Hideki Osawa

Increased Th17-Inducing Activity of CD14+ CD163low Myeloid Cells in Intestinal Lamina Propria of Patients With Crohn’s Disease

BACKGROUND & AIMS Abnormal activity of innate immune cells and T-helper (Th) 17 cells has been implicated in the pathogenesis of autoimmune and inflammatory diseases, including Crohns disease (CD). Intestinal innate immune (myeloid) cells have been found to induce development of Th17 cells in mice, but it is not clear if this occurs in humans or in patients with CD. We investigated whether human intestinal lamina propria cells (LPCs) induce development of Th17 cells and whether these have a role in the pathogenesis of CD. METHODS Normal intestinal mucosa samples were collected from patients with colorectal cancer and noninflamed and inflamed regions of mucosa were collected from patients with CD. LPCs were isolated by enzymatic digestion and analyzed for expression of HLA-DR, lineage markers CD14 and CD163 using flow cytometry. RESULTS Among HLA-DR(high) Lin(-) cells, we identified a subset of CD14(+) CD163(low) cells in intestinal LPCs; this subset expressed Toll-like receptor (TLR) 2, TLR4, and TLR5 mRNAs and produced interleukin (IL)-6, IL-1β, and tumor necrosis factor in response to lipopolysaccharide. In vitro co-culture with naïve T cells revealed that CD14(+) CD163(low) cells induced development of Th17 cells. CD14(+) CD163(low) cells from inflamed regions of mucosa of patients with CD expressed high levels of IL-6, IL-23p19, and tumor necrosis factor mRNAs, and strongly induced Th17 cells. CD14(+) CD163(low) cells from the noninflamed mucosa of patients with CD also had increased abilities to induce Th17 cells compared with those from normal intestinal mucosa. CONCLUSIONS CD14(+) CD163(low) cells in intestinal LPCs from normal intestinal mucosa induce differentiation of naive T cells into Th17 cells; this activity is increased in mucosal samples from patients with CD. These findings show how intestinal myeloid cell types could contribute to pathogenesis of CD and possibly other Th17-associated diseases.

Lypd8 promotes the segregation of flagellated microbiota and colonic epithelia

Colonic epithelial cells are covered by thick inner and outer mucus layers. The inner mucus layer is free of commensal microbiota, which contributes to the maintenance of gut homeostasis. In the small intestine, molecules critical for prevention of bacterial invasion into epithelia such as Paneth-cell-derived anti-microbial peptides and regenerating islet-derived 3 (RegIII) family proteins have been identified. Although there are mucus layers providing physical barriers against the large number of microbiota present in the large intestine, the mechanisms that separate bacteria and colonic epithelia are not fully elucidated. Here we show that Ly6/PLAUR domain containing 8 (Lypd8) protein prevents flagellated microbiota invading the colonic epithelia in mice. Lypd8, selectively expressed in epithelial cells at the uppermost layer of the large intestinal gland, was secreted into the lumen and bound flagellated bacteria including Proteus mirabilis. In the absence of Lypd8, bacteria were present in the inner mucus layer and many flagellated bacteria invaded epithelia. Lypd8−/− mice were highly sensitive to intestinal inflammation induced by dextran sulfate sodium (DSS). Antibiotic elimination of Gram-negative flagellated bacteria restored the bacterial-free state of the inner mucus layer and ameliorated DSS-induced intestinal inflammation in Lypd8−/− mice. Lypd8 bound to flagella and suppressed motility of flagellated bacteria. Thus, Lypd8 mediates segregation of intestinal bacteria and epithelial cells in the colon to preserve intestinal homeostasis.

The effect of Clostridium butyricum MIYAIRI on the prevention of pouchitis and alteration of the microbiota profile in patients with ulcerative colitis

PurposeUlcerative colitis (UC) is a chronic, relapsing, and refractory disorder of the intestine. Total proctocolectomy with ileal pouch anal anastomosis (IPAA) is the preferred and standard surgical procedure for patients’ refractory to medical therapy. Pouchitis is one of the most common long-term complications after IPAA. In the present study, the safety and efficacy of Clostridium butyricum MIYAIRI (CBM) as a probiotic were examined.MethodsA randomized and placebo-controlled study was performed. Seventeen patients were recruited from 2007 to 2013. Nine tablets of MIYA-BM® or placebo were orally administered once daily. The cumulative pouchitis-free survival, pouch condition (using the modified pouch disease activity index), and blood parameters were evaluated. A fecal sample analysis was also performed.ResultsSubjects were randomly allocated to receive MIYA-BM or placebo (9 and 8 subjects, respectively). One subject in the MIYA-BM group and four subjects in the placebo group developed pouchitis. No side effects occurred in either group. Characteristic intestinal flora was observed in each group.ConclusionsOur results suggest that probiotic therapy with CBM achieved favorable results with minimal side effects and might be a useful complementary therapy for the prevention of pouchitis in patients with UC who have undergone IPAA.

