IL-33 has been shown to be a promising biomarker in several types of cancer for tumor detection and as a predictor of prognosis and therapeutic response. Recently, IL-33 was shown to be correlated with a bad prognosis in several types of cancer, although in some cases IL-33 behaves as a tumor suppressor by inducing an immune response. In terms of bad prognosis, high levels of IL-33 were detected in the serum and tumors of patients with glioma (56), gastric cancer (57), hepatocellular carcinoma (58), uterine leiomyoma (59), lung cancer (60), colorectal cancer (61), head and neck squamous cell carcinoma (62), and breast cancer (63), when compared to corresponding healthy tissues. The Cancer Genome Atlas Pan-Cancer analysis project showed and declared that the level of IL-33 expression is altered in only 3% of ∼580 tumors and that the most common genetic alteration is the deletion of the IL-33 gene (64).

Lu and collaborators detected "significantly higher IL-33 expression in glioma tissues than in normal brain tissues through immune-histochemical (IHC) analysis" (56). High IL-33 expression in glioma was correlated with shorter overall survival (OS) and progression-free survival (PFS) (56).  This induces the activation of MEK-ERK, JNK-cJun, and STAT3 signaling pathways, both leading to cell proliferation (65). The expression levels of IL-33 and ST2 proteins were also positively correlated with the expression of Ki-67 in epithelial ovarian cancer tumors and at the metastatic site, and negatively correlated with the patient survival time (66). High expression of IL-33, assessed by IHC staining, was associated with advanced stage clear-cell renal carcinoma and abnormally high amounts of serum IL-33 was detected in patients with hepatocellular carcinoma or gastric cancer. Hence, IL-33 is correlated with a bad prognosis in these types of cancer (57, 58, 67). sST2 was also described as a negative prognostic marker when its serum concentration was associated with OS of patients with hepatocellular carcinoma (68). Nevertheless, this soluble IL-33 receptor can also be associated with a good prognosis in colorectal cancer, as the trapping of soluble IL-33 in the TME inhibits cancer growth and metastases (69). Moreover, the level of IL-33 protein has been inversely correlated with tumor grade and size in patients with pulmonary adenocarcinoma, showing an association of low IL-33 expression level with a poor prognosis (70–72). Likewise, a genome-wide association study unveiled a correlation between high IL-33 expression and a good prognosis in patients with osteosarcoma (73). However, if IL-33 can have a pro-tumor effect by directly targeting cancer cells, the tumor suppressor functions displayed by IL-33 are indirectly promulgated by immune surveillance as we will show hereafter.

# IL-33 AS A PRO-TUMORIGENIC CYTOKINE THROUGH ACTIONS ON CANCER CELLS AND TME

As described above, IL-33 is considered as a prognostic biomarker when expressed in tumors. This intratumoral IL-33 is expressed by cancer cells as well as by other cell components of the TME. For instance, in patients with head and neck squamous cell carcinoma and oral squamous cell carcinoma, intratumoral IL-33 has been shown to be expressed in cancer-associated fibroblasts (CAF) (62, 74). This infiltrating IL-33 has a direct protumorigenic effect on cancer cells and indirect effects on cellular components of the TME.

In the first case, Wang and collaborators showed that the IL-33/ST2 pathway up-regulated membrane glucose transporter 1 in non-small-cell lung cancer cells, enhancing their glucose uptake and glycolysis, thus favoring in vitro outgrowth of human lung cancer and its metastasis in a mouse model (60). By in vitro and in vivo experiments, IL-33 was also shown to be able to promote growth, invasion and migration of gastric cancer and colorectal cancer cells due to the autocrine secretion of several metalloproteases (MMP3, MMP9, MMP2), IL-6 and CXCR4 via the ST2-ERK1/2 pathway (61, 75). Moreover, IL-33 directly targets colon cancer cells and breast cancer cells via JNK-cJun activation, which promotes cell proliferation and therefore tumor growth (65, 76).

The impact of IL-33 on the TME encompasses angiogenesis, matrix remodeling and cytokine/growth factor production by non-epithelial cell components. The IL-33/ST2 signaling pathway, favoring pro-angiogenic VEGF expression in tumor cells and reducing tumor necrosis, is highly involved in mammary tumor growth (77). Concerning matrix modeling, human subepithelial myofibroblasts stimulated in vitro with IL-33 induced the expression of extracellular matrix components and growth factors associated with intestinal tumor progression (78). IL-33-stimulated cancer cells produce cytokines, and TME infiltrating immune cells are also involved in the expression of IL-6 in response to IL-33/ST2 signaling. Likewise, IL-33 stimulates the secretion of cytokines and growth factors in bone marrow myeloid and non-hematopoietic cells, resulting in myeloproliferation of neoplasms (79, 80). Indeed, suppression of IL-33 or high expression of sST2 suppresses IL-33-induced angiogenesis, TH2 responses, macrophage infiltration and M2 macrophage polarization. This negatively regulates tumor growth and metastatic spread of colorectal cancer, for instance through the modification of the TME (69). IL-33 in the TME recruits macrophages and stimulates their production of PGE2, and in turn, macrophage-derived PGE2 stimulates colon tumor development (76). The recruitment of macrophages in the TME might account for the stimulation of CCL2 expression by IL-33-stimulated cancer cells that express ST2, such as human colon cancer cells (81). After recruitment, macrophages are directly induced by IL-33 to be polarized in M2 tumor associated macrophages (TAM) in the TME. Such TAMs are then able to produce IL-10, VEGF, IL-6, and MMP9 which promote proliferation and invasiveness of cancer cells (82–84). Yang and collaborators showed that TAM are recruited by IL-33 in the TME, and IL-33-stimulated TAM can increase intravasation of tumor cells into the circulation at the early stages of metastasis (85). Even in the brain, IL-33 in the TME induces growth of glioma cells and facilitates microglia/macrophage infiltration (86). IL-33-stimulated macrophages are also activated to produce G-CSF, which in turn, boost myeloid-derived suppressor cells (MDSC) from the pro-tumoral TME (87). Indeed, MDSC contribute to tumor-mediated immune escape by suppressing antitumor immune responses. IL-33 released in tumor tissues in breast and colorectal cancer mouse models and in breast cancer patients, has been shown to facilitate MDSC expansion, recruitment and survival in the TME. This role could be due to the induction of an autocrine secretion of GM-CSF (88– 90). Interestingly, another study showed that IL-33 does not affect the number of MDSC but can significantly reduce the differentiation of lineage-negative bone marrow progenitor cells into granulocytic MDSC in tumor-bearing mice. Moreover in the same study, IL-33-treated MDSC were shown to be less immunosuppressive, with a reduced capacity to inhibit T cell proliferation and IFN-γ production, production of reactive oxygen species and their capacity to induce Treg differentiation and expansion (91). IL-33 has a direct effect on Treg cells expressing surface ST2. Indeed, these lymphocytes are constitutively abundant in the intestine and able to prevent dysregulated inflammatory responses to self and environmental stimuli. IL-33 is constitutively expressed in epithelial cells at barrier sites. High levels of IL-33 were also observed in inflamed lesions of patients with inflammatory bowel disease, supporting its role in disease pathogenesis (92, 93). In inflammatory conditions, IL-33 signaling in Treg cells enhances transforming growth factor (TGF)-β1-mediated differentiation. Alternatively, IL-33 may provide a signal necessary for inducing their accumulation and maintenance in inflamed tissues (94). Local accumulation of Treg cells has been described in intestinal tumors preventing tumor clearance in mouse models and in patients. This role may be associated with a reduction of Ecadherin expression, increased β-catenin signaling and IL-33 production by malignant and injured epithelial cells (95). In tumors with low levels of infiltrating Treg cells, administration of IL-33 accelerates tumor growth and occurrence of liver and lung metastasis in breast cancer mouse models, and these models display an intratumoral accumulation of MDSC and Treg cells, as compared to untreated mice (90). Moreover, IL-33 blockade, in addition to abrogating the polarization of TAM, reduces the accumulation of Treg cells in lung tumors of human lung preclinical mouse models (82). However, as inflammation contributes to tumorigenesis, the accumulation of Treg in inflammatory zones must contain inflammation and therefore tumorigenesis. Treg may promote or inhibit tumor development depending on the context, revealing the complex relationship between inflammation, and cancer development. Furthermore, mast cells which also express ST2 receptors and respond to cell injury via IL-33 released from necrotic cells, can secrete leukotrienes and cytokines to initiate pro-inflammatory responses (96). In a colorectal cancer mouse model, IL-33 deficiency reduced mast cell accumulation in tumors. This

deficiency further inhibited the expression of mast cell-derived proteases and cytokines that promote polyposis (78, 96–98). Generally, mast cells accumulate in inflamed gut and in colorectal tumors, and their presence is correlated with a poor prognosis and low overall survival (99, 100). In skin cancers, dermal mast cells are able to respond to UVB-induced IL-33 by releasing IL-10 to protect skin homeostasis after excessive UVB exposure. However, IL-10 may contribute to skin cancer development, as IL-10-deficient mice do not develop skin tumors upon UVB exposure (101, 102).

#### DIRECT OR INDIRECT EFFECTS OF IL-33 AS A TUMOR SUPPRESSOR

Alongside its pro-tumorigenic role, IL-33 can also behave as a tumor suppressor. Only one study has shown a direct anti-tumor effect of this cytokine with the in vitro inhibition of proliferation and induction of apoptosis of MIA PaCa-2, a pancreatic cancer cell line (103). However, its anti-tumor functions were largely associated with the activation of immune effector cells able to lead to tumor clearance. All immune cells express the ST2 receptor and are able to respond to IL-33 stimulation. IL-33 has a significant role in cancer immune-surveillance in primary prostate and lung tumors, which can be lost during the metastatic transition inducing immune escape. The correlation between IL-33 and HLA expression in human tumors using RNAsequencing data of resected prostate tumors was recently shown. The down-regulation of IL-33 during the metastatic process ultimately decreases the functionality of HLA-I and reduces immune-surveillance favoring tumor development (104). In a multivariable analysis, the infiltration of human hepatocellular carcinomas (HCC) by cells expressing IL-33 and by CD8+ T cells was associated with prolonged patient survival. These results led to propose an HCC immune score identifying high- vs. low-risk patients with different gene expression profiles (105). Injection of IL-33 into established murine melanoma or acute myeloid leukemia models inhibits tumor growth in a CD8+ T celldependent manner prolonging the survival of mice. In the first model, the reduction of tumor growth delay was correlated with intratumoral accumulation of CD8+ T cells, and a decrease in the number of immunosuppressive myeloid cells (106). In the second model, the anti-leukemia activity was associated with increased expansion and IFN-γ production of leukemia-reactive CD8+ T cells (107). Moreover, the correlation between decreased IFN-γ secretion and colon cancer aggressiveness, suggests that IL-33 signaling defects may impair the generation of IFN-γ-mediated immunity (108). In soft tissue sarcoma, higher transcriptional levels of IL-33 were also associated with a good prognosis. The expression of IL-33 has also been negatively correlated with the expression of chemokines, such as TGF-β, recruiting Treg and MDSC, and positively correlated with the expression of chemokines that recruit CD8+ T cells which promote antitumor immune responses especially through INF-γ production (109).

It has been shown that IFN-γ-producing cells present in tumors associated with an IL-33 antitumor effect, were CD8+ T cells and NK cells. Indeed, IL-33 expression in several cancers affects the number of CD8+ T cells and NK cells in tumor tissues and the production of IFN-γ/TNF-α, thereby favoring tumor eradication through tumor cell cytolysis (110, 111). This was also shown with the reduction of tumor metastasis in B16 melanoma and Lewis lung carcinoma metastatic models thanks to the transgenic expression of IL-33. In these transgenic mice models, tumor infiltration and CD8+ T lymphocyte and NK cell cytotoxicity was significantly increased compared to non-transgenic mice. Moreover, treatment with recombinant IL-33 increased CD8+ T lymphocyte and NK cell cytotoxicity in vitro (112). CD8+ T cells are also indirectly stimulated by IL-33 through DC. DC maturation is promoted by IL-33 which increases their cross presentation ability particularly during the anti-leukemia or anti-melanoma immune response (107, 113). As mentioned in the introduction, IL-33-activated DC are also able to promote the differentiation of TH17 cells which play an important role in cancer development. TH17 are T helper lymphocytes secreting IL-17 and other inflammatory cytokines, but can also display immunosuppressive activities, therefore mediating context dependent pro- or antitumor responses (114, 115). However, there are no published studies mentioning a direct relationship between TH17 cells and IL-33 in cancers. TH17 cells expressing the ST2 receptor were found to accumulate in the small intestine in bowel diseases where intestinal epithelial cells are the providing source of IL-33, we can therefore stipulate that these cells could play a role in digestive cancers. TH17 cells could have an anti-tumor function with the production of pro-inflammatory cytokines (116) or a pro-tumor role when they can acquire a regulatory phenotype with immunosuppressive properties upon IL-33 activation (117). Furthermore, DC can also drive TH9 cell dependent anti-tumor responses through the expression of Ox40L when activated by IL-33 and stimulated by dectin-1 signaling (118–120).

Finally, the ILC2s which support type 2 immune responses by producing IL-5 and IL-13 in response to IL-33 could also have an antitumor function. Indeed, their tissue-repair function can induce cholangiocarcinoma and liver metastasis (121). ILC2s can also be mobilized from the lung and other tissues thanks to IL-33, to penetrate tumors, mediate immune-surveillance with DC, and promote adaptive cytolytic T cell responses and attraction (122, 123).

