Epithelium-derived cytokines or alarmins, such as interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP), are major players in type 2 immunity and asthma. Here, we demonstrate that TNF-like ligand 1A (TL1A) is an epithelial alarmin, constitutively expressed in alveolar epithelium at steady state in both mice and humans, which cooperates with IL-33 for early induction of IL-9high ILC2s during the initiation of allergic airway inflammation. Upon synergistic activation by IL-33 and TL1A, lung ILC2s acquire a transient IL-9highGATA3low "ILC9" phenotype and produce prodigious amounts of IL-9. A combination of large-scale proteomic analyses, lung intravital microscopy, and adoptive transfer of ILC9 cells revealed that high IL-9 expression distinguishes a multicytokine-producing state-of-activated ILC2s with an increased capacity to initiate IL-5-dependent allergic airway inflammation. Similar to IL-33 and TSLP, TL1A is expressed in airway basal cells in healthy and asthmatic human lungs. Together, these results indicate that TL1A is an epithelium-derived cytokine and an important cofactor of IL-33 in the airways.

BACKGROUND:Allergic asthma is more severe and frequent in women than in men. In male mice, androgens negatively control group 2 innate lymphoid cell (ILC2) development and function by yet unknown mechanisms. OBJECTIVES:We sought to investigate the impact of androgen on ILC2 homeostasis and IL-33-mediated inflammation in female lungs. We evaluated the role of androgen receptor (AR) signaling and the contribution of the putative inhibitory receptor killer cell lectin-like receptor G1 (KLRG1). METHODS:Subcutaneous pellets mimicking physiological levels of androgen were used to treat female mice together with mice expressing a reporter enzyme under the control of androgen response elements and mixed bone marrow chimeras to assess the cell-intrinsic role of AR activation within ILC2s. We generated KLRG1-deficient mice. RESULTS:We established that lung ILC2s express a functionally active AR that can be in vivo targeted with exogenous androgens to negatively control ILC2 homeostasis, proliferation, and function. Androgen signaling upregulated KLRG1 on ILC2s, which inhibited their proliferation on E-cadherin interaction. Despite evidence that KLRG1 impaired the competitive fitness of lung ILC2s during inflammation, KLRG1 deficiency neither alters in vivo ILC2 numbers and functions, nor did it lead to hyperactive ILC2s in either sexes. CONCLUSIONS:AR agonists can be used in vivo to inhibit ILC2 homeostatic numbers and ILC2-dependent lung inflammation through cell-intrinsic AR activation. Although androgen signals in ILC2s to upregulate KLRG1, we demonstrate that KLRG1 is dispensable for androgen-mediated inhibition of pulmonary ILC2s.

IL-9 is key driver of chronic and allergic inflammation at mucosal surfaces, with important roles in the activation and survival of mast cells and type 2 innate lymphoid cells (ILC2s). IL-9-reporter mice have established T cells (Th9) and ILC2s as major sources of IL-9 in vivo. The discovery that TGF-β and IL-4 cooperate for induction of Th9 cells has been an important breakthrough. However, the signals that induce IL-9high ILC2s remain incompletely characterized. Here, we show that IL-33, a critical activator of ILC2s, with important roles in type-2 innate immunity and allergic inflammation (Cayrol, Duval et al., Nature Immunology 2018) synergizes with TNF-family cytokine TL1A to induce prodigious amounts of IL-9 secretion by ILC2s. Unbiased global proteomic analyses revealed that IL-9 was the most-induced protein in ILC2s treated with IL-33 and TL1A. More than 99% of ILC2s expressed IL-9 intracellularly after co-stimulation with IL-33 and TL1A. ILC2s are known to produce very large amounts of IL-5 and IL-13, but ILC2s producing such high levels of IL-9 have not been described previously. Interestingly, IL-9 production by ILC2s was transient and associated with a phenotypic shift characterized by upregulation of IRF4, pSTAT5 and downregulation of ICOS, KLRG1 as well as master transcription factor GATA-3 and its target genes (IL-9R, ST2). Lastly, we detected an expansion of IL-9high ILC2s in the lungs of mice after IL-33/TL1A inhalation and adoptive transfer experiments revealed that IL-9high ILC2s are potent inducers of allergic airway inflammation in vivo. Thus, our study supports the rising plasticity of ILC2s and their critical role in the induction of type-2 inflammatory responses in the lungs.

