Microbiota regulates intratumoral monocytes to promote anti-tumor immune responses.

The gut microbiota has been shown to promote the efficacy of cancer therapy through regulating adaptive immune responses. In this issue of Cell, Lam et al. provide new evidence demonstrating that specific gut bacteria also reprogram the innate immune tumor microenvironment to enhance the efficacy of cancer therapies.

Small intestinal resident eosinophils maintain gut homeostasis following microbial colonization.

The intestine harbors a large population of resident eosinophils, yet the function of intestinal eosinophils has not been explored. Flow cytometry and whole-mount imaging identified eosinophils residing in the lamina propria along the length of the intestine prior to postnatal microbial colonization. Microscopy, transcriptomic analysis, and mass spectrometry of intestinal tissue revealed villus blunting, altered extracellular matrix, decreased epithelial cell turnover, increased gastrointestinal motility, and decreased lipid absorption in eosinophil-deficient mice. Mechanistically, intestinal epithelial cells released IL-33 in a microbiota-dependent manner, which led to eosinophil activation. The colonization of germ-free mice demonstrated that eosinophil activation in response to microbes regulated villous size alterations, macrophage maturation, epithelial barrier integrity, and intestinal transit. Collectively, our findings demonstrate a critical role for eosinophils in facilitating the mutualistic interactions between the host and microbiota and provide a rationale for the functional significance of their early life recruitment in the small intestine.

Fibroblastic reticular cells regulate intestinal inflammation via IL-15-mediated control of group 1 ILCs.

Fibroblastic reticular cells (FRCs) of secondary lymphoid organs form distinct niches for interaction with hematopoietic cells. We found here that production of the cytokine IL-15 by FRCs was essential for the maintenance of group 1 innate lymphoid cells (ILCs) in Peyer's patches and mesenteric lymph nodes. Moreover, FRC-specific ablation of the innate immunological sensing adaptor MyD88 unleashed IL-15 production by FRCs during infection with an enteropathogenic virus, which led to hyperactivation of group 1 ILCs and substantially altered the differentiation of helper T cells. Accelerated clearance of virus by group 1 ILCs precipitated severe intestinal inflammatory disease with commensal dysbiosis, loss of intestinal barrier function and diminished resistance to colonization. In sum, FRCs act as an 'on-demand' immunological 'rheostat' by restraining activation of group 1 ILCs and thereby preventing immunopathological damage in the intestine.

The impact of the gut microbiota on T cell ontogeny in the thymus.

The intestinal microbiota is critical for the development of gut-associated lymphoid tissues, including Peyer's patches and mesenteric lymph nodes, and is instrumental in educating the local as well as systemic immune system. In addition, it also impacts the development and function of peripheral organs, such as liver, lung, and the brain, in health and disease. However, whether and how the intestinal microbiota has an impact on T cell ontogeny in the hymus remains largely unclear. Recently, the impact of molecules and metabolites derived from the intestinal microbiota on T cell ontogeny in the thymus has been investigated in more detail. In this review, we will discuss the recent findings in the emerging field of the gut-thymus axis and we will highlight the current questions and challenges in the field.

Host-microbiota interactions and adaptive immunity.

All mucosal surfaces are colonized with a vast number of microbes, which are essential for stimulating and regulating the immune system. While intrinsic and innate mechanisms exist to promote a strong barrier between the microbiota and the host to ensure compartmentalization, the microbiota is also able to induce robust adaptive immunity. In this review, we discuss the interplay between the microbiota and the adaptive immune system, with a focus on the induction of mucosal and systemic antibody responses and newly defined roles of maternal antibodies. We also highlight recent studies that aim to decipher microbial antigen-specificity of the T-cell compartment.

Macrophages treated with antigen from the tapeworm Hymenolepis diminuta condition CD25+ T cells to suppress colitis.