# CONCLUDING REMARKS

IL-33 therefore appears as a pro-tumorigenic cytokine that can also limit tumor growth through the activation of antitumor immunity. These opposing roles in tumorigenesis, as shown in this review, greatly depend on the IL-33/ST2 signaling in different immune cells. IL-33 is able to promote inflammatory events which contribute to tumorigenesis whilst activating antitumor immune responses. The different events promoted by IL-33 activation of various immune cells which can be found in the TME are summarized in the **Figure 1**. Depending on the tumor context, IL-33 produced in the TME can activate diverse

pro-tumor (green) or anti-tumor (blue) processes leading to the development or to the regression of the tumor. Some cytokines produced by pro-tumor cells such as MDSC or TAM, are also able to produce cytokines which inhibit anti-tumor cells such as all the TH1 cells.

immune cells which are able to promote a pro-tumor effect such as TAM, MDSC, fibroblasts, mast cells, Treg and DC, or to prevent tumor development such as NK cells, CD8+ T cells, iNKT, ILC2, TH9, and TH17. All these cell types produce specific cytokines, chemokines, and other molecules. These conclusions are supported by Wasmer and Krebs' review who demonstrated the multiple functions of IL-33 in different cancer types (124). As many cytokines with immunomodulatory properties, IL-33 has been considered for anticancer immunotherapies. However, knowing its dual role, therapeutic manipulation of this cytokine should be considered with caution. The majority of the studies mentioned propose cancer immunotherapy strategies based on exogenous IL-33 administration. These IL-33 adjuvanted vaccines aim at activating the immune cells involved in the immune response (106, 107, 125–128). IL-33 could also indirectly activate effector T cells. For instance, a replicating viral vector system used in cancer immunotherapy which delivers tumor-associated antigens to DC for efficient cytotoxic T cells priming, depends on IL-33 signaling (129). IL-33 could likewise increase T cell activation to promote graftvs.-leukemia (GVL) reactions while decreasing fatal graft-vs. host- disease (GVHD) (130). Possibly however, ST2 blockade might preserve GVL activity by blocking Treg controlling GVHD (131). Indeed, considering the immunosuppressive protumorigenic role of IL-33, others have proposed to block IL-33 as a novel anticancer strategy (62, 65, 69, 76, 82, 88). In the future, IL-33 targeting in cancer immunotherapies should be considered with caution, especially taking into account the

intricate dual role of this cytokine in cancer as shown in this review.

## AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

#### REFERENCES

#### ACKNOWLEDGMENTS

This work was supported by institutional grants from the Institut National de la Santé et de la Recherche Médicale (INSERM), the Université Toulouse 3, the Centre National de la Recherche Scientifique (CNRS) and the Laboratoire d'Excellence TOUCAN.

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proliferation. J Clin Invest. (2014) 124:3241–51. doi: 10.1172/JCI 73742

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Fournié and Poupot. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Pleiotropic Immunomodulatory Functions of IL-33 and Its Implications in Tumor Immunity

Claudia Afferni 1 , Carla Buccione2 , Sara Andreone2 , Maria Rosaria Galdiero3,4 , Gilda Varricchi 3,4, Gianni Marone3,4,5, Fabrizio Mattei 2 \* and Giovanna Schiavoni 2 \*

1 National Center for Drug Research and Evaluation, Istituto Superiore di Sanità, Rome, Italy, 2 Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy, 3 Department of Translational Medical Sciences and Center for Basic and Clinical Immunology Research (CISI), University of Naples Federico II, Naples, Italy, 4 WAO Center of Excellence, Naples, Italy, 5 Institute of Experimental Endocrinology and Oncology "Gaetano Salvatore", National Research Council, Naples, Italy

#### Edited by:

Hui-Rong Jiang, University of Strathclyde, United Kingdom

#### Reviewed by:

Xiao-Qing Wei, Cardiff University, United Kingdom Padraic Fallon, Trinity College, Dublin, Ireland

> \*Correspondence: Fabrizio Mattei<EMAIL_ADDRESS>Giovanna Schiavoni<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Cytokines and Soluble Mediators in Immunity, a section of the journal Frontiers in Immunology

Received: 24 July 2018 Accepted: 22 October 2018 Published: 13 November 2018

#### Citation:

Afferni C, Buccione C, Andreone S, Galdiero MR, Varricchi G, Marone G, Mattei F and Schiavoni G (2018) The Pleiotropic Immunomodulatory Functions of IL-33 and Its Implications in Tumor Immunity. Front. Immunol. 9:2601. doi: 10.3389/fimmu.2018.02601 Interleukin-33 (IL-33) is a IL-1 family member of cytokines exerting pleiotropic activities. In the steady-state, IL-33 is expressed in the nucleus of epithelial, endothelial, and fibroblast-like cells acting as a nuclear protein. In response to tissue damage, infections or necrosis IL-33 is released in the extracellular space, where it functions as an alarmin for the immune system. Its specific receptor ST2 is expressed by a variety of immune cell types, resulting in the stimulation of a wide range of immune reactions. Recent evidences suggest that different IL-33 isoforms exist, in virtue of proteolytic cleavage or alternative mRNA splicing, with potentially different biological activity and functions. Although initially studied in the context of allergy, infection, and inflammation, over the past decade IL-33 has gained much attention in cancer immunology. Increasing evidences indicate that IL-33 may have opposing functions, promoting, or dampening tumor immunity, depending on the tumor type, site of expression, and local concentration. In this review we will cover the biological functions of IL-33 on various immune cell subsets (e.g., T cells, NK, Treg cells, ILC2, eosinophils, neutrophils, basophils, mast cells, DCs, and macrophages) that affect anti-tumor immune responses in experimental and clinical cancers. We will also discuss the possible implications of diverse IL-33 mutations and isoforms in the anti-tumor activity of the cytokine and as possible clinical biomarkers.

Keywords: IL-33, cancer, immune cell subsets, tumor immunology, IL-33 isoforms

# INTRODUCTION TO IL-33 BIOLOGY

Interleukin-33 (IL-33) is a cytokine member of IL-1 family, including IL-1α, IL-1β, IL-18, and IL-1Ra that are related to each other by receptor structure and signal transduction pathways. These cytokines share a conserved structure of β-trefoil fold comprised of 12 anti-parallel β-strands that are arranged in a three-fold symmetric pattern. The β-barrel core motif is packed by various amounts of helices in each cytokine structure (1). IL-33 was initially described in 2003 by Girard's group as a nuclear protein abundantly expressed in high endothelial venules (HEVs), specialized blood vessels that mediate the entry of lymphocytes into lymphoid organs and therefore named "nuclear factor from high endothelial venules" (NF-HEV) (2).

It is now known that IL-33 is a chromatin-associated nuclear cytokine in vivo through chromatin-binding motif within its Nterminal nuclear domain, suggesting that nuclear localization and binding to histones are important for IL-33 function and regulation (3). Nuclear IL-33 can function as a transcriptional repressor when overexpressed in transfected cells, although there is still no direct evidence that endogenous nuclear IL-33 regulates gene or protein expression (4). IL-33 is constitutively expressed in different human and mouse tissues in the steady-state, including epithelial, endothelial, fibroblast-like cells, and myofibroblasts and its expression can be increased during inflammation (2, 5). After cell stress or necrosis, IL-33 is released into the extracellular space and functions as an endogenous danger signal that alerts the immune system of tissue damage during trauma or infection. Indeed, IL-33 is considered an "alarmin" able to activate different actors of the innate immune system, mediating a variety of immune reactions including anti-cancer immune responses (6). Here, we will review the biological role of IL-33 affecting immune responses with particular emphasis on anti-tumor immunity.

#### IL-33 Isoforms

Similar to IL-1β and IL-18, IL-33 is synthesized in a full-length form (amino acids 1–270) that is found in the nucleus, in the cytosol and outside the cell. As IL-1β and IL-18, IL-33 is cleaved intracellularly by the enzyme caspase-1 before release outside the cell. This process requires the NLRP3 inflammasome, which can be activated in response to endogenous and exogenous danger signals. This NLRP3 inflammasome leads to Caspase-1 activation and, in turn, to IL-33 processing and release (7). When cells undergo necrosis or injury, full-length IL-33 is released in the extracellular space where it is cleaved by inflammatory proteases. During apoptosis, a process that does not trigger inflammation in vivo, IL-33 is cleaved and inactivated by endogenous caspases (8–10). Processing by apoptotic caspases is an important regulatory mechanism that limits or suppresses the pro-inflammatory properties of IL-33 during homeostatic cell turnover. Another regulatory mechanism limiting IL-33 activity is oxidation. Extracellular IL-33 is susceptible to cysteine oxidation that leads to the formation of disulphide bridges, resulting in conformational changes that inhibit the binding to ST2 receptor, thus rapidly inactivating IL-33 following allergen exposure (11).

Recent studies have demonstrated the existence of several human full-length active mRNA splice variants dependent on both the cell type expressing IL-33 and the pathological condition and triggered by diverse stimulations during immune responses (12–14). Of note, inflammatory proteases from neutrophils (proteinase 3, elastase, and cathepsin G) (15), mast cells (chymase, tryptase, and granzyme B) (16), and environmental allergens (17) can process full-length IL-33 into shorter mature forms (18–21 kDa) whose biological activity is 10- to 30-fold more potent than the full-length form (see **Figure 1**). The mature form does not translocate into the nucleus because it lacks the nuclear localization signal found in full-length IL-33 (18, 19). Proteolytic cleavage of IL-33 was shown to induce allergic inflammation in vivo (17) highlighting a novel mechanism by which inflammatory and environmental proteases can amplify allergic inflammation. Of interest, isoform variants as well as cleavage by endogenous and exogenous proteases has been described also for other epithelial-derived cytokines, such as thymic stromal lymphopoietin (TSLP), resulting in pleiotropic functions in health and disease (20). Although both isoforms are biologically active the relative importance of full length and mature IL-33 forms in vivo remains unclear (2, 21). In a mouse model of lung delivery of recombinant adenoviruses encoding IL-33 isoforms the full-length IL-33 induced inflammation in an ST2-independent fashion, but not pulmonary eosinophilia, goblet cell hyperplasia, or Th2 skewing, whereas mature IL-33 induced ST2-dependent Th2-associated effects. Both isoforms had similar effects on gene expression, suggesting that the different effects are due to differential utilization of the ST2 receptor (22). In addition, in a mouse model of DNA cancer vaccine, delivery of either full-length or mature IL-33 as an immunoadjuvant induced potent Th1 and cytotoxic T cell (CTL) associated anti-tumor immunity and complete regression of established TC-1 tumor in mice. Interestingly, the full-length IL-33 was more potent than mature IL-33 in expanding the humoral immune response (23).

### The IL-33/ST2 Axis

IL-33 exert its cytokine activity through binding to its primary specific receptor ST2, which is dependent on the co-receptor, IL-1 receptor accessory protein (IL-1RAcP), and the adaptor protein MyD88 for signaling (24). The crystal structure of IL-33 with ST2 has revealed that surface charge complementarity is crucial for specific binding (25). The gene that encodes for ST2 produces its transmembrane receptor but also produces a soluble form of ST2 (sST2), which acts as a binding decoy for IL-33 and thus downmodulates IL-33 activity during inflammatory responses, such as in experimental allergic asthma (26) and collagen-induced arthritis (27). Most hematopoietic cells express ST2. ILC2s, some Treg cells, and mast cells are the primary tissue-resident cells that constitutively express high levels of ST2, implying that these cells are initial targets of IL-33 (3). Non-hematopoietic cells, including endothelial cells, epithelial cells, and fibroblasts, are reported to express ST2 and respond to IL-33, although the in vivo consequences of signaling in these populations are less well-characterized.

In hematopoietic cells, IL-33 acts primarily on immune cells associated with type 2 and regulatory immune responses, including ILC2s, Th2 cells, eosinophils, mast cells, and basophils, as well as subsets of dendritic cells, myeloid-derived suppressor cells, and Tregs (28). However, it is now clear that the action of IL-33 is not limited to the activation of type-2 immune responses. Indeed, recent studies have revealed important roles of IL-33 in the activation of immune cells involved in type-1 immunity, such as Th1 cells, NK cells, CD8+ T cells, neutrophils, macrophages, B cells, and NKT cells (19, 29, 30). This pleiotropic nature of IL-33 (**Figure 2**) is likely to explain why IL-33 has been implicated in a wide variety of non-allergic diseases, including infectious diseases (fungal, helminth, protozoa, bacterial, and viral infection), cardiovascular diseases, chronic obstructive pulmonary disease (COPD), fibrotic diseases, musculoskeletal diseases, inflammatory bowel diseases, diseases of the central

(ST2) on a plethora of ST2-expressing cells. In other cases this active IL-33 does bind to the soluble form of its receptor (sST2), which acts as a decoy receptor. In this latter event the effect of IL-33 will be suppressed by the formation of an sST2/IL-33 decoy complex. When no apoptotic nor inflammatory enzymes are produced, an uncleaved form of IL-33 is released, with a very low biological activity compared to that showed by IL-33 cleavage products originated by neutrophil and mast cell-derived enzymatic cutting. When the IL-33 is cleaved by certain environmental allergens, their enzymatic activity at the IS site gives rise to multiple peptide products sharing the whole IL-1 like region of IL-33, that does retain the ability to bind ST2. The IL-33 cleavage products herein shown are all equipped with the ST2 binding sequence (inside the IL-1 like region). The secondary fragments lacking the ST2 binding sequence and generated during the cleavage reaction are not depicted and have no effect on IL-33/ST2 binding.

nervous system (Alzheimer), graft vs. host disease (GVHD), obesity, diabetes, and cancer (3).

# IMMUNE CELL TARGETS OF IL-33 AFFECTING TUMOR-IMMUNE RESPONSES

# CD4+ Th Cells

Naïve CD4+ T helper cells constitutively express ST2 and stimulation with IL-33 skews their differentiation toward a Th2 phenotype. CD4+ T cells are needed in the effector phase of a protective antitumor immune response against tumors lacking MHC class II (31). However, human CD4+ T cells can suppress tumors expressing adequate levels of MHC class II and selfantigens on their surface, through secretion of IFN-γ or direct tumor killing (32). Interestingly, Villareal et al. demonstrated that IL-33 can be an effective adjuvant when combined with an HPV16 E6/E7-encoded DNA vaccine, enhancing both antigen specific CD4+ and CD8+ IFN-γ + T cells, and antigen specific IgG concentration in the serum, leading to regression of

established TC-1 tumor in mice (23). In accordance with this study, Mousa Komai-Koma et al. showed that IL-33 may promote CD4+ T helper 1 (Th1) differentiation by a mechanism depending on IL-12 and ST2. IL-33 and IL-12 synergistically increase both ST2 and IL-12R expression in early activated CD4+ T cells. These data indicate that IL-33 promotes Th1 cell development, while it is ineffective on mature Th1 cells (33). A possible explanation for such differences is that ST2 expression is induced only in early-TCR activated naïve CD4+ T cells and is then gradually inhibited when Th1 cells fully mature. Although the signaling pathway by which IL-33 enhances Th1 polarization is still unknown, it is likely that IL-33 inducing Th1 or Th2response depends on the cytokine milieu, in particular the balance of IL-12 and IL-4 levels in vivo (33). Of note, IL-33 also promotes the differentiation of IL-9-producing Th cells (34) which exert potent antitumor immune responses in vivo (35, 36).

