Butyric acid, a short-chain fatty acid and one of the main metabolites of intestinal microbial fermentation of dietary fiber, has been shown to have an important role in maintaining the integrity of the intestinal mucosa, while it also has been shown to exert potent anti-inflammatory effects both in vitro and in vivo. However, the precise mechanisms underlying those effects have not been fully identified. We exposed colonic epithelial cells to butyric acid, then extracted total RNA samples, and subsequently hybridized them to microarray chips. Among the upregulated genes, milk fat globuleepidermal growth factor 8 (MFG-E8) was elevated by approximately fivefold. We previously reported that the potential therapeutic benefits of MFG-E8 in intestinal tissue injury were dependent not only on enhanced clearance of apoptotic cells but also required diverse cellular events for maintaining epithelial integrity. The influence of butyric acid on cell function is often attributed to its inhibition of histone deacetylases (HDACs). We found that acetylation on histone 3 lysine 9 (acetyl-H3K9) around the MFG-E8 promoter was significantly increased with butyric acid exposure. Experimental colitis was induced by administration of dextran sodium sulfate (DSS) in C57BL/6N (MFG-E8 þ / þ ) and MFG-E8 À / À mice. Although the colonic bacterial compositions in wild-type (WT) and MFG-E8 À / À mice were not significantly different, intrarectal administration of butyric acid during an acute phase of colitis attenuated intestinal inflammatory parameters and inhibited body weight loss in the WT mice. Our novel findings suggest that butyric acid has significant antiinflammatory effects partly via MFG-E8 on DSS-induced murine experimental colitis.

The lactogenic hormone prolactin (PRL) regulates milk protein gene expression in mammary glands. To maintain homeostatic balance in the body, milk fat globule epidermal growth factor 8 (MFG-E8) is vital for phagocytic clearance of apoptotic cells. We investigated the effects of PRL on MFG-E8 expression in macrophages by evaluating its promoter function. Macrophages were stimulated with PRL, and the expression of MFG-E8 was determined using real-time PCR and Western blotting. The role of MFG-E8 on phagocytosis of apoptotic cells in PRL-treated macrophages was assessed using microscopy, while the response of PRL to MFG-E8 expression was evaluated using luciferase assay. Following treatment with PRL, significant up-regulations of the PRL receptor and MFG-E8 were observed in macrophages, though PRL-treated macrophages more efficiently engulfed apoptotic cells. The results of MFG-E8 promoter analysis showed considerable up-regulation of promoter activity in macrophages following PRL treatment and results from mutation analysis of the MFG-E8 promoter suggested that the C/EBPbeta binding site was responsible for PRL-induced activation of the MFG-E8 promoter. C/EBPbeta activity was found to be up-regulated in PRL-treated cells as revealed by an electrophoretic mobility shift assay (EMSA). In conclusion, PRL is a potent inducer of MFG-E8 expression in macrophages, while its effect is mediated by the presence of a responsive element in the MFG-E8 promoter.

Summary A unique subset of B cells expressing interleukin‐10 (IL‐10) and transforming growth factor‐β (TGF‐β) plays an essential role in preventing inflammation and autoimmunity. We investigated the presence of this cell subset in intestines and its role in the pathogenesis of ileitis using SAMP1/Yit and age‐matched control AKR/J mice. Mononuclear cells were isolated from mesenteric lymph nodes (MLNs) and the expressions of B220, CD1d, CD5, Toll‐like receptor 4 (TLR4) and TLR9 in isolated cells were analysed. Purified B cells were stimulated with lipopolysaccharide (LPS) or CpG‐DNA, then IL‐10 and TGF‐β1 expressions were examined by enzyme immunoassay and flow cytometry. Production of IL‐1β by TLR‐mediated macrophages co‐cultured with or without purified MLN B cells from SAMP1/Yit and AKR/J mice was evaluated. In addition, interferon‐γ (IFN‐γ) production in intestinal T cells co‐cultured with MLN B cells were also assessed in SAMP1/Yit and AKR/J strains. The production levels of IL‐10 and TGF‐β1 stimulated by LPS and CpG‐DNA were significantly lower in B cells separated from MLNs from the SAMP1/Yit strain. B cells expressing IL‐10 and TGF‐β1 were mainly located in a population characterized by the cell surface marker CD1d+. Interleukin‐1β production by TLR‐activated macrophages co‐cultured with MLN B cells from SAMP1/Yit mice was significantly higher than that of those from AKR/J mice. Interestingly, IFN‐γ production by T cells was noted only when they were co‐cultured with SAMP1/Yit but not the AKR/J B cells. These results are the first to show that disorders of regulatory B‐cell function under innate immune activation may cause disease pathogenesis in a murine model of Crohn’s disease.

CD5(+) B cells may be involved in the pathogenesis of UC, and modulation of this subset by corticosteroid therapy may play a role in the treatment of IBD patients.

Abstract. Milk fat globule epidermal growth factor-8 (MFG-E8) promotes phagocytic clearance of apoptotic cells to maintain normal tissue homeostasis. However, its functions in intestinal inflammatory disorders are unknown. Since the pathogenesis of those disorders are due to abnormal interactions between intestinal epithelial cells (IECs) and microbial pathogens, we analyzed the effects of MFG-E8 on IECs to determine its protective role in murine experimental colitis. Expression of α v β 3 -integrin in Colon-26 cells was examined by RT-PCR and immunostaining. Colon-26 cells were pretreated with recombinant wild-type and mutant MFG-E8 proteins, following stimulation with flagellin as an inducer of innate immunity, and the effects of the recombinant proteins on inhibition of nuclear factor-κB (NF-κB) and inflammatory cytokine production in flagellin-stimulated Colon-26 cells were determined using a luciferase assay and EIA, respectively. Experimental colitis was induced in mice by intrarectal administration of trinitrobenzene sulfonic acid (TNBS). Recombinant proteins were then intrarectally administered into TNBS-induced colitic mice, after which disease activity parameters (body weight, colon length, histological score), as well as interleukin (IL)-6 and MIP-2 levels were determined in inflamed tissues. Flagellin-induced inflammatory cytokine production in vitro was significantly downregulated via α v β 3 -integrin following pretreatment with wild-type MFG-E8 due to inhibition of NF-κB activation. In vivo, intrarectal treatment with wild-type MFG-E8, but not its mutant counterpart, significantly inhibited body weight loss, colon shortening and histological inflammation induced by TNBS administration. Our findings suggest that MFG-E8 has anti-inflammatory effects on flagellin-induced inflamed intestinal epithelial cells and may be a useful therapeutic agent for colitis.