M cell maturation and cDC activation determine the onset of adaptive immune priming in the neonatal Peyer's patch.

Early-life immune development is critical to long-term host health. However, the mechanisms that determine the pace of postnatal immune maturation are not fully resolved. Here, we analyzed mononuclear phagocytes (MNPs) in small intestinal Peyer's patches (PPs), the primary inductive site of intestinal immunity. Conventional type 1 and 2 dendritic cells (cDC1 and cDC2) and RORgt+ antigen-presenting cells (RORgt+ APC) exhibited significant age-dependent changes in subset composition, tissue distribution, and reduced cell maturation, subsequently resulting in a lack in CD4+ T cell priming during the postnatal period. Microbial cues contributed but could not fully explain the discrepancies in MNP maturation. Type I interferon (IFN) accelerated MNP maturation but IFN signaling did not represent the physiological stimulus. Instead, follicle-associated epithelium (FAE) M cell differentiation was required and sufficient to drive postweaning PP MNP maturation. Together, our results highlight the role of FAE M cell differentiation and MNP maturation in postnatal immune development.

Cognate recognition of microbial antigens defines constricted CD4+ T cell receptor repertoires in the inflamed colon.

Key aspects of intestinal T cells, including their antigen specificity and their selection by the microbiota and other intestinal antigens, as well as the contribution of individual T cell clones to regulatory and effector functions, remain unresolved. Here we tracked adoptively transferred T cell populations to specify the interrelation of T cell receptor repertoire and the gut antigenic environment. We show that dominant TCRα clonotypes were shared between interferon-γ- and interleukin-17-producing but not regulatory Foxp3+ T cells. Identical TCRα clonotypes accumulated in the colon of different individuals, whereas antibiotics or defined colonization correlated with the expansion of distinct expanded T cell clonotypes. Our results demonstrate key aspects of intestinal CD4+ T cell activation and suggest that few microbial species exert a dominant effect on the intestinal T cell repertoire during colitis. We speculate that dominant proinflammatory T cell clones might provide a therapeutic target in human inflammatory bowel disease.

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.

B cells and the intestinal microbiome in time, space and place.

The gut immune system is shaped by the continuous interaction with the microbiota. Here we dissect temporal, spatial and contextual layers of gut B cell responses. The microbiota impacts on the selection of the developing pool of pre-immune B cells that serves as substrate for B cell activation, expansion and differentiation. However, various aspects of the gut B cell response display unique features. In particular, occurrence of somatically mutated B cells, chronic gut germinal centers in T cell-deficient settings and polyreactive binding of gut IgA to the microbiota questioned the nature and microbiota-specificity of gut germinal centers. We propose a model to reconcile these observations incorporating recent work demonstrating microbiota-specificity of gut germinal centers. We speculate that adjuvant effects of the microbiota might modify permissiveness for B cell to enter and exit gut germinal centers. We propose that separating aspects of time, space and place facilitate the occasionally puzzling discussion of gut B cell responses to the microbiota.

Mdr1 Saves T Cells from Bile.

The gut contents shape intestinal immune homeostasis, a phenomenon best documented for microbiota-immune interactions. In this issue of Immunity, Cao et al. (2017) show that bile acids trigger T cell-mediated inflammation at their site of active absorption in the ileum, unless cells are protected by the membrane transporter Mdr1.

BALB/c and C57BL/6 Mice Differ in Polyreactive IgA Abundance, which Impacts the Generation of Antigen-Specific IgA and Microbiota Diversity.

The interrelationship between IgAs and microbiota diversity is still unclear. Here we show that BALB/c mice had higher abundance and diversity of IgAs than C57BL/6 mice and that this correlated with increased microbiota diversity. We show that polyreactive IgAs mediated the entrance of non-invasive bacteria to Peyer's patches, independently of CX3CR1(+) phagocytes. This allowed the induction of bacteria-specific IgA and the establishment of a positive feedback loop of IgA production. Cohousing of mice or fecal transplantation had little or no influence on IgA production and had only partial impact on microbiota composition. Germ-free BALB/c, but not C57BL/6, mice already had polyreactive IgAs that influenced microbiota diversity and selection after colonization. Together, these data suggest that genetic predisposition to produce polyreactive IgAs has a strong impact on the generation of antigen-specific IgAs and the selection and maintenance of microbiota diversity.

