T Cells Aggravate
Intestinal Inflammation
      and Fibrosis
                    Eric Ma
    Finlay Lab, Michael Smith Labo...
Introduction: Model
1

2

3

4

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

3

4

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

3

4

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

3

4           ...
Introduction: Model
1

2

3   Salmonella infection of mice is a good model for
    studying the progression inflammation an...
Introduction: T Cells
1

2

3

4

5
Introduction: T Cells
1
            Th2 Antibody-mediated
                immunity
2

3

4

5
Introduction: T Cells
1
            Th2 Antibody-mediated
                immunity
2

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

3           Th1 Cell-mediated
      ...
Mouse Genotypes
1
    Mouse Genotype    Phenotypic Features
2
      C57/BL6 (B6)          Wild-Type
3
                    ...
The Big Q
1

2

3   What is the role of adaptive T cells in intestinal
    inflammation and fibrosis?
4

5
Experimental Design
1

2

3

4

5
Experimental Design
1

2

3

4

5
Experimental Design
1

2         Streptomycin pre-
              treatment
               (Day -1)
3

4

5
Experimental Design
1

2         Streptomycin pre-
              treatment
               (Day -1)
3

4

5
               ...
Experimental Design
1

2                        Streptomycin pre-
                             treatment
                 ...
Histology
1

2   • Gross structure
3   • Certain specific details
    • Stainings:
4
     • Hematoxylin & Eosin
5    • Mass...
Histology: H&E
1             B6   Rag1-/-       TCRa-/-
    Control


2                                        L


3      ...
Histology: H&E
1             B6          Rag1-/-          TCRa-/-
    Control


2                                         ...
Histology: MT
1             B6   Rag1-/-   TCRα-/-
    Control


2

3
    Wk 5




4

5
Histology: MT
1                    B6           Rag1-/-        TCRα-/-
    Control


2

3
    Wk 5




4

5
              ...
Gene Regulation
1

2
    • qPCR: Quantitative Real-Time PCR
3   • Measure of relative production of cytokines
      compar...
5
            4
                        3
                                  2
                                      1
 C  ...
5
    4
          3
                   2
                          1
         C
           tr
        W l
         k
     ...
Pro-fibrotic Cytokines
1          IGF-1               MCP-1


2                                              Rag1-/-
      ...
Pro-fibrotic Cytokines
1          IGF-1               MCP-1


2                                              Rag1-/-
      ...
Immunofluorescence
1

2

3   • Identification of cell types present
    • Co-localization of different cells/
4     proteins...
Immunofluorescence
1        B6, Week 2               Rag1-/-, Week   2

2

3

4

5                  MPO        Salmonella
 ...
Immunofluorescence
1   B6, Week 5              Rag1-/-, Week   5

2

3

4

5     Smooth Muscle Actin       Vimentin
Immunofluorescence
1    B6, Week 5               Rag1-/-, Week   5

2

3

4

5       Smooth Muscle Actin       Vimentin
   ...
Conclusions
1
    • T cell deficient mice exhibit attenuated
      inflammation and fibrosis
2
    • Differential cytokine ex...
Conclusions
1
    • T cell deficient mice exhibit attenuated
       inflammation and fibrosis
2
    • Differential cytokine e...
Outlook
1

2
    • Identify other cell types involved in fibrosis
3   • Flow cytometry analysis to quantify cell types
4   ...
Acknowledgments
1

2   • Finlay Lab (Michael Smith Labs)
     • Guntram A. Grassl
3
     • Yanet Valdez
4    • B. Brett Fi...
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T Cells Aggravate Intestinal Inflammation and Fibrosis

