Published papers

Background: Oral administration of tacrolimus is an effective remission induction therapy for steroid-refractory/dependent ulcerative colitis (UC). Aim: This study aimed to evaluate the short- as well as medium- and long-term effectiveness of tacrolimus therapy. Methods: The medical records of 51 patients treated with tacrolimus for UC at our hospital between July 2009 and December 2011 were reviewed retrospectively. Clinical remission and improvement were defined as a Lichtiger score of 4 or less and as a Lichtiger score of ≤. 10 and a reduction in the score of ≥. 3 compared with the baseline score, respectively. Endoscopic findings were evaluated based on the endoscopic activity index and Mayo endoscopic score. Results: The clinical effectiveness combining clinical remission and improvement was observed in 62.7% of the patients at 3. months. Thirty-six patients underwent colonoscopy at 3. months, and 12 (33.3%) and 10 patients (27.8%) showed Mayo endoscopic scores of 0 and 1, respectively. On Kaplan-Meier analysis, the overall percentage of event-free survivors, who did not require colectomy nor switching to other induction therapy such as infliximab, was 73.0% at 6. months, 49.9% at 1. year, and 37.8% at 2. years. Patients with a Mayo endoscopic score of 0-1 at 3. months showed significantly better medium- and long-term prognosis than those with a score of 2-3 (p<. 0.01). All adverse events, including infections in 2 patients, were reversible. Conclusions: Tacrolimus therapy was effective for inducing clinical and endoscopic remission of steroid-refractory/dependent UC. Endoscopic improvement was associated with favorable medium- and long-term prognosis. © 2013 European Crohn's and Colitis Organisation.

Colitogenic memory CD4+ T cells are important in the pathogenesis of inflammatory bowel disease (IBD). Although memory stem cells with high survival and self-renewal capacity were recently identified in both mice and humans, it is unclear whether a similar subset is present in chronic colitis mice. We sought to identify and purify a long-lived subset of colitogenic memory CD4+ T cells, which may be targets for treatment of IBD. A long-lived subset of colitogenic memory CD4+ T cells was purified using a long-term culture system. The characteristics of these cells were assessed. Interleukin (IL)-7 promoted the in vitro survival for >8 weeks of lamina propria (LP) CD4+ T cells from colitic SCID mice previously injected with CD4+CD45RBhigh T cells. These cells were in a quiescent state and divided a maximum of 5 times in 4 weeks. LP CD4+ T cells expressed higher levels of Bcl-2, integrin-α4β7, CXCR3 and CD25 after than before culture, as well as secreting high concentrations of IL-2 and low concentrations of IFN-γ and IL-17 in response to intestinal bacterial antigens. LP CD4+ T cells from colitic mice cultured with IL-7 for 8 weeks induced more severe colitis than LP CD4+ T cells cultured for 4 weeks. We developed a novel culture system to purify a long-lived, highly pathogenic memory subset from activated LP CD4+ T cells. IL-7 promoted long-term in vitro survival of this subset in a quiescent state. This subset will be a novel, effective target for the treatment of IBD. © 2013 Elsevier B.V.

Interleukin 18 (IL-18) is an IL-1 super family cytokine that is involved in infection, inflammation and autoimmune diseases. Mounting evidence suggests that IL-18 exert a dual role in inflammation and homeostasis. IL-18 can act as a promoter of T cell immunities, such as type 1 and 17 helper T cell responses, and thus enhances T cell-mediated inflammation, whereas IL-18 increases the barrier function and regeneration of epithelial cells and protects the host from inflammatory stimuli. Although the functional role of IL-18 in regulation of inflammation remains controversial, accumulating evidence indicates the contribution of IL-18 to the pathogenesis of inflammatory bowel diseases (IBD). For example, levels of serum and/or mucosal IL-18 and IL-18 binding protein are elevated in the patients with IBD. Furthermore, polymorphisms in IL-18 and IL-18-related molecules, such as the IL-18 receptor and/or an IL-18 activator NLRP3, genes are found in the patients with IBD. Thus, these preclinical data imply that IL-18 can be a novel therapeutic target for the treatment of IBD. In this review, we focus on IL-18 biology and physiological roles in animal models and human IBD, to provide an outline for development of IL-18 blockade strategies.

