1. T Cells Aggravate
Intestinal Inflammation
and Fibrosis
Eric Ma
Finlay Lab, Michael Smith Laboratories
Department of Microbiology and Immunology

2. Introduction: Model
1
2
3
4
5

3. Introduction: Model
1 • Salmonella enterica serovar Typhimurium
• Gram negative
2 • Enteric
3
4
5

4. Introduction: Model
1 • Salmonella enterica serovar Typhimurium
• Gram negative
2 • Enteric
3
4
5

5. Introduction: Model
1 • Salmonella enterica serovar Typhimurium
• Gram negative
2 • Enteric
3
4 • Salmonella: a tool
• Cecal inflammation and fibrosis
5 • Measured parameters similar
to Cronh’s Disease patients

6. Introduction: Model
1
2
3 Salmonella infection of mice is a good model for
studying the progression inflammation and fibrosis
4 in Crohn’s Disease.
5

7. Introduction: T Cells
1
2
3
4
5

8. Introduction: T Cells
1
Th2 Antibody-mediated
immunity
2
3
4
5

9. Introduction: T Cells
1
Th2 Antibody-mediated
immunity
2
3 Th1 Cell-mediated
immunity
4
5

10. Introduction: T Cells
1
Th2 Antibody-mediated
immunity
2
3 Th1 Cell-mediated
immunity
4
Th17 Mucosal immunity,
5 neutrophil recruitment

11. Mouse Genotypes
1
Mouse Genotype Phenotypic Features
2
C57/BL6 (B6) Wild-Type
3
No T and B Cells (i.e. no
Rag1-/-
adaptive immune system)
4
No αβT Cells (i.e. no
TCRα-/-
5 adaptive T cells)

12. The Big Q
1
2
3 What is the role of adaptive T cells in intestinal
inflammation and fibrosis?
4
5

13. Experimental Design
1
2
3
4
5

14. Experimental Design
1
2
3
4
5

15. Experimental Design
1
2 Streptomycin pre-
treatment
(Day -1)
3
4
5

16. Experimental Design
1
2 Streptomycin pre-
treatment
(Day -1)
3
4
5
Oral
infection
(Day 0)

17. Experimental Design
1
2 Streptomycin pre-
treatment
(Day -1)
3
4
• Harvest cecum
5 • Bacterial load
Timecourse: Oral
quantification
0-5 weeks infection
• Histology
• qPCR (Day 0)

18. Histology
1
2 • Gross structure
3 • Certain specific details
• Stainings:
4
• Hematoxylin & Eosin
5 • Massons Trichrome

19. Histology: H&E
1 B6 Rag1-/- TCRa-/-
Control
2 L
3 S
M
S
Wk 5
4 M
L
5

20. Histology: H&E
1 B6 Rag1-/- TCRa-/-
Control
2 L
3 S
M
S
Wk 5
4 M
L
5
T cell-deficient mice exhibit attenuated inflammation

21. Histology: MT
1 B6 Rag1-/- TCRα-/-
Control
2
3
Wk 5
4
5

22. Histology: MT
1 B6 Rag1-/- TCRα-/-
Control
2
3
Wk 5
4
5
T cell-deficient mice exhibit decreased collagen
deposition and fibrosis

23. Gene Regulation
1
2
• qPCR: Quantitative Real-Time PCR
3 • Measure of relative production of cytokines
compared to wild-type uninfected controls.
4 • Pro-inflammatory cytokines
5 • Pro-fibrotic cytokines

24. 5
4
3
2
1
C C
tr tr
W l W l
k k
W 1 W 1
k k
W 3 W 3
k k
5 5
C C
tr
IL-6
tr W l
TNF-a
W l k
k W 1
W 1 k
k W 3
W 3 k
k 5
5
C C
tr tr
W l W l
k k
W 1 W 1
k k
W 3 W 3
k k
5 5
C C
tr tr
W l W l
k
IL-17
k
IFN-g
W 1 W 1
k k
W 3 W 3
k k
5 5
B6
Pro-inflammatory Cytokines
Rag1-/-

25. 5
4
3
2
1
C
tr
W l
k
W 1
k
W 3
k
5
C
tr
W l
IGF-1
k
W 1
k
W 3
k
5
C
tr
W l
k
W 1
k
W 3
k
5
C
tr
W l
k
MCP-1
W 1
k
W 3
k
5
B6
Pro-fibrotic Cytokines
Rag1-/-

26. Pro-fibrotic Cytokines
1 IGF-1 MCP-1
2 Rag1-/-
B6
3
W l
W l
W 1
W 3
5
W 1
W 3
5
tr
tr
W l
W l
W 1
W 3
5
W 1
W 3
5
tr
tr
k
k
k
k
k
k
C
C
k
k
k
k
k
k
C
C
4
Differences in pro-inflammatory and pro-fibrotic
cytokine expression may account for the differing
5 inflammatory and fibrotic phenotypes.

27. Pro-fibrotic Cytokines
1 IGF-1 MCP-1
2 Rag1-/-
B6
3
W l
W l
W 1
W 3
5
W 1
W 3
5
tr
tr
W l
W l
W 1
W 3
5
W 1
W 3
5
tr
tr
k
k
k
k
k
k
C
C
k
k
k
k
k
k
C
C
4
Differences in pro-inflammatory and pro-fibrotic
cytokine expression may account for the differing
5 inflammatory and fibrotic phenotypes.
T cells are responsible for the differences.

28. Immunofluorescence
1
2
3 • Identification of cell types present
• Co-localization of different cells/
4 proteins
5

29. Immunofluorescence
1 B6, Week 2 Rag1-/-, Week 2
2
3
4
5 MPO Salmonella
S. typhimurium co-localize with neutrophils in the
lumen of mice ceca.

30. Immunofluorescence
1 B6, Week 5 Rag1-/-, Week 5
2
3
4
5 Smooth Muscle Actin Vimentin

31. Immunofluorescence
1 B6, Week 5 Rag1-/-, Week 5
2
3
4
5 Smooth Muscle Actin Vimentin
T-cell deficient mice have less numbers of
collagen-depositing fibroblasts.

32. Conclusions
1
• T cell deficient mice exhibit attenuated
inflammation and fibrosis
2
• Differential cytokine expression may explain
3 phenotypic differences
4
• Collagen-depositing fibroblasts are present in
higher numbers in wild-type mice.
5

33. Conclusions
1
• T cell deficient mice exhibit attenuated
inflammation and fibrosis
2
• Differential cytokine expression may explain
3 phenotypic differences
4
• Collagen-depositing fibroblasts are present in
higher numbers in wild-type mice.
5 T cells aggravate intestinal inflammation and fibrosis

34. Outlook
1
2
• Identify other cell types involved in fibrosis
3 • Flow cytometry analysis to quantify cell types
4 • Identify interaction partners
• Elucidate fibrosis mechanism
5

35. Acknowledgments
1
2 • Finlay Lab (Michael Smith Labs)
• Guntram A. Grassl
3
• Yanet Valdez
4 • B. Brett Finlay
5
• Littman Lab (Skirball Inst., New York)
• Gretchen Diehl