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My winning poster for the Rising Stars of Research National Undergraduate Research Poster Competition 2008.
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The Contribution of T Cells to Intestinal Inflammation and FibrosisEric Ma (Asava-Aree)1, Guntram A. Grassl1, B. Brett Finlay1
1Michael Smith Laboratories, The University of British Columbia, Vancouver, British [email protected]; [email protected]; [email protected]
Introduction•Instestinal fibrosis is a major complication in Crohn’s Disease (CD) patients.1
•Mechanisms that lead to intestinal fibrosis and stricture formation are still poorly understood.
•The bacterium Salmonella enterica serovar Typhimurium (S. Typhimurium) causes food poisoning and gastroenteritis in millions of people each year.
•Mice pre-treated with the antibiotic streptomycin prior to infection with S. Typhimurium experience heavy colonization of the cecum and colon with significant colitis.2
•We recently showed that chronic infections with S. Typhimurium lead to severe intestinal fibrosis.3
•Here, we investigated the contribution of T cells to the development of intestinal fibrosis in mice caused by chronic Salmonella-induced colitis, and found that T-cell deficient mice developed attenuated inflammation and fibrosis.
Experimental Approach
Day ‐1 Day 0 Day 7 Day 14 Day 21 Day 28 Day 35
Harvest Organs
Oral
pretreatment
with
streptomycin
Infection with
S. Typhimurium
ΔaroAHomogenize tissue / enumerate bacteria
Histological stains: H&E, Masson's Trichrome to evaluate inflammation/intestinal fibrosis
Immunofluorescent staining to identify and localize cell types
mRNA isolation to perform qPCR to quantify cytokine expression
Treatment Groups:
C57/Bl6 (B6) wild type
control mice
Rag1-/- mice that have
no T and B cells
(4) S. Typhimurium DaroA induces proinflammatory and profibrotic
cytokines
(2) S. Typhimurium DaroA is found primarily in the lumen of infected ceca
Acknowledgments
Conclusions•S. Typhimurium triggers severe fibrosis in the cecum.•Salmonella-induced profibrotic and proinflammatory cytokines are maximally produced at week 3 post-infection.
•Fibroblasts and smooth muscle cells might be the predominant collagen-producing cells.
•T cells enhance intestinal inflammation and fibrosis
(3) Rag1-/- mice display attenuated inflammation and fibrosis
200X
50X
Uni
nfec
ted
Cont
rols
200X
50X
Day
35
B6 Rag1-/-
Masson’s Trichrome
B6 Rag1-/-
H&E
Fig. 3: Masson’s Trichrome (MT) and Hematoxylin & Eosin (H&E) staining of mice ceca from uninfected controls and day 35 infected B6 and Rag1-/- mice. MT staining indicates position of collagen deposition (blue), while H&E staining reveals extent of inflammation and damage. L = Lumen, M = Mucosa, S = Submucosa. Note region of denser collagen deposition (black arrows) with greater edema in the submucosa of B6 ceca compared to Rag1-/- ceca.
Results(1) S. Typhimurium DaroA
chronically colonizes the cecum of B6 and Rag1-/- mice
10 0
10 1
10 2
10 3
10 4
10 5
10 6
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day 7 day 14 day 21 day 28 day 35
S. T
yphi
mur
ium
Bac
teria
l Loa
d (c
fu)
B6
Rag1-/- B6
Rag1-/- B6
Rag1-/- B6
Rag1-/- B6
Rag1-/-
Fig. 1: Bacterial counts of ceca and colons showing similar colonization levels of B6 and Rag1-/- mice; cfu: colony forming units. References
1. Burke J. P., Mulsow J., O’Keane C. et. al. 2007. Fibrogenesis in Crohn’s disease. Am. J. Gastroenterol. 102(2):439-48.
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. Immun. 73(6):3219-27
3. Grassl G. A., Valdez Y., Bergstrom K. S. B. et. al. 2008. Chronic enteric Salmonella infection in mice leads to severe and persistent intestinal fibrosis. Gastroenterology 134(3): 768-80.
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Rag vs B6 Cytokine Graphs.pzf:TNF-a Timecourse graph - Wed Aug 13 14:33:08 2008
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Rag vs B6 Cytokine Graphs.pzf:IL-6 Timecourse graph - Wed Aug 13 14:42:20 2008
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Rag vs B6 Cytokine Graphs.pzf:MCP-1 Timecourse graph - Wed Aug 13 13:33:56 2008
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Rag vs B6 Cytokine Graphs.pzf:IGF-1 Timecourse graph - Wed Aug 13 14:24:47 2008
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Rag vs B6 Cytokine Graphs.pzf:IL-17 Timecourse graph - Wed Aug 13 14:17:01 2008
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Rag vs B6 Cytokine Graphs.pzf:IFN-g Timecourse graph - Wed Aug 13 13:53:44 2008
B6 Rag1-/-
IFN-γ
Fig. 4: Induction of proinflammatory (TNF-α, IL-6, IL-17, IFN-γ) and profibrotic (MCP-1, IGF-1) cytokines in infected ceca was determined by real-time PCR. Data were normalized to GAPDH levels and are relative to an arbitrarily selected uninfected control.
(5) B6 and Rag1-/- ceca have similar numbers of fibroblasts
Fig. 5: Distribution of mesenchymal cell types in ceca of B6 and Rag1-/- mice infected with S. Typhimurium 35 days p.i., differentiated by staining for fibroblast-specific vimentin (V) and α-smooth muscle actin (A) shown at 400X magnification. Similar amounts of V+A- fibroblasts (white arrow) were found in the submucosa and mucosa of B6 and Rag1-/- ceca. S = Submucosa; M = Mucosa
Vimentin α-Smooth Muscle Actin
B6 Rag1-/-
S. Typhimurium MPO
Fig. 2: Salmonella (Salmonella LPS, red) and neutrophils (myeloperoxidase (MPO), green) were primarily found in the lumen of ceca at 14 days post-infection (p.i.). Uninfected controls shown for comparison. DAPI staining of nuclei shown in blue. L = Lumen, M = Mucosa.
B6 Rag1-/-
Uninfected Control
B6 Rag1-/-
Day 14
