Receptor ST2 Research Articles

IL-33/ST2 signaling in monocyte-derived macrophages maintains blood-brain barrier integrity and restricts infarctions early after ischemic stroke

BackgroundBrain microglia and infiltrating monocyte-derived macrophages are vital in preserving blood vessel integrity after stroke. Understanding mechanisms that induce immune cells to adopt vascular-protective phenotypes may hasten the development of stroke treatments. IL-33 is a potent chemokine released from damaged cells, such as CNS glia after stroke. The activation of IL-33/ST2 signaling has been shown to promote neuronal viability and white matter integrity after ischemic stroke. The impact of IL-33/ST2 on blood-brain barrier (BBB) integrity, however, remains unknown. The current study fills this gap and reveals a critical role of IL-33/ST2 signaling in macrophage-mediated BBB protection after stroke.MethodsTransient middle cerebral artery occlusion (tMCAO) was performed to induce ischemic stroke in wildtype (WT) versus ST2 knockout (KO) male mice. IL-33 was applied intranasally to tMCAO mice with or without dietary PLX5622 to deplete microglia/macrophages. ST2 KO versus WT bone marrow or macrophage cell transplantations were used to test the involvement of ST2+ macrophages in BBB integrity. Macrophages were cocultured in transwells with brain endothelial cells (ECs) after oxygen-glucose deprivation (OGD) to test potential direct effects of IL33-treated macrophages on the BBB in vitro.ResultsThe ST2 receptor was expressed in brain ECs, microglia, and infiltrating macrophages. Global KO of ST2 led to more IgG extravasation and loss of ZO-1 in cerebral microvessels 3 days post-tMCAO. Intranasal IL-33 administration reduced BBB leakage and infarct severity in microglia/macrophage competent mice, but not in microglia/macrophage depleted mice. Worse BBB injury was observed after tMCAO in chimeric WT mice reconstituted with ST2 KO bone marrow, and in WT mice whose monocytes were replaced by ST2 KO monocytes. Macrophages treated with IL-33 reduced in vitro barrier leakage and maintained tight junction integrity after OGD. In contrast, IL-33 exerted minimal direct effects on the endothelial barrier in the absence of macrophages. IL-33-treated macrophages demonstrated transcriptional upregulation of an array of protective factors, suggesting a shift towards favorable phenotypes.ConclusionOur results demonstrate that early-stage IL-33/ST2 signaling in infiltrating macrophages reduces the extent of acute BBB disruption after stroke. Intranasal IL-33 administration may represent a new strategy to reduce BBB leakage and infarct severity.

IL-33 protects retinal structure and function via mTOR/S6 signaling pathway in optic nerve crush

This study demonstrated the functions and molecular mechanisms of the IL-33/ST2 axis in experimental optic neuropathy. C57BL/6J mice were used to establish an optic nerve crush (ONC) model. ONC mice were administered with IL-33 intraperitoneal injection, with PBS vehicle as control. Immunofluorescence, quantitative RT-PCR, and western blot techniques were utilized to assess the expression of the IL-33/ST2 axis. The electroretinography (ERG), optical coherence tomography (OCT), H&E, and luxol fast blue were used to assess the structural and functional changes. Western blot was employed to detect the activation of the mTOR/S6 pathway. The IL-33 expression level in the inner nuclear layer of the retina in ONC mice reached its peak on day 3, accompanied by a significant increase in IL-33 receptor ST2 expression. IL-33 treatment promoted the survival of retinal ganglion cells, restored the thickness of inner retina layer (IRL), alleviated the demyelination of the optic nerve, and recovered the decreased amplitude of b-wave in ONC mice. Furthermore, administration of IL-33 activated the mTOR/S6 signaling pathway in RGCs, which was significantly suppressed in the ONC condition. This study indicated that boosting the IL-33/ST2/mTOR/S6 pathway can protect against structural and functional damage to the retina and optic nerve induced by ONC. As a result, the IL-33/ST2 axis holds potential as a therapeutic option for treating various optic neuropathies.

