Neuropilin-1 mediates lung tissue-specific control of ILC2 function in type 2 immunity.

Group 2 innate lymphoid cells (ILC2s) are highly heterogeneous tissue-resident lymphocytes that regulate inflammation and tissue homeostasis in health and disease. However, how these cells integrate into the tissue microenvironment to perform tissue-specific functions is unclear. Here, we show neuropilin-1 (Nrp1), which is induced postnatally and sustained by lung-derived transforming growth factor beta-1 (TGFß1), is a tissue-specific marker of lung ILC2s. Genetic ablation or pharmacological inhibition of Nrp1 suppresses IL-5 and IL-13 production by ILC2s and protects mice from the development of pulmonary fibrosis. Mechanistically, TGFß1-Nrp1 signaling enhances ILC2 function and type 2 immunity by upregulating IL-33 receptor ST2 expression. These findings identify Nrp1 as a tissue-specific regulator of lung-resident ILC2s and highlight Nrp1 as a potential therapeutic target for pulmonary fibrosis.

Environmental allergens trigger type 2 inflammation through ripoptosome activation.

Environmental allergens, including fungi, insects and mites, trigger type 2 immunity; however, the innate sensing mechanisms and initial signaling events remain unclear. Herein, we demonstrate that allergens trigger RIPK1-caspase 8 ripoptosome activation in epithelial cells. The active caspase 8 subsequently engages caspases 3 and 7, which directly mediate intracellular maturation and release of IL-33, a pro-atopy, innate immunity, alarmin cytokine. Mature IL-33 maintained functional interaction with the cognate ST2 receptor and elicited potent pro-atopy inflammatory activity in vitro and in vivo. Inhibiting caspase 8 pharmacologically and deleting murine Il33 and Casp8 each attenuated allergic inflammation in vivo. Clinical data substantiated ripoptosome activation and IL-33 maturation as likely contributors to human allergic inflammation. Our findings reveal an epithelial barrier, allergen-sensing mechanism that converges on the ripoptosome as an intracellular molecular signaling platform, triggering type 2 innate immune responses. These findings have significant implications for understanding and treating human allergic diseases.

Adenovirus vector vaccination reprograms pulmonary fibroblastic niches to support protective inflating memory CD8+ T cells.

Pathogens and vaccines that produce persisting antigens can generate expanded pools of effector memory CD8+ T cells, described as memory inflation. While properties of inflating memory CD8+ T cells have been characterized, the specific cell types and tissue factors responsible for their maintenance remain elusive. Here, we show that clinically applied adenovirus vectors preferentially target fibroblastic stromal cells in cultured human tissues. Moreover, we used cell-type-specific antigen targeting to define critical cells and molecules that sustain long-term antigen presentation and T cell activity after adenovirus vector immunization in mice. While antigen targeting to myeloid cells was insufficient to activate antigen-specific CD8+ T cells, genetic activation of antigen expression in Ccl19-cre-expressing fibroblastic stromal cells induced inflating CD8+ T cells. Local ablation of vector-targeted cells revealed that lung fibroblasts support the protective function and metabolic fitness of inflating memory CD8+ T cells in an interleukin (IL)-33-dependent manner. Collectively, these data define a critical fibroblastic niche that underpins robust protective immunity operating in a clinically important vaccine platform.

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.

An intrinsic role of IL-33 in Treg cell-mediated tumor immunoevasion.

Regulatory T (Treg) cells accumulate into tumors, hindering the success of cancer immunotherapy. Yet, therapeutic targeting of Treg cells shows limited efficacy or leads to autoimmunity. The molecular mechanisms that guide Treg cell stability in tumors remain elusive. In the present study, we identify a cell-intrinsic role of the alarmin interleukin (IL)-33 in the functional stability of Treg cells. Specifically, IL-33-deficient Treg cells demonstrated attenuated suppressive properties in vivo and facilitated tumor regression in a suppression of tumorigenicity 2 receptor (ST2) (IL-33 receptor)-independent fashion. On activation, Il33-/- Treg cells exhibited epigenetic re-programming with increased chromatin accessibility of the Ifng locus, leading to elevated interferon (IFN)-γ production in a nuclear factor (NF)-κB-T-bet-dependent manner. IFN-γ was essential for Treg cell defective function because its ablation restored Il33-/- Treg cell-suppressive properties. Importantly, genetic ablation of Il33 potentiated the therapeutic effect of immunotherapy. Our findings reveal a new and therapeutically important intrinsic role of IL-33 in Treg cell stability in cancer.

