The Role Of CCL20-CCR6 Axis In Respiratory Syncytial Virus Breaking Tolerance Induced By Oral Ovalbumin In Mice

Objective: Previousely,we have established an acute RSV infected mouse which was tolerance to OVA by oral feeding.We found that in this model,the pulmonary inflammation was more severe,and the CCL20 expression in the lung was increased,besides,the CCR6+ inflammatory cell were recruited to the lung.The aim of this study is to further explore the mechanism of CCL20-CCR6 in acute exacerbation of pulmonary inflammation caused by RSV infection.Methods:(1)MLE-12 cell lines were infected by 0,1,5 and 10 MOI of RSV,and then the RNA and protein were collected in 6h,12 h and 24 h post infection,CCL20 mRNA level was detected by RT-qPCR,and the TLR signal pathway factors were detected by western blot.(2)To figure out whether blocking the CCL20-CCR6 axis could ease the inflammation caused by RSV infection in OVA tolerant mice,CCL20 neutralizing antibody was given to the RSV acute infected OVA tolerant mice by intraperitoneal injection,and isotype IgG1 was used as negative control.Three days later,the mice were sacrificed,and ELISA was performed for serum IgE detection,HE staining was used for pulmonary pathology,RT-qPCR was applied to detect the inflammatory cytokines expression.We also collected the BALF,and used Wright staining to observe the kind and number of inflammatory cells.Finally,flow cytometry was performed to investigate the kind and number of lymphocytes in hilar lymph nodes.Results:(1)CCL20 expression was up-regulated by RSV infection in MEL-12 cells,the highest expression was in 12 hours post infection with 5 MOI of RSV,and then CCL20 expression was decreased as time went by.(2)MyD88 protein expression was increased in MLE-12 infected by RSV,so did the phosphorylation of STAT3,IKK? and NF-?B.(3)After CCL20 neutralizing antibody treatment,immune cells include lymphocytes,eosinocytes and neutriphile granulocytes were decreased in BALF,IgE level in serum was also reduced.Inflammatory cell infiltration and airway epithelial hyperplasia in lung tissue were significantly improved,and inflammatory cytokines such as IL-5,IL-13 and Il-7A in lung tissue were decreased,too.What is more,the CCR6+T cells and CCR6+IL-17+ in the hilar lymph nodes were remarkly reduced.Conclusion:(1)RSV infection can increase the expression of CCL20 in epithelial cells,which may be mediated by activation of the TLR signaling pathway.(2)Blocking the CCL20-CCR6 axis can reduce the lung inflammation of RSV mice with acute infection of OVA.