GENES AS POTENTIAL DRUG TARGETS Sciences AGAINST TUBERCULOSIS USING - - PowerPoint PPT Presentation

Faculty of Health Sciences

University of Cape Town

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

HISTORY OF TUBERCULOSIS

IDENTIFICATION OF HOST CANDIDATE GENES AS POTENTIAL DRUG TARGETS AGAINST TUBERCULOSIS USING PULMONARY HISTOPATHOLOGY

A) Basic Leucine Zipper Transcription Factor, ATF-like 2 (Batf2) CONTENT B) Blocking of the alternative activation

• f macrophages via

the IL-4 Receptor alpha chain (IL-4Rα)

IL-4Rα

IMMUNE RESPONSE TO TUBERCULOSIS (1)

NO

BASIC LEUCINE ZIPPER TRANSCRIPTION FACTOR, ATF-LIKE 2 (BATF2)

Part A

 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

BASIC LEUCINE ZIPPER TRANSCRIPTION FACTOR, ATF-LIKE 2 (BATF2)

Transcr script iption ion Factor: Protein that binds to

• ur DNA and

activates genes to bring about certain functions

 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

BASIC LEUCINE ZIPPER TRANSCRIPTION FACTOR, ATF-LIKE 2 (BATF2)

IFN-γ LPS Mtb

Batf2

Confirm the involvement

• f 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

 Lungs were examined using the Nikon NIS-elements Advanced Research Microscope for area of lesion size

METHOD

 Pulmonary histopathology scoring (1-10)  Statistical analysis:

• T-test
• Two-tailed with unequal variance

METHOD

8 3 6

Batf2 -/- (Knockout) Batf2 +/- (Heterozygous) Batf2 +/+ (Normal)

6 Weeks 3 Weeks

RESULTS

BATF2

RESULTS

BATF2

6 Weeks 3 Weeks

Batf2 -/- (Knockout) Batf2 +/- (Heterozygous)

Batf2 +/+ (Normal)

RESULTS

BATF2

RESULTS

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

DISCUSSION

BATF2

BLOCKING OF ALTERNATIVE ACTIVATION OF MACROPHAGES VIA IL-4Rα

Part B

ACTIVATION OF MACROPHAGES

BLOCKING OF ALTERNATIVE ACTIVATION OF MACROPHAGES VIA IL-4Rα

Blocki king g the e IL4Rα could ld inc ncrea rease se availa ilabl ble e arginin inine e for the e production roduction of Nitric tric Ox Oxide ide (NO) O) and nd thus s redu duce ced d susceptib ceptibil ility ity to TB TB

L-arginine

iNOS Arginase NO L-ornithine

Microbicidal Cell Immunity Tissue Damage Wound healing Tissue homeostasis Granuloma Formation

IFN-γ TNF-α LPS IL-4 IL-13 Alternative Activation Classical Activation IL-4Rα

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

• f NO.

AIM

METHOD

Mice

Balb/c (Wildtype), IL-4Rα-/-

• Infected with Mtb HN878 via intranasal and

aerosol route

• Sacrificed after 4 and 18 weeks
• f 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

 Induction of arginase in the absence of IL-4Rα indicates an IL- 4Rα-independent pathway for arginase production  TB can induce production of arginase via the MyD88 pathway (Qualls et.al) IL IL-4R 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

DISCUSSION

IL-4Rα

A) Batf2 is a possible drug target as Batf2 deficient mice showed reduced pulmonary histopathology B) IL IL-4Rα is not a possible drug target due to TB’s ability to alternatively activate macrophages in an IL-4Rα-independent manner

CONCLUSION

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.

ACKNOWLEDGEMENTS

• 1. O'Garra A, Redford PS, McNab FW, Bloom CI, Wilkinson RJ, Berry MP. The immune response in
• tuberculosis. Annual review of immunology. 2013;31:475 -527.
• 2. Karin M, Liu Z, Zandi E. AP-1 function and regulation. Current opinion in cell biology.

1997;9(2):240-6.

• 3. Murphy TL, Tussiwand R, Murphy KM. Specificity through cooperation: BATF -IRF interactions

control immune-regulatory networks. Nature reviews Immunology. 2013;13(7):499 -509.

• 4. Roy S, Guler R, Parihar S, Schmeier S, Kaczkowski B, Nishimura H. Macrophage Batf2/Irf1

regulates inflammatory responses with prolonged survival in tuberculosis in Batf2 deficient

• mice. Unpublished2014.
• 5. Classen A, Lloberas J, Celada A. Macrophage activation: classical versus alternative. Methods

in molecular biology. 2009;531:29 -43.

• 6. Gordon S, Martinez FO. Alternative activation of macrophages: mechanism and functions.
• Immunity. 2010;32(5):593-604.
• 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

• Technology. 2010;3(135).

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.

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