Critical roles of a housekeeping sortase of probiotic Bifidobacterium bifidum in bacterium-host cell crosstalk.

Bifidobacterium bifidum YIT 10347 (BF-1) is adhesive in vitro. Here we studied the molecular aspects of the BF-1 adhesion process. We identified and characterized non-adhesive mutants and found that a class E housekeeping sortase was critical for the adhesion to mucin. These mutants were significantly less adhesive to GCIY cells than was the wild type (WT), which protected GCIY cells against acid treatment more than did a non-adhesive mutant. The non-adhesive mutants aberrantly accumulated precursors of putative sortase-dependent proteins (SDPs). Recombinant SDPs bound to mucin. Disruption of the housekeeping sortase influenced expression of SDPs and pilus components. Mutants defective in a pilin or in an SDP showed the same adhesion properties as WT. Therefore, multiple SDPs and pili seem to work cooperatively to achieve adhesion, and the housekeeping sortase is responsible for cell wall anchoring of its substrates to ensure their proper biological function.

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.

Pulmonary Th17 Antifungal Immunity Is Regulated by the Gut Microbiome.

Commensal microbiota are critical for the development of local immune responses. In this article, we show that gut microbiota can regulate CD4 T cell polarization during pulmonary fungal infections. Vancomycin drinking water significantly decreased lung Th17 cell numbers during acute infection, demonstrating that Gram-positive commensals contribute to systemic inflammation. We next tested a role for RegIIIγ, an IL-22-inducible antimicrobial protein with specificity for Gram-positive bacteria. Following infection, increased accumulation of Th17 cells in the lungs of RegIIIγ(-/-) and Il22(-/-) mice was associated with intestinal segmented filamentous bacteria (SFB) colonization. Although gastrointestinal delivery of rRegIIIγ decreased lung inflammatory gene expression and protected Il22(-/-) mice from weight loss during infection, it had no direct effect on SFB colonization, fungal clearance, or lung Th17 immunity. We further show that vancomycin only decreased lung IL-17 production in mice colonized with SFB. To determine the link between gut microbiota and lung immunity, serum-transfer experiments revealed that IL-1R ligands increase the accumulation of lung Th17 cells. These data suggest that intestinal microbiota, including SFB, can regulate pulmonary adaptive immune responses.

Gut Microbiota Drive Autoimmune Arthritis by Promoting Differentiation and Migration of Peyer's Patch T Follicular Helper Cells.

Gut microbiota profoundly affect gut and systemic diseases, but the mechanism whereby microbiota affect systemic diseases is unclear. It is not known whether specific microbiota regulate T follicular helper (Tfh) cells, whose excessive responses can inflict antibody-mediated autoimmunity. Using the K/BxN autoimmune arthritis model, we demonstrated that Peyer's patch (PP) Tfh cells were essential for gut commensal segmented filamentous bacteria (SFB)-induced systemic arthritis despite the production of auto-antibodies predominantly occurring in systemic lymphoid tissues, not PPs. We determined that SFB, by driving differentiation and egress of PP Tfh cells into systemic sites, boosted systemic Tfh cell and auto-antibody responses that exacerbated arthritis. SFB induced PP Tfh cell differentiation by limiting the access of interleukin 2 to CD4(+) T cells, thereby enhancing Tfh cell master regulator Bcl-6 in a dendritic cell-dependent manner. These findings showed that gut microbiota remotely regulated a systemic disease by driving the induction and egress of gut Tfh cells.

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.

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.

Use of gnotobiotic mice to identify and characterize key microbes responsible for the development of the intestinal immune system.

Symbiosis between intestinal microbiota and the host animal plays an important role in the homeostasis of host physiology. Since the first production of germ-free rodents in 1945, it has become increasingly clear that the intestinal immune system and the biochemical characteristics of epithelial cells differ greatly between conventional and germ-free rodents. However, questions remain about the types of microbes involved and the precise mechanism by which these microbes affect the host physiology. Here, we review experiments designed to answer these questions with the use of gnotobiotic mice. We have determined suitable biochemical and immunological markers for monitoring microbial effects in these mice. Using these markers, we have found clear differences in epithelial cell glycolipid biosynthesis and intraepithelial lymphocyte dynamics between germ-free and conventional mice. Furthermore, we have identified a key microbe that activates the mucosal immune system in the small intestine. This indigenous bacteria, called segmented filamentous bacteria, is a key symbiont in the host-microbiota interplay, including Th17 cell-inducing activity.

Innate lymphoid cells regulate intestinal epithelial cell glycosylation.