The Effect of Metformin Therapy on Serum IL-33 and ST-2 Receptor Levels in Patients With Type 2 Diabetes Mellitus

Introduction: Patients with type 2 had elevated levels of inflammatory biomarkers, including interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF-α). Significant elevations in serum levels of TNF-α and C-reactive protein were seen in individuals with type 2 diabetes mellitus (T2DM). The IL-1 superfamily (IL-1β, IL-18, and IL-33) plays a crucial role in the development of diabetes mellitus by modifying immunological and inflammatory immune responses. Objectives: Little is known about the role of metformin in type 2 diabetes mellitus with respect to serum levels of IL-33 and ST-2 receptors. Therefore, in the present study, we sought toexplore such role. Patients and Methods: This single arm study included 45 type 2 diabetes patients with an age range of 30 to less than 60 years. Patients were given metformin (500 mg orally; for two months then, serum level of interleukin- 33 (IL-33) and ST-2 receptors were evaluated. Results: Treatment resulted in significant reduction of serum the suppression of tumorigenicity suppressor 2 (ST2) from 0.71 ±0.157 µg/mlto 0.59 ±0.11 µg/ml (p<0.001), across with significant increase in serum IL-33 from 0.60 ±0.18 pg/ml to 0.75 ±0.13 pg/ml (p < 0.001). Conclusion: Treatment of type 2 diabetes mellitus using metformin revealed a crucial role for IL-33 and ST-2 receptors in glycemic control since increment in IL-33 and reduction in ST-2 receptors were associated with good glycemic control suggesting an inflammatory role in disease pathogenesis and response to treatment.

IL-33/ST2 enhances MMP-12 expression by macrophages to mediate inflammatory and immune response in IgG4-Related Ophthalmic Disease

IgG4-Related Ophthalmic Disease (IgG4-ROD) is a chronic autoimmune-mediated fibrotic disease that predominantly affects the lacrimal glands, often leading to loss of function in the involved tissues or organs. Recent studies have demonstrated that MMP-12 is highly expressed in IgG4-ROD and plays a significant role in regulating immune responses. In this study, we reviewed nine patients diagnosed with IgG4-ROD based on clinical manifestations and histological analysis, and we investigated the expression of IL-33/ST2 and MMP-12 in IgG4-ROD lacrimal gland tissues using IHC. We found that IL-33 interacts with its specific receptor ST2, both of which are significantly overexpressed in IgG4-ROD tissues. Additionally, we successfully constructed a mouse model by introducing the LatY136F mutation into C57BL/6 mice to mimic IgG4-ROD lacrimal gland involvement, which helped elucidate the mechanisms involved in the induction of MMP-12. Furthermore, immunofluorescence staining confirmed that most MMP-12+ cells were derived from M2 macrophages, and an ELISA assay demonstrated that IL-33 upregulates MMP-12 in IgG4-ROD. Collectively, these data suggest that the IL-33/ST2/MMP-12 signaling pathway is activated in IgG4-ROD, with IL-33/ST2 potentially promoting M2 macrophage polarization and activation to produce MMP-12, which may serve as a novel therapeutic target for IgG4-ROD.

The IL-33/ST2 Axis Protects Retinal Ganglion Cells by Modulating the Astrocyte Response After Optic Nerve Injury.

IL-33 and its receptor ST2 play crucial roles in tissue repair and homeostasis. However, their involvement in optic neuropathy due to trauma and glaucoma remains unclear. Here, we report that IL-33 and ST2 were highly expressed in the mouse optic nerve and retina. Deletion of IL-33 or ST2 exacerbated retinal ganglion cell (RGC) loss, retinal thinning, and nerve fiber degeneration following optic nerve (ON) injury. This heightened retinal neurodegeneration correlated with increased neurotoxic astrocytes in Il33-/- mice. In vitro, rIL-33 mitigated the neurotoxic astrocyte phenotype and reduced the expression of pro-inflammatory factors, thereby alleviating the RGC death induced by neurotoxic astrocyte-conditioned medium in retinal explants. Exogenous IL-33 treatment improved RGC survival in Il33-/- and WT mice after ON injury, but not in ST2-/- mice. Our findings highlight the role of the IL-33/ST2 axis in modulating reactive astrocyte function and providing neuroprotection for RGCs following ON injury.

ST2 + T-Regulatory Cells in Renal Inflammation and Fibrosis after Ischemic Kidney Injury.

