Download Session 4 – Student Presentation 6, Amy Booth

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.