Shinta Mizuno

Mammalian target of rapamycin and glycogen synthase kinase 3 differentially regulate lipopolysaccharide-induced interleukin-12 production in dendritic cells

Phosphoinositide 3-kinase (PI3K) negatively regulates Toll-like receptor (TLR)-mediated interleukin-12 (IL-12) expression in dendritic cells (DCs). We show here that 2 signaling pathways downstream of PI3K, mammalian target of rapamycin (mTOR) and glycogen synthase kinase 3 (GSK3), differentially regulate the expression of IL-12 in lipopolysaccharide (LPS)-stimulated DCs. Rapamycin, an inhibitor of mTOR, enhanced IL-12 production in LPS-stimulated DCs, whereas the activation of mTOR by lentivirus-mediated transduction of a constitutively active form of Rheb suppressed the production of IL-12. The inhibition of protein secretion or deletion of IL-10 cancelled the effect of rapamycin, indicating that mTOR regulates IL-12 expression through an autocrine action of IL-10. In contrast, GSK3 positively regulates IL-12 production through an IL-10-independent pathway. Rapamycin-treated DCs enhanced Th1 induction in vitro compared with untreated DCs. LiCl, an inhibitor of GSK3, suppressed a Th1 response on Leishmania major infection in vivo. These results suggest that mTOR and GSK3 pathways regulate the Th1/Th2 balance though the regulation of IL-12 expression in DCs. The signaling pathway downstream of PI3K would be a good target to modulate the Th1/Th2 balance in immune responses in vivo.

Cross-talk between RORγt+ innate lymphoid cells and intestinal macrophages induces mucosal IL-22 production In Crohn's disease

Background:Interleukin (IL)-22–producing ROR&ggr;t+ innate lymphoid cells (ILCs) play a pivotal role in intestinal immunity. Recent reports demonstrated that ILCs contribute to mucosal protection and intestinal inflammation in mice. In humans, numbers of ROR&ggr;t+ ILCs are significantly increased in the intestine of patients with Crohns disease (CD), suggesting that ILCs may be associated with intestinal inflammation in CD. However, the mechanism by which ILCs are regulated in the intestine of patients with CD is poorly understood. This study aimed to determine the activation mechanism of intestinal ILCs in patients with CD. Methods:CD45+ lineage marker- ILCs were isolated from intestinal lamina propria of patients with CD. ILCs were then subdivided into 4 distinct populations based on the expression of CD56 and CD127. Purified ILC subsets were cocultured with intestinal CD14+ macrophages, and IL-22 production was evaluated. Results:CD127+CD56− and CD127+CD56+ ILC, but not CD127−CD56+ or CD127−CD56− ILC, subsets expressed ROR&ggr;t and produced IL-22. IL-22 production by these ILC subsets was enhanced when ILCs were cocultured with intestinal macrophages. IL-23 or cell-to-cell contact was required for macrophage-mediated activation of ILCs. IL-22 production by ILCs was perturbed in inflamed mucosa compared with noninflamed mucosa. IL-22 induced the expression of Reg1&agr; and Claudin-1 in human intestinal epithelial organoids. Conclusions:ROR&ggr;t+ ILCs might enhance mucosal barrier function through the upregulation of Reg1&agr; through production of IL-22. Although CD14+ macrophages augment intestinal inflammation in patients with CD, macrophages also promote a negative feedback pathway through the activation of IL-22 production by ROR&ggr;t+ ILCs.

Clinical Efficacy and Safety of Oral Qing-Dai in Patients with Ulcerative Colitis: A Single-Center Open-Label Prospective Study

Background/Aims: Chinese herbal medicine Qing-Dai (also known as indigo naturalis) has been used to treat various inflammatory conditions. However, not much has been studied about the use of oral Qing-Dai in the treatment for ulcerative colitis (UC) patients. Studies exploring alternative treatments for UC are of considerable interest. In this study, we aimed at prospectively evaluating the safety and efficacy of Qing-Dai for UC patients. Methods: The open-label, prospective pilot study was conducted at Keio University Hospital. A total of 20 patients with moderate UC activity were enrolled. Oral Qing-Dai in capsule form was taken twice a day (daily dose, 2 g) for 8 weeks. Results: At week 8, the rates of clinical response, clinical remission, and mucosal healing were 72, 33, and 61%, respectively. The clinical and endoscopic scores, CRP levels, and fecal occult blood results were also significantly improved. We observed 2 patients with mild liver dysfunction; 1 patient discontinued due to infectious colitis and 1 patient discontinued due to mild nausea. Conclusion: This is the first prospective study indicating that oral Qing-Dai is effective for inducing remission in patients with moderate UC activity and can be tolerated. Thus, Qing-Dai may be considered an alternative treatment for patients, although further investigation is warranted.

