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Paneth cells contribute to small intestinal homeostasis by secreting antimicrobial peptides and constituting the intestinal stem cell (ISC) niche. Certain T cell–mediated enteropathies are characterized by extensive Paneth cell depletion coincident with mucosal destruction and dysbiosis. In this study, mechanisms of intestinal crypt injury have been investigated by characterizing responses of mouse intestinal organoids (enteroids) in coculture with mouse T lymphocytes. Activated T cells induced enteroid damage, reduced Paneth cell and Lgr5 ⁺ ISC mRNA levels, and induced Paneth cell death through a caspase-3/7–dependent mechanism. IFN-γ mediated these effects, because IFN-γ receptor–null enteroids were unaffected by activated T cells. In mice, administration of IFN-γ induced enteropathy with crypt hyperplasia, villus shortening, Paneth cell depletion, and modified ISC marker expression. IFN-γ exacerbated radiation enteritis, which was ameliorated by treatment with a selective JAK1/2 inhibitor. Thus, IFN-γ induced Paneth cell death and impaired regeneration of small intestinal epithelium in vivo, suggesting that IFN-γ may be a useful target for treating defective mucosal regeneration in enteric inflammation.

The intestine not only plays a role in fundamental processes in digestion and nutrient absorption, but it also has a role in eliminating ingested pathogenic bacteria and viruses. Paneth cells, which reside at the base of small intestinal crypts, secrete α-defensins and contribute to enteric innate immunity through potent microbicidal activities. However, the relationship between food factors and the innate immune functions of Paneth cells remains unknown. Here, we examined whether short-chain fatty acids and amino acids induce α-defensin secretion from Paneth cells in the isolated crypts of small intestine. Butyric acid and leucine elicit α-defensin secretion by Paneth cells, which kills Salmonella typhimurium. We further measured Paneth cell secretion in response to butyric acid and leucine using enteroids, a three-dimensional ex vivo culture system of small intestinal epithelial cells. Paneth cells expressed short-chain fatty acid receptors, Gpr41, Gpr43, and Gpr109a mRNAs for butyric acid, and amino acid transporter Slc7a8 mRNA for leucine. Antagonists of Gpr41 and Slc7a8 inhibited granule secretion by Paneth cells, indicating that these receptor and transporter on Paneth cells induce granule secretion. Our findings suggest that Paneth cells may contribute to intestinal homeostasis by secreting α-defensins in response to certain nutrients or metabolites.

Mouse Paneth cell α-defensins, termed cryptdins, are secreted into the intestinal lumen, have microbicidal activity, and contribute to intestinal innate immunity. Among them, cryptdin-4 (Crp4) has the most potent microbicidal activity. In the intestinal lumen, commensal bacteria colonize and elicit beneficial effects in the host. However, the effects of Crp4 against commensal bacteria are poorly understood. Thus, we investigated the bactericidal activities of Crp4 against commensal bacteria compared to noncommensal bacteria. Oxidized Crp4 showed only minimal or no bactericidal activity against 8 out of 12 commensal bacterial species, including Bifidobacterium bifidum and Lactobacillus casei. We further addressed a role of the conserved disulfide bonds of Crp4 by analyzing reduced Crp4 (r-Crp4). r-Crp4 demonstrated significantly greater bactericidal activities against 7 of 12 commensal bacteria than did oxidized Crp4. Oxidized Crp4 and r-Crp4 elicited equivalently potent bactericidal activities against 11 of the 11 noncommensal bacteria tested, such as Salmonella enterica serovar Typhimurium,and against 5 of 12 commensal bacteria. Furthermore, when r-Crp4 was exposed to a processing enzyme of cryptdins, i.e. MMP-7, r-Crp4 was degraded and the bactericidal activities disappeared. These findings suggest that Crp4 has selective bactericidal activities against intestinal microbiota and that the activities are dependent on the disulfide bonds.

Paneth cells at the base of small intestinal crypts secrete granules containing α-defensins in response to bacteria and maintain the intestinal environment by clearing enteric pathogens and regulating the composition of the intestinal microbiota. However, Paneth cell secretory responses remain debatable and the mechanisms that regulate the secretion are not well understood. Although enteroids, three-dimensional cultures of small intestinal epithelial cells, have proven useful for analyzing intestinal epithelial cell functions including ion transport, their closed structures have imposed limitations to investigating interactions between Paneth cells and the intestinal microbiota. Here, we report that microinjection of bacteria or lipopolysaccharide (LPS) into the enteroid lumen provides an ex vivo system for studying Paneth cell secretion in real-time. The results show that Paneth cells released granules immediately when the apical surfaces of enteroid epithelial cells were exposed to LPS or live bacteria by microinjection. However, Paneth cells did not respond to LPS delivered in culture media to enteroid exterior basolateral surface, although they responded to basolateral carbamyl choline. In addition, Paneth cells replenished their granules after secretion, enabling responses to second stimulation. These findings provide new insight for apically-induced Paneth cell secretory responses in regulating the intestinal environment.

Significance: Paneth cells produce granules containing antimicrobial peptides, such as α-defensin for host defense. Examination of the production and utilization of fucosyltransferase 2 (Fut2) by Paneth cells revealed two distinct subsets: Fut2⁺Paneth cells and Fut2⁻Paneth cells. Compared with Fut2⁻Paneth cells, Fut2⁺Paneth cells were enriched in granules containing antimicrobial peptides. IL-22 and IL-17a were found to be essential for commensal bacteria-dependent Fut2⁺Paneth cell development and function as part of gut defense.