TLR Stimulation of Prostate Tumor Cells of Induces Chemokine-Mediated Recruitment

TLR Stimulation of Prostate Tumor Cells
Induces Chemokine-Mediated Recruitment of
Specific Immune Cell Types
This information is current as
of June 15, 2014.
Roberta Galli, Donatella Starace, Roberta Busà, Daniela F.
Angelini, Alessio Paone, Paola De Cesaris, Antonio
Filippini, Claudio Sette, Luca Battistini, Elio Ziparo and
Anna Riccioli
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Copyright © 2010 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2010; 184:6658-6669; Prepublished online 17
May 2010;
doi: 10.4049/jimmunol.0902401
The Journal of Immunology
TLR Stimulation of Prostate Tumor Cells Induces
Chemokine-Mediated Recruitment of Specific Immune
Cell Types
Roberta Galli,* Donatella Starace,* Roberta Busà,†,‡ Daniela F. Angelini,‡
Alessio Paone,* Paola De Cesaris,x Antonio Filippini,* Claudio Sette,†,‡ Luca Battistini,‡
Elio Ziparo,*,1 and Anna Riccioli*,1
oll-like receptors are a family of transmembrane receptors
that recognize conserved molecular patterns of microbial
origin called pathogen-associated molecular patterns. In
addition, TLRs play an important role in tissue repair and tissue
injury-induced inflammation. In the latter context, TLR ligands can
be microbial or host derived (endogenous). TLRs that recognize
lipid and protein ligands (TLR1, TLR2, TLR4, TLR5, and TLR6) are
expressed on the plasma membrane, whereas TLRs that detect viral
nucleic acids (TLR3, TLR7, and TLR9) are localized in lysosomal
compartments (1). TLRs transduce signals through different
*Department of Histology and Medical Embryology, Institute Pasteur-Foundation
Cenci Bolognetti, “Sapienza” University of Rome; †Section of Anatomy, Department
of Public Health and Cell Biology, University of Rome Tor Vergata; ‡Fondazione
Santa Lucia Istituto di Ricovero e Cura a Carattere Scientifico, Rome; and xDepartment of Experimental Medicine, University of L’Aquila, L’Aquila, Italy
E.Z. and A.R. contributed equally to this work.
Received for publication July 29, 2009. Accepted for publication April 7, 2010.
This work was supported by grants from Ministero dell’Istruzione, dell’Università e
della Ricerca, Progetti di Rilevanza e Interesse Nazionale 2007 (to E.Z.); Associazione Italiana Ricerca sul Cancro, Associazione Internazionale per la Ricerca sul
Cancro, and Fondazione Italiana Sclerosi Multipla (to C.S.); and Progetto Oncologico del Ministero della Salute (to L.B.).
Address correspondence and reprint requests to Dr. Anna Riccioli, Department of
Histology and Medical Embryology, “Sapienza” University of Rome, 00161 Rome,
Italy. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this paper: a.u., arbitrary unit; CEBPB, CCAAT/enhancer
binding protein b; CM, conditioned medium; IRF, IFN regulatory factor; LTA, linfotoxin a; LTB, linfotoxin b; ND, not detectable; PCa, prostate cancer; pNF-kBLuc, NF-kB luciferase reporter vector; poly(I:C), polyinosinic:polycytidylic acid;
PTx, Bordetella pertussis toxin; TIR, Toll/IL-1R; TLR3-DN, TLR3-dominant negative.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
adaptor proteins, which trigger a signaling cascade involving NFkB, MAPKs, JNKs, p38, and ERKs, and IFN regulatory factors
(IRF3, IRF5, and IRF7) (2). Many of the known downstream effects
of TLR signaling occur through nuclear translocation of the transcription factor NF-kB and the subsequent production of cell survival and inflammatory molecules, such as TNF-a and IL-1 and
-6 (3). The classical TLR function is antimicrobial, consisting of
leukocyte recruitment to infected tissues and subsequent induction
of adaptive immune responses. Activation of TLRs on epithelial
cells leads to surface expression of ICAM-1 (4), which, in endothelial cells, plays a key role in leukocyte rolling and adhesion (5).
TLRs were initially thought to be expressed only in immune and
epithelial cells, but recent studies demonstrated that these receptors
are also expressed in some tumor cells and that such expression is
related to tumorigenesis. The role of TLRs in cancer is a matter of
debate because conflicting data argue for TLRs being negative or
positive regulators of cancer [reviewed in Ref. 6]. The administration of TLR agonists was reported to exert strong antineoplastic effects against established tumors in mice and humans (7).
The TLR3 agonist polyinosinic:polycytidylic acid [poly(I:C)] leads
to the apoptosis of tumor cells (8, 9), whereas the TLR4 agonist
LPS is able to kill ancillary cells of the tumor microenvironment,
such as the vascular endothelium (10). TLR activation may also
cause tumor regression by increasing vascular permeability and
through the recruitment of leukocytes, which determines tumor cell
lysis by NK and cytotoxic T cells (6). Accordingly, one of the most
promising effects of TLR stimulation by specific agonists in cancer
therapy is the activation of the adaptive immune system (7, 11).
In contrast, stimulation of some TLRs was shown to enhance
tumor survival and proliferation in vitro (12), suggesting that
stimulation of TLRs favors tumorigenesis. The contradictory evidence that, under certain conditions, inflammation promotes carci-
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TLRs boost antimicrobial response mechanisms by epithelial cells and represent the first line of defense at mucosal sites. In view of
these immunomodulatory properties, TLR stimulation may represent a novel means to activate anticancer immune responses. In
the present study, the ability of TLR ligands to affect the recruitment of different immune cell populations by human prostate
cancer cell lines and the underlying mechanisms were investigated. We showed that LNCaP and DU-145 cells express functionally
active TLR3 and TLR5. Treatment with their respective agonists, polyinosinic:polycytidylic acid and flagellin, rapidly triggered
NF-kB–dependent upregulation of different inflammatory molecules, as assayed by microarray and ELISA. Furthermore, we
demonstrated that conditioned media from polyinosinic:polycytidylic acid- and flagellin-treated LNCaP and DU-145 cells induced
the recruitment of different leukocyte subpopulations, suggesting that TLR stimulation is able to activate the earliest step of
immune response mediated by soluble factors. Interestingly, the more aggressive cancer cell line PC3 expressed TLR3 and TLR5
but failed to respond to TLR agonists in terms of NF-kB activation and the ability to attract immune effectors. Overall, these data
show for the first time that TLR3 and TLR5 stimulation of human prostate cancer cells triggers the production of chemokines,
which, in turn, favor the attraction of immune effectors, thereby representing a tool to enhance the efficacy of conventional
therapies by stimulating anticancer immune responses. The Journal of Immunology, 2010, 184: 6658–6669.
