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. 2008 Dec 30;105(52):20858-63.
doi: 10.1073/pnas.0808723105. Epub 2008 Dec 15.

Paneth Cells Directly Sense Gut Commensals and Maintain Homeostasis at the Intestinal Host-Microbial Interface

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Free PMC article

Paneth Cells Directly Sense Gut Commensals and Maintain Homeostasis at the Intestinal Host-Microbial Interface

Shipra Vaishnava et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The intestinal epithelium is in direct contact with a vast microbiota, yet little is known about how epithelial cells defend the host against the heavy bacterial load. To address this question we studied Paneth cells, a key small intestinal epithelial lineage. We found that Paneth cells directly sense enteric bacteria through cell-autonomous MyD88-dependent toll-like receptor (TLR) activation, triggering expression of multiple antimicrobial factors. Paneth cells were essential for controlling intestinal barrier penetration by commensal and pathogenic bacteria. Furthermore, Paneth cell-intrinsic MyD88 signaling limited bacterial penetration of host tissues, revealing a role for epithelial MyD88 in maintaining intestinal homeostasis. Our findings establish that gut epithelia actively sense enteric bacteria and play an essential role in maintaining host-microbial homeostasis at the mucosal interface.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Intestinal microbiota activate expression of a MyD88-dependent antimicrobial program in Paneth cells. (A and B) Expression of key antimicrobial effectors is MyD88 dependent but NOD2 independent. Gene expression was quantitated by real-time quantitative PCR (Q-PCR) of (A) small intestines (Sm. Int.) of germ-free (gf) wild-type (wt), conventional (cv) wt, cv Nod2−/−, and cv MyD88−/− mice (3–4 mice per group), or of (B) small intestinal Paneth cells isolated by laser capture microdissection. (C) Q-PCR analysis of small intestines of cv mice treated with antibiotics (Abx) to deplete the normal microbiota (3–4 mice per group). (D) Visualization of RegIIIγ expression in small intestinal epithelium. Immunostaining with anti-RegIIIγ antiserum reveals expression of RegIIIγ (red) in both Paneth cells (indicated by arrows) and enterocytes of cv wt mice. Sections were counterstained with Hoechst dye (blue). (Scale bars: 50 μm.) All Q-PCR assays were run in triplicate and are shown as mean values normalized to 18S ribosomal RNA. Relative expression levels were calculated relative to gf controls. Error bars indicate ± SEM. *P < 0.05 compared with cv wt.
Fig. 2.
Fig. 2.
Paneth cells directly sense enteric bacteria through cell-autonomous MyD88 activation. (A) A MyD88 transgene with an N-terminal FLAG epitope fusion was cloned downstream of the Paneth cell-specific Cryptdin-2 (CR2) promoter and introduced into MyD88−/− mice. (B) FLAG-MyD88 (red) and RegIIIγ (red) are expressed in small intestinal crypts of transgenic mice (on a MyD88−/− background, designated CR2-MyD88 Tg mice). RegIIIγ expression in the transgenic mice is reversible by partial depletion of the microflora through administration of broad-spectrum antibiotics (Abx). Sections were counterstained with Hoechst dye (blue). Arrows indicate Paneth cells. (Scale bars: 50 μm.) (C) Expression of antimicrobial factors was quantitated in small intestines of germ-free (gf) wild-type (wt), conventional (cv) wt, cv MyD88−/−, cv CR2-MyD88 Tg mice, or cv Abx-treated transgenic mice. All Q-PCR assays were performed as described in Fig. 1. Error bars indicate ± SEM. *P < 0.05.
Fig. 3.
Fig. 3.
Paneth cell-intrinsic MyD88 expression is sufficient to limit mucosal penetration by commensal and pathogenic bacteria. (A) Commensal bacteria were quantitated in small intestinal lumen and MLN from conventionally reared wt, MyD88−/−, bone marrow chimeric mice, or CR2-MyD88 transgenic mice on the MyD88−/− background (CR2-MyD88 Tg). MyD88−/− → wt, MyD88−/− donor to wt recipient; wt → MyD88−/−, wild-type donor to MyD88−/− recipient. (B) Conventionally raised wt, MyD88−/−, or CR2-MyD88 Tg mice were orally inoculated with 108 cfu of S. typhimurium. Numbers of S. typhimurium in small intestine, MLN, and spleen were quantitated after 48 h. n = 3–4 mice per group, representative of two experiments.
Fig. 4.
Fig. 4.
Paneth cells are essential for limiting mucosal penetration by commensal and pathogenic bacteria. (A) Commensal bacteria were quantitated in conventionally reared Paneth cell-deficient mice (CR2-tox176) transgenic mice and their wild-type (wt) littermates. (B and C) Germ-free CR2-tox176 mice and their wt littermates were orally challenged with 108 cfu of (B) Bacteroides thetaiotaomicron or (C) S. typhimurium, and bacterial numbers were quantitated 48 h later. No commensals or B. thetaiotaomicron were recovered from spleens of wt or CR2-tox176 mice. Error bars indicate ± SEM. n = 3–4 mice per group, representative of two experiments.
Fig. 5.
Fig. 5.
Model of small intestinal Paneth cell function. We have shown that Paneth cells directly sense enteric bacteria through cell-autonomous MyD88 activation and limit bacterial penetration of the mucosal surface. MyD88-dependent sensing triggers expression of a complex antimicrobial program that could function to limit the numbers of bacteria that localize at the mucosal surface, in or beneath the mucus layer. This would in turn limit the numbers of bacteria that are translocated to mesenteric lymph nodes (MLN) via dendritic cells (DCs). In the same way, Paneth cells could inhibit pathogen access to surface niches, thus accounting for the essential role of Paneth cells in limiting mucosal penetration and dissemination of Salmonella.

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