The Role Of MerTK In JEV Infected Monocytes/ Macrophages And Its Regulatory Mechanism

The epidemic Encephalitis B is an acute central nervous system disease infection caused by Japanese Encephalitis Virus（JEV）.It is mainly transmitted by mosquito bites,most of the survivors had severe neurological and psychiatric sequelae.Although attenuated or inactivated JEV vaccines have good protective efficiency,vaccination in epidemic areas cannot fully cover them.Therefore,there are still about 60-70000 cases of Japanese encephalitis every year worldwide.There is no specific and effective therapeutic drug or method for JEV.Therefore,a more in-depth study of JEV infection and pathogenesis will contribute to the discovery of JEV drug targets and the development of treatment strategies.JEV infection can lead to encephalitis,which can be divided into two stages:viremia stage and brain inflammation stage.In the periphery,JEV replicates predominantly in monocytes/macrophages,which can not only replicate within macrophages to produce viruses,but can also rapidly activate macrophages,inducing the production of large amounts of inflammatory and chemokines;These factors not only participate in the anti-virus action of the peripheral immune system,but also can migrate from the peripheral to the central nervous system to intensify the inflammatory response in the brain.How JEV enters monocytes/macrophages and its regulatory mechanisms are still unclear.MerTK,a member of the TAM receptor tyrosine kinase family（TYRO3,Axl,and MerTK）,is highly expressed on a variety of myeloid-derived cells and plays an important role in maintaining histiocytic homeostasis and regulating inflammation.In recent years,some studies have found that MerTK on the host cell membrane can bind to phosphatidylserine on the viral envelope through the ligand Gas6/Pro S1,mimicking the process of phagocytosis of apoptotic cells,thus mediating the entry of virus into cells.Meanwhile,in the development of diseases,MerTK also plays an important role in regulating inflammation repair.At present,the mechanism of MerTK molecule regulating monocyte/macrophage infection and inflammation in JEV induced encephalitis is still unclear.We have conducted the following studies.Objective:Mouse bone marrow-derived macrophage（BMDMs）cells,MerTK^-/-BMDMs cells and macrophage cell line RAW264.7 were isolated and cultured,the purpose of this study was to determine whether MerTK was involved in the process of JEV-infected bone marrow macrophages entry and the regulation of virus replication,as well as the polarization direction of BMDMs.To provide experimental basis for understanding the pathogenesis of JEV.Method:1.Mouse bone marrow cells were isolated and induced to differentiate into BMDMs using supernatant from culture of L929 cells.The purity of BMDMs was Flow cytometry.2.Collect samples of BMDMs and RAW264.7 cells infected with JEV（MOI=0.1）after 12 and 24 hours The expression of JEV m RNA and MerTK m RNA were detected by RT-q PCR at different time points after infection.3.The BMDMS were incubated with JEV（MOI=10）for 40 min,and the unattached virus was washed thoroughly.RT-q PCR was used to detect the differences in JEV m RNA adsorbed by WT and MerTK^-/-mouse BMDMs;After cold incubation at37℃,JEV was initiated and entered the cells.After 1 hour and 4 hours,the differences in JEV m RNA expression between WT and MerTK^-/-groups of mouse BMDMs were detected by RT-q PCR.4.The supernatants and cell samples of BMDMs of WT and MerTK^-/-mice infected with JEV（MOI=0.1）were collected at 12 h and 24 h after infection.The level of JEV m RNA replication was detected by RT-q PCR,and the level of infectious virus particles in cell supernatants was detected by plaque formation assay,RT-q PCR was used to detect the expression of interferon-related genes in BMDMs infected JEV.5.CCK-8 was used to detect the effect of MerTK kinase inhibitor UNC2881 on the activity of BMDMs and determine the working concentration of UNC2881;BMDMs were treated with UNC2881 and infected with JEV,and changes in JEV m RNA expression were detected by RT-q PCR.6.The polarization of JEV-infected BMDMs was detected by Flow cytometry and RT-q PCR,and the polarization of JEV-infected macrophages was detected by Flow cytometry and RT-q PCR Western blot was used to detect the expression of STAT1 and p-STAT1 in WT and MerTK^-/-groups.Results:1.RT-q PCR results showed that JEV infection up-regulated the expression of MerTK in BMDMs and Raw264.7 cells.2.After incubation at 4℃for 40 min and cell washing for 3 times,RT-q PCR results significantly lower than that of WT cells.After incubation at 37℃for 1 hour and 4 hours,the virus RNA level in MerTK^-/-BMDMs was also lower than that in WT cells after washing the cells in the same manner.3.After 12 and 24 hours of JEV infection with BMDMs from MerTK^-/-and WT mice with MOI of 0.1,RT-q PCR and plaque assay results showed that JEV proliferation was significantly reduced in MerTK^-/-BMDMs compared to WT BMDMs at 24 hours.4.The results of RT-q PCR showed that compared with WT BMDMs,MerTK^-/-BMDMs infected with JEV showed significantly upregulated expression of interferon related genes such as IFN-α,MYD88,IFI204,etc.5.RT-q PCR results showed that at 36 hours after JEV infection,the UNC2881（MerTK kinase inhibitor）group significantly reduced the JEV m RNA levels in BMDMs compared to the DMSO treated group.6.After JEV infection,flow cytometry results showed that the expression of BMDMs cell marker CD86 increased while the expression of CD206 decreased;RT-q PCR results showed that the M1 phenotype marker molecules IL-1β,i NOS,TNF-α,IL-6,and CD16 m RNA were significantly increased after JEV infection with BMDMs.7.Flow cytometry results showed that in the case of JEV infection,compared with the WT BMDMs group,the expression of the MerTK^-/-BMDMs cell marker molecule CD86 increased and the expression of CD206 decreased.RT-q PCR results showed that in the case of JEV infection,the expression of IL-1β,i NOS,and TNF-αm RNA in MerTK^-/-BMDMs was significantly upregulated compared to WT BMDMs.Conclusion:1.MerTK molecules are beneficial for virus adsorption and entry in the early stage of JEV infection with BMDMs;2.After JEV enters the cell,MerTK negatively regulates antiviral mechanisms such as interferon expression,thereby promoting JEV proliferation;3.MerTK inhibits M1 polarization in JEV infected BMDMs by regulating the STAT1-SOCS1/3 pathway.This study provides an experimental basis for understanding the regulatory mechanism of JEV infection in monocytes/macrophages.