Published papers

Background: Although the clinical usefulness of leukocytapheresis for patients with inflammatory bowel disease (IBD) has been reported as a selective removal therapy targeting pathogenic immune cells in blood circulation, it remains unclear whether colitogenic CD4+ T cells continuously recirculate in peripheral blood during the chronic phase of colitis. Methods: To resolve this question we conducted a series of in vivo experiments using a murine chronic colitis model induced by adoptive transfer of CD4+CD45RBhigh cells into SCID mice in combination with a parabiosis system. Results: In colitic SCID recipients, first, almost all CD4+ CD45RBhigh donor cells were converted to CD4+CD44highCD62L- IL-7Rα high effector-memory T (TEM) cells at 8 weeks after transfer and were distributed throughout the whole body, including colonic lamina propria, mesenteric lymph nodes, thoracic duct, peripheral blood, spleen, and bone marrow. Second, SCID mice retransferred with the colitic peripheral blood CD4+ T cells developed colitis that is identical to the original colitis. Third, CD4+ cells in parabionts between established colitic RAG-2-/- mice induced by adoptive transfer of Ly5.1+ or Ly5.2+ CD4+CD45RBhigh T cells were well mixed in almost equal proportions at various sites 2 weeks after parabiosis surgery, and the redistribution of Ly5.1+ and Ly5.2+ CD4+ T cells was significantly suppressed in FTY720-treated parabionts. Conclusions: Together, these findings indicate that colitogenic CD4+ TEM cells continuously recirculate in established colitic mice, suggesting that therapeutic approaches targeting systemic CD4+ TEM cells, such as bone marrow transplantation, rather than those targeting only intestinal CD4+ T cells, may be feasible for the treatment of IBD. Copyright © 2008 Crohn's & Colitis Foundation of America, Inc.

2-Amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol hydrochloride (FTY720) suppresses T-cell egress from LN, thereby preventing pathogenic T cells from migrating toward disease sites. However, little is known about whether FTY720 could control the trafficking of T cells without the presence of lymphoid tissues. Here we demonstrate that FTY720 treatment suppresses the recirculation of CD4+ T cells in splenectomized (SPX) lymphotoxin-α-/- (LT-α -/-) mice that lack LN and spleen, as shown by peripheral blood (PB) lymphopenia in FTY720-treated SPX LT-α -/- mice. In a short-term transfer experiment, the cell number of transferred Ly5.1+CD4+ T cells recovered from host FTY720-treated SPX LT-α-/- mice (Ly5.2+) was markedly decreased in PB, but conversely increased in BM. Notably, FTY720 treatment prevented the development of colitis that is otherwise induced in untreated SPX LT-α -/- x RAG-2-/- mice upon transfer of colitic lamina propria CD4+ T cells. In such mice, the number of CD4+ T cells in PB or lamina propria of FTY720-treated SPX LT-α-/- x RAG-2-/- recipients was significantly reduced, but that in the BM was significantly increased as compared with untreated control mice. Altogether, the present results indicate that FTY720 treatment may offer an additional role to direct trafficking of CD4+ T cells in BM, resulting in the prevention of colitis. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA.

Double-balloon endoscopy (DBE) is a new technique that allows diagnosis and treatment throughout the entire small intestine. We have recently developed a method that allows a single operator to perform DBE, by employing "grip and pinch" and "hold (or 'keep') and slide" techniques. This one-person method obviates the need for two operators who must cooperate closely. © Georg Thieme Verlag KG Stuttgart.

IL-7 is a cytokine, which regulates development, maintenance and proliferation of T lymphocytes within the human immune system. Production of IL-7 is observed in a sterile environment such as thymus or bone marrow. However, it is also known that intestinal epithelial cells (IECs) residing in close contact with numerous bacterial stimuli also produce IL-7. Here we show that secretion of IL-7 by IECs is significantly suppressed upon stimulation by various bacterial components, including flagellin. Analysis of the intracellular mechanism by which flagellin regulates IL-7 production revealed that flagellin down-regulates expression of the two major transcripts encoding IL-7. Surprisingly, such function of flagellin was independent from the known transcriptional regulation of the IL-7 gene, as no significant change was observed in the transcriptional activity regulated by the previously identified promoter region. As the stability of IL-7 mRNA also remained unchanged upon flagellin stimulation, results suggested the possible involvement of a yet unknown transcriptional regulation of the IL-7 gene. These results describe a novel regulation of IL-7 production by bacterial stimuli, presumably mediated via Toll-like receptors. The present system might contribute to regulate the local lymphocyte pool, in response to the gut luminal or sub-mucosal bacterial abundance. © 2008 Elsevier Ltd. All rights reserved.

