TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation.

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.

Interleukin-33 (IL-33): A critical review of its biology and the mechanisms involved in its release as a potent extracellular cytokine.

Interleukin-33 (IL-33), a member of the IL-1 family, is an alarmin cytokine with crucial roles in tissue homeostasis and repair, type 2 immunity, allergic and non-allergic inflammation, viral infection, and cancer. IL-33 is abundant in the nuclei of tissue-derived cells, including endothelial cells from blood vessels, epithelial cells from barrier tissues, and fibroblastic stromal cells from various tissues. IL-33 is released upon cell damage or tissue injury and activates Myd88-dependent signaling pathways in cells expressing the ST2 (IL-1RL1) receptor. Analysis of patient samples and studies in murine models support an important role of IL-33/ST2 signaling in allergic inflammation in different tissues (lung, nasopharynx, skin) and diseases (asthma, chronic rhinosinusitis, allergic rhinitis, atopic dermatitis). IL33 and IL1RL1/ST2 are among the most highly replicated susceptibility loci for asthma. However, the IL-33/ST2 pathway is also important in non-allergic inflammation. Indeed, targets of IL-33 include immune cells involved in both type 2 and type 1 immunity and regulatory responses, such as group 2 innate lymphoid cells (ILC2s), mast cells, regulatory T cells (Tregs), Th2 cells, basophils, eosinophils, macrophages, dendritic cells (DCs), neutrophils, Th1 cells, CD8 T cells, NK and iNKT cells. In the main part of this review, we discuss the basic biology of the IL-33 protein (molecular characteristics, nuclear localization, cellular sources in vivo), and its mechanisms of release, and bioactive forms in various contexts. Importantly, we alert the scientific community to the problems of specificity of IL-33 reagents, we explain why studies without specificity controls with IL-33-deficient cells are misleading to the field and lead to unnecessary controversy, and we make recommendations to generate reliable results. In the final part, we review the genetic and environmental regulation of IL-33 in allergic airway inflammation and asthma, and we highlight recent studies showing clinical efficacy of anti-IL-33 antibodies in asthma and chronic obstructive pulmonary disease (COPD).

IL-33, an Alarmin of the IL-1 Family Involved in Allergic and Non Allergic Inflammation: Focus on the Mechanisms of Regulation of Its Activity.

Interleukin-33 (IL-33) is a member of the interleukin-1 (IL-1) family that is expressed in the nuclei of endothelial and epithelial cells of barrier tissues, among others. It functions as an alarm signal that is released upon tissue or cellular injury. IL-33 plays a central role in the initiation and amplification of type 2 innate immune responses and allergic inflammation by activating various target cells expressing its ST2 receptor, including mast cells and type 2 innate lymphoid cells. Depending on the tissue environment, IL-33 plays a wide variety of roles in parasitic and viral host defense, tissue repair and homeostasis. IL-33 has evolved a variety of sophisticated regulatory mechanisms to control its activity, including nuclear sequestration and proteolytic processing. It is involved in many diseases, including allergic, inflammatory and infectious diseases, and is a promising therapeutic target for the treatment of severe asthma. In this review, I will summarize the literature around this fascinating pleiotropic cytokine. In the first part, I will describe the basics of IL-33, from the discovery of interleukin-33 to its function, including its expression, release and signaling pathway. The second part will be devoted to the regulation of IL-33 protein leading to its activation or inactivation.

[Interleukin-33: from biology to potential treatments]. / L'interleukine 33 - De la biologie aux implications thérapeutiques.

Interleukin-33 is a member of the IL-1 cytokine family, expressed in the nucleus of endothelial cells and epithelial cells of barrier tissues. After cellular damage, IL-33 is released in the extracellular space and functions as an alarmin that alerts the immune system. IL-33 plays a critical role in type-2 innate immunity and allergic inflammation, by activating various target cells including mast cells and innate lymphoid cells that secrete high amounts of IL-5 and IL-13, two cytokines involved in allergic reactions. Recent studies suggest that IL-33 can also play other important roles, for example in homeostasis and during viral infection. It is implicated in numerous diseases, including allergic, inflammatory and infectious diseases and it constitutes a promising therapeutic target for treatment of severe asthma.

