Takashi Kusu

Probiotic Bifidobacterium breve Induces IL-10-Producing Tr1 Cells in the Colon

Specific intestinal microbiota has been shown to induce Foxp3+ regulatory T cell development. However, it remains unclear how development of another regulatory T cell subset, Tr1 cells, is regulated in the intestine. Here, we analyzed the role of two probiotic strains of intestinal bacteria, Lactobacillus casei and Bifidobacterium breve in T cell development in the intestine. B. breve, but not L. casei, induced development of IL-10-producing Tr1 cells that express cMaf, IL-21, and Ahr in the large intestine. Intestinal CD103+ dendritic cells (DCs) mediated B. breve-induced development of IL-10-producing T cells. CD103+ DCs from Il10 −/−, Tlr2 −/−, and Myd88 −/− mice showed defective B. breve-induced Tr1 cell development. B. breve-treated CD103+ DCs failed to induce IL-10 production from co-cultured Il27ra −/− T cells. B. breve treatment of Tlr2 −/− mice did not increase IL-10-producing T cells in the colonic lamina propria. Thus, B. breve activates intestinal CD103+ DCs to produce IL-10 and IL-27 via the TLR2/MyD88 pathway thereby inducing IL-10-producing Tr1 cells in the large intestine. Oral B. breve administration ameliorated colitis in immunocompromised mice given naïve CD4+ T cells from wild-type mice, but not Il10 −/− mice. These findings demonstrate that B. breve prevents intestinal inflammation through the induction of intestinal IL-10-producing Tr1 cells.

Commensal microbiota induce LPS hyporesponsiveness in colonic macrophages via the production of IL-10

Several subsets of innate immune cells, all with unique properties, reside within the intestinal lamina propria. However, compared with intestinal dendritic cells (DCs), intestinal macrophages are less well characterized. In this study, we examined the properties of macrophages in the colonic lamina propria (LMφ). Colonic DCs (LDC) showed LPS-induced production of IL-12p40. In contrast, LMφ showed constitutive IL-10 production and unresponsiveness to LPS in terms of inflammatory cytokine production. Comparison of the gene expression profiles between LMφ and LDC revealed that LMφ preferentially expressed IL-10-related genes. LMφ obtained from mice lacking IL-10 or Stat3 showed hyperproduction of tumour necrosis factor (TNF)-α and IL-6 in response to LPS. IL-10 production in the large intestine was mainly induced by LMφ and regulatory T cells and was dependent on the presence of commensal microbiota. Accordingly, LMφ from germ-free mice showed less production of IL-10 and increased levels of LPS-induced TNF-α and IL-6 production. Taken together, these results demonstrate that the activity of LMφ to produce pro-inflammatory cytokines is negatively regulated through commensal microbiota-dependent IL-10 production in the large intestine.

Dietary Folic Acid Promotes Survival of Foxp3+ Regulatory T Cells in the Colon

Dietary compounds as well as commensal microbiota contribute to the generation of a unique gut environment. In this study, we report that dietary folic acid (FA) is required for the maintenance of Foxp3+ regulatory T cells (Tregs) in the colon. Deficiency of FA in the diet resulted in marked reduction of Foxp3+ Tregs selectively in the colon. Blockade of folate receptor 4 and treatment with methotrexate, which inhibits folate metabolic pathways, decreased colonic Foxp3+ Tregs. Compared with splenic Tregs, colonic Tregs were more activated to proliferate vigorously and were highly sensitive to apoptosis. In colonic Tregs derived from mice fed with a FA-deficient diet, expression of anti-apoptotic molecules Bcl-2 and Bcl-xL was severely decreased. A general reduction of peripheral Tregs was induced by a neutralizing Ab against IL-2, but a further decrease by additional FA deficiency was observed exclusively in the colon. Mice fed with an FA-deficient diet exhibited higher susceptibility to intestinal inflammation. These findings reveal the previously unappreciated role of dietary FA in promotion of survival of Foxp3+ Tregs that are in a highly activated state in the colon.