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.

From several years, the anticancer effects of Vγ9 T lymphocytes make these cells good candidates for cancer immunotherapies. However, the proved efficacy of γδ Τ cell‐based cancer immunotherapies in some clinical trials was minimized due to the inherent toxicity of IL‐2, which is essential for the combination therapy with Phosphoantigen (PAg). Recently, we showed that IL‐33, a γ chain receptor‐independent cytokine, was able to induce the in vitro proliferation of PAg‐activated Vγ9 T cells, which were fully functional expressing IFN‐γ and TNF‐α and showing in vitro anti‐tumor cytotoxicity. We proposed IL‐33 as an alternative to IL‐2 for Vγ9 T cell‐based cancer immunotherapies, and have therefore evaluated the efficacy of this cytokine in preclinical investigations. This study shows that human Vγ9 T cells are able to proliferate in a mouse model with the combination of PAg and rhIL‐33, and that IL‐33‐expanded Vγ9 T cells can prevent tumor growth in a mouse lymphoma model.

IL-33 is a nuclear cytokine from the IL-1 family that plays important roles in health and disease. Extracellular IL-33 activates a growing number of target cells, including group 2 innate lymphoid cells, mast cells and regulatory T cells, but it remains unclear whether intracellular nuclear IL-33 has additional functions in the nucleus. Here, we used a global proteomic approach based on high-resolution mass spectrometry to compare the extracellular and intracellular roles of IL-33 in primary human endothelial cells, a major source of IL-33 protein in human tissues. We found that exogenous extracellular IL-33 cytokine induced expression of a distinct set of proteins associated with inflammatory responses in endothelial cells. In contrast, knockdown of endogenous nuclear IL-33 expression using two independent RNA silencing strategies had no reproducible effect on the endothelial cell proteome. These results suggest that IL-33 acts as a cytokine but not as a nuclear factor regulating gene expression in endothelial cells.

The availability of specific stimuli to induce the anticancer cytotoxicity of human TCRV gamma 9-expressing T lymphocytes has allowed the development of gamma delta T cell based cancer immunotherapies. However, the stringent dependence of such strategies on the inherently toxic IL-2 has raised safety concerns for patients, justifying a search for alternative methods for inducing gamma delta T cell stimulation. IL-33 is a gamma-chain receptor-independent cytokine of the IL-1 superfamily that is expressed by endothelial cells from a tumor microenvironment and can sustain Th1 and Th2 immune responses. Therefore, we investigated its ability to support the stimulation of human TCRV gamma 9(+) gamma delta T cells. In this study, we report that IL-33 efficiently sustained the in vitro activation of V gamma 9 T lymphocytes by synthetic phosphoantigens, zoledronate, and a BTN3A1 Ab in the absence of an exogenous supply of IL-2. IL-33 was as potent as IL-2 in allowing the proliferative amplification of V gamma 9 T cells isolated from PBMC following activation by the synthetic phosphoantigen bromohydrin pyrophosphate. IL-33 also induced an identical maturation into TNF-alpha- and IFN-gamma-producing Thl effector memory cells, and IL-33 stimulated cells showed an equivalent cytotoxicity for various tumor cells in vitro. Finally, we found that the bioactivity of IL-33 on the V gamma 9 T cell was indirectly mediated through contact with CD4 T cells and IL-2 production by CD4 T cells and V gamma 9 T cells themselves. These data posit IL-33 as an alternative to IL-2 for V gamma 9 T cell based cancer immunotherapies.

