Cytokines in cerebrospinal fluid and serum as biomarkers are associated with the diagnosis and efficacy evaluation of neurosyphilis

Cytokines in cerebrospinal fluid and serum as biomarkers are associated with the diagnosis and efficacy evaluation of neurosyphilis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cytokines in cerebrospinal fluid and serum as biomarkers are associated with the diagnosis and efficacy evaluation of neurosyphilis Xiao Ying Zhang, Meiduo Gesang, Sha Wu, Jie Fang Chen, Zheng Wang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1562299/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Neurosyphilis remains a chronic infectious disease within central nervous system infected by Treponema pallidum (TP). At present, the diagnosis and efficacy evaluation of neurosyphilis still facing clinical challenges. It has been reported that cytokines, which play multiple roles in neuroinflammation, showed abnormal expression patterns in neurosyphilis. To search for potential biomarkers of neurosyphilis, we analyzed the changes of cytokine profiles in cerebrospinal fluid (CSF) and serum of neurosyphilis. This may provide insight into clinical values for neurosyphilis. Methods Using the quantitative antibody array platform, we measured 80 cytokines expression in the CSF and serum of 16 subjects. 9 patients diagnosed with neurosyphilis, included samples before and after antibiotic treatment. 7 patients with non-inflammatory neurological diseases (NIND) were used as controls. Compared to the differences in cytokine expression between groups and analyzed to the correlations between cytokines and clinical CSF parameters. The ability of valuable biomarkers to diagnosis of neurosyphilis was evaluated by receiver operating characteristic (ROC) curves. Results We observed significantly increased of multiple cytokines (26/80) in CSF of neurosyphilis were significantly increased compared to the NIND group. Concentrations of 15 cytokines were significantly decreased after treatment, of which 11 cytokines were down regulated to the levels were similar to the NIND group. Most CSF cytokine concentrations consistently correlated with clinical parameters, positively related to white blood cells (WBCs, 42/80), total protein (36/80), and albumin quotient (Qalb, 37/80) and negatively related to Glucose level (22/80). More than 40 cytokines concentrations correlated with each other in CSF, most of them were positively correlated. But correlations between CSF cytokine and serum cytokine values were scarce (6/80). The area under the ROC curve (AUC) of G-CSF, ICAM-1, CCL4, CCL19 and IL-12p40 in CSF were all greater than 0.9, with high diagnostic values. Conclusions Our data showed that multiple CSF cytokines were highly expressed of neurosyphilis, and their levels were significantly reduced after antibiotic treatment. Meanwhile, these cytokines were significantly correlated with various CSF parameters. The AUCs of G-CSF, ICAM-1, CCL4, CCL19 and IL-12p40 were all greater than 0.9, suggesting that these cytokines may serve as potential biomarkers for neurosyphilis. Unfortunately, more meaningful results were not observed in the analysis at the serum level. Therefore, along with previous related reports, it is believed that CSF measures are irreplaceable in the current clinical diagnosis, and the combined dynamic detection of multiple cytokines such as G-CSF, ICAM-1, CCL4, CCL19 and IL-12p40 in CSF can serve as a supplementary diagnostic tool for neurosyphilis. Cytokines Chemokine Neurosyphilis Neuro-inflammation Cerebrospinal fluid. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Syphilis is a chronic and systemic sexually transmitted disease caused by TP infection 1 . TP invades the circulatory system and produces an effect on all tissues and organs of the whole body. In severe cases, it may lead to multi-system organic lesions and irreversible damage 2 . Neurosyphilis is TP disseminates to the central nervous system (CNS), causes pathological injury in related brain areas, and results in a series of serious consequences 3 . Most syphilis patients without standardized treatment may develop into neurosyphilis eventually 4 . Neurosyphilis is characterized by complex and diverse clinical manifestations. According to the invasion degree of TP, neurosyphilis can be divided into the early-stage type (only involving meninges and blood vessels) and the late-stage type (involving brain and spinal cord parenchyma) 5 . There is a difference in TP concentrations in peripheral blood and CSF of neurosyphilis patients. Moreover, there may be false-negative results in routine antibody tests 6 7 . In a few patients, syphilis is not even detected in cerebrospinal fluid, with the detection of inflammatory reactions merely, such as increased WBCs and total protein content, etc 5 . These phenomena may also occur in many central nervous system infections and autoimmune diseases 2 . There may be insensitivity to antibiotics in the treatment of neurosyphilis 8 . There exists a difference in the therapeutic effects in patients with different autoimmune statuses and course of the disease. In general, it is considered that long-term alcohol consumption, drug abuse, hypertension, HIV complicated with syphilis are all influential factors of the therapeutic effects 9 . Cytokines are small molecular proteins secreted by cells with extensive biological activities, which can regulate cell proliferation, differentiation and immune response by binding to receptors. After infection with TP, a strong immune response may be triggered in vivo, which may activate relevant immune cells to release a large amount of cytokines. 9 – 11 , The immune response is of great significance for the clearance of TP, which, however, may also induce a local inflammatory response and result in pathological damage 12 . The intensity and type of immune response may have an impact on the severity of syphilis, cellular and humoral immune responses are both involved in the damage process 10 . Without timely treatment, there may be irreversible organic lesions in the brain of patients. Serious inflammatory damage has been detected in the brain tissue of patients with paralytic dementia and gumma 13 . At present, researchers are highly concerned about the expression changes of related cytokines during the exploration of the immune response mechanism of syphilis. Maciej et al. (2013) found that IL-17A and IFN-γ in cerebrospinal fluid of asymptomatic neurosyphilis patients were higher than those of common syphilis patients 14 . Wang et al. (2014) detected the increase of Th17 cells in the cerebrospinal fluid of patients with neurosyphilis, accompanied by an increase in the expression level of IL-17 secreted by Th17 cells as well 15 . Furthermore, Li et al. (2020) found an increase in the expression of IL-10 in the cerebrospinal fluid of neurosyphilis patients with mental symptoms 16 . In the same year, Di et al. (2020) reported lower expression levels of IL-17, TNF-RI, TNF-RII, IL-16, OPN and MCSFR in cerebrospinal fluid of patients with syphilitic serofast state before treatment, yet with higher CCL7, LIF, G-CSF, CCL20 and GH were higher 17 . The immune response is abnormally activated in the cerebrospinal fluid of patients with neurosyphilis. Related cytokines are involved in the inflammatory reaction and progression of neurosyphilis, which has great potential to be novel biomarkers of neurosyphilis. However, it shall be noted that there are greater limitations in the current research. Specifically, there is a difficulty with cerebrospinal fluid sampling. Most studies did not use cerebrospinal fluid samples from non-syphilitic patients as the control. Meanwhile, it is generally believed that neurosyphilis differs from common syphilis in that TP invades the brain region and the blood-brain barrier is disrupted. Nevertheless, some existing studies just enrolled patients with syphilis at various stages roughly without further detailed discussion, ignoring the possible differences in inflammatory response between patients with common syphilis and neurosyphilis; moreover, there is an absence of a comprehensive evaluation of the expression levels of cytokines in cerebrospinal fluid and peripheral blood before and after treatment. For a comprehensive understanding of the distribution of cytokines in the cerebrospinal fluid and peripheral blood in neurosyphilis, the present study was focusing on syphilis patients first-diagnosis with neurological symptoms. By collecting the serum and CSF samples of 9 patients before and after antibiotic treatment, and 7 control, detected the changes of 80 immune-related cytokines by antibody array technology. Materials And Methods 1. Participants characteristics All clinical samples used in this study were recruited at Hangzhou 7th People's Hospital between December 2019 and July 2021. CSF and serum samples were obtained for routine diagnostic workup. The ethics committee of Hangzhou 7th People's Hospital approved this research. Written informed consent was obtained from all participants. The neurosyphilis group consisted of patients who met the diagnostic criteria for neurosyphilis 8 18 . Serological tests: toluidine red unheated serum test (TRUST) and TP particle agglutination (TPPA) test showed both positive; CSF examination: leucocyte count was≥10X10 6 /L, protein concentration was﹥0.45g/L, TRUST or TPPA test showed positive. The patients were not treated with antibiotics or immunosuppressive agents during the preceding 6 months, other chronic inflammatory disorders (such as autoimmunity, systemic inflammation) and underlying acute or chronic disease, HIV infection were also excluded. All patients were hospitalized, non-penicillin-allergic patients received initial treatment with benzathine penicillin (4 million units, intravenously, six times daily for 2 weeks). Penicillin-allergic patients received Ceftriaxone (2 grams, intravenously, once daily for 2 weeks). To prevent Jarisch-Herxheimer reaction, all patients received prednisone (30 mg taken orally, twice daily for 3 days) before the treatment. All patients were returned for follow-up visits at 3, 6 and 12 months after therapy. Lumbar puncture was performed on at the first follow-up visit. If the CSF profile from the previous lumbar puncture was abnormal, it was performed again at subsequent visits. A cohort of patients matched to the neurosyphilis group with NIND was used as controls. They had a lumbar puncture and were diagnosed with dissociative disorder or altered consciousness, but routine blood and cerebrospinal fluid tests were normal. All subjects of control group were negative for HIV and serological tests for syphilis (both TRUST and TPPA negative), and did not have any clinical symptoms consistent with Treponema pallidum infection. 