Comparative Analysis Of JEV Infections In Macrophages With Different Polarized Phenotypes

Japanese encephalitis virus(JEV)is a lead cause of encephalitis,named Japanese encephalitis(JE),which are mainly distributed in southern and southeastern Asia,and become the major viral encephalitis in those areas.According to data of World Health Organization(WHO),JEV infections annually cause death of 10000-15000 worldwide.However,it doesn't mean that all of infections cause disease.In contrast,only a small group of infected people(1:300)shows encephalitis symptoms.Thus,the deaths among infections reveal that JEV can evade host immune defense by certain mechanisms from peripheral infection to dissemination into brain.It has been shown that monocytes in peripheral blood including macrophage and progenitor play a very important role in this process,including: 1)JEV can be isolated from monocytes in peripheral blood;2)JEV can survive in macrophages.Thus,to some extent,the consequence of JEV-macrophage interaction may be the cause of JE.Macrophages are the major immune cell of host,and play important role in innate and adaptive immunity.To note,the biological functions of macrophages is attributed to its activate status of macrophages.To date,in light of activate status,there are M1 macrophages and M2 macrophages,the former mainly differentiated by Th1 cytokines like IFN-?,the latter mainly differentiated by Th2 cytokines like IL-4.In vitro studies have shown that compared with nonimmune cells such as vero,MEF and so on,macrophages can robust inhibit JEV infections.Furthermore,M1 macrophages induced by IFN-? limit JEV infections via production of NO.Though,to date,how M2 macrophages response to JEV infections remain unknown.Interestingly,it has been shown that Th2 immune response play a protective role in JE.Goal: In order to compare the role of M1 macrophage,M2 macrophage,and M0 macrophages in JEV infections,special virulence strains like NJ2008,we set up two parts of experiments,as follows: 1)To clarify characteristics of virulence strain NJ2008 in vivo and in vitro;2)To determine the difference of JEV infections among different activated macrophages.Methods:(1)To clarify infectious characteristics of virulence strain NJ2008 in vivo and in vitro,firstly,the virus stock of NJ2008 was generated by use of C6/36 cell line and its titration was qualified by TCID50 test.Secondly,the survival curve of C57BL/6 was determined after i.p infections with NJ2008 strain.Subsequently,the brain infections of JEV were analyzed by TCID50 test,western blot,and indirect immunofluorescence assay(IFA).Finally,we analyzed infectious characteristics of NJ2008 in vitro,including: 1)comparative analysis of one-step growth curve between BHK-21 and RAW264.7;2)furthermore,comparative analysis of infections between BHK-21 and RAW264.7 by IFA.(2)To determine the difference of JEV infections among different activated macrophages,firstly,RAW264.7 was differentiated into M1 and M2 macrophage by using IFN-? and IL-4 stimulation,respectively.Furthermore,to certify the activation status of macrophages,the expression of hallmark genes including iNOS and Arg1 and cytokines including CXCL-10 and TNF-? was determined by realtime qPCR.Secondly,we compared infectious characteristics of JEV in different activated macrophages by using TCID50 test,IFA and realtime qPCR.Results:(1)For analysis of infectious characteristics of NJ2008 in different activated macrophages,firstly,the titration of viral stocks was calculated by TCID50 test.Our result showed that the titration of viral stock was 8.3×108 TCID50/0.1m L.Secondly,the survival curve of C57BL/6 was determined by infections of 8.3×104 TCID50/100?L/mouse i.p.Our results showed that peak of death happened between day 7 and day 14.During this period,more than 60% of mice were dead.Subsequently,brains of sick mice were harvested for TCID50,western blot,and IFA.Furthermore,our results showed that the viral loads were more than 107 TCID50/brain,consistent with the results of western blot and IFA.Finally,our in vitro results showed that according to one-step growth curve in both BHK-21 and RAW264.7,virus titer reached peak by 24 hpi.Furthermore,compared with virus titer in BHK-21 of each time point,virus titer in RAW264.7 was very low.Furthermore,our data confirmed the above results by detection of NS3 protein by IFA.(2)For comparative analysis of JEV infections in different activated macrophages,firstly,our results showed that compared with PBS-treated group,IFN-? stimulations significantly up-regulated iNOS,one of hallmarks belonging to M1 macrophages,likewise expression of CXCL-10 and TNF-?,indicating that we succeeded to generate M1 macrophages.On the other side,IL-4 stimulations promoted expression of Arg-1,and inhibited expression of CXCL-10 and TNF-?,indicating that the cells became M2 macrophages.Secondly,we analyzed virus infections in the above cells.Although there were no difference in virus titer between M2 macrophages and PBS-treated group,virus titer in M1 macrophages significantly decreased compared with PBS control.Thus,our data indicated that compared with M2 macrophages or PBS-treated group,M1 macrophages pronounced more inhibition of JEV infection,mainly by NO production.Conclusion: Our study systemically analyzed JEV infections in different activated macrophages.Furthermore,our data demonstrated that M1 macrophages pronounced a robust inhibitory effect on JEV infection in comparison of M2 or M0 macrophages.Although M2 macrophages showed less effect on inhibition of JEV infections than M1 macrophages,Th2 immune response played a protective role in JEV brain infections.Whether or not M2 macrophages play a very important role remains unknown.Thus,future studies are needed to determine the question.All together,our results demonstrated that macrophages with different activated phenotypes played differential role in JEV infections.Furthermore,these results provide the basis of understanding the role of macrophage in JEV infections.