Th17 Cell Induction by Adhesion of Microbes to Intestinal Epithelial Cells.

Intestinal Th17 cells are induced and accumulate in response to colonization with a subgroup of intestinal microbes such as segmented filamentous bacteria (SFB) and certain extracellular pathogens. Here, we show that adhesion of microbes to intestinal epithelial cells (ECs) is a critical cue for Th17 induction. Upon monocolonization of germ-free mice or rats with SFB indigenous to mice (M-SFB) or rats (R-SFB), M-SFB and R-SFB showed host-specific adhesion to small intestinal ECs, accompanied by host-specific induction of Th17 cells. Citrobacter rodentium and Escherichia coli O157 triggered similar Th17 responses, whereas adhesion-defective mutants of these microbes failed to do so. Moreover, a mixture of 20 bacterial strains, which were selected and isolated from fecal samples of a patient with ulcerative colitis on the basis of their ability to cause a robust induction of Th17 cells in the mouse colon, also exhibited EC-adhesive characteristics.

Lymphoid-Tissue-Resident Commensal Bacteria Promote Members of the IL-10 Cytokine Family to Establish Mutualism.

Physical separation between the mammalian immune system and commensal bacteria is necessary to limit chronic inflammation. However, selective species of commensal bacteria can reside within intestinal lymphoid tissues of healthy mammals. Here, we demonstrate that lymphoid-tissue-resident commensal bacteria (LRC) colonized murine dendritic cells and modulated their cytokine production. In germ-free and antibiotic-treated mice, LRCs colonized intestinal lymphoid tissues and induced multiple members of the IL-10 cytokine family, including dendritic-cell-derived IL-10 and group 3 innate lymphoid cell (ILC3)-derived IL-22. Notably, IL-10 limited the development of pro-inflammatory Th17 cell responses, and IL-22 production enhanced LRC colonization in the steady state. Furthermore, LRC colonization protected mice from lethal intestinal damage in an IL-10-IL-10R-dependent manner. Collectively, our data reveal a unique host-commensal-bacteria dialog whereby selective subsets of commensal bacteria interact with dendritic cells to facilitate tissue-specific responses that are mutually beneficial for both the host and the microbe.

Induction of intestinal Th17 cells by segmented filamentous bacteria.

The gastrointestinal tract of mammals is inhabited by hundreds of distinct species of commensal microorganisms that exist in a mutualistic relationship with the host. How commensal microbiota influence the host immune system is poorly understood. We show here that colonization of the small intestine of mice with a single commensal microbe, segmented filamentous bacterium (SFB), is sufficient to induce the appearance of CD4(+) T helper cells that produce IL-17 and IL-22 (Th17 cells) in the lamina propria. SFB adhere tightly to the surface of epithelial cells in the terminal ileum of mice with Th17 cells but are absent from mice that have few Th17 cells. Colonization with SFB was correlated with increased expression of genes associated with inflammation and antimicrobial defenses and resulted in enhanced resistance to the intestinal pathogen Citrobacter rodentium. Thus, manipulation of this commensal-regulated pathway may provide new opportunities for enhancing mucosal immunity and treating autoimmune disease.

Gut-residing segmented filamentous bacteria drive autoimmune arthritis via T helper 17 cells.

Commensal microbes can have a substantial impact on autoimmune disorders, but the underlying molecular and cellular mechanisms remain largely unexplored. We report that autoimmune arthritis was strongly attenuated in the K/BxN mouse model under germ-free (GF) conditions, accompanied by reductions in serum autoantibody titers, splenic autoantibody-secreting cells, germinal centers, and the splenic T helper 17 (Th17) cell population. Neutralization of interleukin-17 prevented arthritis development in specific-pathogen-free K/BxN mice resulting from a direct effect of this cytokine on B cells to inhibit germinal center formation. The systemic deficiencies of the GF animals reflected a loss of Th17 cells from the small intestinal lamina propria. Introduction of a single gut-residing species, segmented filamentous bacteria, into GF animals reinstated the lamina propria Th17 cell compartment and production of autoantibodies, and arthritis rapidly ensued. Thus, a single commensal microbe, via its ability to promote a specific Th cell subset, can drive an autoimmune disease.

ATP drives lamina propria T(H)17 cell differentiation.