2. Collection of clinical specimens 5mL of CSF was obtained by lumbar spinal tap, first 2 mL for routine CSF assessment. In addition, 5 mL of whole blood centrifuged at 3000g for 10mins, the serum was collected for Serological tests. Residual CSF and serum were processed according to the collection protocol for CSF and blood pairs for banking 19 and stored at − 80 °C for future cytokine measurements. The average preparation time between sample collection and freezing was 1 hour. Routine CSF analysis included CSF white and red cell count, differential cytology, albumin ratio, total protein and so on. 3. Quantitative measurement of cytokines All CSF and serum samples were screened using the Quantitative antibody array platform purchased from Raybiotech Life, contained 40 Chemokine (QAH-CHE-1) and 40 Inflammation cytokines (QAH-INF-3). The list of the 80 cytokines assayed and detailed detection methods are attached at: www.raybiotech.com/antibody-array. This multiplexed sandwich ELISA-based quantitative array platform enables us to accurately determine the concentration of multiple cytokines simultaneously. It combines the advantages of the high detection sensitivity and specificity of ELISA and the high throughput of arrays. A capture antibody is first bound to the glass surface, and then incubation with the sample for 2 hours, the target cytokine is trapped on the solid surface. They were washed then incubated with a second biotin labeled detection antibody, which can recognize a different epitope of the target cytokine. Subsequently with streptavidin-conjugated Cy3 equivalent dye for 2 hours. The cytokine-antibody-biotin complex can then be visualized using a laser scanner. The intensity was normalized with positive controls from the same glass array. Data was then extracted from the image using a vendor-provided GAL file using compatible microarray analysis software. 4. Statistical analysis All data was plotted and analyzed using GraphPad Prism 8 (GraphPad, San Diego, CA), Microsoft Excel, or OriginPro2018 (OriginLab, Northampton, MA). Data from the antibody array screening assay was used to generate a heatmap which clustered proteins with similar expression patterns together. For clustering, proteins were clustered in an unsupervised manner based on Euclidean distance. GraphPad Prism 8 was used to generate a volcano plot of log 2 fold change of expression versus the −log 10 p-value, as determined by t test. The Shapiro-Wilk and Kolmogorov–Smirnov normality tests were used to test the normal distribution of all data. The t test was used to compare cytokine concentrations between before and after treatment. The Mann–Whitney U test was adopted to compare between NIND and neurosyphilis group. The Spearman or Pearson methods were used for correlation analysis. P values﹤0.05 in 2-sided tests were considered significant. The ability of valuable biomarkers to diagnosis of neurosyphilis was evaluated by ROC curves. Results 1. Clinical characteristics A total of 16 subjects were identified, who were enrolled in this study. 9 patients diagnosed with neurosyphilis. The mean age was 53.78±14.02 years (range: 27-71 years). All patients were non-penicillin-allergic patients who received benzathine penicillin and achieved serological cure or serofast after 6-12 months treatment. In the control group, 7 patients with NIND were recruited from the same hospital. Of them, 2 were diagnosed with migraine, 2 with epilepsy, and 3 with mild cognitive impairment. The mean age was 50.29±12.87 years (range: 27-63 years). TRUST and TPPA tests showed negative. Other infectious diseases (eg. HIV) and autoimmune diseases were excluded from above patients by other tests. Further details are displayed in Table 1. 2. Cytokines antibody array CSF and serum samples were used for the screening of 80 distinct human chemokine and inflammation cytokines using a pre-fabricated, commercially available capture antibody array, including 18 samples from neurosyphilis before and after treatment (Pre- and Post-), 7 samples from NIND. Detailed information on cytokines is provided in Supplementary Table S1. The original data obtained from array screen was processed by Raybiotech software for background removal and normalization, was used to generate heatmaps, comparing NIND and neurosyphilis (Pre- and Post-), using OriginPro (Fig. 1A). The heatmap showed that there were significant differences of some cytokines among the three groups at CSF levels, but not at serum level (data not shown). To better visualize these differences, the volcano plots were generated in GraphPad, plotting fold change (FC) in Pre-therapy vs. NIND, Pre- vs. Post-therapy, Pre-therapy vs. NIND respectively, against the corresponding p-value , the significance threshold of p2 (Fig. 1B). In the CSF level, 11 cytokines (CXCL13, CCL22, G-CSF, CXCL6, CCL3, CXCL10, CCL19, IL-12p40, IL-1a, CCL1, CXCL5) were 5fold change higher and 16 cytokines (ICAM-1, CXCL9, Axl, TARC, TNF RII, IL-7, CCL11, CCL4, CCL8, IL-17, TNFβ, IFN-γ, CXCL12, GM-CSF, CCL24, IL-10) were 2fold change higher in neurosyphilis compared to NIND. After treatment, 5 cytokines (CXCL13, CCL22, G-CSF, CXCL6, CXCL9) were 5fold change decreased and 12 cytokines (IL-12p40, CCL19, CCL27, CCL3, CCL1, ICAM-1, CXCL5, TNF RII, CCL4, IFN-γ, IL-10, TNFβ) were 2fold change decreased in post-therapy compared to Pre-therapy. Moreover, 10 cytokines (CXCL13, CCL22, CXCL10, CCL16, LIGHT, CXCL6, CCL3, CXCL5, CCL11, TARC) were higher and 1 cytokine (CCL27) lower in post-therapy than those in NIND. In the serum level, 3 cytokines (TSLP, CCL20, IL-31) were 2fold change lower and 1 cytokine (OPN) higher in neurosyphilis compared to NIND, and 1 cytokine (CCL20) were 2fold change higher in post-therapy compared to Pre-therapy. After treatment, 2 cytokines (TSLP, IL-31) were lower and 1 cytokine (OPN) higher than those in NIND. Supporting data are detailed in Supplementary Table S2. Concentrations of all CSF cytokines were analyzed by comparing values between the different groups, using the Mann-Whitney U test, or T test. Multiple cytokines (26/27) were overexpressed in neurosyphilis compared to NIND (Fig. 2A), The expression level of IL-1a increased but not significantly (p=0.3577). After antibiotic therapy, the expression of cytokines (15/17) significantly decreased (Fig. 2B), the expression level of 2 cytokines decreased but not significantly (CXCL9, p=0.0977; IFN-γ, p=0.0547). Regrettably, no significantly changes were observed in the serum samples (data not shown). Supporting data are detailed in Supplementary Table S3. 3. Correlation analyses between cytokines We investigated correlations among CSF cytokine concentrations themselves. Among 10 out of 80 cytokines were closely correlated with other cytokines (≥40 were correlated), most of them were positively correlated. The correlations were limited to 15 cytokines ( ≤10 were correlated), which CXCL9ht be regulated more independently. CCL25 was negatively correlated with 8 cytokines, among which the expression of CXCL9 and CCL24 was significantly increased in neurosyphilis patients, suggesting a downregulation of CCL25 during neurosyphilis inflammation (Fig. 3). Supporting data are detailed in Supplementary Table S4. We also examined cytokine correlations between CSF and serum concentrations in order to discriminate to what extent a passive transfer from the periphery into the CSF, or vice versa, might occur. Only 6/80 cytokines showed a significant correlation between CSF and serum values, CCL16, CCL23 and IL-13 concentrations between CSF and serum were positively correlated, NAP-2, IL-6R and IL-12p40 concentrations between CSF and serum were negatively correlated (Fig S1). Supporting data are detailed in Supplementary Table S5. 4. Correlation analyses between cytokine concentrations and CSF parameters We performed correlations among CSF cytokine concentrations and CSF parameters (Fig. 4). Concerning standard CSF parameters, CSF WBCs significantly correlated with 42 cytokines, Qalb significantly correlated with 37 cytokines and CSF-protein significantly correlated with 36 cytokines. The negative correlations were observed between 22 cytokines and Glucose levels. To evaluate distinct effects between cytokines and immune cell subsets, we analyzed the percentage distribution of immune cell subsets. Granulocyte showed a significantly negative correlation with IL-1ra, IL-2, IL-11 and IL-12p40. Lymphocyte showed a significant positive correlation with CXCL16 and TIMP-2. Monocyte showed a significant positive correlation with 11cytokines (CXCL5, CXCL6, NAP-2, PF4, CCL24, G-CSF, ICAM-1, IL-6, IL-12p4, CXCL9 and CCL5) and negatively correlated with CXCL16 and Lymphotactin. Supporting data are detailed in Supplementary Table S6. 5. Diagnostic and therapeutic effect values of cytokines for neurosyphilis. Given the marked elevation of CSF G-CSF, ICAM-1, CCL4, CCL19, CCL1, IL-12p40 and TNF RII in neurosyphilis patients, significant reduction after treatment, and significantly correlated with CSF parameters, we further evaluated these cytokines as biomarkers in neurosyphilis diagnosis by the ROC curve analysis. The AUCs of G-CSF, ICAM-1, CCL4, CCL19 and IL-12p40 in CSF were all greater than 0.9, which were 0.9615, 0.9583, 0.9167, 0.9236 and 0.9127, respectively (Fig. 5). And the optimal cut-off values were defined by the sum of maximum sensitivity and specificity, which were 146.6 pg/ml, 2974 pg/ml, 3.283 pg/ml, 1961 pg/ml and 2.428 pg/ml, respectively. Supporting data are detailed in Supplementary Table S7. Discussion To further clarify the role of the cytokine profile in the inflammatory response of Neurosyphilis, we detected the concentrations of 80 cytokines in CSF and serum of initially diagnosed Syphilis patients with CNS symptoms and NIND patients, changed the cytokine levels were compared before and after the treatment. We performed correlations between CSF cytokine concentrations and clinical diagnostic parameters, including WBCs, total protein, cell percentages, Q Alb and Glucose. The correlations among CSF cytokine concentrations themselves, CSF and serum concentrations were also analyzed. In the present study, it was found that 26 cytokines in CSF of neurosyphilis were significantly increased compared to the NIND group, concentrations of 15 cytokines were significantly decreased after treatment, of which 11 cytokines were down regulated to the levels were similar to the NIND group. Concentrations of 5 cytokines in serum apparently changed, however, the change were not significant. The results of increased expression of CXCL13, CXCL10, CCL11, IL-17, IFN-γ, CCL24 and IL-10 were consistent with the previous researches (see Table 2 10 14 20–27 ). During the follow-up visits, clinical parameters of the CSF returned to normal and the cytokines that were highly expressed in CSF before treatment were significantly reduced except for CXCL13, CXCL10, CCL22, CCL16, CXCL6, CCL3, TARC, LTGHT, CXCL5 and CCL11. Most notably, concentrations of G-CSF, CXCL9, CCL19, IL-12p40, CCL1, ICAM-1, TNF RII, CCL4, TNFβ, IL-10 and IFN-γ were all reduced to the levels not different from those in the NIND group. These results suggested that these cytokines are of great significance to evaluating the therapeutic effect. Analysis of cytokine concentrations and clinical parameters in CSF showed that 22 cytokines were negatively correlated with Glucose levels, which were in line with the clinical observations of decreased relative levels of Glucose caused by intracranial infection. Nearly half of the cytokines were also significantly positively correlated with WBCs (42), total protein (36), and QAlb (37), suggesting that the up-regulation of immune cells in CSF and the destruction of BBB are related to various proinflammatory cytokines, and may play an important role in the progression of the inflammatory response in the brain regions of neurosyphilis. The correlation analysis of cytokine concentrations in CSF