IL-33 Promotes Airway Remodeling In Asthma Through ST2/ERK1/2/MSK1 Signal Pathway

BackgroudBronchial asthma is a chronic inflammatory disease that is involved by inflammatory cells such as eosinophils(EOS),mast cells,T lymphocytes,structure cells such as airway epithelium cells and airway smooth muscle cells,and cellular components.The structure changes of the airway epithelium are usually the main pathological features.Asthma is pathologically characterized by airway hyper-responsiveness,chronic airway inflammation,and airway remodeling.Airway remodeling,resulting in airway narrowing,hyper-responsiveness,and inflammation,is a complex process that involves epithelial metaplasia,deposition of subepithelial collagen,hypertrophy of bronchial smooth muscle,and transformation of fibroblasts to myofibroblasts.The interactions between the epithelium and the underlying mesenchyma,which are jointly referred to as the epithelia-mesenchymal trophic unit(EMTU),are thought to drive pathological remodelling of the airways in asthma.Injury to epithelia in vitro results in increased release of a variety of multifunctional cytokines and growth factors,such as thymic stromal lymphopoietin(TSLP),interleukin-25(IL-25),and interleukin-33(IL-33).Myofibroblast was active by the mediators released by the repairing bronchial epithelium,such as growth factor beta(TGF?)or interleukin-33(IL-33).Myofibroblasts,also called alpha smooth muscle actin(?-SMA)expressing fibroblasts,are mesenchymal cells and known to be the primary source of proinflammatory mediators and extracellular matrix(ECM)proteins,including tenascin,lumican,biglycan,fibronectin and collagens ?,?,and ?,which contribute to the increase in airway remodeling progression.Interleukin-33(IL-33),the eleventh member of the IL-1 family,is expressed by many cell types following pro-inflammatory stimulation and is thought to be released on cell lysis.It has been proposed that IL-33 can directly promotes airway remodeling but the cellular and molecular mechanisms of IL-33 have not been fully understood.IL-33 signaling through ST2 activates at least two independent pathways:the sphingosine kinases(SPHK)pathway and the mitogen-activated protein kinase(MAPK)pathway,which is mediated by the activation of the extracellular signal-regulated protein kinase 1/2(ERK1/2),p38 MAPK and c-Jun N-terminal kinase(JNK).Signaling cascades involving mitogen-activated protein kinases(MAPKs)have been linked to the pathogenesis of asthma inflammation and remodeling.Chemical inhibition of extracellular signal-regulated protein kinase 1/2(ERK1/2)reduced lung eosinophilia in ovalbumin(OVA)-sensitized and-challenged rats and mice.Both chemical and genetic inhibition of ERK1/2 altered the IL-13-induced tissue remodeling responses.MAPKs have also been implicated in IL-33-induced eotaxin production.These data indicate that ERK1/2 signaling may play an important role in IL-33-mediated airway remodeling in asthma.It has been reported that IL-33 was involved in allergic airway inflammation by inducing IL-17F via ERK1/2-MSK1 signaling pathway in bronchial epithelial cells.Taking these above studies together,we hypothesized that IL-33,via ERK1/2/MSK1 signaling,plays a critical role in airway remodeling of asthma.ObjectiveTo further clarify the function of IL-33 on the airway remodeling in asthma,we examined the expression of IL-33 in asthmatic patients and healthy subjects.We also investigated the effect and mechanism of IL-33 on the expression of ?-SMA and collagen I in human lung fibroblast and the effect of the IL-33 blocking on ERK1/2-MSK1 signaling pathway and airway remodeling in an OVA-induced asthma mouse model.MethodsFirstly,expression of IL-33 and ST2 was examined in lung specimens from asthmatic patients.Then,human lung fibroblasts were pretreated with anti-ST2 antibody,U0126 and H89 respectively and then treated with IL-33 recombination protein.Then the activation of extracellular signal-regulated protein kinase 1/2(ERK1/2)and mitogen-and stress-activated protein kinase 1(MSK1)and the expression of alpha smooth muscle actin(?