Obese visceral adipose dendritic cells downregulate regulatory T cell development through IL-33.

Regulatory T cells (Tregs) residing in visceral adipose tissue (VAT) play a pivotal role in regulating tissue inflammation and metabolic dysfunction associated with obesity. However, the specific phenotypic and functional characteristics of Tregs in obese VAT, as well as the regulatory mechanisms shaping them, remain elusive. This study demonstrates that obesity selectively reduces Tregs in VAT, characterized by restrained proliferation, heightened PD-1 expression, and diminished ST2 expression. Additionally, obese VAT displays distinctive maturation of dendritic cells (DCs), marked by elevated expressions of MHC-II, CD86, and PD-L1, which are inversely correlated with VAT Tregs. In an in vitro co-culture experiment, only obese VAT DCs, not macrophages or DCs from subcutaneous adipose tissue (SAT) and spleen, result in decreased Treg differentiation and proliferation. Furthermore, Tregs differentiated by obese VAT DCs exhibit distinct characteristics resembling those of Tregs in obese VAT, such as reduced ST2 and IL-10 expression. Mechanistically, obesity lowers IL-33 production in VAT DCs, contributing to the diminished Treg differentiation. These findings collectively underscore the critical role of VAT DCs in modulating Treg generation and shaping Treg phenotype and function during obesity, potentially contributing to the regulation of VAT Treg populations.

IL-33-ST2 signaling in fibro-adipogenic progenitors alleviates immobilization-induced muscle atrophy in mice.

BACKGROUND: The regenerative and adaptive capacity of skeletal muscles reduces with age, leading to severe disability and frailty in the elderly. Therefore, development of effective therapeutic interventions for muscle wasting is important both medically and socioeconomically. In the present study, we aimed to elucidate the potential contribution of fibro-adipogenic progenitors (FAPs), which are mesenchymal stem cells in skeletal muscles, to immobilization-induced muscle atrophy. METHODS: Young (2-3 months), adult (12-14 months), and aged (20-22 months) mice were used for analysis. Muscle atrophy was induced by immobilizing the hind limbs with a steel wire. FAPs were isolated from the hind limbs on days 0, 3, and 14 after immobilization for transcriptome analysis. The expression of ST2 and IL-33 in FAPs was evaluated by flow cytometry and immunostaining, respectively. To examine the role of IL-33-ST2 signaling in vivo, we intraperitoneally administered recombinant IL-33 or soluble ST2 (sST2) twice a week throughout the 2-week immobilization period. After 2-week immobilization, the tibialis anterior muscles were harvested and the cross-sectional area of muscle fibers was evaluated. RESULTS: The number of FAPs increased with the progression of muscle atrophy after immobilization in all age-groups. Transcriptome analysis of FAPs collected before and after immobilization revealed that Il33 and Il1rl1 transcripts, which encode the IL-33 receptor ST2, were transiently induced in young mice and, to a lesser extent, in aged mice. The number of FAPs positive for ST2 increased after immobilization in young mice. The number of ST2-positive FAPs also increased after immobilization in aged mice, but the difference from the baseline was not statistically significant. Immunostaining for IL-33 in the muscle sections revealed a significant increase in the number of FAPs expressing IL-33 after immobilization. Administration of recombinant IL-33 suppressed immobilization-induced muscle atrophy in aged mice but not in young mice. CONCLUSIONS: Our data reveal a previously unknown protective role of IL-33-ST2 signaling against immobilization-induced muscle atrophy in FAPs and suggest that IL-33-ST2 signaling is a potential new therapeutic target for alleviating disuse muscle atrophy, particularly in older adults.

Association of IL-33 in modeling type-2 airway inflammation and pulmonary emphysema in mice.

We developed pulmonary emphysema and a type 2 airway inflammation overlap mouse model. The bronchoalveolar lavage (BAL) interleukin 13 (IL-13), IL-4, and IL-5 levels in the overlap model were higher than in the pulmonary emphysema model and lower than in the type 2 airway inflammation model, but IL-33 level in the lung was higher than in other models. IL-33 and interferon-γ (IFNγ) in lungs may control the severity of a type 2 airway inflammation in lung.

Interleukin 33 supports squamous cell carcinoma growth via a dual effect on tumour proliferation, migration and invasion, and T cell activation.