# CD4+ Treg Cells

mast cells can produce IL-33.

ST2/IL-33 signaling is known to expand suppressive CD4+ Foxp3+ GATA3+ Treg cells in vivo and in vitro (37). IL-33-expanded Tregs express ST2 and can be found in several immune and non-immune tissues exerting potent suppressor function in a variety of pathological conditions, such as autoimmunity, inflammation, transplantation, and allergy (38). ST2+ Treg expansion can be mediated by IL-33 signaling in DCs, through production of IL-2, which selectively expands ST2+ Tregs (39). In the intestine, particularly rich in ST2+ Treg cells, IL-33 signaling stimulates transforming growth factor (TGF)-β1-mediated differentiation of Treg cells and provides a signal for Treg-cell accumulation and maintenance in inflamed tissues (40). In ApcMin/+ mice, epithelial-derived IL-33 promoted the expansion of ST2+ Treg cells in the colon correlating with increased tumor burden (41, 42). A similar observation was recently reported in the CT26 adenocarcinoma model, where rIL-33 administration to tumor-bearing mice promoted, while IL-33 blockade reduced, the expansion of ST2+ Treg cells in tumor tissue and spleen (43). Moreover, IL-33 blockade reduced accumulation of Treg cells in tumor microenvironment and inhibited tumor growth in a preclinical model of human non-small-cell lung cancer (NSCLC) xenografts (44). In contrast, some studies have reported inhibitory effects of IL-33/ST2 on Treg cells expansion. In a melanoma mouse model, IL-33 was shown to inhibit Treg infiltration in the tumor microenvironment indirectly, through stimulation of MDSCs, which had reduced capacity to induce the differentiation or expansion of Treg cells in vitro (45). A recent study using reciprocal bone marrow chimeras in a mouse model of sporadic colon cancer, genetic ablation of ST2 in both hematopoietic and non-hematopoietic compartments leads to increased tumorinfiltrating ST2+ Foxp3+ Tregs and enhanced colon tumor development (46).

# CD8+ T Cells

Unlike CD4+ T cells, only effector CD8+ T cells or polarized Tc1 cells, but not naïve and early activated CD8+ T cells, express ST2 (47). High expression of ST2 in CD8+ T cells cultured in Tc1 polarizing conditions is regulated by T-bet, a master transcription regulator of Th1 effector functions. Moreover, it was shown that IL-12 and IL-33 synergistically increased Tbet and Blimp1, transcription factors critical for effector fate of CD8+ T cell (47). Recent studies from transplantable solid tumor models have indicated a direct role of exogenous IL-33 in promoting antitumor CD8+ T cell immunity using either IL-33 transgenic mice (48), IL-33 DNA as vaccine adjuvant (23), or IL-33 expressing tumor cells (49). Systemic administration of rIL-33 in melanoma tumor bearing mice, promoted expansion, increased tumor infiltration and effector function of antigenspecific CD8+ IFN-γ + T cells by both a direct or DCsmediated effect (50). In the aggressive C1498 acute myeloid leukemia (AML) model, IL-33 treatment significantly increased the percentage of effector memory liver CD8+ T cells leading to delayed leukemia development and improved overall survival (51). This finding suggest a role of exogenous IL-33 in promoting rapid expansion of the effector memory CD8+ T cell pool, consistent with the results from solid tumor models (23, 48). In this study the increased CD8+ T cells activation level upregulates PD-1/PD-L1 expression in vivo, therefore combination of PD-1 blockade and IL-33 treatment further improves survival of leukemia-bearing mice (51).

### Type-2 Innate Lymphoid Cells

Innate lymphoid cells (ILCs), belonging to the family of innate cells, are characterized by classic lymphoid cell morphology, but lack lineage-specific markers and somatically rearranged antigen receptors. Based on the expression of transcription factors, phenotypic markers, and effector cytokine production profiles, ILCs have been divided into three distinct subclasses: group 1 ILCs, group 2 ILCs, and group 3 ILCs (52). ILC are derived from a common lymphoid progenitor and possess a wide range of cell surface markers, many of which have only recently been elucidated (53). ILC2, originally identified in the mouse and human mesenteric lymph nodes as lineage marker negative, c-kit+, Sca-1+, IL-7Ra+, and ST2+ cells (54), were also found in lung, skin, and gut, while only a small number of circulating ILC2s can be detected in blood (55). They are involved in tissue repair (56), anti-helminth immunity (57), and allergic inflammation (58).These cells are dependent on transcription factor GATA-binding protein 3 for their development and maintenance (59). Activation of ILC2s by alarmins (IL-25, IL-33, and TSLP) secreted by epithelial cells upon cellular stress and tissue damage (55, 60), produce IL-5, IL-13 (54), IL-4, IL-6, IL-9, and amphiregulin which induce Th2 differentiation (61). This group of innate cells was often observed to infiltrate tumors in humans, but their role seems more frequently associated with cancer progression than restriction. Clinical studies suggested that increased numbers of ILC2s in peripheral blood of patients with gastric cancer, could contribute by cytokines they secrete to the immunosuppressive environment maintained by CD4+ T helper 2 (Th2), myeloid-derived suppressor cells (MDSC), and macrophages (62). ILC2s might also induce immune suppression via secretion of amphiregulin (63), which enhances Treg activity in vivo and can thereby inhibit antitumor immune responses induced by DC vaccination (64). The anti-tumoral activity of ILC2 was described for the first time by Ikutani et al. in a mouse model of lung metastatic melanoma. Following tumor induction, administration of rIL-33 induced the development of IL-5 producing ILC2, which recruited and maintained eosinophils responsible for tumor cell death and tumor metastasis prevention (65). Overexpression of IL-33 in several tumor cell lines induced high numbers of ILC2s, when transplanted in mice, with potent anti-tumoral activity. The latter study suggests that local production of IL-33 induces ILC2 to release CXCR2 ligands able to sustain the expression of CXCR2 on tumor cells and induce their apoptosis (66).

### NK and NKT Cells

IL-33 directly activates both human (19) and mouse (30) NKT and NK cells inducing IFN-γ production via cooperation with IL-12, thus contributing to establish Th-1 immunity. During viral infection, IL-33/ST2 axis amplifies the expansion of NK cells and enhances host defense (67, 68). The role of IL-33 on NKT in cancer immunity is unknown. In contrast, a number of reports have analyzed the effects of IL-33 on NK cell expansion and/or activation in tumor-bearing mice. In mouse experimental metastasis models of B16 melanoma and Lewis lung carcinoma, transgenic expression of IL-33 in the host promoted the recruitment of cytotoxic NK cells to the pulmonary site that inhibited metastasis formation (48). In vitro, IL-33 directly activated NK cell cytotoxicity, stimulated NF-κB and up-regulated CD69 expression (48). Furthermore, B16 and 4T1 tumor cells overexpressing IL-33 implanted into syngeneic mice induced IFN-γ + NK cells in tumor tissue that mediated IL-33 anti-tumoral effect (49). Similarly, increased frequencies of CD107a+IFN-γ + NK cells were observed following exogenous administration of IL-33 in spleens and tumors of B16 melanomabearing mice (69). In contrast with these reports, previous studies in 4T1 breast cancer model showed that IL-33/ST2 signaling impairs NK cell activation. ST2-deficient mice bearing 4T1 tumors exhibited increased numbers of activated NK cells (IFNγ + CD27high CD11bhigh, CD69+ KLRG−) and NK cytotoxic activity, with respect to wild-type (WT) counterparts. In vivo depletion of NK cells accelerated 4T1 tumor growth in ST2−/− mice (70). Moreover, exogenous administration of IL-33 to WT 4T1 tumor-bearing mice decreased NK cell activation and cytotoxicity and promoted tumor progression (71), thus suggesting a detrimental role for IL-33 in NK cell-dependent

anti-tumor responses. These contrasting results suggest that IL-33 may exert opposing effects on NK cells within the tumor microenvironment depending on the levels of IL-33 expressed and on the primary target cells.

# Macrophages

Several lines of evidence indicate that IL-33 amplifies the expression of M2 markers on macrophages in vitro and in vivo, thus promoting the suppressor function of tumorassociated macrophages (TAMs). Blockade of IL-33 abrogates the polarization of TAMs into (alternatively activated) M2 macrophages in a model of human non-small-cell lung cancer (44). Expression of IL-33 was found to stimulate the recruitment of M2-like macrophages into the cancer microenvironment in mouse models of colon (41, 72–74) and breast cancer (71) correlating with tumor progression. Of note, IL-33 stimulated macrophages to produce prostaglandin E2, which supported colon cancer stemness (73). Furthermore, in mouse tumor xenografts IL-33 was shown to promote metastasis through recruitment of M2-like TAMs (75). Recently, it was shown in the mouse monocyte/macrophage line RAW264.7 that IL-33 directly induces MMP-9 expression, which facilitates tumor progression, invasion, and angiogenesis (76). These observations indicate that IL-33/ST2 signaling on macrophages promotes M2 polarization, immunosuppression, and tumor progression.

# Dendritic Cells

Although dendritic cells (DCs) express low levels of ST2 on their cell surfaces, they respond to IL-33 by up-regulating MHC-II, CD40, CD80, CD86, OX40L, CCR7, and by increasing production of several cytokines (IL-4, IL-5, IL-13, TNF-α, and IL-1β) and chemokines (CCL17 and CCL22) (77–80). In addition, IL-33-activated DCs promote an atypical Th2-type of immune response inducing IL-5- and IL-13-producing CD4+ T cells in vitro and in vivo (77, 79), which can be further amplified during allergic inflammatory response via ST2 (78, 81). In vivo, IL-33 exposure induces DC recruitment and activation in the lung (78, 79). IL-33 promotes the expansion of DCs from bone marrow (BM), by stimulating the secretion of basophilsderived GM-CSF. However, such IL-33 differentiated BM-DCs expressed low levels of MHC-II but high PD-L1 and PD-L2 immune checkpoints on the surface, and displayed reduced capacity to prime naïve T cells (82). In support of this potential tolerogenic effect, IL-33 has been shown to promote IL-2 secretion by murine DCs, thus supporting the in vitro and in vivo expansion of ST2-expressing Treg cells (39). In mice bearing 4T1 breast cancer IL-33 administration increased the percentage of splenic CD11c+ DCs expressing IL-10 (71). In contrast, in a murine AML model, systemic IL-33 administration promoted DC activation and "licensing" for cross-priming of tumorreactive CD8+ T cells (51). Likewise, in EG7 lymphoma, B16, and inducible BrafV600EPTEN melanoma models exogenous IL-33 activated myeloid DCs within the tumor microenvironment increasing antigen cross-presentation and restoring anti-tumor T cell activity in a ST2, MyD88, and STAT1-dependent manner (50). On the whole, these results suggest that IL-33 depending on the context can stimulate DC antigen presentation and, thus, anti-tumor immune responses, or induce tolerogenic features, thus supporting tumor growth.

## Myeloid-Derived Suppressor Cells

Myeloid-derived suppressor cells (MDSCs) are closely related to granulocytes and monocytes but differ from them in that they are absent in healthy individuals but expand under pathological conditions, such as cancer, exerting potent immune suppressive role (83). Several reports described the ability of IL-33 to expand MDSCs in vivo during tumorigenesis. In 4T1 breast cancer model, IL-33 has been reported to promote MDSC expansion (71). Exogenous administration of IL-33 increased intratumoral and systemic accumulation of CD11b+ Gr-1+ MDSCs that expressed TGF-β1 and IL-13α1R with higher incidence of monocytic vs. granulocytic MDSCs. Fittingly, absence of IL-33/ST2 signaling reduced the accumulation, proliferation, and immunosuppressive ability of MDSCs in tumor-bearing mice (71, 84). Moreover, IL-33 upregulated in MDSCs the expression and activity of arginase-1 in vitro and activated NF-κB and MAPK signaling in vivo, which augment their immunosuppressive ability (84). Conversely, in vitro IL-33 was shown to negatively regulate MDSC development from BM progenitor cells inhibiting the differentiation of granulocytic MDSCs (G-MDSCs), but not of monocytic MDSCs (M-MDSCs). In addition, IL-33-treated BM-derived MDSCs exhibited diminished immunosuppressive capacity, reduced inhibition on T-cell proliferation and IFN-γ production, and diminished production of ROS (45). In B16 melanoma mouse models, IL-33 administration was shown to decrease MDSCs accumulation in the spleen and tumor microenvironment (45, 69). These evidences indicate that IL-33 may promote or halt MDSCs expansion depending on the tumor type.

#### Neutrophils

IL-33 directly acts on murine neutrophils in the lungs (85). Indeed, IL-33-treated neutrophils produced IL-4, IL-5, IL-9, and IL-13 and displayed a distinct gene expression profile in contrast to resting and lipopolysaccharide (LPS)-treated neutrophils. These neutrophils were found in the lungs of ovalbumin (OVA)-induced mouse model of asthma. Adoptive transfer of IL-33-driven neutrophils significantly worsened the severity of the disease in this model (86). In vitro, IL-33 promoted IL-4, IL-5, IL-9, and IL-13 expression in murine neutrophils in time- and dose- dependent manner. IL-33-induced neutrophils expressed high levels of CXCR1, CCR1, IL-1R2, and CXCR2 mRNAs compared with LPS-induced neutrophils. In a mouse model of choriomeningitis virus-induced hepatitis, IL-33 promoted neutrophil recruitment in the liver and dampened liver injury by limiting T-cell activation. Liver neutrophils displayed an immunosuppressive phenotype, characterized by high levels of arginase-1, iNOS and IL-10 (87). Thus, IL-33 may promote inflammatory or regulatory neutrophils, depending on the pathological condition. Little is known about the effects of IL-33 on neutrophils in tumor immunity. In a mouse model of ectopic CT26 colon carcinoma, systemic chemotherapy with irinotecan induced intestinal mucositis, associated with the induction of IL-33, and increased neutrophil accumulation in the intestine. Supernatants from intestine explants treated with irinotecan enhanced migration of neutrophils in vitro in an IL-33/CXCL1/2/CXCR2-dependent manner. Importantly, IL-33 blockade reduced mucositis and enabled prolonged irinotecan treatment of ectopic colon carcinoma leading to a beneficial outcome of the chemotherapy. These results suggest that inhibition of the IL-33/ST2 pathway may represent a novel approach to limit mucositis and improve the effectiveness of chemotherapy (88).