Inhibition of the dipeptidyl peptidase DPP4 (CD26) reveals IL-33-dependent eosinophil-mediated control of tumor growth.

Post-translational modification of chemokines mediated by the dipeptidyl peptidase DPP4 (CD26) has been shown to negatively regulate lymphocyte trafficking, and its inhibition enhances T cell migration and tumor immunity by preserving functional chemokine CXCL10. By extending those initial findings to pre-clinical models of hepatocellular carcinoma and breast cancer, we discovered a distinct mechanism by which inhibition of DPP4 improves anti-tumor responses. Administration of the DPP4 inhibitor sitagliptin resulted in higher concentrations of the chemokine CCL11 and increased migration of eosinophils into solid tumors. Enhanced tumor control was preserved in mice lacking lymphocytes and was ablated after depletion of eosinophils or treatment with degranulation inhibitors. We further demonstrated that tumor-cell expression of the alarmin IL-33 was necessary and sufficient for eosinophil-mediated anti-tumor responses and that this mechanism contributed to the efficacy of checkpoint-inhibitor therapy. These findings provide insight into IL-33- and eosinophil-mediated tumor control, revealed when endogenous mechanisms of DPP4 immunoregulation are inhibited.

IL-1R3 blockade broadly attenuates the functions of six members of the IL-1 family, revealing their contribution to models of disease.

Interleukin (IL)-1R3 is the co-receptor in three signaling pathways that involve six cytokines of the IL-1 family (IL-1α, IL-1ß, IL-33, IL-36α, IL-36ß and IL-36γ). In many diseases, multiple cytokines contribute to disease pathogenesis. For example, in asthma, both IL-33 and IL-1 are of major importance, as are IL-36 and IL-1 in psoriasis. We developed a blocking monoclonal antibody (mAb) to human IL-1R3 (MAB-hR3) and demonstrate here that this antibody specifically inhibits signaling via IL-1, IL-33 and IL-36 in vitro. Also, in three distinct in vivo models of disease (crystal-induced peritonitis, allergic airway inflammation and psoriasis), we found that targeting IL-1R3 with a single mAb to mouse IL-1R3 (MAB-mR3) significantly attenuated heterogeneous cytokine-driven inflammation and disease severity. We conclude that in diseases driven by multiple cytokines, a single antagonistic agent such as a mAb to IL-1R3 is a therapeutic option with considerable translational benefit.

Interferon gamma constrains type 2 lymphocyte niche boundaries during mixed inflammation.

Allergic immunity is orchestrated by group 2 innate lymphoid cells (ILC2s) and type 2 helper T (Th2) cells prominently arrayed at epithelial- and microbial-rich barriers. However, ILC2s and Th2 cells are also present in fibroblast-rich niches within the adventitial layer of larger vessels and similar boundary structures in sterile deep tissues, and it remains unclear whether they undergo dynamic repositioning during immune perturbations. Here, we used thick-section quantitative imaging to show that allergic inflammation drives invasion of lung and liver non-adventitial parenchyma by ILC2s and Th2 cells. However, during concurrent type 1 and type 2 mixed inflammation, IFNγ from broadly distributed type 1 lymphocytes directly blocked both ILC2 parenchymal trafficking and subsequent cell survival. ILC2 and Th2 cell confinement to adventitia limited mortality by the type 1 pathogen Listeria monocytogenes. Our results suggest that the topography of tissue lymphocyte subsets is tightly regulated to promote appropriately timed and balanced immunity.

Environmental allergens induce allergic inflammation through proteolytic maturation of IL-33.