CCR9+ plasmacytoid dendritic cells in the small intestine suppress development of intestinal inflammation in mice.

Almost all mice lacking specific molecules associated with regulatory T cells or barrier function develop intestinal inflammation in the colon, but not in the small intestine (SI). Therefore, intestinal homeostasis of the SI may be tightly controlled by other mechanisms. To determine the role of CCR9(+) plasmacytoid dendritic cells (pDCs) in intestinal homeostasis of the SI we transferred CD4(+)CD45RB(high) T cells into ccr9(-/-)×rag-2(-/-) mice. We showed that CCL25, a counterpart chemokine for CCR9, is constitutively expressed in the SI but not the colon and spleen of rag-2(-/-) or ccr9(-/-)×rag-2(-/-) mice before or after transfer of CD4(+)CD45RB(high) T cells. The ccr9(-/-)×rag-2(-/-) mice did not develop spontaneous intestinal inflammation in the SI and colon. Mice of both genotype where CD4(+)CD45RB(high) T cells were transferred developed colitis. However, the ccr9(-/-)×rag-2(-/-) mice also developed ileitis with marked infiltration of Th1 cells. These results suggest that CCR9(+) pDCs are possibly small, regulatory, antigen-presenting cells of the intestine that suppress intestinal inflammation.

Bifidobacterium-Rich Fecal Donor May Be a Positive Predictor for Successful Fecal Microbiota Transplantation in Patients with Irritable Bowel Syndrome

Background/Aims: Dysbiosis is associated with various systemic disorders including irritable bowel syndrome (IBS). Fecal microbiota transplantation (FMT) might restore intestinal microbial balance. The study aimed to determine the safety and efficacy of FMT in IBS patients, as well as also positive predictors for FMT. Methods: This was a single-arm, open-label study. Eligible patients were diagnosed based on Rome III Diagnostic Criteria. Fecal materials were administered to the patient via colonoscopy. The primary end point was a change in the Bristol stool form scale at 4 weeks after FMT. Recovery to types 3-4 was considered a clinical response. The secondary end point was a change in intestinal microbiota and psychological status using the Hamilton Rating Scale. Results: Ten patients were enrolled. Six patients achieved a clinical response. The diversity of patients 4 weeks after FMT increased significantly compared with patients before FMT, and that of responding patients was significantly higher than non-responder patients. The abundance of Bifidobacterium in effective donors was significantly higher than in ineffective donors and patients. Psychological status of all patients was significantly improved after FMT. Conclusions: FMT for patients with IBS is safe, and relatively effective. Bifidobacterium-rich fecal donor may be a positive predictor for successful FMT. Key Summary: (1) Dysbiosis is associated with various gastrointestinal disorders including IBS. (2) FMT has potential to restore intestinal microbial balance. (3) We showed that FMT improved stool form and psychological status of IBS patients. (4) Bifidobacterium-rich donor efficiently induced symbiosis in IBS patients.

Fecal Microbiota Transplantation for Gastrointestinal Diseases

Fecal microbiota transplantation (FMT) is a treatment to restore the normal microbial composition of the gut by introducing fecal microbiota obtained from a healthy donor into a diseased individual. There has been a growing interest in the use of FMT as a treatment of various diseases including Clostridium difficile infection (CDI), inflammatory bowel disease, and irritable bowel syndrome. Despite the increasing application of FMT, there are no standard protocols. Many aspects of FMT procedures vary regarding donor selection, preparation of fecal materials, recipient preparation, and route of administration. FMT is most successful in treating recurrent CDI. A randomized controlled trial reported a success rate of approximaetly 90%. Ulcerative colitis (UC) is a potentially good indication for FMT, although limited evidence is available on the use of FMT for the treatment of UC. Only several small case series have been reported, and the results in terms of efficacy are inconsistent. FMT can also be used to treat diseases other than gastrointestinal disorders in which the gut microbiota is disturbed, e.g., cardiovascular diseases, autoimmune diseases, and metabolic disorders. There remain many unanswered questions with regard to FMT, and more research is required in this field.