#### Eosinophils

Administration of IL-33 in mice causes massive tissue infiltration of eosinophils and elevations of typical type 2 cytokines such as IL-5, IL-9, and IL-13, contributing to allergy and fibrosis (89). These responses also occurred in RAG knockout mice, suggesting that innate cells, particularly IL-5 producing ILC2s, were the direct target of IL-33 (54). However, IL-33 is now known to act directly on eosinophils leading to upregulation of CCR3, CD69, and CD11b, production of chemokines (CCL17, CXCL2, CXCL3, and CXCL10), and cytokines (IL-6, IL-13, GM-CSF) (90). IL-33 sustained eosinophil survival in vivo in a ST2 dependent manner and via autocrinous production of GM-CSF. Moreover, IL-33 and GM-CSF promoted the production of IL-4 and IL-13 by eosinophils, which in turn favored macrophage polarization toward M2 phenotype (91). In a murine model of melanoma, in vivo expansion of innate IL-5–producing ILC2 cells following systemic IL-33 injection played an important role for eosinophil recruitment and metastasis control. Innate IL-5-producing cells were increased in response to tumor invasion, and their regulation of eosinophils is critical to halt tumor metastasis (65). We reported that in transplantable B16 melanoma models, IL-33 restricted tumor growth and inhibited lung metastasis through recruitment and activation of eosinophils. Indeed, ST2-deficient mice presented an increased metastatic load and reduced lung eosinophilia compared to wild type mice. Depletion of eosinophils completely abolished the anti-tumor effects of IL-33 administration (69). Kim et al. also reported substantial expansion of intratumoral eosinophils in mice transplanted with IL-33-expressing tumor cells (EL4, CT26, and B16); however, their role in IL-33-induced anti-tumor effects was not addressed (66). In our study, IL-33 expanded eosinophils expressed T cell-attracting chemokines and induced NK and CD8+ T cell-recruitment at the subcutaneous tumor site, but not at the lung metastatic site. In addition, IL-33 activated eosinophils in vitro enhancing CD11b, CD69, and granzyme B expression and activating cytotoxic functions against melanoma cells (69). These findings suggest that depending on the tumor site, eosinophils may play an accessory role, supporting the recruitment of tumor-reactive CD8+ T cells (92), or a direct cytotoxic effect against tumor cells. Indeed, IL-33 can potently activate human eosinophils, enhancing adhesion, promoting survival, and inducing ROS production and degranulation (93, 94). Of note, degranulation of eosinophils has been shown in vivo in proximity of tumors (95, 96) and after in vitro stimulation, resulting in efficient killing of target mouse (97) and human (98) tumor cells, highlighting the tumoricidal properties of eosinophils (99).

#### Mast Cells

IL-33 activates its receptor complex (ST2: IL-1RAcP) on human mast cells (100) and basophils (19). IL-33 synergizes with IgEand non-IgE-dependent stimuli to release cytokines from human mast cells (101). Similarly, IL-33 augments substance P-induced vascular endothelial growth factor (VEGF) production from human mast cell lines (102). The latter findings are clinically relevant because VEGFs produced by human mast cells (103) and basophils (104) play an important role in chronic inflammation and in tumor growth (105). We have demonstrated that IL-33 up-regulated the Fcγ receptor type IIa and synergistically enhanced immune complex-triggered activation of human mast cells (106). Collectively, these findings demonstrate that IL-33 can synergistically potentiate the immunologic and nonimmunologic release of mediators from human and rodent mast cells. Single cell analysis demonstrated that IL-33 increased both the number of degranulating and chemokine-producing mast cells and the magnitude of individual mast cell response (107). The relevance of IL-33-mediated mast cell response has been found also in vivo in several pathological conditions, including cancer. Mast cells and basophils are known to infiltrate several types of tumors but, due to the wide range of mediators they release, it is difficult to define their specific pro- or anti-tumoral activity (103). Mast cell activation by IL-33 may occur in a number of tumor types. In skin cancers, mast cells accumulated with IL-33 expressing fibroblasts in UV-exposed murine skin samples (108). In the ApcMin/+ mouse model, IL-33 deficiency reduced tumor burden (109, 110) and decreased mast cell density in polyps as well as suppressed the gene expression of mast cell-derived proteases and cytokines that promote angiogenesis, Treg function, and MDSC recruitment within the tumor microenvironment (111–113).

#### Basophils

Human basophils constitutively express ST2 which is induced by IL-3 (114, 115). Although IL-33 alone failed to directly induce degranulation of human basophils, it exerted priming effects. It enhanced degranulation and IL-4 production in response to IgE cross-linking (114). By contrast, IL-33 alone activated unprimed murine basophilsin vitro (116). Recently, Rivellese and collaborators, using highly purified human basophils, elegantly demonstrated that IL-33 alone induces the release of low but detectable amounts of IL-4 (117). IL-33 synergistically potentiated IL-4 production induced by IL-3 or anti-IgE. Interestingly, IL-33 did not induce basophil degranulation, as evaluated by the membrane expression of CD63, but significantly enhanced IgE-mediated histamine release. Collectively, these findings indicate that IL-33 can activate human basophils presumably through the engagement of ST2. Furthermore, IL-33 can enhance IL-3- and anti-IgE-mediated basophil degranulation, histamine secretion, and cytokine production. The role of basophils in anti-cancer immunity is poorly characterized and the effects of IL-33 in stimulating or regulating basophils responses in cancer are unknown. In mouse models of melanoma, Sektioglu et al. reported that intratumoral basophils enhanced CD8+ T cell infiltration via production of the chemokines CCL3 and CCL4, contributing to tumor rejection following Treg cell depletion (118). Recently, low circulating eosinophils and basophils were associated with poor prognosis in CRC patients (119). Moreover, basophils in tumor-draining lymph nodes of both mice and pancreatic ductal adenocarcinoma patients correlated with Th2 responses in tumors and poor prognosis (120).

#### ROLE OF IL-33 IN CANCER: EVIDENCES FROM EXPERIMENTAL TUMOR MODELS

Accumulating evidences indicate that the axis IL-33/ST2 plays a role in tumor immunity. However, both pro-tumoral and antitumoral functions have been reported and the current literature suggest that IL-33 may differently affect tumor immunity depending on the tumor type, immune cell targets and on cooperating microenvironmental factors (121).

#### Breast Cancer

The majority of reports point to a pro-tumoral role of IL-33 in breast cancer models. In mice, the IL-33/ST2 axis was shown to promote the growth and lung and liver metastases of 4T1 mammary tumors facilitating the intratumoral accumulation of immunosuppressive myeloid (Gr-1+ TGF-β1 + MDSC, IL-10-expressing CD11c+ DCs, and alternatively activated M2 macrophages), ILC2, and Treg cells, while dampening the expansion of activated NK cells (70, 71, 84). Lukic group demonstrated that IL-33/ST2 axis promotes the expression of pro-angiogenic VEGF in tumor cells and attenuates tumor necrosis, thus facilitating mammary tumor growth (122). In contrast, another study reported that transgenic overexpression of IL-33 in 4T1 breast cancer cells reduces tumor growth and metastasis in vivo (49). In vitro, IL-33 acts as a critical tumor promoter during epithelial cell transformation and sustains breast cancer tumorigenesis (123). Recently, it has been reported that IL-33 overexpression in human ER-positive breast cancer cells results in resistance to tamoxifen-induced tumor growth inhibition, by promoting cancer stem cell properties (124). These findings indicate that in breast cancer IL-33/ST2 exert both intrinsic and extrinsic pro-tumoral function favoring tumorigenesis and stemness and reducing anti-tumor immunity.

#### Colorectal Cancer

A number of evidences from mouse models point to an important role of the IL-33/ST2 axis in promoting colorectal cancer (CRC) tumorigenesis, progression and malignancy (125). In the ApcMin/+ mouse model for human familial adenomatous polyposis, abrogation of the IL-33/ST2 axis by knockout of IL-33 (109) or ST2 (41, 110) inhibits proliferation, induces apoptosis, and suppresses angiogenesis, thus decreasing tumor number and size. Accordingly, overexpression of IL-33 in MC-38 mouse CRC cells results in increased in vitro proliferation and enhanced tumor growth and liver metastasis after orthotopic transplant in syngeneic mice through tumor-derived IL-33 induced recruitment of CD11b+ GR1+ and CD11b+ F4/80+ myeloid cells and angiogenesis (126). In addition, expression of IL-33 in human CRC cells promoted their growth and metastasisin vivo and reduced the survival of recipient nude mice (127). Likewise, IL-33/ST2 signaling in CRC tissues promoted the malignant growth and metastatic spread of CRC through modification of the tumor microenvironment (72). In a recent study, it was shown that IL-33 overexpression or exogenous administration of IL-33 to human or murine colon cancer cells enhanced cell growth in vivo and promoted colon cancer cell stemness through an immune-associated mechanism (73). In contrast, some studies have reported an anti-tumoral function of IL-33 in CRC models. Abrogation of ST2 signaling in CT26 mouse adenocarcinoma cells enhanced tumor development after subcutaneous transplant into syngeneic BALB/c mice (74). In the azoxymethane (AOM)/dextran sodium sulfate (DSS) model, IL-33-deficient mice were shown to be highly susceptible to cancer-associated colitis showing increased tumor number, size, and grade, due to a protective function of IL-33 in regulating an IgA-microbiota axis in the intestine (128). Fittingly, in a mouse model of sporadic colon cancer tumorigenesis in the absence of preexisting inflammation lack of IL-33 signaling enhanced colon tumorigenesis, while IL-33 treatment reduced tumor growth in the transplantable MC38 model, via IFN-γmediated antitumor immune response (46). These evidences suggest that in the absence of preexisting chronic or acute inflammation the homeostatic release of IL-33 by dying colon epithelial cells protects against the initiation and development of sporadic colon cancer.

#### Lung Cancer

The IL-33/ST2 axis plays a critical role in various inflammatory lung diseases, including asthma and fibrosis (129). However, few studies have investigated the contribution of this pathway to lung cancer. Akimoto et al. reported that ST2 expression in human and murine lung cancer is inversely correlated with metastatic potential (130). Exposure to IL-33 enhanced oncotic cell death of ST2+ low-metastatic Lewis lung carcinoma cells, but not of ST2− high-metastatic cells, thus suggesting that IL-33 enhances lung cancer progression by selecting for malignant cells. Moreover, in vitro stimulation with IL-33 promoted the migration and invasiveness of human lung A549 cells (131) and enhanced the growth and metastasis of primary non-small-cell lung cancer (NSCLC) cells after xenotransplant into immunodeficient mice (132). Vice versa, IL-33 blockade efficiently inhibited tumor growth of patient-derived NSCLC xenografts, abrogating M2 polarization of macrophages, and reducing the accumulation of Treg cells within the tumor microenvironment (44). In mouse lung tumor models stable transfection of IL-33 gene into tumor cells inhibited tumor growth and metastatic spread in vivo (49, 133). Of note, IL-33 expressing metastatic A9 lung cancer cells, a TC1-derived cell line with spontaneous down-regulation of MHC-I, restored MHC-I expression and immune recognition in mice (133). Although these evidences indicate that IL-33/ST2 expression in lung tissue or cancer cells fuels tumor growth, the contribution of IL-33 to anti-tumor adaptive immune responses against this cancer remains to be established.

#### Melanoma

Accumulating evidences indicate that in melanoma models IL-33 exert anti-tumoral and anti-metastatic effects by conditioning Afferni et al. IL-33 in Tumor Immunology

the local immune environment. Systemic injections of IL-33 significantly inhibit tumor growth in mice bearing subcutaneous B16.F10 melanomas (50, 69) and in BRAFV600EPTEN-inducible melanoma model (50). Dominguez et al. reported that IL-33 both directly stimulated CD8+ T cell expansion and IFN-γ production and activated myeloid dendritic cells (mDCs) increasing antigen cross-presentation. Of note, combination therapy with rIL-33 and agonistic anti-CD40 antibodies demonstrated synergistic anti-tumoral activity in this model (50). Using subcutaneous and experimental metastasis B16 melanoma models, we recently demonstrated that IL-33 inhibits melanoma tumor growth and pulmonary metastasis in vivo in an eosinophil-dependent manner (69). In a previous study, it was shown that IL-33 transgenic mice inhibit tumor metastasis in the B16 melanoma model (48). Likewise, transgenic expression of a secretable mature form of IL-33 by plasmid DNA in B16 tumor cells reduced tumor growth and metastasis in vivo (49). In these models, both NK and CD8+ T cells were required for the anti-tumoral effect of IL-33. Instead, Kim et al. showed that subcutaneous injection of adenoviral vector-transfected B16 melanoma cells engineered to secrete IL-33 did not develop palpable tumors in mice, in a CD8+ T- and NKindependent manner, through expansion of CXCR2 ligandssecreting intratumoral ILC2 (66). Ex vivo explanted IL-33 expressing B16 tumors exhibited higher reactive oxygen species (ROS) levels and increased CXCR2 expression and apoptosis thus suggesting a role for IL-33 in creating a hypoxic tumor microenvironment supporting ILC2-induced apoptosis (66). The explanation for such diverse immune-mediated anti-tumor mechanisms needs further investigations, although it is plausible that different local concentrations of IL-33 within the tumor microenvironment may stimulate different responses.

#### Other Tumors

IL-33 has been reported to exert an anti-tumoral role in other tumor models. In a human papilloma virus (HPV) associated model for cancer immunotherapy IL-33 was shown to act as a potent vaccine adjuvant augmenting Th1 and CD8+ T-cell responses, inducing anti-tumor immunity in vivo (23). Enforced expression of IL-33 in a large collection of murine tumor cell lines, including CT26 colon carcinoma, EL4 lymphoma, B16 melanoma, Lewis lung carcinoma (LLC), A9, and 4T1 lung cancer, results in reduced tumor growth in vivo (49, 66, 133). In a murine acute myeloid leukemia (AML) model administration of IL-33 significantly inhibited leukemia growth and improved mice survival rate in a CD8+ T cell dependent manner (51). Notably, combination of PD-1 checkpoint blockade with IL-33 further prolonged mice survival, and induced complete leukemia regression in 50% of animals. The latter report represents the first evidence for combining IL-33 with immunotherapy targeting immune checkpoint inhibitors. Recently, administration of rhIL-33 was shown to expand Vγ9 T cells improving the therapeutic response to phosphoantigen in preventing tumor growth in a humanized mouse lymphoma model (134). Pro-tumoral effects were observed in several other types of experimental cancers (135). In an organotypic culture model, carcinoma-associated fibroblasts (CAFs) were found to express high levels of IL-33 which promoted cancer invasive behavior of head and neck squamous cell carcinoma (HNSCC) cells (136). In gastric cancer, IL-33 exerted a pro-tumorigenic function inducing cancer cell invasion by stimulating the secretion of MMP-3 and IL-6 via ST2- ERK1/2 pathway (137) and activation of the JNK pathway (138) conferring chemotherapy resistance in vitro. Finally, in mouse models of cholangiocarcinoma (CCA), a malignant neoplasm of the biliary-duct system, administration of IL-33 was found to increase biliary tumorigenesis through an increase of IL-6 expression in tumor tissue (139) and to enhance cholangiocyte proliferation, by increasing the numbers of IL-13-producing ILC2s (140). The effects of IL-33 in experimental cancers are summarized in **Table 1**.

