Akira Mukai

Indigenous opportunistic bacteria inhabit mammalian gut-associated lymphoid tissues and share a mucosal antibody-mediated symbiosis

The indigenous bacteria create natural cohabitation niches together with mucosal Abs in the gastrointestinal (GI) tract. Here we report that opportunistic bacteria, largely Alcaligenes species, specifically inhabit host Peyers patches (PPs) and isolated lymphoid follicles, with the associated preferential induction of antigen-specific mucosal IgA Abs in the GI tract. Alcaligenes were identified as the dominant bacteria on the interior of PPs from naïve, specific-pathogen-free but not from germ-free mice. Oral transfer of intratissue uncultured Alcaligenes into germ-free mice resulted in the presence of Alcaligenes inside the PPs of recipients. This result was further supported by the induction of antigen-specific Ab-producing cells in the mucosal (e.g., PPs) but not systemic compartment (e.g., spleen). The preferential presence of Alcaligenes inside PPs and the associated induction of intestinal secretory IgA Abs were also observed in both monkeys and humans. Localized mucosal Ab-mediated symbiotic immune responses were supported by Alcaligenes-stimulated CD11c+ dendritic cells (DCs) producing the Ab-enhancing cytokines TGF-β, B-cell-activating factor belonging to the TNF family, and IL-6 in PPs. These CD11c+ DCs did not migrate beyond the draining mesenteric lymph nodes. In the absence of antigen-specific mucosal Abs, the presence of Alcaligenes in PPs was greatly diminished. Thus, indigenous opportunistic bacteria uniquely inhabit PPs, leading to PP-DCs-initiated, local antigen-specific Ab production; this may involve the creation of an optimal symbiotic environment on the interior of the PPs.

Functional analysis of agalactosyl IgG in inflammatory bowel disease patients

Background: Agalactosyl immunoglobulin (Ig) G is increased in inflammatory bowel disease (IBD) similarly to rheumatoid arthritis (RA). The lectin complement pathway is shown to be activated through association of agalactosyl IgG with mannan‐binding lectin (MBL) in RA. Functional changes of IgG agalactosylation in IBD, however, have not yet been clarified. Methods: The ratio of the agalactosyl/non‐agalactosyl fraction in fucosylated IgG oligosaccharides (G0F/G2F) and serum MBL levels were analyzed in 59 patients with Crohns disease (CD), 64 ulcerative colitis (UC), and 39 healthy volunteers (HV). The MBL levels associated with serum IgG were analyzed by enzyme‐linked immunosorbent assay. MBL expression in the intestinal mucosa was analyzed by immunohistochemistry. Phagocytosis of sheep red blood cells (SRBC) reacted with either an agalactosyl or non‐agalactosyl SRBC‐specific IgG antibody was determined by flow cytometry. Results: The serum MBL levels were not significantly different among CD, UC, or HV. In patients with CD, the serum MBL levels were negatively correlated with the Crohns Disease Activity Index (CDAI). The levels of MBL associated with agalactosyl IgG were not different from those associated with non‐agalactosyl IgG. Immunoreactivity to MBL was less in the inflamed mucosa compared with the noninflamed mucosa. Phagocytic activity of SRBC was significantly higher in the presence of agalactosyl IgG compared to non‐agalactosyl IgG. Conclusions: Agalactosyl IgG oligosaccharides enhanced antibody‐dependent phagocytosis in vitro but did not activate the lectin complement pathway. Oligosaccharide alterations of IgG are not only a marker of IBD but also functionally modulate the immune function of IBD. (Inflamm Bowel Dis 2010;)

Deficiency of N‐acetylgalactosamine in O‐linked oligosaccharides of IgA is a novel biologic marker for Crohn's disease