Inflammation is a major cause of kidney injury. The Interleukin (IL)-1 family cytokine IL-33 is released from damaged cells and modulates the immune response through its receptor ST2 expressed on many cell types, including regulatory T-cells (Tregs). While a proinflammatory role of IL-33 has been proposed, exogenous IL-33 expanded Tregs and suppressed renal inflammation. However, the contribution of endogenous IL-33/ST2 for the role of Tregs in the resolution of kidney injury has not been investigated. We used murine renal ischemia-reperfusion injury and kidney organoids to delineate the role of the ST2 and amphiregulin (AREG) specifically in Treg cells using targeted deletion. Bulk and single-cell RNA sequencing was performed on flow-sorted Tregs from spleen and CD4 T-cells from post-ischemic kidneys respectively. The protective role of ST2-sufficient Tregs was analyzed using a novel co-culture system of syngeneic kidney organoids and Treg cells under hypoxic conditions. Bulk RNA-sequencing of splenic and single-cell-RNA-sequencing of kidney T-cells showed that ST2+ Tregs are enriched for genes related to Treg proliferation and function. Genes for reparative factors such as AREG were also enriched in ST2+ Tregs. Treg-specific deletion of ST2 or AREG exacerbated kidney injury and fibrosis in the unilateral ischemia reperfusion injury model. In co-culture studies, WT but not ST2-deficient Tregs preserved the hypoxia-induced loss of kidney-organoid viability, which was restored by AREG supplementation. Our study identified the role of the IL-33/ST2 pathway in Tregs for resolution of kidney injury. The transcriptome of ST2+ Tregs was enriched for reparative factors including AREG. Lack of ST2 or AREG in Tregs worsened kidney injury. Tregs protected kidney organoids from hypoxia in ST2 and AREG-dependent manner. Thus Treg-based approaches could be of benefit for resolution of renal injury.

Mast cells contribute to T-cell accumulation in the bronchoalveolar space in mice with IL-33-induced airway inflammation.

Interleukin (IL)-33 released from airway epithelial cells plays a vital role in shaping type 2 immune responses by binding to the ST2 receptor present in many immune cells, including mast cells (MCs). Intranasal administration of IL-33 in mice induces type 2 lung inflammation, an increase in lung MC progenitors, and transepithelial migration of leukocytes to the bronchoalveolar space. The aim of this study was to determine the contribution of MCs in IL-33-induced lung pathology. Four daily intranasal administrations of IL-33 reduced spirometry-like lung function parameters, induced airway hyperresponsiveness, and increased leukocytes in bronchoalveolar lavage fluid (BAL) in an ST2-dependent manner. MC-deficient (Cpa3cre/+) mice, which lack MCs, had intact spirometry-like lung function but slightly reduced airway hyperresponsiveness, possibly related to reduced IL-33 or serotonin. Strikingly, Cpa3cre/+ mice exposed to IL-33 had 50% reduction in BAL T-cells, and CXCL1 and IL-33 were reduced in the lung. Intranasal IL-33 induced CXCR2 expression in T-cells in a MC-independent fashion. Furthermore, IL-33-induced lung MCs were immunopositive for CXCL1 and localized in the epithelium of wild-type mice. These results suggest that MCs are required to sustain intact lung IL-33 and CXCL1 levels in mice with IL-33-induced airway inflammation, thereby facilitating T-cell accumulation in the bronchoalveolar space.

Edwardsiella tarda induces airways inflammation and production of autoantibodies against lung tissues through regulation of the IL-33-ST2 axis.

Chronic obstructive pulmonary disease (COPD) is a highly prevalent chronic respiratory disease characterised by irreversible airways obstruction associated with chronic airways inflammation and remodelling, while the pathogenesis and the mechanistic differences between patients remain to be fully elucidated. We previously reported that alarmin cytokine IL-33 may contribute to the production of autoantibodies against respiratory epithelial cells. Here we expand the hypothesis that pulmonary autoimmune responses induced by airway microbiota also contribute to the progression of COPD. We focused on Edwardsiella tarda which we detected uniquely in the induced sputum of patients with acute exacerbations of COPD. Pernasal challenge of the airways of WT mice with supernatants of cultured E. tarda induced marked, elevated expression of IL-33 in the lung tissues. Immunisation of animals with supernatants of cultured E. tarda resulted in significantly elevated airways inflammation, the formation of tertiary lymphatic structures and significantly elevated proportions of T follicular helper T cells in the lung tissue and mediastinal lymph nodes. Interestingly, such challenge also induced production of IgG autoantibodies directed against lung tissue lysate, alveolar epithelial cell proteins and elastin fragment, while putrescine, one of metabolites generated by the bacterium, might play an important role in the autoantibody production. Furthermore, all of these effects were partly but significantly abrogated in mice with deletion of the IL-33 receptor ST2. Collectively, these data support the hypothesis that COPD is progressed at least partly by airways microbiota such as E. tarda initiating autoimmune attack of the airways epithelium mediated at least partly through the IL-33-ST2 axis.

