Microbiota Sensing by Mincle-Syk Axis in Dendritic Cells Regulates Interleukin-17 and -22 Production and Promotes Intestinal Barrier Integrity.

Production of interleukin-17 (IL-17) and IL-22 by T helper 17 (Th17) cells and group 3 innate lymphoid cells (ILC3s) in response to the gut microbiota ensures maintenance of intestinal barrier function. Here, we examined the mechanisms whereby the immune system detects microbiota in the steady state. A Syk-kinase-coupled signaling pathway in dendritic cells (DCs) was critical for commensal-dependent production of IL-17 and IL-22 by CD4+ T cells. The Syk-coupled C-type lectin receptor Mincle detected mucosal-resident commensals in the Peyer's patches (PPs), triggered IL-6 and IL-23p19 expression, and thereby regulated function of intestinal Th17- and IL-17-secreting ILCs. Mice deficient in Mincle or with selective depletion of Syk in CD11c+ cells had impaired production of intestinal RegIIIγ and IgA and increased systemic translocation of gut microbiota. Consequently, Mincle deficiency led to liver inflammation and deregulated lipid metabolism. Thus, sensing of commensals by Mincle and Syk signaling in CD11c+ cells reinforces intestinal immune barrier and promotes host-microbiota mutualism, preventing systemic inflammation.

Diversification of memory B cells drives the continuous adaptation of secretory antibodies to gut microbiota.

Secretory immunoglobulin A (SIgA) shields the gut epithelium from luminal antigens and contributes to host-microbe symbiosis. However, how antibody responses are regulated to achieve sustained host-microbe interactions is unknown. We found that mice and humans exhibited longitudinal persistence of clonally related B cells in the IgA repertoire despite major changes in the microbiota during antibiotic treatment or infection. Memory B cells recirculated between inductive compartments and were clonally related to plasma cells in gut and mammary glands. Our findings suggest that continuous diversification of memory B cells constitutes a central process for establishing symbiotic host-microbe interactions and offer an explanation of how maternal antibodies are optimized throughout life to protect the newborn.

Intestinal Microbiota Increases Cell Proliferation of Colonic Mucosa in Human-Flora-Associated (HFA) Mice.

Intestinal epithelium renewal strictly depends on fine regulation between cell proliferation, differentiation, and apoptosis. While murine intestinal microbiota has been shown to modify some epithelial cell kinetics parameters, less is known about the role of the human intestinal microbiota. Here, we investigated the rate of intestinal cell proliferation in C3H/HeN germ-free mice associated with human flora (HFA, n = 8), and in germ-free (n = 15) and holoxenic mice (n = 16). One hour before sacrifice, all mice were intraperitoneally inoculated with 5-bromodeoxyuridine (BrdU), and the number of BrdU-positive cells/total cells (labelling index, LI), both in the jejunum and the colon, was evaluated by immunohistochemistry. Samples were also observed by scanning electron microscopy (SEM). Moreover, the microbiota composition in the large bowel of the HFA mice was compared to that of of human donor's fecal sample. No differences in LI were found in the small bowels of the HFA, holoxenic, and germ-free mice. Conversely, the LI in the large bowel of the HFA mice was significantly higher than that in the germ-free and holoxenic counterparts (p = 0.017 and p = 0.048, respectively). In the holoxenic and HFA mice, the SEM analysis disclosed different types of bacteria in close contact with the intestinal epithelium. Finally, the colonic microbiota composition of the HFA mice widely overlapped with that of the human donor in terms of dominant populations, although Bifidobacteria and Lactobacilli disappeared. Despite the small sample size analyzed in this study, these preliminary findings suggest that human intestinal microbiota may promote a high proliferation rate of colonic mucosa. In light of the well-known role of uncontrolled proliferation in colorectal carcinogenesis, these results may deserve further investigation in a larger population study.

Segmented filamentous bacterium uses secondary and tertiary lymphoid tissues to induce gut IgA and specific T helper 17 cell responses.

