Michio Onizawa

Signaling pathway via TNF-α/NF-κB in intestinal epithelial cells may be directly involved in colitis-associated carcinogenesis

Treatment with anti-TNF-alpha MAb has been accepted as a successful maintenance therapy for patients with inflammatory bowel diseases (IBD). Moreover, it has been recently reported that blockade of TNF receptor (TNFR) 1 signaling in infiltrating hematopoietic cells may prevent the development of colitis-associated cancer (CAC). However, it remains unclear whether the TNF-alpha signaling in epithelial cells is involved in the development of CAC. To investigate this, we studied the effects of anti-TNF-alpha MAb in an animal model of CAC by administration of azoxymethane (AOM) followed by sequential dextran sodium sulfate (DSS) ingestion. We observed that the NF-kappaB pathway is activated in colonic epithelia from DSS-administered mice in association with upregulation of TNFR2 rather than TNFR1. Immunoblot analysis also revealed that the TNFR2 upregulation accompanied by the NF-kappaB activation is further complicated in CAC tissues induced in AOM/DSS-administered mice compared with the nontumor area. Such NF-kappaB activity in the epithelial cells is significantly suppressed by the treatment of MP6-XT22, an anti-TNF-alpha MAb. Despite inability to reduce the severity of colitis, sequential administration of MP6-XT22 reduced the numbers and size of tumors in association with the NF-kappaB inactivation. Taken together, present studies suggest that the TNFR2 signaling in intestinal epithelial cells may be directly involved in the development of CAC with persistent colitis and imply that the maintenance therapy with anti-TNF-alpha MAb may prevent the development of CAC in patients with long-standing IBD.

A20 Restricts Ubiquitination of Pro-Interleukin-1β Protein Complexes and Suppresses NLRP3 Inflammasome Activity

Inappropriate inflammasome activation contributes to multiple human diseases, but the mechanisms by which inflammasomes are suppressed are poorly understood. The NF-κB inhibitor A20 is a ubiquitin-modifying enzyme that might be critical in preventing human inflammatory diseases. Here, we report that A20-deficient macrophages, unlike normal cells, exhibit spontaneous NLRP3 inflammasome activity to LPS alone. The kinase RIPK3, but not the adaptor MyD88, is required for this response. In normal cells, A20 constitutively associates with caspase-1 and pro-IL-1β, and NLRP3 activation further promotes A20 recruitment to the inflammasome. Pro-IL-1β also co-immunoprecipitates with RIPK1, RIPK3, caspase-1, and caspase-8 in a complex that is modified with K63-linked and unanchored polyubiquitin. In A20-deficient macrophages, this pro-IL-1β-associated ubiquitination is markedly increased in a RIPK3-dependent manner. Mass spectrometric and mutational analyses reveal that K133 of pro-IL-1β is a physiological ubiquitination site that supports processing. Our study reveals a mechanism by which A20 prevents inflammatory diseases.

The ubiquitin-modifying enzyme A20 restricts ubiquitination of the kinase RIPK3 and protects cells from necroptosis

A20 is an anti-inflammatory protein linked to multiple human diseases; however, the mechanisms by which A20 prevents inflammatory disease are incompletely defined. We found that A20-deficient T cells and fibroblasts were susceptible to caspase-independent and kinase RIPK3–dependent necroptosis. Global deficiency in RIPK3 significantly restored the survival of A20-deficient mice. A20-deficient cells exhibited exaggerated formation of RIPK1-RIPK3 complexes. RIPK3 underwent physiological ubiquitination at Lys5 (K5), and this ubiquitination event supported the formation of RIPK1-RIPK3 complexes. Both the ubiquitination of RIPK3 and formation of the RIPK1-RIPK3 complex required the catalytic cysteine of A20s deubiquitinating motif. Our studies link A20 and the ubiquitination of RIPK3 to necroptotic cell death and suggest additional mechanisms by which A20 might prevent inflammatory disease.

Myosin light chain kinase expression induced via tumor necrosis factor receptor 2 signaling in the epithelial cells regulates the development of colitis-associated carcinogenesis.

It has been suggested that prolonged inflammatory bowel diseases (IBD) may lead to colitis-associated carcinogenesis (CAC). We previously observed that the NF-κB activation in colonic epithelial cells is associated with increased tumor necrosis factor receptor 2 (TNFR2) expression in CAC development. However, the mechanism by which epithelial NF-κB activation leading to CAC is still unclear. Myosin light chain kinase (MLCK) has been reported to be responsible for the epithelial permeability associated with TNF signaling. Therefore we focused on the role of MLCK expression via TNFR2 signaling on CAC development. Pro-tumorigenic cytokines such as IL-1β, IL-6 and MIP-2 production as well as INF-γ and TNF production at the lamina propria were increased in the setting of colitis, and further in tumor tissues in associations with up-regulated TNFR2 and MLCK expressions in the epithelial cells of a CAC model. The up-regulated MLCK expression was observed in TNF-stimulated colonic epithelial cells in a dose-dependent fashion in association with up-regulation of TNFR2. Silencing TNFR2, but not TNFR1, resulted in restoration of epithelial tight junction (TJ) associated with decreased MLCK expression. Antibody-mediated blockade of TNF signaling also resulted in restoration of TJ in association with suppressed MLCK expression, and interestingly, similar results were observed with suppressing TNFR2 and MLCK expressions by inhibiting MLCK in the epithelial cells. Silencing of MLCK also resulted in suppressed TNFR2, but not TNFR1, expression, suggesting that the restored TJ leads to reduced TNFR2 signaling. Such suppression of MLCK as well as blockade of TNFR2 signaling resulted in restored TJ, decreased pro-tumorigenic cytokines and reduced CAC development. These results suggest that MLCK may be a potential target for the prevention of IBD-associated tumor development.