Airway proteomics reveals broad residual anti-inflammatory effects of prednisolone in mepolizumab-treated asthma

BackgroundMepolizumab is an anti-interleukin-5 monoclonal antibody treatment for severe eosinophilic asthma (SEA) that reduces asthma exacerbations. Residual airway inflammation on mepolizumab may lead to persistent exacerbations. Oral corticosteroids remain the main treatment for these residual exacerbations. ObjectiveOur study aimed to explore the corticosteroid-responsiveness of airway inflammation after mepolizumab treatment to find potentially treatable inflammatory mechanisms beyond the IL-5 pathway. MethodThe MAPLE trial was a multi-centre, randomized, double-blind, placebo-controlled, crossover study of 2 weeks of high-dose oral prednisolone treatment at stable state in 27 patients treated with mepolizumab for SEA. We analysed paired sputum (n=16) and plasma (n=25) samples from the MAPLE trial using high-throughput Olink® proteomics. We also analysed additional sputum proteins using ELISA. ResultsIn patients receiving mepolizumab, prednisolone significantly downregulated sputum proteins related to type-2 inflammation and chemotaxis including IL-4, IL-5, IL-13, CCL24, CCL26, EDN, CCL17, CCL22, OX40 receptor, FCER2, and the ST2 receptor. Prednisolone also downregulated cell adhesion molecules, prostaglandin synthases, mast cell tryptases, MMP1, MMP12, and neuroimmune mediators. Neutrophilic pathways were upregulated.Type-2 proteins were also downregulated in plasma, combined with IL-12, IFN-γ, and IP-10. IL-10 and amphiregulin were upregulated. ConclusionAt stable state, prednisolone has broad anti-inflammatory effects on top of mepolizumab. These effects are heterogeneous and may be clinically relevant in residual exacerbations.

Could soluble ST2 levels be used to detect nondipper hypertensive subgroup in newly diagnosed hypertension patients.

ST2 receptor is a member of toll-like/interleukin-1 receptor family. After the activation of IL-33/ST2 signaling pathway clinically detectable amount of soluble form of ST2 (sST2) is released into the circulation. Previous studies showed that sST2 levels were significantly higher in hypertension patients than in controls. In this prospective study, we aimed to analyze this relation and test the predictive accuracy of the sST2 level in diagnosis of nondipping hypertension in newly diagnosed hypertension patients. Three hundred thirty-seven patients (150 normal, 187 hypertension) who presented with symptoms of hypertension were included in the study. All patients underwent 24-h ambulatory blood pressure monitoring and sST2 measurement. Of 187 hypertension patients, 92 of them had nondipping and 95 of them had dipping pattern. sST2 level was significantly higher in nondipping group compared to dipping group and control group (40.79 ± 7.77 vs. 32.47 ± 6.68; P < 0.0001 and 40.79 ± 7.77 vs. 20.09 ± 7.09; P < 0.0001 respectively). Binary logistic regression analysis revealed that; only sST2 level was an independent risk factor for hypertension [P < 0.0001, β: 1.258, odds ratio (OR) (95% confidence interval (CI)): 1.158-1.366]. and also nondipping hypertension [P < 0.0001, β: 1.208, OR (95% CI): 1.108-1.317]. Based on the present study it could be concluded that sST2 level is significantly associated with the newly diagnosed hypertension and nondipping hypertension. Hence it could reliably be used to diagnose hypertension and nondipping hypertension with high sensitivity and specificity.

The IL-33-ST2 axis plays a vital role in endometriosis via promoting epithelial–mesenchymal transition by phosphorylating β-catenin

