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Intrinsic Notch signaling in intestinal epithelial cells restricts secretory cell differentiation. In gut-associated lymphoid tissue (GALT), stromal cells located beneath the follicle-associated epithelium (FAE) abundantly express the Notch ligand delta-like 1 (Dll1). Here, we show that mice lacking Rbpj—a gene encoding a transcription factor implicated in Notch signaling—in intestinal epithelial cells have defective GALT maturation. This defect can be attributed to the expansion of goblet cells, which leads to the down-regulation of CCL20 in FAE. These data demonstrate that epithelial Notch signaling maintained by stromal cells contributes to the full maturation of GALT by restricting secretory cell differentiation in FAE.

Gut commensal microbes shape the mucosal immune system by regulating the differentiation and expansion of several types of T cell. Clostridia, a dominant class of commensal microbe, can induce colonic regulatory T (Treg) cells, which have a central role in the suppression of inflammatory and allergic responses. However, the molecular mechanisms by which commensal microbes induce colonic Treg cells have been unclear. Here we show that a large bowel microbial fermentation product, butyrate, induces the differentiation of colonic Treg cells in mice. A comparative NMR-based metabolome analysis suggests that the luminal concentrations of short-chain fatty acids positively correlates with the number of Treg cells in the colon. Among short-chain fatty acids, butyrate induced the differentiation of Treg cells in vitro and in vivo, and ameliorated the development of colitis induced by adoptive transfer of CD4(+) CD45RB(hi) T cells in Rag1(-/-) mice. Treatment of naive T cells under the Treg-cell-polarizing conditions with butyrate enhanced histone H3 acetylation in the promoter and conserved non-coding sequence regions of the Foxp3 locus, suggesting a possible mechanism for how microbial-derived butyrate regulates the differentiation of Treg cells. Our findings provide new insight into the mechanisms by which host-microbe interactions establish immunological homeostasis in the gut.

Background & Aims In epithelial cells, protein sorting mechanisms regulate localization of plasma membrane proteins that generate and maintain cell polarity. The clathrin-adaptor protein (AP) complex AP-1B is expressed specifically in polarized epithelial cells, where it regulates basolateral sorting of membrane proteins. However, little is known about its physiological significance. Methods We analyzed the intestinal epithelia of mice deficient in Ap1m2 ( Ap1m2 -/- mice), which encodes the AP-1B μ1B subunit, and compared it with 129/B6/CD1 littermates (controls). Notch signaling was inhibited by intraperitoneal injection of dibenzazepine, and β-catenin signaling was inhibited by injection of IWR1. Intestinal tissue samples were collected and analyzed by immunofluorescence analysis. Results Ap1m2 -/- mice developed intestinal epithelial cell hyperplasia. The polarity of intestinal epithelial cells was disrupted, as indicated by the appearance of ectopic microvilli-like structures on the lateral plasma membrane and mislocalization of basolateral membrane proteins, including the low-density lipoprotein receptor and E-cadherin. The E-cadherin−β-catenin complex therefore was disrupted at the adherens junction, resulting in nuclear translocation of β-catenin. This resulted in up-regulation of genes regulated by β-catenin/transcription factor 4 (Tcf4) complex, and increased the proliferation of intestinal epithelial cells. Conclusions AP-1B is required for protein sorting and polarization of intestinal cells in mice. Loss of AP-1B in the intestinal epithelia results in mislocalization of E-cadherin, activation of β-catenin/Tcf4 complex, proliferation, and hyperplasia.

Intestinal epithelial cells (IECs) have important functions as the first line of defense against diverse microorganisms on the luminal surface. Impaired integrity of IEC has been implicated in increasing the risk for inflammatory disorders in the gut. Notch signaling plays a critical role in the maintenance of epithelial integrity by regulating the balance of secretory and absorptive cell lineages, and also by facilitating epithelial cell proliferation. We show in this article that mice harboring IEC-specific deletion of Rbpj (RBP-JΔIEC), a transcription factor that mediates signaling through Notch receptors, spontaneously develop chronic colitis characterized by the accumulation of Th17 cells in colonic lamina propria. Intestinal bacteria are responsible for the development of colitis, because their depletion with antibiotics prevented the development of colitis in RBP-JΔIEC mice. Furthermore, bacterial translocation was evident in the colonic mucosa of RBP-JΔIEC mice before the onset of colitis, suggesting attenuated epithelial barrier functions in these mice. Indeed, RBP-JΔIEC mice displayed increase in intestinal permeability after rectal administration of FITC-dextran. In addition to the defect in physical barrier, loss of Notch signaling led to arrest of epithelial cell turnover caused by downregulation of Hes1, a transcriptional repressor of p27Kip1 and p57Kip2. Thus, epithelial cell-intrinsic Notch signaling ensures integrity and homeostasis of IEC, and this mechanism is required for containment of intestinal inflammation.