Intraepithelial Lymphocytes of the Intestine.

The intestinal epithelium, which segregates the highly stimulatory lumen from the underlying tissue, harbors one of the largest lymphocyte populations in the body, intestinal intraepithelial lymphocytes (IELs). IELs must balance tolerance, resistance, and tissue protection to maintain epithelial homeostasis and barrier integrity. This review discusses the ontogeny, environmental imprinting, T cell receptor (TCR) repertoire, and function of intestinal IELs. Despite distinct developmental pathways, IEL subsets share core traits including an epithelium-adapted profile, innate-like properties, cytotoxic potential, and limited TCR diversity. IELs also receive important developmental and functional cues through interactions with epithelial cells, microbiota, and dietary components. The restricted TCR diversity of IELs suggests that a limited set of intestinal antigens drives IEL responses, with potential functional consequences. Finally, IELs play a key role in promoting homeostatic immunity and epithelial barrier integrity but can become pathogenic upon dysregulation. Therefore, IELs represent intriguing but underexamined therapeutic targets for inflammatory diseases and cancer.

A tissue injury sensing and repair pathway distinct from host pathogen defense.

Pathogen infection and tissue injury are universal insults that disrupt homeostasis. Innate immunity senses microbial infections and induces cytokines/chemokines to activate resistance mechanisms. Here, we show that, in contrast to most pathogen-induced cytokines, interleukin-24 (IL-24) is predominately induced by barrier epithelial progenitors after tissue injury and is independent of microbiome or adaptive immunity. Moreover, Il24 ablation in mice impedes not only epidermal proliferation and re-epithelialization but also capillary and fibroblast regeneration within the dermal wound bed. Conversely, ectopic IL-24 induction in the homeostatic epidermis triggers global epithelial-mesenchymal tissue repair responses. Mechanistically, Il24 expression depends upon both epithelial IL24-receptor/STAT3 signaling and hypoxia-stabilized HIF1α, which converge following injury to trigger autocrine and paracrine signaling involving IL-24-mediated receptor signaling and metabolic regulation. Thus, parallel to innate immune sensing of pathogens to resolve infections, epithelial stem cells sense injury signals to orchestrate IL-24-mediated tissue repair.

Microbiota imbalance induced by dietary sugar disrupts immune-mediated protection from metabolic syndrome.

How intestinal microbes regulate metabolic syndrome is incompletely understood. We show that intestinal microbiota protects against development of obesity, metabolic syndrome, and pre-diabetic phenotypes by inducing commensal-specific Th17 cells. High-fat, high-sugar diet promoted metabolic disease by depleting Th17-inducing microbes, and recovery of commensal Th17 cells restored protection. Microbiota-induced Th17 cells afforded protection by regulating lipid absorption across intestinal epithelium in an IL-17-dependent manner. Diet-induced loss of protective Th17 cells was mediated by the presence of sugar. Eliminating sugar from high-fat diets protected mice from obesity and metabolic syndrome in a manner dependent on commensal-specific Th17 cells. Sugar and ILC3 promoted outgrowth of Faecalibaculum rodentium that displaced Th17-inducing microbiota. These results define dietary and microbiota factors posing risk for metabolic syndrome. They also define a microbiota-dependent mechanism for immuno-pathogenicity of dietary sugar and highlight an elaborate interaction between diet, microbiota, and intestinal immunity in regulation of metabolic disorders.

Enteric pathogens induce tissue tolerance and prevent neuronal loss from subsequent infections.

The enteric nervous system (ENS) controls several intestinal functions including motility and nutrient handling, which can be disrupted by infection-induced neuropathies or neuronal cell death. We investigated possible tolerance mechanisms preventing neuronal loss and disruption in gut motility after pathogen exposure. We found that following enteric infections, muscularis macrophages (MMs) acquire a tissue-protective phenotype that prevents neuronal loss, dysmotility, and maintains energy balance during subsequent challenge with unrelated pathogens. Bacteria-induced neuroprotection relied on activation of gut-projecting sympathetic neurons and signaling via ß2-adrenergic receptors (ß2AR) on MMs. In contrast, helminth-mediated neuroprotection was dependent on T cells and systemic production of interleukin (IL)-4 and IL-13 by eosinophils, which induced arginase-expressing MMs that prevented neuronal loss from an unrelated infection located in a different intestinal region. Collectively, these data suggest that distinct enteric pathogens trigger a state of disease or tissue tolerance that preserves ENS number and functionality.

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.

Stepwise chromatin and transcriptional acquisition of an intraepithelial lymphocyte program.

Mesenteric lymph node (mLN) T cells undergo tissue adaptation upon migrating to intestinal lamina propria and epithelium, ensuring appropriate balance between tolerance and resistance. By combining mouse genetics with single-cell and chromatin analyses, we uncovered the molecular imprinting of gut epithelium on T cells. Transcriptionally, conventional and regulatory (Treg) CD4+ T cells from mLN, lamina propria and intestinal epithelium segregate based on the gut layer they occupy; trajectory analysis suggests a stepwise loss of CD4 programming and acquisition of an intraepithelial profile. Treg cell fate mapping coupled with RNA sequencing and assay for transposase-accessible chromatin followed by sequencing revealed that the Treg cell program shuts down before an intraepithelial program becomes fully accessible at the epithelium. Ablation of CD4-lineage-defining transcription factor ThPOK results in premature acquisition of an intraepithelial lymphocyte profile by mLN Treg cells, partially recapitulating epithelium imprinting. Thus, coordinated replacement of the circulating lymphocyte program with site-specific transcriptional and chromatin changes is necessary for tissue imprinting.

