Enteric Nervous System-Derived IL-18 Orchestrates Mucosal Barrier Immunity.

Mucosal barrier immunity is essential for the maintenance of the commensal microflora and combating invasive bacterial infection. Although immune and epithelial cells are thought to be the canonical orchestrators of this complex equilibrium, here, we show that the enteric nervous system (ENS) plays an essential and non-redundant role in governing the antimicrobial protein (AMP) response. Using confocal microscopy and single-molecule fluorescence in situ mRNA hybridization (smFISH) studies, we observed that intestinal neurons produce the pleiotropic cytokine IL-18. Strikingly, deletion of IL-18 from the enteric neurons alone, but not immune or epithelial cells, rendered mice susceptible to invasive Salmonella typhimurium (S.t.) infection. Mechanistically, unbiased RNA sequencing and single-cell sequencing revealed that enteric neuronal IL-18 is specifically required for homeostatic goblet cell AMP production. Together, we show that neuron-derived IL-18 signaling controls tissue-wide intestinal immunity and has profound consequences on the mucosal barrier and invasive bacterial killing.

Adrenergic Signaling in Muscularis Macrophages Limits Infection-Induced Neuronal Loss.

Enteric-associated neurons (EANs) are closely associated with immune cells and continuously monitor and modulate homeostatic intestinal functions, including motility and nutrient sensing. Bidirectional interactions between neuronal and immune cells are altered during disease processes such as neurodegeneration or irritable bowel syndrome. We investigated the effects of infection-induced inflammation on intrinsic EANs (iEANs) and the role of intestinal muscularis macrophages (MMs) in this context. Using murine models of enteric infections, we observed long-term gastrointestinal symptoms, including reduced motility and loss of excitatory iEANs, which was mediated by a Nlrp6- and Casp11-dependent mechanism, depended on infection history, and could be reversed by manipulation of the microbiota. MMs responded to luminal infection by upregulating a neuroprotective program via ß2-adrenergic receptor (ß2-AR) signaling and mediated neuronal protection through an arginase 1-polyamine axis. Our results identify a mechanism of neuronal death post-infection and point to a role for tissue-resident MMs in limiting neuronal damage.

Diet Diurnally Regulates Small Intestinal Microbiome-Epithelial-Immune Homeostasis and Enteritis.

Throughout a 24-h period, the small intestine (SI) is exposed to diurnally varying food- and microbiome-derived antigenic burdens but maintains a strict immune homeostasis, which when perturbed in genetically susceptible individuals, may lead to Crohn disease. Herein, we demonstrate that dietary content and rhythmicity regulate the diurnally shifting SI epithelial cell (SIEC) transcriptional landscape through modulation of the SI microbiome. We exemplify this concept with SIEC major histocompatibility complex (MHC) class II, which is diurnally modulated by distinct mucosal-adherent SI commensals, while supporting downstream diurnal activity of intra-epithelial IL-10+ lymphocytes regulating the SI barrier function. Disruption of this diurnally regulated diet-microbiome-MHC class II-IL-10-epithelial barrier axis by circadian clock disarrangement, alterations in feeding time or content, or epithelial-specific MHC class II depletion leads to an extensive microbial product influx, driving Crohn-like enteritis. Collectively, we highlight nutritional features that modulate SI microbiome, immunity, and barrier function and identify dietary, epithelial, and immune checkpoints along this axis to be potentially exploitable in future Crohn disease interventions.

Fibroblastic reticular cell lineage convergence in Peyer's patches governs intestinal immunity.

Fibroblastic reticular cells (FRCs) determine the organization of lymphoid organs and control immune cell interactions. While the cellular and molecular mechanisms underlying FRC differentiation in lymph nodes and the splenic white pulp have been elaborated to some extent, in Peyer's patches (PPs) they remain elusive. Using a combination of single-cell transcriptomics and cell fate mapping in advanced mouse models, we found that PP formation in the mouse embryo is initiated by an expansion of perivascular FRC precursors, followed by FRC differentiation from subepithelial progenitors. Single-cell transcriptomics and cell fate mapping confirmed the convergence of perivascular and subepithelial FRC lineages. Furthermore, lineage-specific loss- and gain-of-function approaches revealed that the two FRC lineages synergistically direct PP organization, maintain intestinal microbiome homeostasis and control anticoronavirus immune responses in the gut. Collectively, this study reveals a distinct mosaic patterning program that generates key stromal cell infrastructures for the control of intestinal immunity.

Tuft cell-derived acetylcholine promotes epithelial chloride secretion and intestinal helminth clearance.

