Mechanisms Of Neuromedin U-activated Group 2 Innate Lymphocytes In Sepsis-induced Acute Lung Injury

Objective: Sepsis is a common complication of critical illness and has a high mortality rate,posing a serious threat to human health.Acute lung injury(ALI)is one of the most common complications of sepsis,and the release of large amounts of cytokines is closely related to the severity of tissue injury.However,the immune regulatory processes and the cascade responses of cytokines during ALI are not fully understood.Previous studies have found that interleukin-17A(IL-17A)plays a crucial role in host resistance to pathogenic infections.The level of IL-17 A is significantly up-regulated in the blood of patients with sepsis,which can improve the prognosis of patients.Recent studies revealed that IL-17 A could be secreted by several innate immune cells(e.g.,group 2 innate lymphoid cells(ILC2s),γδ T cells,etc.),and play a vital role in the host’s defense against mucosal infections.However,the regulatory factors and mechanisms of ILC2 s and IL-17 A in ALI are still not fully elucidated.The neuroregulation of ILC2 s has been a hot topic of research in recent years,whether neuromedins are involved in regulating ILC2 s in sepsis-induced ALI is not yet clear.Thus,the aim of this research is to investigate the expression changes and regulatory mechanisms of lung ILC2 s and IL-17 A in sepsis models,and elaborate the inflammatory cascade responses of sepsis and provide theoretical basis for the discovery of therapeutic targets in sepsis.Methods: In our study,wild-type(WT,C57BL/6J)mice were subjected to cecal ligation and puncture(CLP)to induce sepsis.The lungs were isolated at specific time points after CLP,and the levels of ILC2 s,γδ T cells,IL-17 A,neuromedin U(NMU)and NMU receptor 1(NMUR1)were measured by flow cytometry and real-time fluorescence quantitative PCR,respectively.Mouse lung ILC2 s were sorted and cocultured with γδ T cells,and changes in the number of γδ T cells and IL-17 A expression in the coculture system were measured.Exogenous NMU were injected to wild-type septic mice,then the levels of ILC2 s,γδ T cells and IL-17 A were measured.To clarify the regulatory role of NMU on ILC2 s,γδ T cells and downstream,we knocked down the nmur1 gene expressed by ILC2 s using CRISPR/Cas9 method,and incubated the coculture system with NMU,the levels of γδ T cells and IL-17 A were determined.To clarify the effects of ILC2-derived IL-9 on γδ T cells,the Il-9 gene in ILC2 s was knocked down via CRISPR/Cas9,then the number of γδ T cells and concentration of IL-17 A concentration were measured in the coculture system.Results: In septic mice,the protein and m RNA levels of lung IL-17 A were significantly upregulated,and flow cytometry results showed that the cell numbers and percentages of ILC2 s and γδ T cells were significantly upregulated.Further analysis revealed that γδ T cells were the main source of lung IL-17 A in the sepsis model.When lung ILC2 s and γδ T cells were cocultured in vitro,ILC2 s were able to upregulate γδ T cells and promote IL-17 A secretion,suggesting that ILC2 s could exert immune effects by regulating γδ T cells.In addition,we detected significant upregulation of NMU in septic lungs,upregulation of nmur1 expressed by ILC2 s.Lung nmur1 was specifically expressed in ILC2 s but not in γδ T cells.Furthermore,significant upregulation of lung ILC2 s,γδ T cells and IL-17 A were found after exogenous NMU injection.And similar results were obtained in an ex vivo coculture system of ILC2 s and γδ T cells.After knockdown of nmur1 in ILC2 s using CRISPR/Cas9 method,we found that the stimulatory effects of NMU on γδ T cells through ILC2 s were significantly attenuated.The above results suggest that NMU could play a regulatory role by binding to NMUR1 that expressed on the surface of ILC2 s.To further clarify the regulatory mechanism of ILC2 s on γδ T cells,we cocultured lung ILC2 s and γδ T cells in vitro and treated them with NMU,and found that ILC2 s could secrete large amounts of IL-9.After specific knockdown of Il9 in ILC2 s by CRISPR/Cas9 method,ILC2 s were then cocultured with γδ T cells,we found that the stimulatory effects of ILC2 s on γδ T cells and IL-17 A are significantly attenuated.Conclusion: NMU is significantly upregulated in septic lungs.NMU initiates the ILC2 activation by binding to NMUR1,which in turn promotes IL-17A-producing γδ T cell expansion and IL-17 A secretion.ILC2-derived IL-9 plays an important role in mediating γδ T cell expansion and IL-17 A production.This study reveals,for the first time,the function of neuroimmune axis(represented by NMU)during sepsis,and elucidates the important role of innate immune lymphocytes in sepsis.