#### IL-33/ST2 AS A BIOMARKER PREDICTIVE OF CANCER PROGRESSION AND PATIENTS SURVIVAL

The recent literature regarding IL-33 involvement in tumorigenesis is controversial, since IL-33 seems to have dual, pro-inflammatory or protective, roles depending on the cellular and cytokine context. Some studies have shown a positive correlation between IL-33 expression in tumor tissue and a favorable prognosis in cancer patients. For example expression levels of IL-33 and ST2 were significantly down-regulated in both adenocarcinoma and squamous cell carcinoma of lung tissues when compared to adjacent normal lung controls (141). Furthermore, plasma IL-33 levels were elevated during the early stage of lung cancer and decreased with advanced cancer stages, probably due to lung volume reduction containing bronchial epithelium and vascular endothelium as sources of IL-33 (142). The observed decreases indicate that the expression levels of IL-33 are inversely associated with lung cancer progression (143). Of interest, in hepatocellular carcinoma resected tissues expression of IL-33 by intratumoral effector memory CD62L−KLRG1+CD107a+ CD8+ T cells was shown to be a prognostic marker for increased patients survival (144). Serum levels of IL-33 were significantly higher in patients with breast cancer compared to patients with benign breast diseases, so the local expression of IL-33 may be a marker for differentiating malignant from normal/benign tissues (145, 146). IL-33 expression in adjacent tissues also tends to be higher compared to normal tissues, suggesting that adjacent non-cancerous tissues may be similarly relevant to cancers in terms of anti-tumor immunity. Local IL-33 expression may also increase intratumoral accumulation of immunosuppressive lymphoid cells in patients with breast cancer (145, 146). Plasma sST2 levels and nuclear IL-33 expression were found to be increased in endothelial cells in bone marrow biopsies from patients with myeloproliferative neoplasms, whereas low/undetectable levels were found in healthy donors (147). It has been proposed that IL-33 induces the production of cytokines and growth factors that promote myelopoiesis and facilitates the development of leukemia by inducing and/or enhancing the proliferation of hematopoietic progenitors in the

TABLE 1 | Role of IL-33 in experimental tumors.

CRC, colorectal cancer; NSCLC, non-small cell lung cancer; HNSCC, head and neck squamous cell carcinoma; CCA, cholangiocarcinoma; AML, acute myeloid leukemia; CAF, cancer associated fibroblasts.

bone marrow microenvironment from patients with chronic myeloid leukemia (147).

In other clinical conditions, increased IL-33 expression was inversely correlated with the overall survival of cancer patients. Serum IL-33 levels were increased in renal cell carcinoma (RCC) patients compared to healthy volunteers (148). Such over-expression was associated with advanced tumor-lymph node-metastasis (TNM) stage, resulting in reduced survival and increased risk of recurrence in patients. Mechanistically, it has been shown that IL-33 enhances RCC cell growth in vivo and prevents chemotherapy-induced tumor apoptosis in vitro via JNK signaling activation in tumor cells (149). Similarly, both IL-33 and ST2 were up-regulated in ovarian tumors compared to normal ovary and ovarian benign tumors, and the expression levels were further increased in tumor tissues at the metastatic site (150). It has been proposed that IL-33/ST2 axis promotes ovarian cancer migration and metastasis through regulation of ERK and JNK signaling pathways (151). Furthermore, most head and neck squamous cell carcinoma (HNSCC) cases with a low invasion pattern grading score (IPGS) showed low or no expression of IL-33, whereas most HNSCC cases with high IPGS displayed abundant expression of IL-33 in CAFs and in cancer cells. This observation suggests a paracrine effect of IL-33 as a result of the crosstalk between tumor cells and surrounding stromal cells. Hence, CAFs overexpressing IL-33 promote the induction of epithelial-to-mesenchymal transdifferentiation, eventually leading to tumor progression and poor prognosis (136). Higher IL-33 expression was described in glioma tissue compared to normal brain tissues at both transcriptional and translational levels (152, 153). It has been proposed that IL-33 stimulates cell migration through the expression of matrix metalloproteinases (MMP2/MMP9) via the ST2/ NF-κB pathway, thereby promoting cell invasion and tumor growth (154). Higher expression of IL-33 and total ST2 (ST2L and sST2) have also been reported in colorectal cancer (CRC) tissues compared to adjacent normal tissues (127). This observation could be explained considering that inflammatory cytokines are important components of the CRC microenvironment and colon cancer progression is closely related to chronic inflammation. Increased IL-33 expression is observed in poorly differentiated human CRC cells, which is associated with poor survival in patients with metastatic colon cancer (73).

The relation between IL-33 and sST2 serum levels and the survival of patients suffering from liver cirrhosis (LC) and hepatocellular carcinoma (HCC) has not been determined yet. No significant difference in IL-33 serum levels was found in HCC compared to LC. IL-33 levels did not correlate with overall survival or liver function parameters, whereas sST2 levels were significantly elevated in LC and HCC patients, compared to healthy subjects, and were associated with overall survival of HCC. Therefore, its function remains to be clarified (155). Similarly, IL-33 protein levels were significantly lower in gastric cancer tissues than adjacent tissues. These levels were associated to the depth of tumor invasion and the morphology of the tumor, suggesting that IL-33 is involved in the process of inflammatory reaction in the development of gastric cancer, while it is not significantly associated with the overall survival of these patients (156).

#### ANALYSIS OF IL-33 MUTATIONS AND ISOFORMS IN CANCER

IL-33 gene undergoes a certain number of somatic mutations during tumorigenesis. Depending on the exact location in the gene (and protein), the mutation may be putative of some key functional aberrancies that influence the IL-33 global functional properties (**Figure 3**). In general, IL-33 mutations occurred with very low frequencies in all tumors examined (0.072–1.391%). However, some mutations may putatively have a key impact on the functionality of IL-33. IL-33 protein is equipped with three main regions (Nuclear, Central, IL-1-like) with three specific binding and cleavage sequences in each of these domains (**Figure 3**). For example, there are 15 unique mutations in skin cancer patients, with a missense mutation (M52I at nucleotide 6250538 of the chromosome 9) targeting the chromatin binding site (R1). We hypothesize that this amino acid change potentially breaks the ability of IL-33 to bind DNA and thus to exert its regulatory functions. There are additionally 11 unique somatic missense mutations all affecting the IL-1-like domain of the protein (but outside the R3 sequence), that can putatively compromise the IL-33 binding ability to its specific receptor ST2 (3, 157). Another tumor that displays a mutation affecting the ability of IL-33 to bind the

mutations are depicted. (B) Somatic IL-33 gene mutation frequencies calculated by data analyzed in (A) and indicated for each tumor type.

(red box and text) of the IL-33 gene (upper panel) with the location of exons present in each indicated transcript variant (lower panel). Data are obtained by ENSEMBL genome browser (https://www.ensembl.org/). There are 3 isoforms of IL-33 gene, which are: NM\_001199640 (CDDS56563.1), NM\_001199641 (CDDS56564.1), NM\_033439 (CDDS6468.1). (B) Expression of the IL-33 transcript variants indicated in (A) for each tumor compared to the normal tissues. Expression data (T.P.M, transcript per million) are obtained by interrogating the public database for Isoform expression resource analysis (Isoexpresso, http://wiki.tgilab.org/ISOexpresso/main. php). IL-33 transcript variant expression data are not available for skin, blood, brain, and ovarian cancers/normal tissues within Isoexpresso database. The predominant forms for brain and ovarian tumors are variants 1 whereas the predominant form expressed in skin tumors is the transcript variant 3 (resource: Mammalian Transcriptomic Database, http://mtd.cbi.ac.cn/).

chromatin is the uterine cancer with the R48H amino acid change (**Figure 3**). Interestingly, the mutations E121K, H221Y, S225F, and H246Y, all occurring in the IL-1-like domain, are classified as deleterious in terms of IL-33 functional changes for skin cancers as evidenced by the cBioPortal database. Some of these mutations, like the amino acid change H221Y, are also shared with other tumors such as bladder cancer, thus denoting their significance. There is only one somatic missense alteration (G176S) occurring inside the R3 region, shared by colon and blood cancer patients (**Figure 3**). These mutations can putatively disrupt the cleavage site for caspase 3 and 7, thus increasing the IL-33 resistance to these proteases with key consequences in skin tumor survival and progression. Mutations in the cleavage site for inflammatory proteases (R2) occur only in colon and uterine tumors, with the A95V amino acid change (colon) and two mutations in uterine tumor (missense V101L and truncation S111).

As mentioned above, the IL-33 gene is equipped with a splicing system generating three different variants of the IL-33 protein (**Figure 4A**). The first isoform variant is composed by 7 exons, whereas the variants 2 and 3 contain 6 and 4 exons, respectively. The role and the extent of expression of each isoform in cancer is still a field of debate. Nevertheless, public databases such as IsoExpresso are now available, containing expression data of isoforms associated to thousands of human genes involved in cancer progression (158). We then extracted data about the expression of IL-33 isoform variants in different types of tumors and the normal counterparts (**Figure 4B**). The IsoExpresso database revealed a differential expression across the lesions and normal tissues or across the isoform variants

of IL-33. Thyroid, liver, breast, and bladder cancers display a change in isoform expression when compared to normal counterparts. Indeed, variants 2 and 3 are highly expressed in these tumors compared to expression levels observed in normal tissues (**Figure 4B**). Of note, bladder cancer presents a splice site mutation (X157, **Figure 3**), and the presence of this mutation may probably be associated to the observed expression shift of the IL-33 isoform variants in this type of cancer (**Figure 4**). Indeed, in the tumor lesions the mainly expressed isoforms are variant 2 and 3, whereas the normal tissues express the variant 1 only (**Figure 4B**). This strongly suggests that this mutation affects the disappearing of IL-33 exon 4 (**Figure 4A**). In contrast, uterine tumor has been associated with a splice mutation (X115, close to the S111 truncation, **Figure 3**) not affecting the expression of IL-33 splice variants (**Figure 4B**).

#### CONCLUDING REMARKS

The role of IL-33 in tumorigenesis and cancer immunity remains controversial. The current literature indicate that IL-33 expression can be regulated during the progression of distinct types of cancers and that IL-33/ST2 signaling within the tumor microenvironment may differently contribute to tumorigenesis, promoting antitumor responses or mediating tumor growth or metastasis, depending on the nature of the malignant tissue. Further studies are needed to elucidate the role of this pathway at specific time points during cancer development as a possible diagnostic/prognostic marker for patients and to clarify whether IL-33/ST2 blockade may represent a valid approach for adjuvant therapies of established IL-33-dependent tumors.

It has been suggested that IL-33 has opposing effects in tumor immunity depending on its local concentration (121). In this respect, the tumor histotype may crucially determine the amount of IL-33 expressed. In addition, the presence of different IL-33 isoforms may play a crucial role. Thus, it may be possible that under steady-state conditions the homeostatic release of IL-33 by apoptotic epithelial cells may be regulated by caspase

#### REFERENCES

cleavage, thus limiting excessive inflammation. We envisage that, as in several chronic inflammatory diseases, also in tumors IL-33 could exist in several different forms as a result of posttranslational processes, including intracellular and extracellular modification, or as a result of mRNA alternative splicing. There is compelling evidence that different forms and cleavage products of IL-33 can exert dissimilar biological activities. Unfortunately, the identification and the concentrations of the different forms of IL-33 in different tumors is largely unknown. Future studies should characterize the different forms of IL-33 in human and experimental tumors. Moreover, the mutation level of IL-33 gene observed in human solid tumors (**Figure 3**) may be associated with different isoforms and distinct stimulated antitumor immune responses. Lastly, since the new frontier of cancer immunotherapy is the employment of immune checkpoint inhibitors (i.e., against PD-1, PD-L1, CTLA-4) further studies are needed to clarify whether IL-33 conditioning may increase the therapeutic response to checkpoint blockade in cancer patients.

#### AUTHOR CONTRIBUTIONS

GS, CA, and FM have conceived and designed the review. CA, CB, SA, MG, GV, GM, FM, and GS contributed intellectually and to the writing of the submitted version of the manuscript.

## FUNDING

This work was supported in part by grants from AIRC IG 14297 to GS; Italian Ministry of Health RF-2011-02347120 to FM; Regione Campania CISI-Lab Project, CRèME Project, and TIMING Project to GM.

#### ACKNOWLEDGMENTS

The authors wish to thank all colleagues that have indirectly supported this work with ideas, discussion and encouragement. Special thanks to Fabrizio Fiorbianco for artwork.

modulating T cell-dependent responses. Front Immunol. (2015) 6:232. doi: 10.3389/fimmu.2015.00232

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effector-memory CD8+ T cells in resected hepatocellular carcinoma prolong patient survival. Hepatology (2015) 61:1957–67. doi: 10.1002/hep. 27728

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Afferni, Buccione, Andreone, Galdiero, Varricchi, Marone, Mattei and Schiavoni. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Interleukin-33 in Systemic Sclerosis: Expression and Pathogenesis

Liya Li 1,2, Honglin Zhu1,2 \* and Xiaoxia Zuo1,2 \*

*1 Department of Rheumatology and immunology, Xiangya Hospital, Central South University, Changsha, China, 2 The Institute of Rheumatology and Immunology, Central South University, Changsha, China*

Interleukin-33 (IL-33), a member of the IL-1 superfamily, functions as a traditional cytokine and nuclear factor. It is proposed to have an "alarmin" role. IL-33 mediates biological effects by interacting with the ST2 receptor and IL-1 receptor accessory protein, particularly in innate immune cells and T helper 2 cells. Recent articles have described IL-33 as an emerging pro-fibrotic cytokine in the immune system as well as a novel potential target for systemic sclerosis. Here, we review the available information and focus on the pleiotropic expression and pathogenesis of IL-33 in systemic sclerosis, as well as the feasibility of using IL-33 in clinical applications.

