The Effects Of Peripheral Dendritic Cells And CD14+ Monocytes On The Pathogenesis Of Severe Fever With Thrombocytopenia Syndrome

BackgroundSevere fever with thrombocytopenia syndrome(SFTS)is a newly emerging infectious disease caused by a novel bunyavirus which was initially identified in rural areas of China.SFTS was mainly spread in the eastern and central regions of China and was characterized by exaggerated immune activation and the 'cytokine storm,which may lead to multi-organ failure at the late stage of disease,with a reported case fatality rate up to 30%.No specific treatment for SFTS is available,therefore,an understanding of the pathogenesis of SFTSV infection is important.Of note,as key regulators of innate immunity,dendritic cells and monocytes were thought to play a critical role in the pathogenesis of SFTSV infection.ObjectiveIn this study,the frequencies.phenotypes and functions of circulating CD 14+monocytes and DCs,including myeloid dendritic cells(mDCs)and plasmacytoid dendritic cells(pDCs),were examined dynamically by flow cytometry.In addition,we performed a detailed evaluation of the role of the abnormally expressed TLR2 on peripheral CD 14+monocytes as a major determinant of disease progression in SFTS patients to shed some light on the pathogenesis of the disease.Methods1.All confirmed SFTS patients(N = 115)were recruited from the Wuhan Union Hospital in 2015;a series of blood samples was collected from SFTS patients every other day starting at the time of admission.In addition,25 healthy volunteers were recruited for the study.Ethylenediaminetetraacetic acid(EDTA)anti-coagulated peripheral blood samples were collected from healthy subjects and SFTS patients.Peripheral blood mononuclear cells(PBMCs)were separated by density gradient centrifugation with Ficoll-Paque Plus.Patient details,including their clinical history,physical examination findings,and routine hematology laboratory results were collected from the medical records to conduct a retrospective analysis.2.Total RNAs were extracted from PBMCs by using TRIZOL Reagent according to manufacturer's protocol and then subjected to One-step real-time RT-PCR analysis using SYBR green I Kit(Takara,Japan)to detect the expressions of TLR2?TLR3?TLR4?TLR7.TLR8 mRN A.The frequencies,phenotypes,and function of circulating CD 14+monocytes and DCs,including myeloid and plasmacytoid dendritic cells(mDCs and pDCs),'were analyzed by flow cytometry.3.The laboratory parameters and other clinical events related to mortality were assessed by multivariate logistic regression analysis and receiver operating characteristic(ROC)curves.4.Serum levels of interleukin(IL)-6,IL-10,interferon-? and tumor necrosis factor alpha(TNF-a)were detected by ELISA.5.After the PBMCs were challenged with TLR2 agonist,the levels of TNF-a and IFN-y expression in CD 14+ monocytes were measured by FACS analysis.6.The purified monocytes were isolated from PBMC by CD 14 Microbeads.Monocyte-derived dendritic cells(MoDC)were generated in vitro with the presence of interleukin-4(IL-4)and granulocyte macrophage colony-stimulating factor(GM-CSF)upon activation by Pam3C.Costimulatory molecule expression of MoDCs and the activated markers on CD4+T cells after allostimulatory mixed lymphocyte reaction(AMLR)were detected in SFTS patients and healthy controls.Results1.The subsets,frequencies and phenotypes of circulating DCs in SFTS patients and its correlation with the prognosis of SFTS patients:1)The percentages of both mDCs and pDCs in PBMCs were markedly reduced inpatients in the acute phase of SFTS compared with healthy controls and recovery patients Additionally,a significantly decreased percentage of mDCs in PBMCs during the acute phase was observed in patients who died when compared to those who survived(p =0.0454).However,the extent of the decrease in pDCs was similar between patients who died and those who survived.2)The mDCs of acute SFTS patients expressed significantly lower levels of CD80(p<0.0001)and CD86(p<0.0001)when compared to healthy controls and convalescent patients.However,on pDCs,a significant increase in CD80 expression was seen in acute SFTS patients compared with healthy controls(p<0.0001).In contrast,no differences were found in the expression of CD86 on pDCs between SFTS patients and healthy controls.No significant differences were observed in the expression of CD80?CD86 on mDCs and pDCs between patients who died and those who survived.3)The