The Effect And Mechanismof HCMV-IE2 On Macrophage Function By Regulating Autophagy

Objective Human Cytomegalovirus(HCMV)belongs to the β-herpesvirus family.The adult infection rate of HCMV in our country is close to 100%,and the high morbidity and mortality caused by HCMV in suppressing individual immunity and congenital infection is shocking.The IE2 protein is encoded by the UL122 gene,which is one of the most important proteins in regulating viral latency and activation.Autophagy is particularly important for maintaining body growth and development,innate and adaptive immunity.This study will focus on exploring how HCMV-IE2 regulates autophagy and affects the phagocytic function of macrophages.Methods1.Use the drugs PMA,LPS and IFN-γ to induce suspension cells U937 to become M1 macrophages.2.FITC-labeled heat-killed Pseudomonas aeruginosa stimulated U937-derived macrophages with a time gradient(0h,1.5h,3h,6h,12 h,24h).Western Blot detected the duration of the strongest expression of autophagy protein;fluorescent labeling method detected the duration of the highest phagocytosis rate of U937-derived macrophages.3.HCMV AD169 strain was used to infect macrophages with time gradient(0h,24 h,48h,72h),and the expression of IE2 protein was detected by Western Blot.After HCMV infected U937-derived macrophages with the highest IE2 protein expression,FITC-labeled heat-killed bacteria were added.The effect of HCMV-IE2 on autophagy protein expression was detected by Western Blot;the phagocytosis of bacteria by U937-derived macrophages was detected by fluorescent labeling.4.After HCMV infection of U937-derived macrophages,the expression of UL122 gene was inhibited by the drug MND,and the expression of IE2 protein was detected by Western Blot.FITC was added to label heat-killed bacteria,and the effect of HCMV-IE2 on autophagy protein expression was detected by Western Blot;the phagocytosis of bacteria by U937-derived macrophages was detected by fluorescent labeling.5.The pc DNA3.1-IE2 plasmid was transfected into macrophages,and the expression of IE2 protein was detected by Western Blot.FITC-labeled heat-killed bacteria were added,and the effect of HCMV-IE2 on autophagy protein expression was detected by Western Blot;the phagocytosis of FITC-labeled bacteria by U937-derived macrophages was detected by fluorescent labeling.6.PCR screening identified UL122 transgenic positive model mice.C57BL/6 wild mice and UL122 transgenic mice were perfused with live Pseudomonas aeruginosa solution to create a pneumonia model and set a blank control.There were 4 groups in total,with n=6 in each group.Western Blot was used to detect the expression of IE2 protein and autophagy protein in lung tissue of mice in each group;HE staining and blood routine were used to detect the pathological changes of lung tissue of mice.Results1.After adding heat-killed Pseudomonas aeruginosa,U937-derived macrophages had the strongest phagocytosis rate and the highest expression of autophagy protein at 24 h.2.When HCMV infects macrophages for 72 h,the expression of IE2 protein is the strongest.3.The expression of autophagy proteins Beclin 1,ATG3 and LC3-II in U937-derived macrophages: Compared with the control group,the expression of autophagy proteins in HCMV AD169 infection group and IE2 plasmid transfection group The expressions of autophagy proteins increased in the MND drug inhibition group.4.Changes in the phagocytic function of U937-derived macrophages: compared with the control group,the phagocytosis of Pseudomonas aeruginosa by macrophages in the HCMV infection group and pc DNA3.1-IE2 plasmid transfection group decreased;MND drug inhibited the IE2 protein expression group Macrophage phagocytosis of Pseudomonas aeruginosa was increased.5.The prognosis of IE2 transgenic mice Pseudomonas aeruginosa pneumonia was worse than that of C57BL/6 wild-type mice,and had more severe allergic reactions.Conclusion1.Experiments show that HCMV-IE2 inhibits the expression of autophagy proteins both at the cellular level and at the animal level.2.HCMV-IE2 inhibits the autophagy of U937-derived macrophages,thereby inhibiting its phagocytic function of Pseudomonas aeruginosa.3.The UL122 transgenic mice successfully constructed in our research group can stably express the IE2 protein,which overcomes the limitation that the host of HCMV is only human,and removes obstacles for the study of HCMV-IE2 and the immune response of the body and the macrophage immunotherapy of HCMV infection.4.The stable expression of HCMV-IE2 protein in mice inhibits autophagy,thereby promoting the body’s inflammatory response,which is not conducive to the outcome of pneumonia in mice.