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Neuromyelitis optica spectrum disorder (NMOSD) is a severe central nervous system (CNS) autoimmune disease that primarily damages the optic nerves and spinal cord. Group 2 innate lymphoid cells (ILC2) are potent producers of type 2 cytokines that orchestrate immune and inflammatory responses. However, the role of ILC2 in CNS autoimmune diseases remains unknown. In patients with NMOSD, we identified a significant reduction of ILC2 in peripheral blood, which was correlated with disease severity. Using a mouse model of NMOSD induced by intracerebral injection of NMOSD-IgG with complement, we found CNS infiltration of ILC2 mainly expressing interleukin (IL)-5 and IL-13. The depletion of ILC2 led to increased CNS lesion volume, reduced CNS glucose metabolism, and augmented astrocyte injury and demyelination. The exacerbated NMOSD pathology was accompanied by increased accumulation of Iba1 + cells and complement activity in CNS lesions. In addition, the expansion of ILC2 using IL-33 attenuated NMO pathology. Collectively, these findings suggest a beneficial role of ILC2 in NMOSD, which deserves further investigation for future design of immune therapies to treat patients with NMOSD., (© 2021 Federation of American Societies for Experimental Biology.)

Despite interleukin 33 (IL-33) functions as an "alarmin" released from hepatic dead cells in response to tissue damages, the interrelationship between IL-33-mediated hepatocyte autophagy and innate immune response in the acetaminophen (APAP)-induced liver injury (AILI) process remains obscure. This study aimed to explore the regulation of IL-33 on hepatocyte autophagy and macrophage polarization after APAP challenge in vivo and vitro. We found IL-33 released from hepatic necrosis was elevated in the AILI mouse model. Blockage of IL-33 exacerbated liver injury by consuming liver-resident macrophages cells (Kupffer cells, KCs) and promoting hepatic inflammatory factors secretion, such as TNF-α, IL-6 and IL-1β in the early phase of liver injury. Interestingly, IL-33 deficiency further activated hepatocyte autophagy and disrupted M2 macrophage polarization post-APAP challenge in vivo and vitro, which can be reversed by recombinant IL-33 treatment. Mechanistically, administration of IL-33 can directly enhance M2 polarization via PI3K/Akt signaling pathway and activate protective hepatocyte autophagy via AMPKα/mTOR signaling pathway in the AILI process. In conclusion, our data firstly demonstrates that IL-33 exerts protective effects on hepatocytes through the activation of autophagy and functions as an innate immunity regulator mediating macrophage polarization in the early phase of AILI., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Environmental allergen sources such as house dust mites contain proteases, which are frequently allergens themselves. Inhalation with the exogenous proteases, such as a model of protease allergen, papain, to airways evokes release and activation of IL-33, which promotes innate and adaptive allergic airway inflammation and Th2 sensitization in mice. Here, we examine whether epicutaneous (e.c.) vaccination with antigens with and without protease activity shows prophylactic effect on the Th airway sensitization and Th2-medated airway inflammation, which are driven by exogenous or endogenous IL-33. E.c. vaccination with ovalbumin restrained ovalbumin-specific Th2 airway sensitization and/or airway inflammation on subsequent inhalation with ovalbumin plus papain or ovalbumin plus recombinant IL-33. E.c. vaccination with papain or protease inhibitor-treated papain restrained papain-specific Th2 and Th9 airway sensitization, eosinophilia, and infiltration of IL-33-responsive Th2 and group 2 innate lymphoid cells on subsequent inhalation with papain. However, e.c. vaccination with papain but not protease inhibitor-treated papain induced Th17 response in bronchial draining lymph node cells. In conclusions, we demonstrated that e.c. allergen vaccination via intact skin in mice restrained even protease allergen-activated IL-33-driven airway Th2 sensitization to attenuate allergic airway inflammation and that e.c. vaccination with protease allergen attenuated the airway inflammation similar to its derivative lacking the protease activity, although the former but not the latter promoted Th17 development. In addition, the present study suggests that modified allergens, of which Th17-inducing e.c. adjuvant activity such as the protease activity was eliminated, might be preferable for safer clinical applications of the e.c. allergen administration., Competing Interests: Declaration of competing interest The authors state no conflicts of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

IL-33 is constitutively expressed in the skin. Psoriasis is a common skin inflammatory disease. The roles of IL-33 in psoriasis have not been well-elucidated. We identified that keratinocytes (KCs) are the predominant cells expressing IL-33 and its receptor, suppression of tumorigenicity 2, in the skin. KCs actively released IL-33 on psoriasis inflammatory stimuli and induced psoriasis-related cytokine, chemokine, and inflammatory molecules genes transcription in KCs in an autocrine manner. IL-33‒specific deficiency in KCs ameliorated imiquimod-induced psoriatic dermatitis. In addition, intradermal injection of recombinant IL-33 alone induced psoriasis-like dermatitis, which is attributed to the transcriptional upregulation of genes enriched in IL-17, TNF, and chemokine signaling pathway in KCs on recombinant IL-33 stimulation. Our data demonstrate that the autocrine circuit of IL-33 in KCs promotes the progression of psoriatic skin inflammation, and IL-33 is a potential therapeutic target for psoriasis., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

2'3'-cGAMP is known as a nonclassical second messenger and small immune modulator that possesses potent antitumor and antiviral activities via inducing the stimulator of IFN genes-mediated (STING-mediated) signaling pathway. However, its function in regulating type 2 immune responses remains unknown. Therefore, we sought to determine a role of STING activation by 2'3'-cGAMP in type 2 inflammatory reactions in multiple mouse models of eosinophilic asthma. We discovered that 2'3'-cGAMP administration strongly attenuated type 2 lung immunopathology and airway hyperreactivity induced by IL-33 and a fungal allergen, Aspergillus flavus. Mechanistically, upon the respiratory delivery, 2'3'-cGAMP was mainly internalized by alveolar macrophages, in which it activated the STING/IFN regulatory factor 3/type I IFN signaling axis to induce the production of inhibitory factors containing IFN-α, which blocked the IL-33-mediated activation of group 2 innate lymphoid (ILC2) cells in vivo. We further demonstrated that 2'3'-cGAMP directly suppressed the proliferation and function of both human and mouse ILC2 cells in vitro. Taken together, our findings suggest that STING activation by 2'3'-cGAMP in alveolar macrophages and ILC2 cells can negatively regulate type 2 immune responses, implying that the respiratory delivery of 2'3'-cGAMP might be further developed as an alternative strategy for treating type 2 immunopathologic diseases such as eosinophilic asthma.

Hippocampal synaptic plasticity is important for learning and memory formation. Homeostatic synaptic plasticity is a specific form of synaptic plasticity that is induced upon prolonged changes in neuronal activity to maintain network homeostasis. While astrocytes are important regulators of synaptic transmission and plasticity, it is largely unclear how they interact with neurons to regulate synaptic plasticity at the circuit level. Here, we show that neuronal activity blockade selectively increases the expression and secretion of IL-33 (interleukin-33) by astrocytes in the hippocampal cornu ammonis 1 (CA1) subregion. This IL-33 stimulates an increase in excitatory synapses and neurotransmission through the activation of neuronal IL-33 receptor complex and synaptic recruitment of the scaffold protein PSD-95. We found that acute administration of tetrodotoxin in hippocampal slices or inhibition of hippocampal CA1 excitatory neurons by optogenetic manipulation increases IL-33 expression in CA1 astrocytes. Furthermore, IL-33 administration in vivo promotes the formation of functional excitatory synapses in hippocampal CA1 neurons, whereas conditional knockout of IL-33 in CA1 astrocytes decreases the number of excitatory synapses therein. Importantly, blockade of IL-33 and its receptor signaling in vivo by intracerebroventricular administration of its decoy receptor inhibits homeostatic synaptic plasticity in CA1 pyramidal neurons and impairs spatial memory formation in mice. These results collectively reveal an important role of astrocytic IL-33 in mediating the negative-feedback signaling mechanism in homeostatic synaptic plasticity, providing insights into how astrocytes maintain hippocampal network homeostasis., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

