T. pallidum remains one of human pathogens that cannot be cultivated in vitro to-date. There is also no suitable animal model for studying the pathogenesis of syphilis. These obstacles have greatly hindered the effort of elucidating the basic immunobiological traits of syphilis. T. pallidum-specific antibodies do not cause any significant change after treatment and have no detectable protective effect. Patients in the serofast state and patients with latent syphilis show about the same serological response. Serological diagnosis cannot distinguish the serofast state from latent syphilis, and the serofast state is not uncommon clinically. There is no evidence that serofast states are relevant to Treponema pallidum. Therefore, how to treat and identify serofast state is a very important issue for the prevention of syphilis. At the same time, the immune mechanism of syphilis is not clear. Several studies have shown that immunosuppression can occur after infection with T. pallidum. However, the mechanism of immune regulation is not clear.
Recently, miRNAs have been intensively studied as new biomarkers for diagnosis and prognosis in various diseases such as, cancers, heart disease, diabetes, psychosis, and infectious diseases [18-22]. Many researchers studying the expression profile of infectious diseases show that microRNAs play an important role in the host's anti-infective immune response. This study provides the first miRNA expression profile of peripheral blood samples from healthy individuals, untreated syphilis patients, patients in the serofast state, and serologically cured patients. In the present study, we used microarray analysis to assess the differential serum miRNA expression profile in syphilis patients and matched healthy controls. Among the differentially expressed microRNAs identified by microarray analysis, miR-21-5p, miR-19b-3p, miR-16-5p, and miR-142-3p were selected as candidates for further testing using RTq-PCR.
In this study, we found 89 differentially expressed miRNAs. According to the microarray analysis, forty-two miRNAs in untreated syphilis patients were up-regulated relative to healthy individuals, and sixteen miRNAs were down-regulates. Fifteen miRNAs in the serofast state were up-regulated relative to serologically cured patients, and a miRNA was down-regulated. However, we did not find the same differential miRNAs upon examining serum. There were many miRNAs expressed in untreated syphilis patients than in healthy controls, indicating that T. pallidum infection can lead to changes in immune mechanisms.
miRNAs regulate gene expression by binding to complementary site on mRNAs and reducing mRNA stability and translation . miRNAs could regulate gene expression and work as transcription factors by regulating development timing and differentiation of cells. Alteration in miRNA expression may have affected the signaling pathway. Pathway analysis helped us to assess the biological processes in immune responses of miRNAs and target genes. In this study, pathway analysis indicated that predicted target genes for those miRNAs were involved in the mucin type O-glycan biosynthesis, proteoglycans in cancer, endocytosis, adherens junction, pathways in cancer, signaling pathways regulating pluripotency of stem cells, GABAergic synapse, fatty acid biosynthesis, thyroid hormone signaling pathway, adrenergic signaling in cardiomyocytes, morphine addiction, phosphatidylinositol signaling system, arrhythmogenic right ventricular cardiomyopathy (ARVC), pancreatic cancer, glioma, Ras signaling pathway, and axon guidance pathways. Adherence-mediated colonization plays an important role in pathogenesis of microbial infections, particularly those caused by extracellular pathogens responsible for systemic diseases, such as Treponema pallidum subsp. pallidum, the agent of syphilis. Many studies have shown that outer membrane proteins of T. pallidum, such as TP0136, TP0155, and TP0483. could participate in the adhesion mechanism [24,25]. Natural immunity is the first barrier after T. pallidum infection. A variety of lipoproteins of T. pallidum activate phagocytes and dendritic cells (DCs) through CD14, toll-like receptor 1 (TLR1) and TLR2-dependent signaling pathways, and these pathogen-associated pattern molecules (PAMPs) are thought to the main pro-inflammatory factors in the process of T. pallidum infection. Special outer membrane structures of T. pallidum that lack exposed surface lipoprotein cause PAMPs to act as refractory to TLRs or other pattern recognition receptors (PRRs) of macrophages or DCs. In this way, innate immunity cannot be activated, and T. pallidum cannot be cleared by the immune system . The miRNAs of these signaling pathways may be suitable targets for research into syphilis immunomodulation.
The miRNA-gene-network helped us to screen miRNAs important to regulating immune response. In this study, miRNAs might play an important role in the regulation of syphilis immune mechanism, such as miR-195-5p and miR-223. We used RTq-PCR to verify the expression of different miRNAs, significant difference was found in the expression of three miRNAs (hsa-miR-195-5p, hsa-miR-223-3p, hsa-miR-589-3p) of PMBCs of 106 samples. First, we are interested in miR-195-5p. Most studies of miR-195-5p focus on the regulatory role of apoptosis, which could inhibit the expression of some anti-apoptotic proteins [27-30]. One recent study showed that miR-195-5p can inhibit macrophages’ proinflammatory expression . Interestingly miR-195-5p were here found to be differentially expressed between the serofast state and latent syphilis (all of early latent and late latent syphilis, data not shown). Furthermore, miR-195-5p was up-regulated after T.pallidum incubated in PBMCs of Healthy persons. These data suggest that miRNAs are associated with T.pallidum infection. In addition, miRNA-223-3p can inhibit the proinflammatory responses in Helicobacter pylori-infection macrophages. Macrophage-mediated inflammation can activate cellular immunity. We suppose that it affects the syphilis immune mechanism by affecting the apoptosis of immune cells, and it is involved in the regulation of syphilis inflammation.
The causes of the serofast state are the subject of some debate. Some studies maintain it is due to T. pallidum not being completely eliminated. However, there is no evidence that T. pallidum is latent. RT-qPCR data indicate that individual differences in serological cure are small. However, individual differences in the serofast state are more pronounced. We think this is due to the unclear definition of serofast state. Results of serological testing of the serofast state are very similar to those of latent syphilis. In this way, we find the serofast state to be a complicated problem. Studying the regulation of miRNAs on immunity can improve our understanding of the serofast state.
In summary, our results may have suggested that changes in miRNAs expression profiles may be associated with immune tolerance and T. pallidum persistence infection through regulation of target genes or signaling pathways. miRNAs that are differentially expressed among syphilis patients, such as miRNA-195-5p, could be new biomarkers for T. pallidum persistence. They might be a good candidates for investigation of the mechanism for T. pallidum clearance. Blocked or decreased expression of these miRNAs might have some role in the treatment of syphilis and management of the serofast state.