Preliminary Study On The Role Of Low-Density Granulocytes In The Immunopathobiology Of Syphilis

BackgroundSyphilis is a chronic,multi-system damaged,sexually transmitted disease caused by Treponema pallidum（Tp）.In recent years,the incidence of syphilis has been increased worldwide,and ranked the top three of infectious diseases in China.Studies have shown that Tp infection can induce innate and adaptive immune responses,which influence on the progression and outcome of syphilis.As one of the most important innate immune cells,neutrophils play a significant role in Tp clearance.Neutrophils used to be considered as a relatively homogeneous groups of terminally differentiated cells.However,recent researches in neutrophil have challenged this limitation,it is found that neutrophils are heterogeneous cells which contain different subpopulations,and there are differences in morphology,phenotype and function among each subgroup.Among them,the most studied and breakthrough neutrophil subsets in the past decade are Low-density granulocytes（LDGs）.Studies have shown that,LDGs play an important role in the progression of many diseases,such as autoimmune diseases,acute and chronic infectious diseases and tumors.Of note,the proportion of LDGs in peripheral blood mononuclear cells（PBMCs）in AIDS and tuberculosis patients was not only higher than that in healthy controls,but also positively correlated with the severity of the disease.It was found that LDGs can secrete higher levels of pro-inflammatory cytokines than Normal-density granulocytes（NDGs）,which can induce endothelial injury and aggravate tissue damage.Moreover,LDGs can significantly up-regulate the expression of I-IFN genes,and can drive their own function and affect the function of other immune cells by producing key cytokines,such as IFN-γ and IL-10.However,the role of LDGs in the pathogenesis of syphilis has never been reported.Therefore,preliminary explore the role of LDGs in the pathogenesis of syphilis will help to deepen the understanding of the role and function of LDGs and provide new directions for the research of LDGs in syphilis.Part 1Objectives To understand the proportion of LDGs in PBMCs of patients with different stages of syphilis and its correlation with the severity of syphilis.Methods The samples of peripheral blood were collected from177 syphilis patients and 40 healthy volunteers,among which P＆S-S（primary and secondary syphilis）（n=26）;LS（latent syphilis）（n=21）;Serofast（n=55）;ANS（asymptomatic neurosyphilis）（n=31）;SNS（symptomatic neurosyphilis）（n=44）;Non-neurosyphilis group（n=102,Non-NS group,including P＆S-S,LSand Serofast group）,Neurosyphilis（n=75,NS group,including both ANS and SNS group）.Flow cytometry（FCM）was used to detect the proportion of LDGs in PBMCs in above syphilis patients and healthy controls.Results The proportion of LDGs in PBMCs in each stage of syphilis patients（P＆S-S,LS group,ANS group,SNS group and Serofast group）was significantly higher than that in HC group,and the proportion of LDGs in SNS group was the highest,and there were statistically significant difference between groups.And the proportion of LDGs in PBMCs in SNS group was higher than that in P＆S-S group（p=0.0353）.What’s more,the proportion of LDGs in PBMCs in NS group which with severe condition was higher than that of Non-NS group,but the difference was not statistically significant.We found that the proportion of LDGs in PBMCs was correlated with CSF abnormity,which were positively correlated with CSF VDRL titer（r =0.22,p=0.003）,CSF WBC counts（r=0.247,p=0.001）,and CSF protein concentration（r=0.238,p =0.001）.Conclusions 1.The proportion of LDGs in PBMCs in syphilis patients was significantly higher than that in HC group,and SNS group was the highest;2.The proportion of LDGs in PBMCs level was positively correlated with indicators of cerebrospinal fluid abnormalities（including VDRL titer,WBC counts,and protein concentration）in neurosyphilis patients.Part 2Objectives To observe the phagocytosis of LDGs in patients with different stages of syphilis,and further explore the effect of TP stimulation on the phagocytosis of LDGs by TP stimulation experiment in vitro.Methods Isolation and purification of LDGs and Normal-density granulocytes（NDGs）from peripheral blood of syphilis patients at different stages（the number of Secondary syphilis（SS）,SNS and Serofast was 5,5,6 respectively）and healthy volunteers（n=8）,and then co-incubated with E.coil particles;LDGs were pre-stimulated by TP with MOI of 10 and 20,and the stimulated LDGs were co-incubated with E.coil particles.The mean fluorescence intensity（MFI）of LDGs/NDGs engulfing E.coil particles was detected by FCM,and the difference of MFI in each group was compared,to analyze the phagocytosis ability of LDGs/NDGs.Results 1)Compared with HC group,the phagocytosis of LDGs in syphilis patients in each group was generally weaker;Compared with SS group and SNS group,the phagocytosis ability of LDGs in Serofast group was significantly decreased.2)Compared with autologous NDGs,the phagocytosis of LDGs in SNS group and Serofast group was decreased.There was no significant difference in the phagocytosis between LDGs and autologous NDGs in SS group.3)Compared with HC LNR（Ratio of LDGs phagocytosis MFI to NDGs）,patients with syphilis（SS,SNS and Serofast）were significantly reduced（p<0.001）.The comparison results among syphilis groups showed significantly higher LNR in SS group compared with SNS and Serofast（p value was 0.0015,<0.001respectively）.There was no significant difference between SNS group and Serofast group（p>0.05）.4)The changes of phagocytosis of LDGs stimulated by Tp in vitro: compared with the control group,the phagocytosis of LDGs in the TP stimulated group was decreased.Conclusions 1.LDGs phagocytosis in syphilis patients was lower than that in healthy controls,TP infection has a certain inhibitory effect on the phagocytosis of LDGs.2..LDGs phagocytosis of patients with symptomatic neurosyphilis and Serofast syphilis were lower than autologous NDGs.Part 3Objectives To explore the effect of LDGs on the phagocytosis of Mac in engulfing Tp,and its role in the secretion of cytokines of Mac.Methods Tp was cocultured with Mac（Mac group）at MOI = 25.In Mac group,LDGs（Mac+LDGs group）or NDGs（Mac+NDGs group）were added at the ratio of Mac: LDGs（or NDGs）of 1:1 to establish the coculture system.The phagocytosis of Mac on Tp was detected by immunofluorescence microscopy,and the phagocytic rate and phagocytic index were used to reflect the changes of phagocytic function of Mac.FCM was used to detect the levels of cytokines in the supernatant of the coculture system,and the differences among the groups were compared and analyzed.Results 1)Comparison of the function of engulfing Tp in different groups: Compared with Mac group,the phagocytic rate of Mac in Mac+LDGs group was significantly higher.and compared with Mac+NDGs group,the phagocytic rate and phagocytic index of Mac in MAC+LDGs group were significantly higher.There was no significant difference in phagocytic rate and phagocytic index between Mac group and Mac+NDGs group.2)Cytokines in supernatant of co-culture system: Compared with Mac group and Mac+LDGs group,the levels of CXCL10 and IL-8 in Mac+LDGs group were significantly increased.The level of IL-6 in Mac+LDGs group was higher than that in Mac+LDGs group.And the level of IL-1 β in Mac+LDGs group was higher than that in Mac group.There was no significant difference in cytokine secretion between Mac group and Mac+ NDGs group.Conclusions LDGs,but not NDGs,promoted the phagocytosis of TP in Mac,and promoted the secretion of pro-inflammatory cytokine IL-6,IL-1β,IFN-γ and chemokine CXCL10,IL-8 in Mac.