Takashi Kurakawa

Dysbiosis contributes to arthritis development via activation of autoreactive T cells in the intestine.

The intestinal microbiota is involved in the pathogenesis of arthritis. Altered microbiota composition has been demonstrated in patients with rheumatoid arthritis (RA). However, it remains unclear how dysbiosis contributes to the development of arthritis. The aim of this study was to investigate whether altered composition of human intestinal microbiota in RA patients contributes to the development of arthritis.

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.

Generation of colonic IgA-secreting cells in the caecal patch

Gut-associated lymphoid tissues are responsible for the generation of IgA-secreting cells. However, the function of the caecal patch, a lymphoid tissue in the appendix, remains unknown. Here we analyse the role of the caecal patch using germ-free mice colonized with intestinal bacteria after appendectomy. Appendectomized mice show delayed accumulation of IgA(+) cells in the large intestine, but not the small intestine, after colonization. Decreased colonic IgA(+) cells correlate with altered faecal microbiota composition. Experiments using photoconvertible Kaede-expressing mice or adoptive transfer show that the caecal patch IgA(+) cells migrate to the large and small intestines, whereas Peyers patch cells are preferentially recruited to the small intestine. IgA(+) cells in the caecal patch express higher levels of CCR10. Dendritic cells in the caecal patch, but not Peyers patches, induce CCR10 on cocultured B cells. Thus, the caecal patch is a major site for generation of IgA-secreting cells that migrate to the large intestine.

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.

An Improved Method for High Quality Metagenomics DNA Extraction from Human and Environmental Samples

To explore the natural microbial community of any ecosystems by high-resolution molecular approaches including next generation sequencing, it is extremely important to develop a sensitive and reproducible DNA extraction method that facilitate isolation of microbial DNA of sufficient purity and quantity from culturable and uncultured microbial species living in that environment. Proper lysis of heterogeneous community microbial cells without damaging their genomes is a major challenge. In this study, we have developed an improved method for extraction of community DNA from different environmental and human origin samples. We introduced a combination of physical, chemical and mechanical lysis methods for proper lysis of microbial inhabitants. The community microbial DNA was precipitated by using salt and organic solvent. Both the quality and quantity of isolated DNA was compared with the existing methodologies and the supremacy of our method was confirmed. Maximum recovery of genomic DNA in the absence of substantial amount of impurities made the method convenient for nucleic acid extraction. The nucleic acids obtained using this method are suitable for different downstream applications. This improved method has been named as the THSTI method to depict the Institute where the method was developed.

Diversity of Intestinal Clostridium coccoides Group in the Japanese Population, as Demonstrated by Reverse Transcription-Quantitative PCR

We used sensitive rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) to quantify the Clostridium coccoides group, which is a major anaerobic population in the human intestine. For this purpose, the C. coccoides group was classified into 3 subgroups and 19 species for expediency in accordance with the existing database, and specific primers were newly developed to evaluate them. Population levels of the C. coccoides group in human feces determined by RT-qPCR were equivalent to those determined by fluorescence in situ hybridization. RT-qPCR analysis of fecal samples from 96 volunteers (32 young children, 32 adults and 32 elderly) by using the 22 new primer sets together with the C. coccoides group-specific primer setm revealed that (i) total counts obtained as the sum of the 3 subgroups and 19 species were equivalent to the results obtained by using the C. coccoides group-specific primer set; (ii) total C. coccoides-group counts in the elderly were significantly lower than those in young children and adults; (iii) genus Blautia was the most common subgroup in the human intestinal C. coccoides-group populations at all age populations tested; (iv) the prevalences of Fusicatenibacter saccharivorans and genus Dorea were significantly higher in adults than in young children and the elderly; and (v) the prevalences of C. scindens and C. hylemonae, both of which produce secondary bile acid in the human intestine, were significantly higher in the elderly than in young children and adults. Hierarchical clustering and principal component analysis showed clear separation of the bacterial components between adult and elderly populations. Taken together, these data suggest that aging plays an important role in the diversity of C. coccoides-group populations in human intestinal microbiota; changes in this diversity likely influence the health of the host.

Fusicatenibacter saccharivorans gen. nov., sp. nov., isolated from human faeces.