and serum showed that only CCL16, CCL23, and IL-13 in CSF were positively correlated with those in the serum, and NAP-2, IL-6R, and IL- 12p40 were negatively correlated. These findings suggested that the changes in most cytokines in CSF of neurosyphilis didn’t originate from passive exchange in BBB, but more forcefully may be rooted in the inflammatory response of pathogens that invade the brain regions and activate the production and up-regulation of cytokines in local tissues. In addition, CSF cytokines (G-CSF, ICAM-1, CCL4, CCL19, CCL1, IL-12p40, TNFRII) that met the criteria, i.e., cytokines that significantly increased in neurosyphilis patients, decreased to levels with no difference from the NIND group after treatment, and significantly correlated with CSF parameters, were further screened. The ROC curve was used to evaluate the value of these candidates for clinical diagnosis and efficacy evaluation. We found that the AUCs of G-CSF, ICAM-1, CCL4, CCL19 and IL-12p40 in CSF were all greater than 0.9, with high diagnostic values. Although we found CXCL13 was also significantly elevated in the CSF of neurosyphilis in our study, again supporting the possibility of CXCL13 as a diagnostic marker, clinical parameters recovered to normal after antibiotic treatment and CXCL13, however, was still highly expressed in CSF, which suggested that CXCL13 may not serve as a reliable measure to evaluate the treatment effect. But G-CSF, ICAM-1, CCL4, CCL19 and IL-12p40 decreased rapidly after antibiotic treatment, suggesting that these cytokines are suitable for monitoring treatment effects with improved diagnostic accuracy. G-CSF plays an important role in non-specific cellular immunity 28 , G-CSF is less expressed under normal conditions. When bacterial infection occurs, activated endothelial cells, macrophages and other immune cells all produce G-CSF to increase the number and physiological functions of neutrophils and macrophages for the anti-infective immune effect. G-CSF decreases to a normal level after infection is controlled 29 . In the present study, the correlation analysis between cytokines in CSF showed a significant positive correlation between CCL4, CCL19, IL-12p40 and G-CSF, which again demonstrated that G-CSF concentration can serve as a reliable indicator to accurately reflect the presence of bacterial infection in the CNS. IL-12p40 is a member of the IL-12 cytokine family. IL-12 is the most important cytokine mediating cellular immunity and plays a major role in neuroinflammation 21 . IL-12p40 critically involves in the inflammatory response and Th1 polarization of T cells in the initially infection 30 . In the present study, the concentration of IL-12p40 increased significantly in CSF and decreased in serum. Although this decrease was not statistically significant, subsequent correlation analysis revealed a significant negative correlation between the concentration of IL-12p40 in CSF and that in the serum, suggesting that the highly expressed IL-12p40 in CSF may be partially explained by the penetration from the blood due to the impaired BBB. ICAM-1 promotes the activation of endothelial cells and enhances the adhesion between leukocytes, inflammatory cells and endothelial cells via specific binding with receptors, playing an important role in cellular immune responses and inflammatory responses. Activated ICAM-1 can up-regulate various inflammatory factors 31 . In vitro BBB model, cross-linking of ICAM-1 upregulates the expression of the chemokine CCL4 in hCMEC/D3 32 . VE-cadherin, an important downstream effector molecule activated by ICAM-1, is a key component of vascular endothelial cell adhesion junctions as well as one important transmembrane protein. Phosphorylation of VE-cadherin can induce the dissociation of endothelial cell junctions and change the membrane permeability 33 . Overexpression of CCL4 has been widely reported in many CNS diseases, such as MS 31 , AD 34 35 and PD 36 . CCL4 can enhance the adhesion of T cells to brain endothelial cells 37 . Some studies have found that CCL4 can affect the redistribution of VE-cadherin via changing the spatial distribution of VE-cadherin in endothelial cells and destroy the integrity of the endothelial cell barrier 38 , thereby promoting the migration of macromolecules and immune cells and ultimately leading to BBB dysfunction to influence the progression of neuroinflammation 39 . CCL19 is constitutively expressed in the CNS and upregulated in brain homogenates from patients with MS lesions as well as in inflammation, infection, and cancer 40 – 42 . Chemokine receptor CCR7 and its ligands CCL19 and CCL21 play important roles in the regulation of immune cell trafficking by secondary lymphoid tissue. Both CCL19 and CCL21 can induce Ca 2+ mobilization, chemotaxis, and integrin-mediated adhesion, and CCL19 more effectively binds to CCR7 with higher affinity 43 44 . It is known that TLRs is critical in pathogen recognition and the initiation of innate immune and inflammatory responses. Studies have found that in an in vitro BBB model, TLR4 can significantly increase the migration ability of monocytes in response to CCL9 and play a role in neuroinflammation of bacterial infection 45 . Similar trends in expression of ICAM-1, CCL4, and CCL19 in the CSF were noted, suggesting that a cascade reaction occurred in the cytokine production, immune cell recruitment and BBB destruction. The progression of neurosyphilis is associated with the destruction of the BBB and the migration of immune cells from the periphery to the CNS. Limitations exist in the present study: First, the number of enrolled cases was limited, the blood and CSF samples from 7 NIND patients and 9 syphilis patients with CNS symptoms were analyzed. Second, correlation analysis between different CSF clinical measures and cytokines needs to be verified in more studies. Third, during the continuous follow-up of the enrolled patients with neurosyphilis, no patients were found not to respond to the antibiotic therapy, and the assessment of cytokine changes in refractory patients was deficient. Fourth, multivariate analysis cannot be performed due to the small sample size, and the practical value of the cut-off value in the diagnosis and efficacy evaluation of neurosyphilis remains to be evaluated. It was believed that further testing on a larger patient population is still needed. Conclusion Our data showed that the cytokines G-CSF, ICAM-1, CCL4, CCL19 and IL-12p40 were highly expressed in the CSF of neurosyphilis patients, and their levels were significantly reduced after antibiotic treatment. Meanwhile, these cytokines were significantly correlated with various CSF clinical parameters, and the AUCs in ROC analysis were all greater than 0.9, suggesting that G-CSF, ICAM-1, CCL4, CCL19 and IL-12p40 in CSF may serve as potential biomarkers for neurosyphilis. Unfortunately, more meaningful results were not observed in the analysis at the serum level. Along with previous related reports, no new non-invasive detection measures that were expected to replace the traditional CSF measures were discovered from serum cytokine profiling. Therefore, it is believed that CSF measures are irreplaceable in the current clinical diagnosis, and the combined dynamic detection of multiple cytokines such as G-CSF, ICAM-1, CCL4, CCL19 and IL-12p40 in CSF can serve as a supplementary diagnostic tool to neurosyphilis and also can be used to monitor therapeutic effect. Abbreviations TP, Treponema pallidum CSF, cerebrospinal fluid NIND, non-inflammatory neurological diseases ROC, receiver operating characteristic WBCs, white blood cells Qalb, albumin quotient AUC, area under the ROC curve CNS, central nervous system TRUST, toluidine red unheated serum test TPPA, TP particle agglutination test Declarations Ethical approval and consent to participate All patients consented to the scientific use of their biological samples. The ethics committee of Hangzhou 7th People's Hospital approved scientific use of CSF and serum biological samples. Consent for publication Not applicable. Availability of data and materials The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by Project for Hangzhou Medical Disciplines of Excellence & Key Project for Hangzhou Medical Discipline. And the Research Fund of Health Commission of Zhejiang Province (2021429094) also provided support. Authors' contributions Shu Qi Wang and Xiao Ying Zhang conceived and designed this research. Xiao Ying Zhang, Meiduo gesang, Zheng Wang and Jie Fang Chen were responsible for selecting cases and sampling. Shu Mei Chi and Man Jin contributed to the medical records and typed data entry the tables. Shu Qi Wang and Xiao Ying Zhang searched literature. Meiduo Gesang, Shu Qi Wang and Xiao Ying Zhang planned and conducted the experiment. Shu Qi Wang and Ming Fen Song analyzed the data, including statistical tests, figures, and table production. Shu Qi Wang wrote the manuscript and Ming Fen Song corrected the draft of the manuscript. All authors have approved the final manuscript as submitted. Acknowledgements We would like to express our gratitude to all those who helped us during the writing of this study. A special acknowledgement should be shown to Dr Pan Yan and Dr Qian Qian Lin for useful instructions of statistics. References Tel BC, Telli G, Onder S, et al. Investigation of the relationship between chronic montelukast treatment, asthma and depression-like behavior in mice. Exp Ther Med 2021;21(1):27. doi: 10.3892/etm.2020.9459 [published Online First: 2020/12/03] Peeling RW, Mabey D, Kamb ML, et al. Syphilis. Nat Rev Dis Primers 2017;3:17073. doi: 10.1038/nrdp.2017.73 [published Online First: 2017/10/13] Marra CM. Neurosyphilis. 