The human microbiota modulates cancer progression through largely unexplored mechanisms. Defining causal pathways is essential for monitoring and fine-tuning the microbiota to improve cancer treatment. Given that amino acid (aa) metabolism is often dysregulated in cancer, we assessed the role of microbiota pathways that modulate intestinal aa levels on colorectal tumor progression in mice. We found that the Bacteroides gene bo-ansB affects tumor responses to dietary asparagine (Asn) by reducing intestinal Asn levels. In mice receiving dietary Asn, bo-ansB promotes tumor progression by altering tumor-infiltrating CD8+ T cells. Mechanistically, bo-ansB depletes Asn in the tumor microenvironment (TME), suppressing the expression of an Asn transporter (SLC1A5) in CD8+ T cells and impairing their stem-like properties and effector functions. In humans, microbiota-encoded genes contributing to aa depletion are associated with colorectal cancer progression. Collectively, these findings reveal nutrient-dependent modulation of anticancer immunity by the gut microbiota and identify diet-microbiota-cancer crosstalk as a potential therapeutic target.
Food shortages and infectious diseases were constant threats throughout mammalian evolution and often occurred simultaneously. When food availability is reduced, it is unclear how the host adapts to support glucose-demanding immune processes while preventing hypoglycemia. In the context of dietary restriction (DR), we found that glucocorticoids (GCs) aligned naive, effector, and memory T cell populations with the nutritional status of the host. DR-induced GCs promoted naive T cell homing to the bone marrow, which supported their homeostasis at steady state. Following a primary infection, DR-induced GCs rewired immunity to simultaneously uphold pathogen control and systemic glucose homeostasis. GCs achieved this by dampening effector T cells and enhancing the response of neutrophils with reduced glucose dependence. Although the total effector T cell pool was decreased during DR, GCs enriched memory-precursor effector cells to preserve memory formation. Thus, GCs align immunity and metabolic physiology to ensure host fitness when food availability is reduced.
Type 2 immunity has evolved to protect against worms but becomes harmful when activated during allergic inflammation. Group 2 innate lymphoid cells (ILC2s) drive type 2 responses by rapidly secreting IL-5 and IL-13. The alarmins, IL-25, IL-33, and TSLP activate ILC2s and are linked to allergic diseases. However, how alarmins connect to the transcriptional networks driving type 2 effector functions remains elusive. Here, we performed RNA sequencing of ILC2s deficient in IL-25, IL-33, or TSLP pathway and identified the transcription factor Blimp-1 as an IL-33-regulated gene in ILC2s. While Blimp-1 was dispensable for ILC2 development, this transcription factor was required for type 2 cytokine production, driving eosinophilia or promoting worm expulsion. Blimp-1 deficiency resulted in reduced IRF4 expression, while Irf4-deficient ILC2s showed diminished Blimp-1 and IL-33 receptor expression, revealing a reciprocal Blimp-1-IRF4 circuit downstream of the IL-33 receptor. These findings expose the Blimp-1-IRF4 axis as an alarmin-regulated transcriptional network controlling ILC2 effector functions required for type 2 immunity.