Objective Interleukin (IL)-7 is mainly produced in bone marrow (BM) that forms the niche for B cells. We previously demonstrated that BM also retains pathogenic memory CD4 T cells in murine models of inflammatory bowel disease (IBD). However, it remains unknown whether BM-derived IL-7 is sufficient for the development of IBD and which cells form the niche for colitogenic memory CD4 T cells in BM. Design To address these questions, we developed mice in which IL-7 expression was specific for BM, and identified colitis-associated IL-7-expressing mesenchymal stem cells (MSC) in the BM. Results IL-7-/-×RAG-1-/- mice injected with BM cells from IL-7+/+×RAG-1-/- mice, but not from IL-7-/-×RAG- 1-/- mice, expressed IL-7 in BM, but not in their colon, and developed colitis when injected with CD4+CD45RBhigh T cells. Cultured BM MSC stably expressed a higher level of IL-7 than that of primary BM cells. IL-7-sufficient, but not IL-7-deficient, BM MSC supported upregulation of Bcl-2 in, and homeostatic proliferation of, colitogenic memory CD4 T cells in vitro. Notably, IL-7-/-×RAG-1-/- mice transplanted with IL-7- sufficient, but not IL-7-deficient, BM MSC expressed IL-7 in BM, but not in their colon, and developed colitis when transplanted with CD4+CD45RBhigh T cells. Conclusions We demonstrate for the first time that BM MSC are a major source of IL-7 and play a pathological role in IBD by forming the niche for colitogenic CD4 memory T cells in BM.

Masticatory function is significantly lower in individuals with malocclusion than in those with normal occlusion. Although several studies suggest that masticatory function influences gastrointestinal digestive function, the relationship between malocclusion and gastrointestinal symptoms has not been studied extensively. We hypothesised that insufficient masticatory function would increase the functional burden of the stomach and have some influence on the gastrointestinal system. The purpose of this study was to investigate masticatory function and gastric emptying rate in subjects with malocclusion. Eleven healthy dentate female volunteers and eleven female patients with maloc-clusion underwent a 13C-acetate breath test with a liquid meal. Maximum 13CO2 exhalation time (Tmax) was compared statistically between both groups. Masticatory function was assessed by colour-changeable chewing gum. In addition, the frequency scale for the symptoms of gastroeso-phageal reflux disease (FSSG) and questionnaires on food intake were given to both groups. The mean Tmax of the malocclusion group was significantly longer than that of the normal occlusion group (P = 0·007). Masticatory performance, measured by colour-changeable gum and questionnaires, was significantly lower in the malocclusion group than in the normal occlusion group (P = 0·023, P = 0·003). There was no significant difference in the FSSG results between the two groups (P = 0·262). This study suggested that there was a correlation between malocclusion and gastric emptying function in women. © 2013 John Wiley & Sons Ltd.

The objective of this study was to establish a novel prediction system of drug absorption in humans by utilizing human intestinal tissues. Based on the transport index (TI), a newly defined parameter, calculated by taking account of the change in drug concentrations because of precipitation on the apical side and the amounts accumulated in the tissue and transported to the basal side, the absorbability of drugs in rank order as well as the fraction of dose absorbed (Fa) in humans were estimated. Human intestinal tissues taken from ulcerative colitis or Crohn's disease patients were mounted in a mini-Ussing chamber and transport studies were performed to evaluate the permeation of drugs, including FD-4, a very low permeable marker, atenolol, a low permeable marker, and metoprolol, a high permeable marker. Although apparent permeability coefficients calculated by the conventional equation did not reflect human Fa values for FD-4, atenolol, and metoprolol, TI values were well correlated with Fa values, which are described by 100 · [1 - e - f · (TI - α)]. Based on this equation, Fa values in humans for other test drugs were predicted successfully, indicating that our new system utilizing human intestinal tissues would be valuable for predicting oral drug absorption in humans. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.

