The Study On RSV Infected Monocyte Regulating CD4+ T Cell Subsets Proliferation

Respiratory syncytial virus(RSV)is the leading cause of lower respiratory tract infection in infants and children.It can lead to severe respiratory diseases,including pneumonia and bronchitis.It will cause 64 million infection cases and 160,000 death cases each year world-widely.As the pathogenesis of RSV is unclear,so far there are no effective vaccines or specific antiviral drugs for RSV.Therefore,it is urgently needed to study the pathogenesis of RSV.Studies have shown that immune regulating effect of RSV,inducing imbalanced CD4+ T cell immune response,is crucial in the pathogenesis of RSV although the detailed mechanism is unclear.Monocyte is an important component of the human immune defense system and play an important role intissue damage repair,virus infection defence and autoimmune disease.However,the role of monocytes in the pathogenesis of RSV is unclear,especially its relationship with the imbalanced CD4+T cell immune disorder induced by RSV infection need to be elucidated.To investigate whether RSV can regulate CD4+ T cell subsets proliferation through infecting monocytes,RSV infected monocytes were co-culture with CD4+ T cells.After 7 days,samples were harvested and the frequency of Th1,Th2,Th17 and Treg subsets ratio in the proliferated CD4+ T cells were measured.Our results showed that RSV infected monocytes could reduce the frequency of Treg cells and increase the frequency of Th2 cells.But of its effect on Thl and Th17 were not significant.These results suggest that RSV could regulate CD4+ T cell subsets proliferation by infecting monocytes.Monocyte is composed of two subsets,CD 16+ and CD 16-subsets.In order to elucidate the effect of the specific monocyte subsets,we isolated monocytes into two subsets of CD 16+ and CD 16-by sorting flow cytometry.CD 16+ and CD 16-monocytes were infected with RSV in the same titer,and the results showed that CD 16+ monocytes were more efficient infected by RSV.CD 16-and CD 16+monocytes were infected with RSV and co-culture with CD4+ T cells for 7 days,respectively.The effects of two mononuclear cell subsets on the proliferation of CD4+T cells subsets were measureed by flow cytometry.The results showed that RSV infected CD 16+ monocytes could significantly reduce the frequency of Treg cells and increase the freuency of Th2 cells.However,RSV infected CD 16-monocytes have no significant regulation effect.These results suggest that RSV can regulate the proliferation of CD4+ T subsets by infecting CD 16+ monocytes but no CD 16-monocyte.To elucidate the mechanism of the RSV infected CD 16+ monocyte regulating CD4+ T subsets proliferation,we co-cultured RSV infected CD 16+ monocytes with CD4+ T cells,with variety of cytokines neutralizing antibodies(IL-1?,IL-6,IL-10,IL-13,IL-27 and TNF-?)and control IgG.Our results showed that after IL-10 neutralization,the effect on RSV infected CD 16+ monocyte inhibiting Treg proliferation was suppressed.The concentration of cytokines in the supernatant of the co-culture system also were measured,the results showed that there are a significant IL-10 level increasion in the supernatant of RSV infection CD 16+ monocyte.The results suggested that IL-10 might mediate the inhibitory effect of RSV on Treg proliferation;In addition to,after IL-1? or TNF-? neutralization,the effect RSV infected CD 16+ monocyte on Th2 cells proliferateion was suppressed.The results suggested that IL-1? and TNF-? may mediate the effect of RSV on Th2 cells.In addition,in order to further investigate the role of RSV virus in the regulation of immunity,we designed an innovative technique to rapidly construct various cell lines that can induce the expression of RSV proteins.The proteins of RSV virus genes containing PiggyBac are respectively connected to different transposon sites and induced by Cumate,and PiggyBac transposon plasmids were transfected into HEK293 cells,then selected by puromycin,in the end viral proteins expression induced by Cumate.The results of flow cytometry and Western blot showed that when we add the Cumate,the corresponding viral proteins began to express in the cell line,which indicated that the cell lines were constructed successfully.Thus,it is proved that PiggyBac transposon can efficiently construct a cell line which can induce RSV proteins expression.This work laid the foundation for the study of the role of RSV virus proteins in the regulation of immune responses.In summary,we found that RSV could regulate CD4+ T cells subsets proliferation,inhibing Treg subset and increasing Th2 subset,by infection CD 16+monocyte.The regulating effect on Treg subset may be mediated by IL-10,the regulating effect on Th2 subsets may be mediated by IL-1? and TNF-?.In our study,we have found a new pathway of RSV regulating the immune response,and provided new information for elucidating the pathogenesis of RSV and potential targets for the treatment of RSV related diseases.