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  • Induction of cecal inflammation and fibrosis with many parameters similar to Crohn’s Disease patients
  • Induction of cecal inflammation and fibrosis with many parameters similar to Crohn’s Disease patients
  • Induction of cecal inflammation and fibrosis with many parameters similar to Crohn’s Disease patients
  • Induction of cecal inflammation and fibrosis with many parameters similar to Crohn’s Disease patients
  • Induction of cecal inflammation and fibrosis with many parameters similar to Crohn’s Disease patients
  • Induction of cecal inflammation and fibrosis with many parameters similar to Crohn’s Disease patients
  • Change Mickey Mouse to another mouse.
  • Change Mickey Mouse to another mouse.
  • Change Mickey Mouse to another mouse.
  • Change Mickey Mouse to another mouse.
  • Change Mickey Mouse to another mouse.
  • Change Mickey Mouse to another mouse.
  • Change Mickey Mouse to another mouse.
  • Change Mickey Mouse to another mouse.
  • Change Mickey Mouse to another mouse.
  • Change Mickey Mouse to another mouse.
  • Change Mickey Mouse to another mouse.
  • Change Mickey Mouse to another mouse.
  • Change Mickey Mouse to another mouse.
  • Change Mickey Mouse to another mouse.
  • Stress point: same magnification: 50X
    Features: Thickening of the submucosa (S) and mucosa (M), constriction of the lumen (L)
  • Stress point: same magnification: 50X
    Features: deposition of collagen (blue)
  • @NOTE: Introduce the overview of the diagrams first to give a sense of what the data is showing.
    @NOTE: If anybody asks about the other mouse genotype, indicate that it is the same.
  • May put in TCR vs B6 graphs instead of Rag vs B6 graphs.
  • May put in TCR vs B6 graphs instead of Rag vs B6 graphs.
  • Goal: find out where salmonella and neutrophils co-localize.
  • T Cells Aggravate Intestinal Inflammation and Fibrosis

    1. 1. T Cells Aggravate Intestinal Inflammation and Fibrosis Eric Ma Finlay Lab, Michael Smith Laboratories Department of Microbiology and Immunology
    2. 2. Introduction: Model 1 2 3 4 5
    3. 3. Introduction: Model 1 • Salmonella enterica serovar Typhimurium • Gram negative 2 • Enteric 3 4 5
    4. 4. Introduction: Model 1 • Salmonella enterica serovar Typhimurium • Gram negative 2 • Enteric 3 4 5
    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. 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. 7. Introduction: T Cells 1 2 3 4 5
    8. 8. Introduction: T Cells 1 Th2 Antibody-mediated immunity 2 3 4 5
    9. 9. Introduction: T Cells 1 Th2 Antibody-mediated immunity 2 3 Th1 Cell-mediated immunity 4 5
    10. 10. Introduction: T Cells 1 Th2 Antibody-mediated immunity 2 3 Th1 Cell-mediated immunity 4 Th17 Mucosal immunity, 5 neutrophil recruitment
    11. 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. 12. The Big Q 1 2 3 What is the role of adaptive T cells in intestinal inflammation and fibrosis? 4 5
    13. 13. Experimental Design 1 2 3 4 5
    14. 14. Experimental Design 1 2 3 4 5
    15. 15. Experimental Design 1 2 Streptomycin pre- treatment (Day -1) 3 4 5
    16. 16. Experimental Design 1 2 Streptomycin pre- treatment (Day -1) 3 4 5 Oral infection (Day 0)
    17. 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. 18. Histology 1 2 • Gross structure 3 • Certain specific details • Stainings: 4 • Hematoxylin & Eosin 5 • Massons Trichrome
    19. 19. Histology: H&E 1 B6 Rag1-/- TCRa-/- Control 2 L 3 S M S Wk 5 4 M L 5
    20. 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. 21. Histology: MT 1 B6 Rag1-/- TCRα-/- Control 2 3 Wk 5 4 5
    22. 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. 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. 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. 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. 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. 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. 28. Immunofluorescence 1 2 3 • Identification of cell types present • Co-localization of different cells/ 4 proteins 5
    29. 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. 30. Immunofluorescence 1 B6, Week 5 Rag1-/-, Week 5 2 3 4 5 Smooth Muscle Actin Vimentin
    31. 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. 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. 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. 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. 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

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