school of veterinary medicine and science immune responses to mycobacterium avium infection and the...
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School of Veterinary Medicine and Science
Immune Responses to Mycobacterium avium infection and the role of MAPK
pathway in regulation of signal transduction during the infection
Mohammed Shukri Shukur
School of Veterinary Medicine and Science
Diseases caused by Mycobacteria
Mycobacterium sp. Host Disease
M. tuberculosis Human Tuberculosis
M. bovis HumanBovine
Tuberculosis
M. avium complex BirdsImmune compromised human, Bovine
Avian TuberculosisDisseminated Tuberculosis in immune compromised
School of Veterinary Medicine and Science
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
School of Veterinary Medicine and Science
Early interactions with Macrophages
Macrophage
Phagocytosis
• Production of RNS and ROS
• Attraction of other immune cells
• Granuloma formationVAN Crevel et al., 2002
School of Veterinary Medicine and Science
Mammalian cellular immune response to Mycobacteria
IL-1β o regulation of iNOS production, o maturation and acidification of phagosomeo expression of adhesion molecules
CXCl-8 o attraction and activation of many
other immune cells
iNOS bactericidal mechanism
IFN-γ o activation of macrophageo acidification of phagosomeo promotes antigen presentation processo stimulation of granuloma formation
Role of cytokines in mycobacterial infection
TNF-αo phagocyte activation o induction of apoptosiso formation and maintenance of granuloma
School of Veterinary Medicine and Science
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.
School of Veterinary Medicine and Science
Nitric oxide assay
qRT-PCR
Methods
HD11 (Avian Macrophage-like cells)
ELISA
qRT-PCR
PMA
THP-1 (Human monocytic cells)
School of Veterinary Medicine and Science
Bacteria used
M. avium subspecies avium
Abb. Strain Source
Ma.1 NC.08562-02 Chicken
Ma.2 NC.0855-07 Chicken
Ma.3 61.3623.05 Calf
Ma.4 61.4627.08 Calf
Ma.5 61.3728.08 Calf
Ma.6 430/330 Human
Ma.7 446/301 Human
Ma.8 460/132 Human
School of Veterinary Medicine and Science
Examples of differential induction of immune response.
•Level does not correlate with source of strains
•Similar pattern was observed in other cytokines
School of Veterinary Medicine and Science
Examples of differential induction of immune response.
•Level does not correlate with source of isolates
School of Veterinary Medicine and Science
Regulation of signaling transduction
LMHSP65 50S RP
NFκB
Cytokine and chemokine
release
19KDa LP,
LM,
LAM, PIMTriacylated LP
CRSR
MR
Dectin-1Dectin-1
DC-SIGNDC-SIGN
Mycob
acteria
IRAK
TRAF6
MAPKs
MyD88
TLR9
Phagosome
Phagosome
Mycoba
cteria
DNA
TLRs
NFκBIκBaIκBa
Mycobacteria
Diacylated LP
TLR2
TLR4TL
R1/2
TLR6
/2
MycobacteriaMDP
NOD2
Phag
ocyt
osis
School of Veterinary Medicine and Science
Is there a difference between strains and how they signal responses?
MAP kinases importance•Cell Proliferation•Apoptosis•Cytokine biosynthesis•Cytoskeletal reorganization
PD98059ERK 1/2 pathway inhibitor
U0126 a significantly higher affinity for MEK1
SB203580P38 pathway inhibitor
SP600125JNK pathway inhibitor
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 membrane
KAC
AP1 SRE CRE NFkB
c-JUN ELK1 SRF KREB ATF1p50 p65
ATF2
School of Veterinary Medicine and Science
TNF-pg/ml
Ma.1Ma.2Ma.3Ma.4Ma.5Ma.6Ma.7Ma.8LPS
Untreated
0
200
400
600
800
1000
Chicken isolate. Cattle isolate. Human isolate.
¥ ¥ ¥
+
¥¥
¥ ¥
¥
Non-inhibitedPD98059
Inhibition of ERK1/2 by PD98059
•Signal transduction might be dependent upon mycobacterial species.
School of Veterinary Medicine and Science
IL-6
pg/ml
Ma.1Ma.2Ma.3Ma.4Ma.5Ma.6Ma.7Ma.8LPS
Untreated
0
50
100
150
200
250
Chicken isolate. Cattle isolate. Human isolate.
¥¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥
Inhibition of ERK1/2 by U0126
•Signal transduction might be dependent upon cytokine being studied.
CXCL-8
pg/ml
Ma.1Ma.2Ma.3Ma.4Ma.5Ma.6Ma.7Ma.8LPS
Untreated
0
1000
2000
3000
4000
Chicken isolate. Cattle isolate. Human isolate.
**
¥¥
¥+
++
*
Non-inhibitedU0126
School of Veterinary Medicine and Science
IL-1
pg/ml
Ma.1Ma.2Ma.3Ma.4Ma.5Ma.6Ma.7Ma.8LPS
Untreated
0
50
100
150
Chicken isolate. Cattle isolate. Human isolate.
+
+
+
*
¥
¥
Non-inhibitedPD98059
Inhibition of ERK1/2 by PD98059
•Signal transduction might be dependent upon cell type being studied.
IL-1
Fol
d c
han
ge
Ma.1Ma.2Ma.3Ma.4Ma.5Ma.6Ma.7Ma.8LPS
Untreated
0
5
10
15
2050
100
150
Chicken isolate. Cattle isolate. Human isolate.
¥¥ ¥ ¥ ¥ ¥ ¥ ¥
School of Veterinary Medicine and Science
IL-1
pg/ml
Ma.1Ma.2Ma.3Ma.4Ma.5Ma.6Ma.7Ma.8LPS
Untreated
0
100
200
300
400
500
Chicken isolate. Cattle isolate. Human isolate.
SP600125Non-inhibited
+¥
Inhibition of p38 by SB203580 and JNK by SP500125
•A cytokine could be differentially regulated by various pathways.
IL-1
pg/ml
Ma.1Ma.2Ma.3Ma.4Ma.5Ma.6Ma.7Ma.8LPS
Untreated
0
100
200
300
400
500
Chicken isolate. Cattle isolate. Human isolate.
+
+
¥
SB203580Non-inhibited
School of Veterinary Medicine and Science
In vivo experiment
Forty eight three week-old commercial chicks
Group3 inoculated with
Ma.5 (High)
Group2 inoculated with
Ma.4 (High)
Group1 inoculated with
Ma.3 (Low)
(i.v. inoculation)
Birds were allocated into 4 groups, twelve birds each in separate rooms
Three birds from each group euthanized at the day of infection and at 7, 14 and 21 days post infection.
Group4 Control
Blood
Liver
Spleen
CFU
CFU,
CFU,
qPCR,
qPCR,
Histpathology
Histpathology
School of Veterinary Medicine and Science
21 days post infection (Liver)
School of Veterinary Medicine and Science
Ma3-infectedNon-infected
Representative histopathological micrographs from the liver ofchickens 21 days after infection with different M. avium isolates. H&E staining, magnification: X20, scale bar: 50µm.
Lymphocyte infiltration observed in all infected groups
Histopathological lesions
School of Veterinary Medicine and Science
• 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.
Summary and Conclusion
School of Veterinary Medicine and Science
Acknowledgements
Prof. Paul BarrowDr. Mike Jones
Nawzat IssaDr. Sabine TötemeyerDr. Paula WilliamsDr. Belinda WangScott Hulme
Ministry of Higher Education in Kurdistan
School of Veterinary Medicine and Science
Thank you! Any questions?
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