The Feature And Function Of Low-density Neutrophils In Severe Fever With Thrombocytopenia Syndrome

Objective: Severe fever with thrombocytopenia syndrome(SFTS),an emerging infectious disease with a high case-fatality rate,while forming SFTS “inflammation storm”,which caused by a novel bunyavirus named SFTS virus(SFTSV).The number of leukocyte,especially neutrophils were often significantly decreased in SFTS patients.Neutrophils are the first immune cell population recruited to site of pathogenic challenge,which also play an important role in the antiviral immune response.Accumulating evidence supports the existence of distinct neutrophil subsets,low density neutrophils(LDNs)and normal density neutrophils(NDNs).Studies have found that LDNs and NDNs have significantly different morphology and function.The obvious abnormality of SFTS neutrophils is worth investigating.This study is intended to explore the feature,the source of LDNs in SFTS patients and their functional role in the development of SFTS diesease.Methods: The comfirmed SFTS samples were recruited from the department of infectious diseases of Union Hospital,Tongji Medical College,Wuhan.Meanwhile,healthy control subjects were also collected.The proportion and phenotype of LDNs and NDNs were detected in SFTS patients and the healthy control subjects by flow cytometry.SFTS viral load in LDNs,NDNs and serum were assayed by Real-time PCR.The level of IL-17,G-CSF and IL-6,IL-8,IL-10,TNF-α in SFTS patients and healthy control subjects were detected by ELISA kit and Human Cytometric Bead Array(CBA)kit respectively.LDNs,NDNs from SFTS and control neutrophils were cultured in vitro,The concentration of the cytokines(IL-6、IL-8、IL-10、TNF-α)in the supernatants were quantified using CBA Kit.The ability of healthy normal neutrophils(NDNs)to transform into LDNs under SFTS conditions was assessed by flow cytometry.Results: 1.We examined the proportion of LDNs and NDNs in peripheral blood of SFTS patients and healthy subjects.The percentage of LDNs in the PBMCs of SFTS patients was significantly elevated compared to that of normal controls(46.80±14.07% vs 2.63±1.42%,P<0.001).Moreover,the percentage of LDNs increased dramatically to more than 50% in some SFTS patients.But there was no significant difference between the percentage of NDNs and that of normal controls(73.11±21.39 % vs 82.07±9.98 %,P>0.05).2.CD11 b and CD66 b are known as markers of neutrophil activation.We observed that the SFTS-LDNs highly expressed CD11 b,CD66b.The MFI level of CD11b(P<0.05),CD66b(P<0.001)in LDNs were higher than that in NDNs form the SFTS.Moreover,the MFI levels of CD11b(P<0.01)and CD66b(P<0.001)in SFTS-LDNs were significantly higher than that of normal controls.The MFI of CD66b(P<0.001)in SFTS-NDNs was higher than that of normal controls,but there was no significant difference between the MFI of CD11b(P>0.05)in SFTS-NDNs and that of normal controls.3.We explored the source of increased LDNs in SFTS patients.NDNs from healthy controls were incubated in the SFTS serum for different times,we found that the percentage of LDNs in PBMCs displayed the increased tendency with the cultured time dependent,reached almost 90% at 48 h,suggesting that normal NDNs are capable of switching to LDNs.4.We analyzed the correlation between the abnormally high percentage of LDNs in SFTS patients and the SFTSV load in serum.We found that there was no correlation between SFTSV load and percentage of NDNs(P>0.05).But we note that the SFTSV load correlates well with propagation of LDNs(r=0.629,p=0.003).Based on this,we therefore explored the SFTSV loads in LDNs and NDNs from the same SFTS patients.The SFTSV loads in LDNs and NDNs from each patients showed diversity.In some SFTS patients,the SFTSV load was higher in LDNs than that in NDNs,but other patients SFTSV load was lower in LDNs than that in NDNs from the same patients.Combined with the clinical data,we found in patients with SFTSV load in LDNs higher than that in NDNs,the platelet(PLT)decreased(26.71±3.59 vs 73.09±25.89,P=0.002,partial thromboplastin time(APTT)was prolonged(96.26±37.44 vs 46.32±13.87,P=0.001),lactate dehydrogenase(LDH)increased(3722.86±2928.53 vs 625.82±202.1,P=0.004),compared to patients with low SFTSV load in LDNs.In addition,in the group of LDNs with high SFTSV load,60% of patients died.Those patients with low SFTSV load in LDNs were all healed.5.The content of G-CSF in SFTS patients were obviously increased compared to that in control groups(p<0.001).We also found that there was a significant correlation between the level of G-CSF and the percentage of LDNs in SFTS patients(r=0.899,p<0.001).In addition,IL-17 levels in SFTS patients were significantly increased compared to that in control groups(p<0.001).And we observed that there was a positive correlation between the level of IL-17 and the percentage of LDNs in SFTS patients(r=0.813,p<0.01).6.We found that cytokines IL-6(P<0.001)、IL-8(P<0.001)、IL-10(P<0.001)were significantly higher in SFTS patients than that of normal controls.We also found that LDNs secreted high level of IL-6(P<0.001)、IL-8(P<0.001)、TNF-α(P<0.001) than that of NDNs.Conclusion: 1.The high percentage of LDNs in SFTS patients may be a marker of the severity of SFTS.2.SFTS-LDNs and SFTS-NDNs have the same cell phenotype,but SFTS-LDNs expressed a more active neutrophil phenotype than that of SFTS-NDNs.3.NDNs are capable of switching to LDNs in “SFTS-dependent fashion”,and this may become one source of increased percentage of LDNs in PBMCs of SFTS patients.Abnormal high levels of IL-17 and G-CSF in SFTS patients were able to stimulate the release of early immature neutrophils,and could also be an additional source of high precentage of LDNs in peripheral blood PBMCs of patients with SFTS.4.The SFTSV loads of LDNs were much higher than NDNs from severe SFTS patients,the SFTSV loads of LDNs were lower than NDNs from moderated SFTS patients.The level of viral load in LDNs can also be used as an indicator of the severity and prognosis of SFTS.5.The LDNs could secreted increased levels of pro-inflammatory cytokines and have more important role in the formation of SFTS “inflammation storm”.LDNs may play important roles in the severity and prognosis of SFTS.