Gut microbiota: a natural adjuvant for vaccination.

In this issue of Immunity, Oh et al. (2014) reveal an unappreciated facet of how the microbiota influences immune responses. Immunity to nonadjuvanted vaccines depends on Toll-like-receptor-5-mediated sensing of the microbiota.

Immunization against poly-N-acetylglucosamine reduces neutrophil activation and GVHD while sparing microbial diversity.

Microbial invasion into the intestinal mucosa after allogeneic hematopoietic cell transplantation (allo-HCT) triggers neutrophil activation and requires antibiotic interventions to prevent sepsis. However, antibiotics lead to a loss of microbiota diversity, which is connected to a higher incidence of acute graft-versus-host disease (aGVHD). Antimicrobial therapies that eliminate invading bacteria and reduce neutrophil-mediated damage without reducing the diversity of the microbiota are therefore highly desirable. A potential solution would be the use of antimicrobial antibodies that target invading pathogens, ultimately leading to their elimination by innate immune cells. In a mouse model of aGVHD, we investigated the potency of active and passive immunization against the conserved microbial surface polysaccharide poly-N-acetylglucosamine (PNAG) that is expressed on numerous pathogens. Treatment with monoclonal or polyclonal antibodies to PNAG (anti-PNAG) or vaccination against PNAG reduced aGVHD-related mortality. Anti-PNAG treatment did not change the intestinal microbial diversity as determined by 16S ribosomal DNA sequencing. Anti-PNAG treatment reduced myeloperoxidase activation and proliferation of neutrophil granulocytes (neutrophils) in the ileum of mice developing GVHD. In vitro, anti-PNAG treatment showed high antimicrobial activity. The functional role of neutrophils was confirmed by using neutrophil-deficient LysMcreMcl1fl/fl mice that had no survival advantage under anti-PNAG treatment. In summary, the control of invading bacteria by anti-PNAG treatment could be a novel approach to reduce the uncontrolled neutrophil activation that promotes early GVHD and opens a new avenue to interfere with aGVHD without affecting commensal intestinal microbial diversity.

Intestinal tolerance requires gut homing and expansion of FoxP3+ regulatory T cells in the lamina propria.

Tolerance to food antigen manifests in the absence and/or suppression of antigen-specific immune responses locally in the gut but also systemically, a phenomenon known as oral tolerance. Oral tolerance is thought to originate in the gut-draining lymph nodes, which support the generation of FoxP3(+) regulatory T (Treg) cells. Here we use several mouse models to show that Treg cells, after their generation in lymph nodes, need to home to the gut to undergo local expansion to install oral tolerance. Proliferation of Treg cells in the intestine and production of interleukin-10 by gut-resident macrophages was blunted in mice deficient in the chemokine (C-X3-C motif) receptor 1 (CX3CR1). We propose a model of stepwise oral tolerance induction comprising the generation of Treg cells in the gut-draining lymph nodes, followed by migration into the gut and subsequent expansion of Treg cells driven by intestinal macrophages.

Lymph node T cell homeostasis relies on steady state homing of dendritic cells.

Little is known about mechanisms determining the homeostasis of lymphocytes within lymphoid organs. Applying different mouse models, including conditionally proficient Ccr7 gene-targeted mice, we now show that semimature steady state dendritic cells (sDCs) constitutively trafficking into lymph nodes (LNs) were essential contributors to T cell homeostasis in these organs. sDCs provided vascular endothelial growth factor known to support high endothelial venule formation, thus facilitating enhanced homing of T cells to LNs. The presence of sDCs led to increased CCL21 production in T-zone fibroblastic reticular cells. CCL21 is a ligand for CCR7 known to regulate homing as well as retention of T cells in LNs. In addition, we provide evidence that CCL21 binds to the surface of DCs via its heparin-binding domain, further explaining why T cells leave LNs more rapidly in the absence of sDCs. Together, these data reveal multiple roles for sDCs in regulating T cell homeostasis in LNs.

