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Chronic pain is a leading cause of disability, affecting more women than men. Different immune cells contribute to this sexual divergence, but the mechanisms, especially in females, are not well defined. We show that pannexin-1 (Panx1) channels on microglia and T cells differentially cause mechanical allodynia, a debilitating symptom of neuropathic pain. In male rodents, Panx1 drives vascular endothelial growth factor-A (VEGF-A) release from microglia. Cell-specific knockdown of microglial Panx1 or pharmacological blockade of the VEGF receptor attenuated allodynia in nerve-injured males. In females, nerve injury increased spinal CD8 + T cells and leptin levels. Leptin release from female-derived CD8 + T cells was Panx1 dependent, and intrathecal leptin-neutralizing antibody injection sex-specifically reversed allodynia. Adoptive transfer of female-derived CD8 + T cells caused robust allodynia, which was prevented by a leptin-neutralizing antibody or leptin small interfering RNA (siRNA) knockdown. Panx1-targeted approaches may alleviate neuropathic pain in both sexes, while T cell- and leptin-directed treatments could have sex-dependent benefits for women., Competing Interests: Declaration of interests T.T. is cofounder and CEO of AphioTx Inc. G.W.Z. is cofounder and CSO of Zymedyne Therapeutics. T.T. has patents for the use of Panx1-targeting therapies for opioid withdrawal and addiction. G.W.Z. has patents targeting voltage-gated calcium channels for use in chronic pain. The patents do not pertain to, or have relevance for, the current reported findings. There is no direct bearing, and there is no commercial gain from the findings reported in the manuscript., (Copyright © 2025 Elsevier Inc. All rights reserved.)

Background: Despite achieving endoscopic remission, over 20% of inflammatory bowel disease (IBD) patients experience chronic abdominal pain. Visceral pain and the microbiome exhibit sex-dependent interactions, while visceral pain in IBD shows a sex bias. Our aim was to evaluate whether post-inflammatory microbial perturbations contribute to visceral hypersensitivity in a sex-dependent manner., Methods: Males, cycling females, ovariectomized, and sham-operated females were given dextran sodium sulfate to induce colitis and allowed to recover. Germ-free recipients received sex-appropriate and cross-sex fecal microbial transplants (FMT) from post-inflammatory donor mice. Visceral sensitivity was assessed by recording visceromotor responses to colorectal distention. The composition of the microbiota was evaluated via 16S rRNA gene V4 amplicon sequencing, while the metabolome was assessed using targeted (short chain fatty acids - SCFA) and semi-targeted mass spectrometry., Results: Post-inflammatory cycling females developed visceral hyperalgesia when compared to males. This effect was reversed by ovariectomy. Both post-inflammatory males and females exhibited increased SCFA-producing species, but only males had elevated fecal SCFA content. FMT from post-inflammatory females transferred visceral hyperalgesia to both males and females, while FMT from post-inflammatory males could only transfer visceral hyperalgesia to males., Conclusions: Female sex, hormonal status as well as the gut microbiota play a role in pain modulation. Our data highlight the importance of considering biological sex in the evaluation of visceral pain.

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Burkhard, Koegler, Brown, Wilson, Mager, Zucoloto, Thomson, Hebbandi Nanjundappa, Skalosky, Ahmadi, McDonald and Geuking.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (© 2022. The Author(s), under exclusive licence to Society for Mucosal Immunology.)