Essential role of a ThPOK autoregulatory loop in the maintenance of mature CD4+ T cell identity and function.

The transcription factor ThPOK (encoded by the Zbtb7b gene) controls homeostasis and differentiation of mature helper T cells, while opposing their differentiation to CD4+ intraepithelial lymphocytes (IELs) in the intestinal mucosa. Thus CD4 IEL differentiation requires ThPOK transcriptional repression via reactivation of the ThPOK transcriptional silencer element (SilThPOK). In the present study, we describe a new autoregulatory loop whereby ThPOK binds to the SilThPOK to maintain its own long-term expression in CD4 T cells. Disruption of this loop in vivo prevents persistent ThPOK expression, leads to genome-wide changes in chromatin accessibility and derepresses the colonic regulatory T (Treg) cell gene expression signature. This promotes selective differentiation of naive CD4 T cells into GITRloPD-1loCD25lo (Triplelo) Treg cells and conversion to CD4+ IELs in the gut, thereby providing dominant protection from colitis. Hence, the ThPOK autoregulatory loop represents a key mechanism to physiologically control ThPOK expression and T cell differentiation in the gut, with potential therapeutic relevance.

SnapShot: Gut Immune Niches.

The intestinal milieu changes along the proximal to distal axis and across its tissue wall, according to the luminal content and tissue function. Correspondingly, highly specialized immune compartments can be found in each intestinal niche. To view this SnapShot, open or download the PDF.

LXR/ApoE Activation Restricts Innate Immune Suppression in Cancer.

Therapeutic harnessing of adaptive immunity via checkpoint inhibition has transformed the treatment of many cancers. Despite unprecedented long-term responses, most patients do not respond to these therapies. Immunotherapy non-responders often harbor high levels of circulating myeloid-derived suppressor cells (MDSCs)-an immunosuppressive innate cell population. Through genetic and pharmacological approaches, we uncovered a pathway governing MDSC abundance in multiple cancer types. Therapeutic liver-X nuclear receptor (LXR) agonism reduced MDSC abundance in murine models and in patients treated in a first-in-human dose escalation phase 1 trial. MDSC depletion was associated with activation of cytotoxic T lymphocyte (CTL) responses in mice and patients. The LXR transcriptional target ApoE mediated these effects in mice, where LXR/ApoE activation therapy elicited robust anti-tumor responses and also enhanced T cell activation during various immune-based therapies. We implicate the LXR/ApoE axis in the regulation of innate immune suppression and as a target for enhancing the efficacy of cancer immunotherapy in patients.

Can U Turn ILC2s Up?

Innate lymphoid cells (ILCs) are tuned to quickly respond to and amplify tissue-specific signals. Work of three independent groups in Nature uncovers a novel mode of inflammatory communication between ILC2s and neurons at mucosal surfaces.

Intestinal Epithelial and Intraepithelial T Cell Crosstalk Mediates a Dynamic Response to Infection.

Intestinal intraepithelial lymphocytes (IELs) are located at the critical interface between the intestinal lumen, which is chronically exposed to food and microbes, and the core of the body. Using high-resolution microscopy techniques and intersectional genetic tools, we investigated the nature of IEL responses to luminal microbes. We observed that TCRγδ IELs exhibit unique microbiota-dependent location and movement patterns in the epithelial compartment. This behavioral pattern quickly changes upon exposure to different enteric pathogens, resulting in increased interepithelial cell (EC) scanning, expression of antimicrobial genes, and glycolysis. Both dynamic and metabolic changes to γδ IEL depend on pathogen sensing by ECs. Direct modulation of glycolysis is sufficient to change γδ IEL behavior and susceptibility to early pathogen invasion. Our results uncover a coordinated EC-IEL response to enteric infections that modulates lymphocyte energy utilization and dynamics and supports maintenance of the intestinal epithelial barrier. VIDEO ABSTRACT.

A triplex for intestinal protection: Neurons, microbes, and goblet cells.

Diverse intestinal components (e.g., gut-associated neurons, immune cells, gut microbes, and epithelium) are intimately intertwined with each other to maintain homeostasis in the gut. In a recent issue of Cell, Zhang et al. (2022) and Yang et al. (2022) present complementary studies uncovering interactions between nociceptor neurons, gut epithelium, and the microbiome to protect intestinal tissue from inflammation.

Immunity to enteric viruses.