The Journal of Immunology
Materials and Methods
Cell lines and reagents
LNCaP and PC3 PCa cells were maintained in DMEM, whereas DU-145
cells were maintained in RPMI 1640 medium. Both media were supplemented with 2 mM L-glutamine, 100 IU/ml penicillin-streptomycin, and
10% FCS (Sigma-Aldrich, St Louis, MO). Cells were serum starved for 18
h prior to stimulation with poly(I:C) or flagellin (both from InvivoGen, San
Diego, CA) in FCS-free medium. BMS345541 was from Sigma-Aldrich.
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA).
Three micrograms total RNA was used for the RT reaction by using the
SuperScript First-Strand Synthesis System Kit (Invitrogen). Each 50 ml
PCR mixture contained 1 ml cDNA, 25 pmol specific primers, 0.2 mM
29-deoxynucleoside 59-triphosphate mix, 1.5 mM MgCl2, 5 ml 103 PCR
buffer, and 2.5 U Taq DNA polymerase (Invitrogen). For human TLRs and
cytokines, the PCR products were amplified using previously reported
primers (23–28). The specificity of the primers was tested by a GenBank
basic local alignment search tool. A T3 thermocycler PCR system (Biometra Whatman, Goettingen, Germany) was used for the reverse transcription reaction, and this was followed by a 30-cycle PCR program.
Flow cytometry
LNCaP, PC3, and DU-145 cells were detached with 0.05% trypsin/0.02%
EDTA and washed with cold PBS. For detection of TLR5, cells were fixed
and permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences,
Franklin Lakes, NJ), according to the manufacturer’s instructions. Mouse
IgG2a anti-human TLR5 (Imgenex, San Diego, CA) mAb or the appropriate isotypic control mAb was used at 0.5 mg/106 cells for 30 min on ice.
After washing with cold PBS, cells were stained with fluorescein (FITC)conjugated anti-mouse (Sigma-Aldrich). Cells were analyzed with a
Coulter Epics XL flow cytometer (Beckman Coulter, Brea, CA). Cells
were gated using forward versus side scatter to exclude dead cells and
debris. Fluorescence of 104 cells per sample was acquired in logarithmic
mode for visual inspection of the distributions and in linear mode for
quantifying the expression of the relevant molecules by calculating the
mean fluorescence intensity.
Western immunoblotting
Total LNCaP, PC3, and DU-145 cell lysates were prepared by lysing and
scraping the cells off the culture plate with cell lysis buffer (Cell Signaling
Technology, Danvers, MA) containing 1 mg/ml leupeptin and 1 mM PMSF
(Sigma-Aldrich). Protein concentration was determined by using the micro
bicinchonic acid method (Pierce, Rockford, IL). Equal amounts of proteins
(15 mg) were subjected to SDS-PAGE and then transferred onto nitrocellulose. The filters were saturated with 5% nonfat dry milk in TBS.
Rabbit polyclonal Ab against total IkB-a was from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphospecific anti-p65 was from BioSource International (Camarillo, CA); Ab against TLR3 was from Cell
Signaling Technology, and Ab against a-tubulin was from Sigma-Aldrich.
The secondary Abs were HRP-conjugated goat anti-rabbit (Pierce) or goat
anti-mouse (Bio-Rad, Hercules, CA). After incubating with the first and
secondary Abs, the membranes were washed three times for 15 min with
TBS containing 0.1% Tween 20. Ab detection was performed by using the
chemiluminescence system (ECL Advance Western blotting detection kit;
GE Healthcare Technologies, Milan, Italy).
Quantification of secreted chemokines by ELISA
LNCaP cell- or DU-145 cell-conditioned medium (CM) from untreated
cells and from cells treated with 100 ng/ml flagellin or 25 mg/ml poly(I:C)
for 24 h were assayed for the presence of CXCL10, CCL3, and CCL5
using the Single Analyte ELISArray Kit (SABiosciences, Frederick, MD)
and for the presence of IL-8 (R&D Systems Minneapolis, MN), according
to the manufacturer’s instructions.
Luciferase assay
NF-kB luciferase reporter vector (pNF-kB-Luc) was from Stratagene (La
Jolla, CA). TLR3-dominant negative (TLR3-DN), a Toll/IL-1R (TIR)-less
form of the TLR3 gene generated by deleting the TIR domain (450 bp)
(pZERO-hTLR3) was from InvivoGen. One day after plating (1.4 3 105
cells/ml), LNCaP cells were cotransfected by means of Lipofectamine
plus reagent (Invitrogen) with pNF-kB-Luc and a b-galactosidase vector
to normalize for transfection efficiency. In the experiments of TLR3-DN
overexpression, LNCaP and DU-145 cells were cotransfected with TLR3DTIR, pNF-kB-Luc, and b-galactosidase vector using Lipofectamine
LTX with PLUS reagent (Invitrogen). Transfection was stopped after 5 h
by adding DMEM containing 20% FCS. After 18 h, cells were rinsed
with fresh medium before stimulation with 100 ng/ml flagellin and 25 mg/
ml poly(I:C) for 6 h followed by lysing using reporter lysis buffer
(Promega, Madison, WI). Luciferase activity was assayed with a Berthold
luminometer using a luciferase assay kit (Promega), according to the
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nogenesis, whereas in others it exerts antitumor effects, could be
explained by the different intensity and nature of the inflammatory
response. In fact, chronic inflammatory processes are milder than
acute inflammatory responses, which are aimed at inducing pathogen clearance. In most cases, cancer-associated inflammation is
similar to chronic inflammation, including the production of factors
that stimulate tissue repair and cancer cell survival and proliferation. However, if the inflammatory response develops into
acute inflammation, an immune effector mechanism is activated,
and cancer regression takes place (13). Among the different elements that control neoplastic processes, a major role is attributed to
members of the chemokine superfamily, a large number of small m.
