Research On Action Of Toll-like Receptor 2 And 9 In Herpes Simplex Virus Infection

Background:Herpes simplex virus(HSV)infection is a common human viral disease which can involve in oral,eyes,skin,central nervous system and external genitalia.It has become a main public health problem because the incidence rate of herpes simplex virus infection is obviously increasing in the recent 20 years,and the infection is also easy to relapse.Genital herpes(GH)is a common sexually transmitted disease caused by HSV which can infect fetus and neonate through placenta,birth canal or other ways and cause abortion,fetal death,congenital malformation,etc after the infection of pregnant women.Neonatal HSV infection can be left severe consequences.Its mortality rate can be as high as 60%～70%and 95%surviving infants have sequelae if not treated promptly.Herpes simplex virus encephalitis (HSVE)is the most common sporadic encephalitis.It has rapid progression without characteristic manifestations.The mortality rate of the patient infected HSVE without any treatment is more than 70%. From recent research,it has been found that the lip cancer and cervical cancer may be related to HSV infection.Also HSV infection is most common in AIDS patients.Toll-like receptors(TLRs)is a newly found class of important pattern recognition receptors.They can rapidly detects invasion of microbial pathogens,induce inflammatory reaction, play an anti-infection role and regulate the acquired immune response to the pathogens.As to the pattern recognition receptors,many researches focus on TLR2 and TLR9 at present.TLR9 mainly recognize the bacteria and virus containing non methylation CpG motif.TLR2 has broad-spectrum recognition capability.It can not only promote the synthesis and releasing of cytokines and the maturation and differentiation of immune cells,but also has prominent action in regulation the level of body immune response,antivirus and other effects on the invasive pathogens.Presently,the pathogenesis of HSV infection is not very clearly.Overseas research has shown the important effect of TLRs in HSV infection.Many researchers demonstrate that TLR2 can recognize HSV and mediate inflammatory cytokines response to HSV infection by the study using gene knockout mice.Also a strong inflammatory cytokines response mediated by TLR2 can cause host mortality.Research also indicates that HSV can induce IFN-αand IL-12 production to up-regulate costimulatory molecules CD86 by TLR9 dependent and independent pathways.Therefore,we propose the following working hypothesis:TLR2 and TLR9 recognize HSV and activate the TLR signaling pathways,trigger the innate immunity.They activate a transcriptional factor,NF-κB,to stimulate production of inflammatory cytokines through a series of protein cascade reactions, participate and regulate the body immune response and inflammation reaction to HSV.The present related study on HSV and TLRs concentrates primarily in the animal experiments,while rarely involving in medication and clinical study.There is so little technique of pathological change and drug treatment effect in human body infected HSV that unfavorable to disease prevention in clinic.In this study we research on two aspects:HSV infection models in mice and HSV infected patients.We try to interpret the action of TLRs in HSV infection,the relationship between TLRs and cytokines,the change of TLRs and cytokines after antivirus treatment and the difference of pathogenesis between HSVs infected animal models and patients.We try to provide theoretical basis for diagnosis and treatment of HSV infection in clinic and provide a new idea for searching for a new treatment approach and target point.Objective:To study the expression level of TLR2 and TLR9 in the brain tissue of infected mice model and peripheral blood mononuclear cells(PBMC)in patients with HSV infection,and the level of IL-6,IL-8,IL-10,TNF-αand IFN-βin blood serum.The purpose of this study is to investigate the action of TLR2 and TLR9 in the pathogenesis of HSV infection,and explore its possible action and mechanisms of protection of ganciclovir against mice HSVE;to investigate the time-effect relationship of HSV infection,the expression of TLR mRNA and the levels of cytokines;and to understand the seropositivity rate of children herpes simplex virus infection.Methods:Part one and two(animal experiment):The mouse HSVE model was established.The expression levels of TLR2 and TLR9 mRNA in brain tissue of HSVE mice were examined by RT-PCR in different infected time,and the levels of IL-8,TNF-αand IFN-βin mice blood serum were examined meanwhile by ELISA method.The time-effect relationship of HSV infection,expression of TLR9 mRNA and levels of IL-8,TNF-α,IFN-βin blood serum and the influence of these indexes after ganciclovir intervention were observed.Part three (clinical cases research):The expression levels of TLR2 and TLR9 in PBMC in recurrent herpes simplex children and genital herpes patients were examined by flow cytometer by using the whole blood dual spectral