ObjectivesInterleukin 33 (IL-33) is a crucial inflammatory factor that functions as an alarm signal in endometriosis (EMs). Epithelial-mesenchymal transition (EMT), a process related to inflammatory signals, intracellular reactive oxygen species (ROS) production, and lipid peroxidation, have been proposed as potential mechanisms that contribute to the development and progression of EMs. IL-33 is highly upregulated in the ectopic milieu. Moreover, ectopic endometrial cells constitutively express interleukin-33 receptor ST2 (IL-33R). However, the role of IL-33/ST2 in the EMT of EMs remains largely unknown. In this study, we aimed to mechanistically determine the role of IL-33/ST2 in EMs-associated fibrosis.Materials and methodsWe established a non-lethal oxidative stress model to explore the conditions that trigger IL-33 induction. We performed α-smooth muscle actin (α-SMA) protein detection, cell counting kit-8 (CCK-8) assays, and scratch assays to analyze the impact of IL-33 on primary endometrial stromal cells (ESCs) proliferation and invasion. Clinical samples from patients with or without EMs were subjected to immunohistochemical (IHC) and and immunofluorescence(IF) staining to assess the clinical relevance of IL-33 receptor ST2 and EMT-related proteins. Furthermore, we used the ectopic human endometrial epithelial cell line 12Z and normal human epithelial cell line EEC to evaluate the effects of IL-33 on Wnt/β-catenin signaling. The effect of IL-33 on EMT-associated fibrosis was validated in vivo by intraperitoneal injections of IL-33 and antiST2.ResultsWe observed that ectopic milieu, characterized by ROS, TGF-β1, and high level of estrogen, triggers the secretion of IL-33 from ectopic ESCs. Ectopic endometrial lesions exhibited higher level of fibrotic characteristics and ST2 expression than that in the normal endometrium. Exogenous recombinant human (rhIL-33) enhanced ESC migration and survival. Similarly, 12Z cells displayed a higher degree of EMT characteristics with elevated expression of CCN4 and Fra-1, downstream target genes of the WNT/β-catenin pathway, than that observed in EECs. Conversely, blocking IL-33 with neutralizing antibodies, knocking down ST2 or β-catenin with siRNA, and β-catenin dephosphorylation abolished its effects on EMT promotion. In vivo validation demonstrated that IL-33 significantly promotes EMs-related fibrosis through the activation of Wnt/β-catenin signaling.ConclusionOur data strongly support the vital role of the IL-33/ST2 pathway in EMs-associated fibrosis and emphasize the importance of the EMT in the pathophysiology of fibrosis. Targeting the IL-33/ST2/Wnt/β-catenin axis may hold promise as a feasible therapeutic approach for controlling fibrosis in EMs.

BMP9 attenuates IL-33 signaling and prevents endothelial-to-mesenchymal transition in pulmonary arterial hypertension

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Dutch Cardiovascular Alliance - PHAEDRA IMPACT Background Pulmonary Arterial hypertension (PAH) is a rare and progressive disease characterized by increased vascular resistance and remodeling of the pulmonary arteries. Disturbed Transforming growth factor (TGF)-β signaling, often associated with mutations in the gene encoding for the bone morphogenic protein receptor type II (BMPR2) and endothelial-to-mesenchymal transition (EndMT), emerges as a central culprit in the pathogenesis of PAH. Interleukin-33 (IL-33) has been identified as an inducer of EndMT, and its soluble receptor ST2 (sST2) has emerged as a potential biomarker for PAH. Bone morphogenic protein (BMP) 9, a BMPR2 ligand, showing a high affinity for endothelial cells, is being explored as a therapeutic approach. Purpose We hypothesize that a crosstalk between the TGF-β pathway and IL-33, a known promoter of vascular remodeling, occurs in PAH. This study aims to unravel the protective role of BMP9 in PAH, with a specific focus on the involvement of EndMT, a key player in the pathogenesis of vascular diseases. Methods The expression of IL-33 protein levels was assessed in Pulmonary Arterial Endothelial Cells (PAEC) and in lung tissues from experimentally induced PH (Sugen/Hypoxia (SuHx)) or control mice. Signal transduction and EndMT were investigated in PAECs exposed to IL-33, BMP9 and sST2, by gene expression analysis (qPCR), western blotting, ELISA and immunostaining. BMP9 and sST2 levels were measured in plasma from PAH patients by ELISA. Results Elevated IL-33 expression was observed in PAEC from PAH patients and lung tissues from SuHx mice compared to healthy controls. In vitro, BMP9 significantly induced the expression of the IL-33 soluble receptor ST2, while concurrently reducing IL-33 target gene expression. IL-33-induced EndoMT in PAEC was effectively mitigated by pre-incubation with BMP9, proving more efficacious than ST2 neutralizing antibody. Furthermore, a correlation between BMP9 and sST2 levels in plasma from PAH patients was identified. Conclusion Our findings demonstrate that BMP9 offers superior protections against IL-33-induced EndMT in PAECs compared to a ST2 neutralizing antibody. BMP9 induces expression of sST2, leading to IL-33 neutralization and subsequent EndMT inhibition. Therefore, targeting the IL-33 pathway in PAH patients presents a promising therapeutic option for preventing EndMT. This study provides novel insights into the pathogenesis of PAH, revealing a potential therapeutic target for this rare disease.BMP9 inhibits IL-33 downstream signalingBMP9 protects from IL-33-induced EndMT

