Study On The Role Of Receptor Tyrosine Kinase MerTK In Encephalitis Caused By JEV Infection

Viral encephalitis is an important disease that threatens human life and health.Epidemic encephalitis type B,also known as Japanese encephalitis（JE）,is an acute central nervous system infectious disease and brain parenchyma inflammation caused by neurotropic encephalitis B virus（known internationally as Japanese Encephalitis virus,JEV）.There are about 67000 JE cases worldwide every year,and the case fatality rate can be as high as 25%to 30%,and about 30%to 50%of the survivors suffer from severe neurological and psychiatric sequelae.So far,there is no specific and effective treatment for encephalitis B.In recent years,due to the widespread use of JEV vaccine,the incidence of Japanese encephalitis has been greatly reduced,but local outbreaks often occur in some areas.Therefore,JEV infection is still one of the important public health problems in many countries in Asian.Studies have known that after being bitten by mosquitoes carrying JEV,the virus first proliferates in the local skin and lymph nodes,and then passes through viremia twice.In some people,JEV can break through the blood-brain barrier（BBB）and enter the central nervous system（CNS）,causing infection and destruction of neurons,activation of microglia and so on,and finally lead to encephalitis.At present,in the process of JEV-induced encephalitis,which molecules are involved in the maintenance and destruction of the BBB,and the mechanisms of antiviral related molecules and damage molecules in neurons and glial cells are not clear.TAM receptor is a newly discovered subfamily of receptor tyrosine kinases,including Tyro3,MerTK and Axl,which play an important role in the regulation of innate immune system and phagocytosis of apoptotic cells.Recent studies have shown that MerTK is involved in the regulation of pathogen entry and inflammation,especially in maintaining BBB integrity and reducing inflammation in the brain after infection with West Nile virus（WNV）,which belongs to the same genus of JEV.At present,the role of MerTK molecules in the process of encephalitis caused by JEV infection has not been reported.For this end,we have carried out the following research:Objective:1.To study the effect of MerTK molecule on viral replication in Peripheral tissue and the regulation of inflammatory response in the process of encephalitis induced by JEV.2.To study the effect of MerTK molecule on virus replication in brain tissue and the regulation of inflammatory response in the process of encephalitis induced by JEV.Methods:1.To construct a mouse model,MerTK knockout mice and wild-type mice were used as objects,and the survival rate and behavioral changes of mice infected with JEV were determined by plantar injection（10⁶PFU）.2.The mouse model of plantar injection was established,and the viral load and inflammatory response of the peripheral tissues of the two groups of mice infected with JEV were determined by qPCR,Wb and plaque test.3.MerTK knockdown macrophages and MerTK knockout primary macrophages were constructed,and the viral replication and inflammatory response in macrophages infected with JEV were determined by qPCR.4.The mouse models of plantar and intracerebral injection were established,and the viral load and inflammatory response of brain tissue of mice infected with JEV were determined by qPCR,Wb,plaque test and HE staining.5.The model of BBB in vivo and in vitro was constructed,and the changes of BBB permeability in vivo and in vitro after JEV infection were determined by sodium fluorescein method,Wb,tissue immunofluorescence,qPCR and electrical impedance detection.6.MerTK knockdown neuronal cells,astrocytes and microglial cell lines were constructed,and the changes of virus replication of nerve cells with low expression of MerTK after JEV infection were confirmed by qPCR,Wb and plaque test.Results:1.After JEV infection,the morbidity and mortality of MerTK^-/-mice increased significantly,and the symptoms of encephalitis were more obvious.Compared with WT mice,there was no significant difference in virus replication and inflammatory factors in peripheral tissues（blood and spleen）of MerTK^-/-mice infected with JEV.Knockout/knockdown of MerTK could inhibit virus replication and inflammation in macrophages.2.Compared with normal mice,the viral load in the brain tissue of MerTK^-/-mice was higher,and the increase of inflammatory reaction was not significant.In the model of BBB in vivo and in vitro,in the MerTK knockout/knockdown group,the permeability of BBB increased significantly and the expression of tight junction molecules in endothelial cells decreased after JEV infection.Low expression of MerTK in neurons,astrocytes and microglia promoted the replication of JEV.Conclusion:1.MerTK is a protective factor in the process of encephalitis caused by JEV infection.2.Deletion of MerTK molecules has little effect on JEV replication and inflammation in peripheral tissues.3.MerTK molecules regulate the replication of JEV in neurons,astrocytes and microglia by participating in the maintenance of BBB integrity,thus alleviating viral encephalitis caused by JEV.However,it is not obvious in the regulation of inflammatory response.