serum levels of TNF-a,IL-6,and IL-10 were significantly higher in acute SFTS patients,but declined to nearly normal levels in the convalescent phase when compared with healthy controls(p<0.0001,respectively).In addition,significantly higher levels of these three cytokines were found in patients who died compared to those who survived(p<0.0001,respectively).Positive correlations were found to exist between SFTS viral 1 load and TNF-a,IL-6,and IL-10.4)The percentage of mDCs in PBMCs was inversely correlated with cytokine levels and viral load in the acute phase of SFTSV infection.5)It was found that a high level of mDCs was a protective factor for SFTS patients,especially from day 9 after the onset of fever(OR 0.487,95%CI 0.432-0.549;p<0.0001).6)ROC curves and the AUCs were used to assess the capacity of serum viral load at admission and the percentage of mDCs in PBMCs from day 9 after disease onset to predict mortality of SFTS patients.The AUC of mDC%was 0.929(95%CI 0.863-0.996;p<0.0001),and the best diagnostic cut-off point was 0.075%,with a sensitivity of 0.86 and specificity of 0.929.2.The phenotype and function of MoDC in acute SFTS patients:1)After in vitro challenge with Pam3c,the expression of CD86?CD83 on MoDC from acute SFTS patients were significant lower than healthy controls(P<0.05,P<0.01,respectively).In addition,the relative expression of Inc-DC mRNA in MoDC in acute SFTS patients was significantly lower than healthy controls(P=0.0357).2)After allostimulatory mixed lymphocyte reaction(AMLR),the expression of activated marker CD69 on CD4+T cells and the secretion of TNF-??IFN-? in CD4+ T cells in SFTS group were much lower than HC group(P<0.001,P<0.001,P<0.0001).3.The frequencies and phenotypes of circulating CD14+ monocytes in SFTS patients and its correlation with the severity of SFTS patients:1)The expression of TLR2 and TLR4 mRNA in PBMCs from acute SFTS patients were significantly higher than in HCs(P<0.01,P<0.05 respectively).2)Decreased percentage of CD 14+ monocytes were observed in acute SFTS patients when compared to healthy controls and convalescent patients(p<0.0001,p<0.0001);The TLR2 expression on peripheral CD 14+ monocytes was slightly higher in the acute phase compared to HCs and dropped to nearly normal level in the convalescent phase(p<0.05,p<0.05,respectively);However,in the deceased patients,the TLR2 expression was much higher when compared to the surviving cases(p<0.01).3)The TLR2 expression on CD 14+ monocytes was positively correlated with serum viral load,ALT,AST,CK,LDH and ?-GT levels of SFTS patients.4)Compared to that of HCs,CD 14+ monocytes from SFTS patients showed increased TNF-a and IFN-y production before stimulus(P<0.0001,P<0.0001;P<0.0001,P<0.001 respectively).Interestingly,patients in acute phase exhibited a higher sensitivity to Pam3C stimulation than those of HCs,as shown by a much higher increased level of TNF-a and IFN-y production in CD 14+ monocytes when compared to HCs(p<0.01,p<0.05,respectively).Furthermore,we observed that the TNF-a production of CD 14+ monocytes was positively correlated with TLR2 expression level on CD 14+ monocytes after stimulus(r=0.6608,p=0.0375).5)The serum levels of TNF-a and IFN-y in acute SFTS patients were much higher in compared to healthy controls and recovery patients(p<0.0001,p<0.0001;p<0.0001,p<0.0001 respectively).Similarly,significantly higher levels were also observed in deceased SFTS patients when compared to surviving patients(p<0.0001).6)Analysis of the MFI showed that the CD 14+ monocytes from acute SFTS patients express significantly lower level of CD86 when compared to HCs(p<0.01).Likewise,a significant difference in the expression level of CD86 was seen between the surviving and deceased patients(p<0.05).Conclusions1.The present results indicate that SFTS patients have a decresed level of circulating DCs and CD 14+ monocytes,as well as a significant lower expression of surface markers in mDCs.A decreased level of circulating mDCs was closely correlated with the severity of SFTS which could be used as a new prognostic marker to SFTS patients.2.The mDCs in SFTS patients might be functionally impaired which may be partially related to SFTS progression.3.Our findings implicate that upregulation and overresponse of TLR2 in peripheral CD 14+ monocytes may be involved in the pathogenesis of SFTSV infection and may contribute to the disease severity through inducing TNF-a or IFN-y by CD 14+monocytes.