w. proteins that regulate the targeting of leukocytes to inflammatory
sites, binding to seven-transmembrane-domain receptors coupled to
heterotrimeric Gi proteins. Chemokines expressed by tumor cells and
by host cells play a critical role in determining the fate of the developing tumor by regulating the migration of different leukocyte
subtypes (14). The relative proportion of each defense cell type
within the tumor (e.g., macrophages, T cells, NK cells, dendritic
cells, or other leukocyte subtypes) largely dictates the immune profile
at the tumor site; local production of numerous inflammatory mediators is crucial for the recruitment and activation of leukocytes in
addition to macrophages and mast cells (15). In particular, CD8
T cells and some types of innate immune cells, such as NK cells,
can protect against experimental tumor growth (16).
Prostate cancer (PCa) represents one of the most common cancers
diagnosed in males in Western countries. Standard pharmacological
therapy, consisting of ablation of androgens, is initially efficacious,
but most treated patients develop progressive disease and eventually
die of cancer. Consequently, many efforts are being made to identify
novel targets and agents useful for the treatment of this disease.
Although there is emerging evidence that inflammation might be
involved in the etiology of PCa (17), several questions on this issue
remain unanswered. In particular, the possible causes of chronic
inflammation in the prostate, as well as the involvement of inflammatory cells in the process, have not been fully investigated.
Many pathogens, including different bacterial species and viruses,
have been detected in the prostate (18, 19), but only some of them
are associated with inflammation. Interestingly, many bacterial
sequences (20) and a novel viral sequence (21) have been detected
in pathological prostate tissue, although the corresponding infective agents could not be isolated and cultured by traditional
means. These observations open a new field of investigation aimed
at clarifying the possible role of bacterial and viral sequences in the
activation of TLRs that might positively or negatively influence
tumorigenesis and/or neoplastic progression by triggering the innate and adaptive immune responses. In addition, regardless of the
role of TLRs in the initiation of cancer, it is likely that the activation
of TLRs in cancer cells influences tumor progression through interaction with the immune system. In this study, we investigated the
effects of TLR3 and TLR5 activation on the expression of inflammatory molecules and the subsequent recruitment of different
leukocyte populations in LNCaP, DU-145, and PC3 cells, which
display low, moderate, and high metastatic potential, respectively
(22). Our results suggested that TLR3 and TLR5 stimulation in
human PCa cells with different aggressiveness differentially influences innate and adaptive immune responses.
manufacturer’s instructions. Transfection efficiency was evaluated by
5-bromo-4-chloro-3-indolyl b-D-galactoside staining for each experiment
and was always .30%. Data were normalized to protein concentration.
LNCaP cells were treated for 4 h with 100 ng/ml flagellin or for 2 h with
25 mg/ml poly(I:C); total RNAs from untreated and treated cells were
extracted using TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. These RNAs were used as a template to generate
biotin16-UTP–labeled cRNA probes using the True Labeling Kit (Superarray, Bethesda, MD). The cRNA probes were hybridized at 60˚C with the
Superarray Human Inflammatory Cytokines and Receptors Microarray
membranes, and signals were revealed using the SuperArray Detection
Kit. Data from two experiments were analyzed by densitometry using
Scanalyze software (Eisen Laboratory, Stanford University, Stanford, CA).
Chemotaxis assay
Statistical analysis
Statistical differences were determined by the Student t test for paired
samples or by one-way ANOVA followed by the Student t test with the
Bonferroni correction. A p value ,0.05 was considered significant.
LNCaP and DU-145 cells express functionally active TLR3 and
To investigate the expression pattern of TLRs in different PCa cell
lines, we initially performed RT-PCR on total RNA. LNCaP and
DU-145 cells consistently expressed high levels of TLR3 and TLR5
mRNAs (Fig. 1A). In contrast, the other TLRs tested were absent
in both cell lines, with the exception of faint expression of TLR2
in LNCaP cells and TLR6 in DU-145 cells. PC3 cells showed a
different pattern of expression, with high expression of TLR2,
TLR3, TLR4, TLR5, and TLR6 and no expression of TLR7,
TLR8, or TLR9 (Fig. 1A). Next, we tested the expression of TLR3
and TLR5 at the protein level, the only TLRs expressed by the
three cell lines tested. We demonstrated by Western blot that
TLR3 is expressed at similar levels in LNCaP and DU-145 cells,
FIGURE 1. TLR mRNA and protein expression in human PCa cell lines
LNCaP, PC3, and DU-145. A, RT-PCR showing basal expression of the indicated Tlr genes. RT-PCR was performed using specific primers as described in Materials and Methods. B, Western blot analysis of TLR3
expression. Similar results were observed in three independent experiments
(A, B). C, Flow cytometric analysis of TLR5 expression was performed using
mouse anti-TLR5 mAb (white areas) or isotypic mouse IgG (gray areas). The
diagrams are representative of at least three independent experiments.
with a slightly lower expression in PC3 cells (Fig. 1B). Moreover,
LNCaP, PC3, and DU-145 cells were stained with a TLR5-specific
Ab and analyzed by flow cytometry. As shown in Fig. 1C, TLR5
protein was expressed at high levels in all three cell lines.
TLR stimulation leads to the activation of NF-kB in most cells
(30). This transcription factor is retained in the cytoplasm in an
inactive form, associated with the inhibitory protein IkBa. TLR
agonists induce IkBa phosphorylation and its subsequent degradation by the proteasome, allowing NF-kB to enter the nucleus.