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Ann Neurol 2004;55(5):627–38. doi: 10.1002/ana.20049 [published Online First: 2004/05/04] Yoshida R, Nagira M, Kitaura M, et al. Secondary lymphoid-tissue chemokine is a functional ligand for the CC chemokine receptor CCR7. J Biol Chem 1998;273(12):7118–22. doi: 10.1074/jbc.273.12.7118 [published Online First: 1998/04/18] Ott TR, Lio FM, Olshefski D, et al. The N-terminal domain of CCL21 reconstitutes high affinity binding, G protein activation, and chemotactic activity, to the C-terminal domain of CCL19. Biochem Biophys Res Commun 2006;348(3):1089–93. doi: 10.1016/j.bbrc.2006.07.165 [published Online First: 2006/08/15] Paradis A, Bernier S, Dumais N. TLR4 induces CCR7-dependent monocytes transmigration through the blood–brain barrier. Journal of Neuroimmunology 2016;295–296:12–17. doi: 10.1016/j.jneuroim.2016.03.019 Tables Table 1. Characteristics of patients. NIND (n=7) Neurosyphilis (n=9) pre-therapy post-therapy Male (%) 71.43% 66.67% Age (years) 50.29±12.87 53.78±14.02 Serum-TRUST 0 128(512-16) 32(128-8) CSF-TRUST 0 8(32-1) 2(8-1) CSF-WBC (×10 6 /L) 1.314±0.778 31.46±12.77 3.567±1.735 CSF-protein (g/l) 0.449±0.046 1.029±0.408 0.7278±0.182 Data are given as median (range), or frequencies, or mean ± standard deviation. Abbreviations: NIND, non-inflammatory neurological diseases; TRUST, toluidine red unheated serum test; CSF, cerebrospinal fluid; WBCs, white blood cells. Table 2. Well documented cytokines rediscovered in screen were consistent with the previous researches. Syphilis Type Sample Type Molecule Method Supplementary References Neurosyphilis vs. NIND CSF CXCL11↑ MB Lepennetier et al. (2019) 20 CXCL13↑ Neurosyphilis vs. Syphilis Neurosyphilis vs. HC CSF CXCL13↑ ELISA Yan et al. (2017) 21 IL-6↑ IL-10↑ Serum IL-6↑ IL-10↑ Syphilis vs. HC Serum chemerin↑ ELISA Stevenson et al. (2017) 22 8 hours after antibiotic treatment for syphilis Serum IFN-γ↓ ELISA Pastuszczak et al. (2017) 23 TNF-α↓ IL-6↓ Neurosyphilis vs. Syphilis CSF CXCL13↑ Array, ELISA Wang et al. (2016) 10 CXCL10↑ CXCL8↑ Serum CXCL13↓ Neurosyphilis vs. Syphilis CSF uPA↑ Array, ELISA Lu et al. (2016) 24 Sy- vs Asy-mptomatic neurosyphilis CSF IL-17↑ ELISA Wang et al. (2014) 25 Neurosyphilis-HIV vs. Syphilis-HIV CSF CXCL2↑ ELISA Tsai et al. (2014) 26 Neurosyphilis vs. Syphilis CSF IL-17α↑ ELISA Pastuszczak et al. (2013) 14 IFN-γ↑ Neurosyphilis-HIV vs. Syphilis-HIV CSF CXCL13↑ ELISA Marra et al. (2010) 27 Serum CXCL13↑ Abbreviations: NIND, non-inflammatory neurological diseases; MB, magnetic beads; HC, healthy controls; ELISA, enzyme-linked immunosorbent assay; CSF, cerebrospinal fluid. Additional Declarations No competing interests reported. Supplementary Files figureS1.tif supplementarymaterialstables.xlsx Supplementarymaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1562299","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":99558515,"identity":"5e214ea3-b65e-4661-ad9b-7f3b4c8d8ab5","order_by":0,"name":"Xiao Ying Zhang","email":"","orcid":"","institution":"Affiliated Mental Health Center \u0026 Hangzhou Seventh People's Hospital, Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"Ying","lastName":"Zhang","suffix":""},{"id":99558516,"identity":"5fa41262-3fb7-44e1-b099-3b569c89250f","order_by":1,"name":"Meiduo Gesang","email":"","orcid":"","institution":"Affiliated Mental Health Center \u0026 Hangzhou Seventh People's Hospital, Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Meiduo","middleName":"","lastName":"Gesang","suffix":""},{"id":99558517,"identity":"de3201f2-0b57-4e00-8f03-ed93026a6c64","order_by":2,"name":"Sha Wu","email":"","orcid":"","institution":"Affiliated Mental Health Center \u0026 Hangzhou Seventh People's Hospital, Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Sha","middleName":"","lastName":"Wu","suffix":""},{"id":99558518,"identity":"db88b8a1-f81a-4ea8-be05-58124d28faa6","order_by":3,"name":"Jie Fang Chen","email":"","orcid":"","institution":"Affiliated Mental Health Center \u0026 Hangzhou Seventh People's Hospital, Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"Fang","lastName":"Chen","suffix":""},{"id":99558519,"identity":"58daf188-0b4e-4fdb-a2be-a1b527fc15b7","order_by":4,"name":"Zheng Wang","email":"","orcid":"","institution":"Affiliated Mental Health Center \u0026 Hangzhou Seventh People's Hospital, Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zheng","middleName":"","lastName":"Wang","suffix":""},{"id":99558520,"identity":"9c4ab257-318d-4341-88e3-1b76c3614a79","order_by":5,"name":"Shu Mei Chi","email":"","orcid":"","institution":"Affiliated Mental Health Center \u0026 Hangzhou Seventh People's Hospital, Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shu","middleName":"Mei","lastName":"Chi","suffix":""},{"id":99558521,"identity":"fc321ac1-8e31-4c19-801b-f7285d4ac936","order_by":6,"name":"Man Jin","email":"","orcid":"","institution":"Affiliated Mental Health Center \u0026 Hangzhou Seventh People's Hospital, Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Man","middleName":"","lastName":"Jin","suffix":""},{"id":99558522,"identity":"37faad97-2e94-4314-b377-ae1ab01786a8","order_by":7,"name":"Ming Fen Song","email":"","orcid":"","institution":"Affiliated Mental Health Center \u0026 Hangzhou Seventh People's Hospital, Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ming","middleName":"Fen","lastName":"Song","suffix":""},{"id":99558523,"identity":"2180a7e9-9519-47e5-be2e-fd2672d5c7d8","order_by":8,"name":"Shu Qi Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYDACCSBOADHYGxsOfDCwsSNBC8/hxoczCtKSidMCYbg3G/N8OMTYQEiH/OwewxsP2+zy5CMY26RtDA4wM7AfProBnxaDO2eMLRLbkosNbze2SecY3OFj4ElLu4FXi0SOmUTiNubEjXMOgrQ8Y2aQ4DHDq0V+BlhLfeLGGYlt0hYGhxkbCGlhuAHWcjhxvkRiszEDMVoMbqQVWyT+O564gedg48Meg7RkNkJ+kZ+RvPHmjzPVifPb2x8c+PHHxo6f/fAx/A5jgEaNwQEoj42QcrgW+QZilI6CUTAKRsGIBABwdFC4Qtqz2AAAAABJRU5ErkJggg==","orcid":"","institution":"Affiliated Mental Health Center \u0026 Hangzhou Seventh People's Hospital, Zhejiang University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Shu","middleName":"Qi","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2022-04-15 16:59:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1562299/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1562299/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":20609503,"identity":"e615a186-1a26-4219-9398-77068cef21aa","added_by":"auto","created_at":"2022-04-21 15:49:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1964105,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e80 cytokines from neurosyphilis patients and NIND controls\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eMensuration of 80 cytokines in CSF and serum samples from neurosyphilis patients and NIND controls (total=25: NIND=7; Neurosyphilis=18, including pre- and post-treatment samples), using the quantitative antibody array platform. \u003cstrong\u003eA. \u003c/strong\u003eHeatmap of cytokines-group-supervised clustering (the columns) reveals the landscape of protein expression across the 25 CSF samples, as determined from the protein array. The red-green color scheme indicates the expression of each of the 80 cytokines (each row representing one cytokine), with red indicating over-expression and green opposite, black indicating the median expression level for this protein. \u003cstrong\u003eB. \u003c/strong\u003eVolcano plot showing expression differences of 80 cytokines in the CSF and serum, when comparing log2 fold change of protein expression vs. the -log10 P-value, considered biologically and statistically significant. Each dot represents a protein and its average value for that subset. Red dots are significant increases, green dots are significant decreases. All biomarker data was normalized and analyzed using a 2-tailed T test. (Detailed data refer to Table S2)\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1562299/v1/8cce38ff2ca38708b58017cc.png"},{"id":20609506,"identity":"179f1b48-61dd-4017-8f55-1e50fdc54c7d","added_by":"auto","created_at":"2022-04-21 15:49:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":433744,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlot diagrams of CSF cytokine concentrations. \u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eA: \u003c/strong\u003e25 CSF samples, including 7 NIND controls (black) and 9 Neurosyphilis (red), list the significant differences in cytokine concentrations between Neurosyphilis and NIND, including CXCL13, CCL22, G-CSF, CXCL6, CCL3, CXCL10, CCL19, IL-12p40, CCL1, CXCL5, ICAM-1, CXCL9, Axl, TARC, TNF RII, IL-7, CCL11, CCL4, CCL8, IL-17, TNFβ, IFN-γ, CXCL12, GM-CSF, CCL24, IL-10 and IL-1α. Differences between groups were analyzed by T test, or Mann Whitney U test. \u003cstrong\u003eB: \u003c/strong\u003e18 CSF samples of neurosyphilis patients before and after antibiotic treatment, list the significant reduction in cytokine concentrations, including CXCL13, CCL22, G-CSF, CXCL6, CXCL9, IL-12p40, CCL19, CCL27, CCL3, CCL1, ICAM-1, CXCL5, TNF RII, CCL4, IFN-γ, IL-10, TNFβ. Differences between before and after treatment were analyzed by paired t-test. (Detailed data refer to Table S3)\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-1562299/v1/7b2056aa8c7d1e8837e455ab.png"},{"id":20609508,"identity":"6574483e-42ea-4301-a8b5-dbb9092a485e","added_by":"auto","created_at":"2022-04-21 15:49:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1173400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeatmap representing correlations between different CSF cytokines among each other.\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe red-blue color scheme indicates the correlations of each of the 80 cytokines (each cell representing one cytokine), with red indicating positive correlations, blue indicating negative correlations and blank indicating insignificant correlation. The correlation analysis performed by Spearman or Pearson methods. (Detailed data refer to Table S4)\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-1562299/v1/c930a5a20474e7ba55615a6f.png"},{"id":20610496,"identity":"99a14511-5676-4580-8e06-146a63f53c38","added_by":"auto","created_at":"2022-04-21 15:54:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":596096,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeatmap representing correlations between CSF cytokines concentrations and CSF parameters.\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eCSF parameters including white blood cell count (WBCs), Albumin quotient (QAlb), CSF total-protein, Glucose and Immune cell subsets percentage (GRANR=Granulocyte, MONOR=Monocyte, LYMPH=lymphocyte). The red-blue color scheme indicates the correlations between them, with red indicating positive correlations and blue indicating negative correlations, blank indicating insignificant correlation. The correlation analysis performed by Spearman or Pearson methods. (Detailed data refer to Table S6)\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-1562299/v1/094c9e7f72a9aff49f322550.png"},{"id":20609505,"identity":"6f28f14f-850e-44bf-8a76-5df0f683eb7b","added_by":"auto","created_at":"2022-04-21 15:49:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1232435,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClinical diagnostic value of CSF cytokines biomarkers for neurosyphilis. \u003c/strong\u003e\u003c/p\u003e\u003cp\u003eList 7 biomarkers to diagnosis of neurosyphilis were evaluated by receiver-operating characteristic (ROC) curves, including G-CSF, ICAM-1, CCL4, CCL19, CCL1, IL-12p40 and TNFRII. The ROC curve shows the tradeoff between the true detections and false detections. The areas under the ROC curve (AUC) is the percentage of randomly drawn pairs for which the test is correct. The AUCs of G-CSF, ICAM-1, CCL4, CCL19 and IL-12p40 in CSF were all greater than 0.9. (Detailed data refer to Table S7)\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-1562299/v1/7b7bfd8174b19133b5c59554.png"},{"id":21486090,"identity":"b1b4e58a-c9c6-4139-9971-86329c9bae23","added_by":"auto","created_at":"2022-05-15 20:44:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1808987,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1562299/v1/3a400dd1-2eea-4380-b38e-37c274bff90f.pdf"},{"id":20609507,"identity":"fadedbb2-278d-413d-a72c-700f62889c78","added_by":"auto","created_at":"2022-04-21 15:49:48","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3638276,"visible":true,"origin":"","legend":"","description":"","filename":"figureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-1562299/v1/825821d7ceca0324265bcfc2.tif"},{"id":20609502,"identity":"f2a5afb8-83a1-4917-a740-5cd600218d3b","added_by":"auto","created_at":"2022-04-21 15:49:48","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":151693,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterialstables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-1562299/v1/5a4cbb80b8d5635409d6d118.xlsx"},{"id":20609501,"identity":"f32fa7be-af37-46b3-96f5-74d629d5d6e7","added_by":"auto","created_at":"2022-04-21 15:49:48","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":12482,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-1562299/v1/4d1643ba0361cd3da1e7e511.