 2007). In addition, as already mentioned,
1α,25(OH)2D3 is a direct inducer of CAMP, shown to provide benefits in DSS-induced colitis.
Moreover, 1α,25(OH)2D3 and other VDR agonists inhibit in vitro, in monocytes or PBMCs,
TNF-α, IL-6, IL-12p40 and IL-1β after activation by any TLR human ligands (IV).
These results highlight an important role for VDR and VDR agonists in the control of
innate and adaptive immunity as well as in the epithelial membrane integrity in IBD,
identifying the vitamin D system as a potential key regulator of gastrointestinal homeostasis.
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Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD
2. Aims of the study
BPH and IBD are two chronic inflammatory disorders affecting the prostate and the
gastrointestinal tract, respectively. A better understanding of the inflammatory component
involved in their pathogenesis could support a potential therapeutic use of VDR agonists in
these indications.
Therefore, aims of the present thesis were:
To characterize the inflammatory component involved in BPH pathogenesis.
To evaluate in BPH cells the anti-inflammatory properties of elocalcitol, a VDR
agonist proposed for the treatment of BPH.
To identify a potent and safe VDR agonist for a potential use in the treatment of
immunomodulatory properties
To contribute to the understanding of IBD pathogenesis.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
Materials and Methods
3. Materials and Methods
The capital letters (I-V) refer to the manuscripts using the corresponding materials and
methods. In this section, methods used by co-authors will not be described.
3.1 VDR agonists
Crystalline 1α,25(OH)2D3 and its analogues (Fig. 12 and Table 4) were a gift of Dr. Milan
Uskokovic (BioXell Inc. Nutley, NJ, USA). The compounds were reconstituted in 100 %
ethanol, at the concentration of 1 mg/ml and stored in concentrated solutions at -80 °C under
nitrogen atmosphere. 1α,25(OH)2D3 and its analogues were freshly diluted before each
experiment, and the ethanol concentration in the test conditions did not exceed 0.00025%.
Fig. 12. Chemical structures of 1α,25(OH)2D3 and key VDR agonists used in this thesis.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD
Table 4. Summary of VDR agonists used in this thesis. The maximal tolerated dose (MTD)
is established as described below. The capital numbers refer to the text as numbered in the List of
Publications (MW: molecular weight).
Chemical name
1,25-dihydroxy-vitamin D3
cyclopropyl-vitamin D3
cyclopropyl-19-nor-vitamin D3
1α-fluoro-25-hydroxy-16-ene-23yne-20-cyclopropyl-vitamin D3
cyclopropyl-vitamin D3
1α-fluoro-25-hydroxy-16,23Ediene-26,27-bishomo-20-epivitamin D3
BXL-628 /
3-desoxy-1,25-dihydroxy-16-ene23-yne-20-cyclopropyl-vitamin D3
1,25-dihydroxy-16-ene-20cyclopropyl-23-yne-26,27hexafluoro-19-nor-vitamin D3
1,25-dihydroxy-16-ene-20cyclopropyl-23-yne-26,27hexafluoro-vitamin D3
1α-fluoro-25-hydroxy-16-ene-20cyclopropyl-23-yne-26,27hexafluoro-vitamin D3
1,25-dihydroxy-16,23E-diene-20cyclopropyl-26,27-hexafluoro-19nor-vitamin D3
1,25-dihydroxy-16,23E-diene-20cyclopropyl-26,27-hexafluorovitamin D3
1α-fluoro-25-hydroxy-16,23Ediene-20-cyclopropyl-26,27hexafluoro-vitamin D3
1,25-dihydroxy-16-ene-20cyclopropyl-19-nor-vitamin D3
1,25-dihydroxy-16-ene-20cyclopropyl-vitamin D3
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
Materials and Methods
3.2 Cell cultures
3.2.1 Primary prostate cell lines
Primary cell lines derived from BPH patients were used for work I and II. BPH cells were
obtained from prostate tissues derived from three patients, who underwent transurethral
resection of the prostate (TURP) for BPH, after informed consent and approval by the Local
Ethical Committee. Patients did not receive any pharmacological treatment in the 3 months
preceding surgery. The tissue was cut into small fragments and treated overnight with 2
mg/mL bacterial collagenase (700 U/ml). Fragments were than extensively washed in
phosphate-buffered saline (PBS, 140 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM
Na2HPO4.2H2O) and cultured in DMEM-F12 1:1 supplemented with 10% heat-inactivated
FBS, 2 mmol/l glutamine, 100 UI/ml penicillin and 100 mg/ml streptomycin. Cells began to
emerge within 1 week and were used within the 15th passage. BPH cell cultures comprised
fibroblasts (for the most part) and fibromuscular cells and were negative for endothelial and
epithelial markers (I, Fig. 1).
3.2.2 Immortal cell lines
Two cells lines, myelomonocytic THP-1 cells, originally derived form human peripheral
blood from a patient suffering of acute monocytic leukaemia, and an ovarian cancer cell line
(CAOV cells) derived from a human ovarian adenocarcinoma, were used in III. THP-1 cells
were maintained in culture in phenol red-free RPMI 1640 with Glutamax and 10% (v/v) heatinactivated FBS supplemented with 0.1 mg/ml streptomicine and 100 UI/ml penicillin. 24 h
prior the treatment THP-1 (106/ml) were grown overnight in phenol red-free RPMI 1640 with
Glutamax and 5% (v/v) charcoal-treated heat-inactivated FBS with 0.1 mg/ml streptomicine
and 100 UI/ml penicillin. After treatment with the corresponding concentration of VDR
agonist, mRNA was extracted. CAOV cells were maintained in McCoy’s culture media
supplemented with 10% FCS. For metabolism studies, 3x106 cells were seeded in T150
culture bottles and were grown to confluence. Confluent CAOV cells were incubated with 1
μM VDR agonists in 50 ml media containing 10% FCS. The incubations were carried out at
37 °C in a humidified atmosphere under 5% CO2 and were quenched at 24 h with 10 ml of
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3.2.3 Peripheral blood mononuclear cells
Peripheral blood mononuclear cells (PBMCs) purified from whole blood or from buffy coats
were obtained by Ficoll gradient (III, IV and V). PBMCs were isolated from buffy coats of
healthy subjects (through the courtesy of Centro Trasfusionale, Ospedale di Magenta, Milan,
Italy) or blood from IBD patients (in collaboration with Dr. Danese from Istituto Clinico
Humanitas, Milan, Italy) by Ficoll gradient (BioChrome AG, Berlin, Germany).
Blood was collected from from 40 UC patients and 44 CD patients. Diagnosis was
based on clinical, radiological, endoscopic and biopsy findings. Sometimes, PBMCs obtained
from one patient were used only for a set of experiments, being insufficient to carry out all the
programmed experiments. Informed consent for this study, carried out in vitro on PBMCs,
was obtained from all patients.
Briefly, buffy coats or blood were diluted with PBS supplemented with 2.5 mM
EDTA (Sigma-Aldrich, USA) and loaded over a Ficoll-Paque. Density gradient was
centrifuged for 30 min at 2000 rpm at room temperature (RT). About 95% of mononuclear
cells at the interface containing PBMC were collected and washed twice with PBS. PBMC
viability was determined by Trypan blue (Sigma-Aldrich) exclusion test. Cells were always
>95% viable at culture initiation.
3.2.4 Lamina propria mononuclear cells
Lamina propria mononuclear cells were purified from colon or ileum biospy from two CD
and two UC patients (in collaboration with Dr. Danese from Istituto Clinico Humanitas,
Milan, Italy) after informed consent and approval by the Local Ethical Committee.