IL-33 controls IL-22-dependent antibacterial defense by modulating the microbiota

IL-22 plays a critical role in defending against mucosal infections, but how IL-22 production is regulated is incompletely understood. Here, we show that mice lacking IL-33 or its receptor ST2 (IL-1RL1) were more resistant to Streptococcus pneumoniae lung infection than wild-type animals and that single-nucleotide polymorphisms in IL33 and IL1RL1 were associated with pneumococcal pneumonia in humans. The effect of IL-33 on S. pneumoniae infection was mediated by negative regulation of IL-22 production in innate lymphoid cells (ILCs) but independent of ILC2s as well as IL-4 and IL-13 signaling. Moreover, IL-33's influence on IL-22-dependent antibacterial defense was dependent on housing conditions of the mice and mediated by IL-33's modulatory effect on the gut microbiota. Collectively, we provide insight into the bidirectional crosstalk between the innate immune system and the microbiota. We conclude that both genetic and environmental factors influence the gut microbiota, thereby impacting the efficacy of antibacterial immune defense and susceptibility to pneumonia.

Abstract 1116: IL-33 Promotes Cardiovascular Lesion Development In A Murine Model Of Kawasaki Disease By Boosting IL-1β Production

Kawasaki Disease (KD), a pediatric acute febrile systemic vasculitis, is the leading cause of acquired heart diseases in children. Coronary artery aneurysms (CAAs) occur in up to 25% of untreated children, which is reduced to 5% with intravenous immunoglobulin (IVIG) treatment. However, up to 20% of KD patients are refractory to IVIG and at higher risk of developing CAAs. This highlights the need to characterize the immune mechanisms mediating KD to develop more efficient therapies. IL-1β plays a key role in KD pathogenesis. IL-33, a member of the IL-1 cytokine family, is released upon inflammation and tissue damage and exerts its effects by binding to its receptor ST2 ( Il1rl1 ). Circulating levels of IL-33 are elevated in KD patients during the acute phase of the disease. However, if and how IL-33 contributes to cardiovascular lesion development in KD remains unknown. Using the Lactobacillus casei cell wall extract (LCWE) murine model of KD vasculitis, we observed increased Il33 mRNA expression in hearts and abdominal aorta aneurysms of LCWE-injected mice. While ST2 was detected by immunofluorescence in the hearts of both control and LCWE-injected mice, its expression was increased in the abdominal aortas of LCWE-injected mice. In contrast, IL-33 expression was only increased in inflamed cardiovascular tissues of LCWE-injected mice. Single-cell RNA-sequencing and flow cytometric analysis of abdominal aortas, as well as spatial transcriptomics of heart tissues from LCWE-injected mice, indicated that Il33 transcripts were expressed primarily by stromal cells, such as fibroblasts, endothelial cells, and vascular smooth muscle cells. On the other hand, Il1rl1 transcripts were highly expressed by tissue infiltrating immune cells, such as macrophages, eosinophils, and neutrophils. Blocking IL-33, using either Il33 -/- mice or an anti-IL-33 antibody, significantly attenuated LCWE-induced KD vasculitis. In vitro, IL-33 treatment of LCWE-stimulated bone marrow-derived macrophages boosted their IL-1β production. Overall, our results indicate that IL-33, produced by stromal cells, may promote LCWE-induced KD vasculitis by increasing the release of IL-1β by tissue infiltrating immune cells, and this axis could therapeutically be targeted in KD.

Conjugation of IL-33 to Microporous Annealed Particle Scaffolds Enhances Type 2-Like Immune Responses In Vitro and In Vivo.

The inflammatory foreign body response (FBR) is the main driver of biomaterial implant failure. Current strategies to mitigate the onset of a FBR include modification of the implant surface, release of anti-inflammatory drugs, and cell-scale implant porosity. The microporous annealed particle (MAP) scaffold platform is an injectable, porous biomaterial composed of individual microgels, which are annealed in situ to provide a structurally stable scaffold with cell-scale microporosity. MAP scaffold does not induce a discernible foreign body response in vivo and, therefore, can be used a "blank canvas" for biomaterial-mediated immunomodulation. Damage associated molecular patterns (DAMPs), such as IL-33, are potent regulators of type 2 immunity that play an important role in tissue repair. In this manuscript, IL-33 is conjugated to the microgel building-blocks of MAP scaffold to generate a bioactive material (IL33-MAP) capable of stimulating macrophages in vitro via a ST-2 receptor dependent pathway and modulating immune cell recruitment to the implant site in vivo, which indicates an upregulation of a type 2-like immune response and downregulation of a type 1-like immune response.