Macrophages play central roles in immunity as early effectors and modulating adaptive immune reponses; we implicated macrophages in the anticolitic effect of infection with the tapeworm Hymenolepis diminuta. Here, gene arrays revealed that H. diminuta antigen (HdAg) evoked a program in murine macrophages distinct from that elicited by IL-4. Further, HdAg suppressed LPS-evoked release of TNF-α and IL-1ß from macrophages via autocrine IL-10 signaling. In assessing the ability of macrophages treated in vitro with an extract of H. diminuta [M(HdAg)] to affect disease, intravenous, but not peritoneal, injection of M(HdAg) protected wild-type but not RAG1-/- mice from dinitrobenzene sulphonic acid (DNBS)-induced colitis. Administration of splenic CD4+ T cells from in vitro cocultures with M(HdAg), but not those cocultured with M(IL-4) cells, inhibited DNBS-induced colitis; fractionation of the T-cell population indicated that the CD4+CD25+ T cells from cocultures with M(HdAg) drove the suppression of DNBS-induced colitis. Use of IL-4-/- or IL-10-/- CD4+ T cells revealed that neither cytokine alone from the donor cells was essential for the anticolitic effect. These data illustrate that HdAg evokes a unique regulatory program in macrophages, identifies HdAg-evoked IL-10 suppression of macrophage activation, and reveals the ability of HdAg-treated macrophages to educate ( i.e., condition) and mobilize CD4+CD25+ T cells, which could be deployed to treat colonic inflammation.-Reyes, J. L., Lopes, F., Leung, G., Jayme, T. S., Matisz, C. E., Shute, A., Burkhard, R., Carneiro, M., Workentine, M. L., Wang, A., Petri, B., Beck, P. L., Geuking, M. B., McKay, D. M., Macrophages treated with antigen from the tapeworm Hymenolepis diminuta condition CD25+ T cells to suppress colitis.

Intestinal bacterial colonization induces mutualistic regulatory T cell responses.

Mammals harbor a dense commensal microbiota in the colon. Regulatory T (Treg) cells are known to limit microbe-triggered intestinal inflammation and the CD4+ T cell compartment is shaped by the presence of particular microbes or bacterial compounds. It is, however, difficult to distinguish whether these effects reflect true mutualistic immune adaptation to intestinal colonization or rather idiosyncratic immune responses. To investigate truly mutualistic CD4+ T cell adaptation, we used the altered Schaedler flora (ASF). Intestinal colonization resulted in activation and de novo generation of colonic Treg cells. Failure to activate Treg cells resulted in the induction of T helper 17 (Th17) and Th1 cell responses, which was reversed by wild-type Treg cells. Efficient Treg cell induction was also required to maintain intestinal homeostasis upon dextran sulfate sodium-mediated damage in the colon. Thus, microbiota colonization-induced Treg cell responses are a fundamental intrinsic mechanism to induce and maintain host-intestinal microbial T cell mutualism.

The microbiome and immune memory formation.

The microbiota plays an important role in regulating both the innate and adaptive immune systems. Many studies have focused on the ability of microbes to shape the immune system by stimulating B-cell and antibody responses and the differentiation of T helper cell function. However, an important feature of the immune system is its ability to generate memory responses, which provide increased survival for the host. This review will highlight the role of the microbiota in the induction of immune memory with a focus on both adaptive and innate memory as well as vaccine efficacy.

Helminth Antigen-Conditioned Dendritic Cells Generate Anti-Inflammatory Cd4 T Cells Independent of Antigen Presentation via Major Histocompatibility Complex Class II.

