Emerging Functions of IL-33 in Homeostasis and Immunity.

Our understanding of the functions of the IL-1 superfamily cytokine and damage-associated molecular pattern IL-33 continues to evolve with our understanding of homeostasis and immunity. The early findings that IL-33 is a potent driver of type 2 immune responses promoting parasite expulsion, but also inflammatory diseases like allergy and asthma, have been further supported. Yet, as the importance of a type 2 response in tissue repair and homeostasis has emerged, so has the fundamental importance of IL-33 to these processes. In this review, we outline an evolving understanding of IL-33 immunobiology, paying particular attention to how IL-33 directs a network of ST2+ regulatory T cells, reparative and regulatory macrophages, and type 2 innate lymphoid cells that are fundamental to tissue development, homeostasis, and repair.

Intraepithelial mast cells drive gasdermin C-mediated type 2 immunity.

A specialized population of mast cells residing within epithelial layers, currently known as intraepithelial mast cells (IEMCs), was originally observed over a century ago, yet their physiological functions have remained enigmatic. In this study, we unveil an unexpected and crucial role of IEMCs in driving gasdermin C-mediated type 2 immunity. During helminth infection, αEß7 integrin-positive IEMCs engaged in extensive intercellular crosstalk with neighboring intestinal epithelial cells (IECs). Through the action of IEMC-derived proteases, gasdermin C proteins intrinsic to the epithelial cells underwent cleavage, leading to the release of a critical type 2 cytokine, interleukin-33 (IL-33). Notably, mast cell deficiency abolished the gasdermin C-mediated immune cascade initiated by epithelium. These findings shed light on the functions of IEMCs, uncover a previously unrecognized phase of type 2 immunity involving mast cell-epithelial cell crosstalk, and advance our understanding of the cellular mechanisms underlying gasdermin C activation.

Mast cells control lung type 2 inflammation via prostaglandin E2-driven soluble ST2.

Severe asthma and sinus disease are consequences of type 2 inflammation (T2I), mediated by interleukin (IL)-33 signaling through its membrane-bound receptor, ST2. Soluble (s)ST2 reduces available IL-33 and limits T2I, but little is known about its regulation. We demonstrate that prostaglandin E2 (PGE2) drives production of sST2 to limit features of lung T2I. PGE2-deficient mice display diminished sST2. In humans with severe respiratory T2I, urinary PGE2 metabolites correlate with serum sST2. In mice, PGE2 enhanced sST2 secretion by mast cells (MCs). Mice lacking MCs, ST2 expression by MCs, or E prostanoid (EP)2 receptors by MCs showed reduced sST2 lung concentrations and strong T2I. Recombinant sST2 reduced T2I in mice lacking PGE2 or ST2 expression by MCs back to control levels. PGE2 deficiency also reversed the hyperinflammatory phenotype in mice lacking ST2 expression by MCs. PGE2 thus suppresses T2I through MC-derived sST2, explaining the severe T2I observed in low PGE2 states.

Immunomodulatory leptin receptor+ sympathetic perineurial barrier cells protect against obesity by facilitating brown adipose tissue thermogenesis.

Adipose tissues (ATs) are innervated by sympathetic nerves, which drive reduction of fat mass via lipolysis and thermogenesis. Here, we report a population of immunomodulatory leptin receptor-positive (LepR+) sympathetic perineurial barrier cells (SPCs) present in mice and humans, which uniquely co-express Lepr and interleukin-33 (Il33) and ensheath AT sympathetic axon bundles. Brown ATs (BATs) of mice lacking IL-33 in SPCs (SPCΔIl33) had fewer regulatory T (Treg) cells and eosinophils, resulting in increased BAT inflammation. SPCΔIl33 mice were more susceptible to diet-induced obesity, independently of food intake. Furthermore, SPCΔIl33 mice had impaired adaptive thermogenesis and were unresponsive to leptin-induced rescue of metabolic adaptation. We therefore identify LepR+ SPCs as a source of IL-33, which orchestrate an anti-inflammatory BAT environment, preserving sympathetic-mediated thermogenesis and body weight homeostasis. LepR+IL-33+ SPCs provide a cellular link between leptin and immune regulation of body weight, unifying neuroendocrinology and immunometabolism as previously disconnected fields of obesity research.

TCR Transgenic Mice Reveal Stepwise, Multi-site Acquisition of the Distinctive Fat-Treg Phenotype.

