The persistent infiltration of the colon with commensal bacteria likely contributes to the chronic inflammatory state following DSS damage of the epithelium in C57BL/6 mice. Figure 2 Proximity and progression of host/commensal interactions following DSS damage. Sorafenib Tosylate purchase We examined the tissue loads of commensal bacteria over the time course of the in vivo experiment. A significant proportion of the gastrointestinal microbiota is not cultureable making an accurate quantitative assessment of undefined bacteria difficult. We circumvented this problem by adapting two previously defined methods to detect bacteria from intestinal samples [23], [24]. The method was validated in a series of experiments summarized in Figure S2.

Pieces of the colon were removed and treated with gentamicin �C a non-cell permeable antibiotic to reduce bacteria associated with the tissue that had not penetrated inside the cells of the host. In the acute phase of colonic inflammation up to day 8, no significant increase in tissue-associated bacterial numbers was observed (Figure 2B). By day 21 however, tissue-associated bacterial counts were significantly increased. These numbers remained significantly elevated up to day 35. Together, the data presented in Figure 2 demonstrates that DSS damage triggers a progressive infiltration of the host with the commensal flora from the colonic lumen. The bacteria invade deeper tissue layers over time eventually reaching and colonizing the submucosa and muscle layers of the gastrointestinal tract.

Immune cell infiltration following DSS damage and correlation with bacterial penetration Since the cytokine profiles demonstrated temporal regulation correlating with the compartmentalization of the tissue-associated bacteria, we examined the infiltration of specific immune cells into the colon over the time course of the experiment to determine how the residence of commensal bacteria in the colonic tissue correlates with the development of the immune response of the host. We examined levels of selected immune cells in the tissue across the time course of the experiment by IHC (Figure 3). As demonstrated by CD3, F4/80 and GR-1/Ly-6G positive staining, a significant infiltration of the colonic mucosa by T cells, macrophages and granulocytes was observed by day 8, post DSS. These cell types accumulated in the tissue up to day 22�C29. While the markers were apparent in the mucosa on day 8, by day 22 the F4/80 macrophage staining partitioned primarily into the sub-mucosal layer adjacent to the muscle while T cells and granulocytes were recruited to the damaged mucosa and lamina propria. Cilengitide All cell types were visible in the tissue until day 42 of the experiment.