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 (IL-33): A nuclear cytokine from the IL-1 family.

Interleukin-33 (IL-33) is a tissue-derived nuclear cytokine from the IL-1 family abundantly expressed in endothelial cells, epithelial cells and fibroblast-like cells, both during homeostasis and inflammation. It functions as an alarm signal (alarmin) released upon cell injury or tissue damage to alert immune cells expressing the ST2 receptor (IL-1RL1). The major targets of IL-33 in vivo are tissue-resident immune cells such as mast cells, group 2 innate lymphoid cells (ILC2s) and regulatory T cells (Tregs). Other cellular targets include T helper 2 (Th2) cells, eosinophils, basophils, dendritic cells, Th1 cells, CD8+ T cells, NK cells, iNKT cells, B cells, neutrophils and macrophages. IL-33 is thus emerging as a crucial immune modulator with pleiotropic activities in type-2, type-1 and regulatory immune responses, and important roles in allergic, fibrotic, infectious, and chronic inflammatory diseases. The critical function of IL-33/ST2 signaling in allergic inflammation is illustrated by the fact that IL33 and IL1RL1 are among the most highly replicated susceptibility loci for asthma. In this review, we highlight 15 years of discoveries on IL-33 protein, including its molecular characteristics, nuclear localization, bioactive forms, cellular sources, mechanisms of release and regulation by proteases. Importantly, we emphasize data that have been validated using IL-33-deficient cells.

Isolation and Culture of Mouse Lung ILC2s.

Group 2 Innate Lymphoid Cells (ILC2) play an important role in immune responses at barrier surfaces, notably in the lung during airway allergic inflammation or asthma. Several studies have described methods to isolate ILC2s from wild-type naive mice, most of them using cell sorting to obtain a pure population. Here, we describe in detail, a simple, efficient method for isolation and culture of lung mouse ILC2s. Lungs from Rag2-/- mice pretreated with IL-33 are collected and processed into single cell suspensions. Lymphoid cells are then recovered by density gradient separation. Lin-CD45+ cells are selected by depletion of lineage positive cells followed by positive selection of CD45+ cells. Culture of the isolated cells for several days results in a highly purified ILC2 population expressing typical cell surface markers (CD90.2, Sca1, CD25, CD127, and IL-33R). These cells can be expanded in culture for up to 10 days and used for diverse ex vivo assays or in vivo adoptive transfer experiments.

IL-33-expanded human Vγ9Vδ2 T cells have anti-lymphoma effect in a mouse tumor model.

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.

Extracellular IL-33 cytokine, but not endogenous nuclear IL-33, regulates protein expression in endothelial cells.

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.

TCRVγ9 γδ T Cell Response to IL-33: A CD4 T Cell-Dependent Mechanism.

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.

Central domain of IL-33 is cleaved by mast cell proteases for potent activation of group-2 innate lymphoid cells.

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.

IL-33: an alarmin cytokine with crucial roles in innate immunity, inflammation and allergy.

IL-33 is a nuclear cytokine from the IL-1 family constitutively expressed in epithelial barrier tissues and lymphoid organs, which plays important roles in type-2 innate immunity and human asthma. Recent studies indicate that IL-33 induces production of large amounts of IL-5 and IL-13 by group 2 innate lymphoid cells (ILC2s), for initiation of allergic inflammation shortly after exposure to allergens or infection with parasites or viruses. IL-33 appears to function as an alarmin (alarm signal) rapidly released from producing cells upon cellular damage or cellular stress. In this review, we discuss the cellular sources, mode of action and regulation of IL-33, and we highlight its crucial roles in vivo with particular emphasis on results obtained using IL33-deficient mice.

Label-free quantification and shotgun analysis of complex proteomes by one-dimensional SDS-PAGE/NanoLC-MS: evaluation for the large scale analysis of inflammatory human endothelial cells.

