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The mechanism by which the neonicotinoid pesticide clothianidin (CLO) disrupts the intestinal microbiota of experimental animals is unknown. We focused on α-defensins, which are regulators of the intestinal microbiota. Subchronic exposure to CLO induced dysbiosis and reduced short-chain fatty acid-producing bacteria in the intestinal microbiota of mice. Levels of cryptdin-1 (Crp1, a major α-defensin in mice) in feces and cecal contents were lower in the CLO-exposed groups than in control. In Crp1 immunostaining, Paneth cells in the jejunum and ileum of the no-observed-adverse-effect-level CLO-exposed group showed a stronger positive signal than control, likely due to the suppression of Crp1 release. Our results showed that CLO exposure suppresses α-defensin secretion from Paneth cells as part of the mechanism underlying CLO-induced dysbiosis.

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease characterized by fibrosis that develops from fatty liver. Disruption of intestinal microbiota homeostasis, dysbiosis, is associated with fibrosis development in NASH. An antimicrobial peptide α-defensin secreted by Paneth cells in the small intestine is known to regulate composition of the intestinal microbiota. However, involvement of α-defensin in NASH remains unknown. Here, we show that in diet-induced NASH model mice, decrease of fecal α-defensin along with dysbiosis occurs before NASH onset. When α-defensin levels in the intestinal lumen are restored by intravenous administration of R-Spondin1 to induce Paneth cell regeneration or by oral administration of α-defensins, liver fibrosis is ameliorated with dissolving dysbiosis. Furthermore, R-Spondin1 and α-defensin improved liver pathologies together with different features in the intestinal microbiota. These results indicate that decreased α-defensin secretion induces liver fibrosis through dysbiosis, further suggesting Paneth cell α-defensin as a potential therapeutic target for NASH., (© 2023. The Author(s).)

Psychological stress has been reported to relate to dysbiosis, imbalance of the intestinal microbiota composition, and contribute to the onset and exacerbation of depression, though, underlying mechanisms of psychological stress-related dysbiosis have been unknown. It has been previously established that α-defensins, which are effector peptides of innate enteric immunity produced by Paneth cells in the small intestine, play an important role in regulation of the intestinal microbiota. However, the relationship between disruption of intestinal ecosystem and α-defensin under psychological stress is yet to be determined. Here we show using chronic social defeat stress (CSDS), a mouse depression model that (1) the exposure to CSDS significantly reduces α-defensin secretion by Paneth cells and (2) induces dysbiosis and significant composition changes in the intestinal metabolites. Furthermore, (3) they are recovered by administration of α-defensin. These results indicate that α-defensin plays an important role in maintaining homeostasis of the intestinal ecosystem under psychological stress, providing novel insights into the onset mechanism of stress-induced depression, and may further contribute to discovery of treatment targets for depression.

Paneth cells contribute to intestinal innate immunity by sensing bacteria and secreting α-defensin. In Institute of Cancer Research (ICR) mice, α-defensin termed cryptdin (Crp) in Paneth cells consists of six major isoforms, Crp1 to 6. Despite accumulating evidences that α-defensin functions in controlling the intestinal microbiota, topographical localization of Paneth cells in the small intestine in relation to functions of α-defensin remains to be determined. In this study, we examined the expression level of messenger RNA (mRNA) encoding six Crp-isoforms and Crp immunoreactivities using singly isolated crypts together with bactericidal activities of Paneth cell secretions from isolated crypts of duodenum, jejunum, and ileum. Here we showed that levels of Crp mRNAs in the single crypt ranged from 5 x 10 3 to 1 x 10 6 copies per 5 ng RNA. For each Crp isoform, the expression level in ileum was 4 to 50 times higher than that in duodenum and jejunum. Furthermore, immunohistochemical analysis of isolated crypts revealed that the average number of Paneth cell per crypt in the small intestine increased from proximal to distal, three to seven-fold, respectively. Both Crp1 and 4 expressed greater in ileal Paneth cells than those in duodenum or jejunum. Bactericidal activities in secretions of ileal Paneth cell exposed to bacteria were significantly higher than those of duodenum or jejunum. In germ-free mice, Crp expression in each site of the small intestine was attenuated and bactericidal activities released by ileal Paneth cells were decreased compared to those in conventional mice. Taken together, Paneth cells and their α-defensin in adult mouse appeared to be regulated topographically in innate immunity to control intestinal integrity., (Copyright © 2020 Nakamura, Yokoi, Fukaya, Ohira, Shinozaki, Nishida, Kikuchi and Ayabe.)

Crohn's disease (CD) is an intractable inflammatory bowel disease, and dysbiosis, disruption of the intestinal microbiota, is associated with CD pathophysiology. ER stress, disruption of ER homeostasis in Paneth cells of the small intestine, and α-defensin misfolding have been reported in CD patients. Because α-defensins regulate the composition of the intestinal microbiota, their misfolding may cause dysbiosis. However, whether ER stress, α-defensin misfolding, and dysbiosis contribute to the pathophysiology of CD remains unknown. Here, we show that abnormal Paneth cells with markers of ER stress appear in SAMP1/YitFc, a mouse model of CD, along with disease progression. Those mice secrete reduced-form α-defensins that lack disulfide bonds into the intestinal lumen, a condition not found in normal mice, and reduced-form α-defensins correlate with dysbiosis during disease progression. Moreover, administration of reduced-form α-defensins to wild-type mice induces the dysbiosis. These data provide novel insights into CD pathogenesis induced by dysbiosis resulting from Paneth cell α-defensin misfolding and they suggest further that Paneth cells may be potential therapeutic targets., (© 2020 Shimizu et al.)

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.

Paneth cells are intestinal epithelial cells that release antimicrobial peptides, such as α-defensin as part of host defense. Together with mesenchymal cells, Paneth cells provide niche factors for epithelial stem cell homeostasis. Here, we report two subtypes of murine Paneth cells, differentiated by their production and utilization of fucosyltransferase 2 (Fut2), which regulates α(1,2)fucosylation to create cohabitation niches for commensal bacteria and prevent invasion of the intestine by pathogenic bacteria. The majority of Fut2- Paneth cells were localized in the duodenum, whereas the majority of Fut2+ Paneth cells were in the ileum. Fut2+ Paneth cells showed higher granularity and structural complexity than did Fut2- Paneth cells, suggesting that Fut2+ Paneth cells are involved in host defense. Signaling by the commensal bacteria, together with interleukin 22 (IL-22), induced the development of Fut2+ Paneth cells. IL-22 was found to affect the α-defensin secretion system via modulation of Fut2 expression, and IL-17a was found to increase the production of α-defensin in the intestinal tract. Thus, these intestinal cytokines regulate the development and function of Fut2+ Paneth cells as part of gut defense.