Keywords: interleukin-33, ST2, systemic sclerosis, pathogenesis, fibrosis

#### Edited by:

*Fang-Ping Huang, University of Hong Kong, Hong Kong*

#### Reviewed by:

*Piergiuseppe De Berardinis, Istituto di Biochimica delle Proteine (IBP), Italy Angela Bonura, Consiglio Nazionale Delle Ricerche (CNR), Italy*

#### \*Correspondence:

*Honglin Zhu<EMAIL_ADDRESS>Xiaoxia Zuo<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Cytokines and Soluble Mediators in Immunity, a section of the journal Frontiers in Immunology*

Received: *06 January 2018* Accepted: *29 October 2018* Published: *15 November 2018*

#### Citation:

*Li L, Zhu H and Zuo X (2018) Interleukin-33 in Systemic Sclerosis: Expression and Pathogenesis. Front. Immunol. 9:2663. doi: 10.3389/fimmu.2018.02663*

#### INTRODUCTION

Systemic sclerosis (scleroderma, SSc) is a heterogeneous autoimmune disease of unknown etiology, clinically characterized with obliterative microvasculopathy, inflammation, and extensive fibrosis of the skin and multiple organ systems and serologically characterized by the presence of circulating specific autoantibodies. SSc has the highest cause-specific mortality among connective tissue diseases (1, 2), and pulmonary artery hypertension and interstitial lung disease (ILD) are the leading causes of death (3, 4). Therapeutic interventions for SSc mainly involve the comprehensive administration of glucocorticoids and immunosuppressants and targeted treatment. To date, no effective medical intervention has been developed to control and reverse the progression of this fibrotic disease (5). Thus, effective and safe targeted therapies for SSc-related fibrosis are urgently needed. In the pathogenesis of SSc, endothelial damage may be a primary event. SSc also exhibits complex interactions during the transition from fibroblasts to myofibroblasts and non-infective inflammation or autoimmunity.

Interleukin (IL)-33 belongs to the IL-1 superfamily and is widely expressed throughout the human body. During cell damage or tissue injury, IL-33 is released into the extracellular space, wherein it produces endogenous danger signals to alert adjacent cells. This function deems IL-33 as an alarmin. IL-33 also functions as a nuclear factor regulating gene transcription in cytokineexpressing or cytokine-responsive cells (6). IL-33 is known to play crucial roles in inflammation. However, recent studies indicated that IL-33 participates in the development and progression of fibrotic diseases and SSc. Here, we review the profibrotic roles of IL-33 and its related mechanisms and discuss its potential application in the treatment of SSc.

#### BIOLOGICAL CHARACTERISTICS OF IL-33

IL-33, also known as IL-1F11, is a member of the IL-1 superfamily (7) and exhibits dual functionality (8). This cytokine was first identified as a nuclear factor in high endothelial venules in 2003 (9) but was renamed as IL-33 when a study in 2005 demonstrated its role as a specific

**99**

receptor accessory protein complex in ST2+ immune cell membranes results in the activation of nuclear factor-κB transcription factors through the MyD88, IL-1R-associated kinase (IRAK), and tumor necrosis factor receptor associated factor 6 (TRAF6) signaling pathways, leading to the induction of inflammation and profibrosis in pathological cells. IL-33 also functions as a nuclear factor to regulate gene transcription in cytokine-expressing or cytokine-responsive cells. Moreover, sST2 acts as a decoy receptor for IL-33 (full-length IL-33 or mature IL-33), and CNTO-7160 (the first monoclonal anti-ST2 antibody) was designed as a new IL-33 inhibitor. Both of these molecules block the downstream signaling of IL-33.

extracellular ligand for the orphan IL-1 receptor family member ST2 (also known as IL-1RL1, DER4, T1, and FIT-1). ST2 is a member of the Toll-like receptor (TLR)/IL-1 receptor superfamily (10), which has two main isoforms, namely, a short soluble form (sST2) and longer transmembrane form (ST2L), with four isoforms in total, including ST2V and ST2LV (11). The mRNA encoding sST2 is a secretory sequence that is generated by alternative splicing and lacks the sequence encoding the transmembrane domain of ST2L (12).

The IL-33 gene is located on human chromosome 9 (or chromosome 19 in mice) and is transcribed from seven coding exons. The protein is synthesized as a 31-kDa pro-IL-331−−270 (full-length IL-33). Following synthesis, IL-33 is transported into the nucleus as a nuclear factor. Similar to the IL-1 family members IL-1β and IL-18, IL-33 lacks the classic signal sequence necessary for the transport by the endoplasmic reticulum/Golgi secretion pathway (13). Upon natural secretion from pathological cells undergoing necrosis or necroptosis, the full-length IL-33 is cleaved by caspase-3 and caspase-7 to activate apoptotic pathways in the cytoplasm, followed by its release into the extracellular environment (14). Once released into the extracellular matrix, full-length IL-33 is further processed by serine proteases (such as cathepsin-G and elastase) into the 18-kDa IL-33112−−270 (mature IL-33) with increased activity (15, 16), forming a soluble recombinant cytokine in circulation. However, both full-length and mature IL-33 bind to ST2L in ST2+ immune cell membranes

**Abbreviations:** ST2L, ST2 longer transmembrane form; sST2, soluble ST2; ILC2, type 2 innate lymphoid cell; Treg, regulatory T cell; DC, dendritic cell; Th1, T helper 1 cell; CD8+ T cell, CD8-positive T cell; iNK T cell, invariant natural killer T cell; NK cell, natural killer cell; SSc, systemic sclerosis; IRAK, IL-1R-associated kinase; TRAF6, tumor necrosis factor receptor associated factor 6; IL, interleukin; ILD, interstitial lung disease; IL-1RAcP, IL-1 receptor accessory protein; NFκB, nuclear factor-κB; MAPK, mitogen-activated protein kinase; IPF, idiopathic pulmonary fibrosis; TGF, transforming growth factor; TLR, Toll-like receptor; IFN, interferon; IRF-7, IFN regulatory factor 7.

and interact with IL-1 receptor accessory protein (IL-1RAcP), eventually leading to the formation of an IL-33/ST2L/IL-1RAcP

complex. This complex induces signaling pathways through MyD88, IL-1R-associated kinase (IRAK), and tumor necrosis factor receptor associated factor 6 (TRAF6) and activates the canonical nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways (17).

IL-33 is principally produced by stromal cells, including epithelial cells, endothelial cells, fibroblast-like cells, and myofibroblasts of lymphoid as well as non-lymphoid organs, under both steady state and inflammation conditions (18–20). In erythroid progenitor cells, IL-33 is produced during the maturation of red blood cells and released upon haemolysis (21). Innate immune cells expressing ST2 mainly include dendritic cells (DCs), natural killer (NK) cells, eosinophils, basophils, macrophages, and neutrophils (12). Full-length IL-33 predominantly remains inside the cell and regulates the expression of genes, which induce pulmonary inflammation and fibrosis. In contrast, mature IL-33 promotes asthma as well as allergic and anti-parasitic responses through the ST2 receptor and Th2 mechanisms (22).

Mechanisms such as inactivation by oxidation of cysteine residues, nuclear localization or sequestration, and proteolytic processing, and receptor antagonists as well as sST2 have evolved to regulate the expression and activities of IL-33 (12, 14, 15, 23, 24). sST2 is constitutively expressed in the human serum, wherein it acts as a decoy receptor for IL-33 and is not involved in signaling (25, 26). sST2, induced during tissue damage, may restrict the deleterious effects of increased IL-33 level. A novel mechanism for the rapid inactivation of IL-33 protein released from the cell in vivo was reported, wherein an oxidation-driven conformational change involving the formation of two disulphide bonds was observed, resulting in the elimination of ST2-dependent activity and reduction of inflammation, consistent with the mechanism of many other IL-1 family members (23).

# EXPRESSION OF IL-33 AND ST2 IN SSC

According to recent studies, increased IL-33 and sST2 levels have been observed in patients with infections, cardiovascular disorders, allergic diseases, and rheumatic diseases such as systemic lupus erythematosus, rheumatoid arthritis (RA), Wegener's granulomatosis, and Behcet's disease (27–31). The serum levels of sST2 and synovial fluid of IL-33 were higher in patients with RA than in healthy controls and patients with osteoarthritis (27). Furthermore, serum sST2 levels were higher in patients with active, newly diagnosed, anti-neutrophil cytoplasmic antibody-associated vasculitis than in patients in remission, indicative of the marker role for sST2 (32). Furthermore, ST2 and IL-33 were highly expressed around ectopic germinal centers in salivary glands from patients with IgG4-related disease, whereas IL-33 was expressed only in epithelial cells in patients with Sjögren's syndrome and controls (33). Interestingly, the exposure of mice in vivo or human skin samples ex vivo to inflammatory doses of ultraviolet B irradiation induced IL-33 expression within the epidermal and dermal skin layers (34). Proteomic analysis used to determine the extracellular and intracellular roles of IL-33 in primary human endothelial cells revealed the induction of inflammation-related protein expression of the exogenous extracellular IL-33, whereas the knockdown of the endogenous nuclear IL-33 expression had no reproducible effect on the endothelial cell proteome (35).

The results described above support that the expression level and biological role of IL-33 are similar to those of ST2. In general, IL-33 expression is upregulated in inflamed tissues following proinflammatory stimulation, and the role of IL-33 in cells may vary under different pathophysiological conditions. In SSc, with an exception during tissue inflammation, the authors proposed that IL-33 commonly responds to tissue injury and typically affects rapid tissue repair and regeneration (36–38).

In patients with SSc, serum levels of IL-33 and sST2 were elevated (39) and positively correlated with the extent of skin sclerosis (higher in diffused cutaneous SSc than in limited cutaneous SSc), severity of pulmonary interstitial fibrosis, and vascular involvement in SSc development (40– 44). In the lesion skin tissues, IL-33 expression is altered depending on the disease stage. IL-33 is downregulated in most endothelial cells in early SSc but not in late SSc (45). IL-33 produced by activated dermal fibroblasts/myofibroblasts has been implicated in the fibrotic pathology associated with SSc, which is profoundly increased by hypertrophic and mechanical stress (46, 47).

The expression of IL-33 mRNA is reported to increase in the primary pulmonary fibroblasts from patients with SSc-ILD as well as in those from patients with idiopathic pulmonary fibrosis (IPF). The elevated levels of IL-33 in bronchoalveolar lavage fluids may be useful in differentiating IPF from other chronic ILDs (48). In patients with IPF and SSc-ILD, the expression of full-length IL-33 was elevated in the affected lungs, consistent with the observation reported in a bleomycin-induced mouse model. Under the conditions of ST2 gene deficiency, the fulllength IL-33 could stimulate the expression of several non-Th2 cytokines and heat shock protein 70. On the other hand, the matured form of IL-33 was unaffected and instead activated Th2 responses (49). In contrast, the expression of the matured form of IL-33 was enhanced but that of the full-length counterpart reduced in the macrophages of bleomycin-induced mouse lung tissues (50). These findings suggest that the full-length IL-33 may serve as a synergistic pro-inflammatory and pro-fibrotic regulator in the lungs.

# PATHOGENESIS OF IL-33/ST2 IN SSC

Fibrosis, a prominent pathological characteristic of SSc (38), is characterized with a deregulated and uncontrolled repair process. Many molecular and signaling pathways involved in the fibrosis of SSc (51, 52), including transforming growth factor (TGF)-β, TLR4, and interferons (IFNs), are wellstudied. TGF-β is responsible for both physiological and pathological matrix remodeling (53) as well as fibroblastmyofibroblast transformation (54). TLR4 induces pro-fibrotic responses by activating NF-κB signaling through MyD88, IRAK, and TRAF6. The TLR/NF-κB signaling pathways enhance the TGF-β-dependent fibrotic process (55, 56). IFNs generally act as negative regulators of collagen synthesis and TGF-βmediated fibrotic responses, while the mechanism of type I IFN signaling in SSc-promoted fibrosis remains unclear (37, 57). The role of IL-33 in SSc was recently evaluated. In pediatric patients with limited cutaneous SSc, high levels of IL-33 and IFN-γ positively correlated with anti-histone and anti-ssDNA antibodies, indicating that the co-expression of IL-33 and IFNγ may contribute to the pathogenesis of SSc (58). Subcutaneous injection of IL-33 in mice resulted in the development of cutaneous fibrosis, similar to that observed in patients with SSc, including dermal mast cells and skin-infiltrating neutrophils through the suppression of Th1-mediated contact hypersensitivity responses (59). This observation highlights the important roles of IL-33 in SSc. However, the exact mechanisms require further investigation.

Known as a master regulator of pathological fibrosis, TGF-β may be produced by IL-33-induced cells. During the amplification of the alternatively activated M2 macrophage polarization, the IL-33/ST2 pathway was shown to play a significant role (60). IL-33 polarized M2 macrophages to produce IL-13 and TGF-β1 and induced the expansion of type 2 innate lymphoid cells (ILC2s) for the production of IL-13 in vitro and in vivo. ST2 may protect ILC2s from IL-33 stimulation by reducing the production of IL-5 and IL-13 (61). IL-13 is a well-known profibrotic cytokine downstream of IL-33 in the immune system (51).

IFN-γ may play regulatory roles in physiological processes involving IL-33. In type 2 immune responses, IL-33 and ILC2s are central mediators that promote tissue and metabolic homeostasis, whereas IFN-γ suppresses this pathway and promotes inflammatory responses (62). In vivo, the co-expression of IL-33 and IFN-γ in pulmonary fibroblast culture and lungs resulted in the attenuation of IL-33 protein levels (63). IFN-regulated genes may regulate IL-33 gene expression. In both human monocytes and macrophages from C57BL/6 mice, transcriptional activation of the IL-33 gene stimulated by the acute-phase protein serum amyloid A, a TLR2 ligand, may be regulated by IFN regulatory factor 7 (IRF-7) recruited to the IL-33 promoter. Silencing of IRF-7 expression may result in the abrogation of the expression of IL-33 induced by serum amyloid A (64).