Fucosylation of intestinal epithelial cells, catalyzed by fucosyltransferase 2 (Fut2), is a major glycosylation mechanism of host-microbiota symbiosis. Commensal bacteria induce epithelial fucosylation, and epithelial fucose is used as a dietary carbohydrate by many of these bacteria. However, the molecular and cellular mechanisms that regulate the induction of epithelial fucosylation are unknown. Here, we show that type 3 innate lymphoid cells (ILC3) induced intestinal epithelial Fut2 expression and fucosylation in mice. This induction required the cytokines interleukin-22 and lymphotoxin in a commensal bacteria-dependent and -independent manner, respectively. Disruption of intestinal fucosylation led to increased susceptibility to infection by Salmonella typhimurium. Our data reveal a role for ILC3 in shaping the gut microenvironment through the regulation of epithelial glycosylation.

Gender bias in autoimmunity is influenced by microbiota.

Gender bias and the role of sex hormones in autoimmune diseases are well established. In specific pathogen-free nonobese diabetic (NOD) mice, females have 1.3-4.4 times higher incidence of type 1 diabetes (T1D). Germ-free (GF) mice lost the gender bias (female-to-male ratio 1.1-1.2). Gut microbiota differed in males and females, a trend reversed by male castration, confirming that androgens influence gut microbiota. Colonization of GF NOD mice with defined microbiota revealed that some, but not all, lineages overrepresented in male mice supported a gender bias in T1D. Although protection of males did not correlate with blood androgen concentration, hormone-supported expansion of selected microbial lineages may work as a positive-feedback mechanism contributing to the sexual dimorphism of autoimmune diseases. Gene-expression analysis suggested pathways involved in protection of males from T1D by microbiota. Our results favor a two-signal model of gender bias, in which hormones and microbes together trigger protective pathways.

Gut immune maturation depends on colonization with a host-specific microbiota.

Gut microbial induction of host immune maturation exemplifies host-microbe mutualism. We colonized germ-free (GF) mice with mouse microbiota (MMb) or human microbiota (HMb) to determine whether small intestinal immune maturation depends on a coevolved host-specific microbiota. Gut bacterial numbers and phylum abundance were similar in MMb and HMb mice, but bacterial species differed, especially the Firmicutes. HMb mouse intestines had low levels of CD4(+) and CD8(+) T cells, few proliferating T cells, few dendritic cells, and low antimicrobial peptide expression--all characteristics of GF mice. Rat microbiota also failed to fully expand intestinal T cell numbers in mice. Colonizing GF or HMb mice with mouse-segmented filamentous bacteria (SFB) partially restored T cell numbers, suggesting that SFB and other MMb organisms are required for full immune maturation in mice. Importantly, MMb conferred better protection against Salmonella infection than HMb. A host-specific microbiota appears to be critical for a healthy immune system.

Complete genome sequences of rat and mouse segmented filamentous bacteria, a potent inducer of th17 cell differentiation.

Segmented filamentous bacteria (SFB) are noncultivable commensals inhabiting the gut of various vertebrate species and have been shown to induce Th17 cells in mice. We present the complete genome sequences of both rat and mouse SFB isolated from SFB-monocolonized hosts. The rat and mouse SFB genomes each harbor a single circular chromosome of 1.52 and 1.59 Mb encoding 1346 and 1420 protein-coding genes, respectively. The overall nucleotide identity between the two genomes is 86%, and the substitution rate was estimated to be similar to that of the free-living E. coli. SFB genomes encode typical genes for anaerobic fermentation and spore and flagella formation, but lack most of the amino acid biosynthesis enzymes, reminiscent of pathogenic Clostridia, exhibiting large dependency on the host. However, SFB lack most of the clostridial virulence-related genes. Comparative analysis with clostridial genomes suggested possible mechanisms for host responses and specific adaptations in the intestine.

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.

Beneficial effects of probiotic bifidobacterium and galacto-oligosaccharide in patients with ulcerative colitis: a randomized controlled study.

BACKGROUND/AIM: In previous studies, we described the beneficial effects of bifidobacteria-fermented milk in patients with ulcerative colitis (UC). Here, we examined the effects of a live Bifidobacterium breve strain Yakult, a probiotic contained in bifidobacteria-fermented milk, and galacto-oligosaccharide (GOS) as synbiotics in UC patients. METHODS: Forty-one patients with mild to moderate UC were assigned to two groups; one group was treated with the synbiotics and the other was not (control). The treatment group ingested 1 g of the probiotic powder (10(9) CFU/g) three times a day, and 5.5 g of GOS once a day for one year. At the start and the end of this study, colonoscopic index and the amount of myeloperoxidase in a lavage solution were used as disease activity indices. Bacterial counts in the feces at the start and the end of this study were also examined. RESULTS: After a one-year treatment with the synbiotics, the clinical status of the UC patients as assessed by colonoscopy, significantly improved. Furthermore, the amount of myeloperoxidase in the lavage also decreased in these patients after the synbiotic treatment. The synbiotics significantly reduced the fecal counts of Bacteroidaceae and fecal pH. CONCLUSION: Administration of live B. breve strain Yakult and GOS can improve the clinical condition of patients with UC. These results encouraged us to perform a large-scale randomized, placebo-controlled trial.

