Lypd8 promotes the segregation of flagellated microbiota and colonic epithelia

Abstract

Colonic epithelial cells are covered by thick inner and outer mucus layers1,2. The inner mucus layer is free of commensal microbiota, which contributes to the maintenance of gut homeostasis3,4,5,6. In the small intestine, molecules critical for prevention of bacterial invasion into epithelia such as Paneth-cell-derived anti-microbial peptides and regenerating islet-derived 3 (RegIII) family proteins have been identified7,8,9,10,11. Although there are mucus layers providing physical barriers against the large number of microbiota present in the large intestine, the mechanisms that separate bacteria and colonic epithelia are not fully elucidated. Here we show that Ly6/PLAUR domain containing 8 (Lypd8) protein prevents flagellated microbiota invading the colonic epithelia in mice. Lypd8, selectively expressed in epithelial cells at the uppermost layer of the large intestinal gland, was secreted into the lumen and bound flagellated bacteria including Proteus mirabilis. In the absence of Lypd8, bacteria were present in the inner mucus layer and many flagellated bacteria invaded epithelia. Lypd8−/− mice were highly sensitive to intestinal inflammation induced by dextran sulfate sodium (DSS). Antibiotic elimination of Gram-negative flagellated bacteria restored the bacterial-free state of the inner mucus layer and ameliorated DSS-induced intestinal inflammation in Lypd8−/− mice. Lypd8 bound to flagella and suppressed motility of flagellated bacteria. Thus, Lypd8 mediates segregation of intestinal bacteria and epithelial cells in the colon to preserve intestinal homeostasis.

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Figure 1: Molecular characteristics of Lypd8.
Figure 2: Penetration of the colonic epithelia by flagellated bactiera in Lypd8−/− mice.
Figure 3: High sensitivity to intestinal inflammation in Lypd8−/− mice.
Figure 4: Lypd8-dependent suppression of P. mirabilis attachment to epithelial cells.

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Acknowledgements

We thank Y. Fujioka, T. Kondo and Y. Magota for technical assistance, T. Oida for generation of mAb, Y. Matsunaga and J. Takagi for the binding assay, S. Pareek for critical reading of the manuscript, and C. Hidaka for secretarial assistance. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, the Japan Agency for Medical Research and Development.

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Contributions

R.O. planed and performed experiments. H.K. and M.Y. generated mutant mice. M.K., T.Ki., N.K., T.Kus., Y.U. and E.U. performed animal experiments. T.Kur., D.M., K.G., S.N. and T.I. analysed composition of microbiota. T.N., H.W. and K.S. performed scanning electron microscopy. Y.O. generated Lypd8venus mice. H.I., S.B., H.O., H.M. and J.N. performed experiments using human samples. K.T. planned and directed the research and wrote the paper.

Corresponding author

Correspondence to Kiyoshi Takeda.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Lypd8 is a GPI-anchored glycoprotein.

a, b, Quantitative RT–PCR analysis of Lypd8 expression in the colonic epithelia of ICR mice in specific-pathogen-free and germ-free conditions (a), and in colonic epithelia of wild-type, Myd88−/− and Asc−/− mice (b). Values were normalized to Gapdh. Data represent the mean ± s.d. (n = 3 per group). NS, not significant. c, Confocal microscopic image of Caco-2 cells expressing Flag-tagged Lypd8 cultured on transwell membrane, stained with anti-Claudin-1 antibody (red), anti-Flag antibody (green), and DAPI (blue). d, 293T cell lysates transiently expressing Flag-tagged Lypd8 and Flag-tagged mutant Lypd8 (N–D), in which thirteen Asp residues were converted to Asn, were separated with SDS–PAGE and immunoblotted with anti-Flag antibody. e, Flow cytometric analysis of CMT93 cells expressing Flag-tagged Lypd8 before (left) and after (right) treatment with PI-PLC. Source data

Extended Data Figure 2 Lypd8 is expressed in enterocytes.

a, Structures of the Lypd8 gene (middle), the targeting vector (top) and the predicted Cre-IRES-Venus inserted gene (bottom). Grey box, coding exon; black box, non-coding exon. b, Flow cytometry of intestinal epithelial cells isolated from caecum and colon of Lypd8venus mice. Venus+ epithelial cells (Lypd8+) are boxed. c, Colon sections of Lypd8venus mice were counterstained with DAPI to visualize nuclei. Scale bar, 20 μm. Venus+ Lypd8 was expressed at the uppermost layer of the colon epithelia. d, Flow cytometry of intestinal epithelial cells isolated from the colon of Lypd8venus mice stained for CD3. e, Lypd8-expressing colonic epithelial cells (black box in d, Lypd8+) and Lypd8 non-expressing colonic epithelial cells (red box in d, Lypd8) are isolated and analysed for expression of Villin (highly expressed in enterocytes), Muc2 (selectively expressed in goblet cells) and Lgr5 (selectively expressed in intestinal stem cells) by quantitative RT–PCR. Lypd8+ cells expressed Villin, but neither Muc2 nor Lgr5. Values were normalized to Gapdh expression. Data are mean ± s.d. (n = 4 per group). *P < 0.05, ****P < 0.001. Source data

Extended Data Figure 3 Generation of Lypd8−/− mice.