Broad IgG repertoire in patients with chronic rhinosinusitis with nasal polyps regulates proinflammatory IgE responses.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is often characterized by local production of polyclonal IgE idiotypes. Although tissue IgE concentrations can be in the range of several thousand kilounits per liter, the regulatory mechanisms by which IgE-mediated inflammation is controlled in patients with nasal polyps are not well understood. OBJECTIVE: We sought to determine whether locally induced IgG antibodies in patients with nasal polyps can inhibit an IgE-mediated proallergic response. METHODS: Nasal polyp homogenates were collected from patients with grass pollen allergy with CRSwNP and nonallergic control subjects. IgE levels were measured using the Immuno Solid-phase Allergen Chip assay. IgE-containing nasal polyp homogenates with or without IgG depletion were evaluated for their capacity to promote IgE-facilitated allergen presentation, basophil activation, and histamine release. Local IgE and IgG repertoires were evaluated using Immunoglobulin 454 sequencing. RESULTS: We show that IgG plays a key role in controlling IgE-mediated inflammatory responses in patients with nasal polyps. Depletion of IgG from nasal homogenates resulted in an increase in CD23-mediated IgE-facilitated allergen binding to B cells but also enhanced FcεRI-mediated allergen-driven basophil activation and histamine release. A similar response was observed in relation to specific IgE antibodies to Staphylococcus aureus enterotoxins. The capacity of IgG in nasal polyps to limit IgE-mediated inflammation is based on the fact that IgG repertoires widely share the antigen targets with the IgE repertoires in both allergic and nonallergic subjects. CONCLUSION: Polyclonal IgE idiotypes in patients with CRSwNP are functional, promote IgE-mediated proallergic inflammation, and are partially antagonized by corresponding IgG idiotypes. This is most likely due to the fact that IgE and IgG clonotypes are widely shared in patients with nasal polyps.

Secretory IgA in the Coordination of Establishment and Maintenance of the Microbiota.

Starting at birth, the intestinal microbiota changes dramatically from a highly individual collection of microorganisms, dominated by comparably few species, to a mature, competitive, and diverse microbial community. Microbial colonization triggers and accompanies the maturation of the mucosal immune system and ultimately results in a mutually beneficial host-microbe interrelation in the healthy host. Here, we discuss the role of secretory immunoglobulin A (SIgA) during the establishment of the infant microbiota and life-long host-microbial homeostasis. We critically review the published literature on how SIgA affects the enteric microbiota and highlight the accessibility of the infant microbiota to therapeutic intervention.

The Extracellular Domains of IgG1 and T Cell-Derived IL-4/IL-13 Are Critical for the Polyclonal Memory IgE Response In Vivo.

IgE-mediated activation of mast cells and basophils contributes to protective immunity against helminths but also causes allergic responses. The development and persistence of IgE responses are poorly understood, which is in part due to the low number of IgE-producing cells. Here, we used next generation sequencing to uncover a striking overlap between the IgE and IgG1 repertoires in helminth-infected or OVA/alum-immunized wild-type BALB/c mice. The memory IgE response after secondary infection induced a strong increase of IgE+ plasma cells in spleen and lymph nodes. In contrast, germinal center B cells did not increase during secondary infection. Unexpectedly, the memory IgE response was lost in mice where the extracellular part of IgG1 had been replaced with IgE sequences. Adoptive transfer studies revealed that IgG1+ B cells were required and sufficient to constitute the memory IgE response in recipient mice. T cell-derived IL-4/IL-13 was required for the memory IgE response but not for expansion of B cells from memory mice. Together, our results reveal a close relationship between the IgE and IgG1 repertoires in vivo and demonstrate that the memory IgE response is mainly conserved at the level of memory IgG1+ B cells. Therefore, targeting the generation and survival of allergen-specific IgG1+ B cells could lead to development of new therapeutic strategies to treat chronic allergic disorders.

