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. 2014 Dec;147(6):1363-77.e17.
doi: 10.1053/j.gastro.2014.08.033. Epub 2014 Aug 27.

Intestinal epithelial cell toll-like receptor 5 regulates the intestinal microbiota to prevent low-grade inflammation and metabolic syndrome in mice

Benoit Chassaing  1 , Ruth E Ley  2 , Andrew T Gewirtz  3
Affiliations

Intestinal epithelial cell toll-like receptor 5 regulates the intestinal microbiota to prevent low-grade inflammation and metabolic syndrome in mice

Benoit Chassaing et al. Gastroenterology. 2014 Dec.

Abstract

Background & aims: Mice lacking the receptor Toll-like receptor 5 (TLR5-null mice), which recognizes flagellin, have an altered intestinal microbiota composition compared with wild-type mice; they develop low-grade inflammation and metabolic syndrome and are prone to colitis. The relative roles of intestinal epithelial cell (IEC) vs dendritic cell (DC) TLR5 in mediating these phenotypes are not clear; modification of intestinal microbiota composition has been reported to reflect animal husbandry practices rather than loss of TLR5. We generated mice with specific disruption of Tlr5 in IECs or DCs by using a breeding scheme that allows comparison with cohoused siblings as controls.

Methods: We generated C57BL/6 mice with LoxP sites flanking Tlr5. These mice were crossed with mice expressing Cre recombinase, regulated by the villin or CD11c promoters, to generate mice that lacked expression of TLR5 by IECs (TLR5(ΔIEC)) or DCs (TLR5(ΔDC)), respectively. Tlr5(fl/fl) siblings were used as controls. On weaning, mice were housed by sex and genotype or by sex only (genotypes cohoused). Mice were examined for basal phenotypes, including microbiota composition; we also analyzed responses to pathobiont challenge, administration of dextran sodium sulfate, and high-fat diets.

Results: Similar to previous findings from TLR5-null mice, TLR5(ΔIEC) mice had low-grade inflammation (mild splenomegaly, shortened colons, and increased fecal levels of lipocalin 2), metabolic syndrome, and an inability to clear pathobionts and were prone to developing colitis compared with their sibling controls under both housing conditions. Development of this inflammation in the TLR5(ΔIEC) mice was eliminated by administration of antibiotics and associated with alterations in localization of microbiota and levels of fecal lipopolysaccharide and flagellin. The composition of the microbiota clustered more closely according to genotype than housing. Loss of TLR5 from DCs did not associate with development of inflammation-associated phenotypes or alterations in the composition of the microbiota but resulted in complete loss of flagellin-induced production of interleukin-22.

Conclusions: In mice, flagellin activation of TLR5 on DCs leads to production of interleukin-22. Expression of TLR5 on IECs regulates the composition and localization of the intestinal microbiota, preventing diseases associated with intestinal inflammation.

Keywords: IBD; Intestine; LPS; Mouse Model.