Background: Ideal biomarkers are required to be developed for the diagnosis and prediction of the treatment of inflammatory bowel disease (IBD). We have reported that alteration of N‐linked oligosaccharides of immunoglobulin (Ig) G is a novel diagnostic marker of IBD. Oligosaccharide alterations of IgA, however, have not been investigated in IBD patients. Methods: N‐ and O‐linked oligosaccharides of serum IgA purified from 32 patients with Crohns disease (CD), 30 patients with ulcerative colitis (UC), and 30 healthy volunteers (HV) were analyzed with high‐performance liquid chromatography and mass spectrometry. Enzymes related to oligosaccharide attachment were investigated. Results: N‐linked oligosaccharides of IgA were not different between IBD and HV. In contrast, the number of N‐acetylgalactosamines per hinge glycopeptide (GalNAc/HP) in the O‐linked oligosaccharides of IgA was significantly decreased in patients with CD compared with UC and HV. GalNAc/HP had high sensitivity and specificity for discriminating between CD and HV based on receiver operating characteristic analysis. Lower GalNAc/HP was associated with more severe disease activity of CD. Changes in GalNAc/HP levels in 6 weeks after treatment with infliximab were associated with the clinical activity of CD at 30 weeks. GalNAc transferase expression of naïve B cells and extent of GalNAc attachment in IgA were significantly decreased by interleukin‐21 in vitro. Conclusions: The number of GalNAc attached in the IgA O‐linked glycans of CD patients was significantly decreased, and strongly correlated with the clinical activity. Alterations of GalNAc attachment in IgA could be useful as a novel diagnostic and prognostic marker of CD. (Inflamm Bowel Dis 2012;)

Indigo Naturalis ameliorates murine dextran sodium sulfate-induced colitis via aryl hydrocarbon receptor activation.

BackgroundIndigo Naturalis (IN) is used as a traditional herbal medicine for ulcerative colitis (UC). However, the mechanisms of action of IN have not been clarified. We aimed to evaluate the efficacy of IN for ameliorating colonic inflammation. We further investigated the mechanisms of action of IN.MethodsColitis severity was assessed in dextran sodium sulfate-induced colitis and trinitrobenzene sulfonic acid-induced colitis models with or without the oral administration of IN or indigo, which is a known major component of IN. Colonic lamina propria (LP) mononuclear cells isolated from IN-treated mice were analyzed with quantitative reverse transcription polymerase chain reaction (qRT-PCR) and flow cytometry. LP and splenic mononuclear cells cultured in vitro with IN or indigo were also analyzed. The role of the candidate receptor for indigo, the aryl hydrocarbon receptor (AhR), was analyzed using Ahr-deficient mice.ResultsColitis severity was significantly ameliorated in the IN and indigo treatment groups compared with the control group. The mRNA expression levels of interleukin (Il)-10 and Il-22 in the LP lymphocytes were increased by IN treatment. The treatment of splenocytes with IN or indigo increased the expression of anti-inflammatory cytokines and resulted in the expansion of IL-10-producing CD4+ T cells and IL-22-producing CD3−RORγt+ cells, but not CD4+Foxp3+ regulatory T cells. The amelioration of colitis by IN or indigo was abrogated in Ahr-deficient mice, in association with diminished regulatory cytokine production.ConclusionsIN and indigo ameliorated murine colitis through AhR signaling activation, suggesting that AhR could be a promising therapeutic target for UC.

Regulation of anergy-related ubiquitin E3 ligase, GRAIL, in murine models of colitis and patients with Crohn’s disease