Briefly, the epithelium was removed from the lamina propria by incubation with 5 mM
EDTA in HBSS (Gibco Invitrogen, Paisley, UK) for 20 min under gentle shaking. Then, the
mucosal layer was cut in small pieces and digested for 30 min in 0.75 mg/ml collagenase type
2 and 20 µg/ml DNase type 1 (Sigma-Aldrich). Single cell suspensions were obtained by
filtering with 100 µm and 70 µm cell strainers, followed by extensive washing in complete
medium. About 50x106 cells were loaded over a Ficoll-Paque density gradient and centrifuged
for 20 min at 690 g. The interface was collected, washed and then loaded on a Percoll density
gradient (GE healthcare, Sweden) over 46% (v/v) and 100% and centrifuged 30 min at 2000
rpm. The interface containing mononuclear cells and the pellet containing leukocytes were
recovered and washed twice with PBS. Viability was determined by Trypan blue (SigmaAldrich) exclusion test. Cells were always >95% viable at culture initiation.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
Materials and Methods
3.3 In vitro experiments
3.3.1 Mixed lymphocyte reaction
After purification of PBMCs as previously described, the same number (3x105/200 μl/well) of
allogeneic PBMCs from two different donors were cocultured in 96-well flat-bottom plates in
RPMI 1640 with Glutamax and 5% (v/v) heat inactivated Fetal Clone-1 (FC-1, Charles River,
Italy) with 1% non essential amino acids, 0.5 mg/ml gentamicin and 1 mM sodium pyruvate,
in presence of the indicated concentrations of VDR agonists. Cultures were incubated at 37
°C in humidified atmosphere containing 5% CO2. After 5 days, proliferation and IFN-γ were
3.3.2 TLR-activated PBMCs or LPMCs
PBMCs (2x105/200 μl/well) were cultured in complete medium (RPMI 1640 with Glutamax
and 10% (v/v) heat-inactivated FBS (Charles River, Italy) with 1% non essential amino acids,
0.5 mg/ml gentamicin and 1 mM of sodium pyruvate) in 96-well flat-bottom plates in
presence of 100 ng/ml of LPS from Escherichia coli 0111:B4 (Sigma-Aldrich) (III) or with
various TLR agonists (TLR agonist kit, InvivoGen, USA) (Table 5) (I, II, IV and V). In V,
cells were treated in addition with an antibody targeting the IL-10 receptor (1 µg/ml,
CDW210, BD biosciences, USA) or with its corresponding isotype control (rat IgG γ2a, BD
biosciences) at similar concentrations. For mRNA quantification in IV, cells were washed
after 6 h with PBS and lised with RLT buffer (Qiagen, Germany) following the protocol
describer later. After 24 h, culture supernatants were harvested and stored at -80°C (III, IV,
Lamina propria leukocytes (1x105/200 µL/well) were cultured in complete medium
supplemented with 100 UI/ml penicillin, 0.1 mg/ml streptamicin and 0.5 µg/ml amphotericin
B in 96-well round bottom plates in presence of 1 µg/ml coated anti-CD2 (BD biosciences)
and 1 µg/ml soluble anti-CD28 (BD biosciences) in presence or absence of VDR agonists
(IV). Mononuclear LPMCs (2x105/200 μl/well) were cultured as described for the PBMC
with the supplemented medium. Cultures were incubating at 37 °C in humidified atmosphere
containing 5% CO2. At the end of the experiment (24 or 72 h) the supernatants were harvested
and stored at -80°C until analysis.
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Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD
3.3.3 BPH cell activation
BPH cells at 70-85% confluence were cultured in DMEM-F12 1:1 supplemented with 10%
heat-inactivated FBS, 2 mmol/l glutamine, 100 UI/ml penicillin and 100 mg/mLl
streptomycin and stimulated with various TLR agonists (TLR agonist kit, InvivoGen) (Table
5) or with a cocktail of cytokine containing 10 ng/ml IFN-γ, IL-17 and/or TNF-α, (optimal
cytokine concentrations to induce in BPH cells; IL-8 and IL-6 production as determined in
separate experiments) or with the indicated concentrations of IL-8 (BD Biosciences). For
mRNA expression, after 4 h stimulation cells were washed with PBS and then lised with RLT
buffer (Qiagen). After 48 h, cell culture supernatants were analyzed for cytokine and
chemokine production.
Table 5. TLR ligands used for the studies. Source and nature of TLR agonists used and their
specificities. Concentrations used for these studies are those recommended by the manufacturer.
Pam3CSK4 synthetic
Imiquimod synthetic
(type B)
Escherichia coli K12
heat-killed gram positive
double-stranded RNA
bacterial outer wall
100 ng/ml
bacterial flagellar
100 ng/ml
N-terminal part of
lipoprotein LP44
imidazoquinoline amine
single stranded RNA GUrich sequence
unmethylated CpG
0.5 µg/l
108 cells/ml
25 µg/l
100 ng/ml
500 ng/ml
500 ng/ml
5 µg/ml
3.3.4 Enzyme-linked immunosorbent assay (ELISA)
ELISAs for human IFN-γ, TNF-α, IL-6, IL-1β, IL-10 and IL-8 were performed using mAb
pairs and standards provided in the BD OptEIA™ Human ELISA set (BD Biosciences),
according to the manufacturer’s procedures. ELISA for human IL-12/23p40 was performed
using commercially available mAbs and standards (BD Biosciences) according to the
manufacturer’s instructions.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
Materials and Methods
3.3.5 Total RNA purification
Total RNA was extracted using the RNeasy Mini kit (Qiagen). Prior to collection, cells were
washed with ice-cold PBS, after which cells were lysed using 350 μl of RLT buffer and RNA
was extracted. The RNA lysates were collected and diluted with 70% ethanol (1:1 ratio) and
loaded onto a silica membrane (RNeasy mini column, Qiagen). After washing, membranes
were treated with DNase to exclude amplification of genomic DNA. Each step was preceded
by a centrifugation of 15 s at 10000 rpm. Finally, RNAs were eluted with 14 μl of sterile H2O
after a centrifugation at 14000 rpm for 1 min. Purities and RNA concentrations were
measured with a NanoDrop ND-1000 (NanoDrop, USA).
3.3.6 cDNA synthesis
To retrotranscribe 1 μg of total RNA to cDNA in III (Fig. 5), 100 pmol of oligodT18 primer,
20 nmol of dNTPs, 200 pmol of DTT, reverse transcriptase buffer (50 mM Tris–HCl, pH 8.3,
50 KCl, 4 mM MgCl2, 10 mM DTT), 40 U of reverse transcriptase and 40 U of RNAse
Inhibitor (buffer and enzymes from Fermentas, Lithuania) were incubated for 1 h at 37 °C in
40 μl volume. Following synthesis the reverse transcriptase was inactivated for 5 min at 95 °C
and cDNA was diluted 1:10 in sterile H2O.
In I, II and IV, 1 µg of total RNAs were retrotranscribed using reverse transcriptase
buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 30 mM MgCl2 and 5 mM DTT), 5.5mM
MgCl2, 500 µM of each dNTPs, 0.4 U/µl of RNase inhibitor, 2.5 µM of random hexamers and
1.25 U/μl multiscribe Reverse transcriptase (Taqman® Reverse transcription Reagents,
Applied Biosystems) and run under the following thermal cycler parameters: 25 ºC for 10
min, 48 ºC for 30 min, and 95 ºC for 5 min. Each sample was then collected, mixed
thoroughly, aliquoted, and frozen at -80ºC.
3.3.7 Real time PCR
In III (Fig. 5), real-time RT-PCR was performed using a LightCycler® 480 System (Roche)
and FastStart SYBR Green Master mix (Roche). Each reaction was performed using 4 pmol
of specific primers, 4 μl of cDNA template and 1x Mastermix in a volume of 10 μl and the
PCR cycling conditions were: pre-incubation for 10 min at 95 °C, 38 cycles of 20 s at 95 °C,
15 s at 60 °C and 15 s at 72 °C. Fold inductions were calculated using the formula 2(ΔΔCt),
where ΔΔCt is the ΔCt(stimulus)-ΔCt(solvent), ΔCt is Ct(target gene)-Ct(RPLP0), Ct is the
cycle at which the threshold is crossed and RPLP0 is the housekeeping gene ribosomal
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Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD
protein, large, P0. Quality of PCR products were monitored using post-PCR melt curve
In I, II, IV and V, real-time RT-PCR of total cDNA using specific primers were
performed under identical conditions using ABI PRISM 7000 Sequence Detection System
(Applied Biosystems, USA) and TaqMan® chemistry with 2x TaqMan Universal PCR Master
Mix (Applied Biosystems) from the same lot number. The primers used are commercially
available from Applied Biosystems as assays-on-demand. Real-time RT-PCR runs were
performed in 96-well optical plates, each containing 1x TaqMan Universal PCR Master Mix
(Applied Biosystems), 0.4 pmol/µl of appropriate forward and reverse primer and 20 ng
cDNA. The conditions for the amplification were as follows: 1 cycle of 2 min at 50 ºC and 1
cycle of 10 min at 95 ºC, followed by 40 cycles of 15 s at 95 ºC, 1 min at 60 ºC. Data were
acquired at the end of each 60 ºC cycle. Fold inductions were calculated using the formula
2(ΔΔCt), where ΔΔCt is the ΔCt(stimulus)-ΔCt(control), ΔCt is Ct(target gene)-Ct(GAPDH),
Ct is the cycle at which the threshold is crossed and GAPDH is the housekeeping gene
glyceraldehyde 3-phosphate dehydrogenase.
3.4 In vivo experiments
3.4.1 Mice
8-10 week-old C57BL/6 mice (Charles River, Italy) were housed in plastic cages with water
absorbent bedding. Litter was changed at least twice a week. The animal room was
temperature controlled and had a 12 h light/dark cycle. Food and water were supplied ad
libitum. Calcium and vitamin D3 content in the diet were respectively 8785 mg/kg and 1260
UI/kg. All procedures were reviewed and approved by local ethical committee.
3.4.2 Assessment of the MTD
Eight-week-old female C57BL/6 mice (3 mice/group) were dosed orally (0.1 ml/mouse) with
various concentrations of VDR agonists daily for four days. Analogues were formulated in
miglyol for a final concentration of 0.01, 0.03, 0.1, 0.3, 1, 3, 10 30, 100 and 300 μg/kg, when
given at 0.1 ml/mouse po. Blood for serum calcium assay was drawn by tail bleed on day five,
the final day of the study. Serum calcium levels were determined using a colorimetric assay
(Sigma Diagnostics, procedure no. 597). The highest dose of VDR agonist tolerated without
inducing serum calcium levels above 10.7 mg/dl (threshold used in the clinic to define
hypercalcemic patients) was taken as the MTD and expressed in μg/kg.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
Materials and Methods
3.4.3 Induction of experimental colitis
Colitis was induced in 8-10-week-old male C57BL/6 mice (5 mice per group) by
administration of 3% DSS (molecular mass 40 kDa; MP Biomedicals) in filter-purified
(Millipore) drinking water for 5 days, after which the mice were resumed on water for the
remainder of the experiment. Fresh DSS solutions were prepared every two days (Fig. 13).
