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P023. Regulation of colonic inflammation by commensal flora in SAMP1/YitFc mice

D. Ishikawa1, T. Osada1, S. Watanabe1, F. Cominelli2, 1Juntendo University, Gastroenterology, Tokyo, Japan, 2Case Western Reserve University, Gastroenterolgy, Cleveland OH, United States

Background

We have previously shown that Tregs of SAMP1/YitFc (SAMP) whose ileitis is similar to human Crohn's disease are functionally defective in vivo, and their development is dependent on commensal flora. We hypothesized that Tregs in germ-free (GF) SAMP mice may be generated and enhanced by commensal bacteria, and that Tregs play a role in regulating the development of colonic inflammation.

Methods

To test this hypothesis, we administered dextran sodium sulfate (DSS) to GF and SPF mice as well as GF mice which were also treated with oral fecal suspensions from SPF mice.

Results

GF and SPF mice were given 3% DSS for 8 days. Administration of 3% DSS caused gross rectal bleeding and body weight loss in GF mice compared to SPF mice (BW loss: GF mean = −11.1%, SPF mean = −6.7%, p = 0.015, n = 8). In contrast to clinical symptoms, histopathological examination showed decreased severity of colitis in GF mice (Total inflammatory score [TIS]: GF = 10.67, SPF = 18.25, p = 0.0005, n = 8). Colonic cytokine mRNA expression and MPO activity also were also decreased in GF mice compared to SPF mice. Next, 1g of feces collected from SPF mice was suspended in 15 ml of saline, and 0.5 ml of fecal suspension was orally administrated through a gastric tube and mice maintained under SPF conditions, followed by administration of 3.5% DSS for 8 days. Colonized-GF mice significantly lost weight and showed severe clinical symptoms by day 5 (BW loss on day 5: GF = −8.2%, SPF = −4.3%, colonized-GF = −14.7%, p < 0.001, n = 8, 8, 5). However, they rapidly recovered, and by day 8 there was no significant difference in clinical symptoms and histological scores between colonized-GF mice and SPF mice (TIS: GF = 7.0, SPF = 12.6, colonized-GF = 12.6). In a separate group of experiments, administration of 4% DSS for 5 days to colonized-GF mice significantly increased histological scores compared to SPF mice (TIS: GF = 9.91, SPF = 11.83, colonized-GF = 14.40, p < 0.05, n = 12, 12, 9). Using FACS analysis of MLNs from microbial colonized-GF mice, we detected a significant increase in absolute number of CD4+CD25+ Foxp3+ cell subsets on day 8 compared to day 5 post-colitis induction.

Conclusion

The intestinal micro flora plays a critical role in regulating colonic inflammation and injury induced by DSS in SAMP1/YitFc mice. Furthermore, immune responses generated by bacterial colonization rapidly induced Tregs in the periphery, with down regulation of DSS-induced colitis. Our study demonstrates that commensal flora is necessary for the normal development of Treg function and the regulation of DSS-induced colitis in SAMP1/YitFc mice.