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Figures

Figure 1
Figure 1. Generation and verification of mice with tissue specific TLR5 deletion
(A) Schematical representation of the transgene containing TLR5 exon 1 flanked with LoxP sites. (B) Analysis of TLR5 mRNA expression by q-RT-PCR in blood, ling, liver, spleen, kidney, stomach, duodenum, jejunum, ileum, caecum, colon, colonic mucosa and purified CD11c+ splenic cells. (C) Analysis of CXCL1 mRNA expression by q-RT-PCR in the colonic mucosa and CXCL1 protein expression by ELISA in serum. Mice were intra-peritoneally injected with 20μg flagellin (+) or PBS (−). 30 min later, serum and intestinal mRNA were isolated. (D) Analysis of IL22 mRNA expression by q-RT-PCR in the colon and IL22 protein expression by ELISA in the serum of mice treated or not with 20μg of flagellin for 2 hours. Data are the means +/− S.E.M. or points are from individual mice with bar representing the mean. Significance was determined by Student's t-test, * indicates p<0.05 compared to TLR5fl/fl group.
Figure 2
Figure 2. Development of metabolic syndrome and low-grade intestinal inflammation in TLR5ΔIEC but not in TLR5ΔDC mice
(A) Spleen weight, colon length, colon weight, colonic myeloperoxidase levels and fecal levels of the inflammatory marker Lcn2 over time of 15 weeks old TLR5fl/fl and TLR5ΔIEC mice, both males and females. (B) Body weight over time, fat pad weight, food intake measurement, 15h fasting blood glucose concentration, cholesterol and triglyceride concentration in the serum of 15 weeks old TLR5fl/fl and TLR5ΔIEC mice, both males and females. (C) Spleen weight, colon length, colon weight, colonic myeloperoxidase levels and fecal levels of the inflammatory marker Lcn2 over time of 15 weeks old TLR5fl/fl and TLR5ΔDC mice. (D) Body weight over time, fat pad weight, food intake, and 15h fasting blood glucose concentration of 15 weeks old TLR5fl/fl and TLR5ΔDC mice. Data are the means +/− S.E.M. or points are from individual mice with bar representing the mean. Significance was determined by Student's t-test (* indicates p<0.05) or 2-way group ANOVA (# indicates p<0.05) compared to TLR5fl/fl group.
Figure 3
Figure 3. Loss of IEC and DC TLR5 contributes to microbiota management
(A) PCR-based quantification of fecal bacterial load. (B) PCR-based quantification of bacterial load adhered to colonic mucosa. (C) Distances of closest bacteria to intestinal epithelial cells (IEC) per condition over 5 high-powered fields per mouse. (D) Confocal microscopy analysis of microbiota localization; Muc2 (green), actin (purple), bacteria (red), and DNA (Blue). (E) AIEC colonization was determinedin WT, TLR5KO, TLR5fl/fl, TLR5ΔIEC and TLR5ΔDC mice by platting on media containing selective antibiotics. Data are the means +/− S.E.M. and points represent data from individual mice. Significance was determined by Student's t-test, * indicates p<0.05 compared to WT or TLR5fl/flgroup.
Figure 4
Figure 4. Effect of high-fat diet (HFD) and TLR4 deficiency on TLR5ΔIEC metabolic syndrome
(A) Body weight over time, fat pad weight, 15h fasting blood glucose concentration, spleen weight, colon length, colon weight, and liver weight of 14 weeks old TLR5fl/fl and TLR5ΔIEC females mice feed with high-fat diet for 8 weeks. (B) Body weight over time, fat pad weight, 15h fasting blood glucose concentration, food intake measurement, spleen weight, colon length, colon weight, colonic myeloperoxidase levels and fecal levels of the inflammatory marker Lcn2 over time of 15 weeks old TLR4KO-TLR5fl/fl and TLR4KO-TLR5ΔIEC males mice. Data are the means +/− S.E.M. or points are from individual mice with bar representing the mean. Significance was determined by Student's t-test (* indicates p<0.05) or 2-way group ANOVA (# indicates p<0.05) compared to TLR5fl/fl group.
Figure 5
Figure 5. TLR5ΔIEC are prone to chemical-induced colitis
(A) Body weight over time, (B) colonic myeloperoxidase levels, fecal levels of the inflammatory marker Lcn2 over time, spleen weight, colon length, colon weight and colonic secretion of CXCL1 cytokine, and (C) colonic secretion of IL1β and serum IL1β of WT, TLR5KO, TLR5fl/fl and TLR5ΔIEC males mice treated with DSS 4% in drinking water for 7 days. (D) Survival curves of WT, TLR5KO, TLR5fl/fl and TLR5ΔIEC males mice treated with DSS 1.25% in drinking water for 40 days. Data are the means +/− S.E.M. or points are from individual mice with bar representing the mean. Significance was determined by Student's t-test, * indicates p<0.05 compared to WT or TLR5fl/fl group.
Figure 6
Figure 6. TLR5ΔIEC are prone to immune dysregulation-induced colitis
(A) Rectal prolapse incidence (% without), (B) survival curves, (C) spleen weight, colon length and colon weight and (D) colonic myeloperoxidase levels and fecal levels of the inflammatory marker Lcn2 over time of TLR5fl/fl, TLR5ΔIEC, IL10KO-TLR5fl/fl and IL10KO-TLR5ΔIEC males mice. Points are from individual mice with bar representing the mean. Significance was determined by Student's t-test, * indicates p<0.05 compared to TLR5fl/fl group.
Figure 7
Figure 7. TLR5ΔIEC have altered microbiota with increased pro-inflammatory potential
(A) PCR-based quantification of fecal bacterial load, spleen weight, colon length and colon weight colonic myeloperoxidase levels, fecal levels of the inflammatory marker Lcn2 over time, body weight over time, fat pad weight, food intake measurement and 15h fasting blood glucose concentration of 15 weeks old TLR5fl/fl and TLR5ΔIEC mice treated with broad spectrum antibiotics for 14 weeks. (B) Firmicutes / Bacteroidetes ratio of TLR5fl/fl and TLR5ΔIEC mice. (C) Principal coordinates analysis (PCoA) of the UniFrac distance matrix of TLR5fl/fl and TLR5ΔIEC mice at days 0, 42 and 84 post-weaning as well as of mucosa-associated bacteria. (D) Bioactive levels of fecal flagellin and LPS assayed with TLR5 and TLR4 reporter cells. Data are the means +/− S.E.M. or points are from individual mice with bar representing the mean. Significance was determined by Student's t-test, * indicates p<0.05 compared to TLR5fl/fl group.
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