In fibrosis, DCs elevated the expression of IL-33 via TLR/NFκB signaling pathways in response to allergic inflammation, resulting in an increase in the expression levels of MyD88, NFκB1, NF-κB2, and RelA accompanied with NF-κB p65 nuclear translocation, possibly through a potential autocrine regulation. These elevations may be blocked with a TLR5 antibody or NF-κB inhibitor quinazoline and thought to be decreased in DCs from MyD88-knockout mice (65). The deficiency in the NF-κB negative feedback regulator A20 in hyperactive mast cells may result in amplified pro-inflammatory responses downstream of IgE/FcεRI, TLRs, IL-1R, and IL-33R (ST2), thereby exacerbating inflammatory disorders (66). In addition, Th2-stimulated (allergen-specific IgG immune complexes and house dust mites) signaling occurs through FcRγ-associated receptors on DCs to upregulate IL-33 production and induce Th2-mediated allergic airway inflammation (67).

In conclusion, IL-33 functions as a pro-fibrogenic cytokine in the development of SSc. IL-33 may enhance the TGF-βdependent fibrotic process by increasing the production of TGF-β and activate TLR/NF-κB-dependent fibrosis signaling pathways, which are regulated by IFN-γ (**Table 1**).

To determine whether IL-33 is a useful therapeutic target, Locksley et al. described the complexity of using IL-33 and therapeutic strategies for altering IL-33 activities in vivo (68). The framework of IL-33 biology was described as a stepwise process. First, the focal cellular necrosis or other signals induce the release of IL-33 from the nucleus to maintain homeostasis; IL-33 acts on tissue-resident ST2-expressing effector cells such as ILC2s, regulatory T cells (Tregs), and mast cells to create a tissue environment that limits inflammation and promotes a reparative state characterized by tolerance. Second, amplification occurs upon exposure to chronic stimuli such as allergens and repetitive tissue damage, wherein excess extracellular IL-33 leads to multiple self-stimulating cycles of release to promote chronic allergic pathology, fibrosis, and excess stores of IL-33 in the circulation and tissues. The third step is conversion, wherein the activated inflammatory cells and cytokines responsive to the IL-33/ST2 axis play various roles such as killing pathogens, mounting anticancer immune responses, increasing tissue damage, and repressing the type 2-associated immune regulation responses. In patients with SSc, repetitive tissue damage by other pro-fibrotic mediators in fibroblasts and endothelial cells likely suppresses the IL-33 pool increases and regulatory mechanisms. Next, inflammation is amplified, fibrosis occurs, and tissue IL-33 levels increase, ultimately contributing to tissue fibrosis and sclerosis.

Therefore, IL-33 from different sources can be up- or downregulated to exert pleiotropic roles in SSc. Zhao et al. proposed that these apparently contradictory results indicate the presence of an extremely complex process of IL-33 processing and secretion (69). The functional properties of recombinant IL-33 used in previous studies are becoming well-characterized, whereas the cellular sources of IL-33 in natural and stimulated expression remain largely unknown. Additional studies are warranted to explain the differences between in vitro and in vivo results.

#### CLINICAL APPLICATIONS OF IL-33 IN SSC

Various aspects of the clinical applications of IL-33 have been examined. However, few studies have evaluated these effects in patients with SSc. Thus, information may be obtained from studies of other diseases that may be applicable to SSc.

IL-33-responsive ILC2s may promote the restoration of injured skin, lung, and gut cells (70). During the regeneration of injured muscles, fibro-adipogenic progenitor cells are the only known source of IL-33 in muscles. The low level of IL-33 expression in older, injured muscle reduces the recruitment and proliferation effects of non-increased muscle-resident Tregs; after the administration of IL-33, the Treg population increases and

*TGF-*β*, transforming growth factor-*β*; ILC2s, type-2 innate lymphoid cells; IFN, interferon; IRF-7, IFN regulatory factor 7; TLR, Toll-like receptor; NF-*κ*B, nuclear factor-*κ*B; IL, interleukin.*

regeneration is enhanced (71, 72). Furthermore, the upstream and downstream regulation of the IL-33 gene may promote the remodeling of tissues such as nerves and tendons (73, 74).

In general, studies of IL-33 in patients with SSc have indicated that IL-33 is a novel and important pro-fibrogenic cytokine and a potential biomarker for monitoring disease activity (40–45). Genetic polymorphisms in the IL-33 gene may be useful for the prediction of the risk of various diseases. The IL-33 rs7044343 CC genotype was suggested to be associated with an increased risk of developing SSc and a decreased risk of developing RA; the T allele may be a susceptibility marker for premature coronary artery disease and central obesity and possibly involved in the regulation of IL-33 production (53, 75–77). The first monoclonal anti-ST2 antibody, CNTO-7160, was recently designed as a new IL-33 inhibitor; this antibody is being evaluated in phase I clinical trials for the treatment of severe asthma and atopic dermatitis, but no data have been published to date (78).

#### PROSPECTS

The alarmin IL-33 has dual functions of a cytokine and nuclear factor. However, differences in the levels of IL-33 and systemic sST2 indicate intra-individual and inter-individual biological variation, reference changes, and sex-specific differences (79). Moreover, the evaluation of the circulating concentrations of sST2, full-length IL-33, mature IL-33, and complexes of sST2 and IL-33 in the same patients is interesting; measurement of these

#### REFERENCES

four analytes and their ratios may increase the understanding of IL-33-related pathophysiology in various diseases (80).

Recent investigations suggested that IL-33 is a novel profibrogenic cytokine in the development of SSc, mainly because it affects the TLR/NF-κB signaling pathways, and TGF-β1 expression is also regulated by IFN-γ. These effects are crucial for the early diagnosis of pulmonary fibrosis. Whether IL-33 is involved in fibroblast activation alone or in combination with other factors is unclear; however, this molecule is likely a potential biomarker and novel therapy target for managing fibrosis in patients with SSc. Furthermore, the inhibitor of IL-33 (CNTO-7160), currently being examined in clinical trials, may possibly be developed as a new therapy for fibrosis in patients with SSc (78).

### AUTHOR CONTRIBUTIONS

LL devised and wrote the manuscript. HZ and XZ revised the manuscript.

#### FUNDING

This work was supported by The National Key Research and Development Program of China (2016YFC0903900), grants from National Natural Science Foundation of China (81671622, 81701621) and Independent Innovation Projects of Central South University (2018zzst290).

study from the EULAR Scleroderma Trials and Research (EUSTAR) database. Ann Rheum Dis. (2010) 69:1809–15. doi: 10.1136/ard.2009. 114264

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Li, Zhu and Zuo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# IL-33 Prevents MLD-STZ Induction of Diabetes and Attenuate Insulitis in Prediabetic NOD Mice

Sladjana Pavlovic1 \* † , Ivica Petrovic1,2†, Nemanja Jovicic1,3†, Biljana Ljujic1,4 , Marina Miletic Kovacevic3 , Nebojsa Arsenijevic1 and Miodrag L. Lukic1 \*

 *Faculty of Medical Sciences, Center for Molecular Medicine and Stem Cell Research, University of Kragujevac, Kragujevac, Serbia, 2 Department of Pathophysiology, Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia, Department of Histology and Embryology, Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia, Department of Genetics, Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia*

#### Edited by:

*Teizo Yoshimura, Okayama University, Japan*

#### Reviewed by:

*Girdhari Lal, National Centre for Cell Science (NCCS), India Toshihiro Ito, Nara Medical University, Japan*

#### \*Correspondence:

*Sladjana Pavlovic<EMAIL_ADDRESS>Miodrag L. Lukic<EMAIL_ADDRESS>
*†These authors have contributed equally to this work*

#### Specialty section:

*This article was submitted to Cytokines and Soluble Mediators in Immunity, a section of the journal Frontiers in Immunology*

Received: *28 May 2018* Accepted: *26 October 2018* Published: *15 November 2018*

#### Citation:

*Pavlovic S, Petrovic I, Jovicic N, Ljujic B, Miletic Kovacevic M, Arsenijevic N and Lukic ML (2018) IL-33 Prevents MLD-STZ Induction of Diabetes and Attenuate Insulitis in Prediabetic NOD Mice. Front. Immunol. 9:2646. doi: 10.3389/fimmu.2018.02646* Type 1 diabetes is an autoimmune disease caused by the immune-mediated destruction of pancreatic β-cells. Prevention of type 1 diabetes requires early intervention in the autoimmune process against beta-cells of the pancreatic islets of Langerhans, which is believed to result from disordered immunoregulation. CD4+Foxp3+ regulatory T cells (Tregs) participate as one of the most important cell types in limiting the autoimmune process. The aim of this study was to investigate the effect of exogenous IL-33 in multiple low dose streptozotocin (MLD-STZ) induced diabetes and to delineate its role in the induction of protective Tregs in an autoimmune attack. C57BL/6 mice were treated i. p. with five doses of 40 mg/kg STZ and 0.4 µg rIL-33 four times, starting from day 0, 6, or 12 every second day from the day of disease induction. 16 weeks old NOD mice were treated with 6 injections of 0.4 µg/mouse IL-33 (every second day). Glycemia and glycosuria were measured and histological parameters in pancreatic islets were evaluated at the end of experiments. Cellular make up of the pancreatic lymph nodes and islets were evaluated by flow cytometry. IL-33 given simultaneously with the application of STZ completely prevented the development of hyperglycemia, glycosuria and profoundly attenuated mononuclear cell infiltration. IL-33 treatment was accompanied by higher number of IL-13 and IL-5 producing CD4+ T cells and increased presence of ST2+Foxp3+ regulatory T cells in pancreatic lymph nodes and islets. Elimination of Tregs abrogated protective effect of IL-33. We provide evidence that exogenous IL-33 completely prevents the development of T cell mediated inflammation in pancreatic islets and consecutive development of diabetes in C57BL/6 mice by facilitating the induction Treg cells. To extend this finding for possible relevance in spontaneous diabetes, we showed that IL-33 attenuate insulitis in prediabetic NOD mice.

Keywords: IL-33, diabetes, C57BL/6 mice, NOD mice, streptozotocin

# INTRODUCTION

Diabetes mellitus type 1 is a chronic inflammatory disease characterized by the progressive destruction of pancreatic β-cells of Langerhans islets caused by autoimmune processes (1– 3). The development of these autoimmune processes is thought to be the result of disorders of immunoregulation (3). This failure allows Th1/Th17 lymphocytes to trigger a cascade of immune/inflammatory processes in the pancretic islets causing β cell destruction. Both the

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numerical and functional equilibrium between effector and regulatory T lymphocytes in pancreatic infiltrates determine the extent of destruction of β cells (4–6).

IL-33 is a member of the IL-1 cytokine family (7). Receptor for IL-33 is ST2 (IL-33R) molecule that is constitutively expressed on various cells including subpopulation of Foxp3 regulatory cells Th2 lymphocytes, innate lymphoid cells (ILC2), mast cells, basophiles, and eosinophils (8–10). IL-33/ST2 axis is important in several inflammatory diseases (11, 12).

The idea behind this work stems from our previous data on the role of IL-33/ST2 axis in the models of the two Th1 mediated diseases: MLD-STZ and experimental allergic encephalomyelitis (EAE) (5, 13–15). Deletion of ST2 abrogates resistance to EAE in BALB/C mice by enhancing polarization of antigen presenting cells to inflammatory phenotypes and enhancing IL-17 and IFN-γ production. In MLD-STZ diabetes model we showed that resistance to disease in BALB/C mice depends partially on CD4+CD25+Foxp3+ cells (5, 15).

Further, Yuan et al. (16) recently showed that in prediabetic NOD mice a combination of CD122 and IL-33 promotes Tregs abundance and function in pancreatic islets. Finally Ryba-Stanislawowska et al. (17) reported that in vitro IL-33 treatment of Tregs derived from patients with type 1 diabetes resulted in quantitative and qualitative enhancement of their suppressive activity.

Siede et al. (18) have reported that IL-33 receptor expressing Treg cells acquire capacity to produce IL-5 and IL-13 and suppress T effectors cells by producing IL-10. Taken together these data suggested that in vivo treatment of IL-33 may have beneficial effects in MLD-STZ diabetes by promoting Tregs and in particular ST2+ Tregs producing IL-10 and possibly IL-5 and/or IL-13.

MLD-STZ induced diabetes appears to be an experimental model for studying T cell-dependent inflammatory pathology in the islets (19). We used this model to investigate the immunomodulatory capacity of IL-33 and to delineate the mechanisms influencing effectors immune cell functions. Our study has shown that IL-33 prevents MLD-STZ diabetes induction if given at the time of disease induction. If given 6 and 12 days after the disease induction IL-33 can still significantly attenuate development of hyperglycemia. Finally, in order to show relevance of our findings for the development of "spontaneous" diabetes, we looked at the possibility that exogenous IL-33 alter the onset of insulitis in prediabetic NOD mice. IL-33 treated NOD mice showed significantly lower mononuclear cells infiltration but higher percentage and number of CD4+IL-5+, CD4+IL-13+, and CD4+Foxp3+ cells expression in the islets.

This beneficial effect appears to be mainly due to the ability of IL-33 to enhance induction of regulatory CD4+Foxp3+ ST2+ T cells.

# MATERIALS AND METHODS

#### Experimental Animals

C57BL/6 mice male 8–10 week old, housed under conventional conditions and allowed laboratory chow and water ad libitum, were used in the experiments. Within each experiment, animals were matched by age and weight (18–24 g) and randomly divided into groups of 7–8 to receive different treatments. Breeding pairs of NOD mice were purchased from Charles River Laboratories SRL (Calco, Italy) and maintained in specific pathogen free facilities. Female NOD mice at the 16 week of age were used in the experiment. In our animal facilities only 25–35% of NOD mice develop diabetes typically at the 24th week of age. In attempt to standardize the evaluation of IL-33 in these mice we used group of 20 female mice at the age of 16 weeks and 10 of them were treated with IL-33.

All animal procedures were approved by the Ethics Committee for Animal studies of the Faculty of Medical Science, University in Kragujevac.

#### Diabetes Induction

Diabetes was induced by MLD-STZ, as described earlier (20). Briefly, animals received intraperitoneally (i. p.) 40 mg/kg (b.w.) STZ (Sigma-Aldrich, St Louis, MO) dissolved in citrate buffer on pH 4.5 for 5 consecutive days. The daily dose of STZ is determined by the daily weight measurement of each mouse immediately prior to the injection of the substance.

#### Diabetes Evaluation

Clinical diabetes was defined by hyperglycaemia (blood glucose levels > 10.3 mmol/l) and glycosuria (urine glucose levels > 7.1 mmol/l or 128 mg/100 ml) in fasted animals. Blood glucose levels and urine glucose were measured three times per week. Samples were taken from the tail tip after starvation for 4 h. Blood glucose levels (mmol/l) were determined using the Accu-Chek Performa glucometer (Roshe Diagnostics, Mannheim, Germany) and urine glucose was analyzed by Uriscan 2 test strip (YD diagnostics, Korea).

