Project description:Interleukin-33 (IL-33) is a novel member of the IL-1 family of cytokines that plays diverse roles in the regulation of immune responses. IL-33 exerts its effects by binding to a heterodimeric receptor complex consisting of interleukin-1 receptor like 1 (IL1RL1) and an accessory receptor protein IL-1RAcP resulting in the production and release of proinflammatory cytokines. A detailed understanding of the signaling pathways activated by IL-33 remains elusive. To elucidate IL-33 mediated signaling, we performed a global quantitative phosphoproteomic analysis using stable isotope labeling by amino acids in cell culture. Employing anti-phosphotyrosine antibodies and titanium dioxide-based enrichment strategies, we identified 6,207 phosphorylation sites mapping to 2,013 phosphoproteins of which more than 185 phosphosites are regulated by IL-33 stimulation. Our findings will greatly expand the understanding of IL-33 signaling and provide novel therapeutic targets for IL-33/IL-33R-associated diseases in humans.
Project description:IL-33 is a nuclear cytokine from the IL-1 family that plays important roles in health and disease. Under healthy conditions, IL-33 is constitutively expressed to high levels in the nucleus of producing cells in various human and mouse tissues. The extracellular function of IL-33 cytokine has been well documented, but it remains unclear whether intracellular nuclear IL-33 has additional functions in the nucleus. Here, we used a global proteomic approach based on quantification of 5000 individual proteins by high-resolution mass spectrometry to compare the extracellular and intracellular roles of IL-33 in primary human endothelial cells, a major source of IL-33 protein in human tissues. Large-scale analysis of protein expression was performed either after stimulation of the cells with the IL-33 mature form IL-3395-270 (during 6h or 24h) or after siRNA knockdown of intracellular IL-33 (two experiments, each with a different pool of distinct siRNAs, noted siRNA1 and siRNA2). In each case, proteins were fractionated by 1D SDS-PAGE in 12 gel bands, and label-free quantitative analysis was performed. The present dataset contains the files for the two experiments of knockdown of endogenous nuclear IL-33 expression: - RNA silencing strategy 1. Knockdown of endogenous nuclear IL-33 expression was performed with a pool of four distinct siRNAs (Dharmacon ON-TARGETplus SMARTpool IL-33 siRNAs) that have been specifically modified for efficient silencing of the target gene with reduced off-target effects. Cells transfected with these siRNA duplexes (si1) were compared with those transfected with the provided controls (CTsi1). Three independent biological replicates (noted _A, _B, _C) were prepared and analyzed for each condition, leading to 6 different samples. Each of them was fractionated into 12 gel bands analyzed by nanoLC-MS/MS, leading to 72 raw files. - RNA silencing strategy 2. The second knockdown strategy was based on the use of an independent pool of three siRNAs targeting IL-33, predesigned by another provider using new and critical siRNA design rules (Sigma MISSION Predesigned Il-33 siRNAs based on Rosetta siRNA design algorithm). Cells transfected with these siRNA duplexes (si2) were compared with those transfected with the provided controls (CTsi2). Three independent biological replicates (noted _A, _B, _C) were prepared and analyzed for each condition, leading to 6 different samples. Each of them was fractionated into 12 gel bands analyzed by nanoLC-MS/MS, leading to 72 raw files.
Project description:Determination of the molecular mechanism of IL33 on glioma cells Since IL-33 is known to associate with chromatin and regulate transcriptional activity and that nuclear expression of IL-33 increases glioma progression, we determined Nuclear IL-33 regulates the expression and secretion of inflammatory cytokines in glioma cells. Using these parameters 340 genes were induced by the ectopic expression of IL-33 and an additional 377 genes were downregulated. Gene ontology terms over-represented in the genes induced by IL-33 include three major clusters that associate with cytokine activity and inflammation
Project description:Interleukin-33 (IL-33), a member of the IL-1 superfamily cytokines, is an endogenous danger signal and a nuclear-associated cytokine. It is one of the essential mediators of both innate and adaptive immune responses. Aberrant IL-33 signaling has been demonstrated to play a defensive role against various infectious and inflammatory diseases. Although the signaling responses mediated by IL-33 have been previously reported, the temporal signalingdynamicsare yet to be explored. Towards this end,we applied quantitative temporal phosphoproteomics analysis to elucidate pathways and proteins induced by IL-33 in THP1 monocytes. Employing TMT labeling-based quantitation and titanium dioxide (TiO2)-based phosphopeptide enrichment strategy followed by mass spectrometry analysis, we identified 14,515 phosphorylation sites mapping to 4,174 proteins across (0 min to 240 mins)time points.
