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Science 334 (6053): 255-258

Copyright © 2011 by the American Association for the Advancement of Science

The Antibacterial Lectin RegIII{gamma} Promotes the Spatial Segregation of Microbiota and Host in the Intestine

Shipra Vaishnava1, Miwako Yamamoto1, Kari M. Severson1, Kelly A. Ruhn1, Xiaofei Yu1, Omry Koren3, Ruth Ley3, Edward K. Wakeland1, and Lora V. Hooper1,2,*

1 Department of Immunology, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.
2 The Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.
3 Department of Microbiology, Cornell University, Ithaca, NY 14853, USA.


Figure 1
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  		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 4',6-diamidino-2-phenylindole (DAPI) to visualize nuclei (blue). Scale bars, 50 μm. Arrows indicate the distance from the villus tip to the microbiota. Mice were cohoused 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 per 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 per genotype. Data are from three groups of littermates. *, P < 0.05; error bars, mean ±SEM; ns, not significant.

 

Figure 2
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  		Fig. 2. Epithelial cell MyD88 is necessary and sufficient to limit bacterial association with the small intestinal surface. Myd88{Delta}IEC and Vil-Myd88Tg mice were each analyzed alongside cohoused littermates (Myd88fl/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 per genotype; data are from three littermate groups. (C) RegIII{gamma} was detected in distal small intestine with antibodies to RegIII{gamma} (13) (red). Nuclei were counterstained with DAPI (blue). Images are representative of three littermate groups. (D) Q-PCR quantification of RegIII{gamma} transcripts in terminal ileum (N = 5 mice per genotype from three littermate groups). Scale bars, 50 μm; *, P < 0.05; **, P < 0.01; error bars, mean ±SEM; ns, not significant.

 

Figure 3
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  		Fig. 3. RegIII{gamma} limits mucosal surface association by Gram-positive bacteria. (A) Q-PCR analysis of RegIII{gamma} transcripts in terminal ileum of wild-type and RegIII{gamma}–/– mice. N = 3 mice per group. (B) RegIII{gamma} was detected in distal small intestine using a polyclonal antibody raised against a peptide unique to RegIII{gamma} (red). (C) FISH analysis of wild-type and RegIII{gamma}–/– mice using a universal bacterial 16S rRNA gene probe (green). Mice were cohoused littermates from intercrossed RegIII{gamma}+/– 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 to 7 mice per 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 to 7 mice per genotype from five littermate groups. (F) Comparison of cohoused wild-type and RegIII{gamma}–/– 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, mean ±SEM; ns, not significant.

 

Figure 4
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  		Fig. 4. RegIII{gamma}–/– mice show increased activation of adaptive immunity. (A) Immunofluorescence detection of IgA+ cells in terminal ileum of RegIII{gamma}–/– mice and cohoused wild-type littermates. Images are representative of five littermate groups. (B) IgA+ cells were quantified by counting 10 well-oriented crypt-villus units in 10 mice per genotype from five littermate groups. (C) Total fecal IgA was determined by enzyme-linked immunosorbent assay. N = 5 to 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 RegIII{gamma}–/– mice. Small intestinal lamina propria cells were isolated from cohoused RegIII{gamma}–/– and wild-type littermates. Cells were gated on T cell receptor β chain (TCRβ) and CD4 (fig. S8A), and IFN-{gamma}+ cells were quantified as a percentage of this population. The IFN-{gamma} gate was determined based on an isotype control. Representative plots are shown. (E) IFN-{gamma}+ cells as a percentage of the TCRβ+CD4+ cell population. N = 6 mice per group from three groups of littermates; representative of three independent trials. Scale bars, 50 μm; *, P < 0.05; **, P < 0.01; error bars, mean ±SEM; ns, not significant.

 

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