MFG-E8 (milk fat globule-epidermal growth factor 8) deficiency is strongly associated with acquisition of immune-mediated disorders due to the loss of tissue homeostasis. However, comparatively little is known regarding its functions in gastrointestinal tract disorders, in which immune homeostasis is a major concern. Herein, we report altered MFG-E8 expression in inflamed colons during the acute phase of murine experimental colitis and found that treatment with recombinant MFG-E8, but not its arginine-glycine-aspartate mutant counterpart, ameliorated colitis by reducing inflammation and improving disease parameters. To reveal the MFG-E8-mediated antiinflammatory mechanism, we employed an in vitro system, which showed the down-regulation of NF-κB in an LPS-dependent manner. Additionally, MFG-E8 altered αvβ3 integrin-mediated focal adhesion kinase phosphorylation by impeding the binding of one of its potent ligands osteopontin, which becomes activated during colitis. Taken together, our results indicated that MFG-E8 has a novel therapeutic potential for treatment of colitis.

SummarySeveral negative regulatory mechanisms control Toll-like receptor (TLR)-mediated inflammatory responses and restore immune system balance, including the zinc-finger protein A20, a negative regulator of TLR signalling that inhibits nuclear factor kappa B (NF-kB) activity. In the present study, we investigated TLR-5-mediated A20 expression and its role in intestinal epithelial cells (IECs) during inflammation. HCT-15 and HT-29 cells were stimulated with flagellin, then the expressions of A20, interleukin-1 receptorassociated kinase (IRAK-M) and Tollip were evaluated using RNase protection assay. Furthermore, experimental colitis was induced in tlr4-deficient CH3/HeJ mice by administration of dextran sodium sulphate (DSS), then flagellin was injected anally, and the colonic expression of A20 was examined by real-time polymerase chain reaction (PCR) and immunohistochemistry. To confirm flagellin-induced expression of A20, we employed an organ culture system. The role of A20 in flagellin-induced tolerance induction was evaluated in vitro, using a gene knock-down method targeting A20. A20 expression increased rapidly and peaked at 1 h after flagellin stimulation in cultured IECs, then declined gradually to the basal level. In vivo, anal injection of flagellin induced epithelial expression of A20 in injured colonic tissue, whereas flagellin did not cause a significant increase in A20 expression in non-injured normal tissue, which was also confirmed in vitro using the organ culture system. Gene knock-down using A20 siRNA did not influence tolerance induced by restimulation with flagellin. A20 is an early response negative regulator of TLR-5 signalling in IECs that functions during intestinal inflammation. Our results provide new insights into the negative feedback regulation of TLR-5 signalling that maintains the innate immune system in the gut.

SummarySingle immunoglobulin (Ig) interleukin-1R-related molecule (SIGIRR) is an Ig-like membrane protein critical for negative regulation of Toll-like receptor (TLR)-4-mediated signalling. We investigated SIGIRR expression and its regulation mechanism in intestinal epithelial cells (IECs) during inflammation. Endoscopic biopsy specimens were obtained from active and inactive colonic mucosa of ulcerative colitis (UC) patients, then SIGIRR expression was examined using real-time polymerase chain reaction (PCR) and immunohistochemistry (IH). Mice experimental colitis models were established by administrations of sulphonic acid (TNBS) and dextran sodium sulphate (DSS), and epithelial expression of SIGIRR was examined using real-time PCR, IH and flow cytometry. The effects of lipopolysaccharide (LPS) and tumour necrosis factor (TNF)-a on SIGIRR expression were evaluated in vitro using cultured IECs. To elucidate SIGIRR expression regulation in IECs, binding ability of the transcription factor SP1 at the responsive element of the SIGIRR promoter was examined using gel-shift and chromatin immunoprecipitation (ChIP) assays. In human colonic samples, SIGIRR was expressed mainly in IECs at levels significantly higher in inactive compared to active mucosa. In the mice, SIGIRR colonic expression decreased rapidly after colitis development and returned gradually to basal levels. Experimental colitis-mediated down-regulation of SIGIRR in IECs was also confirmed by IH and flow cytometry results. Further, inflammatory conditions induced by TLR ligands and TNF-a caused significant down-regulation of SIGIRR expression in IECs, which was dependent upon decreased SP1 binding at the responsive element of the SIGIRR promoter. We found that SIGIRR is expressed in IECs and serves as a negative regulator to maintain gut innate immunity, which is down-regulated during inflammation by inhibition of an SP1-mediated pathway.