Alternatively, NF-kB can be directly activated through the phosphorylation of the NF-kB p65 subunit (31). To study TLR3 and
TLR5 activity, LNCaP and DU-145 cells were treated for increasing lengths of time with the respective ligands poly(I:C) (25
mg/ml) and flagellin (100 ng/ml), and NF-kB activation was
evaluated. Western blotting of whole lysates showed that in both
cell lines, p65 phosphorylation and parallel degradation of IkBa
were triggered by poly(I:C) and flagellin stimulation, although
with slower p65 phosphorylation kinetics induced by poly(I:C)
compared with flagellin (Fig. 2A). In DU-145 cells, p65 basal
phosphorylation was much higher than in LNCaP cells, and a less
apparent increase was induced by TLR3 and TLR5 stimulation. To
confirm activation of NF-kB by poly(I:C) and flagellin, cells were
transfected with an NF-kB luciferase reporter plasmid. The next
day, cells were challenged with poly(I:C) or flagellin for 2, 4, or
6 h, and lysates were assayed for NF-kB–dependent luciferase
activity. Poly(I:C) and flagellin induced a significant NF-kB acti-
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Two types of cell samples from healthy donors were used for in vitro
transmigration experiments. In one set of experiments, whole blood
deprived of RBCs was used, whereas in another set of experiments, PBMCs
obtained by Ficoll-Hypaque gradient centrifugation (Pharmacia, Uppsala,
Sweden) were depleted of monocytes using CD14 MicroBeads (Miltenyi
Biotec, Auburn, CA) to isolate untouched lymphocytes. Briefly, cells were
assayed for their ability to migrate through a polyvinylpyrrolidone filter
(pore size, 3 mm; Whatman International, Maidstone, U.K.) using Boyden
chambers (NeuroProbe, Gaithersburg, MD). In all, 2 3 106 cells/well were
added to the upper compartment of the chamber, and LNCaP cell or
DU-145 cell CM was placed in the lower compartment. In some experiments, PBMCs were pretreated for 18 h with 100 ng/ml Bordetella pertussis toxin (PTx) (Calbiochem, San Diego, CA). The assembled chambers
were incubated for 5 h at 37˚C. Cells migrated to the lower chamber were
counted using a Thoma counting chamber, labeled with previously defined
optimal concentrations of mAbs (conjugated with the appropriate fluorochrome), and acquired on a CyAN cytometer (Beckman Coulter). The
following Abs were used: CD14 FITC, CD8 FITC, CD19 PE (all from
Immunological Sciences, Rome, Italy); CD56 PE, CD16 Pacific Blue (both
from BD Biosciences); CD8 ECD, CD16 PE-Cy7, CD56 allophycocyanin
(all from Beckman Coulter); CD3 Pacific Blue, CD3 Cascade Yellow, CD4
allophycocyanin (all from DakoCytomation, Glostrup, Denmark); CD19
allophycocyanin-Alexa750, CD4 allophycocyanin-Alexa750 (both from
eBioscience, San Diego, CA); CD45RA ECD (Beckman Coulter); and
CD25 PE-Cy7 (BD Biosciences). Data were compensated and analyzed
using FlowJo software (Tree Star, Ashland, OR). Seven- and eight-color
flow cytometry analysis was performed to discriminate among different
leukocyte subpopulations: CD32/CD142/CD16high/side scatter high for
granulocytes; CD32/CD14+/side scatter high for monocytes; CD32/CD19+
for B cells; CD32/CD56high/CD162 and CD32/CD56dull/CD16+ for cytokine-producing and cytotoxic NK cells, respectively; CD3+/CD4+/
CD25+ for activated CD4 cells; CD3+/CD8+/CD45RA2/CD162 for cytokine-producing CD8 effector cells and memory CD8 cells; and CD3+/
CD8+/CD45RA+/CD16+ for CD8 effector cytotoxic cells (29).
The Journal of Immunology
vation, albeit with different kinetics (Fig. 2B). In LNCaP cells, the
highest level of enhancement was reached as early as 2 h after
flagellin treatment, whereas 4 h were required for maximal activation by poly(I:C). In DU-145 cells, the kinetics of NF-kB activation were delayed, with the maximum increase in luciferase
activity detectable after 6 h of poly(I:C) or flagellin stimulation.
PC3 cells showed a strong basal p65 phosphorylation, with no
additional induction by poly(I:C) or flagellin. Moreover, NF-kB–
dependent luciferase activity in this cell line was also basally high,
and it unexpectedly decreased after stimulation with flagellin.
Altogether, these data demonstrate that TLR3 and TLR5 trigger
activation of NF-kB in LNCaP and DU-145 cells.
Poly(I:C)-triggered NF-kB activation is TLR3 dependent
Exogenous flagellin can interact only with membrane-bound
TLR5, whereas different molecules are involved in dsRNA recognition. In fact, poly(I:C) is able to bind TLR3 localized in the
endosomal membrane, but it can also activate different pathways
mediated by cytosolic sensors (32, 33). To determine the direct
involvement of TLR3 in mediating poly(I:C)-triggered effects,
LNCaP and DU-145 cells were transiently transfected with a
control plasmid or with a vector encoding TLR3-DN (nonfunctional because of deletion of the TIR domain), which competes
with the endogenous functional TLR3. Transfected cells were
treated for 4 h with poly(I:C) and analyzed for NF-kB activation
by luciferase assay. We found that downregulation of TLR3
function by TLR3-DN overexpression significantly reduced poly
(I:C)-induced NF-kB activation in both cell lines (Fig. 3).
TLR3 and TLR5 stimulation leads to differential secretion of
NF-kB is a key regulator of TLR-induced proinflammatory molecules (34, 35). Thus, we sought to analyze whether TLR3/5
ligands induce the production of chemokines in LNCaP and DU-
145 cells through the NF-kB pathway. To this end, we performed
ELISA on CM from LNCaP and DU-145 cells treated for 24 h
with poly(I:C) or flagellin. None of the chemokines tested was
detectable in control LNCaP CM, whereas DU-145 cells constitutively secreted low levels of CCL5 and greater amounts of
IL-8 (Fig. 4). Our results indicated that in both cell lines, poly(I:C)
was more effective than flagellin in the induction of CCL3, CCL5,
and CXCL10, and only in DU-145 cells did both TLR agonists
elicit IL-8 production at the same levels (Fig. 4).