Epithelial cells secrete chloride to regulate water release at mucosal barriers, supporting both homeostatic hydration and the "weep" response that is critical for type 2 immune defense against parasitic worms (helminths). Epithelial tuft cells in the small intestine sense helminths and release cytokines and lipids to activate type 2 immune cells, but whether they regulate epithelial secretion is unknown. Here, we found that tuft cell activation rapidly induced epithelial chloride secretion in the small intestine. This response required tuft cell sensory functions and tuft cell-derived acetylcholine (ACh), which acted directly on neighboring epithelial cells to stimulate chloride secretion, independent of neurons. Maximal tuft cell-induced chloride secretion coincided with immune restriction of helminths, and clearance was delayed in mice lacking tuft cell-derived ACh, despite normal type 2 inflammation. Thus, we have uncovered an epithelium-intrinsic response unit that uses ACh to couple tuft cell sensing to the secretory defenses of neighboring epithelial cells.

The Tuft Cell-ILC2 Circuit Integrates Intestinal Defense and Homeostasis.

The intestinal response to helminth infection is mediated by a recently established type 2 immune circuit that consists of intestinal tuft cells and type 2 innate lymphoid cells (ILC2s). Schneider et al. have discovered that tuft cells sense succinate fermented by Tritrichomonas via GPR91 to drive the IL-25-ILC2-IL-13-dependent immune circuit and intestinal remodeling.

Developmental plasticity allows outside-in immune responses by resident memory T cells.

Central memory T (TCM) cells patrol lymph nodes and perform conventional memory responses on restimulation: proliferation, migration and differentiation into diverse T cell subsets while also self-renewing. Resident memory T (TRM) cells are parked within single organs, share properties with terminal effectors and contribute to rapid host protection. We observed that reactivated TRM cells rejoined the circulating pool. Epigenetic analyses revealed that TRM cells align closely with conventional memory T cell populations, bearing little resemblance to recently activated effectors. Fully differentiated TRM cells isolated from small intestine epithelium exhibited the potential to differentiate into TCM cells, effector memory T cells and TRM cells on recall. Ex-TRM cells, former intestinal TRM cells that rejoined the circulating pool, heritably maintained a predilection for homing back to their tissue of origin on subsequent reactivation and a heightened capacity to redifferentiate into TRM cells. Thus, TRM cells can rejoin the circulation but are advantaged to re-form local TRM when called on.

Mechanosensing by Peyer's patch stroma regulates lymphocyte migration and mucosal antibody responses.

Fibroblastic reticular cells (FRCs) and their specialized collagen fibers termed 'conduits' form fundamental structural units supporting lymphoid tissues. In lymph nodes, conduits are known to transport interstitial fluid and small molecules from afferent lymphatics into the nodal parenchyma. However, the immunological contributions of conduit function have remained elusive. Here, we report that intestinal Peyer's patches (PPs) contain a specialized conduit system that directs the flow of water absorbed across the intestinal epithelium. Notably, PP FRCs responded to conduit fluid flow via the mechanosensitive ion channel Piezo1. Disruption of fluid flow or genetic deficiency of Piezo1 on CCL19-expressing stroma led to profound structural alterations in perivascular FRCs and associated high endothelial venules. This in turn impaired lymphocyte entry into PPs and initiation of mucosal antibody responses. These results identify a critical role for conduit-mediated fluid flow in the maintenance of PP homeostasis and mucosal immunity.

Neuro-immune Interactions Drive Tissue Programming in Intestinal Macrophages.

Proper adaptation to environmental perturbations is essential for tissue homeostasis. In the intestine, diverse environmental cues can be sensed by immune cells, which must balance resistance to microorganisms with tolerance, avoiding excess tissue damage. By applying imaging and transcriptional profiling tools, we interrogated how distinct microenvironments in the gut regulate resident macrophages. We discovered that macrophages exhibit a high degree of gene-expression specialization dependent on their proximity to the gut lumen. Lamina propria macrophages (LpMs) preferentially expressed a pro-inflammatory phenotype when compared to muscularis macrophages (MMs), which displayed a tissue-protective phenotype. Upon luminal bacterial infection, MMs further enhanced tissue-protective programs, and this was attributed to swift activation of extrinsic sympathetic neurons innervating the gut muscularis and norepinephrine signaling to ß2 adrenergic receptors on MMs. Our results reveal unique intra-tissue macrophage specialization and identify neuro-immune communication between enteric neurons and macrophages that induces rapid tissue-protective responses to distal perturbations.