-SMA)and collagen I was determined.In addition,the effect of soluble ST2(sST2)was tested in a mouse asthma model to determine if IL-33 blocking influences airway remodeling of asthma.ResultsExpression of IL-33 and ST2 was upregulated in the airway of patients with asthmaWe examined the expression of IL-33 and its receptor ST2 in lung sections from asthmatic patients and control subjects by immunohistochemistry.IL-33 and ST2 immunoreactivity more evident in airways from asthmatic patients compared with control subjects.rIL-33 upregulated the expression of a-SMA,collagen I and ST2 and the phosphorylation of ERK1/2 and MSK1 in human lung fibroblastCompared to unstimulated cells,the protein levels of ?-SMA and collagen I were significantly enhanced by treatment of cells with rIL-33 in a dose-dependent manner and were maximal at 50 ng/ml and maintain this level thereafter.In addition,rIL-33-induced expression of ?-SMA and collagen I was also increased in a time-dependent manner and the highest level was tested at 24 h.The data suggested IL-33 played a significant role in airway remodeling.ST2 was recently identified as the receptor for IL-33.The expression of ST2 was dramatically increased in response to stimulation with rIL-33(50 ng/ml)for 30 min compared to untreated cells.rIL-33-induced phosphorylation of ERK1/2 and MSK1 compared to cells treated with nonimmune control IgG.Involvement of ST2 in phosphorylation of ERK1/2 and MSK1 and the expression of ?-SMA and collagen I in rIL-33-stimulated human lung fibroblastWestern blotting analysis,cell immunofluorescence,and real time quantitive PCR revealed that pretreatment of the cells with anti-ST2 neutralizing antibody significantly diminished the rIL-33-induced phosphorylation of ERK1/2 and MSK1 compared to cells treated with nonimmune control IgG,and abolished IL-33-induced ?-SMA and collagen I expression.ERK1/2 and MSK1 are involved in rIL-33-induced expression of ?-SMA and collagen I in human lung fibroblastERK1/2 inhibition significantly reduced rIL-33-mediated phosphorylation of MSK1 compared to cells treated with control vehicle.However,MSK1 inhibition had no effect on the phosphorylation level of ERK1/2.Taken together,the data clearly suggested that MSK1 was the downstream kinas of ERK1/2.Pre-treatment of HLF-1 cells with U0126 for 30 min prior to rIL-33 stimulation attenuated rIL-33-induced the expression of ?-SMA and collagen I compared to cells treated with U0124(inactive analog of U0126).Phosphorylation of MSK1 and the expression of ?-SMA and collagen I induced by rIL-33 were also reduced in HLF-1 cells pre-treated with H89 compared to cells with H85(inactive analog of H89)treatment.Similar to protein expression,the mRNA expression of ?-SMA and collagen I induced by rIL-33 in HLF-1 was markedly inhibited by pre-treatment with U0126 or H89.Treatment with sST2 abolished OVA-induced phosphorylation of ERK1/2 and MSKland expression of ?-SMA and collagen I in vivoImmunohistochemistry of lungs and western blot analysis of whole lung lysates shows that expression of IL-33,ST2,p-ERK1/2,p-MSK1 and the airway remodeling markers ?-SMA and collagen I was dramatically increased in airways from OVA-challenged mice compared with the control group,whereas the production of these cytokines except IL-33 was markedly reduced by pretreatment with sST2.ConclusionIL-33 induced the expression of ?-SMA and collagen I via the activation of the ST2/ERK1/2/MSK1 signaling pathway in human lung fibroblast.IL-33 blocking with sST2 abolished the phosphorylation of ERK1/2 and MSK1 and expression of ?-SMA and collagen I observed in OVA-treated mice.Therefore,interference of IL-33/ST2/ERK1/2/MSK1 signaling pathway may present new therapeutic options for asthma.