LNCaP and DU-145 cells stimulated with poly(I:C) and
flagellin recruit different leukocyte subpopulations
The chemokine superfamily plays a major role in the control of
neoplastic processes by regulating the directed trafficking of leukocytes to inflammatory sites and enabling their recruitment from
hematopoietic organs (36–38). The activity of chemokines can
exert significant support or inhibition of processes involved in
tumorigenesis, depending on the malignancy context (14). To
clarify the possible functional role of the chemokines upregulated
by treatment with poly(I:C) or flagellin, we performed a chemotaxis assay using Boyden chambers and evaluated the recruitment
of different immune effector cells across a filter (pore size, 3 mm).
The chamber below the filter was filled with CM from LNCaP or
DU-145 cells stimulated with poly(I:C) or flagellin, and total
human leukocytes from healthy blood donors (PBMCs) were
plated in the upper chamber. After 5 h at 37˚C, the cells migrated
to the lower chamber were counted and stained with Abs directed
to specific surface Ags to discriminate among different leukocyte
subpopulations (see Materials and Methods). As shown in Fig. 5A,
poly(I:C) CM and flagellin CM from LNCaP cells induced a
2-fold increase in the chemotaxis of granulocytes and monocytes
versus control CM. However, only poly(I:C) CM enhanced the
recruitment of T and B lymphocytes and NK cells compared with
control CM. Poly(I:C) CM and flagellin CM from DU-145 cells
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FIGURE 2. Poly(I:C) and flagellin induce p65 phosphorylation, IkBa degradation, and NF-kB activation in LNCaP and DU-145 cells but not in PC3
cells. A, LNCaP, DU-145, and PC3 cells were stimulated with 100 ng/ml flagellin and 25 mg/ml poly(I:C) for different times. Whole-cell lysates (15 mg)
were subjected to Western blot analysis using polyclonal Abs against the phosphorylated form of p65 and against total IkBa. The same filters were reincubated with an anti–a-tubulin Ab as a control for an equal amount of protein loaded. These Western blots are representative of three independent
experiments. B, LNCaP, DU-145, and PC3 cells were cotransfected with pNF-kB-Luc reporter and b-galactosidase vectors; the next day, they were treated
for different lengths of time with 25 mg/ml poly(I:C) or 100 ng/ml flagellin prior to performing the luciferase assay. Statistical analysis was performed
comparing untreated cells (Ctr) and TLR agonist-stimulated cells using the Student paired t test. Data are expressed as luciferase activity normalized for
b-galactosidase activity. Data represent the mean of triplicate samples from three independent experiments and is expressed as the mean 6 SEM. pp ,
0.05; ppp , 0.01. a.u., arbitrary unit.
induced a similar 2-fold increase in the chemotaxis of granulocytes, but they failed to affect monocyte migration (Fig. 5B).
The chemotactic ability of TLR-stimulated DU-145 cells on
lymphocytes and NK cells was considerably lower than that observed for LNCaP cells, although the increment with respect to
control CM was comparable. Intriguingly, the recruitment of
T lymphocytes was mainly enhanced by poly(I:C) CM, and the
recruitment of B lymphocytes was induced only by flagellin CM,
whereas NK cells were recruited by both CMs. We also tested
the chemotactic ability of PC3 CM in control conditions and following TLR3 and TLR5 stimulation. PC3 CM induced a high basal
level of chemotaxis in the analyzed populations that was not enhanced by poly(I:C) or flagellin stimulation (data not shown), in line
with the high basal NF-kB activity and lack of increment by
poly(I:C) and flagellin (Fig. 2). Because chemokines bind to seventransmembrane-domain receptors that are coupled to heterotrimeric
Gi proteins, chemokine-induced migration is completely inhibited
by treatment of the cells with PTx, which inhibits Gai and Ga0 proteins (39). To confirm the nature of the chemotactic activity induced
by TLR agonists, we pretreated total leukocytes with PTx for 18 h
before testing their overall migration in TLR-stimulated LNCaP
CM. We observed that toxin pretreatment blocked the effect induced by TLR stimulation (Fig. 5C), demonstrating the direct role
of TLR-induced chemokines produced by LNCaP cells, the most
TLR-responsive cell line, on leukocyte recruitment.
Subsequently, because T lymphocytes and NK cells have a key
role in antitumor immune defense, we set out to characterize the
TLR3 and TLR5 stimulation induces expression of a
proinflammatory mRNA array in LNCaP cells
Because the strongest chemotactic effect was obtained by using
LNCaP CM, we tested the effect of flagellin and poly(I:C) on a
broad array of cytokines and chemokines by Microarray analysis in
this cell line. RNA extracted from cells stimulated with TLR3 or
TLR5 agonists was hybridized onto chips containing a subset of
113 key genes involved in the inflammatory response. LNCaP cells
were treated with poly(I:C) for 4 h and flagellin for 2 h when NF-kB
activation by these agonists reached its peak (Fig. 2B). Table I and
Fig. 7B show that poly(I:C) and flagellin caused a marked change
in the gene-expression pattern; 26.7% and 15%, respectively, of
tested genes were upregulated. Data obtained for all of the genes
tested are shown in Supplemental Fig. 1. Chemokines belonging
to the CC and CXC subfamilies, which account for 52% of the
upregulated genes, showed a similar pattern of expression following stimulation with both agonists. However, a number of
functionally different chemokines were selectively induced by
poly(I:C) or flagellin (Table I). Moreover, poly(I:C) induced the
expression of several other inflammatory molecules, such as the
cytokines IL-6 and -15, TNF, linfotoxin a, and linfotoxin b (LTB);
the cytokine receptors IL-10RA, -13RA2, and -15RA; the chemokine receptor CXCR4; the antiviral molecule IFN-A2; the
complement component C3; and the transcription factor CCAAT/
enhancer binding protein b, whereas flagellin enhanced only
CXCR4, IL-15 and -15RA, LTB, and TNF.