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., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Squamous cell carcinoma of the tonsil is one of the most frequent cancers of the oropharynx. The escalating rate of tonsil cancer during the last decades is associated with the increase of high risk-human papilloma virus (HR-HPV) infections. While the microbiome in oropharyngeal malignant diseases has been characterized to some extent, the microbial colonization of HR-HPV-associated tonsil cancer remains largely unknown. Using 16S rRNA gene amplicon sequencing, we have characterized the microbiome of human palatine tonsil crypts in patients suffering from HR-HPV-associated tonsil cancer in comparison to a control cohort of adult sleep apnea patients. We found an increased abundance of the phyla Firmicutes and Actinobacteria in tumor patients, whereas the abundance of Spirochetes and Synergistetes was significantly higher in the control cohort. Furthermore, the accumulation of several genera such as Veillonella, Streptococcus and Prevotella_7 in tonsillar crypts was associated with tonsil cancer. In contrast, Fusobacterium, Prevotella and Treponema_2 were enriched in sleep apnea patients. Machine learning-based bacterial species analysis indicated that a particular bacterial composition in tonsillar crypts is tumor-predictive. Species-specific PCR-based validation in extended patient cohorts confirmed that differential abundance of Filifactor alocis and Prevotella melaninogenica is a distinct trait of tonsil cancer. This study shows that tonsil cancer patients harbor a characteristic microbiome in the crypt environment that differs from the microbiome of sleep apnea patients on all phylogenetic levels. Moreover, our analysis indicates that profiling of microbial communities in distinct tonsillar niches provides microbiome-based avenues for the diagnosis of tonsil cancer., Competing Interests: The author(s) declares no competing financial interests., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Eradication of pathogens from the bloodstream is critical to prevent disseminated infections and sepsis. Kupffer cells in the liver form an intravascular firewall that captures and clears pathogens from the blood. Here, we show that the catching and killing of circulating pathogens by Kupffer cells in vivo are promoted by the gut microbiota through commensal-derived D-lactate that reaches the liver via the portal vein. The integrity of this Kupffer cell-mediated intravascular firewall requires continuous crosstalk with gut commensals, as microbiota depletion with antibiotics leads to a failure of pathogen clearance and overwhelming disseminated infection. Furthermore, administration of purified D-lactate to germ-free mice, or gnotobiotic colonization with D-lactate-producing commensals, restores Kupffer cell-mediated pathogen clearance by the liver firewall. Thus, the gut microbiota programs an intravascular immune firewall that protects against the spread of bacterial infections via the bloodstream., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Induction of intestinal T helper cell subsets by commensal members of the intestinal microbiota is an important component of the many immune adaptations required to establish host-microbial homeostasis. Importantly, altered intestinal T helper cell profiles can have pathological consequences that are not limited to intestinal sites. Therefore, microbial-mediated modulation of the intestinal T helper cell profile could have strong therapeutic potentials. However, in order to develop microbial therapies that specifically induce the desired alterations in the intestinal T helper cell compartment one has to first gain a detailed understanding of how microbial composition and the metabolites derived or induced by the microbiota impact on intestinal T helper cell responses. Here we summarize the milestone findings in the field of microbiota-intestinal T helper cell crosstalk with a focus on the role of specific commensal bacteria and their metabolites. We discuss mechanistic mouse studies and are linking these to human studies where possible. Moreover, we highlight recent advances in the field of microbial CD4 T cell epitope mimicry in autoimmune diseases and the role of microbially-induced CD4 T cells in cancer immune checkpoint blockade therapy.

Several species of intestinal bacteria have been associated with enhanced efficacy of checkpoint blockade immunotherapy, but the underlying mechanisms by which the microbiome enhances antitumor immunity are unclear. In this study, we isolated three bacterial species- Bifidobacterium pseudolongum , Lactobacillus johnsonii , and Olsenella species-that significantly enhanced efficacy of immune checkpoint inhibitors in four mouse models of cancer. We found that intestinal B. pseudolongum modulated enhanced immunotherapy response through production of the metabolite inosine. Decreased gut barrier function induced by immunotherapy increased systemic translocation of inosine and activated antitumor T cells. The effect of inosine was dependent on T cell expression of the adenosine A 2A receptor and required costimulation. Collectively, our study identifies a previously unknown microbial metabolite immune pathway activated by immunotherapy that may be exploited to develop microbial-based adjuvant therapies., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Early life exposure to microbes plays an important role in immune system development. Germ-free mice, or mice colonized with a low-diversity microbiota, exhibit high serum IgE levels. An increase in microbial richness, providing it occurs in a critical developmental window early in life, leads to inhibition of this hygiene-induced IgE. However, whether this inhibition is dependent solely on certain microbial species, or is an additive effect of microbial richness, remains to be determined. Here we report that mice colonized with a combination of bacterial species with specific characteristics is required to inhibit IgE levels. These defined characteristics include the presence in early life, acetate production and immunogenicity reflected by induction of IgA. Suppression of IgE did not correlate with production of the short chain fatty acids propionate and butyrate, or induction of peripherally induced Tregs in mucosal tissues. Thus, inhibition of IgE induction can be mediated by specific microbes and their associated metabolic pathways and immunogenic properties., (Copyright © 2020 Wyss, Brown, Thomson, Koegler, Terra, Fan, Ronchi, Bihan, Lewis, Geuking and McCoy.)