IL-33 is strongly involved in several inflammatory and autoimmune disorders with both pro- and anti-inflammatory properties. However, its contribution to chronic autoimmune inflammation, such as rheumatoid arthritis, is ill defined and probably requires tight regulation. In this study, we aimed at deciphering the complex role of IL-33 in a model of rheumatoid arthritis, namely, collagen-induced arthritis (CIA). We report that repeated injections of IL-33 during induction (early) and during development (late) of CIA strongly suppressed clinical and histological signs of arthritis. In contrast, a late IL-33 injection had no effect. The cellular mechanism involved in protection was related to an enhanced type 2 immune response, including the expansion of eosinophils, Th2 cells, and type 2 innate lymphoid cells, associated with an increase in type 2 cytokine levels in the serum of IL-33-treated mice. Moreover, our work strongly highlights the interplay between IL-33 and regulatory T cells (Tregs), demonstrated by the dramatic in vivo increase in Treg frequencies after IL-33 treatment of CIA. More importantly, Tregs from IL-33-treated mice displayed enhanced capacities to suppress IFN-γ production by effector T cells, suggesting that IL-33 not only favors Treg proliferation but also enhances their immunosuppressive properties. In concordance with these observations, we found that IL-33 induced the emergence of a CD39(high) Treg population in a ST2L-dependent manner. Our findings reveal a powerful anti-inflammatory mechanism by which IL-33 administration inhibits arthritis development.

The availability of specific stimuli to induce the anticancer cytotoxicity of human TCRVγ9-expressing T lymphocytes has allowed the development of γδ T cell–based cancer immunotherapies. However, the stringent dependence of such strategies on the inherently toxic IL-2 has raised safety concerns for patients, justifying a search for alternative methods for inducing γδ T cell stimulation. IL-33 is a γ-chain receptor-independent cytokine of the IL-1 superfamily that is expressed by endothelial cells from a tumor microenvironment and can sustain Th1 and Th2 immune responses. Therefore, we investigated its ability to support the stimulation of human TCRVγ9+ γδ T cells. In this study, we report that IL-33 efficiently sustained the in vitro activation of Vγ9 T lymphocytes by synthetic phosphoantigens, zoledronate, and a BTN3A1 Ab in the absence of an exogenous supply of IL-2. IL-33 was as potent as IL-2 in allowing the proliferative amplification of Vγ9 T cells isolated from PBMC following activation by the synthetic phosphoantigen bromohydrin pyrophosphate. IL-33 also induced an identical maturation into TNF-α– and IFN-γ–producing Th1 effector memory cells, and IL-33–stimulated cells showed an equivalent cytotoxicity for various tumor cells in vitro. Finally, we found that the bioactivity of IL-33 on the Vγ9 T cell was indirectly mediated through contact with CD4 T cells and IL-2 production by CD4 T cells and Vγ9 T cells themselves. These data posit IL-33 as an alternative to IL-2 for Vγ9 T cell–based cancer immunotherapies.

Interleukin-33 (IL-33) is an alarmin cytokine from the IL-1 family. IL-33 activates many immune cell types expressing the interleukin 1 receptor-like 1 (IL1RL1) receptor ST2, including group-2 innate lymphoid cells (ILC2s, natural helper cells, nuocytes), the major producers of IL-5 and IL-13 during type-2 innate immune responses and allergic airway inflammation. IL-33 is likely to play a critical role in asthma because the IL33 and ST2/IL1RL1 genes have been reproducibly identified as major susceptibility loci in large-scale genome-wide association studies. A better understanding of the mechanisms regulating IL-33 activity is thus urgently needed. Here, we investigated the role of mast cells, critical effector cells in allergic disorders, known to interact with ILC2s in vivo. We found that serine proteases secreted by activated mast cells (chymase and tryptase) generate mature forms of IL-33 with potent activity on ILC2s. The major forms produced by mast cell proteases, IL-33(95-270), IL-33(107-270), and IL-33(109-270), were 30-fold more potent than full-length human IL-33(1-270) for activation of ILC2s ex vivo. They induced a strong expansion of ILC2s and eosinophils in vivo, associated with elevated concentrations of IL-5 and IL-13. Murine IL-33 is also cleaved by mast cell tryptase, and a tryptase inhibitor reduced IL-33-dependent allergic airway inflammation in vivo. Our study identifies the central cleavage/activation domain of IL-33 (amino acids 66-111) as an important functional domain of the protein and suggests that interference with IL-33 cleavage and activation by mast cell and other inflammatory proteases could be useful to reduce IL-33-mediated responses in allergic asthma and other inflammatory diseases.