Chemokine receptors mediate migration of immune cells into the liver, thereby promoting liver inflammation. C-C motif chemokine receptor (CCR) 9+ macrophages are crucial in the pathogenesis of acute liver inflammation, but the role and underlying mechanisms of this macrophage subset in chronic liver injury and subsequent liver fibrosis are not fully understood. We confirmed that tumor necrosis factor alpha (TNF-α)-producing CCR9+ macrophages accumulated during the initiation of carbon tetrachloride (CCl4)-induced liver injury, and CCR9 deficiency attenuated the degree of liver damage. Accumulation of CCR9+ macrophages persisted prominently during the process of liver fibrosis induced by repetitive CCl4 or thioacetamide (TAA)/leptin administration. Increased CCR9 expression was also found on activated hepatic stellate cells (HSCs). Importantly, experimental liver fibrosis was significantly ameliorated in CCR9-/- mice compared with wild-type (WT) mice, assessed by α-smooth muscle actin (α-SMA) immunostain, Sirius red staining, and messenger RNA (mRNA) expression levels of α-SMA, collagen 1α1, transforming growth factor (TGF)-β1, and tissue inhibitor of metalloproteinase (TIMP)-1. Accumulated CD11b+ macrophages in CCl4-treated WT mice showed marked increases in TNF, NO synthase-2, and TGF-β1 mRNA expression compared with CCR9-/- mice, implying proinflammatory and profibrogenic properties. Hepatic CD11b+ macrophages from CCl4-treated WT mice (i.e., CCR9+ macrophages), but not CD8+ T lymphocytes or non-CD11b+ cells, significantly activated HSCs in vitro compared with those from CCR9-/- mice. TNF-α or TGF-β1 antagonism attenuated CCR9+ macrophage-induced HSC activation. Furthermore, C-C motif chemokine ligand (CCL) 25 mediated migration and, to a lesser extent, activation of HSCs in vitro. Conclusion: Accumulated CD11b+ macrophages are critical for activating HSCs through the CCR9/CCL25 axis and therefore promote liver fibrosis. CCR9 antagonism might be a novel therapeutic target for liver fibrosis. © 2013 American Association for the Study of Liver Diseases.

Aim: Liver macrophages play integral roles in both the progression and resolution of hepatic inflammation and fibrosis, comprising opposing functions that largely coincide with the activation state of nearby hepatic stellate cells (HSC). While cross-talk between HSC and macrophages may be essential at various stages of inflammation and fibrogenesis, many facets of this interaction have yet to be thoroughly explored. Here, we examine the potential roles of HSC-derived signaling molecules as mediators of liver macrophage differentiation. Methods: Human peripheral blood mononuclear cells (PBMC) were differentiated to macrophages in the presence or absence of cultured HSC-derived conditioned media. The phenotype of resulting macrophages was characterized by examination of cell surface marker expression, antigen-presenting capabilities and cytokine secretion. Results: Conditioned media from activated human HSC promoted the differentiation of a unique set of macrophages that differed in morphology and function from both classical (M1) and alternative (M2) macrophages, expressing increased levels of CD14 and CD16, as well as a distinct interleukin (IL)-6high/IL-10low/transforming growth factor (TGF)-βhigh expression profile. These macrophages expressed high levels of CD206, CD209, CD80 and human leukocyte antigen DR, though no significant increases in antigen presentation were apparent. HSC-derived macrophages exhibited specific activation of p38 mitogen-activated protein kinase, and inhibition of this activation by p38 inhibitors during differentiation effectively reversed increases in IL-6 and TGF-β. Conclusion: The present results suggest that HSC-derived signaling molecules promote differentiation of liver macrophages with both pro-inflammatory and profibrotic functions. Furthermore, these effects appear to be mediated, at least partially, in a p38-dependent manner. © 2012 The Japan Society of Hepatology.