Segmented filamentous bacterium (SFB) is a symbiont that drives postnatal maturation of gut adaptive immune responses. In contrast to nonpathogenic E. coli, SFB stimulated vigorous development of Peyer's patches germinal centers but paradoxically induced only a low frequency of specific immunoglobulin A (IgA)-secreting cells with delayed accumulation of somatic mutations. Moreover, blocking Peyer's patch development abolished IgA responses to E. coli, but not to SFB. Indeed, SFB stimulated the postnatal development of isolated lymphoid follicles and tertiary lymphoid tissue, which substituted for Peyer's patches as inductive sites for intestinal IgA and SFB-specific T helper 17 (Th17) cell responses. Strikingly, in mice depleted of gut organized lymphoid tissue, SFB still induced a substantial but nonspecific intestinal Th17 cell response. These results demonstrate that SFB has the remarkable capacity to induce and stimulate multiple types of intestinal lymphoid tissues that cooperate to generate potent IgA and Th17 cell responses displaying only limited target specificity.

Growth and host interaction of mouse segmented filamentous bacteria in vitro.

The gut microbiota plays a crucial role in the maturation of the intestinal mucosal immune system of its host. Within the thousand bacterial species present in the intestine, the symbiont segmented filamentous bacterium (SFB) is unique in its ability to potently stimulate the post-natal maturation of the B- and T-cell compartments and induce a striking increase in the small-intestinal Th17 responses. Unlike other commensals, SFB intimately attaches to absorptive epithelial cells in the ileum and cells overlying Peyer's patches. This colonization does not result in pathology; rather, it protects the host from pathogens. Yet, little is known about the SFB-host interaction that underlies the important immunostimulatory properties of SFB, because SFB have resisted in vitro culturing for more than 50 years. Here we grow mouse SFB outside their host in an SFB-host cell co-culturing system. Single-celled SFB isolated from monocolonized mice undergo filamentation, segmentation, and differentiation to release viable infectious particles, the intracellular offspring, which can colonize mice to induce signature immune responses. In vitro, intracellular offspring can attach to mouse and human host cells and recruit actin. In addition, SFB can potently stimulate the upregulation of host innate defence genes, inflammatory cytokines, and chemokines. In vitro culturing thereby mimics the in vivo niche, provides new insights into SFB growth requirements and their immunostimulatory potential, and makes possible the investigation of the complex developmental stages of SFB and the detailed dissection of the unique SFB-host interaction at the cellular and molecular levels.

Commensal microbiota influence systemic autoimmune responses.

Antinuclear antibodies are a hallmark feature of generalized autoimmune diseases, including systemic lupus erythematosus and systemic sclerosis. However, the processes underlying the loss of tolerance against nuclear self-constituents remain largely unresolved. Using mice deficient in lymphotoxin and Hox11, we report that approximately 25% of mice lacking secondary lymphoid organs spontaneously develop specific antinuclear antibodies. Interestingly, we find this phenotype is not caused by a defect in central tolerance. Rather, cell-specific deletion and in vivo lymphotoxin blockade link these systemic autoimmune responses to the formation of gut-associated lymphoid tissue in the neonatal period of life. We further demonstrate antinuclear antibody production is influenced by the presence of commensal gut flora, in particular increased colonization with segmented filamentous bacteria, and IL-17 receptor signaling. Together, these data indicate that neonatal colonization of gut microbiota influences generalized autoimmunity in adult life.

The key role of segmented filamentous bacteria in the coordinated maturation of gut helper T cell responses.

Microbiota-induced cytokine responses participate in gut homeostasis, but the cytokine balance at steady-state and the role of individual bacterial species in setting the balance remain elusive. Herein, systematic analysis of gnotobiotic mice indicated that colonization by a whole mouse microbiota orchestrated a broad spectrum of proinflammatory T helper 1 (Th1), Th17, and regulatory T cell responses whereas most tested complex microbiota and individual bacteria failed to efficiently stimulate intestinal T cell responses. This function appeared the prerogative of a restricted number of bacteria, the prototype of which is the segmented filamentous bacterium, a nonculturable Clostridia-related species, which could largely recapitulate the coordinated maturation of T cell responses induced by the whole mouse microbiota. This bacterium, already known as a potent inducer of mucosal IgA, likely plays a unique role in the postnatal maturation of gut immune functions. Changes in the infant flora may thus influence the development of host immune responses.

Host interactions with Segmented Filamentous Bacteria: an unusual trade-off that drives the post-natal maturation of the gut immune system.