Interleukin (IL)-17-producing CD4(+) T lymphocytes (T(H)17 cells) constitute a subset of T-helper cells involved in host defence and several immune disorders. An intriguing feature of T(H)17 cells is their selective and constitutive presence in the intestinal lamina propria. Here we show that adenosine 5'-triphosphate (ATP) that can be derived from commensal bacteria activates a unique subset of lamina propria cells, CD70(high)CD11c(low) cells, leading to the differentiation of T(H)17 cells. Germ-free mice exhibit much lower concentrations of luminal ATP, accompanied by fewer lamina propria T(H)17 cells, compared to specific-pathogen-free mice. Systemic or rectal administration of ATP into these germ-free mice results in a marked increase in the number of lamina propria T(H)17 cells. A CD70(high)CD11c(low) subset of the lamina propria cells expresses T(H)17-prone molecules, such as IL-6, IL-23p19 and transforming-growth-factor-beta-activating integrin-alphaV and -beta8, in response to ATP stimulation, and preferentially induces T(H)17 differentiation of co-cultured naive CD4(+) T cells. The critical role of ATP is further underscored by the observation that administration of ATP exacerbates a T-cell-mediated colitis model with enhanced T(H)17 differentiation. These observations highlight the importance of commensal bacteria and ATP for T(H)17 differentiation in health and disease, and offer an explanation of why T(H)17 cells specifically present in the intestinal lamina propria.

Impacts of Habitual Diets Intake on Gut Microbial Counts in Healthy Japanese Adults.

Although diet is an important factor influencing gut microbiota, there are very few studies regarding that relationship in Japanese people. Here, we analyzed the relationship between habitual dietary intake surveyed by food frequency questionnaire and the quantitative features of gut bacteria by quantitative PCR and next generation sequencer in 354 healthy Japanese adults. The α-diversity of gut microbiota was positively correlated with the intake of mushrooms and beans and negatively correlated with the intake of grains. The ß-diversity was significantly associated with the intake of fruits, mushrooms, seaweeds, seafoods, and alcoholic beverages. Multiple linear regression analysis of the relationship between food groups associated with the diversity of gut microbiota and the number of gut bacteria at the genus level found 24 significant associations, including a positive association between alcoholic beverages and the number of Fusobacterium. These results support that habitual dietary intake influenced the diversity of gut microbiota and was strongly associated with the number of specific gut bacteria. These results will help us to understand the complex relationship between habitual diet and gut microbiota of the Japanese.

Intracellular offspring released from SFB filaments are flagellated.

The gut commensal segmented filamentous bacterium (SFB) attaches to the ileal epithelium and potently stimulates the host immune system. Using transmission electron microscopy (TEM), we show that mouse and rat SFB are flagellated above the concave tip at the unicellular intracellular offspring (IO) stage and that flagellation occurs prior to full IO differentiation and release of IOs from SFB filaments. This finding adds a missing link to the SFB life cycle.

Differential effects of two probiotic strains with different bacteriological properties on intestinal gene expression, with special reference to indigenous bacteria.

Probiotics are used for the improvement of gut disorders. To explore the potential of probiotics, a gnotobiotic study using BALB/c mice to analyze epithelial gene expression was performed. Microarray analysis of probiotic strain-monoassociated mice showed that Lactobacillus casei Shirota and Bifidobacterium breve Yakult noticeably affected gene expression in the ileal and colonic epithelial cells, respectively, although to a smaller extent than segmented filamentous bacteria (SFB). Lactobacillus casei Shirota enhanced the gene expression involving defense/immune functions and lipid metabolism more strongly than B. breve Yakult. In the colon, expression of a chloride transporter was slightly enhanced, although downregulation of many genes, such as guanine nucleotide-binding protein, was evident in mice with B. breve Yakult compared with the ones with L. casei Shirota. SFB affected gene expression more strongly than the probiotic strains. In particular, alpha(1-2) fucosyltransferase and pancreatitis-associated protein were significantly enhanced only in SFB-monoassociated mice but not probiotic strain-monoassociated mice. Gene expression of SFB-monoassociated mice was either stimulated or repressed in a manner similar to or opposite that of conventional colonized mice. Taken together, probiotic strains of L. casei Shirota and B. breve Yakult differentially affect epithelial gene expression in the small intestine and colon, respectively.

Genetic variation in the flanking regions of Shiga toxin 2 gene in Shiga toxin-producing Escherichia coli O157:H7 isolated in Japan.