The serotonin receptor 5-HT7R regulates the morphology and migratory properties of dendritic cells.

Dendritic cells are potent antigen-presenting cells endowed with the unique ability to initiate adaptive immune responses upon inflammation. Inflammatory processes are often associated with an increased production of serotonin, which operates by activating specific receptors. However, the functional role of serotonin receptors in regulation of dendritic cell functions is poorly understood. Here, we demonstrate that expression of serotonin receptor 5-HT7 (5-HT7R) as well as its downstream effector Cdc42 is upregulated in dendritic cells upon maturation. Although dendritic cell maturation was independent of 5-HT7R, receptor stimulation affected dendritic cell morphology through Cdc42-mediated signaling. In addition, basal activity of 5-HT7R was required for the proper expression of the chemokine receptor CCR7, which is a key factor that controls dendritic cell migration. Consistent with this, we observed that 5-HT7R enhances chemotactic motility of dendritic cells in vitro by modulating their directionality and migration velocity. Accordingly, migration of dendritic cells in murine colon explants was abolished after pharmacological receptor inhibition. Our results indicate that there is a crucial role for 5-HT7R-Cdc42-mediated signaling in the regulation of dendritic cell morphology and motility, suggesting that 5-HT7R could be a new target for treatment of a variety of inflammatory and immune disorders.

Independent bottlenecks characterize colonization of systemic compartments and gut lymphoid tissue by salmonella.

Vaccination represents an important instrument to control typhoid fever in humans and protects mice from lethal infection with mouse pathogenic serovars of Salmonella species. Mixed infections with tagged Salmonella can be used in combination with probabilistic models to describe the dynamics of the infection process. Here we used mixed oral infections with tagged Salmonella strains to identify bottlenecks in the infection process in naïve and vaccinated mice. We established a next generation sequencing based method to characterize the composition of tagged Salmonella strains which offers a fast and reliable method to characterise the composition of genome-tagged Salmonella strains. We show that initial colonization of Salmonella was distinguished by a non-Darwinian selection of few bacteria setting up the infection independently in gut associated lymphoid tissue and systemic compartments. Colonization of Peyer's patches fuels the sustained spread of bacteria into mesenteric lymph nodes via dendritic cells. In contrast, infection of liver and spleen originated from an independent pool of bacteria. Vaccination only moderately reduced invasion of Peyer's patches but potently uncoupled bacterial populations present in different systemic compartments. Our data indicate that vaccination differentially skews the capacity of Salmonella to colonize systemic and gut immune compartments and provide a framework for the further dissection of infection dynamics.

Analysis of factors contributing to variation in the C57BL/6J fecal microbiota across German animal facilities.

The intestinal microbiota is involved in many physiological processes and it is increasingly recognized that differences in community composition can influence the outcome of a variety of murine models used in biomedical research. In an effort to describe and account for the variation in intestinal microbiota composition across the animal facilities of participating members of the DFG Priority Program 1656 "Intestinal Microbiota", we performed a survey of C57BL/6J mice from 21 different mouse rooms/facilities located at 13 different institutions across Germany. Fresh feces was sampled from five mice per room/facility using standardized procedures, followed by extraction and 16S rRNA gene profiling (V1-V2 region, Illumina MiSeq) at both the DNA and RNA (reverse transcribed to cDNA) level. In order to determine the variables contributing to bacterial community differences, we collected detailed questionnaires of animal husbandry practices and incorporated this information into our analyses. We identified considerable variation in a number of descriptive aspects including the proportions of major phyla, alpha- and beta diversity, all of which displayed significant associations to specific aspects of husbandry. Salient findings include a reduction in alpha diversity with the use of irradiated chow, an increase in inter-individual variability (beta diversity) with respect to barrier access and open cages and an increase in bacterial community divergence with time since importing from a vendor. We further observe a high degree of facility-level individuality, which is likely due to each facility harboring its own unique combination of multiple varying attributes of animal husbandry. While it is important to account and control for such differences between facilities, the documentation of such diversity may also serve as a valuable future resource for investigating the origins of microbial-driven host phenotypes.