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cytokines in cerebrospinal fluid and serum as biomarkers are associated with the diagnosis and efficacy evaluation of neurosyphilis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSyphilis is a chronic and systemic sexually transmitted disease caused by TP infection\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. TP invades the circulatory system and produces an effect on all tissues and organs of the whole body. In severe cases, it may lead to multi-system organic lesions and irreversible damage\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Neurosyphilis is TP disseminates to the central nervous system (CNS), causes pathological injury in related brain areas, and results in a series of serious consequences \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Most syphilis patients without standardized treatment may develop into neurosyphilis eventually\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Neurosyphilis is characterized by complex and diverse clinical manifestations. According to the invasion degree of TP, neurosyphilis can be divided into the early-stage type (only involving meninges and blood vessels) and the late-stage type (involving brain and spinal cord parenchyma)\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. There is a difference in TP concentrations in peripheral blood and CSF of neurosyphilis patients. Moreover, there may be false-negative results in routine antibody tests\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In a few patients, syphilis is not even detected in cerebrospinal fluid, with the detection of inflammatory reactions merely, such as increased WBCs and total protein content, etc\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. These phenomena may also occur in many central nervous system infections and autoimmune diseases\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. There may be insensitivity to antibiotics in the treatment of neurosyphilis\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. There exists a difference in the therapeutic effects in patients with different autoimmune statuses and course of the disease. In general, it is considered that long-term alcohol consumption, drug abuse, hypertension, HIV complicated with syphilis are all influential factors of the therapeutic effects\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCytokines are small molecular proteins secreted by cells with extensive biological activities, which can regulate cell proliferation, differentiation and immune response by binding to receptors. After infection with TP, a strong immune response may be triggered in vivo, which may activate relevant immune cells to release a large amount of cytokines.\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, The immune response is of great significance for the clearance of TP, which, however, may also induce a local inflammatory response and result in pathological damage\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The intensity and type of immune response may have an impact on the severity of syphilis, cellular and humoral immune responses are both involved in the damage process \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Without timely treatment, there may be irreversible organic lesions in the brain of patients. Serious inflammatory damage has been detected in the brain tissue of patients with paralytic dementia and gumma\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAt present, researchers are highly concerned about the expression changes of related cytokines during the exploration of the immune response mechanism of syphilis. Maciej et al. (2013) found that IL-17A and IFN-γ in cerebrospinal fluid of asymptomatic neurosyphilis patients were higher than those of common syphilis patients\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Wang et al. (2014) detected the increase of Th17 cells in the cerebrospinal fluid of patients with neurosyphilis, accompanied by an increase in the expression level of IL-17 secreted by Th17 cells as well \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Furthermore, Li et al. (2020) found an increase in the expression of IL-10 in the cerebrospinal fluid of neurosyphilis patients with mental symptoms\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In the same year, Di et al. (2020) reported lower expression levels of IL-17, TNF-RI, TNF-RII, IL-16, OPN and MCSFR in cerebrospinal fluid of patients with syphilitic serofast state before treatment, yet with higher CCL7, LIF, G-CSF, CCL20 and GH were higher\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe immune response is abnormally activated in the cerebrospinal fluid of patients with neurosyphilis. Related cytokines are involved in the inflammatory reaction and progression of neurosyphilis, which has great potential to be novel biomarkers of neurosyphilis. However, it shall be noted that there are greater limitations in the current research. Specifically, there is a difficulty with cerebrospinal fluid sampling. Most studies did not use cerebrospinal fluid samples from non-syphilitic patients as the control. Meanwhile, it is generally believed that neurosyphilis differs from common syphilis in that TP invades the brain region and the blood-brain barrier is disrupted. Nevertheless, some existing studies just enrolled patients with syphilis at various stages roughly without further detailed discussion, ignoring the possible differences in inflammatory response between patients with common syphilis and neurosyphilis; moreover, there is an absence of a comprehensive evaluation of the expression levels of cytokines in cerebrospinal fluid and peripheral blood before and after treatment.\u003c/p\u003e \u003cp\u003eFor a comprehensive understanding of the distribution of cytokines in the cerebrospinal fluid and peripheral blood in neurosyphilis, the present study was focusing on syphilis patients first-diagnosis with neurological symptoms. By collecting the serum and CSF samples of 9 patients before and after antibiotic treatment, and 7 control, detected the changes of 80 immune-related cytokines by antibody array technology.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003e1. Participants characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll clinical samples used in this study were recruited at Hangzhou 7th People\u0026apos;s Hospital between December 2019 and July 2021. CSF and serum samples were obtained for routine diagnostic workup. The ethics committee of Hangzhou 7th People\u0026apos;s Hospital approved this research. Written informed consent was obtained from all participants. The neurosyphilis group consisted of patients who met the diagnostic criteria for neurosyphilis\u003csup\u003e8\u003c/sup\u003e \u003csup\u003e18\u003c/sup\u003e. Serological tests:\u0026nbsp;toluidine red unheated serum test (TRUST) and TP particle agglutination (TPPA) test showed both positive; CSF examination: leucocyte count was\u0026ge;10X10\u003csup\u003e6\u003c/sup\u003e/L, protein concentration was﹥0.45g/L, TRUST or\u0026nbsp;TPPA test showed positive. The patients were not treated with antibiotics or immunosuppressive agents during the preceding 6 months, other chronic inflammatory disorders (such as autoimmunity, systemic inflammation) and underlying acute or chronic disease, HIV infection were also excluded. All patients were hospitalized, non-penicillin-allergic patients received initial treatment with benzathine penicillin (4 million units, intravenously, six times daily for 2 weeks). Penicillin-allergic patients received Ceftriaxone (2 grams, intravenously, once daily for 2 weeks). To prevent Jarisch-Herxheimer reaction, all patients received prednisone (30 mg taken orally, twice daily for 3 days) before the treatment. All patients were returned for follow-up visits at 3, 6 and 12 months after therapy. Lumbar puncture was performed on at the first follow-up visit. If the CSF profile from the previous lumbar puncture was abnormal, it was performed again at subsequent visits. A cohort of patients matched to the neurosyphilis group with NIND was used as controls. They had a lumbar puncture and were diagnosed with dissociative disorder or altered consciousness, but routine blood and cerebrospinal fluid tests were normal. All subjects of control group were negative for HIV and serological tests for syphilis (both TRUST and\u0026nbsp;TPPA\u0026nbsp;negative), and did not have any clinical symptoms consistent with Treponema pallidum infection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Collection of clinical specimens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e5mL of CSF was obtained by lumbar spinal tap, first 2 mL for routine CSF assessment. In addition, 5 mL of whole blood centrifuged at 3000g for 10mins, the serum was collected for Serological tests. Residual CSF and serum were processed according to the collection protocol for CSF and blood pairs for banking\u003csup\u003e19\u003c/sup\u003e and stored at \u0026minus; 80 \u0026deg;C for future cytokine measurements. The average preparation time between sample collection and freezing was 1 hour. Routine CSF analysis included CSF white and red cell count, differential cytology, albumin ratio, total protein and so on.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Quantitative measurement of cytokines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll CSF and serum samples were screened using the Quantitative antibody array platform purchased from Raybiotech Life, contained 40 Chemokine (QAH-CHE-1) and 40 Inflammation cytokines (QAH-INF-3). The list of the 80 cytokines assayed and detailed detection methods are attached at: www.raybiotech.com/antibody-array. This multiplexed sandwich ELISA-based quantitative array platform enables us to accurately determine the concentration of multiple cytokines simultaneously. It combines the advantages of the high detection sensitivity and specificity of ELISA and the high throughput of arrays. A capture antibody is first bound to the glass surface, and then incubation with the sample for 2 hours, the target cytokine is trapped on the solid surface. They were washed then incubated with a second biotin labeled detection antibody, which can recognize a different epitope of the target cytokine. Subsequently with streptavidin-conjugated Cy3 equivalent dye for 2 hours. The cytokine-antibody-biotin complex can then be visualized using a laser scanner. The intensity was normalized with positive controls from the same glass array. Data was then extracted from the image using a vendor-provided GAL file using compatible microarray analysis software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data was plotted and analyzed using GraphPad Prism 8 (GraphPad, San Diego, CA), Microsoft Excel, or OriginPro2018 (OriginLab, Northampton, MA). Data from the antibody array screening assay was used to generate a heatmap which clustered proteins with similar expression patterns together. For clustering, proteins were clustered in an unsupervised manner based on Euclidean distance. GraphPad Prism 8 was used to generate a volcano plot of log\u003csub\u003e2\u003c/sub\u003e fold change of expression versus the \u0026minus;log\u003csub\u003e10\u003c/sub\u003e p-value, as determined by t test. The Shapiro-Wilk and Kolmogorov\u0026ndash;Smirnov normality tests were used to test the normal distribution of all data. The t test was used to compare cytokine concentrations between before and after treatment. The Mann\u0026ndash;Whitney U test was adopted to compare between NIND and neurosyphilis group. The Spearman or Pearson methods were used for correlation analysis. P values﹤0.05 in 2-sided tests were considered significant. The ability of valuable biomarkers to diagnosis of neurosyphilis was evaluated by ROC curves.