Amelioration of DSS-induced murine colitis by VSL#3 supplementation is primarily associated with changes in ileal microbiota composition.

Inflammatory bowel diseases encompass gastrointestinal illnesses typified by chronic inflammation, loss of epithelial integrity and gastrointestinal microbiota dysbiosis. In an effort to counteract these characteristic perturbations, we used stem cells and/or a probiotic therapy in a murine model of Dextran Sodium Sulfate induced colitis to examine both their efficacy in ameliorating disease and impact on niche-specific microbial communities of the lower GI tract. Colitis was induced in C57BL/6 mice by administering 3% DSS in drinking water for 10 days prior to administering one of three treatment plans: daily probiotic (VSL#3) supplementation for 3 days, a single tail vein injection of 1x106 murine mesenchymal stem cells, or both. Ileal, cecal and colonic sections were collected for microbiota and histological analyses. Microbiota profiling revealed distinct bacterial community compositions in the ileum, cecum and colon of control untreated animals, all of which were predicted in silico to be enriched for a number of discrete KEGG pathways, indicating compositional and functional niche specificity in healthy animals. DSS-treatment perturbed community composition in all three niches with ileal communities exhibiting the greatest change relative to control animals. Each treatment group exhibited treatment-specific alterations in microbiota composition in the lower GI tract, though disease scores were only improved in VSL#3-treated animals. The ileal microbiota were most profoundly altered in composition in this group of animals and characterized by significant Enterobacteriaceae enrichment compared with colitic mice (P < 0.05).

The Development of Colitogenic CD4+ T Cells Is Regulated by IL-7 in Collaboration with NK Cell Function in a Murine Model of Colitis

We previously reported that IL-7−/−RAG−/− mice receiving naive T cells failed to induce colitis. Such abrogation of colitis may be associated with not only incomplete T cell maintenance due to the lack of IL-7, but also with the induction of colitogenic CD4+ T cell apoptosis at an early stage of colitis development. Moreover, NK cells may be associated with the suppression of pathogenic T cells in vivo, and they may induce apoptosis of CD4+ T cells. To further investigate these roles of NK cells, RAG−/− and IL-7−/−RAG−/− mice that had received naive T cells were depleted of NK cells using anti-asialo GM1 and anti-NK1.1 Abs. NK cell depletion at an early stage, but not at a later stage during colitogenic effector memory T cell (TEM) development, resulted in exacerbated colitis in recipient mice even in the absence of IL-7. Increased CD44+CD62L− TEM and unique CD44−CD62L− T cell subsets were observed in the T cell-reconstituted RAG−/− recipients when NK cells were depleted, although Fas, DR5, and IL-7R expressions in this subset differed from those in the CD44+CD62L− TEM subset. NK cell characteristics were the same in the presence or absence of IL-7 in vitro and in vivo. These results suggest that NK cells suppress colitis severity in T cell-reconstituted RAG−/− and IL-7−/−RAG−/− recipient mice through targeting of colitogenic CD4+CD44+CD62L− TEM and, possibly, of the newly observed CD4+CD44−CD62L− subset present at the early stage of T cell development.

A20 and ABIN-1 synergistically preserve intestinal epithelial cell survival

A20 (TNFAIP3) and ABIN-1 (TNIP1) are candidate susceptibility genes for inflammatory bowel disease and other autoimmune or inflammatory diseases, but it is unclear how these proteins interact in vivo to prevent disease. Here we show that intestinal epithelial cell (IEC)-specific deletion of either A20 or ABIN-1 alone leads to negligible IEC loss, whereas simultaneous deletion of both A20 and ABIN-1 leads to rapid IEC death and mouse lethality. Deletion of both A20 and ABIN-1 from enteroids causes spontaneous cell death in the absence of microbes or hematopoietic cells. Studies with enteroids reveal that A20 and ABIN-1 synergistically restrict death by inhibiting TNF-induced caspase 8 activation and RIPK1 kinase activity. Inhibition of RIPK1 kinase activity alone, or caspase inhibition combined with RIPK3 deletion, abrogates IEC death by blocking both apoptosis and necroptosis in A20 and ABIN-1 double-deficient cells. These data show that the disease susceptibility proteins A20 and ABIN-1 synergistically prevent intestinal inflammation by restricting IEC death and preserving tissue integrity.