Environmental enteric dysfunction induces regulatory T cells that inhibit local CD4+ T cell responses and impair oral vaccine efficacy.

Environmental enteric dysfunction (EED) is a gastrointestinal inflammatory disease caused by malnutrition and chronic infection. EED is associated with stunting in children and reduced efficacy of oral vaccines. To study the mechanisms of oral vaccine failure during EED, we developed a microbiota- and diet-dependent mouse EED model. Analysis of E. coli-labile toxin vaccine-specific CD4+ T cells in these mice revealed impaired CD4+ T cell responses in the small intestine and but not the lymph nodes. EED mice exhibited increased frequencies of small intestine-resident RORγT+FOXP3+ regulatory T (Treg) cells. Targeted deletion of RORγT from Treg cells restored small intestinal vaccine-specific CD4 T cell responses and vaccine-mediated protection upon challenge. However, ablation of RORγT+FOXP3+ Treg cells made mice more susceptible to EED-induced stunting. Our findings provide insight into the poor efficacy of oral vaccines in EED and highlight how RORγT+FOXP3+ Treg cells can regulate intestinal immunity while leaving systemic responses intact.

A spatial expression atlas of the adult human proximal small intestine.

The mouse small intestine shows profound variability in gene expression along the crypt-villus axis1,2. Whether similar spatial heterogeneity exists in the adult human gut remains unclear. Here we use spatial transcriptomics, spatial proteomics and single-molecule fluorescence in situ hybridization to reconstruct a comprehensive spatial expression atlas of the adult human proximal small intestine. We describe zonated expression and cell type representation for epithelial, mesenchymal and immune cell types. We find that migrating enterocytes switch from lipid droplet assembly and iron uptake at the villus bottom to chylomicron biosynthesis and iron release at the tip. Villus tip cells are pro-immunogenic, recruiting γδ T cells and macrophages to the tip, in contrast to their immunosuppressive roles in mouse. We also show that the human small intestine contains abundant serrated and branched villi that are enriched at the tops of circular folds. Our study presents a detailed resource for understanding the biology of the adult human small intestine.

Tuft-Cell-Derived Leukotrienes Drive Rapid Anti-helminth Immunity in the Small Intestine but Are Dispensable for Anti-protist Immunity.

Helminths, allergens, and certain protists induce type 2 immune responses, but the underlying mechanisms of immune activation remain poorly understood. In the small intestine, chemosensing by epithelial tuft cells results in the activation of group 2 innate lymphoid cells (ILC2s), which subsequently drive increased tuft cell frequency. This feedforward circuit is essential for intestinal remodeling and helminth clearance. ILC2 activation requires tuft-cell-derived interleukin-25 (IL-25), but whether additional signals regulate the circuit is unclear. Here, we show that tuft cells secrete cysteinyl leukotrienes (cysLTs) to rapidly activate type 2 immunity following chemosensing of helminth infection. CysLTs cooperate with IL-25 to activate ILC2s, and tuft-cell-specific ablation of leukotriene synthesis attenuates type 2 immunity and delays helminth clearance. Conversely, cysLTs are dispensable for the tuft cell response induced by intestinal protists. Our findings identify an additional tuft cell effector function and suggest context-specific regulation of tuft-ILC2 circuits within the small intestine.

Mast cells link immune sensing to antigen-avoidance behaviour.

The physiological functions of mast cells remain largely an enigma. In the context of barrier damage, mast cells are integrated in type 2 immunity and, together with immunoglobulin E (IgE), promote allergic diseases. Allergic symptoms may, however, facilitate expulsion of allergens, toxins and parasites and trigger future antigen avoidance1-3. Here, we show that antigen-specific avoidance behaviour in inbred mice4,5 is critically dependent on mast cells; hence, we identify the immunological sensor cell linking antigen recognition to avoidance behaviour. Avoidance prevented antigen-driven adaptive, innate and mucosal immune activation and inflammation in the stomach and small intestine. Avoidance was IgE dependent, promoted by Th2 cytokines in the immunization phase and by IgE in the execution phase. Mucosal mast cells lining the stomach and small intestine rapidly sensed antigen ingestion. We interrogated potential signalling routes between mast cells and the brain using mutant mice, pharmacological inhibition, neural activity recordings and vagotomy. Inhibition of leukotriene synthesis impaired avoidance, but overall no single pathway interruption completely abrogated avoidance, indicating complex regulation. Collectively, the stage for antigen avoidance is set when adaptive immunity equips mast cells with IgE as a telltale of past immune responses. On subsequent antigen ingestion, mast cells signal termination of antigen intake. Prevention of immunopathology-causing, continuous and futile responses against per se innocuous antigens or of repeated ingestion of toxins through mast-cell-mediated antigen-avoidance behaviour may be an important arm of immunity.

