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Host pathogen interaction

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Page 1: Host pathogen interaction
Page 2: Host pathogen interaction

Introduction

• The host immune response against Mycobacteria

infection depends, to a great extent, on the activation of

infected cells.

• Mycobacteria-specific CD4 IFN gamma

• The thymus is a target for mycobacterial dissemination,

independent of the route of infection used

Page 3: Host pathogen interaction

Hypothesis

• What extent T cell differentiation is preserved

and whether the newly generated T cells, whose

differentiation occurred within infected thymi,

differ from those generated in noninfected

thymi.

Page 4: Host pathogen interaction

Methods

Animal Model:

• C57BL/6 (WT), Nude (B6.Cg-foxn1) and TCR alfa KO

• Famele

• 8-10 week-old

• 106 CFU M. Avium (2447)

Page 5: Host pathogen interaction

Methods

• Thymic transplant

• TCR alfa KO mice removed (10-20min)

– DMEM and XL&KT

• Immunization

– OVA 3T at 1wk intervals (10µg s.c)

• Sacrificed 1wk after lmm

Page 6: Host pathogen interaction

MethodsMeasumment

Histopathology H&EGeneral aspect of organs

Immunofluorescence Image analysis

Slides were visualized using: Abs (anti-rabbit Mycobacterium spp, K5, K8, anti CD11c or anti F4/80Secondary Abs: anti-rat IgG Alexa Flour 594 or Alexa Flour 488, anti-rabbit IgG Alexa Flour 488 and Biotinylated anti hanster IgG + PBCS.No synthase (iNOS)

RT-PCRQuantification of TCR rearrangement excision circles

TREC Quantitaive RT-PCR using TCRA constant gene

ELISPOT The stimuli used Ag85A241-260

IFN-gamawas quantified by ELISA

Flow cytometry cells were labeled CD3, CD4 and CD8

cells were labeled with Abs specific for CD3 (145-2C11), CD4 (RM4-5), and CD8 (53-6.7,)

Statistical analysis The two-tailed Student t test.(P < 0.05)

Differences among the means of experimental groups were analyzed with the two-tailed Student t test

Page 7: Host pathogen interaction

Results

Infected cells within the thymus are CD11c+

• Using Abs specific forK8 (subset of K8+ cell within the medulla) and K5 (subset

of K5+K8+ cell cortico-medullary region scarttered in the cortex )

• As infection progressed, bacteria were typically present within clumps of large

cells, mostly at the cortico-medullary region and within the medulla.

• All infected cell stain for CD11c consisted two populations macrophages

(CD11c+F4/80+) and DC (F4/80-)

Page 8: Host pathogen interaction

Results

Representative thymic sections from M. avium-infected WT mice (20–24 wk

postinfection) stained with Abs specific for CD11c ( B–E),

Page 9: Host pathogen interaction

Results

CD4- CD8- CD3-

CD4+ CD8-+CD3low/-

CD4+ CD8- CD3+

CD4- CD8+ CD3+

Only minor difference detected, in infected or Non-infected after 22wk in 4 thymocyte populations

The mycobacterial infection did not result in altered total

thymic cell number up to 22 wk postinfection.

No differences were detected in the amount of TRECs up to 30 wk postinfection

These results suggest that thymocyte differentiation is maintained throughout chronic mycobacterial infection.

Page 10: Host pathogen interaction

Result

What extent the newly generated T cells,

whose differentiation occurred within infected

thymi, were able to mount a protective

immune response against mycobacterial

infections?

Page 11: Host pathogen interaction

ResultsNude mice have no thymus but have competent T cell precursors, and TCR a mice have a thymus, but their T cell precursors are unable to fully differentiate.

Page 12: Host pathogen interaction

Results

T cells that differentiate within infected thymi have an impaired ability to protect against the same pathogen in peripheral organs.T cells that differentiate within infected thymi have an impaired ability to protect against the same pathogen in peripheral organs.

T cells arising from infected or noninfected thymi were equally able to colonize the periphery of nude recipient mice.

Mice transplanted with noninfected thymi presented almost ten times fewer viable bacteria in the liver and spleen than those transplanted with infected thymi

Page 13: Host pathogen interaction

Resultssplenocytes were stimulated in vitro

The prodution of IFN is smiliar in WT an Non infected

Page 14: Host pathogen interaction

Result

T cells that differentiate in an infected thymus have a reduced ability to produce IFN- g in response to M. avium Ags

The number of IFN gamma producing

splenocytes, when stimulated with Ag85 or

with OVA , was assessed by ELISPOT. Each bar

represents the mean 6 SD of the number o f

IFN-gamma producing cells from four to five

mice per group. pp # 0. 05.

Page 15: Host pathogen interaction

Results

Each bar represente the mean SD

from five to nine mice per group

infected thymis is have minor ability to expressed iNOS

Page 16: Host pathogen interaction

Discussion

• Thymi infected with M. avium retain the ability to generate new T

cells. However, T cells generated within infected thymi are unable

to mount a protective response against M. avium in the periphery.

• The infected and noninfected thymi (or of isolated T cells recovered

from transplanted recipients) have a similarities in the ability to

repopulate the periphery, As well a their indistinguishable ability to

produce IFN-g in response to Con A.

Page 17: Host pathogen interaction

Discussion

Page 18: Host pathogen interaction

Discussion

• T cells that differentiate within infected thymi mount an immune

response to OVA that is similar to that of T cells that differentiate in

noninfected thymi.

• The T cell tolerance observed against M. avium Ags in animals

transplanted with infected thymi seems specific to this pathogen.

– Inability to control bacterial growth

– Reduced aptitude to produce IFN-gamma upon stimulation (mycobacterial

Ags)

T cells are able to mount T cell-specific immune responses

Page 19: Host pathogen interaction

Discussion

• Mycobacteria were detected within macrophages and DCs in

the medulla and at the cortico-medullary region .

Page 20: Host pathogen interaction

Conclusions

During infection with M. avium, infected cells within the thymus induce

tolerance specifically to mycobacterial Ags .

During infection with M. avium, infected cells within the thymus induce

tolerance specifically to mycobacterial Ags .

This is the first report showing induced central tolerance to an infecting pathogen

Knowing that the thymus is also a target organ for

mycobacterial infections in humans, it is now important to

evaluate to what extent the newly generated T cells from

patients infected with M. avium or M. tuberculosis display an

impaired ability to respond to mycobacterial Ags.

Page 21: Host pathogen interaction

Thank you For your attention!