Ecto-Nucleoside Triphosphate Diphosphohydrolase 7 Controls Th17 Cell Responses through Regulation of Luminal ATP in the Small Intestine

Extracellular ATP is released from live cells in controlled conditions, as well as dying cells in inflammatory conditions, and, thereby, regulates T cell responses, including Th17 cell induction. The level of extracellular ATP is closely regulated by ATP hydrolyzing enzymes, such as ecto-nucleoside triphosphate diphosphohydrolases (ENTPDases). ENTPDase1/CD39, which is expressed in immune cells, was shown to regulate immune responses by downregulating the ATP level. In this study, we analyzed the immunomodulatory function of ENTPDase7, which is preferentially expressed in epithelial cells in the small intestine. The targeted deletion of Entpd7 encoding ENTPDase7 in mice resulted in increased ATP levels in the small intestinal lumen. The number of Th17 cells was selectively increased in the small intestinal lamina propria in Entpd7−/− mice. Th17 cells were decreased by oral administration of antibiotics or the ATP antagonist in Entpd7−/− mice, indicating that commensal microbiota-dependent ATP release mediates the enhanced Th17 cell development in the small intestinal lamina propria of Entpd7−/− mice. In accordance with the increased number of small intestinal Th17 cells, Entpd7−/− mice were resistant to oral infection with Citrobacter rodentium. Entpd7−/− mice suffered from severe experimental autoimmune encephalomyelitis, which was associated with increased numbers of CD4+ T cells producing both IL-17 and IFN-γ. Taken together, these findings demonstrate that ENTPDase7 controls the luminal ATP level and, thereby, regulates Th17 cell development in the small intestine.

The Ectoenzyme E-NPP3 Negatively Regulates ATP-Dependent Chronic Allergic Responses by Basophils and Mast Cells

Crosslinking of the immunoglobulin receptor FcεRI activates basophils and mast cells to induce immediate and chronic allergic inflammation. However, it remains unclear how the chronic allergic inflammation is regulated. Here, we showed that ecto-nucleotide pyrophosphatase-phosphodiesterase 3 (E-NPP3), also known as CD203c, rapidly induced by FcεRI crosslinking, negatively regulated chronic allergic inflammation. Basophil and mast cell numbers increased in Enpp3(-/-) mice with augmented serum ATP concentrations. Enpp3(-/-) mice were highly sensitive to chronic allergic pathologies, which was reduced by ATP blockade. FcεRI crosslinking induced ATP secretion from basophils and mast cells, and ATP activated both cells. ATP clearance was impaired in Enpp3(-/-) cells. Enpp3(-/-)P2rx7(-/-) mice showed decreased responses to FcεRI crosslinking. Thus, ATP released by FcεRI crosslinking stimulates basophils and mast cells for further activation causing allergic inflammation. E-NPP3 decreases ATP concentration and suppresses basophil and mast cell activity.

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.

Biological implications of thymectomy for myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disease mediated by autoantibodies to the striated muscle tissue. It is often treated by thymectomy. We review recent studies to investigate the biological implications of thymectomy. In anti-acetylcholine receptor antibody (anti-AchR Ab)-positive patients without a thymoma, abnormal germinal center formation in the thymus seems to play an essential role in the pathogenesis of MG. Specific differentiation of B cells producing anti-AchR Ab takes place uniquely in the thymus, and thymectomy is thought to assist in terminating the provision of high-affinity anti-AchR antibody-producing cells to peripheral organs. Thymectomy is not indicated for anti-AchR Ab-negative MG patients who are antimuscle specific kinase antibody (anti-MuSK Ab)-positive, although some anti-MuSK Ab-negative patients may benefit from the procedure. A thymoma can be considered as an acquired thymus with insufficient function of negative selection. The resection of a thymoma is thought to terminate the production of selfreactive T cells. Thus, the biological implications of thymectomy for MG have been partially revealed. Nevertheless, additional studies are needed to elucidate the ontogeny of T cells that recognize AchR and the mechanism of the activation of anti-AchR antibodies producing B cells.