Stromal cells control the epithelial residence of DCs and memory T cells by regulated activation of TGF-ß.

Cells of the immune system that reside in barrier epithelia provide a first line of defense against pathogens. Langerhans cells (LCs) and CD8(+) tissue-resident memory T cells (TRM cells) require active transforming growth factor-ß1 (TGF-ß) for epidermal residence. Here we found that integrins αvß6 and αvß8 were expressed in non-overlapping patterns by keratinocytes (KCs) and maintained the epidermal residence of LCs and TRM cells by activating latent TGF-ß. Similarly, the residence of dendritic cells and TRM cells in the small intestine epithelium also required αvß6. Treatment of the skin with ultraviolet irradiation decreased integrin expression on KCs and reduced the availability of active TGF-ß, which resulted in LC migration. Our data demonstrated that regulated activation of TGF-ß by stromal cells was able to directly control epithelial residence of cells of the immune system through a novel mechanism of intercellular communication.

Identification and distribution of developing innate lymphoid cells in the fetal mouse intestine.

Fetal lymphoid tissue inducer (LTi) cells are required for lymph node and Peyer's patch (PP) organogenesis, but where these specialized group 3 innate lymphoid cells (ILC3s) develop remains unclear. Here, we identify extrahepatic arginase-1(+) Id2(+) fetal ILC precursors that express a transitional developmental phenotype (ftILCPs) and differentiate into ILC1s, ILC2s and ILC3s in vitro. These cells populate the intestine by embryonic day (E) 13.5 and, before PP organogenesis (E14.5-15), are broadly dispersed in the proximal gut, correlating with regions where PPs first develop. At E16.5, after PP development begins, ftILCPs accumulate at PP anlagen in a lymphotoxin-α-dependent manner. Thus, ftILCPs reside in the intestine during PP development, where they aggregate at PP anlagen after stromal cell activation and become a localized source of ILC populations.

Detection of Succinate by Intestinal Tuft Cells Triggers a Type 2 Innate Immune Circuit.

In the small intestine, type 2 responses are regulated by a signaling circuit that involves tuft cells and group 2 innate lymphoid cells (ILC2s). Here, we identified the microbial metabolite succinate as an activating ligand for small intestinal (SI) tuft cells. Sequencing analyses of tuft cells isolated from the small intestine, gall bladder, colon, thymus, and trachea revealed that expression of tuft cell chemosensory receptors is tissue specific. SI tuft cells expressed the succinate receptor (SUCNR1), and providing succinate in drinking water was sufficient to induce a multifaceted type 2 immune response via the tuft-ILC2 circuit. The helminth Nippostrongylus brasiliensis and a tritrichomonad protist both secreted succinate as a metabolite. In vivo sensing of the tritrichomonad required SUCNR1, whereas N. brasiliensis was SUCNR1 independent. These findings define a paradigm wherein tuft cells monitor microbial metabolites to initiate type 2 immunity and suggest the existence of other sensing pathways triggering the response to helminths.

Enteric tuft cells coordinate timely expulsion of the tapeworm Hymenolepis diminuta from the murine host by coordinating local but not systemic immunity.

Recognizing that enteric tuft cells can signal the presence of nematode parasites, we investigated whether tuft cells are required for the expulsion of the cestode, Hymenolepis diminuta, from the non-permissive mouse host, and in concomitant anti-helminthic responses. BALB/c and C57BL/6 mice infected with H. diminuta expelled the worms by 11 days post-infection (dpi) and displayed DCLK1+ (doublecortin-like kinase 1) tuft cell hyperplasia in the small intestine (not the colon) at 11 dpi. This tuft cell hyperplasia was dependent on IL-4Rα signalling and adaptive immunity, but not the microbiota. Expulsion of H. diminuta was slowed until at least 14 dpi, but not negated, in tuft cell-deficient Pou2f3-/- mice and was accompanied by delayed goblet cell hyperplasia and slowed small bowel transit. Worm antigen and mitogen evoked production of IL-4 and IL-10 by splenocytes from wild-type and Pou2f3-/- mice was not appreciably different, suggesting similar systemic immune reactivity to infection with H. diminuta. Wild-type and Pou2f3-/- mice infected with H. diminuta displayed partial protection against subsequent infection with the nematode Heligmosomoides bakeri. We speculate that, with respect to H. diminuta, enteric tuft cells are important for local immune events driving the rapidity of H. diminuta expulsion but are not critical in initiating or sustaining systemic Th2 responses that provide concomitant immunity against secondary infection with H. bakeri.