Allergic inflammation has crucial roles in allergic diseases such as asthma. It is therefore important to understand why and how the immune system responds to allergens. Here we found that full-length interleukin 33 (IL-33FL), an alarmin cytokine with critical roles in type 2 immunity and asthma, functioned as a protease sensor that detected proteolytic activities associated with various environmental allergens across four kingdoms, including fungi, house dust mites, bacteria and pollens. When exposed to allergen proteases, IL-33FL was rapidly cleaved in its central 'sensor' domain, which led to activation of the production of type 2 cytokines in group 2 innate lymphoid cells. Preventing cleavage of IL-33FL reduced allergic airway inflammation. Our findings reveal a molecular mechanism for the rapid induction of allergic type 2 inflammation following allergen exposure, with important implications for allergic diseases.

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.

Bacteria-Induced Group 2 Innate Lymphoid Cells in the Stomach Provide Immune Protection through Induction of IgA.

The intestinal microbiota shapes and directs immune development locally and systemically, but little is known about whether commensal microbes in the stomach can impact their immunological microenvironment. Here, we report that group 2 innate lymphoid cells (ILC2s) were the predominant ILC subset in the stomach and show that their homeostasis and effector functions were regulated by local commensal communities. Microbes elicited interleukin-7 (IL-7) and IL-33 production in the stomach, which in turn triggered the propagation and activation of ILC2. Stomach ILC2s were also rapidly induced following infection with Helicobacter pylori. ILC2-derived IL-5 resulted in the production of IgA, which coated stomach bacteria in both specific pathogen-free (SPF) and H. pylori-infected mice. Our study thus identifies ILC2-dependent IgA response that is regulated by the commensal microbiota, which is implicated in stomach protection by eliminating IgA-coated bacteria including pathogenic H. pylori.

Interleukin-33 Signaling Controls the Development of Iron-Recycling Macrophages.

Splenic red pulp macrophages (RPMs) contribute to erythrocyte homeostasis and are required for iron recycling. Heme induces the expression of SPIC transcription factor in monocyte-derived macrophages and promotes their differentiation into RPM precursors, pre-RPMs. However, the requirements for differentiation into mature RPMs remain unknown. Here, we have demonstrated that interleukin (IL)-33 associated with erythrocytes and co-cooperated with heme to promote the generation of mature RPMs through activation of the MyD88 adaptor protein and ERK1/2 kinases downstream of the IL-33 receptor, IL1RL1. IL-33- and IL1RL1-deficient mice showed defective iron recycling and increased splenic iron deposition. Gene expression and chromatin accessibility studies revealed a role for GATA transcription factors downstream of IL-33 signaling during the development of pre-RPMs that retained full potential to differentiate into RPMs. Thus, IL-33 instructs the development of RPMs as a response to physiological erythrocyte damage with important implications to iron recycling and iron 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.

Lipid-Droplet Formation Drives Pathogenic Group 2 Innate Lymphoid Cells in Airway Inflammation.

Innate lymphoid cells (ILCs) play an important role in the control and maintenance of barrier immunity. However, chronic activation of ILCs results in immune-mediated pathology. Here, we show that tissue-resident type 2 ILCs (ILC2s) display a distinct metabolic signature upon chronic activation. In the context of allergen-driven airway inflammation, ILC2s increase their uptake of both external lipids and glucose. Externally acquired fatty acids are transiently stored in lipid droplets and converted into phospholipids to promote the proliferation of ILC2s. This metabolic program is imprinted by interleukin-33 (IL-33) and regulated by the genes Pparg and Dgat1, which are both controlled by glucose availability and mTOR signaling. Restricting dietary glucose by feeding mice a ketogenic diet largely ablated ILC2-mediated airway inflammation by impairing fatty acid metabolism and the formation of lipid droplets. Together, these results reveal that pathogenic ILC2 responses require lipid metabolism and identify ketogenic diet as a potent intervention strategy to treat airway inflammation.

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.

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.

HpARI Protein Secreted by a Helminth Parasite Suppresses Interleukin-33.