CX3CR1 is a gatekeeper for intestinal barrier integrity in mice: Limiting steatohepatitis by maintaining intestinal homeostasis.

UNLABELLED: Nonalcoholic fatty liver disease is seen as the hepatic manifestation of the metabolic syndrome and represents the most common liver disease in Western societies. The G protein-coupled chemokine receptor CX3CR1 plays a central role in several metabolic syndrome-related disease manifestations and is involved in maintaining intestinal homeostasis. Because diet-induced intestinal dysbiosis is a driver for nonalcoholic fatty liver disease, we hypothesized that CX3CR1 may influence the development of steatohepatitis. In two independent models of diet-induced steatohepatitis (high-fat diet and methionine/choline-deficient diet), CX3CR1 protected mice from excessive hepatic steatosis and inflammation, as well as systemic glucose intolerance. Lack of Cx3cr1 expression was associated with significantly altered intestinal microbiota composition, which was linked to an impaired intestinal barrier. Concomitantly, endotoxin levels in portal serum and inflammatory macrophages in liver were increased in Cx3cr1-/- mice, indicating an increased inflammatory response. Depletion of intestinal microbiota by administration of broad-spectrum antibiotics suppressed the number of infiltrating macrophages and promoted macrophage polarization in liver. Consequently, antibiotic-treated mice demonstrated a marked improvement of steatohepatitis. CONCLUSION: Microbiota-mediated activation of the innate immune responses through CX3CR1 is crucial for controlling steatohepatitis progression, which recognizes CX3CR1 as an essential gatekeeper in this scenario.

Antigen sampling in the small intestine.

Active sampling of intestinal antigen initiates regulated immune responses that ensure intestinal homeostasis. Several specialized mechanisms transport luminal antigen across the gut epithelium. Epithelium overlying lymphoid compartments is equipped with transcytotic microfold (M) cells that transport particulate material either directly or with the help of dendritic cells (DCs). By contrast, normal villous epithelium transports antigen by means of antigen-shuttling receptors together with phagocytes that scan the gut epithelium and potentially the gut lumen. Here, we examine recent insights into the nature of the epithelial and immune cell types involved in antigen uptake and describe how the process of antigen transport has been visualized by intravital microscopy. These new findings might help optimize antigen delivery systems for mucosal vaccination.

Carbonic anhydrase IV is expressed on IL-5-activated murine eosinophils.

Eosinophilia and its cellular activation are hallmark features of asthma, as well as other allergic/Th2 disorders, yet there are few, if any, reliable surface markers of eosinophil activation. We have used a FACS-based genome-wide screening system to identify transcriptional alterations in murine lung eosinophils recruited and activated by pulmonary allergen exposure. Using a relatively stringent screen with false-positive correction, we identified 82 candidate genes that could serve as eosinophil activation markers and/or pathogenic effector markers in asthma. Carbonic anhydrase IV (Car4) was a top dysregulated gene with 36-fold induction in allergen-elicited pulmonary eosinophils, which was validated by quantitative PCR, immunohistochemistry, and flow cytometry. Eosinophil CAR4 expression was kinetically regulated by IL-5, but not IL-13. IL-5 was both necessary and sufficient for induction of eosinophil CAR4. Although CAR4-deficient mice did not have a defect in eosinophil recruitment to the lung, nor a change in eosinophil pH-buffering capacity, allergen-challenged chimeric mice that contained Car4(-/-) hematopoietic cells aberrantly expressed a series of genes enriched in biological processes involved in epithelial differentiation, keratinization, and anion exchange. In conclusion, we have determined that eosinophils express CAR4 following IL-5 or allergen exposure, and that CAR4 is involved in regulating the lung transcriptome associated with allergic airway inflammation; therefore, CAR4 has potential value for diagnosing and monitoring eosinophilic responses.