Three Gram-stain-positive, obligately anaerobic, non-motile, non-spore-forming, spindle-shaped bacterial strains (HT03-11(T), KO-38 and TT-111), isolated from human faeces were characterized by phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing showed that the strains were highly related to each other genetically (displaying >99 % sequence similarity) and represented a previously unknown subline within the Blautia coccoides rRNA group of organisms (cluster XIVa). The closest phylogenetic neighbours of strain HT03-11(T) were Clostridium bolteae WAL 16351(T) (93.7 % 16S rRNA gene sequence similarity) and Clostridium saccharolyticum WM1(T) (93.7 % similarity). All isolates produced lactic acid, formic acid, acetic acid and succinic acid as fermentation end products from glucose. Their chemotaxonomic properties included lysine as the cell wall diamino acid and C16 : 0, C18 : 1ω7c DMA and C16 : 0 DMA as the major fatty acids. The G+C contents of the genomic DNA were 46.9-47.2 mol% (HPLC). Several phenotypic and chemotaxonomic characteristics could be readily used to differentiate the isolates from phylogenetically related clostridia. Therefore, strains HT03-11(T), KO-38 and TT-111 represent a novel species in a new genus of the family Lachnospiraceae, for which the name Fusicatenibacter saccharivorans gen. nov., sp. nov. is proposed. The type strain of the type species is HT03-11(T) ( = YIT 12554(T) = JCM 18507(T) = DSM 26062(T)).

Intestinal Enterobacteriaceae and Escherichia coli populations in Japanese adults demonstrated by the reverse transcription-quantitative PCR and the clone library analyses.

A primer set specific for Escherichia coli/Shigella 16S rRNA was developed and used for RT-qPCR analysis of fecal samples from 35 healthy adult volunteers in combination with the previously reported primer set specific for Enterobacteriaceae. Enterobacteriaceae and E. coli were present in the 29 out of 35 volunteers tested as intestinal commensals at the average population levels of 10(7.1±0.9) and 10(6.8±0.7)cellsg(-1) of stools (mean±standard deviation), respectively. Among the 7 volunteers, the significant deviation between the count of Enterobacteriaceae and that of E. coli was observed, suggesting non-E. coli/Shigella species were predominant in their Enterobacteriaceae populations. The clone library analysis revealed that the non-E. coli/Shigella populations included Citrobacter freundii, Citrobacter koseri, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae/variicola and Morganella morganii. These data suggested that the RT-qPCR method with the primer sets specific to both Enterobacteriaceae and E. coli/Shigella enabled the accurate enumeration of intestinal E. coli populations and the other Enterobacteriaceae species populations.

Fungal ITS1 Deep-Sequencing Strategies to Reconstruct the Composition of a 26-Species Community and Evaluation of the Gut Mycobiota of Healthy Japanese Individuals

The study of mycobiota remains relatively unexplored due to the lack of sufficient available reference strains and databases compared to those of bacterial microbiome studies. Deep sequencing of Internal Transcribed Spacer (ITS) regions is the de facto standard for fungal diversity analysis. However, results are often biased because of the wide variety of sequence lengths in the ITS regions and the complexity of high-throughput sequencing (HTS) technologies. In this study, a curated ITS database, ntF-ITS1, was constructed. This database can be utilized for the taxonomic assignment of fungal community members. We evaluated the efficacy of strategies for mycobiome analysis by using this database and characterizing a mock fungal community consisting of 26 species representing 15 genera using ITS1 sequencing with three HTS platforms: Illumina MiSeq (MiSeq), Ion Torrent Personal Genome Machine (IonPGM), and Pacific Biosciences (PacBio). Our evaluation demonstrated that PacBio’s circular consensus sequencing with greater than 8 full-passes most accurately reconstructed the composition of the mock community. Using this strategy for deep-sequencing analysis of the gut mycobiota in healthy Japanese individuals revealed two major mycobiota types: a single-species type composed of Candida albicans or Saccharomyces cerevisiae and a multi-species type. In this study, we proposed the best possible processing strategies for the three sequencing platforms, of which, the PacBio platform allowed for the most accurate estimation of the fungal community. The database and methodology described here provide critical tools for the emerging field of mycobiome studies.

Vibrio cholerae/mimicus in fecal microbiota of healthy children in a cholera endemic urban slum setting in Kolkata, India

During a double‐blind, randomized, placebo‐controlled probiotic trial among 3758 children residing in an urban slum in Kolkata, India, Vibrio cholerae/mimicus was detected in fecal microbiota of healthy children. The importance of this finding in the local, regional and global transmission of cholera is discussed.