HER3 expression is enhanced during progression of lung adenocarcinoma without EGFR mutation from stage 0 to IA1: Enhanced HER3 in stage IA1 lung ADC

Activating EGFR mutations, HER2, and HER3 are implicated in lung cancer; however, with the exception of EGFR gene amplification in lung adenocarcinoma harboring EGFR mutations, their involvement in disease progression during the early stages is poorly understood. In this paper, we focused on which receptor is correlated with lung adenocarcinoma progression in the presence or absence of EGFR mutation from stage 0 to IA1.

Collagen gel droplet-embedded culture drug sensitivity test for adjuvant chemotherapy after complete resection of non-small-cell lung cancer

PurposeWe conducted a prospective clinical study to individualize adjuvant chemotherapy after complete resection of non-small-cell lung cancer (NSCLC), based on the drug sensitivity test.MethodsPatients with resectable c-stage IB–IIIA NSCLC were registered between 2005 and 2010. We performed the collagen gel droplet-embedded culture drug sensitivity test (CD-DST) on a fresh surgical specimen to assess in vitro chemosensitivity and evaluated the prognostic outcome after adjuvant chemotherapy with carboplatin/paclitaxel based on the CD-DST.ResultsAmong 92 registered patients, 87 were eligible for inclusion in the analysis. The success rate of CD-DST was 86% and chemosensitivity to carboplatin and/or paclitaxel was evident in 57 (76%) of the 75 patients. Adjuvant chemotherapy was completed in 22 (73%) of 30 patients. The 5-year overall survival rates were 71, 73, and 75% for all, CD-DST success, and chemosensitive patients, respectively. The 5-year disease-free survival and overall survival rates of the chemosensitive patients who completed adjuvant chemotherapy using carboplatin/paclitaxel were 68 and 82%, respectively. The 5-year disease-free survival and overall survival rates of the patients with stage II–IIIA chemosensitive NSCLC were 58 and 75%, respectively. Comparative analyses of the chemosensitive and non-chemosensitive/CD-DST failure groups showed no significant survival difference.ConclusionsCD-DST can be used to evaluate chemosensitivity after lung cancer surgery; however, its clinical efficacy for assessing individualized treatment remains uncertain.

Prophylactic middle lobe fixation for postoperative pulmonary torsion

Background To prevent postoperative middle lobe torsion after a right upper lobectomy, we introduced a novel technique of interlobar fixation using collagen fleece coated with fibrin. In this study, the prophylactic effects of this method on the incidence of postoperative pulmonary torsion were analyzed. Methods Between April 2001 and December 2015, 3786 pulmonary resection procedures (excluding total pneumonectomy) were performed in our institution, and prophylactic interlobar fixation was selectively applied when intraoperative examination indicated that the patient was at high risk of postoperative pulmonary lobe torsion. As a control group, 842 patients who underwent pulmonary resection procedures between January 1996 and April 2001 were reviewed. Results During the study period, 10 (0.3%) patients underwent prophylactic middle lobe fixation (to the lower lobe after a right upper lobectomy in 9, and to the upper lobe after a right lower lobectomy in one). Pulmonary lobar (middle lobe) torsion occurred in only one patient (after right upper lobectomy); thus the incidence of this complication was 0.1% among patients who underwent a right upper lobectomy and 0.03% among all pulmonary resection procedures. The rates during the study period were marginally significantly lower than those in the control period (1.3% and 0.24%, respectively; p = 0.071 and p = 0.087, respectively). Conclusion Prophylactic middle lobe fixation might be useful for preventing postoperative pulmonary middle lobe torsion.