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1. Clinical characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 16 subjects were identified, who were enrolled in this study. 9 patients diagnosed with neurosyphilis. The mean age was 53.78\u0026plusmn;14.02 years (range: 27-71 years). All patients were non-penicillin-allergic patients who received benzathine penicillin and achieved serological cure or serofast after 6-12 months treatment. In the control group, 7 patients with NIND were recruited from the same hospital. Of them, 2 were diagnosed with migraine, 2 with epilepsy, and 3 with mild cognitive impairment. The mean age was 50.29\u0026plusmn;12.87 years (range: 27-63 years). TRUST and TPPA tests showed negative. Other infectious diseases (eg. HIV) and autoimmune diseases were excluded from above patients by other tests.\u0026nbsp;Further details are displayed in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Cytokines antibody array\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCSF and serum samples were used for the screening of 80 distinct human chemokine and inflammation cytokines using a pre-fabricated, commercially available capture antibody array, including 18 samples from neurosyphilis before and after treatment (Pre- and Post-), 7 samples from NIND. Detailed information on cytokines is provided in Supplementary Table S1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe original data obtained from array screen was processed by Raybiotech software for background removal and normalization, was used to generate heatmaps, comparing NIND and neurosyphilis (Pre- and Post-), using OriginPro (Fig. 1A). The heatmap showed that there were significant differences of some cytokines among the three groups at CSF levels, but not at serum level (data not shown).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo better visualize these differences, the volcano plots were generated in GraphPad, plotting fold change (FC) in Pre-therapy vs. NIND, Pre- vs. Post-therapy, Pre-therapy vs. NIND respectively, against the corresponding p-value , the significance threshold of p\u0026lt;0.05 and FC\u0026gt;2 (Fig. 1B). In the CSF level, 11 cytokines (CXCL13, CCL22, G-CSF, CXCL6, CCL3, CXCL10, CCL19, IL-12p40, IL-1a, CCL1, CXCL5) were 5fold change higher and 16 cytokines (ICAM-1, CXCL9, Axl, TARC, TNF RII, IL-7, CCL11, CCL4, CCL8, IL-17, TNF\u0026beta;, IFN-\u0026gamma;, CXCL12, GM-CSF, CCL24, IL-10) were 2fold change higher in neurosyphilis compared to NIND. After treatment, 5 cytokines (CXCL13, CCL22, G-CSF, CXCL6, CXCL9) were 5fold change decreased and 12 cytokines (IL-12p40, CCL19, CCL27, CCL3, CCL1, ICAM-1, CXCL5, TNF RII, CCL4, IFN-\u0026gamma;, IL-10, TNF\u0026beta;) were 2fold change decreased in post-therapy compared to Pre-therapy. Moreover, 10 cytokines (CXCL13, CCL22, CXCL10, CCL16, LIGHT, CXCL6, CCL3, CXCL5, CCL11, TARC) were higher and 1 cytokine (CCL27) lower in post-therapy than those in NIND.\u0026nbsp;In the serum level, 3 cytokines (TSLP, CCL20, IL-31) were 2fold change lower and 1 cytokine (OPN) higher in neurosyphilis compared to NIND, and 1 cytokine (CCL20) were 2fold change higher in post-therapy compared to Pre-therapy. After treatment, 2 cytokines (TSLP, IL-31) were lower and 1 cytokine (OPN) higher than those in NIND.\u0026nbsp;Supporting data are detailed in Supplementary Table S2.\u003c/p\u003e\n\u003cp\u003eConcentrations of all CSF cytokines were analyzed by comparing values between the different groups, using the Mann-Whitney U test, or T test.\u0026nbsp;Multiple cytokines (26/27) were overexpressed in neurosyphilis compared to NIND (Fig. 2A), The expression level of IL-1a increased but not significantly (p=0.3577).\u0026nbsp;After antibiotic therapy, the expression of cytokines (15/17) significantly decreased (Fig. 2B), the expression level of 2 cytokines decreased but not significantly (CXCL9, p=0.0977; IFN-\u0026gamma;, p=0.0547). Regrettably, no significantly changes were observed in the serum samples (data not shown). Supporting data are detailed in Supplementary Table S3.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Correlation analyses between cytokines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe investigated correlations among CSF cytokine concentrations themselves. Among 10 out of 80 cytokines were closely correlated with other cytokines (\u0026ge;40 were correlated), most of them were positively correlated. The correlations were limited to 15 cytokines (\u0026nbsp;\u0026le;10 were correlated), which CXCL9ht be regulated more independently. CCL25 was negatively correlated with 8 cytokines, among which the expression of CXCL9 and CCL24 was significantly increased in neurosyphilis patients, suggesting a downregulation of CCL25 during neurosyphilis inflammation (Fig. 3). Supporting data are detailed in Supplementary Table S4.\u003c/p\u003e\n\u003cp\u003eWe also examined cytokine correlations between CSF and serum concentrations in order to discriminate to what extent a passive transfer from the periphery into the CSF, or vice versa, might occur. Only 6/80 cytokines showed a significant correlation between CSF and serum values, CCL16, CCL23 and IL-13 concentrations between CSF and serum were positively correlated, NAP-2, IL-6R and IL-12p40 concentrations between CSF and serum were negatively correlated\u0026nbsp;(Fig S1).\u0026nbsp;Supporting data are detailed in Supplementary Table S5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Correlation analyses between cytokine concentrations and CSF parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe performed correlations among CSF cytokine concentrations and CSF parameters (Fig. 4). Concerning standard CSF parameters, CSF WBCs significantly correlated with 42 cytokines, Qalb significantly correlated with 37 cytokines and CSF-protein significantly correlated with 36 cytokines. The negative correlations were observed between 22 cytokines and Glucose levels. To evaluate distinct effects between cytokines and immune cell subsets, we analyzed the percentage distribution of immune cell subsets. Granulocyte showed a significantly negative correlation with IL-1ra, IL-2, IL-11 and IL-12p40. Lymphocyte showed a significant positive correlation with CXCL16 and TIMP-2. Monocyte showed a significant positive correlation with 11cytokines (CXCL5, CXCL6, NAP-2, PF4, CCL24, G-CSF, ICAM-1, IL-6, IL-12p4, CXCL9 and CCL5) and negatively correlated with CXCL16 and Lymphotactin. Supporting data are detailed in Supplementary Table S6.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. Diagnostic and therapeutic effect values of cytokines for neurosyphilis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the marked elevation of CSF G-CSF, ICAM-1, CCL4, CCL19, CCL1, IL-12p40 and TNF RII in neurosyphilis patients, significant reduction after treatment, and significantly correlated with CSF parameters, we further evaluated these cytokines as biomarkers in neurosyphilis diagnosis by the ROC curve analysis. The AUCs of G-CSF, ICAM-1, CCL4, CCL19 and IL-12p40 in CSF were all greater than 0.9, which were 0.9615, 0.9583, 0.9167, 0.9236 and 0.9127, respectively (Fig. 5). And the optimal cut-off values were defined by the sum of maximum sensitivity and specificity, which were 146.6 pg/ml, 2974 pg/ml, 3.283 pg/ml, 1961 pg/ml and 2.428 pg/ml, respectively. Supporting data are detailed in Supplementary Table S7.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo further clarify the role of the cytokine profile in the inflammatory response of Neurosyphilis, we detected the concentrations of 80 cytokines in CSF and serum of initially diagnosed Syphilis patients with CNS symptoms and NIND patients, changed the cytokine levels were compared before and after the treatment. We performed correlations between CSF cytokine concentrations and clinical diagnostic parameters, including WBCs, total protein, cell percentages, Q\u003csub\u003eAlb\u003c/sub\u003e and Glucose. The correlations among CSF cytokine concentrations themselves, CSF and serum concentrations were also analyzed.\u003c/p\u003e \u003cp\u003eIn the present study, it was found that 26 cytokines in CSF of neurosyphilis were significantly increased compared to the NIND group, concentrations of 15 cytokines were significantly decreased after treatment, of which 11 cytokines were down regulated to the levels were similar to the NIND group. Concentrations of 5 cytokines in serum apparently changed, however, the change were not significant. The results of increased expression of CXCL13, CXCL10, CCL11, IL-17, IFN-γ, CCL24 and IL-10 were consistent with the previous researches (see Table\u0026nbsp;2\u003csup\u003e10 14 20\u0026ndash;27\u003c/sup\u003e). During the follow-up visits, clinical parameters of the CSF returned to normal and the cytokines that were highly expressed in CSF before treatment were significantly reduced except for CXCL13, CXCL10, CCL22, CCL16, CXCL6, CCL3, TARC, LTGHT, CXCL5 and CCL11. Most notably, concentrations of G-CSF, CXCL9, CCL19, IL-12p40, CCL1, ICAM-1, TNF RII, CCL4, TNFβ, IL-10 and IFN-γ were all reduced to the levels not different from those in the NIND group. These results suggested that these cytokines are of great significance to evaluating the therapeutic effect.\u003c/p\u003e \u003cp\u003eAnalysis of cytokine concentrations and clinical parameters in CSF showed that 22 cytokines were negatively correlated with Glucose levels, which were in line with the clinical observations of decreased relative levels of Glucose caused by intracranial infection. Nearly half of the cytokines were also significantly positively correlated with WBCs (42), total protein (36), and QAlb (37), suggesting that the up-regulation of immune cells in CSF and the destruction of BBB are related to various proinflammatory cytokines, and may play an important role in the progression of the inflammatory response in the brain regions of neurosyphilis. The correlation analysis of cytokine concentrations in CSF and serum showed that only CCL16, CCL23, and IL-13 in CSF were positively correlated with those in the serum, and NAP-2, IL-6R, and IL- 12p40 were negatively correlated. These findings suggested that the changes in most cytokines in CSF of neurosyphilis didn\u0026rsquo;t originate from passive exchange in BBB, but more forcefully may be rooted in the inflammatory response of pathogens that invade the brain regions and activate the production and up-regulation of cytokines in local tissues.