IL-33 induces airway inflammation and hyper-responsiveness in respiratory diseases. Although defined as a therapeutic target, there are limited studies that have comprehensively investigated IL-33-mediated responses in the lungs in vivo. In this study, we characterized immunological and physiological responses induced by intranasal IL-33 challenge, in a mouse model. We identified specific cytokines, IL-4, IL-5, IL-6, IL-10, IP-10 and MIP1-α, that are increased in bronchoalveolar lavage and lung tissues by IL-33. Using transcriptomics (RNA-Seq) we demonstrated that 2279 transcripts were up-regulated and 1378 downregulated (≥ 2-fold, p < 0.01) in lung tissues, in response to IL-33. Bioinformatic interrogation of the RNA-Seq data was used to predict biological pathways and upstream regulators involved in IL-33-mediated responses. We showed that the mRNA and protein of STAT4, a predicted upstream regulator of IL-33-induced transcripts, was significantly enhanced in the lungs following IL-33 challenge. Overall, this study provides specific IL-33-induced molecular targets and endpoints that can be used as a resource for in vivo studies, e.g. in preclinical murine models examining novel interventions to target downstream effects of IL-33., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Alarmins, sequestered self-molecules containing damage-associated molecular patterns, are released during tissue injury to drive innate immune cell proinflammatory responses. Whether endogenous negative regulators controlling early immune responses are also released at the site of injury is poorly understood. Herein, we establish that the stromal cell-derived alarmin interleukin 33 (IL-33) is a local factor that directly restricts the proinflammatory capacity of graft-infiltrating macrophages early after transplantation. By assessing heart transplant recipient samples and using a mouse heart transplant model, we establish that IL-33 is upregulated in allografts to limit chronic rejection. Mouse cardiac transplants lacking IL-33 displayed dramatically accelerated vascular occlusion and subsequent fibrosis, which was not due to altered systemic immune responses. Instead, a lack of graft IL-33 caused local augmentation of proinflammatory iNOS+ macrophages that accelerated graft loss. IL-33 facilitated a metabolic program in macrophages associated with reparative and regulatory functions, and local delivery of IL-33 prevented the chronic rejection of IL-33-deficient cardiac transplants. Therefore, IL-33 represents what we believe is a novel regulatory alarmin in transplantation that limits chronic rejection by restraining the local activation of proinflammatory macrophages. The local delivery of IL-33 in extracellular matrix-based materials may be a promising biologic for chronic rejection prophylaxis.

Interleukin-33 (IL-33) regulates innate and acquired immune response to pathogens, self-antigens and tumors. IL-33 effects on tumors depend on the dose and mode of administration along with the type of malignancy. We studied the effects of IL-33 on the development of primary and metastatic melanoma induced by B16-F1 cell line in C57BL/6 mice. Intraperitoneally applied IL-33 restricts primary tumor growth. When administered intranasally 3 days prior to the intravenous injection of the tumor cells, IL-33 promoted growth of B16-F1 melanoma metastases, while B16-F10 gave massive metastases independently of IL-33. To mimic natural dissemination, we next used a limited number (5 × 10 4 ) of B16-F1 cells intravenously followed by application of IL-33 intraperitoneally. IL-33 increased the size of metastases (10.96 ± 3.96 mm 2 ) when compared to the control group (0.86 ± 0.39 mm 2 ), without changing incidence and number of metastases. IL-33 increased expression of ST2 on both tumor and immune cells in metastases. Also, IL-33 enhanced eosinophils and anti-tumor NK cells in the lung. The striking finding was reduced cytotoxicity of CD8 + T cells derived from metastatic lung of IL-33 injected mice. IL-33 reduced the percentage of TNF-α + and IFN-γ + CD8 + T cells while increasing the frequency of CD8 + T cells that express inhibitory molecules (PD-1, KLRG-1 and CTLA-4). There was a significant accumulation of CD11b + Gr-1 + myeloid suppressor cells and FoxP3 + , IL-10 + and CTLA-4 + regulatory T cells in the metastatic lung of IL-33 injected mice. The relevance of IL-33 for melanoma metastases was also documented in a significantly increased level of serum IL-33 in stage III melanoma patients.

Background: During infection with Leishmania donovani, parasite control is linked to the systemic Th1 immune response, but in infected organs (liver, spleen and bone marrow), the response differs according to the micro-environment. The pleiomorphic cytokine interleukin-33 (IL-33) exerts various roles during infection, either protective or detrimental. In this study, we explored the role of IL-33 in the outcome of Leishmania infection in the spleen., Methods: We used several mouse models, on BALB/c and C57BL/6 (B6) backgrounds, infected with L. donovani and sacrificed at 15, 30 or 60 days after infection and characterized mRNA expression of immune markers, immune cell populations, histological response, and parasite loads., Results: During infection IL-33 and ST2 mRNA increased in parallel in the spleen of wild type (wt) animals and paralleled the immunodetection of ST2+ and IL-33+ cells; their expression was twice as high in BALB/c, compared to B6 mice. Mice treated with twice-weekly injections of rIL-33 had higher splenic parasite burdens on D15 (BALB/c) or on D60 (B6). In BALB/c, IL-33 treatment led to immune exhaustion with abolition of Th1 cytokine expression (IFN-γ and IL-12) in the spleen and higher serum levels of Th2 cytokines (IL-4, IL-5 and IL-13). In B6, IL-33 treatment induced the Treg cell pathway with a dramatic increase of FoxP3 mRNA induction and expression on tissue sections. IL-33-KO mice had lower parasite loads and a higher Th1 response than their wt counterparts., Conclusions: IL-33 appears as a factor of aggravation of the disease in the spleen tissue of mice infected with L. donovani.

Myocardial infarction (MI) is the leading cause of mortality worldwide. Interleukin (IL)-33 (IL-33) is a cytokine present in most cardiac cells and is secreted on necrosis where it acts as a functional ligand for the ST2 receptor. Although IL-33/ST2 axis is protective against various forms of cardiovascular diseases, some studies suggest potential detrimental roles for IL-33 signaling. The aim of the present study was to examine the effect of IL-33 administration on cardiac function post-MI in mice. MI was induced by coronary artery ligation. Mice were treated with IL-33 (1 μg/day) or vehicle for 4 and 7 days. Functional and molecular changes of the left ventricle (LV) were assessed. Single cell suspensions were obtained from bone marrow, heart, spleen, and peripheral blood to assess the immune cells using flow cytometry at 1, 3, and 7 days post-MI in IL-33 or vehicle-treated animals. The results of the present study suggest that IL-33 is effective in activating a type 2 cytokine milieu in the damaged heart, consistent with reduced early inflammatory and pro-fibrotic response. However, IL-33 administration was associated with worsened cardiac function and adverse cardiac remodeling in the MI mouse model. IL-33 administration increased infarct size, LV hypertrophy, cardiomyocyte death, and overall mortality rate due to cardiac rupture. Moreover, IL-33-treated MI mice displayed a significant myocardial eosinophil infiltration at 7 days post-MI when compared with vehicle-treated MI mice. The present study reveals that although IL-33 administration is associated with a reparative phenotype following MI, it worsens cardiac remodeling and promotes heart failure., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Regulatory T (Treg) cells are known as immune regulators to decrease infarct volume and improve outcomes after ischemic stroke. Thus, the strategies for increasing Treg cells in ischemic brain may have the beneficial effects on stroke. In this study, we aim to examine the effect of Interleukin-33 (IL-33) on Treg cell expansion in mouse model of ischemic stroke., Methods: Mice were subjected to 30 min of middle cerebral artery occlusion (MCAO) followed by 24 h, 48 h of 72 h of reperfusion. Recombinant mouse IL-33 (2 μg) was pre-treated intracerebroventricularly at 30 min prior to MCAO. The percentage of Treg cells in ischemic brain, related cytokines and transcription factors, the levels of ST2 receptor, amphiregulin (AREG), and epidermal growth factor receptor (EGFR) were measured., Results: IL-33 treatment can increase the number of Foxp3 + Treg cells in the ischemic brain and the levels of IL-10 and TGF- β1 in serum and brain tissues at MCAO 48 h and 72 h, but not at MCAO 24 h. In the Treg cells separated from ischemic brain tissue following MCAO treated by IL-33, the expression level of ST2 receptor was up-regulated. In addition, IL-33 may increase the mRNA level of transcription factor Foxp3. Correspondingly, IL-33 treatment also elevated the levels of AREG and EGFR at MCAO 48 h and 72 h., Conclusion: We speculated that intracerebroventricular IL-33 can activate the downstream Foxp3 via ST2 receptor to increase Treg proportions in the ischemic brain. The elevated Treg cells produce AREG to activate EGFR located in neurons, which contribute to better outcomes., Competing Interests: Declaration of Competing Interest The authors have no potential conflicts of interest to report concerning this article., (Copyright © 2019 Elsevier B.V. All rights reserved.)