immunofluorescence direct standard method.At the same time,the levels of IL-6,IL-10 and TNF-αwere detected by ELISA method.Part four(epidemiological study):2436 samples of HSV antibody in blood serum of children were detected in clinic service and in-patient department of Hunan Children's Hospital.And all the 434 samples of HSV antibody in neonatal cord blood in four hospitals at that time were detected too.All data were analyzed statistically to use SPSS 11.5.Results:1)The HSV detection rates of HSVE model group and ganciclovir intervention group in mice were both 100%.In HSVE model mice group,the development of disease is more quickly than that in the intervention group and the symptoms were also more serious.The mortality was higher and the virus titer in brain was obviously set up in HSVE model mice group.Ganciclovir had the protection of HSVE mice.2)In HSVE model mice group,the expression levels of TLR2 and TLR9 mRNA in brain tissue were obviously increased compared with the normal group,and gradually increasing with the prolonging of infection time.The levels of IL-8 and TNF-αin blood serum were obviously increasing in all the phases at the same time after infection(P＜0.001). The level of IFN-βbegan to increase at the third day after the infection (P＜0.001),synchronizing with the increasing expression of TLR2 and TLR9 mRNA.The results suggest that TLR2 and TLR9 might be involved in the process of recognition against the virus in HSVE,and in the up-regulation of IL-8,TNF-αand IFN-βin the mouse HSVE model. 3)In GCV intervention mice group,the expression levels of TLR2 and TLR9 mRNA in brain tissue were obviously increased compared with the normal group.The expression level of TLR2 mRNA was gradually decreasing with the prolonging of infection time(P＜0.01),while the expression level of TLR9 mRNA was gradually increasing.Comparing with these in the HSVE model group,the levels of TLR2 mRNA in GCV intervention group had attenuated since the third day after infection.The difference of two groups had significant meaning(P＜0.01).Meanwhile the levels of IL-8 and TNF-αwere obviously decreased at all the phases after infection comparing with the HSVE model groups(P＜0.001).The level of IFN-βwas obviously increased(P＜0.001).The results suggested that TLR2 might be involved in the anti-HSV action of ganciclovir.4)The expression levels of TLR2 and TLR9 were both obviously increased in adult genital herpes patients and children with recurrent herpes simplex (P＜0.001).At the same time,the levels of IL-6 and IL-10 in blood serum were obviously increasing,and the level of TNF-αwas obviously decreased.The difference of two groups had obvious significant meaning (P＜0.001).The results suggested that TLR2 and TLR9 might be participated in the body's recognition of HSV and activated the signal pathway of TLRs and medicated the cytokines reactions of IL-6,IL-10, TNF-α,etc..5)The expression levels of TLR2 and TLR9 in both normal children and children with HSV infection were obviously higher than the adult and adult with GH(P＜0.001).6)The expression levels of TLR2 and TLR9 were both obviously increased in children with staphylococcal scalded skin syndrome(SSSS)(P＜0.05).It suggested that TLR2 and TLR9 might be involved in the process of recognition against staphylococcus.7)The seropositivity rate of HSV was 44.55%(143/321) in all the child patients accepted the HSV antibody test.The seropositivity rate of HSV and HSV1 increased significantly with age (P＜0.05),and the girls' was higher than the boys'(P＜0.05).The seropositivity rate of HSV in the group with a clinical diagnosis of herpes (61.14%)was significantly higher than that in the group without herpes(12.73%).8)The seropositivity rate of HSV in neonatal cord blood was 5.76%(25/434).Conclusion:1)Ganciclovir can effectively inhibit the replication of HSV-1 DNA,and has the protection against mouse HSVE model.The anti-HSV function of Ganciclovir might be related to TLR2.2)TLR2 and TLR9 might be participated in the virus recognition of body with HSVE, trigger the TLRs signal pathway,release cytokines such as IL-8,TNF-αand IFN-βand participate in the natural immune towards HSV as well as the inflammatory process in HSVE.3)In the mice with HSVE,the expression of brain tissue's TLRs mRNA and the level of IFN-βin blood serum are positively correlated with the period of HSV infection.4) TLR2 and TLR9 might be participated in the body's recognition of HSV together in children with recurrent herpes simplex and adults with genital herpes,and activate the signal pathway of TLR.5)The expression levels of TLR2 and TLR9 in the surface of peripheral blood mononuclear cells are significantly higher in children than that in adults.6)TLR2 and TLR9 might be involved in the pathogenesis of SSSS.7)The seropositivity rate of HSV in child patients increases with aging.Children are mainly infected by HSV1,while the infection in neonates are mainly caused by HSV2.8)The detection of HSV antibody in blood serum is helpful to the diagnosis of HSV infection in children.