A recently identified feature of the host response to infection with helminth parasites is suppression of concomitant disease. Dendritic cells (DCs) exposed to antigens from the tapeworm Hymenolepis diminuta significantly reduce the severity of dinitrobenzene sulfonic acid-induced colitis in mice. Here we elucidate mechanisms underlying this cellular immunotherapy. We show a requirement for Ccr7 expression on transferred H. diminuta antigen-treated (HD)-DCs, suggesting that homing to secondary lymphoid tissues is important for suppression of colitis. Furthermore, sodium metaperiodate-sensitive helminth-derived glycans are required to drive the anti-colitic response in recipient mice. Induction of Th2-type cytokines and Gata-3+Cd4+ cells in secondary lymphoid tissues is dependent on major histocompatibility complex class II (MHC II) protein expression on transferred DCs, although remarkably, transfer of MHC II-/- HD-DCs still attenuated dinitrobenzene sulfonic acid-induced colitis in recipient mice. Moreover, transfer of Cd4+ splenic T cells retrieved from mice administered MHC II-/- HD-DCs suppressed dinitrobenzene sulfonic acid-induced colitis in recipient mice. Our studies reveal that HD-DCs can suppress colitis via an alternative MHC II-independent pathway that involves, in part, mobilization of T-cell responses. These data support the utility of HD-DCs in blocking colitis, revealing a requirement for Ccr7 and providing for HD-DC autologous immunotherapy for disease in which MHC II expression and/or function is compromised.

Polyphenic trait promotes liver cancer in a model of epigenetic instability in mice.

Hepatocellular carcinoma (HCC) represents the fifth-most common form of cancer worldwide and carries a high mortality rate attributed to lack of effective treatment. Males are 8 times more likely to develop HCC than females, an effect largely driven by sex hormones, albeit through still poorly understood mechanisms. We previously identified TRIM28 (tripartite protein 28), a scaffold protein capable of recruiting a number of chromatin modifiers, as a crucial mediator of sexual dimorphism in the liver. Trim28hep-/- mice display sex-specific transcriptional deregulation of a wide range of bile and steroid metabolism genes and development of liver adenomas in males. We now demonstrate that obesity and aging precipitate alterations of TRIM28-dependent transcriptional dynamics, leading to a metabolic infection state responsible for highly penetrant male-restricted hepatic carcinogenesis. Molecular analyses implicate aberrant androgen receptor stimulation, biliary acid disturbances, and altered responses to gut microbiota in the pathogenesis of Trim28hep-/- -associated HCC. Correspondingly, androgen deprivation markedly attenuates the frequency and severity of tumors, and raising animals under axenic conditions completely abrogates their abnormal phenotype, even upon high-fat diet challenge. CONCLUSION: This work underpins how discrete polyphenic traits in epigenetically metastable conditions can contribute to a cancer-prone state and more broadly provides new evidence linking hormonal imbalances, metabolic disturbances, gut microbiota, and cancer. (Hepatology 2017;66:235-251).

Detrimental effect of systemic antimicrobial CD4+ T-cell reactivity on gut epithelial integrity.

Healthy host-microbe mutualism relies on compartmentalization and proper regulation of systemic and mucosal immune responses. Nevertheless, the systemic immune system is frequently exposed to bouts of bacteraemia, which can trigger systemic antimicrobial immune reactivity including CD4+ T cells. Low-level bacteraemia can occur when immune compartmentalization is compromised, for example in the presence of innate immune deficiency or following use of non-steroidal anti-inflammatory drugs. We generated an Escherichia coli strain expressing a defined T helper neo-epitope to study systemic antigen-specific antimicrobial CD4+ T cells and their potential involvement in the pathogenisis of inflammatory bowel diseases. We found that the dose of bacteria required for the induction of systemic antimicrobial CD4+ T-cell proliferation was high and not easily reached under physiological conditions. Importantly, however, when intestinal barrier function was compromised by induced damage to the intestinal epithelium, the presence of systemic antimicrobial CD4+ T cells specific for a single neo-antigen resulted in dramatically increased levels of bacterial translocation. This study therefore demonstrates that systemic antimicrobial CD4+ T-cell reactivity might impact adversely on the mucosa under conditions of reduced barrier function and that despite strong mucosal immune regulation, antigen-specific recognition is still sensitive.

Improvement in adiposity with oligofructose is modified by antibiotics in obese rats.