\u003c/p\u003e \u003cp\u003eIn addition, CSF cytokines (G-CSF, ICAM-1, CCL4, CCL19, CCL1, IL-12p40, TNFRII) that met the criteria, i.e., cytokines that significantly increased in neurosyphilis patients, decreased to levels with no difference from the NIND group after treatment, and significantly correlated with CSF parameters, were further screened. The ROC curve was used to evaluate the value of these candidates for clinical diagnosis and efficacy evaluation. We found that the AUCs of G-CSF, ICAM-1, CCL4, CCL19 and IL-12p40 in CSF were all greater than 0.9, with high diagnostic values. Although we found CXCL13 was also significantly elevated in the CSF of neurosyphilis in our study, again supporting the possibility of CXCL13 as a diagnostic marker, clinical parameters recovered to normal after antibiotic treatment and CXCL13, however, was still highly expressed in CSF, which suggested that CXCL13 may not serve as a reliable measure to evaluate the treatment effect. But G-CSF, ICAM-1, CCL4, CCL19 and IL-12p40 decreased rapidly after antibiotic treatment, suggesting that these cytokines are suitable for monitoring treatment effects with improved diagnostic accuracy.\u003c/p\u003e \u003cp\u003eG-CSF plays an important role in non-specific cellular immunity\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, G-CSF is less expressed under normal conditions. When bacterial infection occurs, activated endothelial cells, macrophages and other immune cells all produce G-CSF to increase the number and physiological functions of neutrophils and macrophages for the anti-infective immune effect. G-CSF decreases to a normal level after infection is controlled\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In the present study, the correlation analysis between cytokines in CSF showed a significant positive correlation between CCL4, CCL19, IL-12p40 and G-CSF, which again demonstrated that G-CSF concentration can serve as a reliable indicator to accurately reflect the presence of bacterial infection in the CNS.\u003c/p\u003e \u003cp\u003eIL-12p40 is a member of the IL-12 cytokine family. IL-12 is the most important cytokine mediating cellular immunity and plays a major role in neuroinflammation\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. IL-12p40 critically involves in the inflammatory response and Th1 polarization of T cells in the initially infection\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In the present study, the concentration of IL-12p40 increased significantly in CSF and decreased in serum. Although this decrease was not statistically significant, subsequent correlation analysis revealed a significant negative correlation between the concentration of IL-12p40 in CSF and that in the serum, suggesting that the highly expressed IL-12p40 in CSF may be partially explained by the penetration from the blood due to the impaired BBB.\u003c/p\u003e \u003cp\u003eICAM-1 promotes the activation of endothelial cells and enhances the adhesion between leukocytes, inflammatory cells and endothelial cells via specific binding with receptors, playing an important role in cellular immune responses and inflammatory responses. Activated ICAM-1 can up-regulate various inflammatory factors\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In vitro BBB model, cross-linking of ICAM-1 upregulates the expression of the chemokine CCL4 in hCMEC/D3\u003csup\u003e32\u003c/sup\u003e. VE-cadherin, an important downstream effector molecule activated by ICAM-1, is a key component of vascular endothelial cell adhesion junctions as well as one important transmembrane protein. Phosphorylation of VE-cadherin can induce the dissociation of endothelial cell junctions and change the membrane permeability\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Overexpression of CCL4 has been widely reported in many CNS diseases, such as MS\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, AD \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e and PD\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. CCL4 can enhance the adhesion of T cells to brain endothelial cells\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Some studies have found that CCL4 can affect the redistribution of VE-cadherin via changing the spatial distribution of VE-cadherin in endothelial cells and destroy the integrity of the endothelial cell barrier\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, thereby promoting the migration of macromolecules and immune cells and ultimately leading to BBB dysfunction to influence the progression of neuroinflammation\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. CCL19 is constitutively expressed in the CNS and upregulated in brain homogenates from patients with MS lesions as well as in inflammation, infection, and cancer\u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Chemokine receptor CCR7 and its ligands CCL19 and CCL21 play important roles in the regulation of immune cell trafficking by secondary lymphoid tissue. Both CCL19 and CCL21 can induce Ca\u003csup\u003e2+\u003c/sup\u003e mobilization, chemotaxis, and integrin-mediated adhesion, and CCL19 more effectively binds to CCR7 with higher affinity\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. It is known that TLRs is critical in pathogen recognition and the initiation of innate immune and inflammatory responses. Studies have found that in an in vitro BBB model, TLR4 can significantly increase the migration ability of monocytes in response to CCL9 and play a role in neuroinflammation of bacterial infection\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Similar trends in expression of ICAM-1, CCL4, and CCL19 in the CSF were noted, suggesting that a cascade reaction occurred in the cytokine production, immune cell recruitment and BBB destruction. The progression of neurosyphilis is associated with the destruction of the BBB and the migration of immune cells from the periphery to the CNS.\u003c/p\u003e \u003cp\u003eLimitations exist in the present study: First, the number of enrolled cases was limited, the blood and CSF samples from 7 NIND patients and 9 syphilis patients with CNS symptoms were analyzed. Second, correlation analysis between different CSF clinical measures and cytokines needs to be verified in more studies. Third, during the continuous follow-up of the enrolled patients with neurosyphilis, no patients were found not to respond to the antibiotic therapy, and the assessment of cytokine changes in refractory patients was deficient. Fourth, multivariate analysis cannot be performed due to the small sample size, and the practical value of the cut-off value in the diagnosis and efficacy evaluation of neurosyphilis remains to be evaluated. It was believed that further testing on a larger patient population is still needed.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur data showed that the cytokines G-CSF, ICAM-1, CCL4, CCL19 and IL-12p40 were highly expressed in the CSF of neurosyphilis patients, and their levels were significantly reduced after antibiotic treatment. Meanwhile, these cytokines were significantly correlated with various CSF clinical parameters, and the AUCs in ROC analysis were all greater than 0.9, suggesting that G-CSF, ICAM-1, CCL4, CCL19 and IL-12p40 in CSF may serve as potential biomarkers for neurosyphilis. Unfortunately, more meaningful results were not observed in the analysis at the serum level. Along with previous related reports, no new non-invasive detection measures that were expected to replace the traditional CSF measures were discovered from serum cytokine profiling. Therefore, it is believed that CSF measures are irreplaceable in the current clinical diagnosis, and the combined dynamic detection of multiple cytokines such as G-CSF, ICAM-1, CCL4, CCL19 and IL-12p40 in CSF can serve as a supplementary diagnostic tool to neurosyphilis and also can be used to monitor therapeutic effect.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eTP, Treponema pallidum\u003c/p\u003e\n\u003cp\u003eCSF, cerebrospinal fluid\u003c/p\u003e\n\u003cp\u003eNIND, non-inflammatory neurological diseases\u003c/p\u003e\n\u003cp\u003eROC, receiver operating characteristic\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWBCs, white blood cells\u003c/p\u003e\n\u003cp\u003eQalb, albumin quotient\u003c/p\u003e\n\u003cp\u003eAUC, area under the ROC curve\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCNS, central nervous system\u003c/p\u003e\n\u003cp\u003eTRUST, toluidine red unheated serum test\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTPPA, TP particle agglutination test\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll patients consented to the scientific use of their biological samples. The ethics committee of Hangzhou 7th People's Hospital approved scientific use of CSF and serum biological samples.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Project for Hangzhou Medical Disciplines of Excellence \u0026amp; Key Project for Hangzhou Medical Discipline. And the Research Fund of Health Commission of Zhejiang Province (2021429094) also provided support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShu Qi Wang and Xiao Ying Zhang conceived and designed this research. Xiao Ying Zhang, Meiduo gesang, Zheng Wang and Jie Fang Chen were responsible for selecting cases and sampling. Shu Mei Chi and Man Jin contributed to the medical records and typed data entry the tables. Shu Qi Wang and Xiao Ying Zhang searched literature. Meiduo Gesang, Shu Qi Wang and Xiao Ying Zhang planned and conducted the experiment. Shu Qi Wang and Ming Fen Song analyzed the data, including statistical tests, figures, and table production. Shu Qi Wang wrote the manuscript and Ming Fen Song corrected the draft of the manuscript. All authors have approved the final manuscript as submitted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our gratitude to all those who helped us during the writing of this study. A special acknowledgement should be shown to Dr Pan Yan and Dr Qian Qian Lin for useful instructions of statistics.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTel BC, Telli G, Onder S, et al. Investigation of the relationship between chronic montelukast treatment, asthma and depression-like behavior in mice. 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Journal of Neuroimmunology 2016;295\u0026ndash;296:12\u0026ndash;17. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jneuroim.2016.03.019\u003c/span\u003e\u003cspan address=\"10.1016/j.jneuroim.2016.03.019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Characteristics of patients.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" width=\"32.41379310344828%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" width=\"22.06896551724138%\"\u003e\n \u003cp\u003e\u003cstrong\u003eNIND (n=7)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"45.51724137931034%\"\u003e\n \u003cp\u003e\u003cstrong\u003eNeurosyphilis (n=9)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"48.10606060606061%\"\u003e\n \u003cp\u003e\u003cstrong\u003epre-therapy\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"51.89393939393939%\"\u003e\n \u003cp\u003e\u003cstrong\u003epost-therapy\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.41379310344828%\"\u003e\n \u003cp\u003eMale (%)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.06896551724138%\"\u003e\n \u003cp\u003e71.43%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"45.51724137931034%\"\u003e\n \u003cp\u003e66.67%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.41379310344828%\"\u003e\n \u003cp\u003eAge (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.06896551724138%\"\u003e\n \u003cp\u003e50.29\u0026plusmn;12.