Thrombocytopenia-related Immunological Mechanisms In Hemorrhagic Fever With Renal Syndrome

Background:Hantaan virus(HTNV)is one of the major pathogens causing hemorrhagic fever with renal syndrome(HFRS)in China.Thrombocytopenia is one of the important factors leading to pathological injury,but the mechanisms have not been fully elucidated,and the effect and mechanisms of thrombocytopenia on the body’s antiviral immune response have not been clearly determined either.HFRS are associated with symptoms such as thrombocytopenia and thrombosis,which is similar to heparin-induced thrombocytopenia(HIT)mediated by anti-platelet factor 4/heparin(PF4/H)antibodies.However,the relationship between anti-PF4/H antibodies and thrombocytopenia have not been reported in HFRS.In addition,platelets can adhere to monocytes to form platelet-monocyte aggregates(PMA),which not only causes a decrease in circulating platelet count but also modulates the immune function of monocytes.However,the formation and mechanisms of PMA and the regulatory role and the mechanisms of platelets on monocytes in HFRS caused by HTNV infection are still need to be addressed.Object:This research focuses on the mechanisms of thrombocytopenia and the regulatory roles of platelets on monocytes in HFRS.1)To elucidate the relationship between anti-PF4/H antibodies and thrombocytopenia in HFRS.2)To explore the mechanisms of PMA formation and the regulatory role of platelets on monocytes in PMA of HFRS.Method:1)Enzyme-linked immunosorbent assay(ELISA)was performed to evaluate the plasma levels of anti-PF4/H antibodies in HFRS patients.2)PF4-enhanced platelet activation assay was performed to test the function of anti-PF4/H antibodies in HFRS patients.3)Conventional flow cytometry(CFC)and imaging flow cytometry(IFC)were both used to detect the percentage of CD14~+CD41~+cells(PMA)and surface functional molecules of PMA in HFRS.4)An in vitro co-culture model of HTNV-infected platelet and/or monocytes was established.CFC was used to detect the formation of PMA and the tissue factor(TF)expression of monocyte.The levels of IL-6,IL-8,IL-10,IFN-γand TNF-αin the co-culture supernatant were detected by cytokine microarray.In addition,the adhesion of platelets to monocytes were blocked by neutralizing antibodies,and the changes on PMA formation,TF expression and cytokines production of monocytes were observed as previously described.Results:1)Among 75 HFRS patients,50 patients were negative for anti‐PF4/H antibodies(OD<0.65),21 patients were weakly positive(OD:0.66-0.74)and 4 patients were strongly positive,with OD values greater than 1.50,and the highest was 3.87.There were no correlations between anti-PF4/H antibodies and platelet count(PLT),fibrinogen(Fib),D-dimer(DD)and viral load in HFRS patients.Furthermore,the titers of anti-PF4/H antibodies in 4 strongly positive patients did not show significantly changes with variation of clinical indicators during disease progression.Among 7 HFRS patients received multiple CRRT and/or component transfusions after the onset of symptoms(heparin exposure),6 patients were weakly positive for anti-PF4/H antibodies and 1 patient was strongly positive,but the OD value of anti-PF4/H antibodies did not change obviously after several treatments.Finally,in the PF4-enhanced platelet activation assay,anti-PF4/H antibodies presented in the plasma of 4 strongly positive patients did not activate platelets under the conditions that saline,10μg/m L PF4,0.2 U/m L low molecular weight heparin,or 100IU/m L heparin.2)The percentage of PMA were significantly higher in HFRS patients compared with normal controls and were more pronounced in the convalescent phase and moderate/mild stage of HFRS patients(p<0.05,for all).In addition,the percentage of PMA were positively correlated with PLT and albumin(Alb)(r=0.6624,p<0.0001 and r=0.7420,p=0.0004),and negatively correlated with c-reactive protein(CRP),creatinine(Cr)and viral load(r=-0.6603,p=0.0090;r=-0.4328,p=0.0119 and r=-0.4232,p=0.0101).In the convalescent phase,the percentages of PMA formed with classic and intermediate monocyte subsets were higher than that formed with non-classic monocyte subsets(p<0.05 and p<0.01).Whereas in the normal controls,the percentages of PMA formed with intermediate and non-classic monocyte subsets were higher than that