Rational Design, Synthesis and Evaluation of Oxazolo[4,5-c]-quinolinone Analogs as Novel Interleukin-33 Inhibitors.

Interleukin-33 (IL-33) is an epithelial-derived cytokine that plays an important role in immune-mediated diseases such as asthma, atopic dermatitis, and rheumatoid arthritis. Although IL-33 is considered a potential target for the treatment of allergy-related diseases, no small molecule that inhibits IL-33 has been reported. Based on the structure-activity relationship and in vitro 2D NMR studies employing 15 N-labeled IL-33, we identified that the oxazolo[4,5-c]-quinolinone analog 7 c binds to the interface region of IL-33 and IL-33 receptor (ST2), an orphan receptor of the IL-1 receptor family. Compound 7 c effectively inhibited the production of IL-6 in human mast cells in a dose-dependent manner. Compound 7 c is the first low molecular weight IL-33 inhibitor and may be used as a prototype molecule for structural optimization and investigation of the IL-33/ST2 signaling pathway.

ATP/IL-33-triggered hyperactivation of mast cells results in an amplified production of pro-inflammatory cytokines and eicosanoids.

IL-33 and ATP are alarmins, which are released upon damage of cellular barriers or are actively secreted upon cell stress. Due to high-density expression of the IL-33 receptor T1/ST2 (IL-33R), and the ATP receptor P2X7, mast cells (MCs) are one of the first highly sensitive sentinels recognizing released IL-33 or ATP in damaged peripheral tissues. Whereas IL-33 induces the MyD88-dependent activation of the TAK1-IKK2-NF-κB signalling, ATP induces the Ca2+ -dependent activation of NFAT. Thereby, each signal alone only induces a moderate production of pro-inflammatory cytokines and lipid mediators (LMs). However, MCs, which simultaneously sense (co-sensing) IL-33 and ATP, display an enhanced and prolonged activation of the TAK1-IKK2-NF-κB signalling pathway. This resulted in a massive production of pro-inflammatory cytokines such as IL-2, IL-4, IL-6 and GM-CSF as well as of arachidonic acid-derived cyclooxygenase (COX)-mediated pro-inflammatory prostaglandins (PGs) and thromboxanes (TXs), hallmarks of strong MC activation. Collectively, these data show that co-sensing of ATP and IL-33 results in hyperactivation of MCs, which resembles to MC activation induced by IgE-mediated crosslinking of the FcεRI. Therefore, the IL-33/IL-33R and/or the ATP/P2X7 signalling axis are attractive targets for therapeutical intervention of diseases associated with the loss of integrity of cellular barriers such as allergic and infectious respiratory reactions.

Astegolimab (anti-ST2) efficacy and safety in adults with severe asthma: A randomized clinical trial.

BACKGROUND: The IL-33/ST2 pathway is linked with asthma susceptibility. Inhaled allergens, pollutants, and respiratory viruses, which trigger asthma exacerbations, induce release of IL-33, an epithelial-derived "alarmin." Astegolimab, a human IgG2 mAb, selectively inhibits the IL-33 receptor, ST2. Approved biologic therapies for severe asthma mainly benefit patients with elevated blood eosinophils (type 2-high), but limited options are available for patients with low blood eosinophils (type 2-low). Inhibiting IL-33 signaling may target pathogenic pathways in a wider spectrum of asthmatics. OBJECTIVES: This study evaluated astegolimab efficacy and safety in patients with severe asthma. METHODS: This double-blind, placebo-controlled, dose-ranging study (ZENYATTA [A Study to Assess the Efficacy and Safety of MSTT1041A in Participants With Uncontrolled Severe Asthma]) randomized 502 adults with severe asthma to subcutaneous placebo or 70-mg, 210-mg, or 490-mg doses of astegolimab every 4 weeks. The primary endpoint was the annualized asthma exacerbation rate (AER) at week 54. Enrollment caps ensured ∼30 patients who were eosinophil-high (≥300 cells/µL) and ∼95 patients who were eosinophil-low (<300 cells/µL) per arm. RESULTS: Overall, adjusted AER reductions relative to placebo were 43% (P = .005), 22% (P = .18), and 37% (P = .01) for 490-mg, 210-mg, and 70-mg doses of astegolimab, respectively. Adjusted AER reductions for patients who were eosinophil-low were comparable to reductions in the overall population: 54% (P = .002), 14% (P = .48), and 35% (P = .05) for 490-mg, 210-mg, and 70-mg doses of astegolimab. Adverse events were similar in astegolimab- and placebo-treated groups. CONCLUSIONS: Astegolimab reduced AER in a broad population of patients, including those who were eosinophil-low, with inadequately controlled, severe asthma. Astegolimab was safe and well tolerated.

