IDENTIFICATION OF HOST CANDIDATE GENES AS POTENTIAL DRUG TARGETS AGAINST TUBERCULOSIS USING PULMONARY HISTOPATHOLOGY Amy Booth Supervisor – Dr. Reto Guler Co-supervisors – Marilyn Tyler - Lizette Fick - Frank Brombacher Faculty of Health Sciences University of Cape Town HISTORY OF TUBERCULOSIS IDENTIFICATION OF HOST CANDIDATE GENES AS POTENTIAL DRUG TARGETS AGAINST TUBERCULOSIS USING PULMONARY HISTOPATHOLOGY CONTENT A) Basic Leucine Zipper Transcription Factor, ATF-like 2 (Batf2) B) Blocking of the alternative activation of macrophages via the IL-4 Receptor alpha chain (IL-4Rα) IL-4Rα IMMUNE RESPONSE TO TUBERCULOSIS NO (1) BASIC LEUCINE ZIPPER TRANSCRIPTION FACTOR, ATF-LIKE 2 (BATF2) Part A BASIC LEUCINE ZIPPER TRANSCRIPTION FACTOR, ATF-LIKE 2 (BATF2) Protein encoding gene Suppresses Activator Protein 1 which regulates gene expression in response to cytokines and pathogens (Karin et.al, Murphy et.al.) Induction of genes involved in the early immune response and killing effector functions (Murphy et.al.): Compensates for Batf and Batf3 required for development of (Tussiwand et.al.) TH17 cells CD8+ classical dendritic cells Class-switch recombination in B cells Transcription Factor: Protein that binds to our DNA and activates genes to bring about certain functions BASIC LEUCINE ZIPPER TRANSCRIPTION FACTOR, ATF-LIKE 2 (BATF2) Induced by: Interferon-gamma (IFN-γ) in classically activated macrophages Lipopolysaccharide TB infection in classically activated macrophages further enhances Batf2 expression (Roy et.al) Batf2 is an important gene in the host’s immune response to TB and absence of the Batf2 gene should theoretically increase susceptibility to TB IFN-γ LPS Batf2 Mtb Confirm the involvement of Batf2 in either a protective or detrimental immune response to TB AIM METHOD Mice Wildtype (Batf2+/+), Heterozygous (Batf2+/-), Knockout (Batf2-/-) Infected Intranasally with clinical strain Mtb HN878 Sacrificed after 3 and 6 weeks of infection Lungs were sectioned and stained: H&E METHOD Lungs were examined using the Nikon NIS -elements Advanced Research Microscope for area of lesion size METHOD Pulmonary histopathology scoring (1 -10) 8 Statistical analysis: T-test Two-tailed with unequal variance 6 3 RESULTS BATF2 Batf2 +/+ (Normal) 3 Weeks 6 Weeks Batf2 +/- (Heterozygous) Batf2 -/- (Knockout) RESULTS BATF2 Batf2 +/+ (Normal) Batf2 +/- (Heterozygous) 3 Weeks 6 Weeks Batf2 -/- (Knockout) RESULTS BATF2 RESULTS BATF2 DISCUSSION BATF2 Batf2 deficient mice showed reduced pulmonary histopathology than wild-type mice Reasons: Reduced production of CD8+ T cells Reduced production of IFN-γ (Interferon gamma) Reduced production of Reactive Oxygen Species (ROS), Nitric Oxide (NO) and other inflammatory mediators Reduced production of TNF-α (Tumour Necrosis Factor – alpha) Reduced bacterial presentation Reduced induction of immune cells TB represents the fine balance between protection and immunopathology BLOCKING OF ALTERNATIVE ACTIVATION OF MACROPHAGES VIA IL-4Rα Part B ACTIVATION OF MACROPHAGES BLOCKING OF ALTERNATIVE ACTIVATION OF MACROPHAGES VIA IL-4Rα Classical Activation IFN-γ TNF-α LPS NO Microbicidal Cell Immunity Tissue Damage iNOS L-arginine Arginase L-ornithine Wound healing Tissue homeostasis Granuloma Formation IL-4 IL-13 IL-4Rα Alternative Activation Blocking the IL4Rα could increase available arginine for the production of Nitric Oxide (NO) and thus reduced susceptibility to TB Determine whether blocking the alternative activation of macrophages via the IL-4R-alpha would decrease susceptibility to TB by increasing the available L-arginine for the production of NO. AIM METHOD Mice Balb/c (Wildtype), IL-4Rα-/Infected with Mtb HN878 via intranasal and aerosol route Sacrificed after 4 and 18 weeks of infection Stain: iNOS - indicates classical activation of macrophages Arginase - indicates alternative activation of macrophages RESULTS IL-4Rα RESULTS IL-4Rα RESULTS IL-4Rα RESULTS IL-4Rα DISCUSSION IL-4Rα Induction of arginase in the absence of IL -4Rα indicates an IL4Rα-independent pathway for arginase production TB can induce production of arginase via the MyD88 pathway (Qualls et.al) IL-4Rα deficient mice showed reduced pulmonary histopathology in the late stage of TB infection Reasons: Reduced activation of wound repair and restoration of tissue homeostasis Immunopathology due to increased production of pro-inflammatory Th1 cytokines CONCLUSION A) Batf2 is a possible drug target as Batf2 deficient mice showed reduced pulmonary histopathology B) IL-4Rα is not a possible drug target due to TB’s ability to alternatively activate macrophages in an IL-4Rα-independent manner ACKNOWLEDGEMENTS I would like to acknowledge Dr Reto Guler, Ms Marilyn Tyler and Ms Lizette Fick for their immense assistance and support during this project. Without them, this would not have been possible. REFERENCES 1 . O ' G a r r a A , R e d f o r d P S , M c N a b F W, B l o o m C I , W i l k i n s o n R J , B e r r y M P. T h e i m m u n e r e s p o n s e i n t u b e r c u l o s i s . A n n u a l r e v i e w o f i m m u n o l o g y. 2 0 1 3 ; 31 : 47 5 - 5 27. 2 . K a r i n M , L i u Z , Z a n d i E . A P - 1 f u n c t i o n a n d r e g u l a t i o n . C u r r e n t o p i n i o n i n c e l l b i o l o g y. 1 9 97 ; 9 ( 2 ) : 24 0 - 6 . 3 . M u r p h y T L , Tu s s i w a n d R , M u r p h y K M . S p e c i f i c i t y t h r o u g h c o o p e r a t i o n : B AT F - I R F i n t e r a c t i o n s c o n t r o l i m m u n e - r e g u l a t o r y n e t w o r k s . N a t u r e r e v i e w s I m m u n o l o g y. 2 0 1 3 ; 1 3 ( 7 ) : 4 9 9 - 5 0 9 . 4. Roy S, Guler R, Parihar S, Schmeier S, Kaczkowski B, Nishimura H. Macrophage Batf2/Ir f1 regulates inflammator y responses with prolonged sur vival in tuberculosis in Batf2 deficient m i c e . U n p u b l i s h e d 2 01 4 . 5. Classen A, Lloberas J, Celada A. Macrophage activation: classical versus alternative. Methods i n m o l e c u l a r b i o l o g y. 2 0 0 9 ; 5 31 : 2 9 - 4 3 . 6. Gordon S, Mar tinez FO. Alternative activation of macrophages: mechanism and functions. I m m u n i t y. 2 0 1 0 ; 3 2 ( 5 ) : 5 9 3 - 6 0 4 . 7. Qualls J, Neale G, Amber S, Koo M -S, Ashley D, Huiyan Z, et al. Arginine usage in Mycobacteria infected macrophages depends on autocrine -paracrine cytokine signalling. Cell Signalling Te c h n o l o g y. 2 0 1 0 ; 3 ( 1 3 5 ) . IMAGE REFERENCES Slide 3: www.nature.com Slide 4: http://immunopaedia.org.za/index.php?id=250 Slide 6: www.nature.com Slide 18: Gordon S, Martinez FO. Alternative activation of macrophages: mechanism and functions. Nature Reviews Immunology. 2003;3: 26. Slide 19: Gordon S, Martinez FO. Alternative activation of macrophages: mechanism and functions. Nature Reviews Immunology. 2003;3: 32.
© Copyright 2020