Interleukin (IL)-33 is an important cytokine in the tumour microenvironment; it is known to promote the growth and metastasis of solid cancers, such as gastric, colorectal, ovarian and breast cancer. Our group demonstrated that the IL-33/ST2 pathway enhances the development of squamous cell carcinoma (SCC). Conversely, other researchers have reported that IL-33 inhibits tumour progression. In addition, the crosstalk between IL-33, cancer cells and immune cells in SCC remains unknown. The aim of this study was to investigate the effect of IL-33 on the biology of head and neck SCC lines and to evaluate the impact of IL-33 neutralisation on the T cell response in a preclinical model of SCC. First, we identified epithelial and peritumoural cells as a major local source of IL-33 in human SCC samples. Next, in vitro experiments demonstrated that the addition of IL-33 significantly increased the proliferative index, motility and invasiveness of SCC-25 cells, and downregulated MYC gene expression in SCC cell lines. Finally, IL-33 blockade significantly delayed SCC growth and led to a marked decrease in the severity of skin lesions. Importantly, anti-IL-33 monoclonal antibody therapy increase the percentage of CD4+IFNγ+ T cells and decreased CD4+ and CD8+ T cells secreting IL-4 in tumour-draining lymph nodes. Together, these data suggest that the IL-33/ST2 pathway may be involved in the crosstalk between the tumour and immune cells by modulating the phenotype of head and neck SCC and T cell activity. IL-33 neutralisation may offer a novel therapeutic strategy for SCC.

Impact of TNF and IL-33 Cytokines on Mast Cells in Neuroinflammation.

Mast cells (MCs) are derived from hematopoietic progenitors, mature in vascularized tissues, and participate in innate and acquired immunity. Neuroinflammation is a highly debated topic in the biomedical literature; however, the impact of tumor necrosis factor (TNF) and IL-33 on MCs in the brain has not been widely addressed. MCs can be activated by IgE binding to FcεRI, as well as by different antigens. After activation, MCs mediate various immunological and inflammatory responses through TNF and IL-33. TNF has two receptors: TNFR1, a p55 molecule, and TNFR2, a p75 molecule. This cytokine is the only one of its kind to be stored in the granules of MCs and can also be generated by de novo synthesis via mRNA. In the central nervous system (CNS), TNF is produced almost exclusively by microglial cells, neurons, astrocytes, and, minimally, by endothelial cells. After its release into brain tissue, TNF rapidly induces the adhesion molecules endothelial leukocyte adhesion molecule 1 (ELAM-1), intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1) in endothelial cells. TNF causes the chemoattraction of neutrophils by inducing several molecules, including CXC chemokines (IL-8). Both MCs and microglial cells act as a primary barrier against foreign molecules in the CNS, producing pro-inflammatory cytokines such as IL-33. IL-33 belongs to the IL-1 family, is activated through the ST2L/IL1-RAcP receptor complex, and mediates both the innate and adaptive immune response. IL-33 is a nuclear transcription factor expressed in the brain, where it induces pro-inflammatory cytokines (TNF and IL-1) and chemokines (CCL2, CCL3, CCL5, and CXCL10). Therefore, MCs and microglia in the CNS are a source of pro-inflammatory cytokines, including TNF and IL-33, that mediate many brain diseases. The inhibition of TNF and IL-33 may represent a new therapeutic approach that could complement existing neuroinflammatory therapies.

Role of TRPV1 and TRPA1 in TSLP production in nasal epithelial cells.

BACKGROUND: TRP protein is sensitive to external temperature changes, but its pathogenic mechanism in the upper airway mucosa is still unclear. OBJECTIVE: To investigate the mechanism of TRPV1and TRPA1 in regulating the secretion of inflammatory factors in nasal epithelial cells. METHODS: The expression of TRPV1 and TRPA1 in nasal mucosal epithelial cells was investigated using immunofluorescence assays. Epithelial cells were stimulated with TRPV1 and TRPA1 agonists and antagonists, and changes in Ca2+ release and inflammatory factor secretion in epithelial cells were detected. TSLP secretion stimulated with the calcium chelating agent EGTA was evaluated. The transcription factor NFAT was observed by immunofluorescence staining. RESULTS: TRPV1 and TRPA1 expression was detected in nasal epithelial cells, and Ca2+ influx was increased after stimulation with agonists. After the activation of TRPV1 and TRPA1, the gene expression of TSLP, IL-25, and IL-33 and the protein expression levels of TSLP and IL-33 were increased, and only TSLP could be inhibited by antagonists and siRNAs. After administration of EGTA, the secretion of TSLP was inhibited significantly, and the expression of the transcription factor NFAT in the nucleus was observed after activation of the TRPV1 and TRPA1 proteins in epithelial cells. CONCLUSION: Activation of TRPV1 and TRPA1 on nasal epithelial cells stimulates the generation of TSLP through the Ca2+/NFAT pathway. It also induces upregulation of IL-25 and IL-33 gene expression levels and increased levels of IL-33 protein, leading to the development of airway inflammation.