Soluble suppression of tumorigenicity-2 predicts pneumonia in patients with inhalation injury: Results of a pilot study.

INTRODUCTION: Several mechanisms play a role in the development of pneumonia after inhalation injury. Our aim was to analyze whether higher concentrations of inflammatory markers or of biomarkers of epithelial injury are associated with a higher incidence of pneumonia in patients with inhalation injury. MATERIAL AND METHODS: Secondary analysis of a single-center prospective observational cohort pilot study, performed over a two-year period (2015-2017) at the Burns Unit of the Plastic and Reconstructive Surgery Department of Vall d'Hebron University Hospital. All patients aged 18 with suspected inhalation injury undergoing admission to the Burns Unit were included. Plasma biomarkers of the lung epithelium (RAGE and SP-D), inflammation markers (IL6, IL8), and IL33, as well as soluble suppression of tumorigenicity-2 (sST2) levels, were measured within the first 24 h of admission. RESULTS: Twenty-four patients with inhalation injury were included. Eight (33.3%) developed pneumonia after a median of 7 (4-8) days of hospital stay. Patients with pneumonia presented higher plasma concentrations of sST2 (2853 [2356-3351] ng/mL vs 1352 [865-1839] ng/mL; p < 0.001), IL33 (1.95 [1.31-2.59] pg/mL vs 1.26 [1.07-1.45] pg/mL; p = 0.002) and IL8 (325.7 [221.6-430.0] pg/mL vs 174.1 [95.2-253.0] pg/mL; p = 0.017) on day 1 of inclusion. Plasma sST2 concentration in the first 24 h demonstrated excellent diagnostic accuracy for predicting the occurrence of pneumonia in patients with smoke inhalation (AUROC 0.929 [95%CI 0.818-1.000]). A cutoff point of ≥2825 ng/mL for sST2 had a sensitivity of 75% and a specificity of 100%. The risk ratio of pneumonia in patients with sST2 ≥ 2825 ng/mL was 7.14 ([95% CI 1.56-32.61]; p = 0.016). CONCLUSIONS: Plasma sST2 in the first 24 h of admission predicts the occurrence of pneumonia in patients with inhalation injury.

Osthole attenuates ovalbumin­induced lung inflammation via the inhibition of IL­33/ST2 signaling in asthmatic mice.

Asthma is a common chronic inflammatory airway disease. Recent studies have reported that interleukin (IL)­33 is a potential link between the airway epithelium and Th2­type inflammatory responses, which are closely related to the progression of asthma. The IL­33 receptor, ST2, is highly expressed in group 2 innate lymphoid cells (ILC2s), Th2 cells, mast cells, eosinophils and natural killer (NK) cells. Cnidii Fructus is a Chinese herb with a long history of use in the treatment of asthma in China. Osthole is one of the major components of Cnidii Fructus. The present study examined the anti­asthmatic effects of osthole in mice and aimed to elucidate the underlying mechanisms involving the IL­33/ST2 pathway. BALB/c mice were sensitized and challenged with ovalbumin and then treated with an intraperitoneal injection of osthole (25 and 50 mg/kg). Subsequently, the airway hyper­responsiveness (AHR) and inflammation of the lungs were evaluated. The amounts of IL­4, IL­5, IL­13, interferon (IFN)­Î³ and IL­33 in the bronchoalveolar lavage fluid (BALF) were measured by Luminex assay and their mRNA levels in the lungs were measured by reverse transcription­quantitative PCR. The histopathology of the lungs was performed with H&E, PAS and Masson's staining. The expression of ST2 in the lungs was evaluated by immunohistochemistry. The data demonstrated that osthole markedly reduced AHR and decreased the number of eosinophils and lymphocytes in BALF. It was also observed that osthole significantly inhibited the release of Th2­type cytokines (IL­4, IL­5 and IL­13) and upregulated the IFN­Î³ level in BALF. Moreover, osthole significantly attenuated the IL­33 and ST2 expression in the lungs of asthmatic mice. On the whole, osthole attenuated ovalbumin­induced lung inflammation through the inhibition of IL­33/ST2 signaling in an asthmatic mouse model. These results suggest that osthole is a promising target for the development of an asthma medication.