Maintenance of the intestinal epithelium is based on well-balanced molecular mechanisms that confer the stable and continuous supply of specialized epithelial cell lineages from multipotent progenitors. Lineage commitment decisions in the intestinal epithelium system involve multiple regulatory systems that interplay with each other to establish the cellular identities. Here, we demonstrate that the microRNA system could be involved in intestinal epithelial cell differentiation, and that microRNA-194 (miR-194) is highly induced during this process. To investigate this inducible expression mechanism, we identified the genomic structure of the miR-194-2, -192 gene, one of the inducible class of miR-194 parental genes. Furthermore, we identified its transcriptional regulatory region that contains a consensus-binding motif for hepatocyte nuclear factor-1α (HNF-1α), which is well known as a transcription factor to regulate gene expression in intestinal epithelial cells. By chromatin immunoprecipitation assay and luciferase reporter analysis, we revealed that pri-miR-194-2 expression is controlled by HNF-1α, and its consensus binding region is required for the transcription of pri-miR-194-2 in vivo in an intestinal epithelial cell line, Caco-2. Our observations indicate that microRNA genes could be targets of lineage-specific transcription factors and that microRNAs are regulated by a tissue-specific manner in the intestinal epithelium. Therefore, our work suggests that induced expression of these microRNAs have important roles in intestinal epithelium maturation. Published by Cold Spring Harbor Laboratory Press. Copyright © 2008 RNA Society.

Inflammatory bowel diseases take chronic courses due to the expansion of colitogenic CD4+ cells. However, it is unclear whether the persistent disease is driven by continuous reactivation of colitogenic memory CD4+ cells to generate effector CD4+ cells or by continuous generation of effector CD4+ cells from naïve cells. To clarify this issue, we performed a series of sequential adoptive transfers of Ly5.2+ and Ly5.1+ CD4+CD45RBhigh cells into RAG-2-/- mice at different time points. We show here that the secondarily transferred CD4+CD45RBhigh cells can be converted to CD4+CD44highCD62L-IL-7Rα high effector-memory T cells even in the presence of pre-existing effector-memory CD4+ cells. Although the total cell numbers of CD4+ cells in established colitic mice were consistently equivalent irrespective of the number of primarily transferred cells, the ratio of primarily and secondarily transferred cells was dependent on the ratio of the transferred cell numbers, but not on the order of the transfer. Of note, we found that primarily transferred CD4+ cells produced significantly lower amounts of IFN-γ and IL-17 than CD4+ cells arising from secondary transfer. In conclusion, the continuous generation of colitogenic CD4+ cells that compensate for exhausted CD4+ cells may be one of the mechanisms involved in the persistence of colitis. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inflammatory bowel diseases progress steadily by the expansion of colitogenic CD4+ cells. However, it remains unknown whether colitogenic CD4+ cells are long-living like memory cells or exhausted like effector cells. To assess the longevity of colitogenic lamina propria (LP) CD4+ cells, we performed sequential transfers of LP CD4+ cells from colitic CD4+CD45RBhigh cell-transferred SCID mice into new SCID mice. Although SCID mice transferred with colitic LP CD4+ cells stably developed colitis until at least the sixth transfer, the interval to the development of colitis gradually lengthened as the number of transfers increased. The incidence of colitis gradually decreased after the seventh transfer. Furthermore, non-colitic LP CD4+ cells from mice transferred over seven times expressed significantly higher levels of PD-1 and produced significantly lower amounts of IFN-γ, TNF-α, and IL-17 than colitic LP CD4+ cells recovered after the first transfer. Most notably, we found that re-transfer of non-colitic LP CD4+ cells recovered after multiple transfers prevented the development of colitis in SCID mice co-transferred with CD4+CD45RBhigh cells. Thus, colitogenic LP CD4+ cells may be exhausted over time, become non-functional, convert to regulatory cells, and finally suppress colitis in the process of immunosenescence. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Atoh1 plays a crucial role in intestinal cell differentiation. We have demonstrated that its human homolog Hath1 protein is targeted by the Wnt-GSK3 axis, resulting in the proteasomal degradation in human colon cancer. However, the contribution of Hath1 degradation to the undifferentiated state of colon cancer remains unknown. In this study, we demonstrated that both constitutive expression of mutant Hath1 and stabilization of Hath1 protein by a GSK3 inhibitor in colon cancer cells increased the expression of MUC2 known as a representative function of differentiated goblet cells. This means that Hath1 protein degradation may be required for maintaining the undifferentiated state of colon cancers, and that GSK3 inhibitors have potential for use in cancer therapy. © 2008 Elsevier Inc. All rights reserved.