Vitreous Olink proteomics reveals inflammatory biomarkers for diagnosis and prognosis of traumatic proliferative vitreoretinopathy.

Background: The aim of this study was to identify inflammatory biomarkers in traumatic proliferative vitreoretinopathy (TPVR) patients and further validate the expression curve of particular biomarkers in the rabbit TPVR model. Methods: The Olink Inflammation Panel was used to compare the differentially expressed proteins (DEPs) in the vitreous of TPVR patients 7-14 days after open globe injury (OGI) (N = 19) and macular hole patients (N = 22), followed by correlation analysis between DEPs and clinical signs, protein-protein interaction (PPI) analysis, area under the receiver operating characteristic curve (AUC) analysis, and function enrichment analysis. A TPVR rabbit model was established and expression levels of candidate interleukin family members (IL-6, IL-7, and IL-33) were measured by enzyme-linked immunosorbent assay (ELISA) at 0, 1, 3, 7, 10, 14, and 28 days after OGI. Results: Forty-eight DEPs were detected between the two groups. Correlation analysis showed that CXCL5, EN-RAGE, IL-7, ADA, CD5, CCL25, CASP8, TWEAK, and IL-33 were significantly correlated with clinical signs including ocular wound characteristics, PVR scoring, PVR recurrence, and final visual acuity (R = 0.467-0.699, p < 0.05), and all with optimal AUC values (0.7344-1). Correlations between DEP analysis and PPI analysis further verified that IL-6, IL-7, IL-8, IL-33, HGF, and CXCL5 were highly interactive (combined score: 0.669-0.983). These DEPs were enriched in novel pathways such as cancer signaling pathway (N = 14, p < 0.000). Vitreous levels of IL-6, IL-7, and IL-33 in the rabbit TPVR model displayed consistency with the trend in Olink data, all exhibiting marked differential expression 1 day following the OGI. Conclusion: IL-7, IL-33, EN-RAGE, TWEAK, CXCL5, and CD5 may be potential biomarkers for TPVR pathogenesis and prognosis, and early post-injury may be an ideal time for TPVR intervention targeting interleukin family biomarkers.

Activated Leukocyte Cell Adhesion Molecule Regulates the Expression of Interleukin-33 in RSV Induced Airway Inflammation by Regulating MAPK Signaling Pathways.

PURPOSE: The respiratory syncytial virus (RSV) is a common respiratory virus that causes acute lower respiratory tract infectious diseases, particularly in young children and older individuals. Activated leukocyte cell adhesion molecule (ALCAM) is a membrane glycoprotein expressed in various cell types, including epithelial cells, and is associated with inflammatory responses and various cancers. However, the precise role of ALCAM in RSV-induced airway inflammation remains unclear, and our study aimed to explore this gap in the literature. METHODS: C57BL/6 wild-type, ALCAM knockout mice and airway epithelial cells were infected with RSV and the expression of ALCAM and inflammatory cytokines were measured. We also conducted further experiments using Anti-ALCAM antibody and recombinant ALCAM in airway epithelial cells. RESULTS: The expression levels of ALCAM and inflammatory cytokines increased in both RSV-infected mice and airway epithelial cells. Interestingly, IL-33 expression was significantly reduced in ALCAM-knockdown cells compared to control cells following RSV infection. Anti-ALCAM antibody treatment also reduced IL-33 expression following RSV infection. Furthermore, the phosphorylation of ERK1/2, p38, and JNK was diminished in ALCAM-knockdown cells compared to control cells following RSV infection. Notably, in the control cells, inhibition of these pathways significantly decreased the expression of IL-33. In vivo study also confirmed a reduction in inflammation induced by RSV infection in ALCAM deficient mice compared to wild-type mice. CONCLUSION: These findings demonstrate that ALCAM contributes to RSV-induced airway inflammation at least partly by influencing IL-33 expression through mitogen-activated protein kinase signaling pathways. These results suggest that targeting ALCAM could be a potential therapeutic strategy for alleviating IL-33-associated lung diseases.

