1. Mohammed Shukri Shukur
Immune Responses to Mycobacterium avium infection and the role of MAPK pathway in regulation of signal transduction during the infection
Mohammed Shukri Shukur

2. Diseases caused by Mycobacteria
Mycobacterium sp.
Host
Disease
M. tuberculosis
Human
Tuberculosis
M. bovis
Bovine
M. avium complex
Birds
Immune compromised human, Bovine
Avian Tuberculosis
Disseminated Tuberculosis in immune compromised

3. Why M. avium? Avian mycobacteriosis Similar to mammalian pathology
Disseminated disease in HIV patients
Limited knowledge on the immune processes in poultry
There are differences between disease process between mammalian and avian infections
Progressive disease
Chickens are easier model for study mycobacterial infection

4. Early interactions with Macrophages
Phagocytosis
FIG. 2. Phagocytosis and immune recognition of M. tuberculosis. Various receptors have been identified for phagocyosis of M. tuberculosis
(MTB) by macrophages and dendritic cells: complement receptors are primarily responsible for uptake of opsonized M. tuberculosis; MRs and
scavenger receptors for uptake of nonopsonized M. tuberculosis. TLRs play a central role in immune recognition of M. tuberculosis. In the context
of CD14, TLR2 binds lipoarabinomannan, a heterodimer of TLR2 and TLR6 binds a 19-kDa M. tuberculosis lipoprotein, TLR4 binds to an
undefined heat-labile cell-associated factor, and (possibly) TLR9 binds to M. tuberculosis DNA. After binding to TLRs, common signalling
pathways lead to cell activation and cytokine production. TLRs are expressed not only at the cell surface but also in phagosomes; therefore,
immune activation may occur with or without phagocytosis. On the other hand, phagocytosis alone probably does not lead to immune activation
without the involvement of TLRs.
Production of RNS and ROS
Attraction of other immune cells
Granuloma formation
Macrophage
VAN Crevel et al., 2002

5. Role of cytokines in mycobacterial infection
TNF-α
phagocyte activation
induction of apoptosis
formation and maintenance of granuloma
iNOS bactericidal mechanism
IL-1β
regulation of iNOS production,
maturation and acidification of phagosome
expression of adhesion molecules
IFN-γ
activation of macrophage
acidification of phagosome
promotes antigen presentation process
stimulation of granuloma formation
CXCl-8
attraction and activation of many other immune cells
Mammalian cellular immune response to Mycobacteria

6. Aims of the project
To compare the immunological interactions of M. avium subspecies avium from different human and animal sources with human and avian infection model.
To study the role of various MAP kinases in cellular regulation by M. avium stimulation.
To determine the cytokine response to M. avium strains during infection of chickens.

7. Methods HD11 (Avian Macrophage-like cells)
Nitric oxide
assay
qRT-PCR
THP-1 (Human monocytic cells)
ELISA
PMA
qRT-PCR

8. Bacteria used M. avium subspecies avium Ma.1 NC.08562-02 Chicken Ma.2
Abb.
Strain
Source
Ma.1 NC
Chicken
Ma.2 NC
Ma.3
Calf
Ma.4
Ma.5
Ma.6
430/330
Human
Ma.7
446/301
Ma.8
460/132

9. Examples of differential induction of immune response.
Similar pattern was observed in other cytokines
Level does not correlate with source of strains

10. Examples of differential induction of immune response.
Level does not correlate with source of isolates

11. Cytokine and chemokine
Regulation of signaling transduction LM
HSP65
50S RP
NFκB
Cytokine and chemokine
release
19KDa LP, LM,
LAM, PIM
Triacylated LP CR SR MR
Dectin-1
DC-SIGN
Mycobacteria
IRAK
TRAF6
MAPKs
MyD88
TLR9
Phagosome
DNA
TLRs
IκBa
Diacylated LP
TLR2
TLR4
TLR1/2
TLR6/2
MDP
NOD2
Phagocytosis

12. Is there a difference between strains and how they signal responses?
TLR
CDC42/Rac1
PAK1
MEKK1/4
MKK7 SKK4
JNK
Ras
Raf1
MEK1,2
ERK1/2
PI3 Kinase
PKC
ASK1,TAK1
MKK3,4,6
p38
PC,PLC
Plasma
membrane
Nuclear
KAC
AP1
SRE
CRE
NFkB
c-JUN
ELK1
SRF
KREB
ATF1
p50
p65
ATF2
PD98059
ERK 1/2 pathway inhibitor
U0126
a significantly
higher affinity for MEK1
SB203580
P38 pathway inhibitor
SP600125
JNK pathway inhibitor
MAP kinases importance
Cell Proliferation
Apoptosis
Cytokine biosynthesis
Cytoskeletal reorganization

13. Inhibition of ERK1/2 by PD98059
Signal transduction might be dependent upon mycobacterial species.

14. Inhibition of ERK1/2 by U0126
Signal transduction might be dependent upon cytokine being studied.

15. Inhibition of ERK1/2 by PD98059
Signal transduction might be dependent upon cell type being studied.

16. Inhibition of p38 by SB203580 and JNK by SP500125
A cytokine could be differentially regulated by various pathways.

17. In vivo experiment Forty eight three week-old commercial chicks
Birds were allocated into 4 groups, twelve birds each in separate rooms
(i.v. inoculation)
Group1 inoculated with Ma.3 (Low)
Group2 inoculated with Ma.4 (High)
Group3 inoculated with Ma.5 (High)
Group4 Control
CFU
CFU,
Three birds from each group euthanized at the day of infection and at 7, 14 and 21 days post infection.
Blood
qPCR,
Histpathology
Liver
Spleen

18. 21 days post infection (Liver)

19. Histopathological lesions
Lymphocyte infiltration observed in all infected groups
Non-infected
Ma3-infected
Representative histopathological micrographs from the liver of chickens 21 days after infection with different M. avium isolates.
H&E staining, magnification: X20, scale bar: 50µm.

20. Summary and Conclusion
There are M. avium strains specific differences in induction of pro-inflammatory cytokine expression.
These differences do not appear to relate to source of strains
The differential response does no correlated with granuloma formation.
Multiple signal pathways participate in regulation of the signal transduction during infection.
There are several mechanisms of signal transduction depending upon mycobacterial species, type of cytokine and the type of cells being studied.
Our data support the concept of a general similarity between the immune response to mycobacterial infection in avian species, humans and mice.

21. Education in Kurdistan
Acknowledgements
Prof. Paul Barrow Dr. Mike Jones
Nawzat Issa Dr. Sabine Tötemeyer
Dr. Paula Williams
Dr. Belinda Wang
Scott Hulme
Ministry of Higher
Education in Kurdistan

22. Thank you! Any questions?