Abstract Introduction Pain is a hallmark of infection and inflammation, but the role of pain-sensing nociceptors in tissue immunity is poorly defined. We previously showed that gut-innervating TRPV1+ nociceptors shape the intestinal microbiota to promote protection upon acute injury (Zhang et al., Cell 2022), highlighting their immunoregulatory function. At barrier surfaces, type 2 inflammation is an evolutionarily conserved response driving parasite defense, allergy, and repair, initiated by epithelial and immune sensing and further modulated by neuronal signals. How these diverse inputs are coordinated and integrated, however, is poorly understood. Methods In this study, we utilize mouse chemogenetics, scRNA sequencing, spatial transcriptomics, 3D tissue imaging and computational analyses to interrogate the functional interactions between sensory neurons, chemosensory tuft cells and anti-helminth type 2 immune responses. Results We demonstrated that TRPV1+ nociceptors co-opt chemosensory tuft cells to orchestrate protective anti-helminth type 2 immunity. Chemogenetic silencing of TRPV1+ nociceptors resulted in significantly reduced intestinal tuft cells and defective anti-helminth type 2 immunity. Conversely, chemogenetic activation of TRPV1+ nociceptors lead to enhanced intestinal CGRP+ innervation, increased tuft cell accumulation and protective anti-helminth type 2 immunity. Spatial transcriptomic analysis revealed nociceptor activation stimulated rapid epithelial progenitor proliferation and differentiation associated with tuft cell accumulation. Mechanistically, intestinal epithelial cell-intrinsic expression of CGRP receptors were required for anti-helminth type 2 immunity. Conclusion These findings identify a previously unrecognized neuronal—tuft cell circuit as a key upstream regulator of mucosal type 2 immunity (Zhang et al., Nature, accepted), establishing a new paradigm in which peripheral sensory neurons and chemosensory epithelial cells converge to initiate protective immunity at barrier surfaces. Funding Source 2024 NIDDK K99/R00 Transitional to Independence Award, 2024 MIST Scholar Award, Allen Discovery Center program Topic Categories Mucosal and Regional Immunology (MUC)
Abstract Introduction Advances in single cell technologies have uncovered novel biology and revealed cellular heterogeneity of complex tissues. However, single cell resolution data for the distal small intestine - a critical site of nutrient absorption and immunological tolerance - are limited, particular during states of infection or inflammation. Methods We generated GUTMAP, a single cell transcriptional atlas of more than 500k cells, from ileum and draining lymph node to address this need and provide a community resource for the study and annotation of intestinal data. Multiple established models of intestinal infection were profiled at acute immunological timepoints to catalogue innate responses to diverse pathogens including SFB, Nippostrongylus brasiliensis, Candida albicans, Yersinia pseudotuberculosis, Cryptosporidium parvum, and murine norovirus. To complement these data and integrate transcriptional responses with tissue pathology, we also carried out single cell spatially resolved transcriptomics. Results Using GUTMAP, we compare between infections and resolve 91 transcriptional cell states in the ileum, including infection-driven and pathogen-specific transcriptional modules. We describe unique enterocyte biology associated with Yersinia infection and demonstrate a spatial link between enterocyte phenotype and the formation of pyogranulomas during infection. We further probe the spatially coordinated cell networks and trafficking during diverse responses to reveal conserved patterns in immune architecture across infection systems. Conclusion GUTMAP is a resource for mucosal immunologists that captures single cell biology of the ileum across diverse infections to answer pivotal questions about innate immune biology. Additionally, GUTMAP acts as resource and bioinformatic tool to aid in the annotation of mucosal data sets and a foundational support for future studies of inflammatory dysregulation in the small intestine. Funding Source NIAID - U01AI095608 Topic Categories Mucosal and Regional Immunology (MUC)
Type 2 inflammation at barrier surfaces is an evolutionarily conserved response that promotes immunity to helminth parasites, allergic inflammation and tissue repair1-4. Direct sensing of environmental triggers by epithelial cells initiates type 2 inflammation, and signals derived from neurons can modulate immune responses5-8. However, how diverse sensory inputs from epithelial, neuronal and immune cells are coordinated and integrated remains unclear. Here we identify that TRPV1+ pain-sensing nociceptors co-opt chemosensory epithelial tuft cells to initiate a cascade of tissue responses that drive type 2 inflammation. Chemogenetic silencing or chemical ablation of TRPV1+ nociceptors results in a significant reduction in intestinal tuft cells and defective anti-helminth type 2 immunity. By contrast, chemogenetic activation of TRPV1+ nociceptors leads to remodelling of CGRP+ nerve fibres, significantly increased CGRP expression, enhanced tuft cell accumulation and protective anti-helminth type 2 immunity. Using spatial transcriptomic and single-cell RNA sequencing analyses, we reveal that nociceptor activation promotes rapid epithelial progenitor cell proliferation and differentiation. Mechanistically, intestinal epithelial cell-intrinsic and tuft cell-intrinsic expression of CGRP receptor subunits are required for tuft cell responses and type 2 immunity to helminth infection. Together, these results identify sensory convergence of a neuronal-epithelial tuft cell circuit as a critical upstream determinant of type 2 immunity and tissue adaptation.