Visceral adipose tissue (VAT) hosts a population of regulatory T (Treg) cells, with a unique phenotype, that controls local and systemic inflammation and metabolism. Generation of a T cell receptor transgenic mouse line, wherein VAT Tregs are highly enriched, facilitated study of their provenance, dependencies, and activities. We definitively established a role for T cell receptor specificity, uncovered an unexpected function for the primordial Treg transcription-factor, Foxp3, evidenced a cell-intrinsic role for interleukin-33 receptor, and ordered these dependencies within a coherent scenario. Genesis of the VAT-Treg phenotype entailed a priming step in the spleen, permitting them to exit the lymphoid organs and surveil nonlymphoid tissues, and a final diversification process within VAT, in response to microenvironmental cues. Understanding the principles of tissue-Treg biology is a prerequisite for precision-targeting strategies.

Circumvention of luteolysis reveals parturition pathways in mice dependent upon innate type 2 immunity.

Although mice normally enter labor when their ovaries stop producing progesterone (luteolysis), parturition can also be triggered in this species through uterus-intrinsic pathways potentially analogous to the ones that trigger parturition in humans. Such pathways, however, remain largely undefined in both species. Here, we report that mice deficient in innate type 2 immunity experienced profound parturition delays when manipulated endocrinologically to circumvent luteolysis, thus obliging them to enter labor through uterus-intrinsic pathways. We found that these pathways were in part driven by the alarmin IL-33 produced by uterine interstitial fibroblasts. We also implicated important roles for uterine group 2 innate lymphoid cells, which demonstrated IL-33-dependent activation prior to labor onset, and eosinophils, which displayed evidence of elevated turnover in the prepartum uterus. These findings reveal a role for innate type 2 immunity in controlling the timing of labor onset through a cascade potentially relevant to human parturition.

The alarmin interleukin-33 promotes the expansion and preserves the stemness of Tcf-1+ CD8+ T cells in chronic viral infection.

T cell factor 1 (Tcf-1) expressing CD8+ T cells exhibit stem-like self-renewing capacity, rendering them key for immune defense against chronic viral infection and cancer. Yet, the signals that promote the formation and maintenance of these stem-like CD8+ T cells (CD8+SL) remain poorly defined. Studying CD8+ T cell differentiation in mice with chronic viral infection, we identified the alarmin interleukin-33 (IL-33) as pivotal for the expansion and stem-like functioning of CD8+SL as well as for virus control. IL-33 receptor (ST2)-deficient CD8+ T cells exhibited biased end differentiation and premature loss of Tcf-1. ST2-deficient CD8+SL responses were restored by blockade of type I interferon signaling, suggesting that IL-33 balances IFN-I effects to control CD8+SL formation in chronic infection. IL-33 signals broadly augmented chromatin accessibility in CD8+SL and determined these cells' re-expansion potential. Our study identifies the IL-33-ST2 axis as an important CD8+SL-promoting pathway in the context of chronic viral infection.

Disruption of the IL-33-ST2-AKT signaling axis impairs neurodevelopment by inhibiting microglial metabolic adaptation and phagocytic function.

To accommodate the changing needs of the developing brain, microglia must undergo substantial morphological, phenotypic, and functional reprogramming. Here, we examined whether cellular metabolism regulates microglial function during neurodevelopment. Microglial mitochondria bioenergetics correlated with and were functionally coupled to phagocytic activity in the developing brain. Transcriptional profiling of microglia with diverse metabolic profiles revealed an activation signature wherein the interleukin (IL)-33 signaling axis is associated with phagocytic activity. Genetic perturbation of IL-33 or its receptor ST2 led to microglial dystrophy, impaired synaptic function, and behavioral abnormalities. Conditional deletion of Il33 from astrocytes or Il1rl1, encoding ST2, in microglia increased susceptibility to seizures. Mechanistically, IL-33 promoted mitochondrial activity and phagocytosis in an AKT-dependent manner. Mitochondrial metabolism and AKT activity were temporally regulated in vivo. Thus, a microglia-astrocyte circuit mediated by the IL-33-ST2-AKT signaling axis supports microglial metabolic adaptation and phagocytic function during early development, with implications for neurodevelopmental and neuropsychiatric disorders.

Epithelial STAT6 O-GlcNAcylation drives a concerted anti-helminth alarmin response dependent on tuft cell hyperplasia and Gasdermin C.