A helminth-derived suppressor of ST2 blocks allergic responses.

The IL-33-ST2 pathway is an important initiator of type 2 immune responses. We previously characterised the HpARI protein secreted by the model intestinal nematode Heligmosomoides polygyrus, which binds and blocks IL-33. Here, we identify H. polygyrus Binds Alarmin Receptor and Inhibits (HpBARI) and HpBARI_Hom2, both of which consist of complement control protein (CCP) domains, similarly to the immunomodulatory HpARI and Hp-TGM proteins. HpBARI binds murine ST2, inhibiting cell surface detection of ST2, preventing IL-33-ST2 interactions, and inhibiting IL-33 responses in vitro and in an in vivo mouse model of asthma. In H. polygyrus infection, ST2 detection is abrogated in the peritoneal cavity and lung, consistent with systemic effects of HpBARI. HpBARI_Hom2 also binds human ST2 with high affinity, and effectively blocks human PBMC responses to IL-33. Thus, we show that H. polygyrus blocks the IL-33 pathway via both HpARI which blocks the cytokine, and also HpBARI which blocks the receptor.

ST2 Signaling in the Tumor Microenvironment.

Suppression of tumorigenicity 2 (ST2), also known as interleukin-1 receptor-like 1 (IL1RL1), is one of the natural receptors of IL-33. Three major isoforms, ST2L (transmembrane form), sST2 (soluble form), and ST2V, are generated by alternative splicing. Damage to stromal cells induces necrosis and release of IL-33, which binds to heterodimeric ST2L/IL-1RAcP complex on the membrane of a variety of immune cells. This IL-33/ST2L signal induces transcription of the downstream inflammatory and anti-inflammatory genes by activating diverse intracellular kinases and factors to mount an adequate immune response, even in tumor microenvironment. For example, activation of IL-33/ST2L signal may trigger Th2-dependent M2 macrophage polarization to facilitate tumor progression. Notably, sST2 is a soluble form of ST2 that lacks a transmembrane domain but preserves an extracellular domain similar to ST2L, which acts as a "decoy" receptor for IL-33. sST2 has been shown to involve in the inflammatory tumor microenvironment and the progression of colorectal cancer, non-small cell lung cancer, and gastric cancer. Therefore, targeting the IL-33/ST2 axis becomes a promising new immunotherapy for treatment of many cancers. This chapter reviews the recent findings on IL-33/ST2L signaling in tumor microenvironment, the trafficking mode of sST2, and the pharmacological strategies to target IL-33/ST2 axis for cancer treatment.

Anti-inflammatory mediators ST2 and SIGIRR are induced by diphenyldifluoroketone EF24 in lipopolysaccharide-stimulated dendritic cells.