TLRs that mediate the recognition of pathogen-associated molecular patterns are widely expressed on/in cells of the innate immune system. However, recent findings demonstrate that certain TLRs are also expressed in conventional TCRαβ+ T cells that are critically involved in the acquired immune system, suggesting that TLR ligands can directly modulate T cell function in addition to various innate immune cells. In this study, we report that in a murine model of chronic colitis induced in RAG-2-/- mice by adoptive transfer of CD4+CD45RBhigh T cells, both CD4+CD45RBhigh donor cells and the expanding colitogenic lamina propria CD4+CD44hlgh memory cells expresses a wide variety of TLRs along with MyD88, a key adaptor molecule required for signal transduction through TLRs. Although RAG-2-/- mice transferred with MyD88-/-CD4+CD45RBhigh cells developed colitis, the severity was reduced with the delayed kinetics of clinical course, and the expansion of colitogenic CD4+ T cells was significantly impaired as compared with control mice transferred with MyD88+/+CD4 +CD45RBhigh cells. When RAG-2-/- mice were transferred with the same number of MyD88+/+ (Ly5.1+) and MyDSS-/- (Ly5.2+) CD4+CD45RBhigh cells, MyD88-/-CD4 + T cells showed significantly lower proliferative responses assessed by in vivo CFSE division assay, and also lower expression of antiapoptotic Bcl-2/Bcl-xL molecules and less production of IFN-γ and IL-17, compared with the paired MyD88 +/+CD4+ T cells. Collectively, the MyD88-dependent pathway that controls TLR signaling in T cells may directly promote the proliferation and survival of colitogenic CD4+ T cells to sustain chronic colitis. The Journal of Immunology, 2008, 180: Copyright © 2008 by The American Association of Immunologists, Inc.

HCV culture in vitro results in massive cell death, which suggests the presence of HCV-induced cytopathic effects. Therefore, we investigated its mechanisms and viral nucleotide sequences involved in this effect using HCV-JFH1 cell culture and a newly developed HCV plaque assay technique. The plaque assay developed cytopathic plaques, depending on the titer of the inoculum. In the virus-infected cells, the ER stress markers, GRP78 and phosphorylated eIF2-alpha, were overexpressed. Cells in the plaques were strongly positive for an apoptosis marker, annexin V. Isolated virus subclones from individual plaque showed greater replication efficiency and cytopathogenicity than the parental virus. The plaque-purified virus had 9 amino acid substitutions, of which 5 were clustered in the C terminal of the NS5B region. Taken together, the cytopathic effect of HCV infection involves ER-stress-induced apoptotic cell death. Certain HCV genomic structures may determine the viral replication capacity and cytopathogenicity. © 2007 Elsevier Inc. All rights reserved.

Recent progress in both basic and clinical research has led us to develop sophisticated and effective medical therapy of ulcerative colitis (UC). Although classical agents such as aminosalicylates, corticosteroids and immunomodulatory drugs have remained as the gold standard for decades, their novel formulations and/or dosage regimens have changed their placements in the medical management of UC. In addition, studies have shown that a number of novel therapeutic agents, designed to target specific mechanisms involved in the inflammatory cascade, have efficacy for the treatment of UC and they will have significant clinical impacts in the near future. A clear understanding of the proven and potential benefits of both the standard and emerging therapies will be required for the optimum individual care of patients with varied clinical presentations. Copyright © 2008 S. Karger AG.