Infection by helminth parasites is associated with amelioration of allergic reactivity, but mechanistic insights into this association are lacking. Products secreted by the mouse parasite Heligmosomoides polygyrus suppress type 2 (allergic) immune responses through interference in the interleukin-33 (IL-33) pathway. Here, we identified H. polygyrus Alarmin Release Inhibitor (HpARI), an IL-33-suppressive 26-kDa protein, containing three predicted complement control protein (CCP) modules. In vivo, recombinant HpARI abrogated IL-33, group 2 innate lymphoid cell (ILC2) and eosinophilic responses to Alternaria allergen administration, and diminished eosinophilic responses to Nippostrongylus brasiliensis, increasing parasite burden. HpARI bound directly to both mouse and human IL-33 (in the cytokine's activated state) and also to nuclear DNA via its N-terminal CCP module pair (CCP1/2), tethering active IL-33 within necrotic cells, preventing its release, and forestalling initiation of type 2 allergic responses. Thus, HpARI employs a novel molecular strategy to suppress type 2 immunity in both infection and allergy.

Epidermal Growth Factor Receptor Expression Licenses Type-2 Helper T Cells to Function in a T Cell Receptor-Independent Fashion.

Gastro-intestinal helminth infections trigger the release of interleukin-33 (IL-33), which induces type-2 helper T cells (Th2 cells) at the site of infection to produce IL-13, thereby contributing to host resistance in a T cell receptor (TCR)-independent manner. Here, we show that, as a prerequisite for IL-33-induced IL-13 secretion, Th2 cells required the expression of the epidermal growth factor receptor (EGFR) and of its ligand, amphiregulin, for the formation of a signaling complex between T1/ST2 (the IL-33R) and EGFR. This shared signaling complex allowed IL-33 to induce the EGFR-mediated activation of the MAP-kinase signaling pathway and consequently the expression of IL-13. Lack of EGFR expression on T cells abrogated IL-13 expression in infected tissues and impaired host resistance. EGFR expression on Th2 cells was TCR-signaling dependent, and therefore, our data reveal a mechanism by which antigen presentation controls the innate effector function of Th2 cells at the site of inflammation.

Single-Cell Analysis Uncovers Striking Cellular Heterogeneity of Lung-Infiltrating Regulatory T Cells during Eosinophilic versus Neutrophilic Allergic Airway Inflammation.

Allergic airway inflammation results from uncontrolled immune responses to environmental Ags. Although it is well established that allergic immune responses exhibit a high degree of diversity, driven by primary effector cell types such as eosinophils, neutrophils, or CD4 T cells with distinct effector signatures, the mechanisms responsible for such pathogenesis remain elusive. Foxp3+ regulatory T cells (Tregs) are essential immune regulators during chronic inflammation, including allergic airway inflammation. Emerging evidence suggests that Tregs infiltrating inflamed tissues exhibit distinct phenotypes dependent on the specific tissue sites and can display heterogeneity and tissue residency. Whether diverse allergic airway inflammatory responses influence infiltrating Treg heterogeneity or Treg lung residency has not been explored. We employed an unbiased single-cell RNA sequencing approach to investigate lung-infiltrating Tregs in models of eosinophilic and neutrophilic airway inflammation. We found that lung-infiltrating Tregs are highly heterogeneous, and that Tregs displaying lung-resident phenotypes are significantly different depending on the types of inflammation. Treg expression of ST2, a receptor for alarmin IL-33, was predominantly associated with eosinophilic inflammation and tissue residency. Nevertheless, Treg-specific ST2 deficiency did not affect the development of eosinophilic allergic inflammation or the generation of lung-resident Tregs. These results uncover a stark heterogeneity among Tregs infiltrating the lungs during allergic airway inflammation. The results indicate that varying types of inflammation may give rise to phenotypically distinct lung-resident Tregs, underscoring a (to our knowledge) novel mechanism by which inflammatory cues may shape the composition of infiltrating Tregs, allowing them to regulate inflammatory responses through tissue-adapted mechanisms.