Transcript 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
Human
Bovine
Tuberculosis
M. avium complex
Birds
Immune compromised
human, Bovine
Avian Tuberculosis
Disseminated
Tuberculosis in
immune compromised
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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
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Early interactions with Macrophages
Phagocytosis
• Production of RNS and ROS
• Attraction of other immune cells
• Granuloma formation
Macrophage
VAN Crevel et al., 2002
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Role of cytokines in mycobacterial infection
 TNF-α
o phagocyte activation
o induction of apoptosis
o formation and maintenance of granuloma
 IFN-γ
o activation of macrophage
o acidification of phagosome
o promotes antigen presentation process
o stimulation of granuloma formation
 iNOS bactericidal mechanism
 IL-1β
o regulation of iNOS production,
o maturation and acidification of phagosome
o expression of adhesion molecules
 CXCl-8
o attraction and activation of many
other immune cells
Mammalian cellular immune response to Mycobacteria
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.
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Methods
HD11 (Avian Macrophage-like cells)
Nitric oxide
assay
qRT-PCR
THP-1 (Human monocytic cells)
ELISA
PMA
qRT-PCR
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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
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Examples of differential
induction of immune response.
• Similar pattern was observed in other cytokines
• Level does not correlate with source of strains
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Examples of differential
induction of immune response.
• Level does not correlate with source of isolates
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Regulation of signaling transduction
TLR6/2
Diacylated LP Triacylated LP
LM
HSP65
50S RP
MyD88
IRAK
Mycobacteria
MDP
Phagosome
TRAF6
NOD2
MAPKs
Mycobacteria
Phagosome
IκBa
IκBa
NFκB
Dectin-1
DC-SIGN
Cytokine and
chemokine
release
NFκB
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Is there a difference between
strains and how they signal
responses?
Plasma
membrane
TLR
MAP kinases importance
CDC42/Rac1
PI3 Kinase
• Cell Proliferation
PC,PLC
Ras
• Apoptosis
PAK1
PKC
KAC
• Cytokine biosynthesis
• Cytoskeletal reorganization
Raf1
MEKK1/4
JNK
ERK1/2
U0126
a significantly
higher affinity for MEK1
ASK1,TAK1
SB203580
P38 pathway inhibitor
MKK3,4,6
SP600125
JNK pathway inhibitor
MEK1,2
MKK7 SKK4
PD98059
ERK 1/2 pathway inhibitor
p38
Nuclear
membrane
c-JUN
ATF2
AP1
ELK1 SRF
SRE
KREB
ATF1
CRE
p50
p65
NFkB
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Inhibition of ERK1/2 by PD98059
T N F -
1000
N o n -in h ib ite d
PD98059
p g /m l
800
600
400
¥
+
200
¥
¥
¥
¥
¥
¥
¥
H u m a n iso la te.
d
U
C h ic k e n is o la te . C a ttle iso la te .
n
tr
e
a
L
P
te
S
.8
M
a
.7
M
a
.6
M
a
.5
M
a
.4
M
a
.3
M
a
.2
a
M
M
a
.1
0
• Signal transduction might be dependent upon mycobacterial species.
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Inhibition of ERK1/2 by U0126
IL -6
250
4000
C X C L -8
N o n -in h ib ite d
U 0126
200
3000
*
2000
p g /m l
¥
¥
*
¥
1000
150
100
50
¥
¥
¥
¥
¥
¥
¥
¥
¥
d
e
a
L
P
te
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.4
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.3
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.2
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a
M
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a
d
tr
H u m a n iso la te.
n
C h ic k e n is o la te . C a ttle iso la te .
U
H u m a n iso la te.
U
C h ic k e n is o la te . C a ttle iso la te .
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a
.1
a
.1
0
0
M
p g /m l
+
*
+
+
• Signal transduction might be dependent upon cytokine being studied.
School of Veterinary Medicine and Science
Inhibition of ERK1/2 by PD98059
IL -1 
IL - 1 
150
150
N o n -in h ib ite d
PD98059
100
50
100
F o ld c h a n g e
p g /m l
+
+
50
*
¥
20
15
10
5
¥
¥
+
¥
¥
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¥
¥
¥
d
tr
e
a
L
P
te
S
.8
a
M
M
a
.7
.6
M
a
.5
M
a
.4
a
.3
M
M
a
.2
M
a
M
a
.1
d
H u m a n is o la te .
n
C h ic k e n is o la te . C a ttle is o la te .
U
H u m a n iso la te.
U
C h ic k e n is o la te . C a ttle iso la te .
n
tr
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a
L
P
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a
M
a
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a
.1
.2
0
M
¥
0
• Signal transduction might be dependent upon cell type being
studied.
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Inhibition of p38 by SB203580 and
JNK by SP500125
IL -1 
500
IL -1 
500
¥
+
400
400
N o n -in h ib ite d
+
N o n -in h ib ite d
SP600125
p g /m l
p g /m l
SB203580
300
200
100
300
200
100
¥
0
+
d
M
a
.1
M
a
.2
M
a
.3
M
a
.4
M
a
.5
M
a
.6
M
a
.7
M
a
.8
L
P
H u m a n iso la te.
U
C h ic k e n is o la te . C a ttle iso la te .
n
tr
e
a
L
P
te
S
.8
M
a
.7
M
a
.6
M
a
.5
M
a
.4
M
a
.3
M
a
.2
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M
M
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.1
0
C h ic k e n is o la te . C a ttle iso la te .
H u m a n iso la te.
U
S
n
tr
e
a
te
d
• A cytokine could be differentially regulated by various pathways.
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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)
Three birds from each
group euthanized at the
day of infection and at
7, 14 and 21 days post
infection.
Group3
inoculated with
Ma.5 (High)
Group4
Control
Blood
CFU
Liver
CFU, qPCR, Histpathology
Spleen
CFU, qPCR, Histpathology
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21 days post infection (Liver)
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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.
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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.
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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
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Thank you!
Any questions?
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