Given the intimate link between gut microbiota and host physiology, there is growing interest in understanding the mechanisms by which diet influences gut microbiota and affects human metabolic health. Using antibiotics and the prebiotic oligofructose, which has been shown to counteract excess fat mass, we explored the gut microbiota-dependent effects of oligofructose on body composition and host metabolism. Diet-induced obese male Sprague Dawley rats, fed a background high-fat/sucrose diet, were randomized to one of the following diets for 6 wk: 1) high-energy control; 2) 10% oligofructose; 3) ampicillin; 4) ampicillin + 10% oligofructose; 5) ampicillin/neomycin; or 6) ampicillin/neomycin + 10% oligofructose. Combining oligofructose with ampicillin treatment blunted the decrease in adiposity seen with oligofructose. Although ampicillin did not affect total bacteria, ampicillin impeded oligofructose-induced increases in Bifidobacterium and Lactobacillus In contrast, the combination of ampicillin and neomycin reduced total bacteria but did not abrogate the oligofructose-induced decrease in adiposity. Oligofructose-mediated effects on host adiposity and metabolic health appear to be in part dependent on the presence of specific microbial species within the gut.-Bomhof, M. R., Paul, H. A., Geuking, M. B., Eller, L. K., Reimer, R. A. Improvement in adiposity with oligofructose is modified by antibiotics in obese rats.

Attenuated portal hypertension in germ-free mice: Function of bacterial flora on the development of mesenteric lymphatic and blood vessels.

UNLABELLED: Intestinal bacterial flora may induce splanchnic hemodynamic and histological alterations that are associated with portal hypertension (PH). We hypothesized that experimental PH would be attenuated in the complete absence of intestinal bacteria. We induced prehepatic PH by partial portal vein ligation (PPVL) in germ-free (GF) or mice colonized with altered Schaedler's flora (ASF). After 2 or 7 days, we performed hemodynamic measurements, including portal pressure (PP) and portosystemic shunts (PSS), and collected tissues for histomorphology, microbiology, and gene expression studies. Mice colonized with intestinal microbiota presented significantly higher PP levels after PPVL, compared to GF, mice. Presence of bacterial flora was also associated with significantly increased PSS and spleen weight. However, there were no hemodynamic differences between sham-operated mice in the presence or absence of intestinal flora. Bacterial translocation to the spleen was demonstrated 2 days, but not 7 days, after PPVL. Intestinal lymphatic and blood vessels were more abundant in colonized and in portal hypertensive mice, as compared to GF and sham-operated mice. Expression of the intestinal antimicrobial peptide, angiogenin-4, was suppressed in GF mice, but increased significantly after PPVL, whereas other angiogenic factors remained unchanged. Moreover, colonization of GF mice with ASF 2 days after PPVL led to a significant increase in intestinal blood vessels, compared to controls. The relative increase in PP after PPVL in ASF and specific pathogen-free mice was not significantly different. CONCLUSION: In the complete absence of gut microbial flora PP is normal, but experimental PH is significantly attenuated. Intestinal mucosal lymphatic and blood vessels induced by bacterial colonization may contribute to development of PH.

Microbiota-derived compounds drive steady-state granulopoiesis via MyD88/TICAM signaling.

Neutropenia is probably the strongest known predisposition to infection with otherwise harmless environmental or microbiota-derived species. Because initial swarming of neutrophils at the site of infection occurs within minutes, rather than the hours required to induce "emergency granulopoiesis," the relevance of having high numbers of these cells available at any one time is obvious. We observed that germ-free (GF) animals show delayed clearance of an apathogenic bacterium after systemic challenge. In this article, we show that the size of the bone marrow myeloid cell pool correlates strongly with the complexity of the intestinal microbiota. The effect of colonization can be recapitulated by transferring sterile heat-treated serum from colonized mice into GF wild-type mice. TLR signaling was essential for microbiota-driven myelopoiesis, as microbiota colonization or transferring serum from colonized animals had no effect in GF MyD88(-/-)TICAM1(-/-) mice. Amplification of myelopoiesis occurred in the absence of microbiota-specific IgG production. Thus, very low concentrations of microbial Ags and TLR ligands, well below the threshold required for induction of adaptive immunity, sets the bone marrow myeloid cell pool size. Coevolution of mammals with their microbiota has probably led to a reliance on microbiota-derived signals to provide tonic stimulation to the systemic innate immune system and to maintain vigilance to infection. This suggests that microbiota changes observed in dysbiosis, obesity, or antibiotic therapy may affect the cross talk between hematopoiesis and the microbiota, potentially exacerbating inflammatory or infectious states in the host.