a, Structures of the Lypd8 gene (middle), the targeting vector (top) and the predicted disrupted gene (bottom). Grey box, coding exon; black box, non-coding exon. b, Southern blot analysis of offspring from the heterozygote intercrosses. Genomic DNA was extracted from mouse tails, digested with NcoI, separated by electrophoresis and hybridized with the radiolabelled probe shown in a. c, Quantitative RT–PCR analysis of Lypd8 expression in the colon of wild-type and Lypd8−/− mice. The values were normalized to Gapdh expression. Data are mean ± s.d. (n = 3 per group). Source data

Extended Data Figure 4 Bacteria are present in Lypd8−/− epithelial cell layers.

a, Principal component analysis of faecal microbiota in wild-type and Lypd8−/− mice at genus level (n = 8 mice per genotype). b, Bacterial DNA isolated from luminal contents and PBS-washed colon tissues of wild-type and Lypd8−/− mice was analysed by quantitative PCR using universal primers for bacterial 16S rRNA genes. Data show the total bacterial DNA amounts compared to wild-type mice group, and are presented as mean ± s.d. (n = 6 per group). **P < 0.01. Source data

Extended Data Figure 5 Inflammatory responses were not evident in Lypd8−/− mice reared in an SPF facility.

Expression of inflammatory cytokines and chemokines in the colon of wild-type and Lypd8−/− mice. Data represent the mean ± s.d. (n = 10 per group). Source data

Extended Data Figure 6 Gentamicin treatment ameliorated DSS-induced colitis in Lypd8 mice.

a, A paper disc method was performed to determine the sensitivity of gentamicin and vancomycin to P. mirabilis. be, Wild-type (n = 8) and Lypd8−/− mice (n = 8) were orally administered gentamicin (b, d) or vancomycin (c, e) for 2 weeks and then administered DSS for 5 days. Percentage of weight change (b, c). H&E staining of colon tissues at day 8 (gentamicin) and day 5 (vancomycin) of DSS administration (d, e). Scale bars, 50 μm. Data are shown as mean ± s.d. *P < 0.05, ***P < 0.005. f, Lypd8−/− mice (n = 10) and gentamicin-treated Lypd8−/− mice (n = 10) were orally administered 2% DSS for 5 days and their survival rate was monitored. g, Lypd8−/− mice (n = 10) and vancomycin-treated Lypd8−/− mice (n = 10) were orally administered 2% DSS for 5 days and their survival rate was monitored. Data show survival rate of each group. Source data

Extended Data Figure 7 Lypd8 preferentially bound to in-vitro-cultured P. mirabilis.

a, Representative dot plots of pure preparations of in-vitro-cultured B. sartrii, L. acidophilus, B. breve, E. gallinarum and P. mirabilis incubated with Flag-tagged Lypd8. Numbers within plots indicate percentages of bacteria bound by anti-Flag antibody in the respective areas. b, P. mirabilis was cultured in LB medium with the indicated concentrations of recombinant (r)Lypd8 protein for 6 h at 37 °C. The mean CFU of cultured P. mirabilis are shown (n = 4 per group). Data are mean ± s.d. c, In-vitro-cultured P. mirabilis were incubated with or without Flag-tagged Lypd8 and reacted with indicated 1st and 2nd antibodies. Scanning electron microscopic images of P. mirabilis labelled for immunogold detection of the bound Lypd8 were shown. White arrows indicate gold-particles. Scale bars, 300 nm. Source data

Extended Data Figure 8 Isolation of flagella of P. mirabilis and E.coli.

a, b, Bacterial flagella were isolated from bacterial suspension of P. mirabilis and E.coli by vigorous shaking and ultracentrifugation. Transmission electron microscopic images of flagella fraction and bacterial bodies fraction from P. mirabilis (a) and E.coli (b) are shown. Flagella and bacterial bodies were visualized by negative staining. Scale bars, 2.5 μm. c, After incubation of Flag-tagged LOC69864 protein (10 ng μl−1) with flagella of P. mirabilis (1 μg μl−1), flagella were pelleted by ultracentrifugation. The supernatant (sup) and pellet suspension were separated by SDS–PAGE and immunoblotted with anti-Flag antibody. d, e, Association of mouse TLR5-Fc chimaera protein and flagellin (d) or flagella (e) of P. mirabilis was analysed by ELISA assay. Source data

Extended Data Figure 9 LYPD8 expression in Caco-2 cells and human colon epithelia.

a, Quantitative RT–PCR analysis of LYPD8 expression in Caco-2 cells and human colonic epithelia isolated from normal parts of colonic tissues from colon cancer patients (n = 3). b, Immunoblot analysis for detecting Flag-tagged proteins in the supernatants on Caco-2 cells with or without Flag-tagged Lypd8. Source data

Extended Data Figure 10 Motility of E. coli was inhibited in semisolid agar containing Lypd8 protein.

a, Representative images of E. coli motility in semisolid agar with or without Lypd8 protein 4 h after incubation. b, The radii of the motility halos by E. coli were measured at 4 h after incubation. Data are mean ± s.d. (n = 6 per group). ****P < 0.001. Source data

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This file contains the raw data for Figures 1d and 4f and Extended Data Figures 1d, 3b, 4g, 8c. (PDF 720 kb)

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Okumura, R., Kurakawa, T., Nakano, T. et al. Lypd8 promotes the segregation of flagellated microbiota and colonic epithelia. Nature 532, 117–121 (2016). https://doi.org/10.1038/nature17406

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