Background & Aims: Despite achieving endoscopic remission, more than 20% of inflammatory bowel disease patients experience chronic abdominal pain. These patients have increased rectal transient receptor potential vanilloid-1 receptor (TRPV1) expression, a key transducer of inflammatory pain. Because inflammatory bowel disease patients in remission exhibit dysbiosis and microbial manipulation alters TRPV1 function, our goal was to examine whether microbial perturbation modulated transient receptor potential function in a mouse model., Methods: Mice were given dextran sodium sulfate (DSS) to induce colitis and were allowed to recover. The microbiome was perturbed by using antibiotics as well as fecal microbial transplant (FMT). Visceral and somatic sensitivity were assessed by recording visceromotor responses to colorectal distention and using hot plate/automated Von Frey tests, respectively. Calcium imaging of isolated dorsal root ganglia neurons was used as an in vitro correlate of nociception. The microbiome composition was evaluated via 16S rRNA gene variable region V4 amplicon sequencing, whereas fecal short-chain fatty acids (SCFAs) were assessed by using targeted mass spectrometry., Results: Postinflammatory DSS mice developed visceral and somatic hyperalgesia. Antibiotic administration during DSS recovery induced visceral, but not somatic, hyperalgesia independent of inflammation. FMT of postinflammatory DSS stool into antibiotic-treated mice increased visceral hypersensitivity, whereas FMT of control stool reversed antibiotics' sensitizing effects. Postinflammatory mice exhibited both increased SCFA-producing species and fecal acetate/butyrate content compared with controls. Capsaicin-evoked calcium responses were increased in naive dorsal root ganglion neurons incubated with both sodium butyrate/propionate alone and with colonic supernatants derived from postinflammatory mice., Conclusions: The microbiome plays a central role in postinflammatory visceral hypersensitivity. Microbial-derived SCFAs can sensitize nociceptive neurons and may contribute to the pathogenesis of postinflammatory visceral pain., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Myocarditis can develop into inflammatory cardiomyopathy through chronic stimulation of myosin heavy chain 6-specific T helper (T H )1 and T H 17 cells. However, mechanisms governing the cardiotoxicity programming of heart-specific T cells have remained elusive. Using a mouse model of spontaneous autoimmune myocarditis, we show that progression of myocarditis to lethal heart disease depends on cardiac myosin-specific T H 17 cells imprinted in the intestine by a commensal Bacteroides species peptide mimic. Both the successful prevention of lethal disease in mice by antibiotic therapy and the significantly elevated Bacteroides- specific CD4 + T cell and B cell responses observed in human myocarditis patients suggest that mimic peptides from commensal bacteria can promote inflammatory cardiomyopathy in genetically susceptible individuals. The ability to restrain cardiotoxic T cells through manipulation of the microbiome thereby transforms inflammatory cardiomyopathy into a targetable disease., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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., (© 2019 Australian and New Zealand Society for Immunology Inc.)

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-1β 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.

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., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The trillions of microbes that colonize mucosal surfaces are critical for educating the immune system and microbial-derived signals continually shape and set the tone of immune responses. Although Type 2 immune responses are important for mediating protection from helminth infection they also underlie atopy and allergy. Microbes modulate Type 2 immune responses through effects on Type 2 cytokines, dendritic cells and regulatory T cells. Microbial colonization in the gut, the lung and the skin during an early and critical time period in immune development appears to be of particular importance for tolerance induction and regulation of aberrant Type 2 immune responses. This is illustrated by studies showing microbial alterations in early life that are associated with allergies later in life., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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. Trim28 hep-/- 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 Trim28 hep-/- -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)., (© 2017 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.)