IL-33 and its receptor ST2 are contributing factors to airway inflammation and asthma exacerbation. The IL-33/ST2 signaling pathway is involved in both the onset and the acute exacerbations of asthma. In this study, we address the role of endogenous IL-33 and its autoamplification of the IL-33/ST2 pathway in Ag-dependent and Ag-independent asthma-like models. Wild-type, IL-33 knockout, ST2 knockout mice were either intratracheally administrated with 500 ng of rIL-33 per day for four consecutive days or were sensitized and challenged with OVA over 21 d. In wild-type mice, IL-33 or OVA induced similar airway hyperresponsiveness and eosinophilic airway inflammation. IL-33 induced its own mRNA and ST2L mRNA expression in the lung. IL-33 autoamplified itself and ST2 protein expression in airway epithelial cells. OVA also induced IL-33 and ST2 protein expression. In IL-33 knockout mice, the IL-33- and OVA-induced airway hyperresponsiveness and eosinophilic airway inflammation were both significantly attenuated, whereas IL-33-induced ST2L mRNA expression was preserved, although no autoamplification of IL-33/ST2 pathway was observed. In ST2 knockout mice, IL-33 and OVA induced airway hyperresponsiveness and eosinophilic airway inflammation were both completely diminished, and no IL-33/ST2 autoamplification was observed. These results suggest that endogenous IL-33 and its autoamplification of IL-33/ST2 pathway play an important role in the induction of asthma-like phenotype. Thus an intact IL-33/ST2 pathway is necessary for both Ag-dependent and Ag-independent asthma-like mouse models., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Increasing evidence suggests that IL-33 plays an important role in regulating tumor development. However, conflicting results, obtained from numerous studies, have highlighted the divergent functions of IL-33. The detailed mechanisms by which IL-33 modulates tumor development merit further investigation. Here, we report that IL-33 administration can effectively inhibit the development of pulmonary metastasis of breast cancer in a mouse. In our model, IL-33 promotes the production of TNF-α by macrophages, which increases IL-33 specific receptor (ST2) expression on natural killer (NK) cells and is pivotal in IL-33-induced NK cell activation. IL-33 treatment also facilitates the production of CCL5 in the lung by eosinophils and CD8 + T cells, which mediates the recruitment of NK cells to the tumor microenvironment. The systemic activation and local recruitment of NK cells result in potent tumor rejection in the lung. Our study reports a novel mechanism for the IL-33-meditated suppression of metastatic cancer and provides potential therapeutic strategies for targeting metastatic tumor., (© 2019 UICC.)

Occupational exposure to mechanical vibration can produce the hand-arm vibration syndrome (HAVS), whose most disabling symptom is persistent muscle pain. Unfortunately, the pathophysiology of HAVS pain is still poorly understood, precluding the development of mechanism-based therapies. Since interleukin 33 (IL-33) is essential for inflammation and recovery that follows skeletal muscle injury, we explored its role in muscle pain in a model of HAVS, in adult male rats. Concomitant to mechanical hyperalgesia, an increase in IL-33 in the ipsilateral gastrocnemius muscle was observed 24 hours after vibration. A similar hyperalgesia was produced by intramuscular injection of recombinant rat IL-33 (rrIL-33, 10-300 ng). Intrathecal administration of an oligodeoxynucleotide antisense to IL-33R/ST2 mRNA decreased the expression of ST2 in DRG and attenuated both rrIL-33 and vibration-induced mechanical hyperalgesia. Together these data support the suggestion that IL-33 plays a central role in vibration-induced muscle pain by action, at least in part, on skeletal muscle nociceptors. PERSPECTIVE: Our findings provide evidence of the contribution of IL-33, acting on its canonical receptor, in nociceptors, to muscle pain induced by ergonomic vibration. This suggests that targeting IL-33/ST2 signaling may be a useful strategy for the treatment of muscle pain in HAVS., (Copyright © 2019 United States Association for the Study of Pain, Inc. Published by Elsevier Inc. All rights reserved.)

Group 2 innate lymphoid cells (ILC2s) are rare innate immune cells that accumulate in tissues during allergy and helminth infection, performing critical effector functions that drive type 2 inflammation. ILC2s express ST2, the receptor for the cytokine IL-33, and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), a receptor for the bioactive lipid prostaglandin D 2 (PGD 2 ). The IL-33-ST2 and the PGD 2 -CRTH2 pathways have both been implicated in promoting ILC2 accumulation during type 2 inflammation. However, whether these two pathways coordinate to regulate ILC2 population size in the tissue in vivo remains undefined. In this study, we show that ILC2 accumulation in the murine lung in response to systemic IL-33 treatment was partially dependent on CRTH2. This effect was not a result of reduced ILC2 proliferation, increased apoptosis or cell death, or differences in expression of the ST2 receptor in the absence of CRTH2. Rather, data from adoptive transfer studies suggested that defective accumulation of CRTH2-deficient ILC2s in response to IL-33 was due to altered ILC2 migration patterns. Whereas donor wild-type ILC2s preferentially accumulated in the lungs compared with CRTH2-deficient ILC2s following transfer into IL-33-treated recipients, wild-type and CRTH2-deficient ILC2s accumulated equally in the recipient mediastinal lymph node. These data suggest that CRTH2-dependent effects lie downstream of IL-33, directly affecting the migration of ILC2s into inflamed lung tissues. A better understanding of the complex interactions between the IL-33 and PGD 2 -CRTH2 pathways that regulate ILC2 population size will be useful in understanding how these pathways could be targeted to treat diseases associated with type 2 inflammation., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Toxoplasma gondii is a threat for immunocompromized individuals, and no treatment is available for enhancing immunity against infection. Molecular adjuvants may improve the efficacy of DNA vaccine-induced T cell immunity. Here, we report that cocktailed DNA immunization with ROP5 and ROP18 boosted immune responses induced by a single DNA immunization with ROP5 or ROP18, but also that co-administration of molecular adjuvant IL-33 enhanced immune efficacy induced by this cocktailed DNA vaccination. These improved immune responses were characterized by higher Toxoplasma-specific IgG2a titers, Th1 responses associated with the production of IFN-γ, IL-2, IL-12, as well as cell-mediated activity with higher frequencies of CD8+ and CD4+ T cells. More importantly, this enhanced immunity has the ability to confer remarkable protection against a high dose lethal challenge of the T. gondii RH strain and thus against chronic infection with the T. gondii PRU strain. These data show that IL-33 is a promising immunoadjuvant to facilitate humoral as well as cellular immunity in a vaccine setting against T. gondii, and suggest that it should be evaluated in strategies against other apicomplexan parasites., (© Y.-C. Zhu et al., published by EDP Sciences, 2020.)

Innate lymphoid cells (ILCs) are lymphocytes with critical roles in homeostasis, inflammation, and immunity to pathogens. ILCs are rare relative to other immune cell populations and are primarily defined by lack of expression of markers associated with other immune cell lineages and are predominantly found in mucosal tissues like the gut, lung and skin. They are classified into distinct subsets, ILC1, ILC2, and ILC3, which mirror subsets of CD4 + helper T cells. ILC subsets have distinct cytokine and transcription factor profiles which align with their biological functions, although recently it has emerged that ILC subsets are not phenotypically fixed and exhibit considerable heterogeneity and plasticity in different contexts. Here, we describe protocols for the maintenance, expansion, and induction of plasticity in mouse and human ILC2s. The resulting cells can be used for molecular interrogation of ILC function and biology, both in vivo and in vitro.

