The antibacterial lectin RegIIIgamma promotes the spatial segregation of microbiota and host in the intestine

Abstract

The mammalian intestine is home to ~100 trillion bacteria that perform important metabolic functions for their hosts. The proximity of vast numbers of bacteria to host intestinal tissues raises the question of how symbiotic host-bacterial relationships are maintained without eliciting potentially harmful immune responses. Here, we show that RegIIIγ, a secreted antibacterial lectin, is essential for maintaining a ~50-micrometer zone that physically separates the microbiota from the small intestinal epithelial surface. Loss of host-bacterial segregation in RegIIIγ(-/-) mice was coupled to increased bacterial colonization of the intestinal epithelial surface and enhanced activation of intestinal adaptive immune responses by the microbiota. Together, our findings reveal that RegIIIγ is a fundamental immune mechanism that promotes host-bacterial mutualism by regulating the spatial relationships between microbiota and host.

Fig. 1

MyD88 promotes physical separation of the microbiota and the small intestinal surface. (A) Visualization of microbiota localization relative to the small intestinal mucosal surface by FISH. Sections were hybridized to a probe that recognizes the 16S rRNA genes of all bacteria (green), and counterstained with DAPI to visualize nuclei (blue). Scale bars=50 μm. Arrows indicate the distance from the villus tip to the microbiota. Mice were co-housed littermates from intercrossed Myd88^+/− mice. Sections are representative of >10 groups of littermates. (B) Quantification of fluorescence intensity extending from the villus tip into the lumen (N=5 mice/genotype). (C) Mucosa-associated and luminal bacteria were quantified by Q-PCR determination of 16S rRNA gene copy number in the terminal ileum. N=5 mice/genotype. Data are from three groups of littermates. *, p<0.05; Error bars, ±SEM; ns, not significant.

Fig. 2

Epithelial cell MyD88 is necessary and sufficient to limit bacterial association with the small intestinal surface. Myd88 ^ΔIEC and Vil-Myd88^Tg mice were each analyzed alongside co-housed littermates (Myd88^fl/fl and Myd88^−/−, respectively). (A) FISH analysis of microbiota localization in terminal ileum using a universal bacterial 16S rRNA gene probe. Images are representative of >10 littermate groups. (B) Mucosa-associated and luminal bacteria were quantified by Q-PCR determination of 16S rRNA gene copy number in the terminal ileum. N=5 mice/genotype; data are from three littermate groups. (C) RegIIIγ was detected in distal small intestine with anti-RegIIIγ antiserum (13) (red). Nuclei were counterstained with DAPI (blue). Images are representative of three littermate groups. (D) Q-PCR quantification of RegIIIγ transcripts in terminal ileum (N=5 mice/genotype from three littermate groups). Scale bars=50 μm; *, p<0.05; **, p<0.01; Error bars, ±SEM; ns, not significant.

Fig. 3

RegIIIγ limits mucosal surface association by Gram-positive bacteria. (A) Q-PCR analysis of RegIIIγ transcripts in terminal ileum of wild-type and RegIIIγ^−/− mice. N=3 mice/group. (B) RegIIIγ was detected in distal small intestine using a polyclonal antibody raised against a peptide unique to RegIIIγ (red). (C) FISH analysis of wild-type and RegIIIγ^−/− mice using a universal bacterial 16S rRNA gene probe (green). Mice were co-housed littermates from intercrossed RegIIIγ^+/− mice. Images are representative of five littermate groups. (D) Quantification of total ileal mucosa-associated and luminal bacteria by Q-PCR determination of 16S rRNA gene copy number. N=5–7 mice/genotype from five littermate groups. (E) Q-PCR quantification of specific bacterial groups. Bacteria were recovered from the ileal mucosal surface. Values for each bacterial group are expressed relative to the 16S rDNA levels in wild-type mice. N=5–7 mice/genotype from five littermate groups. (F) Comparison of co-housed wild-type and RegIIIγ^−/− littermates by FISH using an SFB-specific probe (red). Images are representative of five littermate groups. All tissues were counterstained with DAPI (blue). Scale bars=50 μm; *, p<0.05; Error bars, ±SEM; ns, not significant.

Fig. 4

RegIIIγ^−/− mice show increased activation of adaptive immunity. (A) Immunofluorescence detection of IgA⁺ cells in terminal ileum of RegIIIγ^−/− mice and co-housed wildtype littermates. Images are representative of five littermate groups. (B) IgA⁺ cells were quantified by counting 10 well-oriented crypt-villus units in 10 mice/genotype from five littermate groups. (C) Total fecal IgA was determined by ELISA. N=5–7 mice per non-antibiotic-treated group from five littermate groups; N=3 mice per antibiotic-treated (Abx) group from two littermate groups. (D) Flow cytometric analysis of Th1 CD4⁺ T cell frequencies in RegIIIγ^−/− mice. Small intestinal lamina propria cells were isolated from co-housed RegIIIγ^−/− and wild-type littermates. Cells were gated on T cell receptor β chain (TCRβ) and CD4 (fig. S8A), and IFNγ⁺ cells were quantified as a percentage of this population. The IFNγ gate was determined based on an isotype control. Representative plots are shown. (E) IFNγ⁺ cells as a percentage of the TCRβ⁺CD4⁺ cell population. N=6 mice/group from three groups of littermates; representative of three independent trials. Scale bars=50 μm; *, p<0.05; **, p<0.01; Error bars, ±SEM; ns, not significant.