Abstract IL-33 is a chromatin-associated nuclear cytokine from the IL-1 family, which has been linked to many inflammatory diseases. IL-33 signals through the receptor ST2 and drives production of cytokines in innate effector cells and T helper type 2 lymphocytes. IL-33 is constitutively expressed in the nuclei of endothelial cells and in epithelial cells of tissues exposed to the environment. It was initially believed that IL-33, like IL-1β and IL-18, requires processing by caspase-1 for biological activity. On the contrary, we reported that full length IL-33 is biologically active, and that processing by apoptotic caspases results in IL-33 inactivation, rather than activation. We also found that full length IL-33 is released upon cellular damage and we proposed that IL-33 may function, similarly to HMGB1, as an endogenous danger signal. Because IL-33 plays important roles in inflammatory diseases, we hypothesized that IL-33 could be cleaved by proteases released during inflammation. We analyzed the effects of inflammatory proteases from innate effector cells, on IL-33 processing and biological activity. We found that IL-33 can be cleaved and activated by several inflammatory proteases. Interestingly, all forms generated by these proteases are “super-active” IL-33 forms that contain an intact IL1-like cytokine domain. We propose that the inflammatory microenvironment exacerbates disease-associated functions of IL-33 through the generation of “super-active” IL-33 forms.

Interleukin-33 (IL-33) (NF-HEV) is a chromatin-associated nuclear cytokine from the IL-1 family, which has been linked to important diseases, including asthma, rheumatoid arthritis, ulcerative colitis, and cardiovascular diseases. IL-33 signals through the ST2 receptor and drives cytokine production in type 2 innate lymphoid cells (ILCs) (natural helper cells, nuocytes), T-helper (Th)2 lymphocytes, mast cells, basophils, eosinophils, invariant natural killer T (iNKT), and natural killer (NK) cells. We and others recently reported that, unlike IL-1β and IL-18, full-length IL-33 is biologically active independently of caspase-1 cleavage and that processing by caspases results in IL-33 inactivation. We suggested that IL-33, which is released upon cellular damage, may function as an endogenous danger signal or alarmin, similar to IL-1α or high-mobility group box 1 protein (HMGB1). Here, we investigated the possibility that IL-33 activity may be regulated by proteases released during inflammation. Using a combination of in vitro and in vivo approaches, we demonstrate that neutrophil serine proteases cathepsin G and elastase can cleave full-length human IL-33(1-270) and generate mature forms IL-33(95-270), IL-33(99-270), and IL-33(109-270). These forms are produced by activated human neutrophils ex vivo, are biologically active in vivo, and have a ~10-fold higher activity than full-length IL-33 in cellular assays. Murine IL-33 is also cleaved by neutrophil cathepsin G and elastase, and both full-length and cleaved endogenous IL-33 could be detected in the bronchoalveolar lavage fluid in an in vivo model of acute lung injury associated with neutrophil infiltration. We propose that the inflammatory microenvironment may exacerbate disease-associated functions of IL-33 through the generation of highly active mature forms.

IL-33 has recently been identified as a cytokine endowed with pro-Th2 functions, raising the question of its effect on invariant natural killer T cell (iNKT), which are potent IL-4 producers. Here, we report a two-fold increase of iNKT-cell counts in spleen and liver after a 7-day treatment of mice with IL-33, which results from a direct effect, given that purified iNKT cells express the T1/ST2 receptor constitutively and respond to IL-33 by in vitro expansion and functional activation. Conversely to the expected pro-Th2 effect, IL-33 induced a preferential increase in IFN-gamma rather than IL-4 production upon TCR engagement that depended on endogenous IL-12. Moreover, in combination with the pro-inflammatory cytokine IL-12, IL-33 enhanced IFN-gamma production by both iNKT and NK cells. Taken together these data support the conclusion that IL-33 can contribute as a co-stimulatory factor to innate cellular immune responses.