CD4 T cell-secreted IFN-γ in Sjögren's syndrome induces salivary gland epithelial cell ferroptosis.

BACKGROUND: Sjögren's syndrome (SS) is a chronic autoimmune disease that predominantly affects exocrine glands. Previous studies have demonstrated that upregulated interferon-gamma (IFN-γ) in SS triggers ferroptosis in salivary gland epithelial cells (SGECs), resulting in impaired salivary gland secretion. However, the immune cells responsible for secreting IFN-γ remain unclear. Therefore, this study conducted bioinformatics analysis and molecular validation to identify the origin of IFN-γ in SS salivary gland. METHODS: The 'limma' package in R software was utilized to identify differentially expressed genes (DEGs) in the human SS dataset. Subsequently, the identified DEGs were compared with the ferroptosis database and screened through Cytoscape to determine candidate genes. The cellular localization and expression patterns of candidate genes were further confirmed in the salivary gland single-cell RNA sequence (scRNA-seq) data set from healthy control and SS mice. Furthermore, in vitro and in vivo studies were performed to analyze the effect of CD4 T-secreted IFN-γ on SGECs' ferroptosis and functions. RESULTS: Upregulated TLR4, IFNG, and IL33 were screened as candidates ferroptosis ferroptosis-inducing genes in SS salivary glands. The association of IFNG and IL33 with CD4 T cells was established through immune infiltration analysis. The expression of IFN-γ on CD4 T cells was robustly higher compared with that of IL33 as evidenced by scRNA-seq and immunofluorescence co-localization. Subsequent experiments conducted on candidate genes consistently demonstrated the potent ability of IFN-γ to induce SGECs' ferroptosis and inhibit AQP5 expression. CONCLUSIONS: Our findings indicate that CD4 T cell-secreted IFN-γ in SS induces SGECs' ferroptosis and inhibits AQP5 expression.

IL-10 Differentially Promotes Mast Cell Responsiveness to IL-33, Resulting in Enhancement of Type 2 Inflammation and Suppression of Neutrophilia.

Mast cells (MCs) play critical roles in the establishment of allergic diseases. We recently demonstrated an unexpected, proinflammatory role for IL-10 in regulating MC responses. IL-10 enhanced MC activation and promoted IgE-dependent responses during food allergy. However, whether these effects extend to IgE-independent stimuli is not clear. In this article, we demonstrate that IL-10 plays a critical role in driving IL-33-mediated MC responses. IL-10 stimulation enhanced MC expansion and degranulation, ST2 expression, IL-13 production, and phospho-relA upregulation in IL-33-treated cells while suppressing TNF-α. These effects were partly dependent on endogenous IL-10 and further amplified in MCs coactivated with both IL-33 and IgE/Ag. IL-10's divergent effects also extended in vivo. In a MC-dependent model of IL-33-induced neutrophilia, IL-10 treatment enhanced MC responsiveness, leading to suppression of neutrophils and decreased TNF-α. In contrast, during IL-33-induced type 2 inflammation, IL-10 priming exacerbated MC activity, resulting in MC recruitment to various tissues, enhanced ST2 expression, induction of hypothermia, recruitment of eosinophils, and increased MCPT-1 and IL-13 levels. Our data elucidate an important role for IL-10 as an augmenter of IL-33-mediated MC responses, with implications during both allergic diseases and other MC-dependent disorders. IL-10 induction is routinely used as a prognostic marker of disease improvement. Our data suggest instead that IL-10 can enhance ST2 responsiveness in IL-33-activated MCs, with the potential to both aggravate or suppress disease severity depending on the inflammatory context.

Celecoxib attenuates interleukin 33-induced expression of vascular cell adhesion molecule-1 in human ovarian endometriotic stromal cells.