Although intestinal microbiota are essential for the development of T cell-mediated colitis, it remains undetermined whether they enhance or suppress the chronic extraintestinal inflammation that often complicates inflammatory bowel diseases. In this study, we investigate the role of intestinal microbiota in the development of colitis and extraintestinal manifestations in a mouse model in which colitis was induced in SCID mice by adoptive transfer of CD4 +CD45RBhigh T cells. Under specific pathogen-free conditions, these mice developed both colitis and extraintestinal interstitial pneumonia, whereas mice given a mixture of antibiotics did not develop colitis, but, surprisingly, developed Th1/Th17-mediated IP. Irrespective of antibiotic treatment, cotransfer of CD4+CD25+ regulatory T cells suppressed the development of pneumonitis and colitis, with all local CD4 +CD45RBhigh T cell-derived cells converted to CD44 highCD62L-IL-7Rαhigh effector-memory T cells. Retransfer of CD4+ effector-memory T cells from the lungs of antibiotic-treated mice with IP not only induced IP in both antibiotic-treated and -untreated recipients but also induced colitis in the untreated recipients. In summary, we have established a unique model of Th1/Th17-mediated IP in microbiota-free and antibiotic-treated mice. This model may be valuable in investigating the immunological mechanisms underlying extraintestinal disorders in patients with inflammatory bowel disease. Copyright © 2013 by The American Association of Immunologists, Inc.

Imbalance in gut bacterial composition provokes host proinflammatory responses causing diseases such as colitis. Colonization with a mixture of Clostridium species from clusters IV and XIVa was shown to suppress colitis through the induction of IL-10-producing regulatory T (Treg) cells. We demonstrate that a distinct Clostridium strain from cluster I, Clostridium butyricum (CB), prevents acute experimental colitis in mice through induction of IL-10, an anti-inflammatory cytokine. However, while CB treatment had no effect on IL-10 production by T cells, IL-10-producing F4/80+CD11b +CD11cint macrophages accumulated in the inflamed mucosa after CB treatment. CB directly triggered IL-10 production by intestinal macrophages in inflamed mucosa via the TLR2/MyD88 pathway. The colitis-preventing effect of CB was negated in macrophage-specific IL-10-deficient mice, suggesting that induction of IL-10 by intestinal macrophages is crucial for the probiotic action of CB. Collectively, CB promotes IL-10 production by intestinal macrophages in inflamed mucosa, thereby preventing experimental colitis in mice. © 2013 Elsevier Inc.

Bile acids (BAs) play important roles not only in lipid metabolism, but also in signal transduction. TGR5, a transmembrane receptor of BAs, is an immunomodulative factor, but its detailed mechanism remains unclear. Here, we aimed to delineate how BAs operate in immunological responses via the TGR5 pathway in human mononuclear cell lineages. We examined TGR5 expression in human peripheral blood monocytes, several types of in vitro differentiated macrophages (Mφ{symbol}s) and dendritic cells. Mφ{symbol}s differentiated with macrophage colony-stimulating factor and interferon-γ (Mγ-Mφ{symbol}s), which are similar to the human intestinal lamina propria CD14+ Mφ{symbol}s that contribute to Crohn's disease (CD) pathogenesis by production of pro-inflammatory cytokines, highly expressed TGR5 compared with any other type of differentiated Mφ{symbol} and dendritic cells. We also showed that a TGR5 agonist and two types of BAs, deoxycholic acid and lithocholic acid, could inhibit tumour necrosis factor-α production in Mγ-Mφ{symbol}s stimulated by commensal bacterial antigen or lipopolysaccharide. This inhibitory effect was mediated by the TGR5-cAMP pathway to induce phosphorylation of c-Fos that regulated nuclear factor-κB p65 activation. Next, we analysed TGR5 levels in lamina propria mononuclear cells (LPMCs) obtained from the intestinal mucosa of patients with CD. Compared with non-inflammatory bowel disease, inflamed CD LPMCs contained more TGR5 transcripts. Among LPMCs, isolated CD14+ intestinal Mφ{symbol}s from patients with CD expressed TGR5. In isolated intestinal CD14+ Mφ{symbol}s, a TGR5 agonist could inhibit tumour necrosis factor-α production. These results indicate that TGR5 signalling may have the potential to modulate immune responses in inflammatory bowel disease. © 2013 Blackwell Publishing Ltd.