B cell activation in the cecal patches during the development of an experimental colitis model

Although previous studies have suggested that appendix seems to be involved in the colitis, the role of this in the pathogenesis remains unclear. In this study, we assessed the importance of appendiceal lymphoid follicles, specifically the cecal patches (CP) in mice, using an experimental colitis model. Treatment with oxazolone resulted in ulcerations particularly at CP with follicular expansion as well as colitis. The colitis was attenuated by either appendectomy or the absence of mature B cells. We therefore established an intravital imaging system accompanied by the fluorescence resonance energy transfer technology to analyze the dynamic immune response of CP B cells. Our observation revealed frequent Ca2+ signaling in CP B cells during the early phase of colitis development. These findings suggested that the CP B cells may be involved in the pathogenesis of colitis including inflammatory bowel diseases in humans.

NK Cells Regulate CD62L− CD44− T Cell Subset in the Development of Pathogenic T Cells in a Murine Model of Colitis

[Background and Aim] Natural killer (NK) cells are associated with regulation of acquired immune responses related to inflammatory diseases such as experimental autoimmune encephalomyelitis and colitis. However, the detail mechanism of NK cell-regulation of pathogenic T cell in inflammation remains unclear. We therefore examine the role of NK cells in a murine model of chronic colitis. [Methods and Results] Anti-asialo GM1 (ASGM1) Ab was injected into RAG deficient (RAG-/-) mice in order to deplete NK cells, followed by adoptive transfer of CD62L+ CD44(naive) T cells. The NK cell depletion interestingly resulted in an increase of CD62LCD44T cells in the spleen and the mesenteric lymph nodes 5 days after T cell reconstitution despite slight exacerbation of colitis compared to mice without NK cell depletion. Flow cytometric analysis showed the CD62Land CD44T cell subset to be Qa-1DR5Lo IL-7R+, while the CD62LCD44+ effecter/memory T cells (TEM) expressed Qa-1+ DR5Hi IL-7R+, suggesting that CD62LCD44T cells are regulated by NK cells in a different mechanism from that of TEM by NK cells via apoptosis. Neither the cytotoxic activity nor the surface markers of NK cells, such as NKG2A/C/E, NKG2D, Ly49, CD94, KLRG1, CD11b and CD27, were affected in the presence or absence of IL-7. Our interest in IL-7 stems from our previous report that IL-7 deficiency completely abrogates colitis in RAG-/mice receiving naive T cells. It had been suggested that such abrogation of colitis may be associated with not only the lack of IL-7 but also another mechanism by which the development of colitis is suppressed at the early stage. Therefore, anti-ASGM1 Ab was injected into RAG-/IL-7-/(DKO) mice receiving naive T cells. The NK cell depletion at the early stage during the induction of colitis resulted in severe colitis in the DKO mice with associated increase in the production of proinflammatory cytokines such as IFN-γ. [Conclusions]Our study suggests that the development of TEM, which induces chronic inflammation, through CD62LCD44T cell subset may be affected by both the presence of NK cells and IL-7, and regulating these mechanisms could be a potential therapeutic target for IBD.

S1743 Myosin Light Chain Kinase is Associated With Disruption of Epithelial Tight Junction in Colitis-Associated Tumor

BACKGROUND: Mast cells play a key role in the pathophysiology of inflammatory bowel disease (IBD). Tranilast, N-(3,4-dimethoxycinnamoyl) anthranilic acid, a mast cell stabilizer, has been empirically used for IBD in Japan. The mechanism of tranilast in the improvement of IBD, however, has not yet been clearly delineated, and requires further investigation. Recently, heme oxygenase (HO)-1 has attracted attention in the pro-inflammationmechanism of tranilast. Aim: To investigate the role of tranilast for the treatment of IBD, and elucidated the mechanism and involvement of HO-1 in this effect, we administered tranilast intrarectally to dextran sulfate sodium (DSS)-induced colitis in mice. METHODS: Colitis was induced in C57BL/6(B6) mice by adding 3% DSS in drinking water ad libium for 5 days. Tranilast was administered by enema on day 0, day 2, and day 4 after induction of colitis. The disease activity index (DAI) and degree of colon injury were determined to know the clinical course of colitis. Toluidine blue staining was carried out to identify the mast cell. Staining for HO1 with immunofluorescence to evaluate the expression of HO-1 in the colon. Total RNA was extracted from colon specimens with quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Tranilast ameliorated DSS colitis clinically and pathologically with decreased number and degranulation of mast cells in the colon of DSS colitis. mRNA expression was increased for tumor necrosis factor(TNF)-α, interferon(IFN)-γ, interleukin (IL)-6, and decreased that for IL-10 in the colon of DSS colitis. In contrast, tranilast markedly decreased expression of mRNAs for the proinflammatory cytokines, and increased that of the anti-inflammatory cytokines. Moreover, tranilast increased HO-1 expression on colonic epithelial cells as well as on colon infiltrating cells of DSS colitis. CONCLUSION: Tranilast ameliorated DSS colitis by regulating mast cell degranulation, decreasing inflammatory cytokines and increasing anti-inflammatory cytokines. Tranilast might exert these effects partly through enhanced HO-1 expression in the colon, suggesting a potential adjunctive therapy for IBD.