Segmented Filamentous Bacteria (SFB) are present in the gut microbiota of a large number of vertebrate species where they are found intimately attached to the intestinal epithelium. SFB has recently attracted considerable attention due to its outstanding capacity to stimulate innate and adaptive host immune responses without causing pathology. Recent genomic analysis placed SFB between obligate and facultative symbionts, unraveled its highly auxotrophic needs, and provided a rationale for the complex SFB life-style in close contact with the epithelium. Herein, we examine how the SFB life-style may underlie its potent immunostimulatory properties and discuss how the trade-off set up between SFB and its hosts can simultaneously help to establish and maintain the ecological niche of SFB in the intestine and drive the post-natal maturation of the host gut immune barrier.

Trade-off between bile resistance and nutritional competence drives Escherichia coli diversification in the mouse gut.

Bacterial diversification is often observed, but underlying mechanisms are difficult to disentangle and remain generally unknown. Moreover, controlled diversification experiments in ecologically relevant environments are lacking. We studied bacterial diversification in the mammalian gut, one of the most complex bacterial environments, where usually hundreds of species and thousands of bacterial strains stably coexist. Herein we show rapid genetic diversification of an Escherichia coli strain upon colonisation of previously germ-free mice. In addition to the previously described mutations in the EnvZ/OmpR operon, we describe the rapid and systematic selection of mutations in the flagellar flhDC operon and in malT, the transcriptional activator of the maltose regulon. Moreover, within each mouse, the three mutant types coexisted at different levels after one month of colonisation. By combining in vivo studies and determination of the fitness advantages of the selected mutations in controlled in vitro experiments, we provide evidence that the selective forces that drive E. coli diversification in the mouse gut are the presence of bile salts and competition for nutrients. Altogether our results indicate that a trade-off between stress resistance and nutritional competence generates sympatric diversification of the gut microbiota. These results illustrate how experimental evolution in natural environments enables identification of both the selective pressures that organisms face in their natural environment and the diversification mechanisms.

Restricted microbiota and absence of cognate TCR antigen leads to an unbalanced generation of Th17 cells.

Retinoic acid-related orphan receptor (ROR)γt(+) TCRαß(+) cells expressing IL-17, termed Th17 cells, are most abundant in the intestinal lamina propria. Symbiotic microbiota are required for the generation of Th17 cells, but the requirement for microbiota-derived Ag is not documented. In this study, we show that normal numbers of Th17 cells develop in the intestine of mice that express a single TCR in the absence of cognate Ag, whereas the microbiota remains essential for their development. However, such mice, or mice monocolonized with the Th17-inducing segmented filamentous bacteria, fail to induce normal numbers of Foxp3(+) RORγt(+) T cells, the regulatory counterpart of IL-17(+)RORγt(+) T cells. These results demonstrate that a complex microbiota and cognate Ag are required to generate a properly regulated set of RORγt(+) T cells and Th17 cells.

Role of microbiota in postnatal maturation of intestinal T-cell responses.

PURPOSE OF REVIEW: Taking advantage of their rapid growth and capacity for continuous genetic adaptation, prokaryotes have colonized all possible ecological environments on earth, including the body surfaces of eukaryotes and their gastrointestinal tract. The mammalian gut contains a complex community of 10 bacteria with a meta-genome containing 1500-fold more genes than the human genome. The forces that control the relationships between eukaryotic hosts and their intestinal bacterial symbionts have, thus, become a major focus of interest. RECENT FINDINGS: Recent data have highlighted how the dialogue between mammalian hosts and their microbiota stimulates the postnatal maturation of an efficient intestinal barrier that promotes niche colonization by symbiotic bacteria and opposes colonization by pathogens. Herein, we review microbiota-induced T-cell responses and discuss how individual bacteria may shape the balance between regulatory and inflammatory responses. We will also show how host factors might influence the outcome of gut immune responses and affect the structure of the microbiota. SUMMARY: Deciphering host-microbiota reciprocal influence may not only help in understanding the recent outburst of intestinal inflammatory diseases but also point to strategies able to maintain or restore intestinal homeostasis.

Dissecting the genetic components of adaptation of Escherichia coli to the mouse gut.