The liver is a physiological site of immune tolerance, the breakdown of which induces immunity. Liver antigen-presenting cells may be involved in both immune tolerance and activation. Although inflammatory diseases of the liver are frequently associated with inflammatory bowel diseases, the underlying immunological mechanisms remain to be elucidated. Here we report two murine models of inflammatory bowel disease: RAG-2-/- mice adoptively transferred with CD4+CD45RBhigh T cells; and IL-10 -/- mice, accompanied by the infiltration of mononuclear cells in the liver. Notably, CD11b-CD11clowPDCA-1+ plasmacytoid dendritic cells (DCs) abundantly residing in the liver of normal wild-type mice disappeared in colitic CD4+CD45RBhigh T cell-transferred RAG-2-/- mice and IL-10-/- mice in parallel with the emergence of macrophages (Mφs) and conventional DCs (cDCs). Furthermore, liver Mφ/cDCs emerging during intestinal inflammation not only promote the proliferation of naïve CD4+ T cells, but also instruct them to differentiate into IFN-γ-producing Th1 cells in vitro. The emergence of pathological Mφ/cDCs in the liver also occurred in a model of acute dextran sulfate sodium (DSS)-induced colitis under specific pathogen-free conditions, but was canceled in germ-free conditions. Last, the Mφ/cDCs that emerged in acute DSS colitis significantly exacerbated Fas-mediated hepatitis. Collectively, intestinal inflammation skews the composition of antigen-presenting cells in the liver through signaling from commensal bacteria and predisposes the liver to inflammation. © 2014 Mikami et al.

Magnetic resonance enterography (MRE) has been reported to be a useful modality for the evaluation of luminal inflammation and extraintestinal complications in Crohn's disease (CD). A recent study indicated that the diagnostic ability of MRE was comparable to the diagnostic ability of other devices, such as ileocolonoscopy. MRE can be performed repeatedly because there is no radiation exposure. Therefore, MRE is useful as a method of follow-up for younger patients with established CD. It is useful for evaluating the efficacy of medical treatments, such as biologics. MRE can detect small intestinal lesions even if the endoscope does not pass through the stenosis. The concerns of availability of expertise and the costs associated with MRE should be addressed so MRE can be widely used for CD patients in the near future.

Although nonalcoholic steatohepatitis (NASH) is associated with hypercholesterolemia, the underlying mechanisms of this association have not been clarified. We aimed to elucidate the precise role of cholesterol in the pathophysiology of NASH. C57BL/6 mice were fed a control, high-cholesterol (HC), methionine-choline-deficient (MCD), or MCD+HC diet for 12 weeks or a control, HC, high-fat (HF), or HF+HC diet for 24 weeks. Increased cholesterol intake accelerated liver fibrosis in both the mouse models without affecting the degree of hepatocellular injury or Kupffer cell activation. The major causes of the accelerated liver fibrosis involved free cholesterol (FC) accumulation in hepatic stellate cells (HSCs), which increased Toll-like receptor 4 protein (TLR4) levels through suppression of the endosomal-lysosomal degradation pathway of TLR4, and thereby sensitized the cells to transforming growth factor (TGF)β-induced activation by down-regulating the expression of bone morphogenetic protein and activin membrane-bound inhibitor. Mammalian-cell cholesterol levels are regulated by way of a feedback mechanism mediated by sterol regulatory element-binding protein 2 (SREBP2), maintaining cellular cholesterol homeostasis. Nevertheless, HSCs were sensitive to FC accumulation because the high intracellular expression ratio of SREBP cleavage-activating protein (Scap) to insulin-induced gene (Insig) disrupted the SREBP2-mediated feedback regulation of cholesterol homeostasis in these cells. HSC activation subsequently enhanced the disruption of the feedback system by Insig-1 down-regulation. In addition, the suppression of peroxisome proliferator-activated receptor γ signaling accompanying HSC activation enhanced both SREBP2 and microRNA-33a signaling. Consequently, FC accumulation in HSCs increased and further sensitized these cells to TGFβ-induced activation in a vicious cycle, leading to exaggerated liver fibrosis in NASH. Conclusion: These characteristic mechanisms of FC accumulation in HSCs are potential targets to treat liver fibrosis in liver diseases including NASH.

A 58-year-old man was diagnosed as a hepatitis B virus (HBV) carrier approximately 30 years ago. He was diagnosed with renal cell carcinoma when he was 57 years old. Radical nephrectomy was performed, and everolimus was administered to treat his lung metastasis. After beginning the everolimus, intermittent fever, general fatigue, and jaundice developed. He was admitted under a diagnosis of flare (acute exacerbation) of chronic B hepatitis due to HBV reactivation. Despite intensive care, he died of hepatic failure and fungus infection. The autopsy findings were compatible with hepatic failure due to HBV reactivation by everolimus. Antiviral prophylaxis must be taken into consideration before beginning immunosuppressive therapy such as everolimus in HBV carriers. © The Author(s) 2013.