The intestinal epithelial lining provides a critical physical barrier to protect host tissues against environmental insults including dietary antigens, allergens, and microorganisms. Regulation of the host epithelial barrier is coordinated by signals from diverse inputs including immune and neuronal cells; however, how these signals are integrated remains unknown. Here, we identify a novel mechanism of neuro-epithelial communication by which pain-sensing TRPV1+ nociceptors alter the responses of intestinal tuft cells, specialized chemosensory epithelial cells known to promote type 2 immunity against parasites. We demonstrate that chemogenetic silencing of TRPV1+ nociceptors leads to reduced tuft cells and delayed Trichuris clearance. In contrast, activation of nociceptors leads to the rapid accumulation of tuft cells and accelerated clearance of the helminth Trichuris muris. Finally, we show that exogenous administration of the neuropeptide calcitonin gene-related peptide (CGRP), increased during nociceptor activation, can induce tuft cell responses both in vitro and in vivo, and that epithelial CGRP signaling contributes to anti-helminth immunity. Together, these results identify a neuronal-epithelial circuit as a critical upstream determinant of type 2 immunity and tissue adaptation and have important implications for inflammatory conditions associated with defects in the epithelial barrier, including asthma, allergy, and inflammatory bowel diseases (IBD). Supported by the NIH (DK126871, AI151599, AI095466, AI095608, AI142213, AR070116, AI172027, DK132244), the Cure for IBD, Weill Cornell Medicine Jill Roberts Institute, and the Sanders Family and the Rosanne H. Silbermann Foundation. This work is also supported by the Allen Discovery Center program, a Paul G. Allen Frontiers Group advised program of the Paul G. Allen Family Foundation. Mucosal and Regional Immunology (MUC)
Metabolites derived from the intestinal microbiota, including bile acids (BA), extensively modulate vertebrate physiology, including development1, metabolism2, 3-4, immune responses5, 6-7 and cognitive function8. However, to what extent host responses balance the physiological effects of microbiota-derived metabolites remains unclear9,10. Here, using untargeted metabolomics of mouse tissues, we identified a family of BA-methylcysteamine (BA-MCY) conjugates that are abundant in the intestine and dependent on vanin 1 (VNN1), a pantetheinase highly expressed in intestinal tissues. This host-dependent MCY conjugation inverts BA function in the hepatobiliary system. Whereas microbiota-derived free BAs function as agonists of the farnesoid X receptor (FXR) and negatively regulate BA production, BA-MCYs act as potent antagonists of FXR and promote expression of BA biosynthesis genes in vivo. Supplementation with stable-isotope-labelled BA-MCY increased BA production in an FXR-dependent manner, and BA-MCY supplementation in a mouse model of hypercholesteraemia decreased lipid accumulation in the liver, consistent with BA-MCYs acting as intestinal FXR antagonists. The levels of BA-MCY were reduced in microbiota-deficient mice and restored by transplantation of human faecal microbiota. Dietary intervention with inulin fibre further increased levels of both free BAs and BA-MCY levels, indicating that BA-MCY production by the host is regulated by levels of microbiota-derived free BAs. We further show that diverse BA-MCYs are also present in human serum. Together, our results indicate that BA-MCY conjugation by the host balances host-dependent and microbiota-dependent metabolic pathways that regulate FXR-dependent physiology.