OBJECTIVE: Endometriosis is an estrogen-dependent chronic inflammatory disease in women of reproductive age. A review of the literature revealed that cytokines and inflammatory factors are associated with endometriosis-associated infertility. Interleukin 33 (IL-33) is a strong inducer of other pro-inflammatory cytokines. Vascular cell adhesion molecule-1 (VCAM-1) plays a central role in recruiting inflammatory cells, whose expression facilitates leukocyte adhesion and is rapidly induced by pro-inflammatory cytokines. Many studies have indicated that VCAM-1 expression is high in endometriosis; however, whether the expression of VCAM-1 is related to IL-33 is unclear. MATERIALS AND METHODS: Human ovarian endometriotic stromal cells (hOVEN-SCs) were treated with IL-33 to enable investigation of cell characterization, gene and protein expression, and signal pathways. Proliferation potential was measured using an MTT assay. Gene expression was analyzed using reverse transcription-polymerase chain reaction. Protein expression assay was performed using western blot analysis. RESULTS: This study investigated the effects of IL-33 on VCAM-1 and COX-2 expression in hOVEN-SCs. First, the results revealed that the IL-33/ST2/mitogen-activated protein kinase (MAPK) signaling pathway could increase the expression of VCAM-1 and COX-2 in hOVEN-SCs. Second, we discovered that COX-2 expression was essential for IL-33-induced VCAM-1 expression because the effects could be negated through NS398, a selective COX-2 inhibitor. Finally, treatment of IL-33-treated hOVEN-SCs with celecoxib significantly and dose-responsively decreased VCAM-1 expression. CONCLUSION: Taken together, these results indicate that IL-33 can upregulate VCAM-1 expression in hOVEN-SCs through the IL-33/ST2/MAPK/COX-2 signaling pathway and thereby contribute to endometriosis.

Staphylococcus aureus exacerbates dermal IL-33/ILC2 axis activation through evoking RIPK3/MLKL-mediated necroptosis of dry skin.

Atopic dermatitis (AD) is a persistent skin disease typified by symptoms of dry skin and recurrent eczema. Patients with AD are at heightened risk for Staphylococcus aureus infection. Group 2 innate lymphoid cells (ILC2s) are mainly activated by epithelial cell-derived cytokines IL-33 and involved in the pathogenesis of AD. However, little is known about the effect of skin delipidization on the epithelial cell-derived cytokines and dermal ILC2s in AD. In our study, we investigated the mechanism by which S. aureus infection modulates and exacerbates the pathogenesis of dry skin, leading to type 2 inflammation in the context of innate immunity. In vivo, we found that S. aureus infection aggravated delipidization-induced dermal IL-33 release and dermal ILC2 accumulation, which exacerbated skin inflammation. We also noticed that Il33fl/fl K14cre mice and Tlr2-/- mice exhibited attenuated skin inflammation. In vitro, treatment with necroptosis inhibitors reduced IL-33 release from S. aureus-infected keratinocytes. Mechanistically, we observed an increase in the necroptosis-associated kinases, MLKL and RIPK3, in S. aureus-infected mice, indicating that IL-33 release was associated with necroptotic cell death responses. Our results reveal that S. aureus infection-elicited keratinocyte necroptosis contributes to IL-33-mediated type 2 inflammation, which exacerbates the pathogenesis of dry skin.

P2X7 Receptor-Induced Human Mast Cell Degranulation Is Enhanced by Interleukin 33.

MCs are tissue-resident immune cells that strategically reside in barrier organs and respond effectively to a wide range of stimuli, such as IL-33, a mediator released upon epithelial damage. Adenosine triphosphate (ATP) accumulates at sites of tissue injury and is known to modulate MC activities. This study investigated how an inflammatory tissue environment rich in IL-33 modulates the ATP-mediated activation of MCs. Human primary MCs primed with IL-33 displayed a strongly increased response to ATP but not ADP. This resulted in increased degranulation, IL-8 release, and pERK1/2 signalling. Such effects are unique to IL-33 stimulation and not shared by the epithelial alarmin, TSLP. MC exposure to IL-33 also increased membrane expression of purinergic and ATP-binding P2X receptors. The use of selective P2X receptor inhibitors identified P2X7 receptor as the key mediator of the enhanced ATP-induced ERK1/2 signalling and degranulation in IL-33-primed MCs. Whilst the inhibition of P2X1 and P2X4 receptors had no effect on MC degranulation, inhibiting these receptors together with P2X7 resulted in further decreased MC-mediated degranulation. These data therefore point toward the potential mechanisms by which IL-33 contributes to the modulation of ATP-mediated activation in human MCs.

