Study On The Mechanism Of HCMV IE2 Regulating Macrophage Polarization

Currently,human cytomegalovirus(HCMV)has infected more than 70% of the population in the world.In healthy individuals,HCMV infection is usually asymptomatic and controlled by the immune system.HCMV infection can be reactivated when the immune system is compromised,resulting in the occurrence of life-threatening diseases.HCMV evolves with the host and develops strategies to escape the host immune response and survive in the body for a long time.Recent studies have shown that HCMV infection mediates immune escape by regulating the immune activity of macrophages.HCMV IE2 protein is encoded and expressed by IE2 gene,which is very important for effective virus replication.At present,the role of IE2 protein encoded by HCMV in immune escape remains to be unclear.Object: HCMV has strict genus-species specificity,and can only use humans as the sole host of infection.Therefore,there is no animal model for studying the HCMV IE2 protein.In order to break the genus-species specificity of HCMV among species,we constructed UL122 transgenic mouse model to stably express IE2 protein.On this basis,UL122 transgenic mice were used to study the mechanism of immune escape.This paper aims to determine the effect of HCMV IE2 on macrophage polarization and function,and explore the role of GRB2 in it.Methods: A UL122 transgenic mouse model was constructed to stably express IE2 protein.PCR was used to identify the genes in the offspring of transgenic mice and wild-type mice.Male UL122 positive mice with 20-24 weeks old were selected as the experimental group and wild-type mice as the control group(15 in each group).2.Flow cytometry was used to detect the proportion of M1 and M2 macrophages in spleen and bone marrow of UL122 transgenic mice and wild-type mice.3.Neutral red uptake(NRU)was used to evaluate the phagocytic function of macrophages.4.Western blot and real-time fluorescence quantitative PCR were used to detect the expression of GRB2 and IE2 protein and mRNA in spleen and bone marrow of UL122 transgenic mice and wild-type mice.5.Real-time fluorescence quantitative PCR was used to detect the relative expression levels of cytokines related to macrophage such as IL-4,IFN-?,IL-13 and TNF – ? in spleen and bone marrow of UL122 transgenic mice and wild-type mice.Results: 1.A UL122 transgenic mouse model was successfully constructed and IE2 protein was stably expressed.The positive mice identified by PCR technology were used as the experimental group and the wild-type C57 BL / 6 mice as the control group(n = 15 in each group).2.The phagocytic function of macrophages was detected in the bonemarrow and spleen of mice.The results showed that IE2 enhanced the phagocytic function of bone marrow macrophages and spleen macrophages in the experimental group.3.The immunophenotyping of bone marrow macrophages and spleen macrophages showed that compared with the control group,the proportion of M2 spleen macrophages was significantly increased,while M1 macrophages were decreased in the mice expressing IE2,indicating that IE2 induced macrophages were polarized into anti-inflammatory M2 macrophages.4.Western blot and real-time fluorescence quantitative PCR showed that compared with the wild-type control group,the expression level of IE2 and GRB2 mRNA and protein in the spleen and bone marrow of transgenic mice were significantly up-regulated.5.The mRNA expression levels of cytokines related to polarization such as IL-4,IFN-?,IL-13 and TNF-? were evaluated in the spleen and bone marrow of mice.Compared with the control group,the expression levels of IL-4 and IL-13 were up-regulated in the mice expressing IE2,and the level of IL-4 was significantly increased.Conclusions: 1.The successfully constructed UL122 transgenic mice overcome the species specificity and can stably express IE2 protein,which provides an effective way to study the interaction between HCMV and immune system.2.HCMV IE2 can promote the polarization of macrophages to become M2 and increase the expression of cytokines related to polarization such as IL-4 and IL-13.3.The long-term stable expression of IE2 can up-regulate the expression level of GRB2,of which up-regulation can affect the polarization and phagocytosis of macrophages.