Commensal Bacteria-Dependent Indole Production Enhances Epithelial Barrier Function in the Colon

<div><p>Microbiota have been shown to have a great influence on functions of intestinal epithelial cells (ECs). The role of indole as a quorum-sensing (QS) molecule mediating intercellular signals in bacteria has been well appreciated. However, it remains unknown whether indole has beneficial effects on maintaining intestinal barriers <i>in vivo</i>. In this study, we analyzed the effect of indole on ECs using a germ free (GF) mouse model. GF mice showed decreased expression of junctional complex molecules in colonic ECs. The feces of specific pathogen-free (SPF) mice contained a high amount of indole; however the amount was significantly decreased in the feces of GF mice by 27-fold. Oral administration of indole-containing capsules resulted in increased expression of both tight junction (TJ)- and adherens junction (AJ)-associated molecules in colonic ECs in GF mice. In accordance with the increased expression of these junctional complex molecules, GF mice given indole-containing capsules showed higher resistance to dextran sodium sulfate (DSS)-induced colitis. A similar protective effect of indole on DSS-induced epithelial damage was also observed in mice bred in SPF conditions. These findings highlight the beneficial role of indole in establishing an epithelial barrier <i>in vivo</i>.</p></div

Indole-containing capsules promote epithelial barrier function in GF mice.

<p>(A) Feces were collected from SPF mice, and GF mice treated with indole- or MCT- containing capsules. Three mice was analysed in each group. The concentration of indole in the feces was measured by HPLC-FL. Data show mean values ± S.D. of 3 samples. *P<0.05. n.s., not significant. SPF, specific pathogen free; GF, germ free; MCT, Medium-Chain Triglycerides. (B) Real-time quantitative RT-PCR analysis of mRNA expression of <i>Cldn7, Ocln, Tjp1, Ctnnb1</i>, and <i>Cdh1</i> in colonic epithelial cells of GF mice treated with indole- (n = 4) or MCT- (n = 4) containing capsules. Values were normalized to the expression of <i>Gapdh</i>. Data are representative of two independent experiments and show mean values ± S.D. of 4 samples performed in duplicate. *P<0.05. (C) Colonic tissues of GF mice treated with indole- or MCT- containing capsules were stained with anti-occludin antibody. Sections were analyzed using a confocal microscope. Bars, 20 µm. Data are representative of two independent experiments. (D) After oral administration with either indole- (n = 6) or MCT- (n = 6) containing capsules for 2 weeks, GF mice were treated by 4% DSS in drinking water for 3 days. Survival rate of the mice in each group is shown. Data are representative of two independent experiments. MCT, Medium-Chain Triglycerides.</p

Indole and indole metabolites are absent in GF mice.

<p>(A, B) Feces and serum were collected from either SPF (n = 3) or GF (n = 3) mice. The concentration of indole in the feces was measured by HPLC-FL, and the serum concentration of indoxyl sulfate was measured by LC-MS/MS. Data are representative of two independent experiments and show mean values ± S.D. of 3 mice. *P<0.05. SPF, specific pathogen free; GF, germ free.</p

Indole-containing capsules show preventative effect on colitis development in SPF mice.

<p>SPF mice were treated with indole- (n = 7) or MCT- (n = 7) containing capsules for 1 week, and then challenged by 5% DSS for 6 days. Body weight changes relative to the value prior to colitis induction are shown. Data are representative of two independent experiments and mean ± S.E.M of 7 mice at each time point is shown. *P<0.05. MCT, Medium-Chain Triglycerides.</p

Epithelial barrier functions is impaired in GF mice.

<p>(A) Real-time quantitative RT-PCR analysis of mRNA expression of <i>Cldn7, Ocln, Tjp1, Ctnnb1, Cdh1</i> in colonic ECs in SPF (n = 4) or GF (n = 4) mice. Values were normalized to that of <i>Gapdh</i>. Data are representative of two independent experiments and show mean values ± S.D. of 4 samples performed in duplicate. *P<0.05. (B) Mouse colonic tissue was stained with anti-occludin antibody. Sections were analyzed using a confocal microscope. Bars, 50 µm. Data are representative of two independent experiments. (C) SPF (n = 8) or GF (n = 8) mice were administered 4% DSS by drinking water for 3 days. Survival rates of the indicated mice are shown. Body weight changes relative to the value prior to colitis induction are shown. Data are mean ± S.E.M of 8 mice at each time point. SPF, specific pathogen free; GF, germ free.</p