BackgroundAbrogating tolerance is a critical step in the pathogenesis of Crohn’s disease (CD). T cell-anergy is one of the main mechanisms of tolerance and is regulated by the gene related to anergy in lymphocytes (GRAIL). This study investigated the expressions and regulation of GRAIL in CD and murine colitis models.MethodsExpressions of GRAIL mRNA and protein in CD4+ T cells were investigated in the peripheral blood and mucosal tissues of patients with CD, mice with dextran sodium salt (DSS)-induced colitis, and Il-10-deficient mice. MicroRNAs responsible for the regulation of GRAIL were examined by miRNA microarray. GRAIL-overexpressing T cells were intravenously injected in mice with DSS-induced colitis.ResultsThe GRAIL expression was higher in the lamina propria (LP) CD4+ T cells of CD patients than of the control subjects, while it was lower in the peripheral blood CD4+ T cells of the CD patients than of the control subjects. The GRAIL mRNA expression was lower, but the GRAIL protein expression was higher in the LP of colitic mice than that of non-colitic mice. The miRNA microarray identified miR-290-5p as an miRNA that inhibits expression of the GRAIL protein and that is highly expressed in the LP of non-colitic mice. GRAIL-expressing T cells expressed regulatory T cell markers and showed suppressive effects in murine DSS-induced colitis.ConclusionsOur results show that expression of GRAIL is uniquely regulated by the specific miRNA in the intestinal mucosa, and suggest that GRAIL may associate with the pathophysiology of CD.

Incidence of gastric adenocarcinoma among lesions diagnosed as low‐grade adenoma/dysplasia on endoscopic biopsy: A multicenter, prospective, observational study

Differentiation between gastric adenocarcinoma and low‐grade adenoma/dysplasia (LGA) on endoscopic forceps biopsy is difficult. We aim to clarify the incidence of carcinoma in specimens, obtained by endoscopic resection (ER), from cases that had been diagnosed as LGA (Vienna category 3) on endoscopic biopsy.

Peyer's Patches Play a Protective Role in Nonsteroidal Anti-inflammatory Drug-induced Enteropathy in Mice

Background:Peyers patches (PPs) play a major role in mucosal immunity. However, their roles in nonsteroidal anti-inflammatory drug–induced enteropathy are poorly understood. Methods:Wild-type (WT) and PP-null mice were injected with indomethacin. Twenty-four hours later, the cellular profiles and cytokine levels in the PPs, mesenteric lymph nodes (MLNs), and lamina propria (LP) of the small intestine were measured. WT and PP-null mice were given antibiotics before indomethacin treatment to evaluate enteropathy. Naive CD4+ T cells were co-cultured with CD103+ or CD103− dendritic cells (DCs) to analyze the interleukin (IL)-10 expression levels. Finally, WT mice adoptively transferred with CD103+ or CD103− DCs were injected with indomethacin. Results:The proportion of CD103+ DCs in PPs and MLNs and IL-10-expressing CD4+ T cells of PPs and the LP increased after indomethacin treatment. The PP-null mice showed greater indomethacin-induced enteropathy, fewer CD103+ DCs in their MLNs, and lower proportion of IL-10-expressing CD4+ T cells of their LP than WT mice, regardless of commensal bacteria. Naive splenic CD4+ T cells co-cultured with CD103+ DCs isolated from the MLNs of indomethacin-injected WT mice produced a higher amount of IL-10 compared with those co-cultured with CD103− DCs. Moreover, WT mice that received CD103+ DCs showed milder enteropathy than those that received CD103− DCs. Conclusions:PPs play a protective role in nonsteroidal anti-inflammatory drug–induced enteropathy, and this protection is associated with an increase in CD103+ DCs and IL-10-producing CD4+ T cells in the intestine, independent of the commensal bacteria.

Sa1813 Peyer's Patches Play a Protective Role in NSAIDs-Induced Enteropathy

G A A b st ra ct s in the mucous gel layer and a corresponding decrease in Bacteroidetes when compared to the luminal communities. The pattern of segregation was less apparent in the distal inflamed regions of the individual with ulcerative colitis and family-level analysis confirmed increased mucosa-associated levels of Clostridiaceae and Peptostreptococcaceae. Conclusion: These results support the concept of a spatially-stable, individual-specific cohort of bacteria with a consistent luminal-mucosal segregation. This provides a platform for the programmed assessment of luminal-mucosal ecology, which may add another dimension to our understanding of the propagation of inflammation in inflammatory bowel disease.