Mouse models are used extensively to study human health and to investigate the mechanisms underlying human disease. In the past, most animal studies were performed without taking into consideration the impact of the microbiota. However, the microbiota that colonizes all body surfaces, including the gastrointestinal tract, respiratory tract, genitourinary tract, and skin, heavily impacts nearly every aspect of host physiology. When performing studies utilizing mouse models it is critical to understand that the microbiome is heavily impacted by environmental factors, including (but not limited to) food, bedding, caging, and temperature. In addition, stochastic changes in the microbiota can occur over time that also play a role in shaping microbial composition. These factors lead to massive variability in the composition of the microbiota between animal facilities and research institutions, and even within a single facility. Lack of experimental reproducibility between research groups has highlighted the necessity for rigorously controlled experimental designs in order to standardize the microbiota between control and experimental animals. Well controlled experiments are mandatory in order to reduce variability and allow correct interpretation of experimental results, not just of host-microbiome studies but of all mouse models of human disease. The protocols presented are aimed to design experiments that control the microbiota composition between different genetic strains of experimental mice within an animal unit. © 2017 by John Wiley & Sons, Inc., (Copyright © 2017 John Wiley & Sons, Inc.)

The ATP-gated ionotropic P2X7 receptor regulates T follicular helper (Tfh) cell abundance in the Peyer's patches (PPs) of the small intestine; deletion of P2rx7, encoding for P2X7, in Tfh cells results in enhanced IgA secretion and binding to commensal bacteria. Here, we show that Tfh cell activity is important for generating a diverse bacterial community in the gut and that sensing of microbiota-derived extracellular ATP via P2X7 promotes the generation of a proficient gut ecosystem for metabolic homeostasis. The results of this study indicate that Tfh cells play a role in host-microbiota mutualism beyond protecting the intestinal mucosa by induction of affinity-matured IgA and suggest that extracellular ATP constitutes an inter-kingdom signaling molecule important for selecting a beneficial microbial community for the host via P2X7-mediated regulation of B cell help., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2016 The Authors. Immunology Published by John Wiley & Sons Ltd.)

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.

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., (© FASEB.)

The overall composition of the mammalian intestinal microbiota varies between individuals: within each individual there are differences along the length of the intestinal tract related to host nutrition, intestinal motility and secretions. Mucus is a highly regenerative protective lubricant glycoprotein sheet secreted by host intestinal goblet cells; the inner mucus layer is nearly sterile. Here we show that the outer mucus of the large intestine forms a unique microbial niche with distinct communities, including bacteria without specialized mucolytic capability. Bacterial species present in the mucus show differential proliferation and resource utilization compared with the same species in the intestinal lumen, with high recovery of bioavailable iron and consumption of epithelial-derived carbon sources according to their genome-encoded metabolic repertoire. Functional competition for existence in this intimate layer is likely to be a major determinant of microbiota composition and microbial molecular exchange with the host.

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., (© 2015 by the American Association for the Study of Liver Diseases.)

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., (Copyright © 2014 by The American Association of Immunologists, Inc.)

The impact of the gut microbiota on immune homeostasis within the gut and, importantly, also at systemic sites has gained tremendous research interest over the last few years. The intestinal microbiota is an integral component of a fascinating ecosystem that interacts with and benefits its host on several complex levels to achieve a mutualistic relationship. Host-microbial homeostasis involves appropriate immune regulation within the gut mucosa to maintain a healthy gut while preventing uncontrolled immune responses against the beneficial commensal microbiota potentially leading to chronic inflammatory bowel diseases (IBD). Furthermore, recent studies suggest that the microbiota composition might impact on the susceptibility to immune-mediated disorders such as autoimmunity and allergy. Understanding how the microbiota modulates susceptibility to these diseases is an important step toward better prevention or treatment options for such diseases.

Intestinal bacterial metabolites are an important communication tool between the host immune system and the commensal microbiota to establish mutualism. In a recent paper published in Science, Wendy Garrett and her colleagues report an exciting role of the three most abundant microbial-derived short-chain fatty acids (SCFA), acetic acid, propionic acid and butyric acid, in colonic regulatory T cell (cTreg) homeostasis.

