Tadakazu Hisamatsu

GRIM-19 Interacts with Nucleotide Oligomerization Domain 2 and Serves as Downstream Effector of Anti-bacterial Function in Intestinal Epithelial Cells

Nucleotide oligomerization domain 2 (NOD2) functions as a mammalian cytosolic pathogen recognition molecule, and variants have been associated with risk for Crohn disease. We recently demonstrated that NOD2 functions as an anti-bacterial factor limiting survival of intracellular invasive bacteria. To gain further insight into the mechanism of NOD2 activation and signal transduction, we performed yeast two-hybrid screening. We demonstrate that GRIM-19, a protein with homology to the NADPH dehydrogenase complex, interacts with endogenous NOD2 in HT29 cells. GRIM-19 is required for NF-κB activation following NOD2-mediated recognition of bacterial muramyl dipeptide. GRIM-19 also controls pathogen invasion of intestinal epithelial cells. GRIM-19 expression is decreased in inflamed mucosa of patients with inflammatory bowel diseases. GRIM-19 may be a key component in NOD2-mediated innate mucosal responses and serve to regulate intestinal epithelial cell responses to microbes.

Interferon-γ Augments CARD4/NOD1 Gene and Protein Expression through Interferon Regulatory Factor-1 in Intestinal Epithelial Cells

Although intestinal epithelial cells appear to be functionally hyporesponsive to normal intestinal flora, human intestinal epithelial cells can respond to enteroinvasive bacteria and induce an inflammatory response. This initial inflammatory response leads to the recruitment of polymorphonuclear leukocytes to the affected site in vitro and in vivo. CARD4/NOD1 is a potential cytosolic receptor for peptidoglycan in mammalian cells that resembles pathogen-resistant proteins of plants. In this context, CARD4/NOD1 is a candidate for a recognition protein of intracellular bacteria or peptidoglycan in intestinal epithelial cells. In this study, we demonstrate that CARD4/NOD1 is constitutively expressed in intestinal epithelial cell lines and isolated primary intestinal epithelial cells. Interferon-γ (IFNγ), which is a potent pro-inflammatory cytokine in intestinal mucosal inflammation, activates CARD4/NOD1 mRNA transcription in a time- and dose-dependent manner and results in augmentation of CARD4/NOD1 protein in SW480 cells. Promoter analysis of CARD4/NOD1 indicates that interferon regulatory factor-1 (IRF-1) binding motif (–791 to –782) is essential for the effect of IFNγ. Nuclear extracts from SW480 cells treated with IFNγ show specific binding of oligonucleotides corresponding to this IRF-1-binding motif, which was supershifted by anti-IRF-1 antibody in electrophoretic mobility shift assay. Overexpression of IRF-1 protein activates the CARD4/NOD1 promoter but not the deletion mutant of the IRF-1-binding site in a co-transfection assay of IRF-1 expression plasmid with CARD4/NOD1 promoter. These studies suggest that the Th1 cytokine, IFNγ, activates CARD4/NOD1 transcription and regulate innate immune mechanisms in the condition of intestinal mucosal inflammation.

Centaurin β1 Down-regulates Nucleotide-binding Oligomerization Domains 1- and 2-dependent NF-κB Activation