Interleukin-33 (IL-33)/ST2-mediated mast cell activation plays important roles in the pathophysiology of allergic diseases. Hence, pharmacologically targeting the IL-33/ST2 pathway in mast cells could help to treat such diseases. We found that resveratrol inhibits IL-33/ST2-mediated mast cell activation. Resveratrol suppressed IL-33-induced IL-6, IL-13, and TNF-α production in mouse bone marrow-derived mast cells (BMMCs), mouse fetal skin-derived mast cells, and human basophils. Resveratrol also attenuated cytokine expression induced by intranasal administration of IL-33 in mouse lung. IL-33-mediated cytokine production in mast cells requires activation of the NF-κB and MAPK p38-MAPK-activated protein kinase-2/3 (MK2/3)-PI3K/Akt pathway, and resveratrol clearly inhibited IL-33-induced activation of the MK2/3-PI3K/Akt pathway, but not the NF-κB pathway, without affecting p38 in BMMCs. Importantly, resveratrol inhibited the kinase activity of MK2, and an MK2/3 inhibitor recapitulated the suppressive effects of resveratrol. Resveratrol and an MK2/3 inhibitor also inhibited IgE-dependent degranulation and cytokine production in BMMCs, concomitant with suppression of the MK2/3-PI3K/Akt pathway. These findings indicate that resveratrol inhibits both IL-33/ST2-mediated and IgE-dependent mast cell activation principally by targeting the MK2/3-PI3K/Akt axis downstream of p38. Thus, resveratrol may have potential for the prevention and treatment of broad ranges of allergic diseases.

The splenic T-cell immune response to stroke has been identified as an important role in the progression of brain injury following ischemic stroke. Interleukin (IL)-33 as a novel cytokine of IL-1 family has been found to be protective for ischemic brain injury. Here, we determined the contribution of IL-33 to the T-cell immune responses in the spleen after experimental ischemic stroke. Mice were subjected to 30 min of middle cerebral artery occlusion (MCAO) for ischemic stroke induction. Recombinant mouse IL-33 (100 μg/kg) was pre-treated intraperitoneally at 30 min prior to MCAO, then the percentages of T cell subsets, related cytokines and transcription factors in the spleen tissues were measured. Intraperitoneal IL-33 pre-treatment may attenuate neurological deficit scores and infarct volumes after MCAO, which was accompanied by reduced IFN-γ + T cells and increased Foxp3 + T cells in the spleen tissues. Meanwhile, IL-33 pre-treatment could decrease the production of IFN-γ and increase the secretion of IL-4, IL-10 and TGF-β from the spleen at 24 h after MCAO. Additionally, the mRNA level of the transcription factor T-bet was downregulated by IL-33, and the levels of GATA-3 and Foxp3 mRNA were upregulated. These results showed that the long-term protective mechanism of IL-33 in ischemic stroke may be partly associated to its modulation role for splenic T-cell immune responses through inhibiting Th1 response and promoting Treg response, suggesting that IL-33 may be a candidate treatment for human stroke via modulating the peripheral immune system following stroke., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Kidney injury, whether due to ischemic insults or chemotherapeutic agents, is exacerbated by inflammation, whereas Tregs are protective. We recently showed that IL-2 and IL-33, especially as a hybrid cytokine (IL233 - bearing IL-2 and IL-33 activities in one molecule), potentiated Tregs and group 2 innate lymphoid cells (ILC2) to prevent renal injury. Recent studies have indicated a reparative function for Tregs and ILC2. Here, using doxorubicin-induced nephrotoxic renal injury model, we investigated whether IL233 administration either before, late or very late after renal injury can restore kidney structure and function. We found that IL233 treatment even 2-weeks post-doxorubicin completely restored kidney function accompanied with an increase Treg and ILC2 in lymphoid and renal compartments, augmented anti-inflammatory cytokines and attenuated proinflammatory cytokine levels. IL233 treated mice had reduced inflammation, kidney injury (Score values - saline: 3.34 ± 0.334; IL233 pre: 0.42 ± 0.162; IL233 24 hrs: 1.34 ± 0.43; IL233 1 week: 1.2 ± 0.41; IL233 2 week: 0.47 ± 0.37; IL233 24 hrs + PC61: 3.5 ± 0.74) and fibrosis in all treatment regimen as compared to saline controls. Importantly, mice treated with IL233 displayed a reparative program in the kidneys, as evidenced by increased expression of genes for renal progenitor-cells and nephron segments. Our findings present the first evidence of an immunoregulatory cytokine, IL233, which could be a potent therapeutic strategy that augments Treg and ILC2 to not only inhibit renal injury, but also promote regeneration.

Candidate vaccines designed to generate T cell-based immunity are typically vectored by nonpersistent viruses, which largely fail to elicit durable effector memory T cell responses. This limitation can be overcome using recombinant strains of CMV. Proof-of-principle studies have demonstrated the potential benefits of this approach, most notably in the SIV model, but safety concerns require the development of nonreplicating alternatives with comparable immunogenicity. In this study, we show that IL-33 promotes the accumulation and recall kinetics of circulating and tissue-resident memory T cells in mice infected with murine CMV. Using a replication-deficient murine CMV vector, we further show that exogenous IL-33 boosts vaccine-induced memory T cell responses, which protect against subsequent heterologous viral challenge. These data suggest that IL-33 could serve as a useful adjuvant to improve the efficacy of vaccines based on attenuated derivatives of CMV., (Copyright © 2019 The Authors.)

The human Interleukin-33 (IL-33), a member of the IL-1 family, is the cytokine as a cell endogenous alarmin, released by damaged or necrotic barrier cells (endothelial and epithelial cells). The signal transduction of IL-33 relies on recognition and interaction with specific receptor ST2, mainly expressed in immune cells. In both innate and adoptive immunity, IL-33 regulates the homeostasis in response to stress from within/out the microenvironment. Various, even negative biofunctions of IL-33 pathways have now been widely verified in pathogenesis among immunological mechanisms, like Th2-related immune-stimuli, inflammation/infection-induced tissue protectors. A larger versatility in studies of IL-33 on malignancies now focuses on: (1) promoting myeloid-derived suppressor cells (MDSC), (2) intervention toward CD8 + T, Natural Killer (NK) cell infiltration, group 2 innate lymphoid cells (ILC2) proliferation, dendritic cells (DC) activation, and (3) inhibiting tumor growth and/or further metastasis as an immunoadjuvant. Although IL-33 functioned pro-tumorigenically in various cancers, for some cancer types the findings so far are controversial. This review begins from a summarized introduction of IL-33, to its remarkable implications and molecular transduction pathway in malignant neoplasms, ends with latest inspiration for IL-33 in treatment.

Breaking tolerance is a key event leading to autoimmunity, but the exact mechanisms responsible for this remain uncertain. Here we show that the alarmin IL-33 is able to drive the generation of autoantibodies through induction of the B cell survival factor BAFF. A temporary, short-term increase in IL-33 results in a primary (IgM) response to self-antigens. This transient DNA-specific autoantibody response was dependent on the induction of BAFF. Notably, radiation resistant cells and not myeloid cells, such as neutrophils or dendritic cells were the major source of BAFF and were critical in driving the autoantibody response. Chronic exposure to IL-33 elicited dramatic increases in BAFF levels and resulted in elevated numbers of B and T follicular helper cells as well as germinal center formation. We also observed class-switching from an IgM to an IgG DNA-specific autoantibody response. Collectively, the results provide novel insights into a potential mechanism for breaking immune-tolerance via IL-33-mediated induction of BAFF.