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FIGURE 3. Poly(I:C) fails to induce NF-kB activation in LNCaP and
DU-145 cell lines transfected with TLR3-DN plasmid. Both cell lines were
transiently transfected with CMV (control plasmid) or TLR3-DN. Subsequently, cells were treated or not with poly(I:C) for 4 h prior to luciferase
assay. b-galactosidase assay was used to normalize for transfection efficiency. Data are expressed as fold of induction in TLR agonist-treated
versus untreated samples. Statistical analysis was performed by comparing
poly(I:C)-stimulated CMV-transfected cells with poly(I:C)-stimulated
TLR3-DN–transfected cells. Data represent the mean of triplicate samples
from three independent experiments 6 SEM. pp # 0.05; ppp , 0.01,
Student paired t test.
subpopulations of lymphocytes and NK cells that are selectively
recruited by TLR-stimulated LNCaP and DU-145 cells. To this end,
PBLs from healthy donors were isolated by Ficoll gradient and
magnetic depletion of the monocyte population (see Materials and
Methods) and subjected to chemotaxis assay.
Preliminarily, because it is well known that flagellin and poly
(I:C) affect various functions of lymphocytes and NK cells (40, 41),
we assayed DMEM + 25 mg/ml poly(I:C) and DMEM + 100
ng/ml flagellin, incubated 24 h at 37˚C, for their chemotactic
activity. Direct treatment of the PBLs with flagellin or poly(I:C)
did not enhance chemotaxis (data not shown).
As for LNCaP cells, flagellin CM had no effect on lymphocyte
migration, whereas poly(I:C) CM from LNCaP cells induced the
migration ∼70% of the total CD4+ T lymphocytes (Fig. 6, upper
left panel), the most represented T cell subset in the upper
chamber, with a 3-fold increase compared with control CM. For
the less represented subsets, such as activated CD4+ T cells and
CD4+CD25high T cells (regulatory T cells), poly(I:C) CM induced
an increase in their migration, although the absolute number of
regulatory T cells was very low. As for CD4+ T lymphocyte migration with DU-145 CM, poly(I:C) CM, and flagellin CM, they
all induced a significant increase in migration compared with the
very low rate observed with control CM (Fig. 6, upper right
panel). With regard to total CD8+ T cells, 83% were attracted by
LNCaP poly(I:C) CM (Fig. 6, middle left panel). A detailed
analysis of the migrated CD8+ cells showed that the total fraction
of the major cytotoxic population of CD8+ T cells and 73% of
memory cytokine-producing cells were attracted by poly(I:C) CM
(Fig. 6, middle left panel). Interestingly, 100% of NK lymphocytes, both cytokine producing and cytotoxic cells, were recruited
by poly(I:C) CM (Fig. 6, lower left panel).
With regard to CM from DU-145 cells, flagellin and poly(I:C)
CM induced a significant recruitment of total CD8+ memory
cytokine-producing T and NK cells (Fig. 6, middle and lower right
panels); however, the rate of migration of T and NK cell subpopulations was less than that induced by CM from untreated and
TLR-stimulated LNCaP cells.
The Journal of Immunology
To determine whether these effects on gene expression were
dependent on NF-kB activity, we used the specific IKK inhibitor
BMS345541 (42), which efficiently blocked NF-kB activation
induced by both TLR agonists in reporter assays (Fig. 7A). Cells
were pretreated 1 h with the inhibitor (10 mM) before stimulation
with poly(I:C) for 4 h prior to total RNA extraction. As shown in
Fig. 7B and Supplemental Fig. 1, pretreatment with BMS345541
dramatically suppressed the expression of inflammatory genes
induced by poly(I:C), indicating a direct involvement of NF-kB
in their upregulation. The same degree of inhibition was observed
on the flagellin-induced genes tested by RT-PCR (data not shown).
To confirm the microarray data, upregulation of some key inflammatory cytokines and chemokines after flagellin and poly
(I:C) treatment was assayed by RT-PCR. In agreement with results
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FIGURE 4. Stimulation with agonists of TLR3 and
TLR5 modulate chemokine production. LNCaP or
DU-145 cells were treated for 24 h with poly(I:C) or
flagellin, CM were collected, and ELISA assay was
performed as described in Materials and Methods.
Data represent the mean 6 SEM of quadruplicate
samples from two independent experiments. Statistical analysis was performed by comparing chemokine levels in TLR-stimulated CM versus control
CM, when detectable. pp # 0.05; ppp # 0.01, Student t test. ND, not detectable.
obtained by microarray, we observed that both TLR agonists induced a strong increase in IL-8 and TNF-a expression, whereas
only poly(I:C) upregulated IL-6 and CXCL9 mRNA (Fig. 7C).
TLRs play an important role in cancer development, a concept
supported by the association of numerous polymorphisms in TLRs
with human cancer in a variety of organs, including the prostate
(43). In this study, we investigated the expression of TLRs in
different human PCa cell lines (LNCaP, DU-145, and PC3) and
showed that only TLR3 and TLR5 are shared by the three cell
lines. Moreover, we demonstrated, for the first time in prostate
tumor cells, that ligand-induced activation of TLR3 and TLR5
leads to selective migration of leukocytes through upregulation
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FIGURE 5. LNCaP and DU-145 cells stimulated with poly(I:C) and flagellin produce chemokines that induce differential leukocyte chemotaxis. LNCaP
(A) or DU-145 (B) cells were stimulated with poly(I:C) (25 mg/ml) or flagellin (100 ng/ml). After 24 h, CM were collected from stimulated and unstimulated (Ctr) cells and used for a chemotaxis assay (using Boyden transwell cell culture chambers) on total leukocytes from healthy blood donors.
