The Role Of Monocyte Subsets And Their Functional Changes In The Occurrence Of Syphilis

Background:Syphilis is a chronic infectious disease caused by Treponema pallidum（T.pallidum）.Syphilis is primarily transmitted through sexual contact,which can induce chronic inflammation to multiple system organs throughout the body.Previous studies showed that the host would activate immune responses to eliminate T.pallidum once infected.However,T.pallidum can escape host immune clearance in multiple ways and eventually causes a chronic infection.However,the mechanisms underlying the immune evasion have not yet been fully understood.Studies showed that T.pallidum could achieve immune evasion in several ways such as antigen alternation,virulence variation,or host immune response suppression.Monocytes（M₀）play important roles in the innate immune response,which can quickly recognize the invaded T.pallidum via its Toll-like receptor 2（TLR2）.However,T.pallidum can escape the phagocytosis and the removal by the monocyte-macrophage system.The proportion imbalance of monocyte subsets has been reported in syphilis patients,which was mainly manifested as an increase of intermediate monocytes.However,the role of this subset in the pathogenesis of syphilis is largely unknown.T cell immunoglobulin and mucin domain 3（Tim-3）,programmed cell death-receptor1（PD-1）,and programmed cell death-ligand 1（PD-L1）have been reported as three important immune regulatory molecules,which mediate the immune escape of tumor cells,autoimmune diseases and infectious diseases.Besides T cells,they are also expressed on innate immune cells,such as monocytes.The proportion imbalance of monocyte subsets and the abnormal expression of Tim-3,PD-1,and PD-L1 on monocyte may affect the function of monocytes and facilitate the immune escape of pathogens in infectious diseases.Previously studies have shown that both monocytes in the peripheral blood and macrophages in the tissue played important roles in the development of syphilis.Therefore,we infer that the proportion imbalance of monocyte subsets and the abnormal expression of Tim-3,PD-1,and PD-L1 on monocytes may contribute to the pathogenesis of syphilis.In this study,we collected the peripheral blood samples from syphilis patients with different clinical manifestations and conducted the following studies:1.To investigate the changes of the proportion of monocyte subsets and the expression levels of Tim-3 and PD-L1 in syphilis patients with different disease courses.2.To explore the effect of the imbalance of monocyte subsets on the differentiation of Tregs in syphilis patients.3.To verify the regulatory role of Tim-3 and PD-L1 on monocytes function in syphilis patients.Part 1 Association of the proportion of peripheral monocyte subsets,expression levels of Tim-3 and PD-L1 with different clinical stages of syphilis patientsObjectives:Syphilis with diverse clinical manifestations is a chronic sexually transmitted disease caused by T.pallidum.Studies have shown that the monocyte-macrophage system plays an important role in the clearness of T.pallidum.This part aims to explore the role of frequency alternation in circulating monocyte subsets,the expression levels of Tim-3,PD-L1,and HLA-DR,and functional changes of monocytes in the pathogenesis of syphilis.Methods:Peripheral blood samples from 35 healthy controls and 189 syphilis patients（35 secondary syphilis,30 latent syphilis,51 serofast,34 asymptomatic neurosyphilis,and 39 symptomatic neurosyphilis）were collected.12 cases of neurosyphilis were followed up before and after treatment.In addition,15 cases of patients with syphilis whose serum TRUST titer turned negative after treatment were collected.Flow cytometry was used to detect the distribution of circulating monocyte subsets,and the expression levels of Tim-3,PD-L1,PD-1 and HLA-DR on monocytes and their subsets.The expression and distribution of IL-10 and TGF-βin circulating monocytes and their subsets from syphilis patients were also detected by flow cytometry.Results:Flow cytometry analysis showed that the proportion of CD14⁺⁺CD16^-（classical）subset decreased and the proportion of CD14⁺⁺CD16⁺（intermediate）monocytes increased in syphilis groups except for the serofast group,especially in secondary syphilis and the symptomatic neurosyphilis groups（p<0.001）.The proportion of the intermediate