Objective: Chronic rhinosinusitis (CRS) is a complicated disease with several variants caused by different cellular and molecular mechanisms. The characterization of this heterogeneity supports the definition that the disease consists of many endotypes, such as eosinophilic and neutrophilic CRS, and so on. This study aimed to explore group 2 innate lymphoid cells (ILC2s) in neutrophilic CRS without nasal polyps (CRSsNP) and with nasal polyps (CRSwNP), and evaluate ILC2s across characteristics of the disease., Methods: Nasal biopsy samples were obtained from normal subjects or subjects with CRSsNP or CRSwNP during surgery. ILC2s were sorted and purified as CD45 + Lin - CD127 + CD4 - CD8 - CRTH2 + CD161 + cells through flow cytometry, and were compared among three groups of subjects. Then, these samples were cultured in vitro, and inflammatory factors were assessed in tissue cultures. After that, human recombinant (rm) interleukin (IL)-33 or IL-17 were administered into the cultures, and we again examined relevant inflammatory substances., Results: ILC2s were upregulated in neutrophilic CRSsNP and CRSwNP patients, and there were no statistical differences between them. Eosinophil cation protein (ECP), myeloperoxidase (MPO), IL-25, IL-33, IL-5, IL-13, interferon (IFN)-γ and IL-17 were increased in the cultures, however, only concentrations of MPO, IFN-γ and IL-17 were enhanced in CRSwNP tissues compared to CRSsNP ones. After administration of rmIL-33, ECP, IL-5 and IL-13 were all increased in tissues from CRSsNP and CRSwNP patients, however, there were no significant differences between them. Finally, we evaluated concentrations of several above inflammatory factors after the treatment of rmIL-17, and found that MPO and IFN-γ were enhanced in these two phenotypes of patients, and were elevated significantly in CRSwNP tissue cultures., Conclusion: These findings show that ILC2s might be inactivated in neutrophilic CRSsNP and CRSwNP based on this pilot study.

Asthma is one of the most common immunological diseases and is characterized by airway hyperresponsiveness (AHR), mucus overproduction, and airway eosinophilia. Although mouse models have provided insight into the mechanisms by which type-2 cytokines induce asthmatic airway inflammation, differences between the rodent and human immune systems hamper efforts to improve understanding of human allergic diseases. In this study, we aim to establish a preclinical animal model of asthmatic airway inflammation using humanized IL-3/GM-CSF or IL-3/GM-CSF/IL-5 Tg NOD/Shi-scid-IL2rγnull (NOG) mice and investigate the roles of human type-2 immune responses in the asthmatic mice. Several important characteristics of asthma - such as AHR, goblet cell hyperplasia, T cell infiltration, IL-13 production, and periostin secretion - were induced in IL-3/GM-CSF Tg mice by intratracheally administered human IL-33. In addition to these characteristics, human eosinophilic inflammation was observed in IL-3/GM-CSF/IL-5 Tg mice. The asthmatic mechanisms of the humanized mice were driven by activation of human Th2 and mast cells by IL-33 stimulation. Furthermore, treatment of the humanized mice with an anti-human IL-13 antibody significantly suppressed these characteristics. Therefore, the humanized mice may enhance our understanding of the pathophysiology of allergic disorders and facilitate the preclinical development of new therapeutics for IL-33-mediated type-2 inflammation in asthma.

Signaling through IL-2/IL-15Rβ (CD122) is essential for the differentiation and function of T cells and NK cells. A mAb against CD122 has been implicated to suppress autoimmune type 1 diabetes (T1D) development in animal models. However, the mechanisms remain poorly understood. We find that in vivo administration of an anti-CD122 mAb (CD122 blockade) restores immune tolerance in nonobese diabetic (NOD) mice via multiple mechanisms. First, CD122 blockade selectively ablates pathogenic NK cells and memory phenotype CD8+ T cells from pancreatic islets. In contrast, islet CD4+Foxp3+ Tregs are only mildly affected. Second, CD122 blockade suppresses IFN-γ production in islet immune cells. Third, CD122 blockade inhibits the conversion of islet Th17 cells into diabetogenic Th1 cells. Furthermore, a combination of anti-CD122 mAb and Treg-trophic cytokines (IL-2 or IL-33) enhances the abundance and function of islet Tregs. In summary, these data provide crucial mechanistic insights into CD122 blockade-mediated immunoregulation and support therapeutic benefits of this combinational treatment in T1D.

Toll-interacting protein (Tollip) is a key negative regulator of innate immunity by preventing excessive proinflammatory responses. Tollip genetic variation has been associated with airflow limitation in asthma subjects and Tollip expression. Whether Tollip regulates lung inflammation in a type 2 cytokine milieu (e.g., IL-13) is unclear. Our goal was to determine the in vivo role of Tollip in IL-13-mediated lung eosinophilic inflammation and the underlying mechanisms. Tollip-knockout (KO) and wild-type (WT) mice were inoculated intranasally with recombinant mouse IL-13 protein to examine lung inflammation. To determine how Tollip regulates inflammation, alveolar macrophages and bone marrow-derived macrophages from Tollip KO and WT mice were cultured with or without IL-13 and/or IL-33. IL-13-treated Tollip KO mice significantly increased lung eosinophilic inflammation and eotaxin-2 (CCL24) levels compared with the WT mice. IL-13- treated Tollip KO (vs. WT) macrophages, in the absence and particularly in the presence of IL-33, increased expression of the IL-33 receptor ST2L and CCL24, which was in part dependent on enhanced activation of interleukin (IL)-1 receptor-associated kinase 1 (IRAK1) and signal transducer and activator of transcription 6 (STAT6). Our results suggest that Tollip downregulates IL-13-mediated pulmonary eosinophilia in part through inhibiting the activity of the ST2L/IL-33/IRAK1 axis and STAT6., (© 2018 S. Karger AG, Basel.)

The calcineurin inhibitor cyclosporine A (CsA) suppresses the immune system but promotes hypertension, vascular dysfunction, and renal damage. CsA decreases regulatory T cells and this contributes to the development of hypertension. However, CsA's effects on another important regulatory immune cell subset, myeloid-derived suppressor cells (MDSCs), is unknown. We hypothesized that augmenting MDSCs would ameliorate the CsA-induced hypertension and vascular and renal injury and dysfunction and that CsA reduces MDSCs in mice. Daily interleukin-33 treatment, which increased MDSC levels, completely prevented CsA-induced hypertension and vascular and renal toxicity. Adoptive transfer of MDSCs from control mice into CsA-treated mice after hypertension was established dose-dependently reduced blood pressure and vascular and glomerular injury. CsA treatment of aortas and kidneys isolated from control mice for 24 hours decreased relaxation responses and increased inflammation, respectively, and these effects were prevented by the presence of MDSCs. MDSCs also prevented the CsA-induced increase in fibronectin in microvascular and glomerular endothelial cells. Last, CsA dose-dependently reduced the number of MDSCs by inhibiting calcineurin and preventing cell proliferation, as other direct calcineurin signaling pathway inhibitors had the same dose-dependent effect. These data suggest that augmenting MDSCs can reduce the cardiovascular and renal toxicity and hypertension caused by CsA., (© 2017 American Heart Association, Inc.)

T-helper type 2 (Th2) cytokines, including interleukin (IL)-13 and IL-4, produced in adipose tissue, are critical regulators of intra-adipose and systemic lipid and glucose metabolism. Furthermore, IL-13 is a potential therapy for insulin resistance in obese mouse models. Here, we examined mediators produced by adipocytes that are responsible for regulating systemic glucose homeostasis in response to Th2 cytokines. We used RNA sequencing data analysis of cultured adipocytes to screen factors secreted in response to recombinant IL-13. Recombinant IL-13 induced expression of growth differentiation factor 15 (GDF15) via the Janus kinase-activated STAT6 pathway. In vivo administration of α-galactosylceramide or IL-33 increased IL-4 and IL-13 production, thereby increasing GDF15 levels in adipose tissue and in plasma of mice; however, these responses were abrogated in STAT6 knockout mice. Moreover, administration of recombinant IL-13 to wild-type mice fed a high-fat diet (HFD) improved glucose intolerance; this was not the case for GDF15 knockout mice fed the HFD. Taken together, these data suggest that GDF15 is required for IL-13-induced improvement of glucose intolerance in mice fed an HFD. Thus, beneficial effects of Th2 cytokines on systemic glucose metabolism and insulin sensitivity are mediated by GDF15. These findings open up a potential pharmacological route for reversing insulin resistance associated with obesity., (© 2017 by the American Diabetes Association.)

