My winning poster for the Rising Stars of Research National Undergraduate Research Poster Competition 2008.

1. The Contribution of T Cells to Intestinal Inflammation and Fibrosis
Eric Ma (Asava-Aree) , Guntram A. Grassl , B. Brett Finlay 1 1 1
1
Michael Smith Laboratories, The University of British Columbia, Vancouver, British Columbia
ericasav@interchange.ubc.ca; guntram@msl.ubc.ca; bfinlay@interchange.ubc.ca Rag vs B6 Cytokine Graphs.pzf:TNF-a Timecourse graph - Wed Aug 13 14:33:08 2008
Rag vs B6 Cytokine Graphs.pzf:IL-6 Timecourse graph - Wed Aug 13 14:42:
Introduction (2) S. Typhimurium DaroA is found primarily in the lumen of (4) S. Typhimurium DaroA induces
•Instestinal fibrosis is a major complication in Crohn’s infected ceca proinflammatory and profibrotic
Disease (CD) patients.1 Uninfected Control Day 14
•Mechanisms that lead to intestinal fibrosis and stricture B6 Rag1-/- B6 Rag1-/-
cytokines
formation are still poorly understood. TNF-α IL-6
•The bacterium Salmonella enterica serovar
Rag vs B6 Cytokine Graphs.pzf:IL-17 Timecourse graph - Wed Aug 13 14:17:01 2008
12 100 Rag vs B6 Cytokine Graphs.pzf:IFN-g Timecourse graph - Wed Aug 13 13:
11
Typhimurium (S. Typhimurium) causes food poisoning 10
Induction
Induction
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and gastroenteritis in millions of people each year.
Fold
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Fold
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•Mice pre-treated with the antibiotic streptomycin prior 4
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to infection with S. Typhimurium experience heavy 2
1
0 0
colonization of the cecum and colon with significant
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colitis.2
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B6 Rag1-/- B6 Rag1-/-
•We recently showed that chronic infections with S. S. Typhimurium MPO
Typhimurium lead to severe intestinal fibrosis.3 IL-17 IFN-γ
Fig. 2: Salmonella (Salmonella LPS, red) and neutrophils (myeloperoxidase (MPO), green) were primarily Rag vs B6 Cytokine Graphs.pzf:MCP-1 Timecourse graph - Wed Aug 13 13:33:56 2008
•Here, we investigated the contribution of T cells to the found in the lumen of ceca at 14 days post-infection (p.i.). Uninfected controls shown for comparison.
70
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450
400
Rag vs B6 Cytokine Graphs.pzf:IGF-1 Timecourse graph - Wed Aug 13 14:2
Induction
development of intestinal fibrosis in mice caused by
Induction
DAPI staining of nuclei shown in blue. L = Lumen, M = Mucosa. 50
350
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chronic Salmonella-induced colitis, and found that T-cell
Fold
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deficient mice developed attenuated inflammation 30
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and fibrosis.
(3) Rag1 mice display attenuated inflammation and fibrosis
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-/- 10
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0 0
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Experimental Approach
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Masson’s Trichrome H&E B6 Rag1-/- B6 Rag1-/-
B6 Rag1-/- B6 Rag1-/-
MCP-1 IGF-1
45 5.5
40 5.0
Induction
Induction
35 4.5
30 4.0
Fold
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Fold
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50X
Harvest Organs
Uninfected Controls
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15 2.0
1.5
10
Day ‐1 Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 5
1.0
0.5
Oral Infection with 0 0.0
pretreatment S. Typhimurium Homogenize tissue / enumerate bacteria
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with
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ΔaroA
streptomycin
Histological stains: H&E, Masson's B6 Rag1-/- B6 Rag1-/-
Trichrome to evaluate inflammation/
Treatment Groups: intestinal fibrosis Fig. 4: Induction of proinflammatory (TNF-α, IL-6,
IL-17, IFN-γ) and profibrotic (MCP-1, IGF-1) cytokines in
200X
C57/Bl6 (B6) wild type
control mice Immunofluorescent staining to identify
and localize cell types infected ceca was determined by real-time PCR. Data
Rag1-/- mice that have
no T and B cells
were normalized to GAPDH levels and are relative to an
mRNA isolation to perform qPCR to arbitrarily selected uninfected control.
quantify cytokine expression
Results (5) B6 and Rag1 ceca have -/-
similar numbers of fibroblasts
50X
(1) S. Typhimurium DaroA B6 Rag1-/-
chronically colonizes the cecum
Day 35
of B6 and Rag1 mice
-/-
day 7 day 14 day 21 day 28 day 35
200X
Bacterial Load (cfu)
S. Typhimurium
10 9
10 8
10 7 Vimentin α-Smooth Muscle Actin
10 6 Fig. 3: Masson’s Trichrome (MT) and Hematoxylin & Eosin (H&E) staining of mice ceca from uninfected
10 5
controls and day 35 infected B6 and Rag1-/- mice. MT staining indicates position of collagen deposition Fig. 5: Distribution of mesenchymal cell types in ceca
10 4
(blue), while H&E staining reveals extent of inflammation and damage. L = Lumen, M = Mucosa, S = of B6 and Rag1-/- mice infected with S. Typhimurium 35
10 3
Submucosa. Note region of denser collagen deposition (black arrows) with greater edema in the submucosa days p.i., differentiated by staining for fibroblast-specific
10 2
10 1 of B6 ceca compared to Rag1-/- ceca. vimentin (V) and α-smooth muscle actin (A) shown at
10 0 400X magnification. Similar amounts of V+A- fibroblasts
(white arrow) were found in the submucosa and mucosa
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B6
B6
B6
B6
B6
-/
-/
-/
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-/
g1
g1
g1
g1
g1
of B6 and Rag1-/- ceca. S = Submucosa; M = Mucosa
Conclusions
Ra
Ra
Ra
Ra
Ra
Fig. 1: Bacterial counts of ceca and colons showing similar
•S. Typhimurium triggers severe fibrosis in the cecum.
References
colonization levels of B6 and Rag1-/- mice; cfu: colony
forming units.
•Salmonella-induced profibrotic and proinflammatory cytokines are 1. Burke J. P., Mulsow J., O’Keane C. et. al. 2007. Fibrogenesis in
maximally produced at week 3 post-infection. Crohn’s disease. Am. J. Gastroenterol. 102(2):439-48.
Acknowledgments •Fibroblasts and smooth muscle cells might be the predominant collagen-
2. Coburn B., Li Y., Owen D. et. al. 2005. Salmonella enterica serovar
Typhimurium pathogenicity island 2 is necessary for complete
virulence in a mouse model of infectious enterocolitis. Infect.
producing cells. Immun. 73(6):3219-27
3. Grassl G. A., Valdez Y., Bergstrom K. S. B. et. al. 2008. Chronic
•T cells enhance intestinal inflammation and fibrosis enteric Salmonella infection in mice leads to severe and persistent
intestinal fibrosis. Gastroenterology 134(3): 768-80.