Gene Expression Profiles And MicroRNA Expression Profiles Of Peripheral Blood Mononuclear Cells From Patients With Early Syphilis And Serofast State After Therapy

Syphilis is a multistage, sexually transmitted disease caused by the obligate human pathogen Treponema pallidum subsp. pallidum(Tp). Despite the robust nature of the humoral and cellular immune responses associated with the disease, weeks to months may elapse before the host gains control of the infection. Moreover, in the absence of antibiotic treatment, containment is often incomplete and relapses are common. However, immunological determinants of host-pathogen interactions resulting in disease progression, latency or pathogen clearance remain poorly understood.On the other hand, patients with syphilis who have not achieved the desired serological response and remain serofast represent a clinical challenge. These nontreponemal antibodies sometimes persist for the lifetime of the patient. The factors that predict serological response after syphilis treatment have not been well-defined, and whether the serofast condition represents persistent Tp latent infection or variability in host immune response against Tp is unclear.During the past decade, genome-wide and functional genetic approaches(host GWAS studies, host siRNA screens, host gene expression profiling, host epigenomic profiling and host miRNA profiling) are widely used to uncover new mechanisms by which the human immune system attempts to control infection and how pathogens elude these mechanisms. PBMCs comprise several cell types such as CD4+and CD8+T lymphocytes (70%), B lymphocytes (15%), natural killer cells (10%), monocytes (5%) and dendritic cells (-1%). PBMC-based gene expression analysis have been a valuable research tools and will become increasingly useful and possibly indispensable for clinical diagnostics. MicroRNAs are small endogenously expressed RNAs that have emerged as important constituents of gene regulatory networks in the immune system. Notably, peripheral blood mononuclear cells (PBMC) gene expression profiles and miRNAs expression profiles have been useful to pinpoint genes and pathways that may be involved in the pathogenesis of infectious diseases. To date, they have not been used to analyse the molecular immune mechanisms of Tp infection and serological response after therapy.In this study we performed genome-wide gene expression profiling and microRNA expression profiling analysis of PBMCs from subjects with SS/ELS/HC, LS/SC/SF/HC to identify unique immunological network signatures of different clinical status of Tp infection and different serological response after syphilis therapy. The identified signatures will provide new insight into the immune mechanisms involved in Tp infection and serological response after therapy. The current study was divided into four parts as follows: Chapter one:Gene expression profiles of peripheral blood mononuclear cells from patients with secondary syphilis and early latent syphilisObjective To analyse gene expression profiling characteristics of PBMCs from patients with secondary syphilis and early latent syphilis and to to gain insights into the host molecular immune mechanisms involved in Treponema pallidum infection. Methods Using genome-based high-throughput RNA-sequencing technology, we comprehensively determined the transcriptional difference in PBMCs from12individuals divided into three groups:4secondary syphilis patients,4early latent syphilis patients and4healthy controls. RT-PCR of selected genes was used to validate RNA-seq data. Results When compared to healthy controls, the expression levels of78genes were differentially expressed in patients with secondary syphilis, among which16genes associated with the immune system. The up-regulation of differentially expressed genes like pro-inflammatory cytokines and related receptors, such as IL17C, IL21, IL-31receptor A (IL31RA) were identified. Also up-regulated were transcripts for the CD4+T lymphocytes activation markers CD38, B lymphocytes activation markers MS4A4A, Fc-mediated phagocytosis receptors (FCyRIA, FCyR3B), complement (C2, SERPING1). Increased expression of immune-related genes and decreased expression of type I IFN-inducible genes were found in ELS individuals compared with SS individuals. Conclusion Our results indicated that during syphilis progression, systemic innate and adaptive immune effector molecules, specifically IFN-inducible genes, are involved in the host clearance mechanism.. Chapter two:MiRNAs expression profiles of peripheral blood mononuclear cells from patients with secondary syphilis and early latent syphilisObjective To analyse miRNA expression profiling characteristics of PBMCs from patients with secondary syphilis and early syphilis and to to gain insights into the host molecular immune mechanisms involved in Treponema pallidum infection. Methods Using genome-based high-throughput Illumina sequencing technology, we comprehensively determined the miRNAs chang of PBMCs from12individuals divided into three groups:4secondary syphilis patients,4early latent syphilis patients and4healthy controls. RT-PCR of selected genes was used to validate RNA-seq data. Results When compared to healthy controls, miRNAs expression signature in PBMC from patients with secondary Syphilis:miR-125a-3p, miR-1306-5p, miR-339-5p, miR-708-5p and miR-874-3p were downregulated while miR-4433-3p and miR-4667-5p were upregulated in SS patients. Functionally immune-related miRNAs (miR-1273h、miR-1291、miR-150、miR-181a、miR-185、miR-296、miR-331、miR-361、 miR-3690、miR-532、miR-589、miR-660、miR-6735、miR-6813、miR-874、miR-92a) were differentially altered between SS subjects and ELS subjects. Conclusion Present results has identified dynamic fluctuations in the expression of miRNAs related to immunity and inflammation in PBMCs from patients during syphilis progression, indicating the role of miRNAs in this disease.Chapter three:Analysis of factors predicting the serological response after early syphilis treatmentObjective To identify the factors associated with serological response after treatment of early syphilis. Methods A total of226HIV-negative patients with treated syphilis were enrolled to conduct a retrospective study. Multivariate analyses were used to identify factors associated with a serological cure and serofast state in syphilis patients one year after therapy. Results One year after the recommended therapy,152patients achieved a serological cure, and74patients (32.4%) remained in the serofast state. The serological serofast rate increased in the order of primary, secondary, early latent, and late latent syphilis. The multivariate logistic analysis indicated that factors including female patients(OR=2.517,95%CI:1.159-5.464), non-penicillin treatment (OR=4.959,95%CI:1.650-14.901) and a baseline RPR titre≦1:16(OR=4.370,95%CI:1.311-14.562) were associated with serological serofast state. Primary or secondary syphilis and younger patients were associated with an increased likelihood of a serological cure(both P <0.05).Conclusion Serological cure or serological serofast at1year after syphilis treatment is significantly associated with syphilis stage, the grug used and baseline RPR titer.Chapter four:Gene expression profiles of peripheral blood mononuclear cells from patients with serofast state after therapyObjective To analyse gene expression profiling characteristics of PBMCs from patients with serofast state after therapy and to identify the molecular immune mechanisms implicated in serofast state pathogenesis. Methods Using genome-based high-throughput RNA-sequencing technology, we comprehensively determined the transcriptional difference in PBMCs from32 individuals divided into three groups:8serological cure syphilis patients,8serological serofast syphilis patients,8latent syphilis patients and8healthy controls. Results When compared to serological cure syphilis patients and latent syphilis patients, the expression levels of hundreds genes, IRF7and VSTM1included, were differentially expressed in patients with serological serofast syphilis. The differentially regulated genes belong to diverse functional classes and include genes involved in cytokines, signal transduction, inflammation, immune response, apoptosis. Hundreds of genes displayed significant changes in expression level between serologicalserofast male syphilis patients and serologicalserofast female syphilis patients Conclusion The obtained gene expression signature in PBMCs from different individuals indicated that these differentially expressed genes may play a role in serofast state pathology.