We previously demonstrated that IL-7 is produced by intestinal goblet cells and is essential for the persistence of colitis. It is well known, however, that goblet cells are decreased or depleted in the chronically inflamed mucosa of animal colitis models or human inflammatory bowel diseases. Thus, in this study, we assess whether intestinal IL-7 is surely required for the persistence of colitis using a RAG-1/2-/- colitis model induced by the adoptive transfer of CD4+CD45RBhigh T cells in combination with parabiosis system. Surprisingly, both IL-7-/- x RAG-1-/- and IL-7+/+ x RAG-1-/- host mice developed colitis 4 wk after parabiosis to a similar extent of colitic IL-7+/+ x RAG-1 -/- donor mice that were previously transferred with CD4 +CD45RBhigh T cells. Of note, although the number of CD4+ T cells recovered from the spleen or the bone marrow of IL-7-/- x RAG-1-/- host mice was significantly decreased compared with that of IL-7+/+ x RAG-1-/- host mice, an equivalent number of CD4+ T cells was recovered from the lamina propria of both mice, indicating that the expansion of CD4+ T cells in the spleen or in the bone marrow is dependent on IL-7, but not in the lamina propria. Development of colitis was never observed in parabionts between IL-7+/+ x RAG-1-/- host and noncolitic IL-7-/- x RAG-1-/- donor mice that were transferred with CD4 +CD45RBhigh T cells. Collectively, systemic, but not intestinal, IL-7 is essential for the persistence of colitis, suggesting that therapeutic approaches targeting the systemic IL-7/IL-7R signaling pathway may be feasible in the treatment of inflammatory bowel diseases. Copyright © 2007 by The American Association of Immunologists, Inc.

It has been recently demonstrated that NKG2D is an activating costimulatory receptor on natural killer (NK) cells, natural killer T (NKT) cells, activated CD8+ T cells, and γδ T cells, which respond to cellular stress, such as inflammation, transformation, and infection. Here we show that intestinal inflammation in colitic SCID mice induced by adoptive transfer of CD4+CD45RBhigh T cells is characterized by significant increase of CD4+NKG2D+ T cells and constitutive expression of NKG2D ligands, such as H60, Mult-1, and Rae-1, by lamina propria CD11c + dendritic cells. Furthermore, treatment with nondepleting and neutralizing anti-NKG2D MAb after transfer of CD4+CD45RB high T cells into SCID mice significantly suppressed wasting disease with colitis, abrogated leukocyte infiltration, and reduced production of IFN-γ by lamina propria CD4+ T cells. These findings demonstrate that NKG2D signaling pathway is critically involved in CD4 + T cell-mediated disease progression and suggest a new therapeutic target for inflammatory bowel diseases. Copyright © 2008 the American Physiological Society.

It is well known that immune responses in the intestine remain in a state of controlled inflammation, suggesting that not only does active suppression by regulatory T (TREG) cells play an important role in the normal intestinal homeostasis, but also that its dysregulation of immune response leads to the development of inflammatory bowel disease. In this study, we demonstrate that murine CD4+CD25+ T cells residing in the intestinal lamina propria (LP) constitutively express CTLA-4, glucocorticoid-induced TNFR, and Foxp3 and suppress proliferation of responder CD4+ T cells in vitro. Furthermore, cotransfer of intestinal LP CD4+CD25+ T cells prevents the development of chronic colitis induced by adoptive transfer of CD4+CD45RBhigh T cells into SCID mice. When lymphotoxin (LT)α-deficient intercrossed Rag2 double knockout mice (LTα-/- × Rag2-/-), which lack mesenteric lymph nodes and Peyer's patches, are transferred with CD4+CD45RB high T cells, they develop severe wasting disease and chronic colitis despite the delayed kinetics as compared with the control LTα +/+ × Rag2-/- mice transferred with CD4 +CD45RBhigh T cells. Of note, when a mixture of splenic CD4+CD25+ TREG cells and CD4 +CD45RBhigh T cells are transferred into LTα-/- × Rag2-/- recipients, CD4 +CD25+ TREG cells migrate into the colon and prevent the development of colitis in LTα-/- × Rag2 -/- recipients as well as in the control LTα+/+ × Rag2-/- recipients. These results suggest that the intestinal LP harboring CD4+CD25+ TREG cells contributes to the intestinal immune suppression. Copyright © 2007 by The American Association of lmmunologists, Inc.