3% DSS
Days after VDR agonist
Fig. 13. DSS chemical structure and experimental set-up for the induction of colitis. Mice
were pretreated with VDR agonists 24 h before addition of 3% DSS in drinking water. At day 0, 3%
DSS was administered ad libitum in drinking water for 5 days and then removed until the end of the
experiment. VDR agonists were administered daily intra-rectally in miglyol, until the end of the
3.4.4 Administration of VDR agonists
For local treatment, 1 μg/kg of BXL-62 and 0.3µg/kg of 1α,25(OH)2D3, were dissolved in oil
(miglyol 812,Sasol Germany GmbH) and 60µL were administered rectally to slightly
anaesthetized mice through a 3.5 F catheter carefully inserted into the rectum. The catheter tip
was inserted 4 cm proximal to the anal verge. To ensure distribution of the solution within the
entire colon and caecum, mice were held in a vertical position for 1 min after the instillation.
Treatments started one day prior DSS administration and continued every day thereafter for
the duration of the experiment. Miglyol alone was administered as vehicle control. Prior
observations confirmed that intra-rectal miglyol administration does not modify the
bodyweight loss from mice undergoing DSS-induced colitis.
3.4.5 Assessment of inflammation
All mice were observed daily for signs of gross toxicity, consistency of stools (formed, soft,
mixed and diarrhea) and presence of gross blood (Haemoccult Sensa, Beckman Coulter,
USA). The presence of blood was graded using a score of 0 for no color; 1 for a very light
blue color taking over 30 seconds to appear; 2 for a blue color developing about 30 seconds; 3
for an immediate change in color occurring in less than 30 seconds and 4 for gross blood
observable on the slide. Daily, mice were weighted and the body weight (BW) loss was
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Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD
graded using a score of 0 for gain or no bodyweight loss; 1 for BW loss from 1 to 4.9%; 2 for
BW loss from 5.0 to 9.9; 3 for BW loss from 10.0 to 15 and 4 for BW loss over 15. For each
group, the disease activity index (DAI) was determined by combining scores of BW loss,
haemoccult positivity and stool consistency (Table 6).
On day 9, animals were euthanized by CO2 inhalation and necropsied. The colon was
gently stretched and the distance from the colon-cecal junction to the end of the distal rectum
was measured. Serum calcium levels were determined using a colorimetric assay (Sigma
Diagnostics, procedure no. 597).
Table 6. Scoring system for the disease activity index (DAI). The mean scores for each
parameter and for each mouse represent the DAI score.
Weight loss (%)
gain or 0
less than 5
less than 10
less than 15
more than 15
Stool consistency
mixed (soft and liquid)
Bloody stool (haemoccult test)
no color
light blue
positivity around 30 s
positivity lower than 30 s
gross bleeding
3.4.6 Histology
Entire colons were fixed in 4% paraformaldehyde for at least 48 h and were processed by the
laboratory of Pathology Unit, "L. Sacco" Department Clinical Sciences, Milano. Colon tissues
were embedded in paraffin and three µm sections were cut and stained with hematoxylin and
eosin. Sections were scored blindly for the anus, the descendant and ascendant colon. For
each section, inflammatory infiltrate, ulcerative lesion and regenerative hyperplasia were
evaluated. In addition, the area with the histological lesion was measured. Total histological
score was calculated as detailed in Table 7.
Table 7. Scoring system for the histology score. Ulceration and regeneration were not
graded, however the inflammation of the external wall (phlogosis) was quantified from absence to
intensive infiltration. The total score represents the sum of all the four parameters.
Ulceration Regeneration Phlogosis
Lesion lenght (mm)
less than 5
less than 10
less than 15
less than 20
20 and more
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Materials and Methods
3.5 Statistical analysis
Data were analyzed with Graph Prism version 5.0 and curves were generated with the
appropriated nonlinear fit regression. Statistical significance was determined by using the
appropriate analysis of variance followed by a post-hoc test for multiple variable analyses or
by the appropriate t-test for one to one comparison. Data had to follow a normal distribution
before being tested for significance. Differences were considered significant at P < 0.05.
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Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD
4. Results
4.1 BPH cells can act as non-professional APCs to induce chronic prostate
BPH, the most common age-related disease of the male, occurs clinically in about half of all
men at 70 years of age (Webber 2006). A variety of growth factors and inflammatory
chemokines like IL-8 have been implicated in the pathogenesis of BPH (Ropiquet, Giri et al.
1999; Giri and Ittmann 2001).
However, the complex regulatory mechanisms of growth control in BPH are still
incompletely understood. Inflammatory infiltrates in BPH have been found to consist
primarily of T cells, mostly CD4+CD45RO+ cells, B cells and macrophages (Theyer, Kramer
et al. 1992). Up-regulation of several pro-inflammatory cytokines has been described in BPH,
in particular IL-2 and IFN-γ (Steiner, Stix et al. 2003), IL-15 (Handisurya, Steiner et al. 2001)
and IL-17 (Steiner, Newman et al. 2003), leading to the hypothesis that BPH may represent an
“immune inflammatory” disease (Kramer and Marberger 2006). This is an attractive
hypothesis, because the association of BPH with chronic inflammation could offer a sound
framework to understand the pathogenesis of the disease. However, immune mechanisms
leading to chronic inflammation in BPH have not yet been clearly defined.
In order to further characterize the immune mechanisms behind BPH pathogenesis, we
have examined the capacity of prostate stromal cells obtained from BPH tissue to actively
contribute to the organ specific inflammatory process by acting as APCs or as targets of TLR
agonists, leading to the production of pro-inflammatory cytokines and chemokines able to
mediate prostate chronic inflammation and hyperplasia.
Analysis of TLR transcript expression by real-time RT-PCR on BPH cells shows that
the 10 members of the family identified in human are constitutively expressed and are
functional. With the exception of TLR9, BPH cells stimulated by any TLR agonist (Table 5)
show a strong induction of pro-inflammatory cytokine (IL-8 and IL-6) and chemokine
(CXCL10) production (I, Fig. 2B).
The capacity of BPH cells to act as APCs was first examined by expression of MHC
class II and co-stimulatory molecules, such as CD40, CD80, CD86 and CD134 on their
surface. BPH cells constitutively express both MHC-I and -II molecules and co-stimulatory
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
molecules, which are strongly up-regulated by a 48 h incubation with IFN-γ, as detected by
cytofluorometry (I, Fig. 3A) and confirmed by confocal microscopy (I, Fig. 3B and Fig. 5A).
Then, we examined ability of BPH cells to present alloantigen to alloreactive CD4+ T
cells. Constitutive expression of MHC-II molecules by BPH cells, albeit relatively low, is
already sufficient to induce proliferation of carboxyfluorescein succinimidyl ester (CFSE)labeled alloreactive CD4+ T cells, which is increased following IFN-γ treatment of BPH cells
(I, Fig. 4A). Alloreactive CD4+ T cells co-cultured with IFN-γ-stimulated BPH cells produce
not only IFN-γ but also IL-17 (I, Fig. 4D).
Interestingly, IL-17 and IFN-γ produce by BPH-stimulated CD4+ T cells markedly upregulate production of IL-8, CXCL10 and IL-6 (I, Fig. 7). In addition, IL-8 is expressed in situ
by epithelial and stromal prostate cells, and is functional, as shown by the recruitment of
CXCR1- and CXCR2-positive leukocyte, as well as CD15+ neutrophils (I, Fig. 8). IL-8mediated BPH cell growth can be induced by a combination of IFN-γ and IL-17, thus
establishing a possible relationship between the T-cell response induced by BPH cells and
prostate cell growth (I, Fig. 9).
In conclusion, our results show that human prostate cells can act as APCs, i.e. they are
able to stimulate alloreactive CD4+ T cells to produce IFN-γ and IL-17. The induction of a
BPH cell-driven autoimmune response, as well as triggering of TLRs expressed by BPH cells,
up-regulate production of IL-8, IL-6 and CXCL10, which are key factors sustaining prostate
inflammation, recruiting inflammatory leukocytes and promoting prostate cell hyperplasia.