Migrated human leukocytes were collected, labeled with Abs recognizing leukocyte subpopulations, and analyzed by flow cytometry. Significant differences in the leukocyte migration rates induced by poly(I:C) CM or flagellin CM (versus Ctr CM) are shown. ANOVA with Bonferroni posttest. C, Migration
of total leukocytes pretreated or not for 16 h with PTx (100 ng/ml) prior to stimulation with CM from untreated LNCaP cells or TLR agonist-treated LNCaP
cells. Statistical analysis was performed by comparing chemotactic response in the presence versus the absence of PTx in poly(I:C) and flagellin CMs. Data
represent the mean 6 SEM of a quadruplicate from a representative experiment, which was replicated three times. pp # 0.05; ppp # 0.01, ANOVA with
Bonferroni posttest.
of different chemokine patterns. Several recent reports on the
expression of TLRs in human tumors, including prostate carcinomas (44), suggested conflicting roles for the function of TLRs
in human cancer (45–47). Infection- or injury-induced inflammation can promote tumorigenesis, owing to chronic tissue
damage with subsequent tissue repair that could evolve in
The Journal of Immunology
uncontrolled cell proliferation. On the other hand, cancer growth
can mimic tissue damage, and this can trigger TLR-dependent
production of numerous inflammatory mediators associated with
the recruitment and activation of leukocytes, particularly mast
cells, macrophages, and neutrophils with tumor-promoting properties (48). Conversely, TLR agonists were shown to directly kill
tumor cells or to cause tumors to regress indirectly by recruiting
NK and cytotoxic T cells (6). In this article, we demonstrated that
stimulation of TLR3 and TLR5 significantly upregulated chemokines and other inflammatory molecules in LNCaP and DU-145
cells, but not in PC3 cells (data not shown). Importantly, the induction of such factors determines oriented migration of different
immune cell subpopulations, suggesting that TLR stimulation is
able to activate the earliest step of immune response mediated by
soluble factors in the prostate. Our data showed that LNCaP cells
stimulated with both TLR agonists triggered chemotaxis of T
lymphocytes and NK cells much more effectively than did DU-
145 cells, whereas granulocyte migration was induced by both cell
lines at the same extent. On the contrary, CM from PC3 cells
stimulated with poly(I:C) or flagellin did not enhance chemotaxis
of leukocyte subpopulations. We also demonstrated that the chemotactic effect of TLR-stimulated media depended on chemokines, because pretreatment of PBMCs with PTx, which
specifically inhibits G protein-mediated signaling and, thus, chemokine effects, blocked the migration of leukocytes. These processes depend on the NF-kB pathway because pretreatment with a
specific inhibitor completely abolished the upregulation of inflammatory cytokines and chemokines induced by TLR stimulation. These results are in line with the proposed role of NF-kB
in the regulation of the inflammatory process in innate and
adaptive immune responses, through the local production of
proinflammatory factors (49). Noteworthy, in PC3 cells, NF-kB is
constitutively activated at high levels and was not induced further
by poly(I:C) or flagellin treatment, consistent with the lack of
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FIGURE 6. Different lymphocyte subpopulations are attracted by LNCaP or DU-145 CM. PBLs and NK cells from healthy blood donors were isolated
by granulocyte and monocyte depletion. T lymphocytes and NK cells were used for chemotaxis assay with CM from LNCaP or DU-145 cells treated or not
with 25 mg/ml poly(I:C) or 100 ng/ml flagellin. The total number of cells plated in Boyden chambers (white boxes) and cells migrated after 5 h at 37˚C
(other boxes) were labeled with Abs against markers specific for NK and T subpopulations, as described in Materials and Methods, and analyzed by flow
cytometry. Values are from a representative experiment that was replicated three times. Each value represents the mean 6 SEM of a quadruplicate experiment. Statistical analysis was performed by comparing the chemotactic response induced by control CM versus TLR-stimulated CM. pp # 0.05,
ANOVA with Bonferroni posttest.
Table I. Upregulation in gene expression induced by poly(I:C) and flagellin in LNCaP cells
Average Fold Change
Poly(I:C) versus Controla
Flagellin versus Controla
Key Immune Target Cells
Monocytes, mast cells
Th17, dendritic cells
CD8 T cells, NK cells
CD8 T cells, monocytes, NK cells
Monocytes, NK cells
Th1 cells, naive and cytotoxic CD8 T cells
Monocytes, NK cells
Th1 cells, CD8 T cells, mast cells
Th1 cells, NK cells
CD8 T cells, Th1 cells, NK cells
Neutrophils, NK cells
Other Inflammatory Molecules
Transcription factor
Receptor binding
Malignant cell growth
Induction of apoptosis; inflammatory response
Integral to membrane; receptor activity
Receptor activity
Leukocyte activation
Receptor activity
Pro- or antiproliferative
Immune response; induction of apoptosis
Inflammatory response
Inflammation; antiapoptotic response; tumor
Ratio of the mean expression between control and poly(I:C) or flagellin samples of two independent experiments.
Fold changes could not be determined because cDNA hybridization signal was undetectable in control arrays but was clearly detectable in poly(I:C)
and flagellin arrays.
CEBPB, CCAAT/enhancer binding protein b; LTA, linfotoxin a.
immune cell chemotactic response to CM from TLR-stimulated
PC3 cells.
Regarding the possible TLR5 function in cancer cells, an in vivo
approach recently showed that peri-tumoral flagellin treatment of
tumor xenografts of human colon cancer cells increased necrosis,
leading to significant tumor regression (50). In contrast, tumor
growth was accelerated when flagellin was administered at the
time of mammary tumor implantation (51). Given the bivalent
effect of flagellin on cell survival or tumor necrosis, which is strictly
dependent on the cancer model and on the immunological context,
we set out to study its effect on the modulation of a broad array of
inflammatory mediators in LNCaP cells. Flagellin induced the expression of several chemokines involved in the migration of neutrophils and monocytes (CXCL2, CXCL3, CXCL8, CCL2, and
CX3CL1), whereas it weakly induced or failed to upregulate chemokines involved in the migration of NK and T cells (CCL3, CCL4,
CCL5, CCL8, CXCL9, and CXCL10). Consequently, CM from
LNCaP and DU-145 cells stimulated with flagellin are able to specifically recruit mainly neutrophils and monocytes. Interestingly,
CM from flagellin-stimulated DU-145 cells, but not from LNCaP
cells, further induced T and NK cell migration, indicating that
flagellin can differentially influence the immunomodulatory function of PCa cells at different stages of malignancy. Neutrophil infiltration is a key factor eliciting antitumor activity in several tumor
models (52). Interestingly, it was described that blocking TLR5/
MyD88-mediated signaling in human colon cancer xenografts
suppresses neutrophil infiltration and tumor necrosis, which are
associated with enhanced tumor growth (50). These data strongly
suggest that TLR5 engagement by flagellin mediates innate im-
munity and elicits potent antitumor activity. To our knowledge, this
is the first demonstration that the PCa cells LNCaP and DU-145
express TLR5, suggesting that this receptor might be exploited as a
therapeutic target. In this regard, the potential role of flagellin in
activating an immune response in cancer immunotherapy is under
investigation in melanoma and renal carcinoma (53).