We recently demonstrated that the pattern recognition receptors (PRRs) toll-like receptor 2 (TLR2), TLR4, and CD14 are expressed in mouse colonic epithelium in a compartmentalized manner. Here we report the localization of TLR5, the receptor for bacterial flagellin, and its distinctive down-regulation during experimental colitis. Guts from normal BALB/c mice and those with dextran sodium sulfate (DSS)-induced colitis were compared. Each gut was divided into seven segments (stomach, small intestine [three parts], and colon [three parts]), and epithelial cells and crypt units were collected by scraping and EDTA treatment, respectively. Northern blotting showed that TLR5 mRNA was preferentially expressed in the epithelium of the proximal colon in normal mice. Laser capture microdissection coupled to reverse transcriptase PCR confirmed this localization. TLR5 protein expression reflected mRNA expression, as evidenced by Western blotting. In mice with acute colitis, inflammation occurred mainly in the distal colon. Interestingly, while TLR2, TLR4, and CD14 were up-regulated in the inflamed colon, TLR5 was down-regulated at both the mRNA and protein levels. Decreased TLR5 expression was more evident during chronic colitis. Additional in vitro studies using a mouse cell line, Colon-26, showed that gamma interferon (IFN-␥) time-and dose-dependently down-regulates TLR5. In conclusion, epithelial cells, mainly in the proximal colon, constitutively express TLR5. TLR5 expression is down-regulated in vivo during acute and chronic DSS-induced colitis, in contrast to the expression of TLR2, TLR4, and CD14. The mechanism governing TLR5 regulation may therefore differ from that controlling other PRRs. Finally, IFN-␥ may be involved in down-regulating TLR5 expression.The concept of a highly efficient innate immune system that recognizes potential pathogens by detecting lipopolysaccharides (LPSs), peptidoglycans, lipopeptides, flagellin, or many other highly preserved and unvarying structural molecules (27) is now widely accepted, and innate immunity acts as the firstline defense against pathogenic microorganisms in mammals. Although the innate immune system is basically activated by pathogen-related molecules, this activation seems to be driven by several kinds of strategically regulated pattern recognition receptors (PRRs). Because of the variety of PRRs and their sophisticated intracellular signal transduction systems, the innate immune system is increasingly regarded as highly complex.Since the discovery of human toll (22), several new receptors and ligands have been recognized, and more are constantly being added to the arena. Toll-like receptors (TLRs) are transmembrane proteins that share a leucine-rich repeat ectodomain, which confers high specificity for particular ligands (4). TLRs also share an intracellular domain which contains a toll-interleukin-1 (IL-1) receptor homology domain common to the IL-1 receptor family and the IL-18 receptor (36). Ligand binding to TLRs triggers activation of the IL-1 receptor-associated k...

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.

Regardless of how inflammatory bowel disease (IBD) is defined, the term "genetic susceptibility" is always included. Due to substantial progress in the characterization of susceptible genes that interact with environmental influences, a number of review articles offering the latest insights continue to be presented. To date, more than 30 novel IBD susceptible loci have been found, while several promising associations between IBD and gene variants have also been identified and replicated effectively. The present review highlights recent insights regarding linkage analysis and genome-wide association presented in studies of IBD susceptible genes, which provide additional evidence supporting their involvement in disease pathogenesis, based on linking to innate immune systems as a result of interactions with intestinal microbial flora. An improved understanding of IBD genetics will promote the identification of novel therapeutic agents, making it possible to identify environmental factors related to intestinal inflammation.

Various therapies are used for inflammatory bowel diseases (IBD), though none seem to be extremely effective. AP-1 is a major transcription factor that upregulates genes involved in immune and proinflammatory responses. We investigated decoy oligodeoxynucleotide (ODN) targeting AP-1 to prevent dextran sulfate sodium (DSS)-induced colitis in mice. Functional efficacies of synthetic decoy and scrambled ODNs were evaluated in vitro by a reporter gene luciferase assay and measuring flagellin-induced IL-8 expression by HCT-15 cells transfected with ODNs. Experimental colitis was induced in mice with a 2.5% DSS solution in drinking water for 7 days, and decoy or scrambled ODNs were intraperitoneally injected from days 2 to 5. Colitis was assessed by weight loss, colon length, histopathology, and detection of myeloperoxidase (MPO), IL-1b, and TNF-a in colon tissue. Therapeutic effects of AP-1 and NF-kB decoy ODNs were compared. Transfection of AP-1 decoy ODN inhibited AP-1 transcriptional activity in reporter assays and flagellin-induced IL-8 production in vitro. In mice, AP-1 decoy ODN, but not scrambled ODN, significantly inhibited weight loss, colon shortening, and histological inflammation induced by DSS. Further, AP-1 decoy ODN decreased MPO, IL-1b, and TNF-a in colonic tissue of mice with DSS-induced colitis. The AP-1 decoy therapeutic effect was comparable to that of NF-kB decoy ODN, which also significantly decreased intestinal inflammation. Double-strand decoy ODN targeting AP-1 effectively attenuated intestinal inflammation associated with experimental colitis in mice, indicating the potential of targeting proinflammatory transcription factors in new therapies for IBD.

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.