Asthma is a complex disease that is promoted by dysregulated immunity and the presence of many cytokine and lipid mediators. Despite this, there is a paucity of data demonstrating the combined effects of multiple mediators in asthma pathogenesis. Group 2 innate lymphoid cells (ILC2s) have recently been shown to play important roles in the initiation of allergic inflammation; however, it is unclear whether lipid mediators, such as cysteinyl leukotrienes (CysLTs), which are present in asthma, could further amplify the effects of IL-33 on ILC2 activation and lung inflammation. In this article, we show that airway challenges with the parent CysLT, leukotriene C4 (LTC4), given in combination with low-dose IL-33 to naive wild-type mice, led to synergistic increases in airway Th2 cytokines, eosinophilia, and peribronchial inflammation compared with IL-33 alone. Further, the numbers of proliferating and cytokine-producing lung ILC2s were increased after challenge with both LTC4 and IL-33. Levels of CysLT1R, CysLT2R, and candidate leukotriene E4 receptor P2Y12 mRNAs were increased in ILC2s. The synergistic effect of LTC4 with IL-33 was completely dependent upon CysLT1R, because CysLT1R -/- mice, but not CysLT2R -/- mice, had abrogated responses. Further, CysLTs directly potentiated IL-5 and IL-13 production from purified ILC2s stimulated with IL-33 and resulted in NFAT1 nuclear translocation. Finally, CysLT1R -/- mice had reduced lung eosinophils and ILC2 responses after exposure to the fungal allergen Alternaria alternata Thus, CysLT1R promotes LTC4- and Alternaria -induced ILC2 activation and lung inflammation. These findings suggest that multiple pathways likely exist in asthma to activate ILC2s and propagate inflammatory responses., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Approximately half of all atopic dermatitis (AD) patients subsequently develop asthma, particularly those with severe AD. This association, suggesting a role for AD as an entry point for subsequent allergic disease, is a phenomenon known as the "atopic march". While the underlying cause of the atopic march remains unknown, recent evidence suggests that epithelial cell (EC)-derived cytokines play a major role. We showed that mice exposed to antigen through the skin, in the presence of IL-33, developed antigen-specific airway inflammation when later challenged in the lung. IL-33 signaling was dispensable during effector/challenge phase. These data reveal critical roles for IL-33 in the "atopic march" and will offer a new therapeutic target in the treatment and prevention of allergic asthma.

Accumulated data have shown that alternatively activated macrophage exerts a modulatory role in many diseases, including colitis. Interleukin-33 (IL-33), a critical modulator in adaptive and innate immune, has been implicated in autoimmunity and inflammation. Previously, we have reported that IL-33 functions as a protective modulator in TNBS-induced colitis, which is closely related to a Th1-to-Th2/Treg switch. Here, we present novel evidence suggesting that IL-33 primes macrophage into alternatively activated macrophages (AAM) in TNBS-induced colitis. The strong polarized effect of IL-33 was tightly associated with the markedly increased induction of Th2-type cytokines. To confirm the beneficial effects of AAM induced by IL-33, peritoneal AAMs isolated from IL-33-treated mice were transferred to recipient mice with TNBS colitis. The adoptive transfer resulted in prominent inhibition of disease activity and inflammatory cytokines in the TNBS-treated mice. In conclusion, our data provide clear evidence that IL-33 plays a protective role in TNBS-induced colitis, which is closely related to AAM polarization.

The objective of the study is to investigate the role and specific molecular mechanism of interleukin-33 (IL-33) acted on acute lung injury (ALI) induced by lipopolysaccharide (LPS). C57BL/6 mice intratracheally instilled LPS to induce ALI model. The mice were randomly divided into three groups: the sham operation group (Sham), ALI group (ALI), and pretreatment with IL-33 of ALI group (IL-33). By observing the survival rate, inflammatory cytokines in bronchoalveolar lavage fluid (BALF), myeloperoxidase (MPO) levels in lung tissue, lung histopathological examination, pulmonary capillary leakage, lung wet/dry (W/D) weight ratio, fibrosis levels in lung tissue, and associated pathways changes among the different groups, comparing to explore the role of IL-33 pretreatment on ALI mice and the possible molecular mechanisms. IL-33 pretreatment overall decreased the survival rate of ALI mice. IL-33 aggravated inflammation reaction showing as increasing the release of proinflammatory cytokines TNF-α and IL-6, increasing MPO levels in lung tissue, and aggravating lung pathology injury. In addition, IL-33 pretreatment further destroyed adherens junctions (AJs) by increasing the phosphorylation of VE-cadherin, resulting in the concomitantly pulmonary capillary barrier damage and pulmonary edema. During this process, mitogen-activated protein kinase (MAPK) pathways further activated. However, IL-33 pretreatment had no significant impact on collagen content of lung tissue. Our results indicated that IL-33 aggravated inflammatory reaction and increased microvascular permeability, but had little effect on pulmonary fibrosis, associated with the further activation of MAPK family proteins in the process. To sum up, IL-33 decreased survival rate and aggravated LPS-induced ALI.

Myeloid-derived suppressor cells (MDSCs) contribute to tumor-mediated immune escape by suppressing antitumor immune responses. Interleukin-33 (IL-33) is capable of regulating various immune cell populations; however, the effects of IL-33 on the differentiation of MDSCs have not been well characterized. In this study, we evaluated the effects of IL-33 on MDSCs and found that IL-33 significantly reduced the differentiation of lineage-negative bone marrow progenitor cells into granulocytic MDSCs (G-MDSCs). IL-33-treated MDSCs exhibited diminished immunosuppressive capacity; reduced inhibition on T-cell proliferation and interferon-γ production, and diminished production of reactive oxygen species. However, IL-33 treatment did not affect the frequency of monocytic MDSCs (M-MDSCs) or their production of nitric oxide and expression of arginase-1. Additionally, compared with control MDSCs, IL-33-treated MDSCs had reduced capacity to induce the differentiation or expansion of Treg cells. Moreover, in vivo IL-33 administration significantly decreased MDSCs and G-MDSCs accumulation in the spleen and tumor microenvironment. Also, despite increasing CD4 + and CD8 + T-cell infiltration, IL-33 administration markedly decreased Treg-cell population in tumor microenvironment. Taken together, our findings indicate that IL-33 reduces the frequency and immunosuppressive activity of G-MDSCs and ultimately the extent of tumor growth.

Allergic asthma is a chronic inflammatory lung disease mediated by type 2 cytokines produced by T helper 2 (Th2) cells as well as the recently discovered group 2 innate lymphoid cells (ILC2). Due to a lack of unique markers, the accurate phenotypic characterization and quantification of ILC2 requires a comprehensive panel of fluorescently labeled antibodies. The markers that are currently used to characterize ILC2 have not been standardized and often vary between research groups, which poses significant challenges when comparing data. Intranasal administration of the pro-inflammatory cytokine IL-33 in mice is associated with strong, Th2 cell-independent ILC2 activation. ILC2 are also activated in mouse models of allergic asthma based on the physiologically relevant house dust mite (HDM) allergen, which parallel eosinophilic airway inflammation observed in asthma patients. Here, we describe the analysis of ILC2 by flow cytometry in these two commonly used allergic airway inflammation models in the mouse.