The Aquaporin 3 Polymorphism (rs17553719) Is Associated with Sepsis Survival and Correlated with IL-33 Secretion.

Sepsis is a common life-threatening disease caused by dysregulated immune response and metabolic acidosis which lead to organ failure. An abnormal expression of aquaporins plays an important role in organ failure. Additionally, genetic variants in aquaporins impact on the outcome in sepsis. Thus, we investigated the polymorphism (rs17553719) and expression of aquaporin-3 (AQP3) and correlated these measurements with the survival of sepsis patients. Accordingly, we collected blood samples on several days (plus clinical data) from 265 sepsis patients who stayed in different ICUs in Germany. Serum plasma, DNA, and RNA were then separated to detect the promotor genotypes of AQP3 mRNA expression of AQP3 and several cytokines. The results showed that the homozygote CC genotype exhibited a significant decrease in 30-day survival (38.9%) compared to the CT (66.15%) and TT genotypes (76.3%) (p = 0.003). Moreover, AQP3 mRNA expression was significantly higher and nearly doubled in the CC compared to the CT (p = 0.0044) and TT genotypes (p = 0.018) on the day of study inclusion. This was accompanied by an increased IL-33 concentration in the CC genotype (day 0: p = 0.0026 and day 3: p = 0.008). In summary, the C allele of the AQP3 polymorphism (rs17553719) shows an association with increased AQP3 expression and IL-33 concentration accompanied by decreased survival in patients with sepsis.

Investigating the impact of antibiotic-induced dysbiosis on protection from Clostridium difficile colitis by mouse colonic innate lymphoid cells.

Innate lymphoid cells (ILCs) play a critical role in maintaining intestinal health in homeostatic and diseased conditions. During Clostridium difficile infection (CDI), IL-33 activates ILC2 to protect from colonic damage and mortality. The function of IL-33 and ILC is tightly regulated by the intestinal microbiota. We set out to determine the impact of antibiotic-induced disruption of the microbiome on ILC function. Our goal was to understand antibiotic-induced changes in ILC function on susceptibility to C. difficile colitis in a mouse model. We utilized high-throughput single-cell RNAseq to investigate the phenotypic features of colonic ILC at baseline, after antibiotic administration with or without IL-33 treatment. We identified a heterogeneous landscape of colonic ILCs with gene signatures of inflammatory, anti-inflammatory, migratory, progenitor, plastic, and antigen-presenting ILCs. Antibiotic treatment decreased ILC2 while coordinately increasing ILC1 and ILC3 phenotypes. Notably, Ifng+, Ccl5+, and Il23r+ ILC increased after antibiotics. IL-33 treatment counteracted the antibiotic effect by downregulating ILC1 and ILC3 and activating ILC2. In addition, IL-33 treatment markedly induced the expression of type 2 genes, including Areg and Il5. Finally, we identified amphiregulin, produced by ILC2, as protective during C. difficile infection. Together, our data expand our understanding of how antibiotics induce susceptibility to C. difficile colitis through their impact on ILC subsets and function.IMPORTANCEClostridium difficile infection (CDI) accounts for around 500,000 symptomatic cases and over 20,000 deaths annually in the United States alone. A major risk factor of CDI is antibiotic-induced dysbiosis of the gut. Microbiota-regulated IL-33 and innate lymphoid cells (ILCs) are important in determining the outcomes of C. difficile infection. Understanding how antibiotic and IL-33 treatment alter the phenotype of colon ILCs is important to identify potential therapeutics. Here, we performed single-cell RNAseq of mouse colon ILCs collected at baseline, after antibiotic treatment, and after IL-33 treatment. We identified heterogeneous subpopulations of all three ILC subtypes in the mouse colon. Our analysis revealed several potential pathways of antibiotic-mediated increased susceptibility to intestinal infection. Our discovery that Areg is abundantly expressed by ILCs, and the protection of mice from CDI by amphiregulin treatment, suggests that the amphiregulin-epidermal growth factor receptor pathway is a potential therapeutic target for treating intestinal colitis.

