The intestinal tract contains many commensal bacteria that modulate various physiological host functions. Dysbiosis of commensal bacteria triggers dysfunction of the intestinal epithelial barrier, leading to the induction or aggravation of intestinal inflammation. To elucidate whether microRNA plays a role in commensal microbiome-dependent intestinal epithelial barrier regulation, we compared transcripts in intestinal epithelial cells (IECs) from conventional and germ-free mice and found that commensal bacteria induced the expression of miR-21-5p in IECs. miR-21-5p increased intestinal epithelial permeability and up-regulated ADP ribosylation factor 4 (ARF4), a small GTPase, in the IEC line Caco-2. We also found that ARF4 expression was up-regulated upon suppression of phosphatase and tensin homolog () and programmed cell death 4 (), which are known miR-21-5p targets, by RNAi. Furthermore, ARF4 expression in epithelial cells of the large intestine was higher in conventional mice than in germ-free mice. ARF4 suppression in the IEC line increased the expression of tight junction proteins and decreased intestinal epithelial permeability. These results indicate that commensal microbiome-dependent miR-21-5p expression in IECs regulates intestinal epithelial permeability via ARF4, which may therefore represent a target for preventing or managing dysfunction of the intestinal epithelial barrier.

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α-Defensin 5 is important to both maintenance of a gut microbiota and host immunity. While previous reports have shown that gut bacteria are able to upregulate α-defensin 5 through Toll-like receptor signaling, we demonstrate here that α-defensin 5 expression can also be regulated by microbial metabolites. Among these, lactate appeared to significantly suppress α-defensin 5 gene transcription. Actually, fractions of <3 kD compounds obtained from the ceca of SPF mice were suppressed α-defensin 5 gene transcription at specific concentrations. Our results also suggest that cecal content may include as yet unidentified factors that can enhance α-defensin 5 expression. Our data point to a novel function for the gut microbial metabolites in controlling the expression of antimicrobial peptides in the intestine.

Immune responses against gut microbiota should be minimized to avoid unnecessary inflammation at mucosal surface. In this study, we analyzed the expression patterns of Toll-interacting protein (Tollip), an inhibitor of TLRs and IL-1 family cytokine-related intracellular signaling, in intestinal epithelial cells (IECs). Comparable mRNA expression was observed in murine small and large IECs (S-IECs and L-IECs). However, Tollip protein was only detected in L-IECs, but not in S-IECs. Similar results were obtained in germ-free mice, indicating that L-IEC-specific TOLLIP expression does not depend on bacterial colonization. Next, to understand the mechanisms underlying the post-transcriptional repression of Tollip, 3´-UTR-mediated translational regulation was evaluated. The region +1876/+2398 was responsible for the repression of Tollip expression. This region included the target sequence of miR-31. The inhibition of miR-31 restored the 3´-UTR-meditaed translational repression. In addition, miR-31 expression was significantly higher in S-IECs than in L-IECs, suggesting that miR-31 represses the translation of Tollip mRNA in S-IECs. Collectively, we conclude that the translation of Tollip is inhibited in S-IECs, at least in part, by miR-31 to yield L-IEC-specific high-level expression of the Tollip protein, which may contribute to the maintenance of intestinal homeostasis.

The crystal structure of a high-temperature disordered form, y form, ofp-chlorobenzamide (C7H~CINO), and a reversible solid-phase transformation occurring between a and y forms at about 44 °C were investigated by X-rays. Crystal data at 50°C are: a = 5.015 (4), b = 5.530 (4), c = 14.707 (7) A, a = 97.53 (6), fl = 113.03 (6), y = 95.67 (6) °, Z = 2, triclinic with space group Pi. Intensity data were collected by the equiinclination Weissenberg technique. The photographs showed a marked diffuse scattering associated with reciprocal lattice points with h = n + ~ (n integer). The 'averaged structure' was described in terms of four structural units; three of them, P, Q and R, with equal proportions are deduced from the structure of the a form by taking their unit-cell origins at 0, ~a and ~}a along a, and the remaining unit, V, has positional parameters very close to the mean values of those for the above three units. The final R value was 0.113 with the probability 0.75 of finding V in the crystal. The diffuse scattering patterns were well interpreted in terms of a random distribution of four kinds of 'domains', each. consisting of any one of the above four structures. The domain containing 216 molecules has dimensions 15, 66 and 58/k along a, b and e' respectively, where c' = 6a + 9b + 3e. A plausible process for the phase transformation occurring between the a and y forms consists of two types of molecular movements: one is reorientation and repositioning of molecules in a molecular chain along a, and the other is cooperative displacements of the chains along a. It was found that the y form undergoes a reversible solid-phase transformation at about 70°C to another disordered form, form.