Pathogenic enteric viruses are a major cause of morbidity and mortality, particularly among children in developing countries. The host response to enteric viruses occurs primarily within the mucosa, where the intestinal immune system must balance protection against pathogens with tissue protection and tolerance to harmless commensal bacteria and food. Here, we summarize current knowledge in natural immunity to enteric viruses, highlighting specialized features of the intestinal immune system. We further discuss how knowledge of intestinal anti-viral mechanisms can be translated into vaccine development with particular focus on immunization in the oral route. Research reveals that the intestine is a complex interface between enteric viruses and the host where environmental factors influence susceptibility and immunity to infection, while viral infections can have lasting implications for host health. A deeper mechanistic understanding of enteric anti-viral immunity with this broader context can ultimately lead to better vaccines for existing and emerging viruses.

Newly recruited intraepithelial Ly6A+CCR9+CD4+ T cells protect against enteric viral infection.

The intestinal epithelium comprises the body's largest surface exposed to viruses. Additionally, the gut epithelium hosts a large population of intraepithelial T lymphocytes, or IELs, although their role in resistance against viral infections remains elusive. By fate-mapping T cells recruited to the murine intestine, we observed an accumulation of newly recruited CD4+ T cells after infection with murine norovirus CR6 and adenovirus type-2 (AdV), but not reovirus. CR6- and AdV-recruited intraepithelial CD4+ T cells co-expressed Ly6A and chemokine receptor CCR9, exhibited T helper 1 and cytotoxic profiles, and conferred protection against AdV in vivo and in an organoid model in an IFN-γ-dependent manner. Ablation of the T cell receptor (TCR) or the transcription factor ThPOK in CD4+ T cells prior to AdV infection prevented viral control, while TCR ablation during infection did not impact viral clearance. These results uncover a protective role for intraepithelial Ly6A+CCR9+CD4+ T cells against enteric adenovirus.

Genetic tracing reveals transcription factor Foxp3-dependent and Foxp3-independent functionality of peripherally induced Treg cells.

Regulatory T (Treg) cells expressing the transcription factor Foxp3 are an essential suppressive T cell lineage of dual origin: Foxp3 induction in thymocytes and mature CD4+ T cells gives rise to thymic (tTreg) and peripheral (pTreg) Treg cells, respectively. While tTreg cells suppress autoimmunity, pTreg cells enforce tolerance to food and commensal microbiota. However, the role of Foxp3 in pTreg cells and the mechanisms supporting their differentiation remain poorly understood. Here, we used genetic tracing to identify microbiota-induced pTreg cells and found that many of their distinguishing features were Foxp3 independent. Lineage-committed, microbiota-dependent pTreg-like cells persisted in the colon in the absence of Foxp3. While Foxp3 was critical for the suppression of a Th17 cell program, colitis, and mastocytosis, pTreg cells suppressed colonic effector T cell expansion in a Foxp3-independent manner. Thus, Foxp3 and the tolerogenic signals that precede and promote its expression independently confer distinct facets of pTreg functionality.

Neuro-Immune Interactions at Barrier Surfaces.

Multidirectional interactions between the nervous and immune systems have been documented in homeostasis and pathologies ranging from multiple sclerosis to autism, and from leukemia to acute and chronic inflammation. Recent studies have addressed this crosstalk using cell-specific targeting, novel sequencing, imaging, and analytical tools, shedding light on unappreciated mechanisms of neuro-immune regulation. This Review focuses on neuro-immune interactions at barrier surfaces-mostly the gut, but also including the skin and the airways, areas densely populated by neurons and immune cells that constantly sense and adapt to tissue-specific environmental challenges.

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.

"A Sledgehammer Breaks Glass but Forges Steel": Bacteria Adhesion Shapes Gut Immunity.

Gut bacteria are known to affect immune cell development, but most intestinal lymphocytes have no direct contact with luminal bacteria. Two studies by Atarashi et al. and Sano et al. shed light on how bacterial adhesion can cue intestinal epithelial cells to direct differentiation of gut T cells.

Universal recording of immune cell interactions in vivo.

Immune cells rely on transient physical interactions with other immune and non-immune populations to regulate their function1. To study these 'kiss-and-run' interactions directly in vivo, we previously developed LIPSTIC (labelling immune partnerships by SorTagging intercellular contacts)2, an approach that uses enzymatic transfer of a labelled substrate between the molecular partners CD40L and CD40 to label interacting cells. Reliance on this pathway limited the use of LIPSTIC to measuring interactions between CD4+ T helper cells and antigen-presenting cells, however. Here we report the development of a universal version of LIPSTIC (uLIPSTIC), which can record physical interactions both among immune cells and between immune and non-immune populations irrespective of the receptors and ligands involved. We show that uLIPSTIC can be used, among other things, to monitor the priming of CD8+ T cells by dendritic cells, reveal the steady-state cellular partners of regulatory T cells and identify germinal centre-resident T follicular helper cells on the basis of their ability to interact cognately with germinal centre B cells. By coupling uLIPSTIC with single-cell transcriptomics, we build a catalogue of the immune populations that physically interact with intestinal epithelial cells at the steady state and profile the evolution of the interactome of lymphocytic choriomeningitis virus-specific CD8+ T cells in multiple organs following systemic infection. Thus, uLIPSTIC provides a broadly useful technology for measuring and understanding cell-cell interactions across multiple biological systems.