P065 Effects of gut bacteria on the intestinal immune cell composition and barrier function

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Background: Inflammatory bowel diseases (IBD) go along with a dysbiosis of intestinal microbiota. Little is known about the effect of these changes on the local immune cell subsets and intestinal integrity.

The aim was to determine the consequences of changes in the intestinal microbiota compositions for epithelial integrity and immune cell composition in the gut.

Methods: The immune cell phenotype in the gut of germ free (GF), specific pathogen free (SPF) mice and GF mice colonised with SPF microbiota at the age of 5 weeks was assessed in health and inflammation. The phenotype of lamina propria mononuclear cells (LPMC) was determined by flow cytometry and immunohistochemistry. LPMC were stimulated with lipopolysaccharides LPS) in the presence of Brefeldin A to assess intracellular tumor necrosis factor (TNF) α-expression by flow cytometry.

Acute colitis was induced by dextran sodium sulphate (DSS). Colonic barrier function was assessed by electrophysiology using the Ussing chamber. Supernatants of cultures of ex vivo isolated colon tissue were analysed regarding cytokine production by Cytometric Bead Array. To confirm successful colonisation ceacal content was sequenced for 16 S ribosomal RNA. Mucins were analysed using periodic acid-Schiff reagent/Alcian blue staining on histological sections.

Results: In the terminal ileum of GF mice the number of T cells was profoundly decreased. The amount of Ly6C+ monocytes as well as F4/80+ CD11b+ macrophages were decreased in the distal part of the ileum. The immune cell composition in the healthy colon was similar in GF and SPF mice. However, colonic macrophages from GF mice showed an increased TNFα-expression after LPS-stimulation compared to macrophages from SPF mice. Furthermore, in GF mice the total resistance of the colon was decreased accompanied by an increased 3H-Mannitol flux suggesting a barrier dysfunction. Less mucin was expressed by the intestine of GF mice.

GF mice died following the exposure to DSS, whereas SPF mice survived and showed signs of colitis. Colonisation rescued GF mice from death. However, the inflammation score and the expression of monocyte chemoattractant protein-1 and interleukin-6 in the colon were higher in SPF than in colonised mice whereas the immune cell composition as well as intestinal microbiota was similar in these mice.

Conclusions: The microbiota is essential for the development of the colonic integrity and local immune cell composition in the ileum. The dysbiosis might play a role in the pathogenesis of IBD and could provide a potential target for therapeutic intervention.

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