monocytes decreased after two treatments in 12 follow-up patients with neurosyphilis.There was no significant difference in the frequency of intermediate monocytes in patients whose serum TRUST titer was negative after treatment.The expression of Tim-3 was decreased and PD-L1 was increased in secondary syphilis,latent syphilis and neurosyphilis groups.Compared with control,there was no significant difference in the expression of PD-1.The further analyses of Tim-3 and PD-L1 on each subset showed that the expression of Tim-3 decreased in three subsets.But no significant difference was found for PD-L1 on other two subsets compared with the control group（p>0.05）except on classical monocytes in patients with secondary syphilis.The expression of HLA-DR on monocytes and three subsets from syphilis patients showed that HLA-DR were down-regulated in classical and intermediate subsets from symptomatic neurosyphilis group.Furthermore,we found that the levels of TGF-βand IL-10 of in monocytes from syphilis patients were increased.IL-10 was mainly secreted by the classical monocytes,while TGF-βwas primarily secreted by the intermediate monocytes.The correlation analysis showed that the frequency of intermediate monocytes is positively correlated with serum TRUST titer（r=0.328,p<0.001）.Conclusions:1.The imbalance of monocyte subsets in patients with syphilis was manifested as the decreased proportion of CD14⁺⁺CD16^-monocytes and the increased proportion of CD14⁺⁺CD16⁺monocytes,especially in secondary syphilis and neurosyphilis.Furthermore,the proportion of intermediate subset was positively correlated with serum TRUST titer,suggesting that the increased proportion of the intermediate subgroup might be related to the activity of syphilis.2.The expression of Tim-3 was down-regulated on monocytes,especially on the classic and intermediate subsets from secondary syphilis and neurosyphilis groups,and PD-L1 was increased on monocytes,suggesting that T.pallidum may change the function of monocytes by regulating the expression of Tim-3 and PD-L1.3.The HLA-DR on the classical and intermediate monocytes was down-regulated in neurosyphilis,which might involve the occurrence of neurosyphilis.Part 2 The influence of the proportion imbalance of monocyte subsets on Tregs differentiation in syphilis patientsObjectives: Regulatory T cells（Tregs）are a specialized subpopulation of T cells that act to suppress immune response.Studies have reported that monocyte subsets can participate in the regulation of Tregs cell differentiation by secreting cytokines.This part aims to investigate the effect of the imbalance of monocyte subsets on the Tregs and other T cells subsets differentiation caused by T.pallidum infection.Methods: Blood samples from 13 healthy subjects and 3 cases of secondary syphilis patients were collected.Tp47（T.pallidum extracorporeal membrane protein 47）was used to stimulate PBMC from healthy donors and then the distribution of monocyte subsets was detected by flow cytometry.Besides,the levels of IL-1β,TNF-α,IL-6,IL-8,IL-10,IL-12,IFN-γ and TGF-β were detected by LEGEND plex multiple factor kit and ELISA.The effect of monocytes stimulated by Tp47 on Tregs differentiation was detected by flow cytometry after co-culture with lymphocytes.The intermediate subset from the secondary syphilis and naive T cells from healthy controls were isolated,and then cultured them together.The cytokines in culture supernatant were detected by LEGEND plex multiple factor kit and ELISA.Results: In vitro,CD14++CD16+ monocytes were increased when PBMC were stimulated by Tp47.Tp47 could promote monocytes to secrete cytokines including IL-1β,TNF-α,IL-6,IL-8,IL-10 and TGF-β,and inhibit the secretion of IFN-γ.The differentiation of Tregs was induced by monocytes stimulated by Tp47.The intermediate monocytes isolated from secondary syphilis could induce T cells to differentiate into Tregs and Th2 subgroups.Conclusions: T.pallidum infection could cause the imbalance of monocyte subsets and increase the secretion of anti-inflammatory factors including IL-10 and TGF-β,which may promote the differentiation of Tregs.In addition,the intermediate monocytes could facilitate Th2 subgroup differentiation,except for Tregs.Part 3 Tim-3 and PD-L1 regulate the function of monocytes in patients with syphilisObjectives: Monocytes（M0）are one of the