Active secretion of IL-33 from astrocytes is dependent on TMED10 and promotes central nervous system homeostasis

Interleukin-33 (IL-33), secreted by astrocytes, regulates the synapse development in the spinal cord and hippocampus and suppresses autoimmune disease in the central nervous system (CNS). However, the mechanism of unconventional protein secretion of this cytokine remains unclear. In this study, we found that IFN-γ promotes the active secretion of IL-33 from astrocytes, and the active secretion of IL-33 from cytoplasm to extracellular space was dependent on interaction with transmembrane emp24 domain 10 (TMED10) via the IL-1 like cytokine domain in astrocytes. Knockout of Il-33 or its receptor St2 induced hippocampal astrocyte activation and depressive-like disorder in naive mice, as well as increased spinal cord astrocyte activation and polarization to a neurotoxic reactive subtype and aggravated passive experimental autoimmune encephalomyelitis (EAE). Our results have identified that IL-33 is actively secreted by astrocytes through the unconventional protein secretion pathway facilitated by TMED10 channels. This process helps maintain CNS homeostasis by inhibiting astrocyte activation.

IL-33 Reduces Saturated Fatty Acid Accumulation in Mouse Atherosclerotic Foci.

The cellular and molecular mechanisms of atherosclerosis are still unclear. Type 2 innate lymphocytes (ILC2) exhibit anti-inflammatory properties and protect against atherosclerosis. This study aimed to elucidate the pathogenesis of atherosclerosis development using atherosclerosis model mice (ApoE KO mice) and mice deficient in IL-33 receptor ST2 (ApoEST2 DKO mice). Sixteen-week-old male ApoE KO and ApoEST2 DKO mice were subjected to an 8-week regimen of a high-fat, high-sucrose diet. Atherosclerotic foci were assessed histologically at the aortic valve ring. Chronic inflammation was assessed using flow cytometry and real-time polymerase chain reaction. In addition, saturated fatty acids (palmitic acid) and IL-33 were administered to human aortic endothelial cells (HAECs) to assess fatty acid metabolism. ApoEST2 DKO mice with attenuated ILC2 had significantly worse atherosclerosis than ApoE KO mice. The levels of saturated fatty acids, including palmitic acid, were significantly elevated in the arteries and serum of ApoEST2 DKO mice. Furthermore, on treating HAECs with saturated fatty acids with or without IL-33, the Oil Red O staining area significantly decreased in the IL-33-treated group compared to that in the non-treated group. IL-33 potentially prevented the accumulation of saturated fatty acids within atherosclerotic foci.

IL-33/soluble ST2 axis is associated with radiation-induced cardiac injury.

Radiotherapy for treating breast cancer is associated with cardiac damage. This study aimed to investigate the role of the interleukin (IL)-33/soluble receptor ST2 (sST2) axis in radiation-induced cardiac injury. Expressions of IL-33 and sST2 were detected in breast cancer patients following radiotherapy, radiation-induced cardiac damaged mice model, and cardiomyocytes using quantitative real-time PCR (qRT-PCR) and immunohistochemical assay. Cardiac injury was evaluated through an ultrasound imaging system and hematoxylin & eosin staining. The transcriptional factor was assessed using dual-luciferase reporter assay and chromatin immunoprecipitation. The results indicated that IL-33 and sST2 were highly expressed in breast cancer patients, which further elevated post-6 months but reduced after 12 months of radiotherapy. Radiation induces cardiac dysfunction and elevated IL-33 and sST2 levels in a time-dependent manner. However, silencing of IL-33 decreased sST2 expression to alleviate radiation-induced cardiac dysfunction. The IL-33 could be transcriptional activated by TCF7L2 by binding to IL33 promoter sites, which mutation alleviated cardiomyocyte injury caused by radiation. Additionally, radiation treatment resulted in higher levels of TCF7L2, IL-33, and sST2 in cardiomyocytes, and TCF7L2 knockdown reduced IL-33 and sST2 expression. In conclusion, TCF7L2 transcriptional-activated IL-33 mediated sST2 to regulate radiation-induced cardiac damage, providing novel insights into radiotherapy-induced cardiac damage.