Distal Immunization and Systemic Cytokines Establish a Transient Immune Alert State in the Intestine.

Conventionally, immune responses are studied in the context of inflamed tissues and their corresponding draining lymph nodes (LNs). However, little is known about the effects of systemic inflammatory signals generated during local inflammation on distal tissues and nondraining LNs. Using a mouse model of cutaneous immunization, we found that systemic inflammatory stimuli triggered a rapid and selective distal response in the small intestine and the mesenteric LN (mesLN). This consisted of increased permeability of intestinal blood vessels and lymphatic drainage of bloodborne solutes into the mesLN, enhanced activation and migration of intestinal dendritic cells, as well as amplified T cell responses in the mesLNs to systemic but not orally derived Ags. Mechanistically, we found that the small intestine endothelial cells preferentially expressed molecules involved in TNF-α signaling and that TNF-α blockade markedly diminished distal intestinal responses to cutaneous immunization. Together, these findings reveal that the intestinal immune system is rapidly and selectively activated in response to inflammatory cues regardless of their origin, thus identifying an additional layer of defense and enhanced surveillance of a key barrier organ at constant risk of pathogen encounter.

Gut microorganisms act together to exacerbate inflammation in spinal cords.

Accumulating evidence indicates that gut microorganisms have a pathogenic role in autoimmune diseases, including in multiple sclerosis1. Studies of experimental autoimmune encephalomyelitis (an animal model of multiple sclerosis)2,3, as well as human studies4-6, have implicated gut microorganisms in the development or severity of multiple sclerosis. However, it remains unclear how gut microorganisms act on the inflammation of extra-intestinal tissues such as the spinal cord. Here we show that two distinct signals from gut microorganisms coordinately activate autoreactive T cells in the small intestine that respond specifically to myelin oligodendrocyte glycoprotein (MOG). After induction of experimental autoimmune encephalomyelitis in mice, MOG-specific CD4+ T cells are observed in the small intestine. Experiments using germ-free mice that were monocolonized with microorganisms from the small intestine demonstrated that a newly isolated strain in the family Erysipelotrichaceae acts similarly to an adjuvant to enhance the responses of T helper 17 cells. Shotgun sequencing of the contents of the small intestine revealed a strain of Lactobacillus reuteri that possesses peptides that potentially mimic MOG. Mice that were co-colonized with these two strains showed experimental autoimmune encephalomyelitis symptoms that were more severe than those of germ-free or monocolonized mice. These data suggest that the synergistic effects that result from the presence of these microorganisms should be considered in the pathogenicity of multiple sclerosis, and that further study of these microorganisms may lead to preventive strategies for this disease.

Intestinal tissue-resident memory T cells maintain distinct identity from circulating memory T cells after in vitro restimulation.

Resident memory T (TRM) cells have been recently established as an important subset of memory T cells that provide early and essential protection against reinfection in the absence of circulating memory T cells. Recent findings showing that TRM expand in vivo after repeated antigenic stimulation indicate that these memory T cells are not terminally differentiated. This suggests an opportunity for in vitro TRM expansion to apply in an immunotherapy setting. However, it has also been shown that TRM may not maintain their identity and form circulating memory T cells after in vivo restimulation. Therefore, we set out to determine how TRM respond to antigenic activation in culture. Using Listeria monocytogenes and LCMV infection models, we found that TRM from the intraepithelial compartment of the small intestine expand in vitro after antigenic stimulation and subsequent resting in homeostatic cytokines. A large fraction of the expanded TRM retained their phenotype, including the expression of key TRM markers CD69 and CD103 (ITGAE). The optimal culture of TRM required low O2 pressure to maintain the expression of these and other TRM-associated molecules. Expanded TRM retained their effector capacity to produce cytokines after restimulation, but did not acquire a highly glycolytic profile indicative of effector T cells. The proteomic analysis confirmed TRM profile retention, including expression of TRM-related transcription factors, tissue retention factors, adhesion molecules, and enzymes involved in fatty acid metabolism. Collectively, our data indicate that limiting oxygen conditions supports in vitro expansion of TRM cells that maintain their TRM phenotype, at least in part, suggesting an opportunity for therapeutic strategies that require in vitro expansion of TRM.