Chikara Kadota

A20 is an early responding negative regulator of Toll-like receptor 5 signalling in intestinal epithelial cells during inflammation.

Several 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‐κB) 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 receptor‐associated 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.

A Japanese case of eosinophilic esophagitis

Eosinophilic esophagitis (EE) is a rarely diagnosed condition involving eosinophilic infiltration of the esophageal mucosa. Here we present a case of EE in a 69-year-old Japanese man, who presented with abdominal pain, appetite loss, and a history of bronchial asthma. Laboratory findings included peripheral eosinophilia and an increased serum immunoglobulin E level. Computed tomography showed diffuse severe thickening of the esophageal wall, and a barium esophagogram revealed a small caliber of the middle and lower portion of the esophagus, without normal peristaltic contractions. Endoscopy of the esophagus showed a pale mucosa, with adherent whitish exudates resembling fungal infection, and prominent ring-like contractions. Histologic examination of a biopsy specimen revealed marked eosinophil infiltration into the esophageal mucosa. Endoscopic ultrasonography (EUS) demonstrated marked circumferential thickening of the esophageal submucosal layer, and an esophageal manometry study showed a high percentage of ineffective esophageal peristalsis and high-amplitude esophageal body contractions. EUS findings showed no change even after oral corticosteroid therapy, although the histological findings were improved. This is thought to be the first documented Japanese case of EE. EE should be considered in the differential diagnosis in cases of esophageal motility disturbance, even if the patients do not complain of dysphagia.

Epithelial toll-like receptor 5 is constitutively localized in the mouse cecum and exhibits distinctive down-regulation during experimental colitis.

ABSTRACT 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-γ) 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-γ may be involved in down-regulating TLR5 expression.

Down-regulation of single immunoglobulin interleukin-1R-related molecule (SIGIRR)/TIR8 expression in intestinal epithelial cells during inflammation

Single 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)‐α 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‐α 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.

Decoy oligodeoxynucleotide targeting activator protein-1 (AP-1) attenuates intestinal inflammation in murine experimental colitis

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-1β, and TNF-α in colon tissue. Therapeutic effects of AP-1 and NF-κB 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-1β, and TNF-α in colonic tissue of mice with DSS-induced colitis. The AP-1 decoy therapeutic effect was comparable to that of NF-κB 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.

Prolactin induces MFG-E8 production in macrophages via transcription factor C/EBPβ-dependent pathway

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/EBPβ binding site was responsible for PRL-induced activation of the MFG-E8 promoter. C/EBPβ 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.

Alterations of peripheral blood CD5+ B cells in inflammatory bowel disease

Objective. CD5+ B cells comprise a unique subset of B cells that modulates innate as well as autoimmune systems. The aim of this study was to investigate alterations of the circulating CD5+ B-cell subset in patients with inflammatory bowel disease (IBD) by evaluating various clinical parameters, including therapeutic regimens. Material and methods. Thirty-four patients with ulcerative colitis (UC), 19 patients with Crohns disease (CD), and 46 healthy control subjects were enrolled in this study. CD5+ B cells in peripheral blood collected from each subject were analyzed by flow cytometry. Multiple regression analysis was carried out to evaluate the factors related to the circulating CD5+ B-cell subset in the IBD patients. In an in vitro examination, dexamethasone-induced apoptosis in peripheral blood B cells was examined by detecting cell surface binding of the annexin-V antibody. Results. Age and gender in the control subjects did not influence the circulating CD5+ B-cell subset. Multiple regression analysis showed that the presence of UC, corticosteroid therapy, and number of white blood cells in peripheral blood each had a significant influence in decreasing the number of circulating CD5+ B cells in the IBD patients. Furthermore, in vitro results showed that dexamethasone treatment significantly induced apoptosis in CD5+ B cells, though apoptosis was similarly observed in CD5− B cells. Conclusions. 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.

S1741 Milk Fat Globule EGF-8 Attenuates Intestinal Inflammation in Murine Experimental Colitis Via Inhibition of NF-κB Activation

(Background and Aim) Milk fat globule epidermal growth factor-8 (MFG-E8) promotes efficient phagocytic clearance of apoptotic cells to maintain normal tissue homeostasis and modulate immune responses. Colitis is characterized by severe colonic inflammation with marked elevation of proinflammatory responses through involvement of the major transcription factor NF-κB. We investigated the role of MFG-E8 to improve the pathogenesis of murine experimental colitis. (Materials and Methods) Experimental acute colitis was induced in 7-week-old male BALB/c mice by giving a 2.5% dextran sulfate sodium (DSS) solution for 7 days or a single dose intrarectal administration of trinitrobenzene sulfonic acid (TNBS) (100 μl, 100 mg/kg) dissolved in 40% ethanol. MFG-E8 expression in the distal colon was checked during injury and healing stages. Further, murine wild-type and RGD mutant MFGE8 were purified with a mammalian expression system, and the functional effects of purified MFG-E8 on peritoneal macrophages were investigated based on the phagocytic potential of apoptotic thymocytes. Recombinant proteins were injected into the tail veins of mice with colitis induced by DSS or TNBS, after which a disease activity index (body weight, colon length, and histological scores), and pro-inflammatory cytokine and myeloperoxidase (MPO) profiles in inflamed tissues were determined. The In Vitro effects of recombinant MFG-E8 on inhibition of NF-κB and pro-inflammatory cytokine production were also investigated using luciferase, an electrophoreticmobility shift assay (EMSA), and an enzyme immunoassay. (Results)MFG-E8 was elevated during the healing stage after stopping theDSS administration. An increased phagocytic potential of apoptotic thymocytes was found, which indicated the functional efficiency of the purified recombinant MFG-E8. In the experimental colitis model mice, treatment with the wild-type, but not mutant MFG-E8, significantly inhibited weight loss and shortening of the colon induced by DSS or TNBS administration. In addition, histological examinations showed that lamina propria infiltration by polymorphonuclear and mononuclear cells, as well as crypt epithelial damage were markedly decreased in the wildtype recombinant MFG-E8-treated mice. MFG-E8 treatment also significantly decreased the contents of IL-1β, TNF-α, and MPO in colonic tissues of DSSand TNBS-induced colitis mice. In Vitro, transcription factor NF-κB and its regulated pro-inflammatory cytokines were inhibited in MFG-E8 treated cultured macrophages. (Conclusion) Our findings suggest that MFG-E8 is a potent therapeutic agent to attenuate intestinal inflammation in murine experimental colitis.