The epithelium is an integral component of mucosal barrier and host immunity. Following helminth infection, the intestinal epithelial cells secrete "alarmin" cytokines, such as interleukin-25 (IL-25) and IL-33, to initiate the type 2 immune responses for helminth expulsion and tolerance. However, it is unknown how helminth infection and the resulting cytokine milieu drive epithelial remodeling and orchestrate alarmin secretion. Here, we report that epithelial O-linked N-Acetylglucosamine (O-GlcNAc) protein modification was induced upon helminth infections. By modifying and activating the transcription factor STAT6, O-GlcNAc transferase promoted the transcription of lineage-defining Pou2f3 in tuft cell differentiation and IL-25 production. Meanwhile, STAT6 O-GlcNAcylation activated the expression of Gsdmc family genes. The membrane pore formed by GSDMC facilitated the unconventional secretion of IL-33. GSDMC-mediated IL-33 secretion was indispensable for effective anti-helminth immunity and contributed to induced intestinal inflammation. Protein O-GlcNAcylation can be harnessed for future treatment of type 2 inflammation-associated human diseases.

Interleukin-33-activated neuropeptide CGRP-producing memory Th2 cells cooperate with somatosensory neurons to induce conjunctival itch.

Allergic conjunctivitis is a chronic inflammatory disease that is characterized by severe itch in the conjunctiva, but how neuro-immune interactions shape the pathogenesis of severe itch remains unclear. We identified a subset of memory-type pathogenic Th2 cells that preferentially expressed Il1rl1-encoding ST2 and Calca-encoding calcitonin-gene-related peptide (CGRP) in the inflammatory conjunctiva using a single-cell analysis. The IL-33-ST2 axis in memory Th2 cells controlled the axonal elongation of the peripheral sensory C-fiber and the induction of severe itch. Pharmacological blockade and genetic deletion of CGRP signaling in vivo attenuated scratching behavior. The analysis of giant papillae from patients with severe allergic conjunctivitis revealed ectopic lymphoid structure formation with the accumulation of IL-33-producing epithelial cells and CGRP-producing pathogenic CD4+ T cells accompanied by peripheral nerve elongation. Thus, the IL-33-ST2-CGRP axis directs severe itch with neuro-reconstruction in the inflammatory conjunctiva and is a potential therapeutic target for severe itch in allergic conjunctivitis.

Interleukin-33 Promotes Serotonin Release from Enterochromaffin Cells for Intestinal Homeostasis.

The gastrointestinal tract is known as the largest endocrine organ that encounters and integrates various immune stimulations and neuronal responses due to constant environmental challenges. Enterochromaffin (EC) cells, which function as chemosensors on the gut epithelium, are known to translate environmental cues into serotonin (5-HT) production, contributing to intestinal physiology. However, how immune signals participate in gut sensation and neuroendocrine response remains unclear. Interleukin-33 (IL-33) acts as an alarmin cytokine by alerting the system of potential environmental stresses. We here demonstrate that IL-33 induced instantaneous peristaltic movement and facilitated Trichuris muris expulsion. We found that IL-33 could be sensed by EC cells, inducing release of 5-HT. IL-33-mediated 5-HT release activated enteric neurons, subsequently promoting gut motility. Mechanistically, IL-33 triggered calcium influx via a non-canonical signaling pathway specifically in EC cells to induce 5-HT secretion. Our data establish an immune-neuroendocrine axis in calibrating rapid 5-HT release for intestinal homeostasis.

Interleukin-33 Induces the Enzyme Tryptophan Hydroxylase 1 to Promote Inflammatory Group 2 Innate Lymphoid Cell-Mediated Immunity.

Group 2 innate lymphoid cells (ILC2s) regulate immunity, inflammation, and tissue homeostasis. Two distinct subsets of ILC2s have been described: steady-state natural ILC2s and inflammatory ILC2s, which are elicited following helminth infection. However, how tissue-specific cues regulate these two subsets of ILC2s and their effector functions remains elusive. Here, we report that interleukin-33 (IL-33) promotes the generation of inflammatory ILC2s (ILC2INFLAM) via induction of the enzyme tryptophan hydroxylase 1 (Tph1). Tph1 expression was upregulated in ILC2s upon activation with IL-33 or following helminth infection in an IL-33-dependent manner. Conditional deletion of Tph1 in lymphocytes resulted in selective impairment of ILC2INFLAM responses and increased susceptibility to helminth infection. Further, RNA sequencing analysis revealed altered gene expression in Tph1 deficient ILC2s including inducible T cell co-stimulator (Icos). Collectively, these data reveal a previously unrecognized function for IL-33, Tph1, and ICOS in promoting inflammatory ILC2 responses and type 2 immunity at mucosal barriers.