To perform differential studies of complex protein mixtures, strategies for reproducible and accurate quantification are needed. Here, we evaluated a quantitative proteomic workflow based on nanoLC-MS/MS analysis on an LTQ-Orbitrap-VELOS mass spectrometer and label-free quantification using the MFPaQ software. In such label-free quantitative studies, a compromise has to be found between two requirements: repeatability of sample processing and MS measurements, allowing an accurate quantification, and high proteomic coverage of the sample, allowing quantification of minor species. The latter is generally achieved through sample fractionation, which may induce experimental bias during the label-free comparison of samples processed, and analyzed independently. In this work, we wanted to evaluate the performances of MS intensity-based label-free quantification when a complex protein sample is fractionated by one-dimensional SDS-PAGE. We first tested the efficiency of the analysis without protein fractionation and could achieve quite good quantitative repeatability in single-run analysis (median coefficient of variation of 5%, 99% proteins with coefficient of variation <48%). We show that sample fractionation by one-dimensional SDS-PAGE is associated with a moderate decrease of quantitative measurement repeatability while largely improving the depth of proteomic coverage. We then applied the method for a large scale proteomic study of the human endothelial cell response to inflammatory cytokines, such as TNFα, interferon γ, and IL1ß, which allowed us to finely decipher at the proteomic level the biological pathways involved in endothelial cell response to proinflammatory cytokines.

IL-33 is processed into mature bioactive forms by neutrophil elastase and cathepsin G.

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-1ß 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.

Mechanisms of IL-33 processing and secretion: differences and similarities between IL-1 family members.

Interleukin-33 (IL-33) is the latest member of the IL-1 family that has become very attractive since the discovery of its major target cells, the innate lymphoid cells type 2 (ILC2), involved in the initiation of the type 2 immune response (secretion of IL-5 and IL-13) during parasitic infection and allergic diseases such as asthma. IL-33 is a chromatin-associated protein as it possesses in its N-terminus, a chromatin-binding domain, and is constitutively expressed in the nuclei of endothelial cells and in epithelial cells of tissues exposed to the environment. It is however, essential for IL-33 to be extracellularly released to bind to its receptor ST2 through the C-terminus portion of the protein in order to induce the inflammatory and type 2 responses. Like other IL-1 family members, IL-33 does not possess any signal peptide and may be released through unconventional secretory mechanisms or following cell damage and necrosis. It was initially believed that IL-33, like IL-1ß and IL-18, requires processing by caspase-1 to be released, and for biological activity. On the contrary, full length IL-33 is biologically active, and processing by caspases results rather in IL-33 inactivation. Moreover, it has been recently shown that the bioactivity of IL-33 can be increased by inflammatory proteases secreted in the microenvironment, similarly to IL-1α, IL-1ß and IL-18. This review will summarize recent progress on how IL-33 is released and processed compared with the other IL-1 family members, and how the immune cells recruited to the site of injury can regulate the disease-associated function of IL-33.

Direct interaction between causative genes of DYT1 and DYT6 primary dystonia.

Primary dystonia is a movement disorder characterized by sustained muscle contractions and in which dystonia is the only or predominant clinical feature. TOR1A(DYT1) and the transcription factor THAP1(DYT6) are the only genes identified thus far for primary dystonia. Using electromobility shift assays and chromatin immunoprecipitation (ChIP) quantitative polymerase chain reaction (qPCR), we demonstrate a physical interaction between THAP1 and the TOR1A promoter that is abolished by pathophysiologic mutations. Our findings provide the first evidence that causative genes for primary dystonia intersect in a common pathway and raise the possibility of developing novel therapies targeting this pathway.

The THAP-zinc finger protein THAP1 associates with coactivator HCF-1 and O-GlcNAc transferase: a link between DYT6 and DYT3 dystonias.