Circulating miR-199a-3p as a novel serum biomarker for colorectal cancer

Serum microRNAs (miRNAs) have been shown to have potential for cancer diagnosis. The main objective of the present study was to identify a novel serum miRNA biomarker from patients with colorectal cancer (CRC). Microarray analysis of miRNA expression was performed using paired pre-operative and post-operative serum from 10 CRC patients. Expression of two miRNAs (let-7a and miR-199a-3p) was significantly decreased in the post-operative serum when compared to levels in the pre-operative serum (P=0.015 and 0.029, respectively). Quantitative real-time polymerase chain reaction (qRT-PCR) confirmed the decrease in the miRNAs in an extended number (n=30) of paired serum samples. Next, we examined the serum let-7a level in 32 non-cancer patients and 84 CRC patients but we found no significant difference (P=0.120). In contrast, miR199a-3p expression was significantly higher in the CRC patients than that in the non-cancer patients (P=0.016). Furthermore, clinical and pathological survey indicated that high expression of miR-199a-3p was significantly associated with deep wall invasion. Our data suggest that circulating miR-199a-3p could be a novel serum biomarker for CRC.

A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids

Summary Gut epithelial organoids are routinely used to investigate intestinal biology; however, current culture methods are not amenable to genetic manipulation, and it is difficult to generate sufficient numbers for high-throughput studies. Here, we present an improved culture system of human induced pluripotent stem cell (iPSC)-derived intestinal organoids involving four methodological advances. (1) We adopted a lentiviral vector to readily establish and optimize conditioned medium for human intestinal organoid culture. (2) We obtained intestinal organoids from human iPSCs more efficiently by supplementing WNT3A and fibroblast growth factor 2 to induce differentiation into definitive endoderm. (3) Using 2D culture, followed by re-establishment of organoids, we achieved an efficient transduction of exogenous genes in organoids. (4) We investigated suspension organoid culture without scaffolds for easier harvesting and assays. These techniques enable us to develop, maintain, and expand intestinal organoids readily and quickly at low cost, facilitating high-throughput screening of pathogenic factors and candidate treatments for gastrointestinal diseases.

Eosinophil depletion suppresses radiation-induced small intestinal fibrosis

Eosinophils are a potential target for treating radiation-induced intestinal fibrosis. Eosinophils foster fibrosis Cancer patients receiving abdominal radiation can develop radiation-induced intestinal fibrosis (RIF). Takemura et al. observed eosinophils in the small intestine of RIF patients and irradiated mice. Lymphocyte-deficient mice still developed RIF, but eosinophils were indispensible. They probed the mechanism of interaction between stromal cells, and recruited eosinophils, and showed depletion of eosinophils with two types of antibodies that ameliorated RIF. As anti-eosinophil antibodies are moving forward clinically for other diseases, they may easily be repurposed to prevent RIF in cancer patients. Radiation-induced intestinal fibrosis (RIF) is a serious complication after abdominal radiotherapy for pelvic tumor or peritoneal metastasis. Herein, we show that RIF is mediated by eosinophil interactions with α-smooth muscle actin–positive (α-SMA+) stromal cells. Abdominal irradiation caused RIF especially in the submucosa (SM) of the small intestine, which was associated with the excessive accumulation of eosinophils in both human and mouse. Eosinophil-deficient mice showed markedly ameliorated RIF, suggesting the importance of eosinophils. After abdominal irradiation, chronic crypt cell death caused elevation of extracellular adenosine triphosphate, which in turn activated expression of C-C motif chemokine 11 (CCL11) by pericryptal α-SMA+ cells in the SM to attract eosinophils in mice. Inhibition of C-C chemokine receptor 3 (CCR3) by genetic deficiency or neutralizing antibody (Ab) treatment suppressed eosinophil accumulation in the SM after irradiation in mice, suggesting a critical role of the CCL11/CCR3 axis in the eosinophil recruitment. Activated α-SMA+ cells also expressed granulocyte-macrophage colony-stimulating factor (GM-CSF) to activate eosinophils. Transforming growth factor-β1 from GM-CSF–stimulated eosinophils promoted collagen expression by α-SMA+ cells. In translational studies, treatment with a newly developed interleukin-5 receptor α–targeting Ab, analogous to the human agent benralizumab, depleted intestinal eosinophils and suppressed RIF in mice. Collectively, we identified eosinophils as a crucial factor in the pathogenesis of RIF and showed potential therapeutic strategies for RIF by targeting eosinophils.