important components of host defense system and the abnormal expression Tim-3,PD-1 and PD-L1 on monocytes and their subgroups could affect the function of monocytes and participate in immune evasion.This part aims to explore the expression changes of Tim-3,PD-1 and PD-L1 on monocytes and their effect on the function of monocytes during T.pallidum infection.Methods: Blood samples were collected from 12 healthy controls,70 syphilis patients（14 secondary syphilis,14 latent syphilis,14 serofast,14 asymptomatic neurosyphilis and 14 symptomatic neurosyphilis）,10 neurosyphilis before and after treatment.The phagocytosis of monocytes was evaluated by detecting their ability to phagocytose E.coil particles.The effects of blocking Tim-3 and PD-L1 on the phagocytosis of monocytes in syphilis patients were detected by flow cytometry.In vitro,peripheral blood monocytes from healthy controls were stimulated with different concentrations of Tp47,and the expression of Tim-3,PD-1 and PD-L1 were detected by flow cytometry.After stimulation with Tp47,the changes in phagocytosis capacity of monocytes were detected by flow cytometry.After Tim-3 and PD-L1 were blocked by the neutralizing antibodies,phagocytosis of these monocytes and the expression of above cytokines in the supernatant were detected.When monocytes were stimulated by Tp47 and blocked Tim-3 and PD-L1 with neutralizing antibodies,the changes of HLADR and co-stimulatory molecules CD40,CD80 and CD86 on monocytes were investigated.Furthermore,the effect of Tp47 on lymphocytes proliferation was detected by CFSE labeling.The expression of Tim-3 on macrophages derived from monocytes stimulated by T.pallidum was detected by Western Blot,and the effect of blocking the Tim-3 pathway on the phagocytic function of macrophages was detected by immunofluorescence.Results: 1.Compared with the control group,the phagocytosis of monocytes from secondary syphilis,latent syphilis,asymptomatic neurosyphilis and symptomatic neurosyphilis groups were decreased（p<0.05）.In addition,the phagocytosis of monocytes was increased after treatment.2.After blocking the Tim-3 pathway,the phagocytic function of monocytes was further decreased（p<0.05）.However,the blocking of PD-L1 by neutralizing antibody had no significant effect on the phagocytic function of monocytes（p>0.05）.3.After Tp47 stimulated and blocked Tim-3 on monocytes,IL-1β,TNF-α,IL-6 and IL-10 secretion increased.After blockade of PDL1 on monocytes,IL-1β,TNF-α,IL-6,IL-10 and TGF-β secretion decreased,especially IL-10.4.Tp47 could up-regulate the expression levels of CD40 and CD80 and downregulate the expression levels of CD86 on monocytes.5.After blocking the Tim-3 pathway,the expression levels of CD40 and CD80 in monocytes were increased（p<0.05）,and the expression level of CD86 was lower than that of the control group（p<0.05）.However,the expression levels of CD40 and CD80 were decreased after blocking PD-L1,and no significant change was found for CD86.6.The effect of blocking the Tim-3 and PD-L1 pathways on the expression of HLA-DR receptor on monocytes was no significant.7.Lymphocyte proliferation experiment showed that the proliferation ability of lymphocytes was decreased after blocking monocyte Tim-3 receptor（p<0.01）,while the proliferation ability of lymphocytes was promoted by blocking PD-L1（p<0.01）.T.pallidum could down-regulate the expression of Tim-3 on macrophages derived from monocytes,and the ability of macrophages to phagocytose T.pallidum might be inhibited by blocking Tim-3.Conclusions: The phagocytic capacity of circulating monocytes from syphilis patients was decreased,and the phagocytic capacity was further decreased after blocking Tim-3.The secretion of inflammatory factors could be affected by blocking the pathways of Tim-3 and PD-L1 on monocytes,especially IL-10.The down-regulation of Tim-3 and the increased expression of PD-L1 both promoted the secretion of IL-10.In syphilis patients,especially in secondary syphilis and neurosyphilis,the down-regulated expression of Tim-3 and the increased expression of PD-L1 could inhibit the proliferation of lymphocytes,cause the abnormal phagocytosis of monocytes and secrete anti-inflammation cytokines primarily.The down-regulation of Tim-3 and upregulation of PD-L1 might be related to the immune escape of T.pallidum and the chronic infection of the disease.