Immunological Mechanism Of Thrombocytopenia In HFRS Patients

Objective and methods:Hemonrrhagic fever with renal syndrome(HFRS)caused by Hantaan virus(HTNV)infection.Fever,bleeding,thrombocytopenia and acute renal insufficiency are the main clinical manifestations of HFRS patients.The main mechanisms of thrombocytopenia caused by viral infections are considered to be the destruction of platelets after direct contact with the virus,the reduction of platelet production,the immune destruction of platelet-virus complexes,and the inhibition of platelet function.However,the mechanism of thrombocytopenia caused by HTNV infection is still unclear.Growth arrest specific protein 6(Gas6),and milk fat globule epidermal growth factor 8(MFG-E8)belong to the opsonin family,which phagocyting apoptotic cells associated to the clearance of apoptotic cells.In the present study,we collected blood samples from HFRS patients with different course of disease through cooperation with the department of infectious diseases of Tangdu hospital of the second affiliated hospital of our school.Immunofluorescence staining,flow cytometry,and western-blot techniques were enrolled to detect platelet activation and apoptosis in HFRS.Gas6,and MFG-E8,in plasma of HFRS patients,were analyzed for their correlation with clinical indicators.Immunofluorescence staining and flow cytometry were used to analyze the receptors of Gas6 and MFG-E8 on monocytes in HFRS patients.The fluorescence staining and confocal microscope were used to detect the colocalization of opsonin and receptor in THP-1 cell activated by HTNV infection.Immunofluorescence staining and flow cytometry were performed to analyze the effects of MFG-E8 and?v?3 or?v?5neutralizing antibodies on the phagocytosis of phagocytic cells and their molecular mechanisms.Sandwish ELISA was used to detect the CX3CL1 and GM-CSF in the plasma of HFRS patients,which might participate in the up-regulation of MFG-E8expression and the correlation with clinical indicators.Furthermore,the expression of CX3CR1 on THP-1 cells were also measured,which will be the cell model used in the future.Results:Using fluorescence-labeled Annexin-V and flow cytometry,it was found that the proportion of PS~+platelets in the acute phase of HFRS patients was significantly higher(14.31±11.43%)than the convalsescent phase(8.98±5.52%)(p<0.05),and normal control(6.52±2.41%)(p<0.01).The activation level of 17 kD Caspase 3 was significantly higher in the acute phase than that in the convalsescent phase and the normal control group.The average level of Gas6 in the acute phase was 19155±1211pg/mL,which was much higher than that of convalsescent phase(10359±7761pg/mL)(p<0.01)and control group(5430±485.4 pg/mL)(p<0.001).The plasma levels of MFG-E8 in patients with the acute phase of HFRS(8953±507.6pg/mL)were significantly higher than those in the convalsescent phase(6162±433.5pg/mL)(p<0.01)and normal control group(5082±654.8pg/mL)(p<0.001).Gas6 levels in the patients with critical/severe disease(15626±1845pg/mL)were significantly higher than that of mild/moderate group(12954±943pg/mL)(p<0.05).Both of Gas6(p<0.001,r=-0.4766)and MFG-E8(p<0.001,r=-0.4573)were negatively correlated with platelet count(PLT).The frequency of Gas6receptor Tyro3 on the monocytes of HFRS patients in the acute phase of HFRS patients was 3.74 times that of the normal control group,MFG-E8 receptor integrin?v?3 and?v?5on the monocytes of HFRS patients was significantly increased in normal control group4.12,and 4.56 times respectively(p<0.05).Using in vitro THP-1 cell activation model,it was found that Gas6 and receptor Tyro3 can co-localize on the cell membrane.The phagocytosis rate in the phagocytic cell system with the addition of MFG-E8(24.12±4.23%)was increased higher than that before addition(4.78±2.69%)(p<0.01),While the phagocytosis rate was decreased after the addition of?v?3 and?v?5neutralizing antibody,and the inhibition rate was 68.5%,the neutralizing antibody of?v?3or?v?5 had a synergistic effect on the inhibition of phagocytosis.The level of CX3CL1 in plasma of HFRS were elevated in the acute phase(2.277±1.082ng/mL)compared with those in convalsescent phase(1.135±0.926ng/mL)(p<0.01)and normal control(0.6453±0.496ng/mL)(p<0.01).CX3CL1 levels in the plasma of patients with HFRS delineated into different disease severity groups,in the patients with critical/severe disease(1.969±1.278ng/mL)were significantly higher than that of mild/moderate group(1.361±0.9632ng/mL)(p<0 05),and was negatively correlated with PLT(p<0.001,r=-0.5844).The expression of CX3CR1 in THP-1 cells was 78.6%,which can be used as a subsequent cell model for the regulation of MFG-E8.Conclusions:HFRS early monocytes may be involved in the immunological mechanisms of thrombocytopenia through the phagocytic PS~+activation or apoptosis of platelets by Gas6 and MFG-E8.Our study provided novel insights into the immunological mechanisms of thrombocytopenia in HFRS and contributed important theoretical and experimental evidence to the research field.