Neutrophil-derived oxidative stress contributes to skin inflammation and scratching in a mouse model of allergic contact dermatitis via triggering pro-inflammatory cytokine and pruritogen production in skin

Allergic contact dermatitis (ACD) is a common skin disease featured with skin inflammation and a mixed itch/pain sensation. The itch/pain causes the desire to scratch, affecting both physical and psychological aspects of patients. Nevertheless, the mechanisms underlying itch/pain sensation of ACD still remain elusive. Here, we found that oxidative stress and oxidation-related injury were remarkably increased in the inflamed skin of a mouse model of ACD. Reducing oxidative stress significantly attenuated itch/pain-related scratching, allokonesis and skin inflammation. RNA-Sequencing reveals oxidative stress contributes to a series of skin biological processes, including inflammation and immune response. Attenuating oxidative stress reduces overproduction of IL-1β and IL-33, two critical cytokines involved in inflammation and pain/itch, in the inflamed skin of model mice. Exogenously injecting H2O2 into the neck skin of naïve mice triggered IL-33 overproduction in skin keratinocytes and induced scratching, which was reduced in mice deficient in IL-33 receptor ST2. ACD model mice showed remarkable neutrophil infiltration in the inflamed skin. Blocking neutrophil infiltration reduced oxidative stress and attenuated scratching and skin inflammation. Therefore, our study reveals a critical contribution of neutrophil-derived oxidative stress to skin inflammation and itch/pain-related scratching of ACD model mice via mechanisms involving the triggering of IL-33 overproduction in skin keratinocytes. Targeting skin oxidative stress may represent an effective therapy for ameliorating ACD.

Abstract 2700: Therapeutic IL-33 enhances the antigen presentation function of ILC2s to exert anti-tumor immune activity

Abstract IL-33 regulates the function of ILC2s through ST2. However, the role of IL-33, especially exogenous IL-33, on ILC2s in tumors is controversial depending on the cytokines they secrete. Here we report that exogenous, but not endogenous, IL-33 significantly inhibits the progression of a variety of mouse solid tumors, including melanoma, colorectal cancer, lung adenoma, and breast cancer. By inoculating tumor cells in Il1rl1−/- and NSG mice, we found that the tumor-suppressive effect of IL-33 on tumors was dependent on the expression of ST2 receptors and the presence of lymphocytes. Regarding tumor-infiltrating immune cells, exogenous IL-33 significantly increases the proportions of ST2+ Tregs (1.10 ± 0.20% vs. 1.74 ± 0.36%), eosinophils (6.00 ± 1.33% vs. 22.73 ± 6.51%), Granzyme B+ CD8+ T cells (4.32 ± 2.77% vs. 11.53 ± 6.81%), and bulk ILC2s (0.08 ± 0.04% vs. 5.31 ± 1.33%) and IA/IE+ ILC2s (0.02 ± 0.01% vs. 3.67 ± 1.03%). In draining lymph nodes, IL-33 also significantly increases the proportions of ILC2s (0.03 ± 0.02‰ vs. 6.37 ± 1.71‰) and IA/IE+ ILC2s (0.003 ± 0.003‰ vs. 4.38 ± 1.21‰). Importantly, single-cell transcriptomic analysis on tumor-infiltrating ILC2s highlights the expression of genes related with antigen processing and presentation including H2-K1, H2-Q7, B2m, Hspa1a, Hspa1b, Creb1, Hspa4, Tap1 and Tap2. We testified this finding by DQ-OVA experiment and found that IL-33 could promote ILC2s to up-take and process exogenous antigens (17.78 ± 0.75 fold vs. 22.02 ± 0.75 fold). The antigen presentation function of ILC2s was further established by mixed lymphocyte culture where BALB/c ILC2s potently stimulated C57BL/6 CD8+ T cell proliferation, which was further augmented by IL-33 treatment. Taken together, our data demonstrate an anti-tumor function of therapeutic IL-33 that elevates the number and antigen presentation function of ILC2s. Citation Format: Jie Liu, Zhenchu Feng, Hongyan Wei, Chang Li, Xiaoshi Li, Wenlong Chen, Yixi Ke, Minjun Wang, Lantian Liu, Cunni Zheng, Quan Liu. Therapeutic IL-33 enhances the antigen presentation function of ILC2s to exert anti-tumor immune activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2700.