The objective of this study was to investigate the effect of EF24, an NF-κB-inhibitor, on the expression of negative regulators in IL-1R pathway, namely ST2 and SIGIRR. Murine JAWS II dendritic cells (DC) were challenged with lipopolysaccharide (LPS, 100 ng/ml) for 4 h, followed by treatment with 10 µM EF24 for 1 h. ST2 and SIGIRR expression was monitored by qRT-PCR and immunoblotting. ST2L and MyD88 interaction was studied by co-immunoprecipitation, and IL-33, a ST2L ligand, was assayed by ELISA. Activation of transcription factor SP1 was examined by confocal microscopy, immunoblotting, and EMSA. The effect of EF24 on accumulation of ubiquitinated proteins in DCs and proteolysis of fluorogenic peptides by purified proteasome was studied. We found that EF24 upregulated the expression of ST2 and SIGIRR and decreased the interaction of the membrane-bound ST2 (ST2L) with MyD88, and significantly reduced IL-33 levels in LPS-stimulated DCs. Simultaneously it increased the activation of transcription factor SP1and restored the basal level of ubiquitinated proteins in LPS-stimulated DCs. Moreover, EF24 inhibited trypsin- and chymotrypsin-like activity of proteasome by directly interacting with 26S proteasome. The results suggest that EF24 activates endogenous anti-inflammatory arm of IL-1R signaling, most likely by stabilizing SP1 against proteasomal degradation.

Resveratrol inhibits IL-33-mediated mast cell activation by targeting the MK2/3-PI3K/Akt axis.

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

ST2 blockade mitigates peritoneal fibrosis induced by TGF-ß and high glucose.

Peritoneal fibrosis (PF) is an intractable complication of peritoneal dialysis (PD) that leads to peritoneal membrane failure. This study investigated the role of suppression of tumorigenicity (ST)2 in PF using patient samples along with mouse and cell-based models. Baseline dialysate soluble (s)ST2 level in patients measured 1 month after PD initiation was 2063.4 ± 2457.8 pg/mL; patients who switched to haemodialysis had elevated sST2 levels in peritoneal effluent (1576.2 ± 199.9 pg/mL, P = .03), which was associated with PD failure (P = .04). Baseline sST2 showed good performance in predicting PD failure (area under the receiver operating characteristic curve = 0.780, P = .001). In mice with chlorhexidine gluconate-induced PF, ST2 was expressed in fibroblasts and mesothelial cells within submesothelial zones. In primary cultured human peritoneal mesothelial cells (HPMCs), transforming growth factor-ß treatment increased ST2, fibronectin, ß-galactosidase and Snail protein levels and decreased E-cadherin level. Anti-ST2 antibody administration reversed the up-regulation of ST2 and fibronectin expression; it also reduced fibrosis induced by high glucose (100 mmol/L) in HPMCs. Thus, high ST2 level in dialysate is a marker for fibrosis and inflammation during peritoneal injury, and blocking ST2 may be an effective therapeutic strategy for renal preservation.

T2 Biologics for Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a significant cause of morbidity and mortality worldwide. In contrast to other chronic diseases, COPD is increasing in prevalence and is projected to be the third leading cause of death and disability worldwide by 2030. Recent advances in understanding the underlying pathophysiology of COPD has led to the development of novel targeted therapies (biologics and small molecules) that address the underlying pathophysiology of the disease. In severe asthma, biologics targeting type 2 (T2)- mediated immunity have been successful and have changed the treatment paradigm. In contrast, no biologics are currently licensed for the treatment of COPD. Those targeting non-T2 pathways have not demonstrated efficacy and in some cases raised concerns related to safety. With the increasing recognition of the eosinophil and perhaps T2-immunity possibly playing a role in a subgroup of patients with COPD, T2 biologics, specifically anti-IL-5(R), have been tested and demonstrated modest reductions in exacerbation frequency. Potential benefit was related to the baseline blood eosinophil count. These benefits were small compared with asthma. Thus, whether a subgroup of COPD sufferers might respond to anti-IL-5 or other T2-directed biologics remains to be fully addressed and requires further investigation.

Increased Expression of sST2 in Early HIV Infected Patients Attenuated the IL-33 Induced T Cell Responses.