formed with classic monocyte subsets(p<0.05 and p<0.01).And the percentage of PMA formed with classic monocyte subsets in moderate/mild stage of HFRS patients was also higher than that in normal controls(p<0.05).Additionally,there was no statistical difference of the mean fluorescence intensity(MFI)of CD41 on monocytes between HFRS patients and normal controls detected by CFC(p>0.05,for all).In contrast,the results of IFC showed that the number of platelets adhered on monocytes in the convalescent phase of HFRS patients were significantly increased compared to acute phase and normal controls.The expression of CD62P on platelets in PMA was significantly higher in HFRS patients compared to normal controls and was more pronounced in the convalescent phase of HFRS patients(p<0.05,for all).Importantly,the expression of CD62P on platelets in PMA was positively correlated with PMA formation(r=0.5504,p=0.0005).Application of IFC to observe the morphological features of PMA formation revealed the co-expression of CD14 and CD41(overlay)in HFRS patients and the formation of PMA in a CD62P-dependent and CD62P-non-dependent manner.Besides,the plasma levels of CD62P and CD40L were significantly increased in HFRS patients compared to normal subjects and were more pronounced in the convalescent phase and moderate/mild stage of HFRS patients(p<0.05,for all).Furthermore,the plasma levels of CD62P were positively correlated with PLT and Alb(r=0.3895,p=0.0009 and r=0.3791,p=0.0324),and negatively correlated with blood urea nitrogrn(BUN),Cr and white blood cell(WBC)(r=-0.2663,p=0.0453;r=-0.3129,p=0.0094 and r=-0.2764,p=0.0236).The plasma levels of CD40L were positively correlated with PLT(r=0.4169,p=0.0003),and negatively correlated with BUN,Cr and WBC(r=-0.2669,p=0.0447;r=-0.2727,p=0.0244 and r=-0.2739,p=0.0249).Finally,the expression of CD62P and CD40L on platelets,and PSGL-1(CD162)and CD40 on monocytes infected by HTNV in vitro were higher than those in uninfected group(p<0.05,for all).Increased PMA formation and TF expression on monocytes were detected in monocytes co-cultured with HTNV-infected platelets compared to monocytes co-cultured with uninfected platelets(p<0.05,for both).In contrast,there were no significant changes in PMA formation and TF expression on monocytes between platelets co-cultured with HTNV-infected monocytes group and platelets co-cultured with uninfected monocytes(p>0.05,for all).When anti-CD62P or anti-CD40L neutralizing antibodies were added to the co-culture system of monocytes and HTNV-infected platelets,the percentage of PMA was significantly reduced in both cases(p<0.001 and p<0.01),but the TF expression of monocytes was not significantly changed in either case(p>0.05,for both).Increased levels of IL-6,IL-8,IL-10 and IFN-γ(p<0.05,for all)and decreased levels of TNF-α(p<0.01)were valued in supernatants of monocytes co-cultured with HTNV-infected platelets compared to monocytes co-cultured with uninfected platelets.Meanwhile,increased levels of IL-6,IL-8 and TNF-αwere valued in supernatants of HTNV-infected monocytes co-cultured with uninfected platelets compared to HTNV infected monocytes alone(p<0.05,for all).Moreover,HTNV-infected monocytes secreted increased levels of IL-8 but decreased levels of IFN-γcompared to uninfected monocytes(p<0.01 and p<0.001).When anti-CD62P or anti-CD40L neutralizing antibodies were added to the co-culture system of monocytes and HTNV-infected platelets,anti-CD62P neutralizing antibodies significantly inhibited the secretion of IL-6,IL-8,IL-10 and IFN-γand increased the secretion of TNF-α(p<0.05,for all).whereas,anti-CD40L neutralizing antibodies inhibited the secretion of IL-6,IL-10 and IFN-γand increased the secretion of TNF-α(p<0.05,for all).Conclusion:1)HTNV infection can result in the rare development of anti-PF4/H antibodies in HFRS patients with no pathological effect in thrombocytopenia and their production were not associated with either HTNV RNA or heparin exposure.2)PMA formation was increased after HTNV infection and might play a protective role in the disease progression of HFRS.Platelets participate in PMA formation through surface CD62P and CD40L binding to PSGL-1(CD162)and CD40 on the surface of monocytes,and finely regulate the cytokine production of monocytes,which involved in anti-HTNV infection response.