Impairment Of Host Innate And Humoral Immune Response Caused By SFTS Virus Infection

Severe Fever with Thrombocytopenia Syndrome(SFTS)is an emerging infectious diseasecharacterized with high fever,thrombocytopenia,leukocytopenia,gastrointestinal symptoms,hemorrhage,and multiple organ failure.Its high mortality in hospitalized patients has been reported ranging from 12%to 30%,and its increasing prevalence has become a persistent public health threat in the East Asia.The etiological agent of SFTS has been identified as a novel member of Bunyaviridae in 2010.To date,no vaccines are available and specific therapeutic intervention is lacking.The immunological pathogenesis of the disease,including the regulation of innate immune and specific humoral immune response,as well as its relationship with the prognosis of SFTS patients,is yet to be elucidated.To gain further insight into the immune mechanisms of SFTS and develop effective prevention and therapeutic interventions,we conducted a comprehensive and systemic study taking the advantage of a relatively large patient cohort admitted in Nanjing Drum-Tower hospital in recent six years.Recent researches on thepathogenesis of SFTS virus infection mainly focused on two aspects:cytokine storm-mediated immune activation and mechanisms of impairment of innate immune response.Some researchesdemonstrated that cytokine storm-mediated immune activation characterized by a drastic increase of inflammatory cytokines such as IL-6,TNF-?,IFN-? isclosely associated with the clinical outcome of SFTS patients.However,the results of various studies displayed quiet different regulatory profiles of inflammatory cytokines.Experiments performed in vitro showed that virus encoded NSs and N proteins could impair the signaling pathway of innate immune response in monocytes and suppress type I interferon response.However,the regulation and cellular mechanisms of innate immune response in SFTS patients has been poorly understood.Hence,in the first part of our study,we investigated the modulation of cytokines and three types of interferons,immune related gene expression in peripheral blood cells,and dynamic changes of circulating dendritic cells during the acute phase of SFTSV infection.Our research reveals strong correlationsbetween disease severity and persistent high level viremia,elevated serum IL-6 and TNF-?,decline of serum IFN-? and IL-1? level,reduction of myeloid dendritic cells(mDCs)and suppressed Toll like receptor 3 expression in monocytes and mDCs.Although the innate immune response serves as the first line of host defense against pathogens,the final resolution of viral infection still largely depends on the estabilishment of adaptive immune response,including specific humoral immune.Despite few investigations about the serological response to viral nucleocapsid protein in the convalescent SFTS patients have been reported in recent years,the role of serological response specific to membrane glycoproteins Gn and Gc in the elimination of viral infection,as well as the regulation of cell components and related cytokines involved in the humoral response have been poorly understood.Because Gn and Gc are two transmembrane proteins with high glycosylation,they are difficult to been expressed in vitro and seldom reported before our study.Considering the targets of neutralizing antibodies are usually located at the membrane glycoprotein,elucidating the dynamic profiles of the serological response to Gn and Gc during acute phase of SFTSV infection is extremely important for further clarifying the mechanisms of disease pathogenesis,and also for developing the diagnostic and interventional strategies.Besides serological study,we also carried on the phenotypic and functional investigation of antibody-secreting cells in peripheral blood of SFTS patients,applying multiparametric flow cytometer and Elispot assay which captures virus specific IgG.B-cell dependent immunity is regulated by antigen presenting cells(APCs)and follicular helper T cells(Tfh).In our previous study,we found that mDCs were impaired in the fatal cases.Therefore,it's necessary to illustrate the differentiation and function of dendritic cells,as well as the impact on the activation of helper T cells during SFTSV infection.Finally,we determined relevant cytokines in SFTS patients' sera using a highly sensitive Milliplex chip.The main results of the research are as follows:1.Thefirst part of the study:(1)Clinical characteristic of SFTS patients shows dysfunction in hemostasis,coagulation and multi-organ injuries associated with the progress of SFTS,and high serum viral load closely correlated with key clinical parameters.(2)The measurement of three types of interferons and major pro-inflammatory cytokines in sera of SFTS patients demonstrated that the serum levelof IFN-? and IL-1? were significantly reduced in the fatal patients,and inverselycorrelated with the increase of the disease severity.However,the serum IL-6 and TNF-? dramatically increased as the severity of the disease increased.(3)Transcriptional profile of immune related genes in peripheral monocytes of SFTS patients was analyzed using quantitative mRNA microarray chip.We found that compared with other major pattern recognition receptors,mRNAs of TLR3in the monocytes manifested the most significant down-modulation as the severity of the disease increases.Meanwhile,mRNAs of interferon regulatory factors 3 and 7(IRF3 and IRF7)exhibited similar regulatory patterns as TLR3.IFN-?1 mRNAs in monocytes were upregulated in survived patients,but down-regulated together with IFN-?1 in the fatal patient.(4)Analysis of kinetics of dendritic cells in patients'peripheral blood during acute SFTS virus infection demonstrated that the survived patients,but not the deceased,manifested steady increase of the CD11c+CD123population,indicating that the impeded differentiation of mDC was involved in the aggravation of disease pathogenesis.