The habitat, double life, citizenship, and forgetfulness of IgA.

Immunoglobulin A (IgA) is the main secretory immunoglobulin of mucous membranes and is powerfully induced by the presence of commensal microbes in the intestine. B cells undergo class switch recombination to IgA in the mucosa-associated lymphoid tissues, particularly mesenteric lymph nodes (MLNs) and Peyer's patches, through both T-dependent and T-independent pathways. IgA B cells primed in the mucosa traffic from the intestinal lymphoid structures, initially through the lymphatics and then join the bloodstream, to home back to the intestinal mucosa as IgA-secreting plasma cells. Once induced, anti-bacterial IgA can be extremely long-lived but is replaced if there is induction of additional IgA specificities by other microbes. The mucosal immune system is anatomically separated from the systemic immune system by the MLNs, which act as a firewall to prevent penetration of live intestinal bacteria to systemic sites. Dendritic cells sample intestinal bacteria and induce B cells to switch to IgA. In contrast, intestinal macrophages are adept at killing extracellular bacteria and are able to clear bacteria that have crossed the mucus and epithelial barriers. There is both a continuum between innate and adaptive immune mechanisms and compartmentalization of the mucosal immune system from systemic immunity that function to preserve host microbial mutualism.

Homeland security: IgA immunity at the frontiers of the body.

IgA is the most abundant immunoglobulin produced in mammals, and is mostly secreted across mucous membranes. At these frontiers, which are constantly assaulted by pathogenic and commensal microbes, IgA provides part of a layered system of immune protection. In this review, we describe how IgA induction occurs through both T-dependent and T-independent mechanisms, and how IgA is generated against the prodigious load of commensal microbes after mucosal dendritic cells (DCs) have sampled a tiny fraction of the microbial consortia in the intestinal lumen. To function in this hostile environment, IgA must be induced behind the 'firewall' of the mesenteric lymph nodes to generate responses that integrate microbial stimuli, rather than the classical prime-boost effects characteristic of systemic immunity.

Intestinal colonization regulates systemic anti-commensal immune sensitivity and hyperreactivity.

Healthy host-microbial mutualism with our intestinal microbiota relies to a large degree on compartmentalization and careful regulation of adaptive mucosal and systemic anti-microbial immune responses. However, commensal intestinal bacteria are never exclusively or permanently restricted to the intestinal lumen and regularly reach the systemic circulation. This results in various degrees of commensal bacteremia that needs to be appropriately dealt with by the systemic immune system. While most intestinal commensal bacteria, except for pathobionts or opportunistic pathogen, have evolved to be non-pathogenic, this does not mean that they are non-immunogenic. Mucosal immune adaptation is carefully controlled and regulated to avoid an inflammatory response, but the systemic immune system usually responds differently and more vigorously to systemic bacteremia. Here we show that germ-free mice have increased systemic immune sensitivity and display anti-commensal hyperreactivity in response to the addition of a single defined T helper cell epitope to the outer membrane porin C (OmpC) of a commensal Escherichia coli strain demonstrated by increased E. coli-specific T cell-dependent IgG responses following systemic priming. This increased systemic immune sensitivity was not observed in mice colonized with a defined microbiota at birth indicating that intestinal commensal colonization also regulates systemic, and not only mucosal, anti-commensal responses. The observed increased immunogenicity of the E. coli strain with the modified OmpC protein was not due to a loss of function and associated metabolic changes as a control E. coli strain without OmpC did not display increased immunogenicity.