THAP1 is a sequence-specific DNA binding factor that regulates cell proliferation through modulation of target genes such as the cell cycle-specific gene RRM1. Mutations in the THAP1 DNA binding domain, an atypical zinc finger (THAP-zf), have recently been found to cause DYT6 dystonia, a neurological disease characterized by twisting movements and abnormal postures. In this study, we report that THAP1 shares sequence characteristics, in vivo expression patterns and protein partners with THAP3, another THAP-zf protein. Proteomic analyses identified HCF-1, a potent transcriptional coactivator and cell cycle regulator, and O-GlcNAc transferase (OGT), the enzyme that catalyzes the addition of O-GlcNAc, as major cellular partners of THAP3. THAP3 interacts with HCF-1 through a consensus HCF-1-binding motif (HBM), a motif that is also present in THAP1. Accordingly, THAP1 was found to bind HCF-1 in vitro and to associate with HCF-1 and OGT in vivo. THAP1 and THAP3 belong to a large family of HCF-1 binding factors since seven other members of the human THAP-zf protein family were identified, which harbor evolutionary conserved HBMs and bind to HCF-1. Chromatin immunoprecipitation (ChIP) assays and RNA interference experiments showed that endogenous THAP1 mediates the recruitment of HCF-1 to the RRM1 promoter during endothelial cell proliferation and that HCF-1 is essential for transcriptional activation of RRM1. Together, our findings suggest HCF-1 is an important cofactor for THAP1. Interestingly, our results also provide an unexpected link between DYT6 and DYT3 (X-linked dystonia-parkinsonism) dystonias because the gene encoding the THAP1/DYT6 protein partner OGT maps within the DYT3 critical region on Xq13.1.

The IL-1-like cytokine IL-33 is inactivated after maturation by caspase-1.

IL-33 is a chromatin-associated cytokine of the IL-1 family that has recently been linked to many diseases, including asthma, rheumatoid arthritis, atherosclerosis, and cardiovascular diseases. IL-33 signals through the IL-1 receptor-related protein ST2 and drives production of pro-inflammatory and T helper type 2-associated cytokines in mast cells, T helper type 2 lymphocytes, basophils, eosinophils, invariant natural killer T cells, and natural killer cells. It is currently believed that IL-33, like IL-1beta and IL-18, requires processing by caspase-1 to a mature form (IL-33(112-270)) for biological activity. Contrary to the current belief, we report here that full-length IL-33(1-270) is active and that processing by caspase-1 results in IL-33 inactivation, rather than activation. We show that full-length IL-33(1-270) binds and activates ST2, similarly to IL-33(112-270), and that cleavage by caspase-1 does not occur at the site initially proposed (Ser(111)), but rather after residue Asp(178) between the fourth and fifth predicted beta-strands of the IL-1-like domain. Surprisingly, the caspase-1 cleavage site (DGVD(178)G) is similar to the consensus site of cleavage by caspase-3, and IL-33 is also a substrate for this apoptotic caspase. Interestingly, we found that full-length IL-33, which is constitutively expressed to high levels by endothelial cells in most normal human tissues, can be released in the extracellular space after endothelial cell damage or mechanical injury. We speculate that IL-33 may function, similarly to the prototypical alarmins HMGB1 and IL-1alpha, as an endogenous danger signal to alert cells of the innate immune system of tissue damage during trauma or infection.

Molecular mimicry between IL-33 and KSHV for attachment to chromatin through the H2A-H2B acidic pocket.

Interleukin-33 (IL-33) is an IL-1-like ligand for the ST2 receptor that stimulates the production of Th2-associated cytokines. Recently, we showed that IL-33 is a chromatin-associated factor in the nucleus of endothelial cells in vivo. Here, we report the identification of a short IL-33 chromatin-binding peptide that shares striking similarities with a motif found in Kaposi sarcoma herpesvirus LANA (latency-associated nuclear antigen), which is responsible for the attachment of viral genomes to mitotic chromosomes. Similar to LANA, the IL-33 peptide docks into the acidic pocket formed by the H2A-H2B dimer at the nucleosomal surface and regulates chromatin compaction by promoting nucleosome-nucleosome interactions. Taken together, our data provide important new insights into the nuclear roles of IL-33, and show a unique example of molecular mimicry of a chromatin-associated cytokine by a DNA tumour virus. In addition, the data provide, to the best of our knowledge, the first demonstration of the existence of non-histone cellular factors that bind to the acidic pocket of the nucleosome.