4.2 VDR agonist elocalcitol inhibits IL-8-dependent BPH cell proliferation
and inflammatory response
Little data assessing the clinical response of anti-inflammatory therapy in BPH is
available, but treatment with elocalcitol has been found to arrest BPH development (Colli,
Rigatti et al. 2006). VDR agonists, by promoting innate immunity and regulating adaptive
immune responses, exert anti-inflammatory and immunoregulatory properties potentially
useful in the treatment of diseases characterized by chronic inflammation and cell
proliferation (Adorini and Penna 2008). The prostate is a target organ of VDR agonists and
represents an extrarenal synthesis site of 1α,25(OH)2D3 (Flanagan, Young et al. 2006), but its
capacity to respond to VDR agonists has been mostly probed clinically for the treatment of
prostate cancer (Deeb, Trump et al. 2007). Based on the marked inhibitory activity of the
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Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD
VDR agonist elocalcitol on basal and growth factor induced proliferation of human prostate
cells (Crescioli, Ferruzzi et al. 2004), its anti-inflammatory properties in the treatment of EAP
induced by injection of prostate homogenatecomplete Freund’s adjuvant in NOD male mice
were tested (Penna, Amuchastegui et al. 2006). Administration of elocalcitol, at
normocalcemic doses, for 2 weeks in already established EAP inhibits significantly the
intraprostatic cell infiltrate, with reduced cell proliferation and increased apoptosis of resident
and infiltrating cells (Penna, Amuchastegui et al. 2006). Th1 cell responses are decreased as
well as production of IL-17 (Penna, Amuchastegui et al. 2006) emphasizing the potential of
VDR agonists in the treatment of immuno-mediated diseases of the prostate (Adorini, Penna
et al. 2007).
To provide an understanding of the mechanism of action of elocalcitol potency in
BPH, we analyzed in publication II its anti-proliferative and anti-inflammatory properties in
IL-8 dependent mechanisms.
In study II, we demonstrated that the increased expression of IL-8 and VDR transcripts
promoted by T cell-derived inflammatory cytokines in BPH cells renders them more
susceptible to the inhibitory action of elocalcitol (II, Fig. 1A). Moreover, elocalcitol inhibits
dose dependently IL-8 production by BPH cells more effectively than calcitriol, whereas the
5-α reductase inhibitor finasteride has no effect on IL-8 production induced by proinflammatory cytokines in BPH cells (II, Fig. 1C). In addition, increasing concentrations (1017
to 10-6 M) of elocalcitol inhibit dose-dependently BPH cells proliferation induced by 10 nM
IL-8 (II, Fig. 2B).
Treatment with elocalcitol of cytokine-stimulated BPH cells does not significantly
affect COX-1 expression but it significantly reduces, with an average decrease of about 50%,
both COX-2 expression and PGE2 production (II, Fig. 3B and Fig. 4). To explain COX-2
inhibition, we further analyzed the NF-κB translocation after treatment with elocalcitol.
Confocal microscopic analysis clearly showed translocation of NF-κB p65 to the nucleus in
cytokine-stimulated BPH cells, whereas this is mostly retained into the cytoplasm in BPH
cells treated with elocalcitol before cytokine addition (II, Fig. 5). Thus, elocalcitol inhibits
NF-κB p65 nuclear translocation in BPH cells stimulated by inflammatory cytokines leading
to inhibition of pro-inflammatory cytokine production.
Activation of NF-κB signaling through the RhoA/ROCK pathway induces IL-8
production in a number of different cell types, including cervical stromal cells (Shimizu,
Tahara et al. 2007), Kaposi sarcoma (Shepard, Yang et al. 2001; Zhao, Kuhnt-Moore et al.
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
2003), endothelial cells (Hippenstiel, Soeth et al. 2000) and colonic epithelial cells
(Hippenstiel, Soeth et al. 2000). As RhoA activation results in its translocation to the plasma
membrane (Somlyo and Somlyo 2003), we investigated by Western blot analysis its
subcellular distribution (membrane vs. cytosol) in BPH cells, under basal conditions and after
a 48 h stimulation with IL-8 (10 nM) in combination or not with elocalcitol (10-8 M). We
could show that IL-8 stimulated RhoA membrane translocation was completely prevented by
elocalcitol (II, Fig. 6A lower panel). The results were confirmed by confocal microscopy (II,
Fig. 6B). These effects are consistent with the role of this calcium-sensitising signaling in the
regulation of the inflammatory processes (Segain, Raingeard de la Bletiere et al. 2003)
through the activation of NF-κB (Montaner, Perona et al. 1998) and the induction of IL-8
secretion (Shimizu, Tahara et al. 2007).
Thus, these data provide a mechanistic explanation for the role of IL-8 in BPH
pathogenesis, showing that a combination of Th1 and Th17 cell-derived inflammatory
cytokines can markedly stimulate its secretion by BPH stromal cells. Moreover, our results
suggest an IL-8-dependent link bridging inflammatory response and cell proliferation in BPH
pathogenesis, which can be targeted by elocalcitol via multiple mechanisms of action.
4.3 Potent
cyclopropyl-vitamin D3 (BXL-62) in inflammatory bowel disease models
Additions of 16-ene and/or a 20-epi moeity to vitamin D3 are known to represent an important
chemical tools to increase the potency of VDR agonists with physiological calcium
homeostasis (Uskokovic, Manchand et al. 2006). 20-epi-1α,25(OH)2D3 or 16-ene1α,25(OH)2D3 exhibit anti-proliferative activity from 200-5000 fold greater than the natural
hormone coupled to more potent anti-inflammatory properties in a variety of cancer cell line
and human keratinocytes (Uskokovic, Norman et al. 2001; Uskokovic, Manchand et al. 2006).
Medicinal chemistry approaches lead to the proposition that a C20-cyclopropyl group
could represent an interesting alternative to mimic the methyl group (Uskokovic, Manchand et
al. 2006). These 20-cyclopropyl VDR agonists show a higher potency in the inhibition of
IFN-γ production in MLR assays, with a 10-times lower capacity to induce hypercalcemia in
mice (Uskokovic, Manchand et al. 2006).
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A previous study combined these two modifications and showed that, among paired
16-ene-20-cyclopropyl VDR agonists, 1α,25(OH)2-16-ene-20-cyclopropyl-vitamin D3 (BXL62) is the most potent compound inhibiting IFN-γ in MLR assays (Uskokovic, Manchand et
al. 2006).
We undertook study III to evaluate the potency of 16-ene-20-cyclopropyl VDR
agonists not only for IFN-γ inhibition in the MLR assay, but to extend our analysis to TNF-α
inhibition in LPS-induced PBMC activation. We confirmed that BXL-62 is the most potent
compound among the VDR agonists tested in the inhibition of both IFN-γ and TNF-α (III,
Table1 and Fig. 6). BXL-62 is also more potent, compared to the natural hormone calcitriol,
in the inhibition of two other key pro-inflammatory cytokines, IL-12/23p40 and IL-6 (III, Fig.
6). In addition, BXL-62 induces VDR primary response genes at a concentration 15 times
lower than the natural hormone both in primary cells (PBMCs) (III, Fig. 4) and an immortal
cell line (THP-1) without modification of its kinetic (III, Fig. 5). Moreover, despite the potent
anti-inflammatory properties of BXL-62, this VDR agonist present a better safety as shown
by its maximum tolerated dose 3 times higher than the natural hormone, confirming that this
compound represents a potent VDR agonist (III, Fig. 8) with reduced calcemic liability
compared to calcitriol.
VDR expression is required to control inflammation of spontaneous and induced
colitis mice model as demonstrated by exacerbation of the symptoms in Il-10/Vdr double
deficient and in DSS-induced colitis mice (Froicu, Weaver et al. 2003; Froicu and Cantorna
2007). In addition, 1α,25(OH)2D3 has been shown to ameliorate spontaneous colitis, an effect
mediated by direct and indirect inhibition of TNF-α (Zhu, Mahon et al. 2005) . Recently, a
pilot clinical study was performed in IBD patients, showing short-term beneficial effects on
bone metabolism and on disease activity after a one year administration of 1α,25(OH)2D3
(Miheller, Muzes et al. 2009).
Recently, efficacy of VDR analogues were proposed in the IBD context, as the VDR
agonist 19-nor-14,20-bisepi-23-yne-1α,25(OH)2-vitamin D3 (TX527) showed inhibition of
TNF-α production and proliferation in PBMCs from CD patients (Stio, Martinesi et al. 2007).
In mice models, the VDR agonist 22-ene-25-oxa-1α,25(OH)2D3 (ZK156979) was previously
shown to improve symptoms in TNBS-induced colitis at normocalcemic doses by inhibiting
TNF-α production and increasing level of anti-inflammatory cytokine (Daniel, Radeke et al.
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2006). We undertook the project IV to support by pre-clinical data the potential used of our
VDR agonist, BXL-62, for the treatment of IBD.
To confirm the potential of this VDR agonist in the treatment of IBD, we have first
studied its activity in the induction of VDR primary response genes in PBMCs purified from
IBD patients. A dose-response titration of BXL-62 and 1α,25(OH)2D3 in unstimulated
PBMCs from IBD patients shows a significantly higher increase of transcripts encoding
CYP24A1 and CAMP induced by BXL-62 compared to 1α,25(OH)2D3 (IV, Fig. 1). These
results confirm those obtained in PBMCs from healthy individuals (III), and demonstrate that
PBMCs from IBD patients present an unaltered VDR-dependent signaling machinery.
We also confirmed anti-inflammatory activites in LPS-activated PBMCs purified from
IBD patients as observed in healthy donors (IV, Fig. 2B), but we also demonstrated that this
protein inhibition is the consequence of an earlier mRNA inhibition (IV, Fig. 2A). We also
extended this analysis and showed that in IBD patients, VDR agonists are able to inhibit proinflammatory cytokine production after any TLR agonist activation (Table 5) (IV, Fig. 3).
