The Roles Of Neuromedin U In Asthmatic Type-2 Inflammation

Objective:Bronchial asthma is a disease characterized by chronic airway inflammation,which is associated with type-2 cytokine mediated inflammatory responses.Increasing evidences from animal model or cell line-based studies suggested that neuromedin U(NMU),a specific group of neuropeptides,was emerged as a potential pro-inflammatory mediator in type-2 inflammation.However,the roles of NMU in human immune response and asthma has not been elucidated yet.In this study,we aimed to investigate the potential modulatory effect of NMU and its receptor on human asthmatic type-2 inflammation.Materials and Methods:Patients meeting definition of the global initiative for asthma(GINA)with sputum eosinophil counts and healthy control subjects were recruited from John Radcliffe Hospital or Churchill Hospital,Oxford.Peripheral blood was collected and used for flow cytometry to detect the expression levels of NMUR1.Samples for paired peripheral blood and lung resection tissue from same individuals were collected by Oxford Radcliffe Biobank at John Radcliffe hospital,Oxford at the day of surgery.Flow cytometry was used to compare the expression levels of NMUR1 on bloodcirculating and lung-resident type-2 cells.Furthermore,Th2、Tc2、ILC2s were sorted from peripheral blood mononuclear cells and expanded in vitro.Cultured type-2 cells were treated with serial concentrations of human NMU(NMU-25),NMU-25 combined with PGD2,LTE4,alarmin(IL-25/IL-33/TSLP)or individually,respectively.The IL5 and IL13 m RNA levels were measured with q PCR,while the IL-5 and IL-13 protein levels released in the supernatants were examined with ELISA.To confirm the effect of NMU25 on eosinophil migration,eosinophils were purified from fresh blood.Chemotaxis assay for purified eosinophils was conducted ex vivo.Results:Our data,for the first time,demonstrated that:1.NMU receptor 1(NMUR1)was expressed in human(both healthy controls and asthmatics)type-2 cells and eosinophils but not in B cells,neutrophils,basophils or monocytes.The expression levels were no differences among these three type-2 cells.2.The amounts of Th2 and Tc2 cells were increased in eosinophilic asthmatics compared with healthy controls.The expression levels of NMUR1 in ILC2 s of healthy controls were higher than that of asthmatics,while differences of expression levels in Th2 and Tc2 between healthy and asthma phenotypes were ignorable.3.NMUR1 expression levels up-regulated in lung-resident type-2 T cells compared with their circulating counterparts.In addition,the rates of effector T cells in tissue-resident NMUR1 positive Th2/Tc2 cells increased compared with that in blood counterparts.4.In vitro experiments showed that NMU-25 induced type-2 cytokine production in type-2 cells at both gene level and protein level,in a dose dependent manner.Compared to PGD2 or IL-25+IL-33+TSLP treatment individually,combination with NMU-25 increased the release of IL-5 and/or IL-13 in all three types of cells.However,up-regulation of type-2 cytokine in ILC2 s only was detected,regarding to combination of NMU-25 and LTE4.5.Besides,our ex vivo data showed that NMU-25 induced cell migration of eosinophils.Conclusion:Briefly,our data demonstrated that NMUR1 was expressed in human type-2 cells and eosinophils,the expression levels were no obvious differences among three types of type-2 cells,but up-regulated in lung resident type-2 cells.NMU-25 elicited type-2 cytokine production in human type-2 cells and induced eosinophil migration.NMU-25 also enhanced type-2 cell activity in response to other stimuli,particularly PGD2 and alarmin.Taken together,these data indicated that NMU could contribute to type-2 inflammation mediated asthma in human via pro-inflammatory effects on type-2cells and eosinophils.