Glucagonlike peptide-1 (GLP-1) promotes insulin release; however, the relationship between the GLP-1 signal and chronic pancreatitis is not well understood. Here we focus on chemokine (C-C motif) ligand 2 (CCL2) and its receptor (CCR2) axis, which regulates various immune cells, including macrophages, to clarify the mechanism of GLP-1-mediated insulin secretion in chronic pancreatitis in mice. One and multiple series of repetitive cerulein administrations were used to induce acute and chronic cerulein pancreatitis, respectively. Acute cerulein-administered CCR2-knockout (KO) mice showed suppressed infiltration of CD11b+Gr-1low macrophages and pancreatic inflammation and significantly upregulated insulin secretion compared with paired wild-type (WT) mice. However, chronic cerulein-administered CCR2-KO mice showed significantly increased infiltration of CD11b+/ Gr-1 -and CD11b+/Gr-1high cells, but not CD11b+/Gr-1low cells, in pancreas with severe inflammation and significantly decreased insulin secretion compared with their WT counterparts. Furthermore, although serum GLP-1 levels in chronic cerulein-administered WT and CCR2-KO mice were comparably upregulated after cerulein administrations, GLP-1 receptor levels in pancreases of chronic ceruleinadministered CCR2-KO mice were significantly lower than in paired WT mice. Nevertheless, a significantly higher hyperglycemia level in chronic cerulein-administered CCR2-KO mice was markedly restored by treatment with a GLP-1 analog to a level comparable to the paired WT mice. Collectively, the CCR2/CCL2 axis-mediated CD11b+-cell migration to the pancreas is critically involved in chronic pancreatitismediated hyperglycemia through the modulation of GLP-1 receptor expression and insulin secretion. © 2013 the American Physiological Society.

Ability of a new scoring system for alcoholic hepatitis (AH) (Japan Alcoholic Hepatitis Score: J AS) to predict outcome was confirmed in the data of patients with AH in 2011. Twenty six hospitals provided full demographic data on 59 patients with AH (Moderate 26, Alive 22, Dead 4; Severe 33, Alive 16, Dead 17). There was no report of death in patients with mild AH (J AS score was 7 and less). Prevalence of renal failure, DIC and gastrointestinal bleeding was higher in patients who were died. Cr and PT (INR) were higher in severe AH patients who were died. These results suggest that JAS allows the stratification of the risk of death in AH, and can help the management of patients with AH. If there is any 3 points factor, intensive care such as corticosteroids, granulocytes/monocytes apheresis, plasma exchange and haemodialysis is recommended. © 2014 The Japan Society of Hepatology.

Background and Aims: Infliximab (IFX) is a monoclonal antibody used to treat patients with Crohn's disease (CD). Intra-abdominal abscess formation is a major complication of CD with negative effects on patient prognosis. We have analyzed risk factors for abscess formation in CD patients treated with IFX. Methods: CD patients who received IFX between January 2000 and April 2011 at Keio University Hospital were analyzed retrospectively. Risk factors for abscess formation were assessed by univariate and multivariate logistic regression analyses. Results: Intra-abdominal abscess was seen in 15 of 258 patients. Univariate analyses showed serum C-reactive protein (CRP) concentration at 14 weeks after initiation of IFX (p = 0.021), serum albumin concentration at week 0 (p = 0.022) and week 14 (p = 0.004), the presence of anal lesions (p = 0.036), progression of intestine deformation (p = 0.015) and early loss of response to IFX (p < 0.0001) to be risk factors. Multivariate analysis showed that CRP concentration at 14 weeks [odds ratio (OR) 1.361] and loss of IFX response within 6 months (OR 5.361) were independent risk factors. Conclusions: Abscess formation should be suspected in patients with symptoms of CD recurrence during IFX therapy. Uncontrolled CRP concentration and early loss of response to IFX are risk factors. © 2014 S. Karger AG, Basel.