Microbiotal influence on T cell subset development.

The mammalian alimentary tract harbors hundreds of bacterial species that constitute the indigenous microbial flora. The indigenous microbial flora has long been appreciated for its role in host immune system development. Recent reports suggest that components of the microbial flora differentially affect the proportion and number of functionally distinct subsets of T cells in the intestine. Substantial changes in the composition of the microbiota are associated with inflammatory bowel disease. This review will discuss the importance of individual species of microbial flora in the induction of T cell subsets, particularly Th17 cells and regulatory T (Treg) cells in the intestine.

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.

Proinflammatory T-cell responses to gut microbiota promote experimental autoimmune encephalomyelitis.

Although the effects of commensal bacteria on intestinal immune development seem to be profound, it remains speculative whether the gut microbiota influences extraintestinal biological functions. Multiple sclerosis (MS) is a devastating autoimmune disease leading to progressive deterioration of neurological function. Although the cause of MS is unknown, microorganisms seem to be important for the onset and/or progression of disease. However, it is unclear how microbial colonization, either symbiotic or infectious, affects autoimmunity. Herein, we investigate a role for the microbiota during the induction of experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Mice maintained under germ-free conditions develop significantly attenuated EAE compared with conventionally colonized mice. Germ-free animals, induced for EAE, produce lower levels of the proinflammatory cytokines IFN-γ and IL-17A in both the intestine and spinal cord but display a reciprocal increase in CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). Mechanistically, we show that gut dendritic cells from germ-free animals are reduced in the ability to stimulate proinflammatory T cell responses. Intestinal colonization with segmented filamentous bacteria (SFB) is known to promote IL-17 production in the gut; here, we show that SFBs also induced IL-17A-producing CD4(+) T cells (Th17) in the CNS. Remarkably, germ-free animals harboring SFBs alone developed EAE, showing that gut bacteria can affect neurologic inflammation. These findings reveal that the intestinal microbiota profoundly impacts the balance between pro- and antiinflammatory immune responses during EAE and suggest that modulation of gut bacteria may provide therapeutic targets for extraintestinal inflammatory diseases such as MS.

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.

Indigenous opportunistic bacteria inhabit mammalian gut-associated lymphoid tissues and share a mucosal antibody-mediated symbiosis.

The indigenous bacteria create natural cohabitation niches together with mucosal Abs in the gastrointestinal (GI) tract. Here we report that opportunistic bacteria, largely Alcaligenes species, specifically inhabit host Peyer's patches (PPs) and isolated lymphoid follicles, with the associated preferential induction of antigen-specific mucosal IgA Abs in the GI tract. Alcaligenes were identified as the dominant bacteria on the interior of PPs from naïve, specific-pathogen-free but not from germ-free mice. Oral transfer of intratissue uncultured Alcaligenes into germ-free mice resulted in the presence of Alcaligenes inside the PPs of recipients. This result was further supported by the induction of antigen-specific Ab-producing cells in the mucosal (e.g., PPs) but not systemic compartment (e.g., spleen). The preferential presence of Alcaligenes inside PPs and the associated induction of intestinal secretory IgA Abs were also observed in both monkeys and humans. Localized mucosal Ab-mediated symbiotic immune responses were supported by Alcaligenes-stimulated CD11c(+) dendritic cells (DCs) producing the Ab-enhancing cytokines TGF-beta, B-cell-activating factor belonging to the TNF family, and IL-6 in PPs. These CD11c(+) DCs did not migrate beyond the draining mesenteric lymph nodes. In the absence of antigen-specific mucosal Abs, the presence of Alcaligenes in PPs was greatly diminished. Thus, indigenous opportunistic bacteria uniquely inhabit PPs, leading to PP-DCs-initiated, local antigen-specific Ab production; this may involve the creation of an optimal symbiotic environment on the interior of the PPs.

Immune responses to gut microbiota-commensals and pathogens.

The mammalian alimentary tract harbors hundreds of species of commensal microorganisms that intimately interact with the host immune system. Within the gut, the immune system actively reacts with potentially pathogenic microbes, while simultaneously remaining ignorant towards the vast majority of non-pathogenic microbiota. The disruption of this delicate balance results in inflammatory bowel diseases. In this review, we describe the recent advances in our understanding of how host-microbiota interactions shape the immune system and how they affect the responses against pathogenic bacteria.

Effect of Lactobacillus casei on Streptococcus bovis in faecal flora.

Bacteraemia caused by Streptococcus bovis is often associated with colorectal tumours. Also, experimental studies have been proposed that S bovis acts as a promoter of tumours. We report the case of a man with colon adenoma who had a high proportion of S bovis in his faecal flora. He was treated with a Lactobacillus casei preparation (BLP) and the effect on the faecal flora was examined. L casei reduced the proportion of S bovis (from 43% down to 9%), and the effect continued after the administration of BLP was stopped. Our data indicate that BLP can repress the excessive colonisation of S bovis.