Microbial exposure following birth profoundly impacts mammalian immune system development. Microbiota alterations are associated with increased incidence of allergic and autoimmune disorders with elevated serum IgE as a hallmark. The previously reported abnormally high serum IgE levels in germ-free mice suggests that immunoregulatory signals from microbiota are required to control basal IgE levels. We report that germ-free mice and those with low-diversity microbiota develop elevated serum IgE levels in early life. B cells in neonatal germ-free mice undergo isotype switching to IgE at mucosal sites in a CD4 T-cell- and IL-4-dependent manner. A critical level of microbial diversity following birth is required in order to inhibit IgE induction. Elevated IgE levels in germ-free mice lead to increased mast-cell-surface-bound IgE and exaggerated oral-induced systemic anaphylaxis. Thus, appropriate intestinal microbial stimuli during early life are critical for inducing an immunoregulatory network that protects from induction of IgE at mucosal sites., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Thymic stromal lymphopoietin (TSLP) is constitutively expressed in the intestine and is known to regulate inflammation in models of colitis. We show that steady-state TSLP expression requires intestinal bacteria and has an important role in limiting the expansion of colonic T helper type 17 (Th17) cells. Inappropriate expansion of the colonic Th17 cells occurred in response to an entirely benign intestinal microbiota, as determined following the colonization of germ-free C57BL/6 or TSLPR(-/-) mice with the altered Schaedler flora (ASF). TSLP-TSLPR (TSLP receptor) interactions also promoted the expansion of colonic Helios(-)Foxp3(+) regulatory T cells, necessary for the control of inappropriate Th17 responses following ASF bacterial colonization. In summary, these data reveal an important role for TSLP-TSLPR signaling in promoting steady-state mutualistic T-cell responses following intestinal bacterial colonization.

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., (Copyright © 2012. Published by Elsevier Ltd.)

The large production of immunoglobulin (Ig)A is energetically costly. The fact that evolution retained this apparent luxury of intestinal class switch recombination to IgA within the human population strongly indicates that there must be a critical specific function of IgA for survival of the species. The function of IgA has been investigated in a series of different models that will be discussed here. While IgA has clear protective functions against toxins or in the context of intestinal viral infections, the function of IgA specific for non-pathogenic commensal bacteria remains unclear. In the context of the current literature we present a hypothesis where secretory IgA integrates as an additional layer of immune function into the continuum of intestinal CD4 T cell responses, to achieve a mutualistic relationship between the intestinal commensal microbiota and the host., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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., (© 2011 John Wiley & Sons A/S.)

Healthy individuals live in peaceful co-existence with an immense load of intestinal bacteria. This symbiosis is advantageous for both the host and the bacteria. For the host it provides access to otherwise undigestible nutrients and colonization resistance against pathogens. In return the bacteria receive an excellent nutrient habitat. The mucosal immune adaptations to the presence of this commensal intestinal microflora are manifold. Although bacterial colonization has clear systemic consequences, such as maturation of the immune system, it is striking that the mutualistic adaptive (T and B cells) and innate immune responses are precisely compartmentalized to the mucosal immune system. Here we summarize the mechanisms of mucosal immune compartmentalization and its importance for a healthy host-microbiota mutualism.

How a mutualistic relationship between the intestinal microbiota and intestinal T cell compartments is established is important, as a breakdown of intestinal T cell homeostasis may cause inflammatory bowel diseases. A number of studies have shown that different bacterial species modulate the intestinal CD4(+) T cell compartment in different ways. We performed mechanistic in vivo studies that demonstrated the crucial requirement for regulatory T cells (Treg) and interleukin-10 (IL-10) in the induction of intestinal T cell homeostasis even following colonization with a completely benign microbiota. In the absence of a functional Treg response or IL-10 receptor signaling, the same bacteria that induced a Treg response in wild-type animals now induced T helper type 17 responses, without intestinal inflammation. Therefore, Treg, IL-10 and Th17 are crucial regulatory mechanisms in the intestine not only for controlling inflammation, but also to establish a continuum of CD4(+) T cell homeostasis upon commensal colonization.

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.

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The lower intestine of adult mammals is densely colonized with nonpathogenic (commensal) microbes. Gut bacteria induce protective immune responses, which ensure host-microbial mutualism. The continuous presence of commensal intestinal bacteria has made it difficult to study mucosal immune dynamics. Here, we report a reversible germ-free colonization system in mice that is independent of diet or antibiotic manipulation. A slow (more than 14 days) onset of a long-lived (half-life over 16 weeks), highly specific anticommensal immunoglobulin A (IgA) response in germ-free mice was observed. Ongoing commensal exposure in colonized mice rapidly abrogated this response. Sequential doses lacked a classical prime-boost effect seen in systemic vaccination, but specific IgA induction occurred as a stepwise response to current bacterial exposure, such that the antibody repertoire matched the existing commensal content.