The distal small intestine plays vital roles in host physiology by regulating nutrient and fluid homeostasis. Despite being impacted in Crohn's disease and a major target for a range of infections, we know relatively little about the complexity of cellular responses and cell-cell communication in the ileum during infection. Single cell and spatial transcriptomics have emerged as powerful technologies to study tissue heterogeneity in the gut, but these tools have focused on the large intestine, in part due to the accessibility of this tissue for biopsies and its importance in cancer. Here we present GutPath, an atlas of over 500,000 single cells with RNA and protein expression profiles for 91 cell states in the ileum across diverse infectious archetypes. We show that GutPath accurately captures established immune responses to infection while revealing pathogen-specific responses in enterocytes. To highlight the discovery potential of this atlas, we identify a novel enterocyte cell state present during Yersinia pseudotuberculosis infection that is spatially linked to bacterial load and tissue pathology. GutPath establishes a much-needed resource for the immunology community that will accelerate the study of the transcriptional diversity of cellular landscapes in the small intestine.
Sensing and recognition are key properties of both the immune and nervous systems. In the immune system, pattern recognition or antigen-specific receptors represent classic motifs in innate and adaptive immunity, respectively. In the nervous system, there is a major anatomic division between how we sense stimuli from within the body (vagal sensory nervous system) and the outside world (somatosensory nervous system). However, in the last 5 years, there has been an explosion of discoveries revealing interactions between the immune and the sensory nervous systems that govern an array of physiologic and pathologic processes including allergy, infection, autoimmunity, regeneration, cancer, and beyond. Herein, we highlight recent advances that demonstrate how peripheral sensory neuroimmunology has emerged as a powerful field that provides new insights into classic immunologic processes including immune hypersensitivity, inflammation, and tissue homeostasis.
Microbiota-derived bile acids (BAs) are associated with host biology/disease, yet their causal effects remain largely undefined. Herein, we speculate that characterizing previously undefined microbiota-derived BAs would uncover previously unknown BA-sensing receptors and their biological functions. We integrated BA metabolomics and microbial genetics to functionally profile >200 putative microbiota BA metabolic genes. We identified 56 less-characterized BAs, many of which are detected in humans/mammals. Notably, a subset of these BAs are potent antagonists of the human androgen receptor (hAR). They inhibit AR-related gene expression and are human-relevant. As a proof-of-principle, we demonstrate that one of these BAs suppresses tumor progression and potentiates the efficacy of anti-PD-1 treatment in an AR-dependent manner. Our findings show that an approach combining bioinformatics, BA metabolomics, and microbial genetics can expand our knowledge of the microbiota metabolic potential and reveal an unexpected microbiota BA-AR interaction and its role in regulating host biology.
Neuro–immune circuits regulate innate and adaptive immunity at barrier surfaces. However, the differential impact of these circuits on proinflammatory versus tissue-protective responses remains poorly defined. We demonstrate that enteric neurons produce calcitonin gene-related peptide-related adrenomedullin 2 (ADM2) and identify a previously unrecognized role for the ADM2 pathway in promoting intestinal tissue-protective functions of group 2 innate lymphoid cells (ILC2s). Genomic or ILC2-intrinsic deletion of ADM2 receptor subunits resulted in a significant reduction in tissue-protective ILC2 responses, defective amphiregulin (AREG) production and increased susceptibility to intestinal damage and inflammation. Conversely, therapeutic delivery of recombinant ADM2 elicited tissue-protective AREG+ ILC2s and limited intestinal inflammation. Expression of genes encoding human ADM2 receptor (CALCRL and RAMP3) was altered in participants with inflammatory bowel diseases and associated with reduced expression of AREG in ILC2s. Collectively, these findings identify that the ADM2–ADM2 receptor pathway can promote tissue-protective functions of ILC2s in the context of intestinal damage and inflammation. Artis and colleagues show that enteric neurons produce CGRP-related ADM2 to promote intestinal tissue-protective functions in ILC2s.
