Study On Characteristics Of Peripheral Blood Immune Cells In Patients With Severe Fever With Thrombocytopenia Syndrome

Background:Severe fever with thrombocytopenia syndrome(SFTS)is an emerging viral hemorrhagic fever that was first reported in China in 2009,and subsequently reported in Taiwan of China,South Korea,Japan,Vietnam,and Myanmar,with a high fatality rate ranging from 12%to 50%.The causative pathogen of SFTS belongs to the Bandavirus genus in the family Phenuiviridae,named Dabie bandavirus,also known as SFTSV(SFTS virus).Tick is the main transmission vector of SFTS.There are also several case reports of human-to-human transmission through close contact with patient secretions.Due to its high mortality rate and the wide distribution of tick vectors,SFTS has posed a potential threat to public health.In 2017,SFTS was listed as one of the top ten infectious diseases requiring urgent research by the World Health Organization.Currently,the pathogenic mechanism of SFTSV remains to be elucidated,and there is a lack of approved specific therapeutic drugs or protective vaccines against SFTSV.The dysregulated immune response of patients is closely related to the progression and poor prognosis of SFTS.Therefore,it is extremely urgent to investigate the immune patterns associated with pathophysiology involving SFTS.Identification of the peripheral immune responses in patients may provide important clues for understanding the pathogenesis of SFTS,improving clinical prognosis prediction,and developing novel antiviral therapies.Objective:(1).To clarify the differences in the type and composition of peripheral blood immune cell subsets in patients with surviving and deceased SFTS.(2).To reveal the compositional changes and related functional characteristics of peripheral blood immune cell subsets in the progression of SFTS patients.(3).To describe the relationship between the changes of monocyte,B cell and T cell subsets in peripheral blood and disease progression.The purpose is to identify immune indicators related to predicting disease progression,provide important clues for the study of the pathogenesis of SFTS,and clinical treatment of the disease.Methods:Laboratory-confirmed patients with SFTSV infection and healthy controls were prospectively enrolled in the sentinel hospital,People’s Liberation Army No.990 Hospital in Xinyang City,Henan Province,during the same period.Whole blood and serum samples were collected.Peripheral blood mononuclear cells(PBMC)were extracted from the whole blood and subjected to transcriptome sequencing by using 10×Genomics single-cell RNA sequencing(sc RNA-seq)technology.Then,based on the sequencing results,principal component analysis and the UMAP method were used for dimensionality reduction and clustering.Differentially expressed gene and pathway enrichment analysis were performed to explore the expression of immune-related genes in different immune cells during infection.SFTSV infection rate in immune cells was determined by flow cytometry.A highly sensitive ELISA via single-molecule array(Simoa)technology was used to quantitatively detect IFN-αin serum,and the Luminex flexible multi-analyte profiling(x MAP)technology was used to detect concentrations of cytokines,chemokines,and complement factors in serum.Correlation analysis was used to clarify their relationship with disease progression and prognosis.Moreover,a retrospective study was conducted to explore the clinical efficacy of IFN-αin the treatment of SFTS.Results:A total of 15 hospitalized patients with laboratory-confirmed SFTSV infection and 4 healthy controls(HCs)were enrolled in this study.We performed 10×Genomics single-cell RNA sequencing(sc RNA-seq)on 27 peripheral blood mononuclear cell(PBMC)samples.Eight of 11 patients who eventually survived from the disease were sampled twice:once during the acute phase(viral load cycle threshold value≤30,AS group)and once during the recovered phase(viral load cycle threshold value>30,RS group).Four patients who eventually deceased from SFTSV infection were sampled once during the acute infection(AD group).After quality control and normalization of the sequencing raw data,we sequenced 139,885 high-quality cells with an average of 5,185cells per sample and a median of 1,932 genes detected per cell.Using unbiased clustering and uniform manifold approximation and projection(UMAP)analysis,we identified 23clusters with distinct transcriptomic signatures.Using automated cluster annotation combined with data-derived marker-gene-based manual annotation to annotate clusters,we identified 9 canonical immune cell types:CD14~+monocytes,CD16~+monocytes,B cells,plasmablasts,CD4~+T cells,dividing lymphocytes,CD8~+T cells,NK cells,DC cells.We observed different cell compositions across four disease conditions.CD4~+T,CD8~+T,and DC cells were depleted,while plasmablasts and dividing lymphocytes were enhanced in SFTS patients compared to HCs.Within SFTS groups,CD14~+monocytes and dividing lymphocytes were further augmented,while plasmablasts were decreased in the recovered patients(RS group)in comparison with the acute patients(AS or AD group).Given that the different cell compositions were correlated with four disease conditions,we next analyzed monocytes,B cells,T and NK cells with more granularity.Seven subsets of monocytes were reclustered,with the phenotypic shift of classical monocytes(CD14~+CD16~-)into intermediate monocytes(CD14~+CD16~+).Notably,the intermediate monocytes were the most common in the deceased patients.Eight subsets of B cells were reclustered.The proportion of na(?)ve B and memory B cells decreased,and the proportion of active B cell subsets,including IFN-I-inducible B cells,germinal center B cells,preplasmablasts,plasma cells,IFN-I-inducible plasmablasts cells,and plasmablasts increased post SFTSV infection.Significant expansion of overall plasmablasts at the acute infection was verified by