The Role Of OX40/OX40L In ILC2s-regulated CD4~+T Cell Proliferation And Activation During RSV Infection

Objective:Respiratory syncytial virus(RSV)is the main pathogen of causing lower respiratory tract infection in infants and people with low-immunity,it is also an important cause of allergic asthma.Recent studies found that ILC2s(Group 2 innate lymphoid cells)not only can produce IL-4,IL-5,IL-9,IL-13 cytokines to mediated airway inflammation and airway remodeling in patients with asthma,ILC2s also can regulate the biological activity of the adaptive immune cells such as CD4~+Th2 cells and then affect the formulation and development of asthma.The studies showed that ILC2s affect CD4~+T cells function in a contact-dependent manner,and OX40/OX40L plays a crucial role in ILC2s-regulated CD4~+T cell proliferation and activation.However,whether OX40/OX40L is involved in the proliferation and activation of CD4~+T cells regulated by ILC2s during RSV-induced airway inflammation,it's still not clear.Thus,in the present study,by using BALB/c mice modle with acute RSV infection and through experiments in vivo and in vitro,aim to confirm ILC2s regulate the proliferation and activation of CD4~+T cells;clarify the role of OX40/OX40L in ILC2s-regulated CD4~+T cell activation and its related mechanisms;and further understand the role of OX40/OX40L signaling pathway in the development of asthma.Methods:BALB/C mice with acute RSV infection experiment models were established;the number of CD4~+T cells,IFN-?~+CD4~+T cells,IL-4~+CD4~+T cells,and IL-5~+CD4~+T cells in the lung were determined by flow cytometry,the expression of IFN-?,IL-4 and IL-5 mRNAs in lung CD4~+T cells were determined by real-time PCR,to confirm RSV infection induce the proliferation and activation of CD4~+T cells.Adoptive transfer ILC2s experiments were performed to identify the role of ILC2s on the proliferation and activation of CD4~+T cells in RSV-induced airway inflammation.Transwell experiments were performed to identify ILC2s influence the proliferation and activation of CD4~+T cells in a contact-dependent manner.OX40 expression on CD4~+T cells and OX40L expression on ILC2s were detected by flow cytometry,real-time RT-PCR,and western blot,respectively,Block OX40/OX40L to confirm ILC2s regulate the proliferation and activation of CD4~+T cells through OX40/OX40L.Results:1.RSV infection mediates the proliferation and activation of CD4~+T cells in lung tissue:RSV infection not only increased the number of CD4~+T cells,IFN-?~+CD4~+T cells,IL-4~+CD4~+T cells,and IL-5~+CD4~+T cells in the lung,but also increased the expression levels of IFN-?,IL-4 and IL-5 mRNA in lung CD4~+T cells significantly,suggesting that RSV infection mediates the proliferation and activation of CD4~+T cells.2.ILC2s promote the proliferation and activation of CD4~+T cells in lung tissue during RSV infection:compared with non-transferred ILC2 mice,the number of CD4~+T cells and its cytokines mRNA expression were increased significantly after transfer ILC2s into BALB/C mice before RSV infection,suggesting that ILC2s promotes the proliferation and activation of CD4~+T cells during RSV infection.3.ILC2s promotes the proliferation and activation of CD4~+T cells in a contact-dependent manner during RSV infection:we used a transwell culture syetem with CD4~+T cells in the upper chamber and ILC2s in the bottom chamber by a culture insert,We found that compared with CD4~+T cells cultured alone,the numer of CD4~+T cells and the mRNA expression of its cytokines were significantly increased when co-culture with ILC2s,while significantly reduced when co-culture in the transwell system.4.The OX40?OX40L expression on CD4~+T cells?ILC2s in lung tissue were significantly increased after RSV infection:RSV infection not only significantly augmented the number of OX40~+CD4~+T cells and OX40L~+ILC2s,but also increased the mRNA and protein expression of OX40?OX40L in lung CD4~+T cells?ILC2s,suggesting that OX40/OX40L pathway may play a role in the activation of CD4~+T cells mediated by ILC2s.5.OX40/OX40L is involved in the proliferation and activation of CD4~+T cells regulated by ILC2s:Treatment with anti-OX40L in the CD4~+T cells and ILC2s co-culture system,The relative expression against?-actin of IFN-?,IL-2,IL-13 mRNA in CD4~+T cells was shown to be down-regulated compared to without anti-OX40L.Conclusion:ILC2s regulate the proliferation and activation of CD4~+T cells through OX40/OX40L during RSV-induced airway inflammation.