Vascular traffic control of neutrophil recruitment to the liver by microbiota-endothelium crosstalk.

During bloodstream infections, neutrophils home to the liver as part of an intravascular immune response to eradicate blood-borne pathogens, but the mechanisms regulating this crucial response are unknown. Using in vivo imaging of neutrophil trafficking in germ-free and gnotobiotic mice, we demonstrate that the intestinal microbiota guides neutrophil homing to the liver in response to infection mediated by the microbial metabolite D-lactate. Commensal-derived D-lactate augments neutrophil adhesion in the liver independent of granulopoiesis in bone marrow or neutrophil maturation and activation in blood. Instead, gut-to-liver D-lactate signaling primes liver endothelial cells to upregulate adhesion molecule expression in response to infection and promote neutrophil adherence. Targeted correction of microbiota D-lactate production in a model of antibiotic-induced dysbiosis restores neutrophil homing to the liver and reduces bacteremia in a model of Staphylococcus aureus infection. These findings reveal long-distance traffic control of neutrophil recruitment to the liver by microbiota-endothelium crosstalk.

Long-distance relationships - regulation of systemic host defense against infections by the gut microbiota.

Despite compartmentalization within the lumen of the gastrointestinal tract, the gut microbiota has a far-reaching influence on immune cell development and function throughout the body. This long-distance relationship is crucial for immune homeostasis, including effective host defense against invading pathogens that cause systemic infections. Herein, we review new insights into how commensal microbes that are spatially restricted to the gut lumen can engage in long-distance relationships with innate and adaptive immune cells at systemic sites to fortify host defenses against infections. In addition, we explore the consequences of intestinal dysbiosis on impaired host defense and immune-mediated pathology during infections, including emerging evidence linking dysbiosis with aberrant systemic inflammation and immune-mediated organ damage in sepsis. As such, therapeutic modification of the gut microbiota is an emerging target for interventions to prevent and/or treat systemic infections and sepsis by harnessing the long-distance relationships between gut microbes and systemic immunity.

Immunoglobulin A: a bridge between innate and adaptive immunity.

PURPOSE OF REVIEW: The review summarizes the recent progress that has been made in understanding the function of immunoglobulin A (IgA) in promoting a healthy mutualism with the commensal microbiota and protecting against pathogens. Although IgA is by far the most abundant antibody produced by mammals, direct experimental evidence for its function is still lacking. RECENT FINDINGS: IgA is the predominant antibody induced in response to intestinal colonization with commensal bacteria: even fish have been shown to have a mucosal immunoglobulin (IgT), which is produced in the mucosa and coats commensals in the intestinal lumen. Recent studies indicate that intestinal IgA can be highly specific to the inducing commensals. Priming of IgA also appears to be a long-lasting response dependent on the overall dose (integral) of the bacteria sampled rather than exhibiting prime-boost effects normally observed with systemic immunoglobulin responses. Not only is human IgA highly mutated, but a mouse model with deficient hypermutation but intact class-switch recombination also shows that this mutation process (presumably leading to better anticommensal affinities) is important for IgA protection at the mucosal surface. It has been shown that some IgA can be induced independently of T cells through stimulation by epithelial cell and plasmacytoid dendritic cell cytokines including BAFF and APRIL, although the relative roles of the T-dependent and T-independent IgA pathways in generating mucosal protection are still unclear. SUMMARY: Protection at mucosal surfaces through the secretion of antibodies is a phylogenetically ancient function. Mammals can produce high and low-affinity IgA against their commensal microbes via T-cell-dependent and T-cell-independent pathways to contribute to host microbial mutualism. The process of improving IgA affinity to intestinal luminal contents through somatic hypermutation of immunoglobulin genes improves the level of protection at the mucosal surface and such mutations are abundant in human IgA sequences.