Project description:To validate the modulation of inflammatory genes by ectopic IL-33, we performed Gene expression analysis using the nCounter Mouse v2 Inflammation Panel. This result validated a significant increase in a number of anti-inflammatory cytokines in the IL-33+ xenografts that were absent in the DNLS xenografts
Project description:Background: IL-33, a cytokine with pleiotropic functions, is elevated in serum of patients with hepatocellular carcinoma (HCC). This study investigated effects of local IL-33 expression in resected HCC on patient survival and on immunological and molecular tumor microenvironment. Methods: Tissue of resected HCCs was stained for H&E, masson trichrome, alpha smooth muscle actin, IL-33, CD8 and IL-13 and analysed by flow cytometry. Besides histomorphologic evaluation, the immunohistochemical stainings were analysed for the respective cell numbers separately for tumor area, infiltrative margin and distant liver stroma. These findings were correlated with clinical data and patient outcome. Further, gene expression of different HCC risk groups was compared using Micro Arrays. Results: In multivariable analysis, infiltration of HCCs by IL-33+ cells (P=0.032) and CD8+ cells (P=0.014) both independently were associated with prolonged patient survival. Flow cytometry demonstrated that cytotoxically active CD8+CD62L-KLRG1+CD107a+ effectory-memory cells are the main producers of IL-33 in these HCC patients. Using infiltration by IL-33+ and CD8+ cells as two separate factors, a HCC immune score (HCCIS) was designed and evaluated that stratified patient survival (P=0.0004). This HCCIS identified high and low risk patients who differ in gene expression profiles (P<0.001). Conclusion: Infiltration of HCCs by IL-33+ and CD8+ cells is independently associated with prolonged patient survival. We suggest that this is due to an induction of highly effective cytotoxically active CD8+CD62L-KLRG1+CD107a+ effector-memory cells producing IL-33. Based on these two independent factors we established a HCC immune score that provides risk stratification for HCC patients and can be used in the clinical setting. To investigate if HCCIS 0 high risk and HCCIS 2 low risk tumors exhibit a distinct molecular environment and gene expression pattern, RNA from fresh tumor tissue was isolated and analyzed by whole genome Microarray 4 patients with low risk tumors (HCCIS 2) were compared to 4 patients with high risk tumors (HCCIS 0).
Project description:Mast cells are tissue resident granulocytes which are most abundant at the interface between tissues and the external environment, such as around blood vessels, in the skin or mucosal surfaces in the lungs and gut. Pathologically they are involved in allergic reactions and anaphylaxis, however they may also play protective roles in responses to some infections, particularly to pathogenic helminths. Mast cells also express high levels of the IL-33 receptor, which like TLRs, activates Myd88 dependent signalling pathways to drive de novo cytokine production in mast cells.IL-33 is a member of the IL-1 family known to stimulate a number of immune cell types including mast cells. IL-33 is a strong activator of de novo cytokine production in mast cells without inducing degranulation, although it has also been shown to synergise with other signals to promote degranulation. Bone Marrow-Derived Mast cells (BMMCs) were cultured as described previously [27]. Briefly, bone marrow was flushed in PBS and the cells pelleted by centrifugation. Cells were cultured at 1 million cells per ml in RPMI 1640 supplemented with 10% FBS (Biosera/Labtech), 5 mM l‐Glutamine (GIBCO Life Technologies), 100 U/ml Penicillin (GIBCO Life Technologies), 100 μg/ml Streptomycin (GIBCO Life Technologies), 25 mM HEPES (Lonza), 1 mM sodium pyruvate (Lonza), 1X nonessential amino acids (Lonza), 50 μM 2‐mercaptoethanol and 30 ng/ml IL‐3 (PeproTech). Cells were passaged twice per week and used between passage 12 and 16. 4 independent BMMC cultures were either stimulated with 10 ng/ml IL-33 for 48 hours or left unstimulated, followed by single shot LC-MS analysis.
Project description:To know target gene of IL-33 full-length, we generated IL-33 full length and IL-33 cytokine construct. After overexpression each construct in cell line, we did ChIP assay and analyzed ChIP-seq. As a result, we found different gene regulation by IL-33