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Intestinal epithelial cells separate subepithelial tissues from luminal environment formed with food, incoming pathogens, and resident intestinal microbiota, etc., and elicit various intestinal function. Enteroid, a three-dimensional culture system of small intestinal epithelial cells, has been widely used for analyzing the intestinal function, further a transgenic enteroid was developed to investigate the mo-lecular mechanisms. However, conventional transgenic enteroid production method, which transfer gene into single stem cells, has limitations including low efficiency and time-consuming. Here we show that by gene transfer into small intestinal isolated crypts maintaining stem cell niche, a transgenic enteroid was obtained quickly and efficiently. Isolated crypts were transfected by lentiviral vector without separating into single cells, and transgenic enteroid composed of all lineages of intestinal epithelial cells was generated at day 7 with yield of 56%, maintaining the intestinal function in drug transport and innate immunity. Our efficient and simple transgenic enteroid generation method enables high-throughput investigation of intestinal epithelial cells and contributes to understanding intestinal function.(c) 2022 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Paneth cells and Lgr5+ intestinal stem cells (Lgr5+ ISCs) constitute the stem cell niche and maintain small intestinal epithelial integrity by recognizing various niche factors derived from subepithelial cells and external antigens. Although it has been known that interferon-gamma (IFN-gamma), a Th1 cytokine, is associated with intestinal epithelial disruption during inflammation as a niche factor, dynamics of Paneth cells and Lgr5+ ISCs in response to IFN-gamma remain to be understood. Here we show that CAG-tdTomato;Lgr5-EGFP (CT-LE) mice generated in this study enable to identify Paneth cells and Lgr5+ ISCs separately by fluorescence signals. Lgr5+ ISCs underwent cell death a little earlier than Paneth cells in response to IFN-gamma by simultaneous tracking using CT-LE mice. In addition, the timing of cell death in most Paneth cells overlapped with Lgr5+ ISCs, suggesting that Paneth cell depletion is induced directly by IFN-gamma. Taken together, we established a novel simultaneous stem cell niche tracking method and clarified the involvement of both Paneth cells and Lgr5+ ISCs in stem cell niche damage induced by IFN-gamma, further contribute to understanding the mechanism for maintaining intestinal homeostasis by stem cell niche. (C) 2021 The Authors. Published by Elsevier Inc.

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 alpha -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 alpha -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 alpha -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 alpha -defensin. These results indicate that alpha -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 alpha-defensin. In Institute of Cancer Research (ICR) mice, alpha-defensin termed cryptdin (Crp) in Paneth cells consists of six major isoforms, Crp1 to 6. Despite accumulating evidences that alpha-defensin functions in controlling the intestinal microbiota, topographical localization of Paneth cells in the small intestine in relation to functions of alpha-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 alpha-defensin in adult mouse appeared to be regulated topographically in innate immunity to control intestinal integrity.

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 alpha-defensin misfolding have been reported in CD patients. Because alpha-defensins regulate the composition of the intestinal microbiota, their misfolding may cause dysbiosis. However, whether ER stress, alpha-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 alpha-defensins that lack disulfide bonds into the intestinal lumen, a condition not found in normal mice, and reduced-form alpha-defensins correlate with dysbiosis during disease progression. Moreover, administration of reduced-form alpha-defensins to wild-type mice induces the dysbiosis. These data provide novel insights into CD pathogenesis induced by dysbiosis resulting from Paneth cell alpha-defensin misfolding and they suggest further that Paneth cells may be potential therapeutic targets.

The intestinal microbial ecosystem is actively regulated by Paneth cell-derived antimicrobial peptides such as α-defensins. Various disorders, including graft-versus-host disease (GVHD), disrupt Paneth cell functions, resulting in unfavorably altered intestinal microbiota (dysbiosis), which further accelerates the underlying diseases. Current strategies to restore the gut ecosystem are bacteriotherapy such as fecal microbiota transplantation and probiotics, and no physiological approach has been developed so far. In this study, we demonstrate a novel approach to restore gut microbial ecology by Wnt agonist R-Spondin1 (R-Spo1) or recombinant α-defensin in mice. R-Spo1 stimulates intestinal stem cells to differentiate to Paneth cells and enhances luminal secretion of α-defensins. Administration of R-Spo1 or recombinant α-defensin prevents GVHD-mediated dysbiosis, thus representing a novel and physiological approach at modifying the gut ecosystem to restore intestinal homeostasis and host-microbiota cross talk toward therapeutic benefits.