While pleiotropic adaptive mutations are thought to be central for evolution, little is known on the downstream molecular effects allowing adaptation to complex ecologically relevant environments. Here we show that Escherichia coli MG1655 adapts rapidly to the intestine of germ-free mice by single point mutations in EnvZ/OmpR two-component signal transduction system, which controls more than 100 genes. The selective advantage conferred by the mutations that modulate EnvZ/OmpR activities was the result of their independent and additive effects on flagellin expression and permeability. These results obtained in vivo thus suggest that global regulators may have evolved to coordinate activities that need to be fine-tuned simultaneously during adaptation to complex environments and that mutations in such regulators permit adjustment of the boundaries of physiological adaptation when switching between two very distinct environments.

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.

Gnotobiotic mouse immune response induced by Bifidobacterium sp. strains isolated from infants.

Bifidobacterium, which is a dominant genus in infants' fecal flora and can be used as a probiotic, has shown beneficial effects in various pathologies, including allergic diseases, but its role in immunity has so far been little known. Numerous studies have shown the crucial role of the initial intestinal colonization in the development of the intestinal immune system, and bifidobacteria could play a major role in this process. For a better understanding of the effect of Bifidobacterium on the immune system, we aimed at determining the impact of Bifidobacterium on the T-helper 1 (T(H)1)/T(H)2 balance by using gnotobiotic mice. Germfree mice were inoculated with Bifidobacterium longum NCC2705, whose genome is sequenced, and with nine Bifidobacterium strains isolated from infants' fecal flora. Five days after inoculation, mice were killed. Transforming growth factor beta1 (TGF-beta1), interleukin-4 (IL-4), IL-10, and gamma interferon (IFN-gamma) gene expressions in the ileum and IFN-gamma, tumor necrosis factor alpha (TNF-alpha), IL-10, IL-4, and IL-5 secretions by splenocytes cultivated for 48 h with concanavalin A were quantified. Two Bifidobacterium species had no effect (B. adolescentis) or little effect (B. breve) on the immune system. Bifidobacterium bifidum, Bifidobacterium dentium, and one B. longum strain induced T(H)1 and T(H)2 cytokines at the systemic and intestinal levels. One B. longum strain induced a T(H)2 orientation with high levels of IL-4 and IL-10, both secreted by splenocytes, and of TGF-beta gene expression in the ileum. The other two strains induced T(H)1 orientations with high levels of IFN-gamma and TNF-alpha splenocyte secretions. Bifidobacterium's capacity to stimulate immunity is species specific, but its influence on the orientation of the immune system is strain specific.

Modulation of the gut microbiota to improve innate resistance.

One major benefit from the association of hosts with the complex microbial communities that establish at body surfaces is the resistance to pathogen infection. This protective role of symbiotic microbes is becoming ever more relevant, given the alarming rise of multidrug-resistant pathogens and severe infections in patients following extensive antibiotic treatment. Herein, we highlight some recent mechanistic studies that have provided insights into how the highly dynamic dialogue amongst intestinal bacteria and between intestinal bacteria and their host can contribute to protect the host against pathogens in and outside the gut. We then discuss how delineating the rules of this dialogue can help design strategies to modulate the microbiota and improve host resistance to infections.

Segmented filamentous bacteria, Th17 inducers and helpers in a hostile world.

The Th17 cell composition in the murine gut is strikingly dependent on the presence of the commensal segmented filamentous bacteria (SFB). SFB potently stimulates innate and adaptive immune responses and protects the host from pathogens both in and outside of the gut, partly due to its unique ability to promote a Th17-fostering environment. Recent work has highlighted the role of the tight adherence of SFB to the intestinal surface in mediating the potent immunostimulatory potential of SFB. Progress has also been made in our understanding of how SFB fosters this protective immune environment on the cellular and molecular level. This review focuses on the ability of SFB to specifically stimulate Th17 immunity.

Human Gut Symbiont Roseburia hominis Promotes and Regulates Innate Immunity.