Macrophages reprogramming improves immunotherapy of IL-33 in peritoneal metastasis of gastric cancer.

Peritoneal metastasis (PM) has a suppressive tumor immune microenvironment (TIME) that limits the effects of immunotherapy. This study aimed to investigate the immunomodulatory effects of intraperitoneal administration of IL-33, a cytokine that is reported to potentiate antitumor immunity and inhibit metastasis. We found survival was significantly prolonged in patients with high IL-33 mRNA expression. In immunocompetent mice, intraperitoneal administration of IL-33 could induce a celiac inflammatory environment, activate immunologic effector cells, and reverse the immunosuppressive tumor microenvironment, which effectively delayed tumor progression and PM of gastric cancer. Mechanistically, IL-33 could induce M2 polarization by activating p38-GATA-binding protein 3 signaling. IL-33 combined with anti-CSF1R or p38 inhibitor to regulate tumor-associated macrophages (TAMs) had a synergistic antitumor effect. Inducing a local inflammatory milieu by IL-33 administration provided a novel approach for treating peritoneal metastasis, which, when combined with TAM reprogramming to reshape TIME, can achieve better treatment efficacy.

MiR-548t-5p regulates pancreatic ductal adenocarcinoma metastasis through an IL-33-dependent crosstalk between cancer cells and M2 macrophages.

IL-33 has been associated with pro- and anticancer functions in cancer. However, its role in pancreatic cancer metastasis remains unknown. This study aimed to explore the role of miR-548t-5p/IL-33 axis in the metastasis of pancreatic cancer. Luciferase activity assay, qRT-PCR, Western blot and ELISA were performed to prove whether IL-33 is the target of miR-548t-5p. In vivo metastasis assay and cellular transwell assay were performed to explore the role of miR-548t-5p/IL-33 axis in the invasion and metastasis of pancreatic cancer. Co-culture experiments and immunohistochemistry were performed to observe whether IL-33 affects cell invasion and metastasis dependent on the involvement of M2 macrophages. THP-1 cell induction experiment and flow cytometry were performed to explore the effect of IL-33 on macrophage polarization. CCK-8, colony formation, cell apoptosis, cell cycle, cell wound healing and transwell assay were performed to investigate the effect of IL-33 induced M2 macrophages on cell malignant biological behavior by coculturing pancreatic cancer cells with the conditioned medium (CM) from macrophages. We found that miR-548t-5p regulated the expression and secretion of IL-33 in pancreatic cancer cells by directly targeting IL-33 mRNA. IL-33 secreted by cancer cells promoted the recruitment and activation of macrophages to a M2-like phenotype. In turn, IL-33 induced M2 macrophages promoted the migration and invasion of cancer cells. Moreover, IL-33 affected pancreatic cancer cell invasion dependent on the involvement of M2 macrophages in the co-culture system. Thus, our study suggested that manipulation of this IL-33-dependent crosstalk has a therapeutic potential for the treatment of pancreatic cancer metastasis.

Dexamethasone Suppresses IL-33-exacerbated Malignant Phenotype of U87MG Glioblastoma Cells via NF-κB and MAPK Signaling Pathways.

BACKGROUND: Interleukin (IL)-33 is highly expressed in glioblastoma (GBM) and promotes tumor progression. Targeting IL-33 may be an effective strategy for the treatment of GBM. Dexamethasone (DEX) is a controversial drug routinely used clinically in GBM therapy. Whether DEX has an effect on IL-33 is unknown. This study aimed to investigate the effect of DEX on IL-33 and the molecular mechanisms involved. METHODS: U87MG cells were induced by tumor necrosis factor (TNF)-α to express IL-33 and then treated with DEX. The mRNA levels of IL-33, NF-κB p65, ERK1/2, and p38 were determined by real-time quantitative PCR. The expression of IL-33, IkBα (a specific inhibitor of NF-κB) and MKP-1 (a negative regulator of MAPK), as well as the phosphorylation of NF-κB, ERK1/2 and p38 MAPK, were detected by Western blotting. The secretion of IL-33 was measured by ELISA. The proliferation, migration and invasion of U87MG cells were detected by CCK8 and transwell assays, respectively. RESULTS: DEX significantly reduced TNF-α-induced production of IL-33 in U87MG cells, which was dependent on inhibiting the activation of the NF-κB, ERK1/2 and p38 MAPK signaling pathways, and was accompanied by the increased expression of IkBα but not MKP-1. Furthermore, the proliferation, migration and invasion of U87MG cells exacerbated by IL-33 were suppressed by DEX. CONCLUSION: DEX inhibited the production and tumor-promoting function of IL-33. Whether DEX can benefit GBM patients remains controversial. Our results suggest that GBM patients with high IL-33 expression may benefit from DEX treatment and deserve further investigation.