In agreement with the multifaceted effects of TLR activation, our
results in LNCaP cells demonstrated that the TLR3 agonist poly(I:
C) induced the expression of a pattern of cytokines and chemokines
different from that induced by flagellin treatment. In addition to the
upregulation of chemokines involved in neutrophil and monocyte
recruitment, we report that poly(I:C) treatment induced the
upregulation of the chemokines CCL3, CCL4, CCL5, CCL8,
CXCL9, and CXCL10, specifically interacting with chemokine
receptors CCR1, CCR4, CCR5, and CXCR3, which were expressed
by freshly isolated primary NK cells and CD8 T cells (54, 55). In
accordance with this chemokine panel, such interaction in our
model induced massive NK and CD8 T cell chemotaxis. The
importance of CXCR3 expression in NK cells and CTLs was recently highlighted by the observation that the therapeutic effect of
immunochemotherapy is abrogated in Cxcr32/2 mice (56). In
fact, CD8 cytotoxic T cells and NK cells can play an important
role in antitumor defenses (57, 58). Recent studies showed that
NK cell activity is controlled through a balance between inhibitory
and stimulatory receptor signals. One of the activating NK and
CD8 TCRs, the NKG2D receptor, may have the ability to specifically target tumor cells for killing through recognition of its
ligands, which include the MHC class I chain-related A and B
molecules (59). Interestingly, LNCaP cells display high expression
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Protein Function
The Journal of Immunology
of MHC class I chain-related A and B molecules (60), suggesting
that the increased recruitment of NK and CD8 T cells as a result of
poly(I:C) stimulation may play a part in activating these immune
cells. The importance of NK activity in PCa was recently demonstrated in TRAMP mice, an in vivo model of PCa (61). Indeed,
we previously demonstrated that poly(I:C) exerts direct antiproliferative and apoptotic effects on LNCaP cells after 24–48 h of
stimulation (8), a rather slow kinetics compared with the very
early onset of the upregulation of inflammatory molecules. These
two kinetically different effects of poly(I:C) on LNCaP cells
converge toward an antineoplastic final effect. By exerting a direct
proapoptotic effect on LNCaP cells (8) and an immune-mediated
effect due to the recruitment of NK cells and cytotoxic CD8 cells
(present data), poly(I:C) is a potentially valid therapeutic agent in
PCa, likely capable of improving conventional therapies because it
may fulfill different criteria that were recently proposed (62).
It was recently demonstrated in in vivo murine tumor models that
the TLR3 agonist polyadenylic acid–polyuridylic acid induces the
production of CCL5 and CXCL10 (CXCR3 ligand) in the tumor
parenchyma. Moreover, CCL5 blockade improved the efficacy of
immunochemotherapy [vaccination followed by conventional
chemotherapy in combination with polyadenylic acid–polyuridylic
acid injection], whereas CXCR3 blockade abolished its beneficial
effects, indicating that TLR3 stimulation can induce tumor cells to
produce a range of chemokines that reinforce immunostimulatory
or immunosuppressive effect, thus influencing the therapeutic response (56). Accordingly, our data show for the first time that
following TLR3 and TLR5 stimulation, human PCa cells produce
chemokines with potentially opposite effects on immune system
The challenge for further investigation will be to exploit poly(I:
C) and flagellin in in vivo experiments to clarify the consequences
of immune cell subpopulation recruitment over neoplastic progression in PCa. Intriguingly, the exploitation of TLRs for cancer
immunotherapy and vaccines is promising; in particular, a clinical
trial for the treatment of ovarian cancer is in progress exploiting
poly(I:C) as an adjuvant together with a vaccine (63).
Altogether, our data indicated that TLR3 and TLR5 were
expressed and functionally active in LNCaP and DU-145 PCa cells,
whereas TLR3/5 stimulation did not affect NF-kB activation, cytokine/chemokine expression, or immune cell recruitment in PC3
cells. This suggests that PC3 cells, which represent the most aggressive castration-resistant stage of prostate malignancy, are not
able to activate an antitumor immune response. In conclusion, the
ability to mount an immune response following TLR stimulation
seems to be inversely correlated with neoplastic progression.
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FIGURE 7. Poly(I:C) and flagellin induce differential mRNA expression of proinflammatory molecules via NF-kB activation. A, LNCaP cells were
cotransfected with pNF-kB-Luc reporter and b-galactosidase vectors, and the next day they were stimulated for 4 h with 25 mg/ml poly(I:C) or 100 ng/ml
flagellin with or without 10 mM BMS345541 pretreatment. Subsequently, cell lysates were subjected to luciferase assay. Statistical analysis was performed
by comparing luciferase activity of TLR-stimulated cells with BMS345541-pretreated TLR-stimulated cells. ppp , 0.01, Student paired t test. Transfection
efficiency was evaluated by b-galactosidase assay. Data 6 SEM represent the mean of triplicate samples from three independent experiments. B, Total RNA
from TLR agonist-stimulated LNCaP cells, pretreated or not with BMS345541 for 4 h, were labeled and analyzed on the Superarray Human Inflammatory
Cytokines and Receptors chip. Examples of five genes modulated by poly(I:C) or flagellin with or without BMS345541 treatment are circled. C, LNCaP
cells were treated with 25 mg/ml poly(I:C) or 100 ng/ml flagellin for 4 and 2 h, respectively. Total RNAs were subjected to RT-PCR using specific primers
for IL-6 (462 bp), IL-8 (494 bp), TNF-a (166 bp), and CXCL9 (168 bp). Actin (350 bp) was used as a loading control. Results are representative of three
independent experiments.
We thank Prof. Fioretta Palombi for helpful discussion and critical reading
of the manuscript and Dr. Fabrizio Padula for assistance with cytometric
The authors have no financial conflicts of interest.
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