Cold-responsive adipocyte progenitors couple adrenergic signaling to immune cell activation to promote beige adipocyte accrual.

The full array of cold-responsive cell types within white adipose tissue that drive thermogenic beige adipocyte biogenesis remains undefined. We demonstrate that acute cold challenge elicits striking transcriptomic changes specifically within DPP4+ PDGFRß+ adipocyte precursor cells, including a ß-adrenergic receptor CREB-mediated induction in the expression of the prothermogenic cytokine, Il33 Doxycycline-inducible deletion of Il33 in PDGFRß+ cells at the onset of cold exposure attenuates ILC2 accumulation and beige adipocyte accrual. These studies highlight the multifaceted roles for adipocyte progenitors and the ability of select mesenchymal subpopulations to relay neuronal signals to tissue-resident immune cells in order to regulate tissue plasticity.

Neuropeptide CGRP Limits Group 2 Innate Lymphoid Cell Responses and Constrains Type 2 Inflammation.

Innate lymphocytes maintain tissue homeostasis at mucosal barriers, with group 2 innate lymphoid cells (ILC2s) producing type 2 cytokines and controlling helminth infection. While the molecular understanding of ILC2 responses has advanced, the complexity of microenvironmental factors impacting ILC2s is becoming increasingly apparent. Herein, we used single-cell analysis to explore the diversity of gene expression among lung lymphocytes during helminth infection. Following infection, we identified a subset of ILC2s that preferentially expressed Il5-encoding interleukin (IL)-5, together with Calca-encoding calcitonin gene-related peptide (CGRP) and its cognate receptor components. CGRP in concert with IL-33 and neuromedin U (NMU) supported IL-5 but constrained IL-13 expression and ILC2 proliferation. Without CGRP signaling, ILC2 responses and worm expulsion were enhanced. Collectively, these data point to CGRP as a context-dependent negative regulatory factor that shapes innate lymphocyte responses to alarmins and neuropeptides during type 2 innate immune responses.

Adventitial Stromal Cells Define Group 2 Innate Lymphoid Cell Tissue Niches.

Type 2 lymphocytes promote both physiologic tissue remodeling and allergic pathology, yet their physical tissue niches are poorly described. Here, we used quantitative imaging to define the tissue niches of group 2 innate lymphoid cells (ILC2s), which are critical instigators of type 2 immunity. We identified a dominant adventitial niche around lung bronchi and larger vessels in multiple tissues, where ILC2s localized with subsets of dendritic and regulatory T cells. However, ILC2s were most intimately associated with adventitial stromal cells (ASCs), a mesenchymal fibroblast-like subset that expresses interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP). In vitro, ASCs produced TSLP that supported ILC2 accumulation and activation. ILC2s and IL-13 drove reciprocal ASC expansion and IL-33 expression. During helminth infection, ASC depletion impaired lung ILC2 and Th2 cell accumulation and function, which are in part dependent on ASC-derived IL-33. These data indicate that adventitial niches are conserved sites where ASCs regulate type 2 lymphocyte expansion and function.

Aryl Hydrocarbon Receptor Signaling Cell Intrinsically Inhibits Intestinal Group 2 Innate Lymphoid Cell Function.

Innate lymphoid cells (ILCs) are important for mucosal immunity. The intestine harbors all ILC subsets, but how these cells are balanced to achieve immune homeostasis and mount appropriate responses during infection remains elusive. Here, we show that aryl hydrocarbon receptor (Ahr) expression in the gut regulates ILC balance. Among ILCs, Ahr is most highly expressed by gut ILC2s and controls chromatin accessibility at the Ahr locus via positive feedback. Ahr signaling suppresses Gfi1 transcription-factor-mediated expression of the interleukin-33 (IL-33) receptor ST2 in ILC2s and expression of ILC2 effector molecules IL-5, IL-13, and amphiregulin in a cell-intrinsic manner. Ablation of Ahr enhances anti-helminth immunity in the gut, whereas genetic or pharmacological activation of Ahr suppresses ILC2 function but enhances ILC3 maintenance to protect the host from Citrobacter rodentium infection. Thus, the host regulates the gut ILC2-ILC3 balance by engaging the Ahr pathway to mount appropriate immunity against various pathogens.

Amphiregulin-Producing Pathogenic Memory T Helper 2 Cells Instruct Eosinophils to Secrete Osteopontin and Facilitate Airway Fibrosis.