Centaurin β1 (CENTB1), a GTPase-activating protein, is a member of the ADP-ribosylation factor family encoded by a gene located on the short arm of human chromosome 17. A yeast two-hybrid screen first suggested a direct interaction between CENTB1 and NOD2. Co-immunoprecipitation experiments confirmed direct interaction between CENTB1 and NOD2 and demonstrated similar interaction between CENTB1 and NOD1. We also demonstrate that endogenous CENTB1 interacts with endogenous NOD2 and NOD1 in SW480 and HT-29 intestinal epithelial cells. CENTB1 partially co-localized with NOD2 and NOD1 proteins in the cytoplasm of mammalian cells. CENTB1 expression in epithelial cells was highly induced by tumor necrosis factor α, interleukin 1β, and the NOD1 and NOD2 ligands (γ-d-glutamyl-meso-diaminopimelic acid and muramyl dipeptide, respectively). In addition, CENTB1 mRNA level is increased in the inflamed mucosa of patients with inflammatory bowel disease. Functionally, CENTB1 overexpression inhibited NOD1- and NOD2-dependent activation of NF-κB, whereas small inhibitory RNA against CENTB1 increased NF-κB activation following NOD1- or NOD2-mediated recognition of the bacterial components γ-d-glutamyl-meso-diaminopimelic acid and muramyl dipeptide, respectively. In contrast, CENTB1 had no effect on NF-κB activation induced by Toll-like receptors. In conclusion, CENTB1 selectively down-regulates NF-κB activation via NODs pathways, creating a “feedback” loop and suggesting a novel role of CENTB1 in innate immune responses to bacteria and inflammatory responses.

MDP-NOD2 stimulation induces HNP-1 secretion, which contributes to NOD2 antibacterial function.

Background:Human neutrophil peptide 1 (HNP‐1) is a defensin with antibacterial activity secreted by various cells as a component of the innate immune host defense. NOD2 is a cytoplasmic protein that recognizes bacterial derived muramyl dipeptide, and is involved in bacterial clearance. The aim of the present study was to investigate the relationship between antibacterial activity of NOD2 and HNP‐1 expression in epithelial cell lines. Methods:Gentamicin protection assay using Salmonella typhimurium was performed in Caco‐2 cells. The mRNA level was determined by quantitative reverse‐transcription polymerase chain reaction (RT‐PCR) and defensin expression was assessed by Western blot and enzyme‐linked immunosorbent assay (ELISA). Nuclear factor‐&kgr;B activation was assessed using pIV luciferase and Renilla plasmids. A NOD2 mutant was generated by site‐directed mutagenesis. Results:Among the defensins tested, only HNP‐1 expression is induced in colonic epithelial model HCT116 cells after MDP‐LD stimulation. HNP‐1 secretion is significantly increased after MDP‐LD stimulation in the cell supernatant of intestinal epithelial cells expressing endogenous NOD2, but not in cells that lack endogenous NOD2 expression. HNP‐1 is required for NOD2‐dependent NF‐&kgr;B activation after MDP‐LD stimulation since hnp‐1 siRNA transfection abrogated the response to MDP‐LD stimulation. The antibacterial function of NOD2 against S. typhimurium was impaired when expression of HNP‐1 was blocked by siRNA. Conclusions:HNP‐1 secretion depends on NOD2 stimulation by MDP‐LD and contributes to antibacterial activity in intestinal epithelial cells expressing endogenous NOD2, but not NOD2 3020insC mutant associated with increased susceptibility to Crohns disease. (Inflamm Bowel Dis 2009)

Advances in Endoscopy in Inflammatory Bowel Disease

Endoscopic assessment of mucosal lesions has emerged as an important concept of disease activity in inflammatory bowel disease (IBD), and recently mucosal healing has generally been regarded as a therapeutic goal not only in ulcerative colitis (UC) but also in Crohn’s disease (CD). Several pieces of evidence have now accumulated to show that mucosal healing determined by endoscopy can alter the course of IBD, as it is associated with sustained clinical remission, and reduced rates of hospitalization and surgical resection. Generally, clinical activity indices established in IBD are mainly determined based on subjective/objective signs and the results of laboratory tests. However, those indices sometimes lead to discrepancy compared with endoscopic indices. Although endoscopy has been rarely investigated as a predictor of the clinical course of IBD, there is now growing evidence that morphological examination, including endoscopy, may help to identify among IBD patients those who should be treated with more intensive treatments. Furthermore, as demonstrated in a recent study assessing early intervention with combination of biologics and immunomodulators, endoscopy may help to select patients who will obtain the best results with early intervention. This chapter summarizes the role of endoscopy in IBD by introducing several modalities such as colonoscopy, balloon-assisted enteroscopy, and video capsule endoscopy, as well as CT colonography and MR enterography.