Interleukin (IL)-33 is a novel IL-1 family member, and its administration has been associated with promotion of T helper type-2 (Th2) cell activity and cytokines, particularly IL-4 and IL-5 in vivo. Recently, IL-33 was shown to increase CD4(+)Foxp3(+) regulatory T cells (Tregs) and to suppress levels of the Th1-type cytokine IFN-γ in allogeneic heart transplantation in mice. Therefore, we hypothesized that IL-33 and leflunomide (Lef) could prolong graft survival in the concordant mouse-to-rat heart transplantation model. In this model, xenografts undergo acute humoral xenograft rejection (AHXR) typically on day 3 or cell-mediated rejection approximately on day 7 if AHXR is inhibited by Lef treatment. Recipients were treated with Lef (n=6), IL-33 (n=6), IL-33 combined with Lef (n=6), or left untreated (n=6) for survival studies. Heart grafts were monitored until they stopped beating. Mouse heterotopic grafts were performed, and recipients were sacrificed on days 2 and 7 for histological and flow cytometric analyses. The combination of IL-33 and Lef significantly prolonged the grafts from 17.3±2.3 to 2.8±0.4 days, compared to untreated controls. IL-33 administration with Lef, while facilitating Th2-associated cytokines (IL-4 on day 2 but not day 7), also decreased IFN-γ on day 2 and day 7, compared with Lef treatment only. Furthermore, IL-33 with Lef administration caused an expansion of suppressive CD4(+)Foxp3(+) Tregs in rats. The IL-33 and Lef combination therapy resulted in significantly prolonged graft survival, associated with markedly decreased Th1 cells and increased IL-10 levels. In addition, the combination therapy significantly decreased the percentage of CD-45(+) B cells on days 2 and 7, compared with monotherapy. These findings reveal a new immunoregulatory property of IL-33. Specifically, it facilitates regulatory cells, particularly functional CD4(+)Foxp3(+) Tregs that underlie IL-33-mediated cardiac xenograft survival. Moreover, it can decrease Th1 cells and cytokine expression of Th1 T cells in xenograft recipients, for example IFN-γ.

Recent research findings have provided convincing evidence indicating a role for Interleukin-33 (IL-33) signalling pathway in a number of central nervous system (CNS) diseases including multiple sclerosis (MS) and Alzheimer's disease. However, the exact function of IL-33 molecule within the CNS under normal and pathological conditions is currently unknown. In this study, we have mapped cellular expression of IL-33 and its receptor ST2 by immunohistochemistry in the brain tissues of MS patients and appropriate controls; and investigated the functional significance of these findings in vitro using a myelinating culture system. Our results demonstrate that IL-33 is expressed by neurons, astrocytes and microglia as well as oligodendrocytes, while ST2 is expressed in the lesions by oligodendrocytes and within and around axons. Furthermore, the expression levels and patterns of IL-33 and ST2 in the lesions of acute and chronic MS patient brain samples are enhanced compared with the healthy brain tissues. Finally, our data using rat myelinating co-cultures suggest that IL-33 may play an important role in MS development by inhibiting CNS myelination.

Alzheimer's disease (AD) is a devastating condition with no known effective treatment. AD is characterized by memory loss as well as impaired locomotor ability, reasoning, and judgment. Emerging evidence suggests that the innate immune response plays a major role in the pathogenesis of AD. In AD, the accumulation of β-amyloid (Aβ) in the brain perturbs physiological functions of the brain, including synaptic and neuronal dysfunction, microglial activation, and neuronal loss. Serum levels of soluble ST2 (sST2), a decoy receptor for interleukin (IL)-33, increase in patients with mild cognitive impairment, suggesting that impaired IL-33/ST2 signaling may contribute to the pathogenesis of AD. Therefore, we investigated the potential therapeutic role of IL-33 in AD, using transgenic mouse models. Here we report that IL-33 administration reverses synaptic plasticity impairment and memory deficits in APP/PS1 mice. IL-33 administration reduces soluble Aβ levels and amyloid plaque deposition by promoting the recruitment and Aβ phagocytic activity of microglia; this is mediated by ST2/p38 signaling activation. Furthermore, IL-33 injection modulates the innate immune response by polarizing microglia/macrophages toward an antiinflammatory phenotype and reducing the expression of proinflammatory genes, including IL-1β, IL-6, and NLRP3, in the cortices of APP/PS1 mice. Collectively, our results demonstrate a potential therapeutic role for IL-33 in AD.

The T-helper cell type 2-promoting cytokine interleukin-33 (IL-33) has been implicated in asthma pathogenesis. Angiogenesis is a feature of airways remodelling in asthma. We hypothesized that IL-33 induces airways angiogenesis and expression of angiogenic factors in an established murine surrogate of asthma. In the present study, BALB/c mice were subjected to serial intranasal challenge with IL-33 alone for up to 70 days. In parallel, ovalbumin (OVA) -sensitized mice were subjected to serial intranasal challenge with OVA or normal saline to serve as positive and negative controls, respectively. Immunohistochemical analysis of expression of von Willebrand factor and erythroblast transformation-specific-related gene, both blood vessel markers, and angiogenic factors angiogenin, insulin-like growth factor-1, endothelin-1, epidermal growth factor and amphiregulin was performed in lung sections ex vivo. An established in-house assay was used to test whether IL-33 was able to induce microvessel formation by human vascular endothelial cells. Results showed that serial intranasal challenge of mice with IL-33 or OVA resulted in proliferation of peribronchial von Willebrand factor-positive blood vessels to a degree closely related to the total expression of the angiogenic factors amphiregulin, angiogenin, endothelin-1, epidermal growth factor and insulin-like growth factor-1. IL-33 also induced microvessel formation by human endothelial cells in a concentration-dependent fashion in vitro. Our data are consistent with the hypothesis that IL-33 has the capacity to induce angiogenesis at least partly by increasing local expression of multiple angiogenic factors in an allergen-independent murine asthma surrogate, and consequently that IL-33 or its receptor is a potential novel molecular target for asthma therapy., (© 2016 John Wiley & Sons Ltd.)

Background: The transcription factor promyelocytic leukemia zinc finger (PLZF) is transiently expressed during development of type 2 innate lymphoid cells (ILC2s) but is not present at the mature stage. We hypothesized that PLZF-deficient ILC2s have functional defects in the innate allergic response and represent a tool for studying innate immunity in a mouse with a functional adaptive immune response., Objective: We determined the consequences of PLZF deficiency on ILC2 function in response to innate and adaptive immune stimuli by using PLZF(-/-) mice and mixed wild-type:PLZF(-/-) bone marrow chimeras., Methods: PLZF(-/-) mice, wild-type littermates, or mixed bone marrow chimeras were treated with the protease allergen papain or the cytokines IL-25 and IL-33 or infected with the helminth Nippostrongylus brasiliensis to induce innate type 2 allergic responses. Mice were sensitized with intraperitoneal ovalbumin-alum, followed by intranasal challenge with ovalbumin alone, to induce adaptive TH2 responses. Lungs were analyzed for immune cell subsets, and alveolar lavage fluid was analyzed for ILC2-derived cytokines. In addition, ILC2s were stimulated ex vivo for their capacity to release type 2 cytokines., Results: PLZF-deficient lung ILC2s exhibit a cell-intrinsic defect in the secretion of IL-5 and IL-13 in response to innate stimuli, resulting in defective recruitment of eosinophils and goblet cell hyperplasia. In contrast, the adaptive allergic inflammatory response to ovalbumin and alum was unimpaired., Conclusions: PLZF expression at the innate lymphoid cell precursor stage has a long-range effect on the functional properties of mature ILC2s and highlights the importance of these cells for innate allergic responses in otherwise immunocompetent mice., (Copyright © 2015 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