These anti-inflammatory properties have been extended to lymphocytes purified from the
inflamed target tissue, as shown by inhibition of TNF-α and IFN-γ production after activation
of lymphocytes purified from LPMCs activated by antibodies targeting CD2 and CD28,
potent activators of T cell responses (Targan, Deem et al. 1995) (IV, Fig. 4).
To validate a potential use of BXL-62 as a therapeutic agent in IBD, we have analyzed
its in vivo efficacy, compared to 1α,25(OH)2D3, in the DSS-induced colitis model, as
previously described (Froicu and Cantorna 2007). Daily intra-rectal administration of BXL-62
prevents the body weight loss following DSS administration (IV, Fig. 5A) and induces a
significant improvement in stool consistency (Fig. 5B) and in visible fecal blood (IV, Fig.
5C). In contrast, 1α,25(OH)2D3 treatment does not affect body weight loss (IV, Fig. 5A),
improves stool consistency only early on in the disease course (IV, Fig. 5B) and ameliorates
the bloody stool score (IV, Fig. 5C). The DAI summarizing the daily parameters observed,
confirms that BXL-62 ameliorates significantly colitis symptoms, from day 4 until the end of
the experiment, while 1α,25(OH)2D3 treatment leads to significant amelioration of disease
only at day 4 (IV, Fig. 5D). At day 10 after initiation of DSS administration, BXL-62 shows a
colon shortening significantly reduced compared to vehicle (IV, Fig. 6A) accompanied with a
decrease of total colon lesions (IV, Fig. 6B). To control the treatment safety, we measured
serum calcium levels and show that ten days of consecutive intra rectal administrations of
VDR agonists do not induce hyper-calceamia in mice undergoing DSS-induced colitis (IV,
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Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD
Fig. 5A). In addition, BXL-62 activity is dose dependent and at equimolar dosage, BXL-62
conserved is higher potency than the natural hormone to ameliorate the stool consistency (IV,
Fig. 6A) and the visible blood (IV, Fig. 6B).
In conclusion, the strongest potential of BXL-62, compared to 1α,25(OH)2D3, to
inhibit pro-inflammatory cytokines, in PBMCs and LPMCs from IBD patients, combined
with its in vivo efficacy for amelioration of colitis symptoms in an experimental colitis model,
suggest its potential use as a future therapy for IBD.
4.4 24-oxo BXL-62 metabolite exerts biological activities similar to its
parent compound
Modification of the catabolism of VDR agonists was proposed to explain, at least in
part, their increase of potency compared to 1α,25(OH)2D3 (Lemire, Archer et al. 1994), as
demonstrated by accumulation of a 24-oxo active metabolite from the 20-epi and 16-eneVDR agonists (Lemire, Archer et al. 1994; Siu-Caldera, Clark et al. 1996; Campbell, Reddy
et al. 1997). However, little is known about 20-cyclopropyl-16-ene VDR agonists that exhibit
stronger potency in proliferation inhibition and anti-inflammatory properties (Uskokovic,
Manchand et al. 2006). Metabolic studies performed on a rat osteosarcoma cell line proposed
that BXL-62 is protected, as the 16-ene-vitamin D3, from the C23 hydroxylation by
CYP24A1, resulting to an accumulation of the 24-oxo metabolite (Uskokovic, Manchand et
al. 2006). This observation could explain the higher potency of BXL-62, since 24-oxo-16-enevitamin D3 isolated from a culture supernatant presents similar anti-inflammatory activities
than its parent compound (Lemire, Archer et al. 1994; Siu-Caldera, Rao et al. 2001). We
undertook the study IV to achieve the characterization and synthesis of this 24-oxo metabolite
and compared its activity to its parent VDR agonist.
In order to confirm the previous observation (Uskokovic, Manchand et al. 2006),
human cells derived from an ovarian carcinoma were treated 24 h with BXL-62 or
1α,25(OH)2D3 and their metabolites where identified by high pressure liquid chromatography.
The pattern of metabolism of 1α,25(OH)2D3 into its various metabolites is similar to the
previously reported (Reddy and Tserng 1989), but the pattern of the metabolism of BXL-62 is
different. While 1α,25(OH)2D3 pattern presents 23-hydroxylate and 25-hydroxylate
metabolites (III, Fig. 2 upper panel), they are absent from the BXL-62 profile (III, Fig. 2
lower panel). The BXL-62 pattern shows a higher amount of 24-oxo metabolite compared to
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
1α,25(OH)2D3 (III, Fig. 2 lower panel). Mass spectrometry analysis allowed us to characterize
definitively this 24-oxo metabolite as 16-ene-20-cyclopropyl-24-oxo-1α,25(OH)2D3 (BXL143) (III, Fig. 3). Finally, in order to confirm that the potency of BXL-62 could be due to the
accumulation of its 24-oxo metabolite, the chemical synthesis of BXL-143 was carried out
(III, scheme 1).
Characterization and synthesis of the 24-oxo metabolite from the active VDR agonist
BXL-62 allowed us to compare their activity in order to confirm that BXL-62 activity could
pass through accumulation of BXL-143. We first showed a similar ability of BXL-62 and its
24-oxo metabolite to induce primary VDR target genes, CYP24A1 and CAMP, in two
different human cell culture systems, PBMCs (III, Fig. 4A) and THP-1 cells (III, Fig. 5B). To
compare the anti-inflammatory properties of these VDR agonists, we performed two different
assays, the MLR and the LPS-induced PBMC activation. In these two different assays, BXL62 and BXL-143 present similar inhibition of pro-inflammatory cytokine production,
including IFN-γ (III, Fig. 6A), TNF-α (III, Fig. 6B), IL-12/23p40 (III, Fig. 7A) and IL-6 (III,
Fig. 7B). Both compounds exhibit these activities at a lower dose than the natural hormone,
indicating that BXL-62 activity could be mediated by accumulation of its 24-oxo metabolite.
Interestingly, the normocalcemic activity of BXL-62 seems to be also a consequence
of its specific metabolism as shown by the lower calcemic activity of its 24-oxo metabolite
compared to its parent compound (III, Fig. 8).
In conclusion, the potent VDR agonist BXL-62 is metabolized into a stable 24-oxo
metabolite (BXL-143), which resists further metabolism. As a result, BXL-143 accumulates
in tissues. By combining our observations of equipotency between BXL-62 and BXL-143 in
transcript regulation and similar inhibition of cytokine production, we conclude that
accumulation of BXL-143 can explain the strong potency of BXL-62, supporting the concept
that specific differences in the target tissue metabolism of VDR agonists can play a critical
role to increase their potency.
4.5 Specific IL-10 production deficiency in inflammatory bowel disease
patients compared to healthy controls
Recent evidence supports the key role of innate immunity in the IBD onset, as shown
by increased severity of chemically-induced colitis in TLR adaptor protein Myd88 KO vs.
wild type mice (Araki, Kanai et al. 2005) and the description of the PRR member Nod2 as a
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Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD
susceptible gene for CD (Cho and Abraham 2007). CD patients presenting the Nod2 mutation
3020insC were associated with a lower production of IL-10 in the periphery after costimulation with MDP and TLR1/2 agonist (Noguchi, Homma et al. 2009) confirming
potential higher disease susceptibility in patients presenting Nod2 and IL-10 polymorphism
(Marrakchi, Moussa et al. 2009). These observations highlight the potential link between
innate immune system and IL-10 deficiency in IBD pathogenesis.
Genetic studies using Il-10-deficient (Kuhn, Lohler et al. 1993) and IL-10 transgenic
mice (Hagenbaugh, Sharma et al. 1997) established the unequivocal importance of IL-10 in
controlling inflammation initiated and perpetuated by pro-inflammatory signals in acute and
chronic diseases such as IBD (Moore, de Waal Malefyt et al. 2001). Despite the presence of
polymorphisms in IBD patients (Aithal, Craggs et al. 2001), IL-10 regulatory properties for
the inhibition of pro-inflammatory cytokine production seems to be maintained (Schmit,
Carol et al. 2002). Interestingly, IL-10 production is shown to be lower in PHA-stimulatedLPMCs derived from inflamed colonic mucosal from IBD patients compared to its noninflamed conterpart (Gasche, Bakos et al. 2000). We undertook study V to further analyze the
potential correlation between IL-10 production and IBD pathogenesis. We focussed on the IL10 production in the periphery and the inflamed tissue after TLR agonist stimulation.
Previous observations have shown that TLR4 and TLR5 are the two stimuli, among
the TLR agonist family tested, producing higher level of pro-inflammatory cytokine after 24 h
activation (IV, Fig. 3). We demonstrated a significantly lower IL-10 production in both UC
and CD compared to normal control, only in PBMCs stimulated by TLR4 agonist (V, Fig.
1A). This defect is not present for pro-inflammatory cytokines, such as TNF-α (V, Fig. 1), IL6 and IL-12/23p40 (V, Fig. 3). These data provide the first evidence that PBMCs purified
from IBD patients present a reduction of IL-10 production specifically after TLR4
As previously described (Schmit, Carol et al. 2002), IL-10 is able to regulate proinflammatory cytokine production since the blocking of its receptor by blocking antibody
induced an up-regulation of TNF-α, ΙL-6 or IL-12/23p40 production after stimulation with
TLR4 and TLR5 agonists (V, Fig. 2). While PBMCs stimulated with TLR5 in presence of the
IL-10R blocking antibody respond with a significant increase of IL-10 production in both
IBD subtypes and normal controls (V, Fig. 2A, right panel), PBMCs purified from IBD
patients present a deficit in IL-10 production after TLR4 stimulation, while normal controls
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
fail to present this defect (V, Fig. 2A, right panel). This observation confirms that IL-10
production is specifically defective in TLR4-stimulated PBMCs from IBD patients.