A 61-years-old man started the combination treatment with telaprevir, peginterferon-alfa 2b, and ribavirin against chronic hepatitis C after the cure of hepatocellular carcinoma. Telaprevir was reduced to 1500 mgday due to renal dysfunction but the serum HCV-RNA was disappeared on the 15th day. On the 18th day, he visited our hospital with severe epigastralgia and a high fever. He was diagnosed acute pancreatitis by elevation of his serum amylase (2905 IUL) and diffuse swelling of pancreas. We suspected the drug-induced pancreatitis and stopped telaprevir. Although peginterferon and ribavirin were continued for the treating acute pancreatitis, his HCV-RNA became positive again. Therefore we restarted telaprevir with plenty of informed consent after recovery of the pancreatitis. On the 10th day after the resume of telaprevir, the recurrence of the pancreatitis occurred and we stopped any anti-HCV treatment. These twice pancreatitis were immediately improved by the suspension of telaprevir and the administration of antibiotics and gabexate mesilate. We here report this case of telaprevir induced acute pancreatitis confirmed by the re-administration of telaprevir. © 2013 The Japan Society of Hepatology.

Retinoid-related orphan receptor (ROR) γt is known to be related to the development and function of various immunological compartments in the liver, such as Th17 cells, natural killer T (NKT) cells, and innate lymphoid cells (ILCs). We evaluated the roles of RORγt-expressing cells in mouse acute hepatitis model using RORγt deficient (RORγt-/-) mice and RAG-2 and RORγt double deficient (RAG-2-/- × RORγt-/-) mice. Acute hepatitis was induced in mice by injection with carbon tetrachloride (CCl4), to investigate the regulation of liver inflammation by RORγt-expressing cells. We detected RORC expression in three compartments, CD4+ T cells, NKT cells, and lineage marker-negative SCA-1+Thy1high ILCs, of the liver of wild type (WT) mice. CCl4-treated RORγt-/- mice developed liver damage in spite of lack of RORγt-dependent cells, but with reduced infiltration of macrophages compared with WT mice. In this regard, ILCs were significantly decreased in RAG-2-/- × RORγt-/- mice that lacked T and NKT cells. Surprisingly, RAG-2-/- × RORγt-/- mice developed significantly severer CCl4-induced hepatitis compared with RAG-2-/- mice, in accordance with the fact that hepatic ILCs failed to produce IL-22. Lastly, anti-Thy1 monoclonal antibody (mAb), but not anti-NK1.1 mAb or anti-asialo GM1 Ab administration exacerbated liver damage in RAG-2-/- mice with the depletion of liver ILCs. Collectively, hepatic RORγt-dependent ILCs play a part of protective roles in hepatic immune response in mice. © 2013 Matsumoto et al.

Although the precise etiologies of inflammatory bowel disease (IBD) (ulcerative colitis and Crohn's disease) remain obscure, several reports have indicated that dysfunction of the mucosal immune system plays an important role in its pathogenesis. Recent progress with genome-wide association studies has identified many IBD susceptibility genes. In individuals with genetic risk, abnormal interactions between the host immune system and gut flora, and dysregulation of cellular responses such as autophagy and ER stress, induce an abnormal host immune response in the gut resulting in intestinal inflammation. Research progress animal models in IBD, and in human IBD, has identified several key molecules in IBD pathogenesis such as TNFα and adhesion molecules, and molecular targeting therapies based on these molecules have been developed. Here, we review immunological aspects in IBD pathogenesis and the development of immunoregulatory therapy. © 2012 Elsevier Inc.

Background: Although accumulating studies in Japan show that cytapheresis (CAP) therapy is safe and effective for the induction of remission of moderate or severe ulcerative colitis (UC), the long-term prognosis of UC patients treated with CAP is unknown. The aim of this study was to determine the long-term prognosis of UC patients treated with CAP. Methods: Ninety patients treated previously with CAP and followed for more than 3. years were evaluated. The rates of operation, readmission, and use or dose-up of corticosteroid were analyzed as long-term prognosis. Results: Following the first course of CAP treatment, 64% of patients showed clinical improvement (> 4-point decrease in the clinical activity index (CAI)), and 49% of patients achieved clinical remission (CAI ≤ 4). Longer disease duration and lower age at the first CAP treatment correlated significantly with the therapeutic effects of CAP (p= 0.003 and 0.035, respectively). The rates of operation and readmission were significantly lower in patients who showed previous clinical effects of CAP than in those who did not respond to CAP. The rates of operation and readmission were also significantly lower in patients whose treatment was combined with immunomodulators after the initiation of CAP than in patients who did not use immunomodulators. Importantly, the second course of CAP was also effective in most of the patients who showed a clinical response to the first CAP. Conclusions: Patients who achieve remission after the first CAP therapy may have a good long-term prognosis and a good response to a second CAP therapy even after relapse. © 2012 European Crohn's and Colitis Organisation.