Sequential engagement of adhesion molecules and cytokine receptors impacts both piecemeal and anaphylactic degranulation of human basophils.

Basophils are rare granulocytes in circulation which home to tissues in a process depending on rolling, adhesion and cytokine exposure. However, it is still unclear how these steps affect basophil degranulation. Our aim was to imitate these processes associated with homing by sequential crosslinking of adhesion molecules and cytokine exposure and evaluate the effect on basophil piecemeal (PMD) and anaphylactic degranulation (AND). Blood donors with or without allergic asthma were recruited from an ongoing cohort study. Basophils were subjected to CD62L-, CD49d- or CD11b crosslinking and IL-3 or IL-33 stimulation in different orders followed by anti-IgE and fMLP stimulation. Basophil CD203c and CD63 expression were analysed by flow cytometry to determine PMD and AND, respectively. IL-3 induced PMD in basophils and combined with CD62L- or CD11b crosslinking, IL-3 potentiated the degranulation regardless of sequential order. IL-3 priming followed by adhesion molecule crosslinking induced AND and potentiated the effect of anti-IgE. CD62L- and CD11b crosslinking did not further potentiate this effect. CD49d crosslinking followed by IL-3 increased CD63 expression following anti-IgE. IL-3 potentiated the effect of fMLP on AND while adhesion molecule crosslinking did not. IL-33 had impact on PMD only when followed by adhesion molecule crosslinking but did not potentiate neither IgE-dependent nor IgE-independent degranulation. Our data indicate that sequential interactions between basophils, cytokines and adhesion molecule ligands have a decisive effect on basophil degranulation and that these interactions are operational for fine-tuning the activity of tissue dwelling basophils. These data should be considered when the effect of different pharmaceutical on basophil function is studied.

Hepatocyte GPCR signaling regulates IRF3 to control hepatic stellate cell transdifferentiation.

BACKGROUND: Interferon Regulatory Factor 3 (IRF3) is a transcription factor that plays a crucial role in the innate immune response by recognizing and responding to foreign antigens. Recently, its roles in sterile conditions are being studied, as in metabolic and fibrotic diseases. However, the search on the upstream regulator for efficient pharmacological targeting is yet to be fully explored. Here, we show that G protein-coupled receptors (GPCRs) can regulate IRF3 phosphorylation through of GPCR-Gα protein interaction. RESULTS: IRF3 and target genes were strongly associated with fibrosis markers in liver fibrosis patients and models. Conditioned media from MIHA hepatocytes overexpressing IRF3 induced fibrogenic activation of LX-2 hepatic stellate cells (HSCs). In an overexpression library screening using active mutant Gα subunits and Phos-tag immunoblotting, Gαs was found out to strongly phosphorylate IRF3. Stimulation of Gαs by glucagon or epinephrine or by Gαs-specific designed GPCR phosphorylated IRF3. Protein kinase A (PKA) signaling was primarily responsible for IRF3 phosphorylation and Interleukin 33 (IL-33) expression downstream of Gαs. PKA phosphorylated IRF3 on a previously unrecognized residue and did not require reported upstream kinases such as TANK-binding kinase 1 (TBK1). Activation of Gαs signaling by glucagon induced IL-33 production in hepatocytes. Conditioned media from the hepatocytes activated HSCs, as indicated by α-SMA and COL1A1 expression, and this was reversed by pre-treatment of the media with IL-33 neutralizing antibody. CONCLUSIONS: Gαs-coupled GPCR signaling increases IRF3 phosphorylation through cAMP-mediated activation of PKA. This leads to an increase of IL-33 expression, which further contributes to HSC activation. Our findings that hepatocyte GPCR signaling regulates IRF3 to control hepatic stellate cell transdifferentiation provides an insight for understanding the complex intercellular communication during liver fibrosis progression and suggests therapeutic opportunities for the disease. Video Abstract.