As we have previously observed, TNF-α and IL-12/23p40 production by TLR4 or
TLR5 PBMCs-activated are inhibited by 10 nM of 1α,25(OH)2D3 (IV, Fig. 3). In study V we
show that this anti-inflammatory effect is independent of IL-10 activity, since 1α,25(OH)2D3
inhibits TNF-α and IL-12/23p40 production in presence of the blocking IL-10R antibody (V,
Fig. 4). These observations are similar for IL-12/23p40 inhibition after TLR4 stimulation (V,
Fig. 4).
IL-10 regulatory properties for the inhibition of pro-inflammatory cytokine production
is maintained, since IL-10 in LPMCs purified from IBD patients are able to inhibit IFN-γ and
TNF-α production (Schmit, Carol et al. 2002). We next wanted to study the IL-10 production
in the tissue site target of the inflammation. We purified LPMCs from four IBDs patients and
performed a similar experiment than with PBMCs. Surprisingly, IL-10 production is
increased after IL-10R blocking in LPMCs stimulated with LPS and Flagellin. These results
present an intriguing disconcordance with the observations in PBMCs (V, Fig 5).
These results highlight a specific TLR-induced defect in the periphery of IBD patients,
inducing a lower production of IL-10, an important anti-inflammatory cytokine. Despite the
lack of lower IL-10 production in LPMCs from the inflamed tissue, these findings contribute
to confirm that IL-10 represents a key cytokine in the pathogenesis of IBD.
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5. Discussion
5.1 Prostatic cells as inducers and targets of chronic inflammation
decompensation are still unclear, but it is likely that both increased urethral narrowing and
bladder smooth muscle overactivity are involved (Andersson and Arner 2004). Hence, in the
development of BPH and its related symptoms, at least three distinct components can be
defined: a static component, related to the overgrowth of the prostate gland, a dynamic
component, associated with smooth muscle hypercontractility, and an inflammatory
component. The static component is mostly responsible for obstructive symptoms, because
the enlarged prostate is a mechanic obstacle to the physiological urinary outflow resulting in
complaints of weak stream, intermittent urinary flow and/or straining to void. The dynamic
component is responsible for the occurrence of storage (irritative) symptoms as urinary
frequency, urgency and nocturia. Recently, chronic inflammation has emerged as the third
component of BPH pathogenesis, taking part with the androgen receptor signaling in the
induction of the tissue remodelling typical of the advanced stages of the disease and the
prostatic inflammatory infiltrates observed in a large percentage of BPH surgical specimens
(Nickel 2008). Previous work in our laboratory has shown significantly increased levels of the
pro-inflammatory cytokines IL-1α, IL-1β, IL-6 and IL-12p70 and the chemokines CCL1,
CCL4, CCL22 and IL-8 in the seminal plasma from BPH patients (Penna, Mondaini et al.
2007). The concomitant increase of several inflammatory cytokines and chemokines in BPH
patients is consistent with an important chronic inflammatory component in disease
pathogenesis and expression profiling data demonstrate a strong correlation between
inflammation and symptomatic BPH (Prakash, Pirozzi et al. 2002).
Results in study I demonstrate for the first time that IL-8-mediated BPH cell growth
can be induced by a combination of IFN-γ and IL-17, thus establishing a possible relationship
between the T-cell response induced by BPH cells and prostate cell growth. Therefore, these
data provide a mechanistic explanation for the role of IL-8 in BPH pathogenesis, showing that
a combination of Th1 and Th17 cell-derived inflammatory cytokines can markedly stimulate
its secretion by BPH stromal cells. Thus, BPH can be seen as a form of chronic prostatitis,
whose pathogenesis may be triggered by infection. The release of prostatic self-antigens
following tissue damage may sensitise the immune system and start autoimmune responses
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
and among pro-inflammatory cytokines and chemokines produced by the prostatic
microenvironment, stromal-derived IL-8 may be considered a key link between chronic
inflammation and stromal cell proliferation.
5.2 VDR agonists inhibit intraprostatic inflammatory responses
Pharmacological management of BPH is a novel application of vitamin D analogs,
prompted by the detection of VDR expression in cultured prostatic and bladder stromal cells
derived from BPH patients. Reducing prostate overgrowth by decreasing intra-prostatic
androgen signaling, without directly interfering with systemic androgen action, would obviate
the adverse systemic side effects of anti-androgens, such as 5α-reductase inhibitors. In
addition, VDR agonists can modulate the dynamic component of LUTS pathogenesis, and
exert anti-inflammatory activities. Thus, this class of agents could represent an interesting
therapeutic option for the pharmacological treatment of BPH.
We have already discussed in study I that among the pro-inflammatory cytokines and
chemokines produced by the prostatic microenvironment, stromal-derived IL-8 may be
considered a key link between chronic inflammation and stromal cell proliferation. Consistent
with an IL-8-dependent link bridging inflammatory response and cell growth in BPH cells,
the VDR agonist elocalcitol, a well-defined anti-inflammatory agent inhibiting prostate
growth in experimental models (Crescioli, Ferruzzi et al. 2004) and arresting prostate growth
in BPH patients (Colli, Rigatti et al. 2006) has been shown to inhibit IL-8-mediated prostate
growth and inflammation through multiple mechanisms of action (Penna, Amuchastegui et al.
2006; Morelli, Vignozzi et al. 2007).
VDR expression is promoted by T cell-derived inflammatory cytokines in BPH cells,
rendering them more susceptible to the inhibitory action of elocalcitol. In addition, elocalcitol
inhibits IL-8 production induced by pro-inflammatory cytokines secreted by prostateinfiltrating CD4+ T cells (IFN-γ, IL-17 and TNF-α) in human prostatic stromal cells,
accompanied by reduced COX-2 expression and PGE2 production and by arrest of the nuclear
translocation of NF-κB, a transcriptional factor that directly regulates IL-8 production.
Elocalcitol dose-dependently counteracts IL-8-dependent BPH cell proliferation via
inhibition of the RhoA/ROCK pathway. These effects are consistent with the role of this
calcium-sensitising signaling in the regulation of the inflammatory processes (Segain,
Raingeard de la Bletiere et al. 2003), through the activation of NF-κB (Montaner, Perona et
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Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD
al. 1998) and the induction of IL-8 secretion (Zhao, Kuhnt-Moore et al. 2003). In particular,
the effect on this calcium-sensitizing pathway may represent a common denominator for the
therapeutic efficacy of this drug on all the three components of BPH: static, dynamic and
inflammatory. Consistent with the role of RhoA/ROCK signaling in regulating human and rat
bladder contraction and tone, in particular in generating involuntary contractions (Peters,
Schmidt et al. 2006), elocalcitol affects bladder contractility via inhibition of the calcium
sensitizing RhoA/ROCK pathway by interfering with RhoA activation (Morelli, Vignozzi et
al. 2007). The reduction of RhoA/ROCK-mediated inappropriate bladder contraction induced
by elocalcitol does not interfere with the overall detrusor motility, preventing urinary
retention due to voiding impairment. In conclusion, combined results on elocalcitol activity
indicate possible beneficial effects of elocalcitol on bladder overactivity by two mechanisms:
counteracting the enhanced expression and signaling of growth factors involved in bladder
smooth muscle hypertrophy and hyperplasia (Crescioli, Morelli et al. 2005) and increasing the
contractile efficiency of bladder muscle cells through the modulation of smooth muscle gene
expression and the down-regulation of smooth muscle myofilament sensitization to calcium
(Morelli, Vignozzi et al. 2007).
Based on these preclinical premises, a phase IIa, double blind, placebo-controlled
study has shown clear efficacy signals on the primary endpoint (mean volume voided per
micturition) and on symptoms, including frequency, nocturia and incontinence episodes
(Colli, Digesu et al. 2007). A multi-center phase IIb trial shows that elocalcitol arrests prostate
growth and positive effects on the secondary endpoints of LUTS (ie, urgency and frequency
of urination, and nocturia) and International Prostate Symptom Score (IPSS) were also
observed (Fibbi, Penna et al. 2009). Unfortunately, the additional potential benefits of
elocalcitol in men with BPH and associated LUTS and/or bladder outlet obstruction were
further obviated because of negative results from a phase IIb study in OAB. Despite these
results, elocalcitol will still have the status of 'first-in-target-class' of vitamin D3 analogs,
given its encouraging data on several endpoints (Tiwari 2009).
