school of veterinary medicine and science immune responses to mycobacterium avium infection and the...

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


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


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


Early interactions with Macrophages

Macrophage

Phagocytosis

• Production of RNS and ROS

• Attraction of other immune cells

• Granuloma formationVAN Crevel et al., 2002


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


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.


Nitric oxide assay

qRT-PCR

Methods

HD11 (Avian Macrophage-like cells)

ELISA

qRT-PCR

PMA

THP-1 (Human monocytic cells)


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


Examples of differential induction of immune response.

•Level does not correlate with source of strains

•Similar pattern was observed in other cytokines


Examples of differential induction of immune response.

•Level does not correlate with source of isolates


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


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


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.


IL-6

pg/ml


Untreated

0

50

100

150

200

250


¥¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥

Inhibition of ERK1/2 by U0126

•Signal transduction might be dependent upon cytokine being studied.

CXCL-8

pg/ml


Untreated

0

1000

2000

3000

4000


**

¥¥

¥+

++

*

Non-inhibitedU0126


IL-1

pg/ml


Untreated

0

50

100

150


+

+

+

*

¥

¥

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


Untreated

0

5

10

15

2050

100

150


¥¥ ¥ ¥ ¥ ¥ ¥ ¥


IL-1

pg/ml


Untreated

0

100

200

300

400

500


SP600125Non-inhibited

+¥

Inhibition of p38 by SB203580 and JNK by SP500125

•A cytokine could be differentially regulated by various pathways.

IL-1

pg/ml


Untreated

0

100

200

300

400

500


+

+

¥

SB203580Non-inhibited


In vivo experiment

Forty eight three week-old commercial chicks

Group3 inoculated with

Ma.5 (High)


Ma.4 (High)


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


21 days post infection (Liver)


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


• 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


Acknowledgements

Prof. Paul BarrowDr. Mike Jones

Nawzat IssaDr. Sabine TötemeyerDr. Paula WilliamsDr. Belinda WangScott Hulme

Ministry of Higher Education in Kurdistan


Thank you! Any questions?

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