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

Macrophage

Phagocytosis

• Production of RNS and ROS

• Attraction of other immune cells

• Granuloma formation VAN Crevel et al., 2002

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Mammalian cellular immune response to Mycobacteria

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

iNOS bactericidal mechanism

IFN-γ

o activation of macrophage o acidification of phagosome o promotes antigen presentation process o stimulation of granuloma formation

Role of cytokines in mycobacterial infection

TNF-α

o phagocyte activation o induction of apoptosis o formation and maintenance of granuloma

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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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Nitric oxide assay

qRT-PCR

Methods

HD11 (Avian Macrophage-like cells)

ELISA

qRT-PCR

PMA

THP-1 (Human monocytic cells)

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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.

• Level does not correlate with source of strains

• Similar pattern was observed in other cytokines

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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

LM HSP65 50S RP

NFκB

Cytokine and chemokine

release

Triacylated LP

Dectin-1

DC-SIGN

Myc

ob

act

eria

IRAK

TRAF6

MAPKs

MyD88

Phagosome

Phagosome

NFκB IκBa IκBa

Diacylated LP

TLR

6/2

Mycobacteria MDP

NOD2

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Is there a difference between strains and how they signal responses?

MAP kinases importance

• Cell Proliferation

• Apoptosis

• Cytokine biosynthesis

• Cytoskeletal reorganization

PD98059 ERK 1/2 pathway inhibitor U0126 a significantly higher affinity for MEK1 SB203580 P38 pathway inhibitor SP600125 JNK 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 ATF1 p50 p65

ATF2

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T N F -p

g/m

l

Ma.1

Ma.2

Ma.3

Ma.4

Ma.5

Ma.6

Ma.7

Ma.8

LP

S

Un

treate

d

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

C h ick en iso la te . C attle iso la te . H u m an iso la te .

¥ ¥ ¥

+

¥

¥

¥¥

¥

N o n -in h ib ite d

P D 9 8 0 5 9

Inhibition of ERK1/2 by PD98059

• Signal transduction might be dependent upon mycobacterial species.

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IL -6

pg

/ml

Ma.1

Ma.2

Ma.3

Ma.4

Ma.5

Ma.6

Ma.7

Ma.8

LP

S

Un

treate

d

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

C h ick en iso la te . C attle iso la te . H u m an iso la te .

¥¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥

Inhibition of ERK1/2 by U0126

• Signal transduction might be dependent upon cytokine being studied.

C X C L -8

pg

/ml

Ma.1

Ma.2

Ma.3

Ma.4

Ma.5

Ma.6

Ma.7

Ma.8

LP

S

Un

treate

d

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

C h ick en iso la te . C attle iso la te . H u m an iso la te .

**

¥¥

¥+

++

*

N o n -in h ib ite d

U 0 1 26

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IL -1

pg

/ml

Ma.1

Ma.2

Ma.3

Ma.4

Ma.5

Ma.6

Ma.7

Ma.8

LP

S

Un

treate

d

0

5 0

1 0 0

1 5 0

C h ick en iso la te . C attle iso la te . H u m an iso la te .

+

+

+

*

¥

¥

N o n -in h ib ite d

P D 9 8 0 5 9

Inhibition of ERK1/2 by PD98059

• Signal transduction might be dependent upon cell type being studied.

IL -1

Fo

ld c

ha

ng

e

Ma.1

Ma.2

Ma.3

Ma.4

Ma.5

Ma.6

Ma.7

Ma.8

LP

S

Un

treate

d

0

5

1 0

1 5

2 0

5 0

1 0 0

1 5 0

C h ick en iso la te . C attle iso la te . H um an iso la te .

¥¥ ¥ ¥ ¥ ¥ ¥ ¥

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IL -1

pg

/ml

Ma.1

Ma.2

Ma.3

Ma.4

Ma.5

Ma.6

Ma.7

Ma.8

LP

S

Un

treate

d

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

C h ick en iso la te . C attle iso la te . H u m an iso la te .

S P 6 0 0 1 2 5

N o n -in h ib ite d

+¥

Inhibition of p38 by SB203580 and JNK by SP500125

• A cytokine could be differentially regulated by various pathways.

IL -1

pg

/ml

Ma.1

Ma.2

Ma.3

Ma.4

Ma.5

Ma.6

Ma.7

Ma.8

LP

S

Un

treate

d

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

C h ick en iso la te . C attle iso la te . H u m an iso la te .

+

+

¥

S B 2 0 3 5 8 0

N o n -in h ib ite d

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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

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21 days post infection (Liver)

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Ma3-infected Non-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.

Lymphocyte infiltration observed in all infected groups

Histopathological lesions

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• 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

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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

School of Veterinary Medicine and Science

Thank you!

Any questions?