Commensal bacteria in the lower intestine of mammals are 10 times as numerous as the body's cells. We investigated the relative importance of different immune mechanisms in limiting the spread of the intestinal microbiota. Here, we reveal a flexible continuum between innate and adaptive immune function in containing commensal microbes. Mice deficient in critical innate immune functions such as Toll-like receptor signaling or oxidative burst production spontaneously produce high-titer serum antibodies against their commensal microbiota. These antibody responses are functionally essential to maintain host-commensal mutualism in vivo in the face of innate immune deficiency. Spontaneous hyper-activation of adaptive immunity against the intestinal microbiota, secondary to innate immune deficiency, may clarify the underlying mechanisms of inflammatory diseases where immune dysfunction is implicated.

Mammals coexist with an extremely dense microbiota in the lower intestine. Despite the constant challenge of small numbers of microbes penetrating the intestinal surface epithelium, it is very unusual for these organisms to cause disease. In this review article, we present the different mucosal firewalls that contain and allow mutualism with the intestinal microbiota.

Retroviruses have the potential to acquire host cell-derived genetic material during reverse transcription and can integrate into the genomes of larger, more complex DNA viruses. In contrast, RNA viruses were believed not to integrate into the host's genome under any circumstances. We found that illegitimate recombination between an exogenous nonretroviral RNA virus, lymphocytic choriomeningitis virus, and the endogenous intracisternal A-type particle (IAP) retrotransposon occurred and led to reverse transcription of exogenous viral RNA. The resulting complementary DNA was integrated into the host's genome with an IAP element. Thus, RNA viruses should be closely scrutinized for any capacity to interact with endogenous retroviral elements before their approval for therapeutic use in humans.

CD8+ T cells are crucial for the control of intracellular pathogens such as viruses and some bacteria. Using lymphocytic choriomeningitis virus (LCMV) infection of mice--the prototypic arenavirus evolutionarily closely related to human Lassa fever and South American hemorrhagic fever viruses, we have shown previously that the kinetics of Ag presentation determine immunodominance of the LCMV-specific CTL response due to progressive exhaustion of LCMV nucleoprotein (NP)-specific CTL upon increasing viral load. In this study, we provide evidence that CTL against early LCMV NP-derived epitopes are more important in virus control than those against late glycoprotein-derived epitopes. We show that mice that are tolerant to all NP-derived T cell epitopes are severely compromised in their ability to control larger inocula of LCMV, supporting our hypothesis that CD8+ T cells specific for early viral Ags play a major role in acute virus control. Thus, the kinetics with which virus-derived T cell epitopes are presented has a strong impact on the efficacy of the antiviral immunity. This aspect should be taken into consideration for the development of vaccines.

IgE induction by parasites and allergens is antigen driven and cognate T cell help dependent. We demonstrate that spontaneously produced IgE in T cell-deficient and germ-free wild-type (wt) mice is composed of natural specificities and induced by a mechanism independent of MHC class II (MHC II) cognate help. This does not require secondary lymphoid structures or germinal center formation, although some bystander T cell-derived IL-4 is necessary. The pathway of spontaneous IgE production is not inhibited by regulatory T cells and increases with age to constitute significant serum concentrations, even in naive animals.

Rare cases of stable allograft acceptance after discontinuation of immunosuppression are often accompanied by macrochimerism (> 1% donor cells in blood) or microchimerism (< 1% donor cells in blood). Here, we have investigated whether persistence of donor cells is the cause or the consequence of long-lasting CTL unresponsiveness. We found that engraftment of splenocytes bearing a single foreign MHC class I-restricted epitope resulted in lifelong donor cell microchimerism and specific CTL unresponsiveness. This status was reversed in a strictly time- and thymus-dependent fashion when the engrafted cells were experimentally removed. The results presented herein show that microchimerism actively maintains CTL unresponsiveness toward a minor histocompatibility antigen by deleting the specific repertoire and thus excluding dominant, T cell extrinsic mechanisms of CTL unresponsiveness independent of systemically persisting donor cell antigen.