Macrophages and Dendritic Cells Emerge in the Liver during Intestinal Inflammation and Predispose the Liver to Inflammation

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.

Dysregulated balance of retinoid-related orphan receptor γt-dependent innate lymphoid cells is involved in the pathogenesis of chronic DSS-induced colitis.

Retinoid-related orphan receptor (ROR) γt-expressing and IL-22-producing NKp46(+) innate lymphoid (ILC22) cells reside in the lamina propria of the intestine in mice, suggesting that ILC22 cells contribute to host defense during intestinal damage in models of colitis in mice. Nevertheless, another set of pathological interferon (IFN)-γ and/or IL-17A-producing innate lymphoid cells (ILC1 and ICL17) may participate in the pathogenesis in different models of colitis. We here showed that RORγt(+)IL-22(+) ILC22 cells were localized in Thy-1(high)SCA-1(high) and/or Thy-1(high)SCA-1(low) subpopulations of the intestine in normal and dextran sodium sulfate (DSS)-induced colitic RORγt-sufficient Rag-2(-/-) mice. RORγt-deficient Rag-2(-/-) mice developed more severe DSS-induced colitis accompanied with lower expression of REG3β and REG3γ in the colon, but with a lower ratio and absolute number of IFN-γ-producing ILC1 cells as compared to control RORγt-sufficient Rag-2(-/-) mice. Collectively, not only the presence of ILC22 cells but also the balance of protective and pathogenic ILCs may be involved in the prevention of colitis.

Leucine-rich Alpha-2 glycoprotein is a serum biomarker of mucosal healing in ulcerative colitis

Background and Aims: Although several noninvasive and easily accessible biomarkers for inflammatory bowel disease [IBD] are available, their sensitivity and specificity are not adequate to be used as single markers and do not overrule the need for endoscopic evaluation. We previously reported that serum leucine-rich alpha-2 glycoprotein [LRG] was a novel biomarker for rheumatoid arthritis and IBD. We herein investigated whether LRG could indicate endoscopic activity in patients with ulcerative colitis [UC]. Methods: Serum LRG concentrations were determined by enzyme-linked immunosorbent assay [ELISA] in consecutive 129 patients with UC in two tertiary care hospitals, and associations of LRG with clinical and endoscopic activities were evaluated. Clinical activity index [CAI] < 6 was defined as clinical remission, and mucosal healing [MH] and complete mucosal healing were defined as Matts’ endoscopic grades of 1 or 2 and grade of 1, respectively. Results: Serum LRG levels were significantly increased and correlated with clinical and endoscopic activities in patients with UC. LRG levels were associated with both clinical and endoscopic activities even in patients with normal serum C-reactive protein [CRP] levels. Furthermore, LRG levels were significantly lower in patients with complete MH and deep remission. Serial measurements of LRG levels in a subset of patients demonstrated that LRG was significantly elevated during the endoscopically active stage compared with that during the MH stage. Conclusions: Serum LRG is a novel biomarker for detecting MH during disease course in patients with UC and a surrogate marker of endoscopic inflammation in patients with normal CRP levels.

Ulcerative colitis endoscopic index of severity is associated with long-term prognosis in ulcerative colitis patients treated with infliximab

Ulcerative colitis (UC) is a chronic inflammatory bowel disease that is characterized by periods of remission and episodes of relapse. Mucosal healing is an emerging therapeutic target in UC and various scoring systems have been used. The UC endoscopic index of severity (UCEIS) is the only validated endoscopic index at present, with minimum interobserver variation. Correlation of UCEIS scores after treatment and clinical outcomes of UC has not been examined. In the present study, we aimed to evaluate the usefulness of UCEIS after treatment with infliximab.