Host Response To Treponema Pallidum In Neurosyphilis Patients

Background The damage on nervous system caused by Treponema pallidum(Tp)infection is irreversible.The incidence of syphilis and neurosyphilis has undergone increase every year since 21St century.Neurosyphilis can be developed in any stage of syphilis.Currently,the host response caused by Tp still remains unknown.Recently,many IncRNAs were found to be involved in the regulation of immune response triggered by pathogens.Up to date,there is no study published about if IncRNAs in CD4+T cells can function in Tp infection,and if IncRNA expression profile would be changed.Objective To analyze host response to Tp in neurosyphilis patients and analyze the IncRNA expression profile of neurosyphilis patients,to provide an idea to screen valuable biomarkers for identifying neurosyphilis and syphilis non-neurosyphilis,to screen IncRNAs which can render impact on CD4+T cells,and to gain insight on their underlying mechanism.Methods1.The clinical characteristics and laboratory parameters were analyzed by employing flow cytometry,enzyme-linked immunosorbent assay,metabolomics analysis to assess host responses induced by Tp in neurosyphilis patients.Host responses included cellular immune response,humoral immune response,and metabolic changes.2.Neurosyphilis patients were subjected to lncRNA and mRNA expression profile analysis by using Arraystar Human LncRNA Microarray V4.0.Based on the lncRNA expression profile,target genes were identified.Over-expressed target gene of lentivirus vector was prepared and co-cultured with Jurkat cells to analyze the changes in these cells.Chromatin fractionation was performed to identify the location of target gene in cells.RNA-pull down combined with MS was conducted to obtain the protein profile which can be interact with target lncRNA and to further identify the involved proteins.RIP experiment was conduct to further confirm if these proteins can be combined with target lncRNA.Results1.Host responses induced by Tp in neurosyphilis patients(1)Humoral immune response in neurosyphilis patientsThe abnormal humoral immunity was observed on neurosyphilis patients.The content of CXCL13 secreted by peripheral blood B cells was significantly higher than that of peripheral blood of patients with syphilis non-neurosyphilis and healthy controls(P=0.084,P<0.001).The CXCL13 quotient(QCXCL13)was also significantly higher than syphilis/non-neurosyphilis patients(P=0.002)and non-syphilitic patients without neurological infection(P＝0.003).The area under curve of ROC by cerebrospinal fluid(CSF)CXCL13 and QCXCL13 were 0.831 and 0.758,respectively;When the cut-off value was set to 4.871 pg/mL and 2.408 pg/mL,the sensitivity for distinguishing neurosyphilis patients and syphilis patients were 80.0%and 87.5%;the specificity for distinguishing neurosyphilis patients and syphilis patients were 81.4%and 69.2%,respectively.In addition,IL-17 and IFN-y in CSF of neurosyphilis patients were significantly higher than those with syphilis/non-neurosyphilis patients(P<0.05).(2)Cellular immune response in neurosyphilis patientsThe abnormal cell immunity was observed on neurosyphilis patients.CD3+CD4+T cell counts and CD3+CD4+/CD3+CD8+ratio in blood of neurosyphilis patients were significantly higher than syphilis/non-neurosyphilis patients;NK cell counts were significantly lower than syphilis/non-neurosyphilis patients.The content of Th2 cells and Th9 cells in CD4+T cell subsets in blood of neurosyphilis patients were significantly lower than that in syphilis/non-neurosyphilis patients(4.7%vs.9.0%,P=0.002;0.5%vs.1.0%,P=0.038);CD8+IFN-γ+cells in CD8+T cell subsets in blood of neurosyphilis patients were significantly higher than syphilis patients(P=0.049),and on the contrary,CD8+IL4+cells and CD8+IL17+cells were significantly lower in neurosyphilis patients when comparing with syphilis patients.Both RPR titer and TPPA titer in CSF were inversely correlated with CD8+IL17+cells.