(5)Dynamic analysis of TLR3 expression by flow cytometry demonstrated that TLR3 expressions by mDCsand monocytes in deceased group were gradually downregulated during the acute phase,as compared with the survived group upregulated.the regulation of TLR3 could probably be the mechanism involved in the impaired innate immune by SFTS virus infection.2.The second part of the study:(1)We constructed the expression system of Gn and Gc,then the two major viral structural glycoproteins were expressed in mammalian cells and purified.Through detection of serological response specific to Gn and NP during the three-week period after symptom onset,we found that the absence of both NP-and Gn-specific IgG antibodies in the deceased patients,indicating that the defective serological responses to the pathogen are associated with the fatal outcome.Meanwhile,persistently elevated viremia was correlated with the deficiency of specific IgG response in deceased patients.Neutralization assay further proved that the serum of the convalescent patient and Gn-immuned camel,but not of the deceased patient,manifested the effective neutralization potency.Interestingly,none of the patient sera reacted with Gc.It is not clear if the absence of Gc reactivity reflects the poor immunogenicity of Gc or the Gc does not express native epitopes that can be recognized by serum antibodies.(2)We analyzed various B cell subtypes in PBMCs by flow cytometry.The results showed that the proportion of naive and marginal-zone like B cells within total peripheral B cells of the deceased group was significantly lower than that of the survived group and the healthy control in the three-week period post symptom onset.Two important effector B cell subsets,PB,and MB cells,in both survived and deceased patients were significantly higher than that of the healthy control.Surprisingly,the fraction of PBs inthe deceased group increased significantly more than the survived group.The frequency of IgD-CD27-subset notably increased with the disease aggravation in the deceased group.(3)In order to further estimate the differentiation and function of antibody-secreting cells,we performed two expeirments in the current study.At first,we examined immunoglobulin expression by both PB and MB subsets by intracellular staining of IgG and IgM using another gating strategy(CD3?CD19?CD27?CD38).The kinetics of PBs and MBs as defined by CD38 expression were highly consistent with the result of previous analysis.Meaningfully,class-switched IgM-IgG+PBs inthe survived patients exhibited steady elevation as compared with the drastic decline inthe deceased patients in acute phase of SFTS.To illustrate the functionalregulation of ASC in SFTS patients,we performed an Elispot assay which captures virus specific IgG after cell stimulation in vitro.The result also demonstrated that the deceased patient failed to produce Gn-specific IgG.Based on above results,we imply that the ASCs of the deceased patients fail to undergo IgG class-switch recombination(CSR)as the result of SFTSV infection.(4)Apoptosis analysis of peripheral monocytes demonstrated that monocytes underwent severe apoptosis and necrosis at an early stage of SFTSV infection.We further investigated the differentiation and function of mDCs by flow cytometry.The robust differentiation of mDCs in the survived patients,not in the deceased,was observed at the 2nd week,and sustained to the 3rd week post symptom onset.Additionally,the expression of CD86+on mDCs and the proportion of CD80+CD86+/mDCs of the survived patients were significantly higher than that of the deceased patients.Therefore,Ag presenting functionof mDCs was involved in the progress of SFTS.In vitro infection assay demonstrated that SFTSV could cause significant apoptosis of mDC and impair its antigen presentation function.(5)Expression analysis of HLA-DR and CD80 on peripheral total B cells and major subsets revealed that in the deceased patients,the HLA-DR expression on total B cells,MBs,MZ B and CD27'IgD-B cellswas significantly more down-regulated than that in the survived patients.Meanwhile,the proportion of CD80+HLA-DR+cells and the CD80 expression in total B cells,MBs and MZ B cells but not in naive and CD27'IgD' B cells differed between the deceased and the survived groups.Thus,theseevidencessuggestthat SFTSV infection causes the impairment of B cell antigen presentation that results in the defective humoral response in the deceased patients.(6)Though the dynamic analysis of peripheral follicular CD4+T cells of SFTS patients during acute phase,we found that the number of pTfh in the survived,but not the deceased patients showed robust increase in the 1st week,as compared with that in the healthy control.After ex vivo stimulation on PBMC,the proportion of IL-21+ pTfh was significantly higher in the survived patients than that in the deceased patients in the 1st and 2nd weeks post symptom onset.These evidences indicated that the effective differentiation of pTfh occurred at the early stage of SFTSV infection in survived patients,not in deceased individuals.CD4+B helper assay indicated that the function of pTfh in the deceased patients was significantly inhibited.(7)We determined serum T-helper-related cytokines in SFTS patients'sera using a highly sensitive Milliplex chip toreveal the dynamic profile of the cytokine regulationduring the three weeks of acute infection.In the 1st week,serum levels of IFN-y,IL-23,IL-12 and IL-10 increased dramatically in the deceased patients,but serum level of GM-CSF decreased significantly compared to in the survived patients.In the 2nd week,IFN-?,IL-23,IL-12 and IL-10 showed declining trend in the deceased group,accompanied by IL-6 and TNF-?markedly elevated.In the 3rd week,serum IL-1? and IL-4 in the survived patients markedly elevated.Based on the above results,we can conclude that dysfunction of innate immune response and pro-inflammatory cytokine storm are both involved in the disease pathogenesis.The effective differentiation of myeloid dendritic cells plays essential role in the elimination of viremia.The regulation of TLR3 could probably be the molecular mechanism involved in the impaired innate immune by SFTS virus infection.In the investigation of specific humoral immune to SFTS virus infection,the absence of both serum IgM and IgG specific to NP,and the absence of IgG specific to Gn are closely associated with the fatal outcome of SFTS patients.The deficiency of humoral response is caused by global disruption of B cell immunity,including the impeded maturation of mDC,impaired antigen presentation function of mDC and B cell and the failure of Tfh differentiation at the early phase of SFTS.In conclusion,the current study points to the roles of NP-and/or Gn-specific antibody response in the control of viral pathogenesis and shed light to the potential target for vaccine or therapeutic antibody development.