The tuft cell-group 2 innate lymphoid cell (ILC2) circuit orchestrates rapid type 2 responses upon detecting microbially derived succinate and luminal helminths. Our findings delineate key mechanistic steps involving IP3R2 engagement and Ca2+ flux, governing interleukin-25 (IL-25) production by tuft cells triggered by succinate detection. While IL-17RB has a pivotal intrinsic role in ILC2 activation, it exerts a regulatory function in tuft cells. Tuft cells exhibit constitutive Il25 expression, placing them in an anticipatory state that facilitates rapid production of IL-25 protein for ILC2 activation. Tuft cell IL-17RB is crucial for restraining IL-25 bioavailability, preventing excessive tonic ILC2 stimulation due to basal Il25 expression. Supraoptimal ILC2 stimulation by IL-25 resulting from tuft cell Il17rb deficiency or prolonged succinate exposure induces a state of hypoproliferation in ILC2s, also observed in chronic helminth infection. Our study offers critical insights into the regulatory dynamics of IL-25 in this circuit, highlighting the delicate tuning required for responses to diverse luminal states.
Visceral pain disorders such as interstitial cystitis/bladder pain syndrome (IC/BPS) and irritable bowel syndrome (IBS) often manifest concurrently in the bladder and colon. Yet, the mechanistic basis of such comorbidities and the transmission of neural hypersensitivity across organ systems has remained a mystery. Here, we identify a mast cell-sensory neuron circuit that initiates bladder inflammation and simultaneously propagates neural hypersensitivity to the colon in a murine model of IC/BPS. We unveil anatomic heterogeneity of mast cells in relation to nociceptors in the bladder and their critical dependence on Mas-related G protein-coupled receptor B2 (MrgprB2) to promote visceral hypersensitivity. Employing retrograde neuronal tracing, in vivo calcium imaging, and intersectional genetics, we uncover a population of polyorganic sensory neurons that simultaneously innervate multiple organs and exhibit functional convergence. Importantly, using humanized mice, we demonstrate that pharmacological blockade of mast cell-expressed MRGPRX2, the human ortholog of MrgprB2, attenuates both bladder pathology and colonic hypersensitivity. Our studies reveal evolutionarily conserved neuroimmune mechanisms by which immune cells can directly convey signals from one organ to another through sensory neurons, in the absence of physical proximity, representing a new therapeutic paradigm.
Tertiary lymphoid structures (TLSs) are de novo ectopic lymphoid aggregates that regulate immunity in chronically inflamed tissues, including tumours. Although TLSs form due to inflammation-triggered activation of the lymphotoxin (LT)–LTβ receptor (LTβR) pathway1, the inflammatory signals and cells that induce TLSs remain incompletely identified. Here we show that interleukin-33 (IL-33), the alarmin released by inflamed tissues2, induces TLSs. In mice, Il33 deficiency severely attenuates inflammation- and LTβR-activation-induced TLSs in models of colitis and pancreatic ductal adenocarcinoma (PDAC). In PDAC, the alarmin domain of IL-33 activates group 2 innate lymphoid cells (ILC2s) expressing LT that engage putative LTβR+ myeloid organizer cells to initiate tertiary lymphoneogenesis. Notably, lymphoneogenic ILC2s migrate to PDACs from the gut, can be mobilized to PDACs in different tissues and are modulated by gut microbiota. Furthermore, we detect putative lymphoneogenic ILC2s and IL-33-expressing cells within TLSs in human PDAC that correlate with improved prognosis. To harness this lymphoneogenic pathway for immunotherapy, we engineer a recombinant human IL-33 protein that expands intratumoural lymphoneogenic ILC2s and TLSs and demonstrates enhanced anti-tumour activity in PDAC mice. In summary, we identify the molecules and cells of a druggable pathway that induces inflammation-triggered TLSs. More broadly, we reveal a lymphoneogenic function for alarmins and ILC2s. IL-33 induces tertiary lymphoid structures.
