Neuromedin U Contributes To The Proliferation And Activation Of Pulmonary Group 2 Innate Lymphoid Cells During Acute Respiratory Syncytial Virus Infection In Mice

Objective:Respiratory syncytial virus(RSV)can cause bronchiolitis or pneumonia in infants,immunocompromised persons and the elderly.In severe cases,it can induce asthma-like symptoms and even allergic diseases.This may be related to the secretion of Th2 cytokines by Th2 cells induced by RSV infection.Group 2 innate lymphoid cells(ILC2s),which are widely distributed in the lung tissue,can participate in maintaining the pulmonary homeostasis through the Th2-type cytokines.The activation mechanism remains to be explored.In RSV-induced airway inflammation,ILC2s produce IL-13 via the IL-33/ST2 pathway,which leads to eosinophilic infiltration and airway hyperresponse,promotes the establishment of Th2-type immune response,induces and aggravates airway inflammation.The respiratory system is highly neuromodulated,while the airways are dominated by autonomic nerves from the parasympathetic nervous system.Besides cytokines and chemokines,neurotransmitters such as neuropeptides and neurotrophic factors are also involved in the regulation of airway function.Recently,more and more attention has been paid to the role of neuro-immune regulation in the maintenance of homeostasis.Secreted by cholinergic neurons,Neuronmendin U(NMU),a neuropeptide is a member of the neurointerleukin family,involved in a variety of bodily processes.NMU has immunomodulatory ability and is involved in the secretion of inflammatory cytokines.There are two different receptors for NMU,NMUR1 and NMUR2,both of which belong to the G-protein coupled receptor group A.Most studies believe that NMUR1 is mainly expressed in the periphery,while NMUR2 is mainly expressed in the central nervous system.Several studies have shown that peripheral NMU plays a biological role through NMUR1.So,during the process of RSV infection,whether there is an increase in NMU secretion;Whether NMU has a regulatory effect on ILC2s;what is the mechanism?Is NMUR1 involved?During the RSV acute infection model in mice,the use of such experimental methods as HE staining,immunofluorescence,Real-time PCR,Werstern blot,ELISA and flow cytometry,this study aimed to explore the role and mechanism of NMU on the proliferation and activation of ILC2s in the process of acute airway inflammation induced by RSV,in order to deepen the understanding of nerve-immune regulation on airway inflammation caused by viral infection.It provided an experimental basis to promote the diagnosis and treatment of non-allergic lung infections induced by viral infections.Method:1.Real-time PCR and Western blot were used to detect the mRNA and protein expression levels of NMU in the lungs during RSV infection.2.Primary neurons were isolated and cultured,and RSV stimulation was given in vitro.Real-time PCR and Western blot were used to detect the expression levels of NMU and TLRs,and the optimal multiplicity of infection index was selected.TLRs-specific agonists and inhibitors were used to co-culture with RSV and neurons in vitro.Real-time PCR and Western blot were used to detect the expression of NMU.3.In the in vivo NMU stimulation-blocking experiment,the inflammatory infiltration of the lung was observed by HE staining,and the inflammatory cells were collected by the classification technology of alveolar lavage fluid to evaluate the changes in the inflammatory response in the lung,and the expression level of cytokines was detected by ELISA.The amount and activity of ILC2s were determined by flow cytometry.4.In vitro experiment,NMU was used to stimulate ILC2s under RSV infection,cell supernatant and intracellular RNA were extracted,and the expression level of inflammatory cytokines was detected by ELISA,Real-time PCR and flow cytometry.5.In in vivo and in vitro experiments,flow cytometry was used to analyze the expression of NMUR1~+in ILC2s during RSV infection;ILC2s were sorted after RSV infection,and the expression level of NMUR1 was detected by Real-time PCR and Western blot.Immunofluorescence was used to observe the expression of NMUR1 after NMU stimulation of ILC2s in vitro,and the average fluorescence intensity was analyzed.The expression level of NMUR1 in ILC2s was determined by flow cytometry.6.Different concentrations of NMUR1-blocking antibodies were co-cultured with NMU and ILC2s selected by infected RSV,and the expression levels of inflammatory cytokines were detected by flow cytometry and ELISA.7.The signaling protein inhibitors were co-cultured with NMU and ILC2s,and the expression level of inflammatory cytokines was detected by Real-time PCR.Results:1.Real-time PCR and Western blot were used to detect the increased expression of NMU mRNA and protein in the lung during RSV infection.2.Primary neurons were isolated and cultured,and RSV stimulation was given in vitro.Real-time PCR technique was used to select MOI:10 as the optimal multiplicity of infection index.Real-time PCR and Western blot analysis showed that the expression of TLR4/TLR7 were increased during the process.TLR4/TLR7 specific agonists and inhibitors were used to co-culture with RSV and neurons in vitro.Real-time PCR and Western blot detection showed that the expression of NMU was increased in the agonist group,while the expression of NMU was lower in the inhibitor group.3.In the in vivo NMU stimulation-blocking experiment,HE staining observed that compared with the RSV group,lung inflammation in the NMU stimulation group was more serious,and inflammation in the NMU blocking group was reduced.The total number of inflammatory cells was significantly increased in the NMU stimulation group,especially the number of lymphocytes and eosinophils,while the number of inflammatory cells was decreased in the blocking group.The expression levels of inflammatory cells in alveolar lavage fluid were detected by ELISA,and the expression levels of IL-5/IL-13 were increased in the NMU stimulation group,but decreased in the NMU blocking group.Flow cytometry showed that compared with the RSV group,the number of ILC2s and the percentage of IL-5/IL-13 secretion were increased in the NMU stimulation group,and the reverse was observed in the NMU blocking group.4.In in vitro experiment,NMU was used to stimulate ILC2s after RSV infection,cell supernatant and intracellular RNA were extracted,and the optimal infection concentration was selected by ELISA;ELISA,Real-time PCR and flow cytometry showed that both IL-5 and IL-13 were highly expressed in the NMU stimulation group.5.Through in vivo and in vitro experiments,flow cytometry analysis showed that the expression of NMUR1 in ILC2s was increased during RSV infection,and increased significantly during the acute infection stage;Real-time PCR and Western blot analysis showed that the expression of NMUR1 was increased during RSV infection,and increased significantly in the acute infection stage.In vitro experiment,immunofluorescence was used to observe the expression of NMUR1 in ILC2 stimulated by NMU in vitro,and the mean fluorescence intensity analysis indicated that NMUR1was significantly expressed in ILC2s stimulated by NMU.Flow cytometry also indicated that NMUR1 was significantly expressed in ILC2s in NMU stimulation group.6.Different concentrations of NMUR1-blocking antibodies were co-cultured with NMU and ILC2s infected by RSV.Flow cytometry and ELISA were used to detect that the expression levels of IL-5 and IL-13 in the NMUR1-blocking group decreased,and the blocking effect was enhanced with the increase of the concentration.7.The signal protein blockers were co-cultured with NMU and ILC2s sorted after RSV infection,and the mRNA relative expression levels of IL-5 and IL-13 were decreased by Real-time PCR detection,suggesting that NMU may activate ILC2s through PI3K/MEK signaling pathway.Conclusion:1.RSV infection promotes the secretion of NMU in the lung;2.RSV induces neuron secretion of NMU and depends on TLR4/TLR7 pathway;3.NMU regulates the proliferation and activation of ILC2s during RSV infection;4.NMU stimulates ILC2s through NMUR1 and via PI3K/MEK pathway.