(3)Metabolite changes in neurosyphilis patientsMetabolic abnormalities were found by performed metabolomics analysis in neurosyphilis patients.The L-gulono-gamma-lactone,D-mannose,and hypoxanthine levels were significantly decreased in neurosyphilis patients compared with syphilis patients with fold changes of 0.517,0.872,and 0.745(P<0.05),respectively.In contrast,an 87.4-fold change in N-acetyl-L-tyrosine levels was observed in neurosyphilis patients compared with syphilis patients(P<0.05),and an approximately 7.5 times increase was found in neurosyphilis patients compared to patients without syphilis(0.05<P<0.10),which is a potential diagnostic indicator.The KEGG pathway analysis showed seven metabolic pathways that were enriched in neurosyphilis patients compared to syphilis patients;these pathways included tryptophan metabolism,biosynthesis of unsaturated fatty acids,fatty acid biosynthesis,lysosome,ABC transporters,fructose and mannose metabolism,and galactose metabolism.2.LncRNA expression profiles and functions of CD4+T lymphocytes in neurosyphilis patients.The lncRNA microarray analysis showed that 393 lncRNAs and 287 mRNA were significantly up-regulated,whereas 287 lncRNAs and 331 mRNA were significantly down-regulated(fold change>1.5,P<0.05)in the neurosyphilis subjects;They can be further classified into six classes:bidirectional lncRNAs,exon sense-overlapping lncRNAs,intergenic lncRNAs,intron sense-overlapping lncRNAs,intronic antisense lncRNAs,and natural antisense lncRNAs.The most striking finding in the analysis was the significantly altered expression of adjacent mRNA by 59 differentially expressed lncRNAs.After GO and KEGG analysis,lncRNA ENST00000421645 was selected for further analysis.A Gene Set Enrichment Analysis(GSEA)was conducted and revealed that samples with high expression of lncRNA ENST00000421645 has enriched in following terms:cell cycle,ubiquitin mediated proteolysis,slicing,T-cell receptor signaling pathway.Cell localization showed that lncRNA ENST00000421645 was located in the cytoplasma of Jurkat cells.Owing to the up-regulation on lncRNA ENST00000421645 in CD4+T cells of blood collected from neurosyphilis patients,we constructed an over-expressed lncRNA ENST00000421645 lentivirus vector to transfect Jurkat cells,and found it can improve the IFN-y secretion in Jurkat cells.IFN-γ mRNA in Lv-1645 cells was 2.5 times higher than that of Lv-vector cells,and IFN-γ protein level was 3.7 times higher in Lv-1645 cells.By using MS identification and analysis on the RNA pull down product of lncRNA ENST00000421645,155 proteins were differentially expressed.By combining MS and RNA pull down technique,we found that lncRNA ENST00000421645 can be bound with PCM-1 protein.Conclusions1.The response to Tp in neurosyphilis patients include include the following content.(1)Abnormal humoral immunity was found in neurosyphilis patients due to invasion of Tp.Cerebrospinal fluid CXCL13,IL-17 and IFN-y are involved in the humoral immune response in neurosyphilis patients.The CSF CXCL13 and QCXCL13 could serve as valuable biomarkers for differentiating neurosyphilis from non-neurosyphilis/syphilis in HIV-negative patients.(2)Abnormal cellular immunity was found in neurosyphilis patients due to invasion of Tp.The main contributing cells are CD3+CD4+T cells and CD8+IFN-γ+cells which participate in the pathogenesis of neurosyphilis.(3)Metabolic abnormalities were found in neurosyphilis patients due to invasion of Tp.This study was the first to identify significant differential metabolites between neurosyphilis patients and non-neurosyphilis/syphilis patients,N-acetyl-L-tyrosine was the most significant increases in neurosyphilis patients which may be used as differential diagnostic indicators for neurosyphilis which can improve the diagnosis of neurosyphilis.Differentially expressed metabolites are enriched into some overlapping pathways,the most important of which is the ABC transporter;most overlapping pathways may be associated with alternative carbon sources for Tp.Metabolites with significant differences;its potential role in the development of neurosyphilis deserves further exploration.2.The present study acquired the lncRNA and mRNA expression profile from CD4+T cells of neurosyphilis patients which were different from healthy controls.Differentially expressed lncRNAs were involved in the biological processes which are triggered by Tp in CD4+T cells.LncRNA ENST00000421645 located in the cytoplasm of CD4+T cells,and binded to the PCM-1 protein.Protein binding may promote an increase in IFN-y secretion by regulating PCM-1 protein which participate in humoral and cellular immune responses induced by Tp.This topic provides a new perspective for the study of the mechanism of neurosyphilis,and provides new ideas for its research and treatment.