T cell responses were less functional and persisted in an exhausted state in chronic HIV infection. Even in early phase of HIV infection, the dysfunction of HIV-specific T cells can be observed in rapid progressors, but the underlying mechanisms are not fully understood. Cytokines play a central role in regulating T cell function. In this study, we sought to elucidate whether IL-33/ST2 axis plays roles in the regulation of T cell function in HIV infection. We found that the level of IL-33 was upregulated in early HIV-infected patients compared with that in healthy controls and has a trend associated with disease progression. In vitro study shows that IL-33 promotes the expression of IFN-γ by Gag stimulated CD4+ and CD8+T cells from HIV-infected patients to a certain extent. However, soluble ST2 (sST2), a decoy receptor of IL-33, was also increased in early HIV infected patients, especially in those with progressive infection. We found that anti-ST2 antibodies attenuated the effect of IL-33 to CD4+ and CD8+T cells. Our data indicates that elevated expression of IL-33 in early HIV infection has the potential to enhance the function of T cells, but the upregulated sST2 weakens the activity of IL-33, which may indirectly contribute to the dysfunction of T cells and rapid disease progression. This data broadens the understanding of HIV pathogenesis and provides critical information for HIV intervention.

Suppression of Tumorigenicity 2 in Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Soluble suppression of tumorigenicity 2 (sST2) receptor is a biomarker that is elevated in certain systemic inflammatory diseases. Comorbidity-driven microvascular inflammation is postulated to play a key role in heart failure with preserved ejection fraction (HFpEF) pathophysiology, but data on how sST2 relates to clinical characteristics or inflammatory conditions or biomarkers in HFpEF are limited. We sought to determine circulating levels and clinical correlates of sST2 in HFpEF. METHODS AND RESULTS: At enrollment, patients (n=174) from the Phosphodiesterase-5 Inhibition to Improve Clinical Status And Exercise Capacity in Diastolic Heart Failure (RELAX) trial of sildenafil in HFpEF had sST2 levels measured. Clinical characteristics; cardiac structure and function; exercise performance; and biomarkers of neurohumoral activation, systemic inflammation and fibrosis, and myocardial necrosis were assessed in relation to sST2 levels. Median sST2 levels in male and female HFpEF patients were 36.7 ng/mL (range 30.9-49.2 ng/mL; reference range 4-31 ng/mL) and 30.8 ng/mL (range 25.3-39.3 ng/mL; reference range 2-21 ng/mL), respectively. Among HFpEF patients, higher sST2 levels were associated with the presence of diabetes mellitus; atrial fibrillation; renal dysfunction; right ventricular pressure overload and dysfunction; systemic congestion; exercise intolerance; and biomarkers of systemic inflammation and fibrosis, neurohumoral activation, and myocardial necrosis (P<0.05 for all). sST2 was not associated with left ventricular structure or left ventricular systolic or diastolic function. CONCLUSIONS: In HFpEF, sST2 levels were associated with proinflammatory comorbidities, right ventricular pressure overload and dysfunction, and systemic congestion but not with left ventricular geometry or function. These data suggest that ST2 may be a marker of systemic inflammation in HFpEF and potentially of extracardiac origin. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00763867.

Monoclonal antibodies targeting ST2L Domain 1 or Domain 3 differentially modulate IL-33-induced cytokine release by human mast cell and basophilic cell lines.

The cell-surface receptor ST2L triggers cytokine release by immune cells upon exposure to its ligand IL-33. To study the effect of ST2L-dependent signaling in different cell types, we generated antagonist antibodies that bind different receptor domains. We sought to characterize their activities in vitro using both transfected cells as well as basophil and mast cell lines that endogenously express the ST2L receptor. We found that antibodies binding Domain 1 versus Domain 3 of ST2L differentially impacted IL-33-induced cytokine release by mast cells but not the basophilic cell line. Analysis of gene expression in each cell type in the presence and absence of the Domain 1 and Domain 3 mAbs revealed distinct signaling pathways triggered in response to IL-33 as well as to each anti-ST2L antibody. We concluded that perturbing the ST2L/IL-33/IL-1RAcP complex using antibodies directed to different domains of ST2L have a cell-type-specific impact on cytokine release, and may indicate the association of additional receptors to the ST2L/IL-33/IL-1RAcP complex in mast cells.