Memory T cells provide long-lasting protective immunity, and distinct subpopulations of memory T cells drive chronic inflammatory diseases such as asthma. Asthma is a chronic allergic inflammatory disease with airway remodeling including fibrotic changes. The immunological mechanisms that induce airway fibrotic changes remain unknown. We found that interleukin-33 (IL-33) enhanced amphiregulin production by the IL-33 receptor, ST2hi memory T helper 2 (Th2) cells. Amphiregulin-epidermal growth factor receptor (EGFR)-mediated signaling directly reprogramed eosinophils to an inflammatory state with enhanced production of osteopontin, a key profibrotic immunomodulatory protein. IL-5-producing memory Th2 cells and amphiregulin-producing memory Th2 cells appeared to cooperate to establish lung fibrosis. The analysis of polyps from patients with eosinophilic chronic rhinosinusitis revealed fibrosis with accumulation of amphiregulin-producing CRTH2hiCD161hiCD45RO+CD4+ Th2 cells and osteopontin-producing eosinophils. Thus, the IL-33-amphiregulin-osteopontin axis directs fibrotic responses in eosinophilic airway inflammation and is a potential target for the treatment of fibrosis induced by chronic allergic disorders.

Lung γδ T Cells Mediate Protective Responses during Neonatal Influenza Infection that Are Associated with Type 2 Immunity.

Compared to adults, infants suffer higher rates of hospitalization, severe clinical complications, and mortality due to influenza infection. We found that γδ T cells protected neonatal mice against mortality during influenza infection. γδ T cell deficiency did not alter viral clearance or interferon-γ production. Instead, neonatal influenza infection induced the accumulation of interleukin-17A (IL-17A)-producing γδ T cells, which was associated with IL-33 production by lung epithelial cells. Neonates lacking IL-17A-expressing γδ T cells or Il33 had higher mortality upon influenza infection. γδ T cells and IL-33 promoted lung infiltration of group 2 innate lymphoid cells and regulatory T cells, resulting in increased amphiregulin secretion and tissue repair. In influenza-infected children, IL-17A, IL-33, and amphiregulin expression were correlated, and increased IL-17A levels in nasal aspirates were associated with better clinical outcomes. Our results indicate that γδ T cells are required in influenza-infected neonates to initiate protective immunity and mediate lung homeostasis.

Tissue-Restricted Adaptive Type 2 Immunity Is Orchestrated by Expression of the Costimulatory Molecule OX40L on Group 2 Innate Lymphoid Cells.

The local regulation of type 2 immunity relies on dialog between the epithelium and the innate and adaptive immune cells. Here we found that alarmin-induced expression of the co-stimulatory molecule OX40L on group 2 innate lymphoid cells (ILC2s) provided tissue-restricted T cell co-stimulation that was indispensable for Th2 and regulatory T (Treg) cell responses in the lung and adipose tissue. Interleukin (IL)-33 administration resulted in organ-specific surface expression of OX40L on ILC2s and the concomitant expansion of Th2 and Treg cells, which was abolished upon deletion of OX40L on ILC2s (Il7raCre/+Tnfsf4fl/fl mice). Moreover, Il7raCre/+Tnfsf4fl/fl mice failed to mount effective Th2 and Treg cell responses and corresponding adaptive type 2 pulmonary inflammation arising from Nippostrongylus brasiliensis infection or allergen exposure. Thus, the increased expression of OX40L in response to IL-33 acts as a licensing signal in the orchestration of tissue-specific adaptive type 2 immunity, without which this response fails to establish.

IL-33 controls IL-22-dependent antibacterial defense by modulating the microbiota.

IL-22 plays a critical role in defending against mucosal infections, but how IL-22 production is regulated is incompletely understood. Here, we show that mice lacking IL-33 or its receptor ST2 (IL-1RL1) were more resistant to Streptococcus pneumoniae lung infection than wild-type animals and that single-nucleotide polymorphisms in IL33 and IL1RL1 were associated with pneumococcal pneumonia in humans. The effect of IL-33 on S. pneumoniae infection was mediated by negative regulation of IL-22 production in innate lymphoid cells (ILCs) but independent of ILC2s as well as IL-4 and IL-13 signaling. Moreover, IL-33's influence on IL-22-dependent antibacterial defense was dependent on housing conditions of the mice and mediated by IL-33's modulatory effect on the gut microbiota. Collectively, we provide insight into the bidirectional crosstalk between the innate immune system and the microbiota. We conclude that both genetic and environmental factors influence the gut microbiota, thereby impacting the efficacy of antibacterial immune defense and susceptibility to pneumonia.