flow cytometry analysis,whereas no significant differences were observed between the surviving and the deceased patients on both sc RNA-seq or flow cytometry data.In addition,the proportion of IFN-I-induced plasmablasts was significantly increased in the deceased patients.Likewise,we identified an SFTSV-infected plasmablast population via flow cytometry,with its frequency in proportion to IFN-I-induced plasmablasts and significantly higher in the deceased patients in comparison with the survivors.These findings indicated that IFN-I-induced plasmablasts might be the target cells of SFTSV infection.We reclustered T and NK cells into 13 subsets based on canonical markers.Three cell subsets,including IFN-I inducible CD4~+T,CD8~+GZMH~-effector T,and CD56~+CD160~-NK were overrepresented in the deceased patients compared to the survivors.We profiled the expression of genes encoding cytotoxicity and exhaustion markers and identified highly exhausted cellular status in SFTS patients.In detail,a higher exhaustion module score was observed in deceased patients compared to the survivors,with CD8~+GZMH~-effector T,CD8~+GNLY~-effector T,and CD56~+CD160~+NKT as the most exhausted cell subsets.This data suggested that the highly exhausted status of CD8~+T and NKT cells might be relate to SFTSV infection and aggravating disease.We performed differentially expressed genes(DEGs)and pathway enrichment analysis,and found that high enrichment of type I interferon(IFN-I)signaling across most cell types of SFTS patients relative to HCs.Notably,the ISG module score was increased in most immune cell types,with the highest level in CD14~+monocytes,followed by NK cells and plasmablasts.In particular,a higher ISG score trended towards a positive correlation with the older age of SFTS patients and a negative correlation with the long delay from symptom onset to sampling,both phenotypes were significantly related to adverse outcomes.By evaluating the serially collected serum samples of the 15 SFTS patients using Simoa technology,we noted that serum IFN-αconcentrations were significantly increased in the AD group than the AS group,which nevertheless returned to baseline levels at about 12 days since symptom onset for the RS group.Strikingly,IFN-αconcentrations were elevated in proportion to both ISG expression and viral load,as well as to death-related cytokines or chemokines,such as CXCL10,CXCL12,CXCL16,CCL2,CCL8,CCL11,CLL19,and IL6.These findings collectively suggested the potential role of robust IFN-I signaling in the induction of cytokine storm,rather than benefiting viral clearance at the early stage of SFTSV infection.The intermediate monocytes exhibited high expression of ISGs(i.e.,MT2A,IFIT1,ISG15,GBP1,and IFIT3)and IFN-dependent chemokines(i.e.,CXCL10).Indeed,the intermediate monocytes displayed more pronounced complement activation.Based on the measurement of the SFTS case cohort,secretion of C3b/i C3b and complement factor D were remarkably induced out of 12 complement components in the deceased SFTS.Additionally,disseminated intravascular coagulation(DIC)that is related to complement activation,occurred in three out of four deceased patients,which suggests complement overactivation that increases the procoagulant response could be exacerbating the disease in patients with SFTS.We revealed that enhanced IFN-I response was significantly associated with fatal outcomes of SFTS patients.Given the concern that there might be an adverse clinical effect of using IFN-αtreatment,we performed a retrospective clinical study to analyze the clinical efficiency of IFN-αtreatment.We found that IFN-αtherapy neither improved patient survival nor affected the clearance of viremia.The laboratory indicators that conferred significant risk for death of SFTS also had no accelerated recovery to normal.Conclusions:SFTSV infection leads to a significant difference in the type and composition of peripheral blood immune cells.Our study unveiled that the reprogrammed peripheral immune responses were featured by monocyte phenotypic along with complement activation,expansion of plasmablasts and exhausted T cells,which all were related to fatal outcome in SFTS patients.The intermediate monocytes and IFN-I-inducible plasmablast may serve as the main target of SFTSV infection in the peripheral blood.Complement activation of intermediate monocytes was related to DIC.The highly exhausted status of CD8~+T and NKT cells was related to SFTSV infection and in aggravating the disease.In addition,we found that the IFN-I signaling pathway was correlated with the poor prognosis of the disease,suggesting IFN-I plays an important role in SFTSV infection.The expression levels of ISG and IFN-αcould be used as biomarkers for the prediction of death outcomes.The retrospective clinical investigation on a large cohort showed no apparent advantage or even adverse effect of IFN-αin treating SFTS.It could be a therapeutic strategy by inhibition of IFN-I signaling and depletion of IFN-I-producing cells to bring the exaggerating immune responses back to balance,especially for those with fatal risk.In summary,our study provides insight into the immunotherapies of SFTS.Significance and innovation:In this study,we described a complete and comprehensive single-cell landscape of peripheral immune cells in patients with SFTS by using 10×Genomics sc RNA-seq technology.Enhanced IFN-I signaling pathway in PBMC might be used as a feature of disease progression.We also found that intermediate monocytes and IFN-I-induced plasmablasts might act as the main target cells for SFTSV infection,and the infection of both cells was related to the over-activation of the IFN-I signaling pathway.We performed a retrospective study to investigate the efficacy of IFN-αon treating SFTS.Our study may provide important clues for understanding the immunopathogenesis of SFTS and developing a potential solution for SFTS immunotherapies.