5.3 TLR specific deficiency for IL-10 production
IL-10 represents clearly a key cytokine in the pathogenesis of IBD as demonstrated by Il-10
deficient mice that develop spontaneous colitis (Kuhn, Lohler et al. 1993). In addition, an
inappropriate regulation of IL-10 production in the periphery could represent an important
event for the pathogenesis since transgenic CD4+CD45RBhigh T cell, purified from
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splenocytes producing high level of IL-10, transfered in a SCID mice recipient do not exhibit
wasting colitis symptoms as observed in transfer with wild type T cells (Hagenbaugh, Sharma
et al. 1997). Consistent with this observation, T cell-induced severe colitis was totally
abrogated in mice treated with recombinant IL-10 (Powrie, Leach et al. 1994). Recently, the
concept of lower IL-10 levels circulating in sera of IBD patients was correlated with the
presence of neutralizing antibodies (Ebert, Schott et al. 2009), but despite some report
showing that IL-10 production is lower in 3020insC Nod2 mutated patients after MDP and
TLR2 co-activation (Noguchi, Homma et al. 2009), no clear evidence of an impaired of IL-10
secretion in the periphery was reported.
In study V, we demonstrated that IL-10 production in PBMCs purified from IBD
patients is defective after TLR4 stimulation but not after TLR5 stimulation. Surprisingly,
LPMCs do not seem to exhibit this defect. These findings highlight a possible difference in
response to TLR4 in the periphery and in tissue site of inflammation in IBD patients,
confirming that IL-10 production in the periphery from IBD patients could play a key role in
the disease onset.
Compared to PBMCs, lamina propria macrophages present a low expression of TLR2
and TLR4 (Hausmann, Kiessling et al. 2002) and isolated lamina propria macrophages do not
express CD14 and are unresponsive to LPS (Smith, Smythies et al. 2001). In addition, colonic
epithelial cells express low levels of TLR4 and MD2 and are poorly responsive to LPS
(Abreu, Vora et al. 2001; Naik, Kelly et al. 2001; Suzuki, Hisamatsu et al. 2003). These
findings support the concept that in humans the TLR4 co-receptor CD14 promoter
polymorphisms could contribute to disease development (de Buhr, Hedrich et al. 2009).
In the DSS-induced colitis model, evidence suggests that TLR4 may play a partial role
in the development of severe disease, at least in certain mouse strains (Lange, Delbro et al.
1996). TLR4 mutation in C3H/HeJ mice renders them more susceptible to the spontaneous
development of chronic colitis (Elson, Cong et al. 2000) suggesting, as observed in humans,
that genetic factors can influence the relative importance of TLR4 in the development of
colitis (Sepulveda, Beltran et al. 2008).
A role for TLR4 in the development of chronic colitis has been demonstrated clearly
in mice that have a myeloid-specific deletion of Stat3 and present enhanced Th1 responses
and develop chronic colitis, probably due to the inability of myeloid cells to respond to IL-10
(Takeda, Clausen et al. 1999). In these mice, colitis development seems to depend on the
presence of TLR4, IL-12/23p40 and T cells, because conditional Stat3 KO mice that are also
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Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD
deficient in any of these molecules or cells do not develop colitis (Takeda, Clausen et al.
1999). These observations were confirmed by the blocking of TLR4 complex signaling that
result in decreased intestinal inflammation in the Mdr1a-deficient mice, another model of
colitis that is characterized by the presence of Th1-type T cells (Panwala, Jones et al. 1998)
These data demonstrate that TLR4 signaling in response to the presence of bacteria in
periphery could have an important role in the onset of IBD by reducing production of IL-10, a
potent anti-inflammatory cytokine.
5.4 BXL-62 ameliorates symptoms in experimental model of colitis
Because supraphysiologic concentrations of 1α,25(OH)2D3 and its analogues are usually
required to exert immunosuppressive and anti-proliferative effects, more than 3000 vitamin
D3 analogues with several different structural modifications have been synthesized during the
past two decades (Deluca and Cantorna 2001; Uskokovic, Norman et al. 2001; Uskokovic,
Manchand et al. 2006). We have already discussed about elocalcitol that presents an enhanced
biological activity with less calcemic liability compared to 1α,25(OH)2D3 (Colli, Rigatti et al.
2006). Previous studies showed that the potency of 20-cyclopropyl-vitamin D3 analogues can
be increased both in terms of their calcemic and immunomodulatory activities by the addition
of a 16-ene moiety (Uskokovic, Manchand et al. 2006).
In study III we have further characterized the potency of 16-ene-20-cyclopropyl
family members with respect to their anti-inflammatory properties by studying the inhibition
of IFN-γ production in MLR assays and TNF-α in LPS-activated PBMCs. We have identified
BXL-62 as the most potent anti-inflammatory compound among all the members of the 16ene-20-cyclopropyl family tested. The MLR assay is used as an in vitro model of adaptive
immunity and is widely applied to monitor diseases, such as AIDS (Clerici, Stocks et al.
1989) to predict transplant rejection, especially in renal transplantation (Kerman, Susskind et
al. 1997) and to screen for novel immunosuppressive drugs (Matsumoto, Marui et al. 1993).
One of the mechanisms regulating innate immunity involves the TLRs (Athman and Philpott
2004), so in our experiments we focused on TLR4, because its ligand, the bacterial endotoxin
LPS, is a potent inducer of immune responses (Medzhitov 2007). We have analyzed the proinflammatory cytokine TNF-α, which is produced after the activation of TLR4. TNF-α plays
a key role in many disorders, such as rheumatoid arthritis, acute lung injury and IBD (Bradley
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
2008). Anti-TNF-α biologicals are clinically used for the treatment of several autoimmune
diseases including IBD (Baumgart and Sandborn 2007).
Our observations, documenting the capacity our selected VDR agonists to markedly
inhibit TNF-α, suggest this class of agents as a potential treatment of IBD. In order to validate
its potentcy in IBD context, we wanted to confirm in vitro potency of our selected VDR
agonist in PBMCs purified from IBD patients.
Inhibition of pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-12/23p40, by
VDR agonists after any TLR activation in PBMCs purified from IBD patients could represent
an important mechanism for the regulation of the abnormal immune response to the bacteria,
as commonly though as starting point of the disease in genetic predisposed individual (Danese
and Fiocchi 2006). Additionaly, our VDR agonist present an important regulation of adaptive
immunity, since these three pro-inflammatory cytokines, especially IL-12/23p40, are potent
regulators of Th1 and Th17-mediated T cell response (Steinman 2007).
Pro-inflammatory cytokine production is controlled by the inducible NF-κB, which
binds to regulatory regions inflammatory genes and up-regulates the transcription of proinflammatory cytokines (Oeth, Parry et al. 1994). NF-κB nuclear translocation is the main
event downstream the TLR, inducing transcription of pro-inflammatory genes. Coupled to the
direct transcript inhibition mediated by the VDR, the indirect effect on NF-κB confirmed the
potency of this agonist as alternative of the biological agent in the regulation of the proinflammatory response mediated by TLR members (Lips 1996). Finally, since IL-10
represents an important cytokine for IBD onset, we demonstrated that despite a deficiency
observed in PBMCs from IBD patients, the VDR agonist BXL-62 maintains its antiinflammatory poteny for TNF-α and IL-12/23p40.
No study has been previously carried out on anti-inflammatory properties exterted by
VDR agonists on human LPMCs purified from inflamed tissue. To extend our previous
results, it was interesting to study the anti-inflammatory potency of BXL-62 on cells purified
from tissues directly involved in disease pathogenesis. A similar inhibition of proinflammatory cytokines was observed in PBMCs and LPMCs, indicating that VDR
expression in LMPCs is functional.
These results are promising to further determine the potency of BXL-62 in the DSSinduced colitis model.
DSS-induced colitis is a model of wasting disease based on epithelial damage
mimicking the increase of the permeability leading to the abnormal presentation of the
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Laverny Gilles: Identification of a potent and safe VDR agonist for the treatment of IBD
bacteria flora (Xavier and Podolsky 2007). Recent work has shown the important role of the
VDR in the onset of spontaneous and induced colitis in mice and the amelioration of DSSinduced colitis symptoms after intra-rectal injection of calcitriol (2.5 µg/kg) (Cantorna,
Munsick et al. 2000; Froicu, Weaver et al. 2003; Zhu, Mahon et al. 2005; Froicu and
Cantorna 2007). It was interesting to see that in this model, the amelioration was directly
mediated by TNF-α inhibition (Zhu, Mahon et al. 2005).
In study IV, we demonstrated that at the maximum tolerated dose established by four
consecutive oral administrations of compound, intra-rectal administration of BXL-62 (1
µg/kg, 20 ng/mouse) prevents symptoms of colitis while calcitriol (0.3 µg/kg, 6.7 ng/mice)
failed to ameliorate disease. The strong BXL-62 efficacy on the fecal blood could be
explained by the anti-inflammatory activity of the compound but also by the critical role of
VDR in the maintenance of the integrity if the intestinal mucosal barrier, as demonstrated by a
greater loss of intestinal transepithelial electric resistance in Vdr KO mice compared to wild
type (Kong, Zhang et al. 2008). The regulation of inflammation, confirmed by an increase of
the colon length after BXL-62 treatment, could result, as previously discussed, in a stronger
effect of this compound in the regulation of TLR-mediated inflammation.
Taken together, these pre-clinical in vitro and in vivo data confirm the potential
therapeutic used of VDR agonists for the treatment of IBD.