CD103+ Dendritic Cell Function Is Altered in the Colons of Patients with Ulcerative Colitis

Background: Human intestinal innate myeloid cells can be divided into 3 subsets: HLA-DRhighCD14+ cells, HLA-DRhighCD103+ dendritic cells (DCs), and HLA-DRhighCD14−CD103− cells. CD103+ DCs generate Treg cells and Th17 cells in the ileum, but their function in the colon remains largely unknown. This study characterized CD103+ DCs in the colon and investigated whether these cells are implicated in the pathogenesis of ulcerative colitis (UC). Methods: Normal intestinal mucosa was obtained from intact sites of patients with colorectal cancer (n = 24). Noninflamed and inflamed colonic tissues were obtained from surgically resected specimens of patients with UC (n = 13). Among Lin−CD45+HLA-DRhigh intestinal lamina propria cells, CD14+ cells and CD103+ DCs were sorted and analyzed for microRNA expression of cytokines and toll-like receptors by quantitative real-time polymerase chain reaction. In addition, IL-4/IL-5/IL-13/IL-17/IFN-&ggr; production and Foxp3 expression by naive T cells cultured with CD14+ cells and CD103+ DCs were analyzed. Results: CD103+ DCs in the normal colon showed lower expression of toll-like receptors and proinflammatory cytokines than CD14+ cells. Coculture with naive T cells revealed that CD103+ DCs generated Treg cells. CD103+ DCs from patients with UC did not generate Treg cells, but they induced IFN-&ggr;-, IL-13-, and IL-17-producing CD4+ T cells and showed higher expression of IL6 (P < 0.0001), IL23A (P < 0.05), IL12p35 (P < 0.05), and TNF (P < 0.05). Conclusions: In patients with UC, CD103+ DCs show the impaired ability to generate Treg cells, but exhibit a colitogenic function inducing Th1/Th2/Th17 responses. These findings show how human CD103+ DCs could contribute to the pathogenesis of UC.

Full-length LGR5-positive cells have chemoresistant characteristics in colorectal cancer.

Background:Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a target of Wnt signalling and considered both a cancer stem cell marker and intestinal stem cell marker. We found first some splice variants of LGR5 in human intestine and elucidated the functional feature of full-length LGR5 (LGR5FL).Methods:Reverse transcript PCR using mRNA extracted from intestine revealed the existence of LGR5 splice variants. We designed an antibody that recognises only LGR5FL and assessed immunohistochemically the distribution of LGR5FL-positive cells and Ki-67-positive cells in clinical samples.Results:Two LGR5 splice variants were expressed in the human intestine crypt cells; one lacked exon 5 and the other lacked exons 5–8. Only LGR5FL appeared during cell cycle arrest, whereas the transcript variants appeared when the cell cycle was proceeding. Immunohistochemistry and in situ hybridisation showed that LGR5FL-positive cells were negative for Ki-67. Comparing prechemotherapy and post-chemotherapy specimens, the population of LGR5FL-positive cells significantly increased with therapy (P<0.01).Conclusions:The function of LGR5FL-positive cells had low cell proliferative ability compared with the cells, which expressed splice variants of LGR5 and remained after chemotherapy. Designing therapeutic strategies that target LGR5FL-positive cells seems to be important in colorectal cancer.

Surgical Management of Perforated Gastrointestinal Posttransplantation Lymphoproliferative Disorder After Heart Transplantation

Posttransplantation lymphoproliferative disorder (PTLD) is a relatively rare and life-threatening complication after organ transplantation. From 1999 to 2012, 45 adult patients underwent heart transplantation at our hospital. Two of the patients developed PTLD after transplantation and required emergency surgery due to intestinal perforation. These cases were informative regarding the adequate surgical management of such cases. Both cases revealed Epstein-Barr virus-related PTLD. The optimal treatment of PTLD remains controversial, and PTLD with gastrointestinal perforation could be critical because the patients are already debilitated and immunocompromised after transplantation. Therefore, the nonspecific abdominal symptoms can be diagnostic for PTLD, and proper surgical intervention should be performed immediately. We present these two suggestive and rare cases in regard to the management of perforation with PTLD and a review of literature.