We found frequent IS1 integration nearby the stx(2) gene during in vitro mutagenesis of an stx(2) variant, stx(2vhd). To examine the possibility that such insertions have been contributing to generate new stx(2) variants, we screened 86 strains of Escherichia coli O157:H7 isolated in Japan for variations in the ca. 4-kb region flanking the stx(2) locus using PCR methods. Two major classes were identified based on the PCR amplicon size. DNA sequence analysis revealed that the stx(2) subtype of the two classes were stx(2) (referred to as stx(2-EDL933)) and stx(2vhd). IS1203v insertions were found in three stx(2vhd)-positive strains and two stx(2-EDL933)-positive strains, and no other insertions were found. These results suggest that the DNA sequences surrounding the stx(2) genes are preferably integrated by IS1203v in wild-type Shiga toxin-producing E. coli strains.

Comparison of Bifidobacterium breve strain Yakult transcriptomes in germ-free mice with those in fecal cultures.

Bifidobacteria are beneficial to human health, but the mechanism remains unknown. We employed oligonucleotide microarrays to identify the Bifidobacterium breve strain Yakult (BbrY) genes up-regulated specifically in mouse intestine. Based on BbrY transcriptional responses in germ-free mice and in fecal cultures, k-means clustering picked up 93 genes that were up-regulated in the mouse intestine and thereafter Venn analysis to exclude genes that were up-regulated in both the mouse intestine and the fecal culture classified 45 genes as up-regulated specifically in the mouse intestine. Most of those genes are involved in sugar transport or sugar liberation, although the functions of several genes are unknown. Most of these genes are clustered on the BbrY genome and appear to be organized into operons. Expressions of several genes were further investigated by real time PCR, revealing that their expression profiles were identical in the mouse cecum and colon. The up-regulation of genes involved in sugar liberalization and uptake suggests that BbrY could possibly maintain energy homeostasis inside the mouse intestine, which contains low quantities of readily fermentable sugars.

Induction of colonic regulatory T cells by indigenous Clostridium species.

CD4(+) T regulatory cells (T(regs)), which express the Foxp3 transcription factor, play a critical role in the maintenance of immune homeostasis. Here, we show that in mice, T(regs) were most abundant in the colonic mucosa. The spore-forming component of indigenous intestinal microbiota, particularly clusters IV and XIVa of the genus Clostridium, promoted T(reg) cell accumulation. Colonization of mice by a defined mix of Clostridium strains provided an environment rich in transforming growth factor-ß and affected Foxp3(+) T(reg) number and function in the colon. Oral inoculation of Clostridium during the early life of conventionally reared mice resulted in resistance to colitis and systemic immunoglobulin E responses in adult mice, suggesting a new therapeutic approach to autoimmunity and allergy.

Anti-inflammatory activity of probiotic Bifidobacterium: enhancement of IL-10 production in peripheral blood mononuclear cells from ulcerative colitis patients and inhibition of IL-8 secretion in HT-29 cells.

AIM: To determine the anti-inflammatory activity of probiotic Bifidobacteria in Bifidobacteria-fermented milk (BFM) which is effective against active ulcerative colitis (UC) and exacerbations of UC, and to explore the immunoregulatory mechanisms. METHODS: Peripheral blood mononuclear cells (PBMNC) from UC patients or HT-29 cells were co-cultured with heat-killed probiotic bacteria or culture supernatant of Bifidobacterium breve strain Yakult (BbrY) or Bifidobacterium bifidum strain Yakult (BbiY) to estimate the amount of IL-10 or IL-8 secreted. RESULTS: Both strains of probiotic Bifidobacteria contained in the BFM induced IL-10 production in PBMNC from UC patients, though BbrY was more effective than BbiY. Conditioned medium (CM) and DNA of both strains inhibited IL-8 secretion in HT-29 cells stimulated with TNF-alpha, whereas no such effect was observed with heat-killed bacteria. The inhibitory effect of CM derived from BbiY was greater than that of CM derived from BbrY. DNAs of the two strains had a comparable inhibitory activity against the secretion of IL-8. CM of BbiY induced a repression of IL-8 gene expression with a higher expression of IkappaB-zeta mRNA 4 h after culture of HT-29 cells compared to that in the absence of CM. CONCLUSION: Probiotic Bifidobacterium strains in BFM enhance IL-10 production in PBMNC and inhibit IL-8 secretion in intestinal epithelial cells, suggesting that BFM has anti-inflammatory effects against ulcerative colitis.