Lymphocyte apoptosis plays a pivotal role in sepsis-induced immunosuppression and is the primary cause of high mortality rates. Interleukin-33 is a member of the interleukin-1 family that is involved in the polarization of T cells toward a T helper 2-cell phenotype and may regulate apoptotic cell death. The objective of the present study was to assess the effects of interleukin-33 on T lymphocyte apoptosis in sepsis and determine the mechanisms involved. Sepsis was induced in C57BL/6 mice via a cecal ligation and puncture. Mice were infused with recombinant interleukin-33 protein at 1h and 6h after surgery. The mortality rates were evaluated over the subsequent 7 days. In a separate experiment, mice were sacrificed 24h after surgery. Bacterial burdens in the blood and peritoneal cavity were calculated to assess the bacterial clearance. Liver, lung and renal pathology were observed via transmission electron microscopy. The serum levels of interleukin-6, interleukin-10, interleukin-17, interferon-γ and tumor necrosis factor-α were measured via enzyme-linked immunosorbent assays. The number of T and B lymphocytes, the percentage of apoptotic cells and the expression of Fas, Bcl-2, caspase-3, caspase-8 and caspase-9 in CD3(+) T lymphocytes were analyzed by flow cytometry. Interleukin-33 enhanced bacterial clearance, attenuated the severity of organ damage and improved the survival of septic mice. Interleukin-33 decreased the levels of interleukin-6, interleukin-10, interferon-γ and tumor necrosis factor-α, and it increased the levels of interleukin-17. Interleukin-33 also inhibited the apoptosis of CD4(+) and CD8(+) T lymphocytes and CD19(+) B cells in the spleen. The number of CD3(+) T cells was higher and the expression of active caspase-3, caspase-8 and caspase-9 was lower in the interleukin-33 group compared to the CLP group. The expression of Fas was lower and the expression of Bcl-2 was higher in the interleukin-33 group than in the CLP group. Interleukin-33 prevented apoptosis of T lymphocytes and improved survival in a mouse model of sepsis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Thymic-derived, regulatory T cells (Treg) represent a subset of CD4(+) T cells that are required for normal immune homeostasis and suppression of unwanted responses against self-antigens (Ags) that prevent autoimmunity. Their role as immune regulators and potent ability to suppress T cell responses has been the focus of intense investigations aimed at utilizing these cells therapeutically, particularly in the settings of autoimmunity and transplantation. Many methods for expanding Treg have been described; however, efforts to generate large numbers of Treg for use in vivo often compromise their suppressor function or rely on the induction of Treg rather than their expansion. Our recent studies have focused on the barrier tissue-derived cytokine IL-33, a recently described IL-1 family member. IL-33 has emerged as a multifunctional protein, with reported roles in driving potent Type 1 and Type 2 immunity, as well as facilitating profound Treg expansion in vitro and in vivo. IL-33-expanded Treg express the IL-33 receptor (R) ST2, and express classical markers associated with Treg phenotype and suppressor function. They suppress both CD4(+) and CD8(+) T cell proliferation and effector functions in vitro, and Treg expressing ST2 have been identified as important regulators of detrimental immune responses in vivo. In the present chapter, we detail methods for expanding significant numbers of Treg using IL-33 both in vitro and in vivo that may potentially be used to promote/maintain organ transplant tolerance or suppress autoimmunity.

Cerebral stroke induces massive Th1-shifted inflammation both in the brain and the periphery, contributing to the outcome of stroke. A Th1-type response is neurotoxic whereas a Th2-type response is accompanied by secretion of anti-inflammatory cytokines, such as interleukin-4 (IL-4). Interleukin-33 (IL-33) is a cytokine known to induce a shift towards the Th2-type immune response, polarize macrophages/microglia towards the M2-type, and induce production of anti-inflammatory cytokines. We found that the plasma levels of the inhibitory IL-33 receptor, sST2, are increased in human stroke and correlate with a worsened stroke outcome, suggesting an insufficient IL-33-driven Th2-type response. In mouse, peripheral administration of IL-33 reduced stroke-induced cell death and improved the sensitivity of the contralateral front paw at 5days post injury. The IL-33-treated mice had increased levels of IL-4 in the spleen and in the peri-ischemic area of the cortex. Neutralization of IL-4 by administration of an IL-4 antibody partially prevented the IL-33-mediated protection. IL-33 treatment also reduced astrocytic activation in the peri-ischemic area and increased the number of Arginase-1 immunopositive microglia/macrophages at the lesion site. In human T-cells, IL-33 treatment induced IL-4 secretion, and the conditioned media from IL-33-exposed T-cells reduced astrocytic activation. This study demonstrates that IL-33 is protective against ischemic insult by induction of IL-4 secretion and may represent a novel therapeutic approach for the treatment of stroke., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In present research work, for the first time, we demonstrate that neuropathogenesis in HIV-1 clade B and C infection is associated with IL-33 and ST2 dysregulation, that is, implication toward neuropathogenesis. It is known that neuropathogenesis of HIV infected individuals is clade dependent. Proinflammatory cytokines and related receptors play a significant role in the complex regulatory mechanisms of neuropathogenesis in HIV-1 infection. Among them, IL-33 is an inflammatory cytokine expressed in the central nervous system (CNS) and activates microglia cells and may affect neuroimmune inflammatory processes involved in HIV neuropathogenesis. Beside this, IL-33 receptor (ST2) plays a role in neuroinflammatory processes through the modulation of the biological action of IL-33. quantitative real time PCR (qRT-PCR), ELISA, Western blot (WB), and flow cytometry experiments were performed to elucidate the role of IL-33/ST2 in HIV neuropathogenesis in CNS cells. Apoptosis and mechanisms of IL-33 in neuronal cells were studied using caspase-3 assay and RT-PCR. Results of the studies suggest that the infection in CNS cells with HIV-1 clade B resulted in higher levels of IL-33/ST2L expression compared to HIV-1 clade C infection. Furthermore, higher concentrations of IL-33 were associated with a decrease in myocyte enhancer factor 2C (MEF2C) expression, a transcription factor that regulates synaptic function, and an increase in apoptosis, NOD2, and SLC11A1 in clade B infection. This led to neuroinflammation which dysregulates synaptic function and apoptosis. These parameters are common in neuroAIDS provoked by HIV infection.

Inflammation is regarded as one of the major hallmarks of tumors, and has a very close relationship with gastric cancer. Interleukin-33 (IL-33), a new member of the IL-1 family, plays an important role in both inflammatory disease and tumors. The present study was designed to explore the effects of IL-33 on the proliferation, drug sensitivity, and the invasiveness of gastric cancer cells in vitro. IL-33 at concentrations lower than 100 pg/ml did not alter the inhibitory rate of gastric cancer cells. Moreover, IL-33 at these low concentrations protected against platinum-induced apoptosis in various gastric cancer cell lines, yet not in normal gastric epithelial cells. We also found that IL-33 increased the activation of the JNK pathway, and enhanced the expression of ST2. Furthermore, SP600125, a selective inhibitor of the JNK pathway, significantly blocked the protective effects of IL-33 in gastric cancer cells. In addition, Matrigel invasion assay showed that IL-33 markedly promoted gastric cancer cell invasion. In conclusion, the present study demonstrated that IL-33 protected against platinum-induced apoptosis and promoted cell invasion via activation of the JNK pathway in gastric cancer cells. In light of the prevalence of platinum-based chemotherapeutics in the treatment of gastric cancer, our results suggest that the level of IL-33 should be monitored during the treatment of gastric cancer, particularly when using platinum-based chemotherapeutics.

Ischemic stroke causes brain injury with activation of an inflammatory response that can contribute to clinical impairment. As a novel cytokine of the interleukin-1 (IL-1) family, IL-33 has been suggested to be involved in regulating pathophysiology and inflammatory responses in the central nervous system (CNS). However, the role and underlying mechanisms of IL-33 in ischemic stroke remain poorly understood. Here, adult male C57BL/6 mice were subjected to middle cerebral artery occlusion (MCAO) for stroke induction. The MCAO procedure resulted in the enhanced Th1 and Th17 immune responses from 6h after transient cerebral ischemia/reperfusion even up to day 3. Meanwhile, the protein and mRNA level of IL-33 expression was significantly decreased at 6h and 72 h, but not at 24h after MCAO. Moreover, recombinant mouse IL-33 administration substantially attenuated ischemic brain damage and neurological deficit at 24h and 72 h, but not at 6h after MCAO. Interestingly, the reduced CNS inflammation in IL-33-treated MCAO mice may be at least partly due to an induced immuno-shift of Th cells from Th1 to Th2 response and suppressing Th17 immune response. These findings demonstrate that IL-33 can play a protective role after MCAO and may be a new target for therapy of ischemic stroke., (Copyright © 2014 Elsevier B.V. All rights reserved.)