5.5 24-oxo metabolite accumulation, a key event for BXL-62 potency
It is now well established that 1α,25(OH)2D3 is metabolized by CYP24A1, a multi-catalytic
enzyme, in various target tissues (Deeb, Trump et al. 2007). Over a decade ago, differences
were identified in the metabolism between 1α,25(OH)2-16-ene-vitamin D3 and 1α,25(OH)2D3
and it was recognized that minor alterations in the structure of 1α,25(OH)2D3 can produce
major changes in its target tissue metabolism, which allows efficient C24-hydroxylation and
C24-oxidation but not C23-hydroxylation (Lemire, Archer et al. 1994; Siu-Caldera, Clark et
al. 1996). As a result, the 24-oxo metabolite of 1α,25(OH)2-16-ene-vitamin D3 accumulates in
increasing amounts in target tissues when compared to the corresponding 24-oxo metabolite
of 1α,25(OH)2D3. The biological activity of the stable 24-oxo metabolite of 1α,25(OH)2-16ene-vitamin D3 seems to be similar to some of the actions of 1α,25(OH)2-16-ene-vitamin D3,
such as induction of cell growth inhibition and promotion of RWLeu-4 human myeloid
leukemic cell differentiation (Siu-Caldera, Clark et al. 1996). These data thus support the
Kuopio Univ. Publ. C. Nat. and Environ. Sci. 264:1-135 (2009)
concept that one of the important mechanisms responsible for the increase in the potency of
some of the vitamin D analogues can be due to their metabolism through alternative pathways
leading to the production of stable and bioactive metabolites. On the basis of these earlier
observations, the metabolism of 1α,25(OH)2-20-cyclopropyl-vitamin D3 with that of BXL-62
was compared, showing that 1α,25(OH)2-20-cyclopropyl-vitamin D3 is rapidly metabolized
through three different pathways (24-oxidation, C3-epimerization and C1-esterification
pathways) (Uskokovic, Manchand et al. 2006), with a pattern of metabolism similar to that of
1α,25(OH)2-20-epi-vitamin D3 (Siu-Caldera, Sekimoto et al. 1999). Conversely, BXL-62 is
mainly metabolized through only two pathways (C24-oxidation and C3-epimerization
In study III, we have investigated the metabolism of 1α,25(OH)2-20-cyclopropyl-16ene-vitamin D3 in human ovarian carcinoma derived cell line and confirmed that the 24-oxo
metabolite BXL-143 is indeed the final, stable metabolite of BXL-62. The chemical synthesis
of BXL-143 allowed us to assess its various biological properties. We demonstrated that VDR
primary response gene induction, as well as inhibition of pro-inflammatory cytokine
production, are similar between BXL-62 and BXL-143. Expression of CAMP, a primary VDR
target gene, is also modulated by BXL-62 and BXL-143 in the same way as that of CYP24A1
(Wang, Nestel et al. 2004). As CAMP plays an important role in innate immunity (Wang,
Nestel et al. 2004; Liu, Stenger et al. 2006; Schauber, Dorschner et al. 2007), these data
indicate comparable properties for BXL-62 and BXL-143 in the regulation of innate immune
Collectively, these findings indicate that the strong potency of BXL-62 can be
explained by the accumulation of its stable 24-oxo metabolite that displays immunoregulatory
and anti-inflammatory properties superimposable to those exerted by BXL-62 itself.
However, we found that the MTD of BXL-143 is three times higher than its parent compound
BXL-62, indicating that the potency of the metabolite compared to the parent compound to
induce hypercalcemia was reduced. Based on these findings, we have proposed that BXL-143
represents a superior anti-inflammatory agent to its parent BXL-62, due to its wider
therapeutic window.
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6. Summary and conclusions
In conclusion, the five publications, on which this thesis is based, contribute to the
comprehension of the pathogenesis of two chronic inflammatory disorders, BPH and IBD.
The developments of alternative therapeutic agents are critical for ameliorating the patient’s
quality of life and represent an important challenge. Work in this thesis on patient samples
and animal experimental models proposes VDR agonists as promising therapeutic agents that
should be further investigated and developed.
Chronic inflammation induced by stromal BPH cells
In study I, we show that stromal cell purified from biopsies from patients suffering of BPH
express functional receptors involving in innate immunity, the TLRs, as well as the complex
machinery required for induction of adaptive immune responses. We demonstrate that TLR
activation of stromal BPH cells induces pro-inflammatory cytokine production, such as IL-8,
which promotes BPH cells growth, and CXCL10 that recruits imflammatory leukocytes.
Additionally, BPH cells can activate CD4+ T cells inducing production of pro-inflammatory
cytokines typical of Th1 and Th17-mediated immune responses, such as IFN-γ and IL-17.
Thus, in study I we provide, for the first time, direct demonstration of the immunological
mechanisms leading to an inflammatory component in BPH pathogenesis, and highlight
possible new therapeutic agents for this disease.
Elocalcitol inhibit inflammatory reponse in BPH cells
Following the discovery of an inflammatory component in BPH, we studied in publication II
the inhibitory potency of elocalcitol, a VDR agonist proposed for BPH treatment. We
demonstrated that IL-8-dependent BPH cell growth is inhibited by elocalcitol, as well as proinflammatory factors, such as COX-2 and PGE2. These inhibitiory activities are directly
mediated by a reduction of the nuclear translocation of NF-κB and a down-regulation of Rho/Rho kinase pathway. Thus, elocalcitol could ameliorate static, dynamic, and inflammatory
components of BPH pathogenesis.
IBD patients present in the periphery a IL-10 production defect
In study V, we investigated the production of anti- and pro-inflammatory cytokines after
TLR4 and TLR5 stimulation in the periphery and the tissue site of inflammation in IBD
patients. We demonstrated that despite a similar pro-inflammatory cytokine production profile
in the periphery between IBD patients and healthy controls, the production of the anti-
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Summary and conclusions
inflammatory cytokine IL-10 is defective in PBMCs from IBD patients stimulated by a TLR4
agonist. We could not confirm this IL-10 defect in the inflamed tissue, highlighting the
potential relevance of peripheral IL-10 production in IBD pathogenesis.
BXL-62 as a potent anti-inflammatory VDR agonist in IBD
Following the definition of BXL-62 as the most potent anti-inflammatory VDR agonist
among its family members tested, we decided to study its potency to ameliorate colitis
symptoms both in mononuclear cells purified from patients and in a chemically-induced
colitis model in the mouse. We confirmed the strong anti-inflammatory potency of this VDR
agonist compared to the natural hormone in IBD patients, both in the periphery and in
inflamed intestinal tissue. We demonstrated also that VDR agonists are able to inhibit proinflammatory cytokines after stimulation by any TLR agonist, confirming VDR agonists as
potent regulators of the abnormal bacteria recognition. In addition, DSS-induced colitis
symptoms in mice were significantly more ameliorated by daily treatment with BXL-62
compared to the natural ligand, confirming a potential interest for its development as a
therapeutics agent in IBD.
BXL-62 potency is mediated by its 24-oxo metabolite accumulation
In study III, we confirm previous observations and show that BXL-62 is protected from
CYP24A1 hydroxylation, leading to an accumulation of a 24-oxo-BXL-62 (BXL-143). This
VDR agonist shows similar VDR primary response gene induction and anti-inflammatory
potency to its parent compound, suggesting that BXL-62 activity is mediated by this 24-oxo
metabolite. Additionaly, the latter compound exhibits a maximum tolerated dose 3 times
lower than its parent compound, suggesting BXL-143 as an even more interesting VDR
General conclusion
In conclusion, these studies confirm elocalcitol as a potent VDR agonist and a potential drug
to ameliorate BPH. Elocalcitol targets BPH cells, inhibiting proliferation and inflammation
induced by their capacity to act as APCs and to recognize bacterial components via TLRs. In
addition, we characterize a novel potent VDR agonist, BXL-62, that exhibits strong antiinflammatory and immunoregulatory properties both in innate and adaptive immunity, and
propose this VDR agonist as a potential therapeutic agent for IBD, a chronic inflammatory
disease of the gastrointestinal tract.
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7. Future aspects
IBD is a complex disease where the cross talk between environmental and genetic factors
contributes to disease pathogenesis. The precise ethiology of IBD remains unknown, despite
all the progress in genome-wide association studies. The current understanding of IBD
pathogenesis has nevertheless permitted the development of effective therapies, although none
of them leads to complete remission. Drugs with the capacity to regulate abnormal
inflammatory responses could lead to the selection of novel candidates. In this context, in
addition to promising pre-clinical activity in diverse inflammatory models, VDR agonists
represent potential candidates for the treatment of IBD, among other chronic inflammatory
The VDR agonist BXL-62 identified in this thesis could represent an interesting
candidate for future development. Further characterization of its potency in spontaneous and
induced colitis models and the confirmation of its mechanisms of action mediated by its 24oxo metabolite will lead to the possible identification of novel treatment opportunities, since
undesirable effects induced by VDR agonists appear to be predictable and manageable.
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Appendix: Original publications
Publication I
Human Benign Prostatic Hyperplasia Stromal Cells as Inducers
and Targets of Chronic Immuno-mediated Inflammation
Penna G, Fibbi B, Amuchastegui S, Cossetti C, Aquilano F, Laverny G,
Gacci M, Crescioli C, Maggi M, Adorini
Journal of Immunology 182(7):4056-64