OBJECTIVE: Roseburia hominis is a flagellated gut anaerobic bacterium belonging to the Lachnospiraceae family within the Firmicutes phylum. A significant decrease of R. hominis colonization in the gut of ulcerative colitis patients has recently been demonstrated. In this work, we have investigated the mechanisms of R. hominis-host cross talk using both murine and in vitro models. DESIGN: The complete genome sequence of R. hominis A2-183 was determined. C3H/HeN germ-free mice were mono-colonized with R. hominis, and the host-microbe interaction was studied using histology, transcriptome analyses and FACS. Further investigations were performed in vitro and using the TLR5KO and DSS-colitis murine models. RESULTS: In the bacterium, R. hominis, host gut colonization upregulated genes involved in conjugation/mobilization, metabolism, motility, and chemotaxis. In the host cells, bacterial colonization upregulated genes related to antimicrobial peptides, gut barrier function, toll-like receptors (TLR) signaling, and T cell biology. CD4+CD25+FoxP3+ T cell numbers increased in the lamina propria of both mono-associated and conventional mice treated with R. hominis. Treatment with the R. hominis bacterium provided protection against DSS-induced colitis. The role of flagellin in host-bacterium interaction was also investigated. CONCLUSION: Mono-association of mice with R. hominis bacteria results in specific bidirectional gene expression patterns. A set of genes thought to be important for host colonization are induced in R. hominis, while the host cells respond by strengthening gut barrier function and enhancing Treg population expansion, possibly via TLR5-flagellin signaling. Our data reveal the immunomodulatory properties of R. hominis that could be useful for the control and treatment of gut inflammation.

Stimulation of immunity without alteration of oral tolerance in mice fed with heat-treated fermented infant formula.

OBJECTIVES: Little information is available on the properties of fermented milk formula intended to healthy infants. This study analyzes the effect of long-term ingestion of a heat-treated, fermented milk formula on the development of oral tolerance or systemic immune response to soluble antigens in mice. MATERIALS AND METHODS: The C3H/HeN mice, fed with a heat-treated fermented (Bifidobacterium breve C50 and Streptococcus thermophilus 065) infant formula (htFF) or a matched control diet (control), were immunized with ovalbumin (OVA) with or without gavage of 20 mg OVA to induce tolerance or immunity, respectively. Systemic and local anti-OVA immune responses and intestinal barrier function were measured after 5 to 6 weeks. RESULTS: Oral tolerance to OVA developed similarly in htFF- and control-fed mice, attested to by the downregulation of OVA-specific immunoglobulin (Ig) G and IgE after oral OVA administration. In contrast, immunization with OVA led to significantly higher titers in htFF-fed mice than in control-fed mice (log2 IgG titers, 16.45 +/- 1.24 and 15.46 +/- 0.79, respectively; P = 0.012). Jejunal interferon gamma, interleukin 12p40 and interleukin 10 expressions were significantly higher in tolerized mice fed with htFF compared with those fed with the control diet. Mucosal to serosal intact horseradish peroxidase fluxes were lower in htFF-fed mice than in control-fed mice (39 +/- 8 and 118 +/- 38 ng/h x cm2, respectively; P < 0.0001), indicating that the htFF diet reinforces intestinal barrier capacity to macromolecules. CONCLUSIONS: In mice, htFF strengthens intestinal barrier and enhances systemic immune responses to antigens without interfering with the development of oral tolerance, suggesting a potential beneficial effect in host defence and vaccination.

[Gut microbiota and development of the immune system]. / Microbiote intestinal et développement du système immunitaire.

During their long co-evolution, bacteria and their animal host have developed mutualistic interactions that are regulated by the immune system of the host. A dialogue between bacteria and the host immune system is initiated at birth during microbial colonization. This colonization induces the recruitment of multiple immune cell types that cooperate with the intestinal epithelium to construct a barrier capable of confining the microbes within the intestinal lumen. Regulatory mechanisms avoid deleterious inflammatory reactions that would harm both the host and its microbiota. In mouse, homeostatic activation of the intestinal immune system is recapitulated by a small number of bacteria, and more particularly by the segmented filamentous bacteria.

MUCOSAL IMMUNOLOGY. The microbiota regulates type 2 immunity through RORγt⁺ T cells.

Changes to the symbiotic microbiota early in life, or the absence of it, can lead to exacerbated type 2 immunity and allergic inflammations. Although it is unclear how the microbiota regulates type 2 immunity, it is a strong inducer of proinflammatory T helper 17 (T(H)17) cells and regulatory T cells (T(regs)) in the intestine. Here, we report that microbiota-induced T(regs) express the nuclear hormone receptor RORγt and differentiate along a pathway that also leads to T(H)17 cells. In the absence of RORγt(+) T(regs), T(H)2-driven defense against helminths is more efficient, whereas T(H)2-associated pathology is exacerbated. Thus, the microbiota regulates type 2 responses through the induction of type 3 RORγt(+) T(regs) and T(H)17 cells and acts as a key factor in balancing immune responses at mucosal surfaces.