Background: Eosinophilic esophagitis (EoE) is a chronic, type 2 inflammatory disease that is increasing in incidence and has substantial morbidity. Despite being clinically defined as a food allergy, the molecular details of food antigen presentation and recognition by the immune system are unknown. Objective: The objective of this study was to identify and characterize the molecular basis of milk antigen presentation and T cell recognition in a patient with EoE milk allergy. Methods: Milk-expanded TCR clonotypes were identified using ex vivo stimulation followed by single cell RNA with linked, paired TRA and TRB sequencing. HLA restriction and antigen specificity of TCRs were identified using a combination of lentiviral expression, HLA sequencing, antibody blockade, and a peptide library screen. Results: We isolated a β-casein specific TCR clonotype (eoeTCR-4) and determined its HLA restriction (HLA-DRB1*07:01) and cognate antigen (β-casein AA 59-78). EoeTCR-4 was not detected among a larger group of subjects and is likely private to EoE Subject 1. Conclusion: In conjunction with the parent manuscript, this companion article provides the first molecular identity of food antigen presentation and immune recognition in EoE. ### Competing Interest Statement JD, JMS, and DAH hold a patent on the use of functional T cell assays for the identification of EoE-causal foods. The remaining authors have no conflicts of interest to disclose.
Diet alters both the composition and metabolic output of the intestinal microbiota, which has a major impact on the nature of immune responses. However, most microbiota-derived metabolites, their regulation by diet, and their immunoregulatory effects remain unknown. Employing untargeted metabolomics in mice, we show that an inulin fiber diet induces a significant shift in the levels of various microbial metabolites, notably elevated levels of bile acids. This metabolomic shift is associated with type 2 cytokine responses in several mucosal and visceral sites, characterized by elevated IL-33 in stromal cells, IL-5 production by group 2 innate lymphoid cells, and tissue eosinophilia. This eosinophilia is microbiota-dependent and can be reproduced by the administration of specific bile acids. Deletion of microbial metabolic genes or host bile acid receptors abolishes the eosinophilia. The fiber-induced eosinophils have distinct tissue-specific roles. In the mucosa, they promote defense against helminth infection but exacerbate disease outcomes during intestinal damage and inflammation. On the other hand, the fiber-induced eosinophils in visceral adipose tissues promote browning of white adipocytes and reduce tissue adiposity. Together, our studies identify novel molecular and cellular targets of diet- and microbiota-mediated immunoregulation with implications for the development of emerging precision nutrition and therapeutics for infection, inflammation, and metabolic health. NIH/K99AI173660, AI172027, DK132244; Crohn’s & Colitis Foundation Mucosal and Regional Immunology (MUC)
The intestinal barrier serves as a boundary between the mucosal immune system in the lamina propria and the external environment of the intestinal lumen, which contains a diverse array of microorganisms and ingested environmental factors, including pathogens, food antigens, toxins, and other foreign substances. This barrier has a central role in regulating the controlled interaction between luminal factors and the intestinal immune system. Disruptions of intestinal epithelial cells, which serve as a physical barrier, or the antimicrobial peptides and mucins they produce, which act as a chemical barrier, can lead to a leaky gut. In this state, the intestinal wall is unable to efficiently separate the intestinal flora and luminal contents from the intestinal immune system. The subsequent activation of the immune system has an important role in the pathogenesis of inflammatory bowel disease, as well as in metabolic dysfunction-associated steatohepatitis, primary sclerosing cholangitis, and colorectal cancer. Dysregulated intestinal barrier integrity has also been described in patients with chronic inflammatory diseases outside the gastrointestinal tract, including rheumatoid arthritis and neurodegenerative disorders. Mechanistic studies of barrier dysfunction have revealed that the subsequent local activation and systemic circulation of activated immune cells and the cytokines they secrete, as well as extracellular vesicles, promote proinflammatory processes within and outside the gastrointestinal tract. In this Review, we summarise these findings and highlight several new therapeutic concepts currently being developed that attempt to control inflammatory processes via direct or indirect modulation of intestinal barrier function.