# Glucose Tolerance Test (GTT)

GTT analyses were performed both in C57BL/6 and NOD mice at the end of experiment. To this end food was withheld 16 h before testing. Animals were weighed and injected with 2 g/kg of glucose (i.p.). Glucose concentrations were measured before and at 0, 15, 30, 60, and 120 min after glucose injection.

#### IL-33 Application

C57BL/6 mice received exogenous mouse IL-33 (0.4 µg/injection; eBioscience) intraperitoneally, according to the following scheme: I group: days 0, 2, 4, and 6; II group: days 6, 8, 10, and 12; III group: days 12, 14, 16, and 18. Control animals were treated with intraperitoneally PBS + citrate buffer (CB) solution or IL-33 + citrate buffer solution at the same time interval.

The effects of IL-33 on development of periinsulitis and insulitis in prediabetic NOD mice were evaluated. Sixteen weeks old animals that were all normoglycemic, free of glycosuria and with normal GTT test were treated with 6 injections of 0.4 µg/mouse IL-33 (every second day) and sacrificed for histological analysis at 18th week of age.

#### Cyclophosphamide (CY) Administration

CY was administered at a dose 200 mg/kg of body weight twice on 5 and 7th day of the treatment. The first CY injection was given ∼8 h after the last administration of STZ as a precaution against a possible interaction of CY with STZ (21).

#### Histological Examination of Pancreata

Pancreata of all groups were excised and placed in 10% buffered formaldehyde fixative solution overnight at room temperature. Pancreatic tissue paraffin sections were stained with hematoxylin-eosin were used for the analysis of lymphocytic infiltrates in the Langerhans pancreatic islets by light microscope (BX51; Olympus, Japan) using a magnification lens of 40 × (14). Histological analysis of the distribution of inflammatory cell infiltrate in pancreatic islets was performed in blinded fashion by two independent observers (MM and SP). Insulitis was graded and a mean insulitis score was calculated as described previously (22).

### Isolation of Lymphoid Cells for Phenotypic Assessment

Pancreatic draining lymph nodes were removed, and singlecell suspensions were obtained by mechanical disruption. After removing pancreatic lymph nodes, pancreas was processed through three steps: in situ perfusion with collagenase, pancreatic digestion, and isolation of the islet. The cells were separated according to the protocol as describe elsewhere (23) and analyzed by flow cytofluorimetry. Data was shown as percentage of mononuclear cells and absolute number of cells per islets from one pancreas.

### Flow Cytometric Analysis

Cells suspensions were prepared from lymph nodes and pancreatic islets. Single-cell suspensions were labeled with fluorochrome-conjugated monoclonal antibodies: anti-mouse CD3, CD4, CD8, ST2, and CXCR3 (BD Biosciences), CD11c and CD11b antibodies (BioLegend, San Diego, CA) or with isotype-matched control and analyzed on a FACSCalibur (BD) using CELLQUEST software (BD). The intracellular staining was performed with lymph node cells incubated for 6 h in the presence of Phorbol 12-myristate13-acetate (50 ng/ml) (Sigma, USA), Ionomycin (Sigma, USA) (500 ng/ml), and GolgyStop (BD Pharmingen) at 37◦C, 5% CO2, stained with anti-CD4 monoclonal antibodies or appropriate isotype controls, fixed and permeabilized with a Cytofix/Cytoperm solution. Intracellular staining was performed using monoclonal antibodies: IFN-γ, IL-17, IL-10, IL-5, IL-13, IL-2, and Foxp3 (BD Biosciences) or appropriate negative controls. Cells were analyzed with the FACSCalibur Flow Cytometer (BD Biosciences), and analysis was conducted with FlowJo (Tree Star).

#### Statistical Analysis

All variables were continuous and values were described by the means ± SEM. In order to determine differences in the mean values of continuous variables with a normal distribution of values, parametric Student's t-test was used, and its non-parametric alternative Mann-Whitney test if data did not follow a normal distribution. All data were analyzed using the statistical program SPSS version 20 (SPSS Inc., Chicago, IL) where p-value < 0.05 was considered statistically significant.

## RESULTS

## IL-33 Treatment Prevents Diabetes Induction in C57BL/6 Mice as Evaluated by Glycemia, Glucose Tolerance Test, Glycosuria, and Islet Infiltration

Present study was undertaken to analyze the effect of exogenous IL-33 on the onset and the development of type 1 diabetes as evaluated by glycemia, glucose tolerance test, glycosuria, and islet infiltration. C57BL/6 mice were injected with four injections of IL-33 or PBS starting from the day 0 as described in material and methods section (I group). As shown in **Figure 1**, exogenous IL-33 showed strong suppressive effects on diabetes induction and no biochemical parameters of the disease onset were noticed. Significant difference in values of glycemia was observed from the day 15 and remained until the end of the experiment (**Figure 1A**).

We examined blood sugar values of the experimental mice after glucose loading using a glucose tolerance test. All measurements were conducted within 120 min in line with the schedule: 0, 15, 30, 60, and 120. Assessing the differences between the group that received IL-33 from the day 0 and the control group, we found significant differences in all five time points. Significances (p-values) according to the schedule of measurements were as follows: 0.004, 0.039, 0.003, < 0.001, and 0.013, respectively (**Figure 1B**). Control animals did not develop hyperglycemia by the end of the experiment (**Figures 1A**,**B**).

We also evaluated the onset and the differences in levels of glycosuria between the observed groups of animals. Measurements were performed on days 10, 14, 18, 22, 24, 26, and 28. First two measurements (days 10 and 14) did not detect glucose in animal's urine in either group. Glycosuria occurred on the day 18 in mice treated with STZ only. Complete prevention of glycosuria was achieved by administration IL-33 (**Figures 1C**,**D**).

The degree of insulitis was graded according to Hall et al. (22) and Pejnovic et al. (24): normal islet, score 1; perivascular/periductal infiltration, score 2; peri-insulitis, score 3; mild insulitis (< 25% of the islet infiltrated), score 4; and severe insulitis (more than 25% of the islet infiltrated), score 5. The histological examination of the pancreata revealed differences in the intensity of mononuclear cells infiltration in the islets. No islets with severe insulitis were detected in the group of mice treated with IL-33, while the control group of mice had 47.06% of islets with severe insulitis (p < 0.001). The percentage of total intact islets was 58.82% in IL-33 treated mice compared to control group of mice with 5.8% of healthy islets (p < 0.001) (**Figures 1E**,**F**).

FIGURE 1 | Concomitant treatment with recombinant IL-33 completely abrogates induction of diabetes by MLD-STZ. Effect of four injections of 0.4 µg/mouse IL-33 on glycemia (A), GTT test (B), and glycosuria (C,D). Histology of the islets showed highly significant (*p* < 0.001) decrease of mononuclear cells influx in IL-33 treated group compared to control group (E,F). An analysis of lymphocytic infiltrates in Langerhans's pancreatic islands was performed by light microscope using a magnifying lens of 40 X.

# Exogenous IL-33 Particularly Enhances the Number of CD4+Foxp3+ Cells in Pancreatic Lymph Nodes in C57BL/6 Mice

IL-33 did not cause significant differences in the total number and percentage of CD4+ (**Figures 2A**,**B**) and CD8+ lymphocytes (**Figures 2C**,**D**) in pancreatic lymph nodes of examined animals. There were no significant differences in percentage and total number of IFN-γ producing CD4+ T lymphocytes between the group that were treated with IL-33 and the group that were treated with STZ only (**Figures 2E**,**F**). IL-33 treatment significantly reduced the percentage of CD8 lymphocytes producing IFN-γ (p = 0.011) (**Figure 2G**). Likewise, the absolute number of CD8+IFN-γ + cells was also significantly lower in the group of mice treated with IL-33, compared to the control group (p = 0.029) (**Figure 2H**). There was no significant difference in the percentage and number of IL-17 producing CD4+cells (**Figures 2I**,**J**). IL-33 treatment significantly increased the percentage (p = 0.005) and the total number (p = 0.038) of CD4+IL-5+ cells (**Figures 2K**,**L**) and the percentage and total number of CD4+IL-13+ cells (p = 0.02, p = 0.028, respectively) (**Figures 2M**,**N**).

We noticed a significant increase in the percentage (p = 0.038) and number (p = 0.037) of regulatory CD4+Foxp3+ cells in the group treated with IL-33 (**Figures 3A**,**B**). Further, the IL-33 treatment led to highly significant increase of the percentage and the number of Treg cells expressing ST2 molecule (p < 0.001) (**Figures 3C**,**D**). Exogenous IL-33 significantly increased percentage and number of CD4+Foxp3+ST2+IL-10+ (p = 0.037, p < 0.001, respectively) (**Figures 3E**,**F**), CD4+Foxp3+ST2+IL-13+ (p < 0.001, p < 0.001, respectively) (**Figures 3G**,**H**) and percentage of CD4+Foxp3+ST2+IL-5+ (p < 0.001) (**Figure 3J**) cells in pancreatic lymph nodes. However, IL-33 treatment did not increase the percentage and number of CD11c+ cells (**Figures 3K**,**L**), but significantly increased percentage of CD11c+ population that produces IL-2 which is important for the survival of Treg cells (p = 0.023; **Figure 3M**). Dot plots relevant for **Figures 2**, **3** are given in **Supplementary Figure S1**.

### IL-33 Treatment Leads to Significant Decrease in CXCR3+ Diabetogenic Cells in the Islets

We also investigated mononuclear infiltrate in pancreatic islets at the end of the experiments (**Figure 4**). There was no difference in the percentage and number of CD4+ cells (**Figures 4A,B**) and CD8+ cells (**Figures 4C,D**). Results showed significantly decreased percentage (p = 0.029) and number (p < 0.001) of effectors CD4+CXCR3+ cells in the group of mice treated with IL-33 (**Figures 4G,H**). Similarly, we noticed lower percentage of CD8+CXCR3+ cells (**Figures 4I,J**) and higher percentage of CD4+IL-10+ cells (**Figures 4K,L**) but differences did not reach statistical significance. The treatment with IL-33 induced higher percentage (p = 0.042) and total number (p = 0.038) of CD4+Foxp3+ST2+ (**Figures 4O,P**) in mononuclear cell population in the islets. Total number of CD4+Foxp3+ST2+IL-10+ cell was also increased (p = 0.038) **(Figures 4Q,R**). Furthermore, we have shown that exogenous IL-33 increased the percentage and the total number of tolerogenic dendritic CD11b+CD11c+ cells (p = 0.013, p < 0.001, respectively) (**Figures 4S,T**) in mononuclear cell population in the islets. Dot plots relevant for **Figure 4** are given in **Supplementary Figure S2**.

### Low Dose of Cyclophosphamide (CY) Affects Regulatory Cells and Attenuates Protective Effect of IL-33 in Autoimmunity Including Type 1 Diabetes

We and others have shown previously that in mice (15) and rats (25) low dose of CY enhances diabetes induction by affecting regulatory cells. Therefore, we tested by day 18 whether protective effect of IL-33 will be affected by pretreatment with CY. There was significant difference in the streptozotocin induced level of glycemia between the groups treated with IL-33 + CY and IL-33 only (p < 0.001). Animals treated with STZ + PBS showed high glycemia (23 ± 2.16) vs. animals treated with IL-33+CB or PBS+CB (glycemia 6.8 ± 0.36; 7.0 ± 0.28) which did not develop disease (**Figure 5A)**.

Phenotyping of the cells in pancreatic lymph nodes at the end point of the experiment confirmed the low total number of CD4+Foxp3+ positive cells (p = 0.013) (**Figure 5C**) as well as CD4+Foxp3+ST2+ cells (p = 0.008) (**Figure 5D**) after CY treatment that correlated with higher glycemia. The percentage of CD4+Foxp3+ positive cells was also increased in pancreatic islets (p = 0.004) (**Figure 5E**). These data suggest that CD4+Foxp3+ (ST2+) cells are major downregulatory cells induced by IL-33 in MLD-STZ diabetes.

### IL-33 Downregulates Diabetes If Given After the Onset of Disease

Exogenous IL-33 treatment, applied after MLD-STZ, had also antidiabetogenic effects in mice received IL-33 from the day 6 and 12, respectively (**Figure 6**). In the groups of mice that received IL-33 from day 6, two out of 7 animals and from day 12 three out of 7 developed hyperglycemia presenting the partial effect of IL-33 if given after the onset of disease. There was significant difference in glycemia level between IL-33 treated and control animals (**Figures 6A**,**B**).

GTT results were significantly different when comparing the group that received IL-33 from the day 6 to control with the exception of GTT measurement in minute 15 showing similar results within the groups. Quantifications of GTT in remaining four-time intervals were significantly different with p-values in minute 0, 0.008; min 30, 0.005; min 60, 0.002; min 120, 0.023 (**Figure 6C**). Comparing group that received IL-33 from the day 12 with control, the only result with similar GTT levels was evaluated in min 30. Remaining four of scheduled measurements revealed significant differences with p-values of 0.004, 0.034, 0.001, and 0.004, respectively (**Figure 6D**).

Initiated on the day 6 and 12, IL-33 caused a development of a low level of glycosuria. Those low levels were significantly different (p = 0.011 from day 18) when IL-33 was initiated on the day 6 and remained different until the end of the experiment on

percentage and total number of CD4+Foxp3+ (A,B), CD4+Foxp3+ST2+ (C,D), CD4+Foxp3+ST2+IL-10+ (E,F), CD4+Foxp3+ST2+IL-13+ (G,H), CD4+Foxp3+ST2+IL-5+ (I,J), CD11c+ (K,L), CD11c+IL-2+ (M,N) were examined. The animals were treated with 0.4 µg/injection IL-33 together with MLD-STZ (i.p. 40 mg/kg for 5 consecutive days) or of an equimolar dose of PBS or with 0.4 µg/injection IL-33 together with citrate buffer. Cells were obtained from pancreatic lymph nodes on day 28 after diabetes induction. Data from two individual experiments with at least 8 mice per group are shown as mean ± SEM; by paired *t*-test when compared with values obtained with phosphate-buffered saline.

the day 28 (p = 0.044) when compared with MLD-STZ treated mice (**Figures 6E**,**F**).

#### IL-33 Treatment Attenuates Insulitis in Prediabetic NOD Mice