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"45.51724137931034%\"\u003e\n \u003cp\u003e53.78\u0026plusmn;14.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.41379310344828%\"\u003e\n \u003cp\u003eSerum-TRUST\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.06896551724138%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.896551724137932%\"\u003e\n \u003cp\u003e128(512-16)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.620689655172413%\"\u003e\n \u003cp\u003e32(128-8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.41379310344828%\"\u003e\n \u003cp\u003eCSF-TRUST\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.06896551724138%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.896551724137932%\"\u003e\n \u003cp\u003e8(32-1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.620689655172413%\"\u003e\n \u003cp\u003e2(8-1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.41379310344828%\"\u003e\n \u003cp\u003eCSF-WBC (\u0026times;10\u003csup\u003e6\u003c/sup\u003e/L)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.06896551724138%\"\u003e\n \u003cp\u003e1.314\u0026plusmn;0.778\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.896551724137932%\"\u003e\n \u003cp\u003e31.46\u0026plusmn;12.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.620689655172413%\"\u003e\n \u003cp\u003e3.567\u0026plusmn;1.735\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.41379310344828%\"\u003e\n \u003cp\u003eCSF-protein (g/l)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.06896551724138%\"\u003e\n \u003cp\u003e0.449\u0026plusmn;0.046\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.896551724137932%\"\u003e\n \u003cp\u003e1.029\u0026plusmn;0.408\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.620689655172413%\"\u003e\n \u003cp\u003e0.7278\u0026plusmn;0.182\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eData are given as median (range), or frequencies, or mean \u0026plusmn; standard deviation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAbbreviations: NIND, non-inflammatory neurological diseases; TRUST, toluidine red unheated serum test; CSF, cerebrospinal fluid; WBCs, white blood cells.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Well documented cytokines rediscovered in screen were consistent with the previous researches.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.779761904761905%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSyphilis Type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.9688%;\" width=\"11.30952380952381%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSample Type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.1445%;\" width=\"12.648809523809524%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMolecule\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.6934%;\" width=\"15.476190476190476%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMethod\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.1816%;\" width=\"26.785714285714285%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSupplementary References\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"33.779761904761905%\"\u003e\n \u003cp\u003eNeurosyphilis vs. NIND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 16.9688%;\" valign=\"top\" width=\"11.30952380952381%\"\u003e\n \u003cp\u003eCSF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.1445%;\" valign=\"top\" width=\"12.648809523809524%\"\u003e\n \u003cp\u003eCXCL11\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 12.6934%;\" valign=\"top\" width=\"15.476190476190476%\"\u003e\n \u003cp\u003eMB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 22.1816%;\" valign=\"top\" width=\"26.785714285714285%\"\u003e\n \u003cp\u003eLepennetier et al. (2019)\u003csup\u003e20\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.1445%;\" valign=\"top\" width=\"100%\"\u003e\n \u003cp\u003eCXCL13\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" width=\"33.779761904761905%\"\u003e\n \u003cp\u003eNeurosyphilis vs. Syphilis\u003c/p\u003e\n \u003cp\u003eNeurosyphilis vs. HC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 16.9688%;\" valign=\"top\" width=\"11.30952380952381%\"\u003e\n \u003cp\u003eCSF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.1445%;\" valign=\"top\" width=\"12.648809523809524%\"\u003e\n \u003cp\u003eCXCL13\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" style=\"width: 12.6934%;\" valign=\"top\" width=\"15.476190476190476%\"\u003e\n \u003cp\u003eELISA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" style=\"width: 22.1816%;\" valign=\"top\" width=\"26.785714285714285%\"\u003e\n \u003cp\u003eYan et al. (2017)\u003csup\u003e21\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.1445%;\" valign=\"top\" width=\"100%\"\u003e\n \u003cp\u003eIL-6\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.1445%;\" valign=\"top\" width=\"100%\"\u003e\n \u003cp\u003eIL-10\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 16.9688%;\" valign=\"top\" width=\"47.20496894409938%\"\u003e\n \u003cp\u003eSerum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.1445%;\" valign=\"top\" width=\"52.79503105590062%\"\u003e\n \u003cp\u003eIL-6\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.1445%;\" valign=\"top\" width=\"100%\"\u003e\n \u003cp\u003eIL-10\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.779761904761905%\"\u003e\n \u003cp\u003eSyphilis vs. HC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.9688%;\" valign=\"top\" width=\"11.30952380952381%\"\u003e\n \u003cp\u003eSerum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.1445%;\" valign=\"top\" width=\"12.648809523809524%\"\u003e\n \u003cp\u003echemerin\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.6934%;\" valign=\"top\" width=\"15.476190476190476%\"\u003e\n \u003cp\u003eELISA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.1816%;\" valign=\"top\" width=\"26.785714285714285%\"\u003e\n \u003cp\u003eStevenson et al. (2017)\u003csup\u003e22\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" width=\"33.779761904761905%\"\u003e\n \u003cp\u003e8 hours after antibiotic treatment for syphilis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 16.9688%;\" valign=\"top\" width=\"11.30952380952381%\"\u003e\n \u003cp\u003eSerum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.1445%;\" valign=\"top\" width=\"12.648809523809524%\"\u003e\n \u003cp\u003eIFN-\u0026gamma;\u0026darr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 12.6934%;\" valign=\"top\" width=\"15.476190476190476%\"\u003e\n \u003cp\u003eELISA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 22.1816%;\" valign=\"top\" width=\"26.785714285714285%\"\u003e\n \u003cp\u003ePastuszczak et al. (2017)\u003csup\u003e23\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.1445%;\" valign=\"top\" width=\"100%\"\u003e\n \u003cp\u003eTNF-\u0026alpha;\u0026darr;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.1445%;\" valign=\"top\" width=\"100%\"\u003e\n \u003cp\u003eIL-6\u0026darr;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"4\" valign=\"top\" width=\"33.779761904761905%\"\u003e\n \u003cp\u003eNeurosyphilis vs. Syphilis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 16.9688%;\" valign=\"top\" width=\"11.30952380952381%\"\u003e\n \u003cp\u003eCSF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.1445%;\" valign=\"top\" width=\"12.648809523809524%\"\u003e\n \u003cp\u003eCXCL13\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 12.6934%;\" valign=\"top\" width=\"15.476190476190476%\"\u003e\n \u003cp\u003eArray, ELISA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"4\" style=\"width: 22.1816%;\" valign=\"top\" width=\"26.785714285714285%\"\u003e\n \u003cp\u003eWang et al. (2016)\u003csup\u003e10\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.1445%;\" valign=\"top\" width=\"100%\"\u003e\n \u003cp\u003eCXCL10\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.1445%;\" valign=\"top\" width=\"100%\"\u003e\n \u003cp\u003eCXCL8\u0026uarr;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16.9688%;\" valign=\"top\" width=\"47.20496894409938%\"\u003e\n \u003cp\u003eSerum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.1445%;\" valign=\"top\" width=\"52.79503105590062%\"\u003e\n \u003cp\u003eCXCL13\u0026darr;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.779761904761905%\"\u003e\n \u003cp\u003eNeurosyphilis vs. Syphilis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.9688%;\" valign=\"top\" width=\"11.30952380952381%\"\u003e\n \u003cp\u003eCSF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.1445%;\" valign=\"top\" width=\"12.648809523809524%\"\u003e\n \u003cp\u003euPA\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.6934%;\" valign=\"top\" width=\"15.476190476190476%\"\u003e\n \u003cp\u003eArray, ELISA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.1816%;\" valign=\"top\" width=\"26.785714285714285%\"\u003e\n \u003cp\u003eLu et al. (2016)\u003csup\u003e24\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.779761904761905%\"\u003e\n \u003cp\u003eSy- vs Asy-mptomatic neurosyphilis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.9688%;\" valign=\"top\" width=\"11.30952380952381%\"\u003e\n \u003cp\u003eCSF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.1445%;\" valign=\"top\" width=\"12.648809523809524%\"\u003e\n \u003cp\u003eIL-17\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.6934%;\" valign=\"top\" width=\"15.476190476190476%\"\u003e\n \u003cp\u003eELISA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.1816%;\" valign=\"top\" width=\"26.785714285714285%\"\u003e\n \u003cp\u003eWang et al. (2014)\u003csup\u003e25\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.779761904761905%\"\u003e\n \u003cp\u003eNeurosyphilis-HIV vs. Syphilis-HIV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.9688%;\" valign=\"top\" width=\"11.30952380952381%\"\u003e\n \u003cp\u003eCSF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.1445%;\" valign=\"top\" width=\"12.648809523809524%\"\u003e\n \u003cp\u003eCXCL2\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.6934%;\" valign=\"top\" width=\"15.476190476190476%\"\u003e\n \u003cp\u003eELISA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.1816%;\" valign=\"top\" width=\"26.785714285714285%\"\u003e\n \u003cp\u003eTsai et al. (2014)\u003csup\u003e26\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"33.779761904761905%\"\u003e\n \u003cp\u003eNeurosyphilis vs. Syphilis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 16.9688%;\" valign=\"top\" width=\"11.30952380952381%\"\u003e\n \u003cp\u003eCSF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.1445%;\" valign=\"top\" width=\"12.648809523809524%\"\u003e\n \u003cp\u003eIL-17\u0026alpha;\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 12.6934%;\" valign=\"top\" width=\"15.476190476190476%\"\u003e\n \u003cp\u003eELISA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 22.1816%;\" valign=\"top\" width=\"26.785714285714285%\"\u003e\n \u003cp\u003ePastuszczak et al. (2013)\u003csup\u003e14\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.1445%;\" valign=\"top\" width=\"100%\"\u003e\n \u003cp\u003eIFN-\u0026gamma;\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"33.779761904761905%\"\u003e\n \u003cp\u003eNeurosyphilis-HIV vs. Syphilis-HIV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 16.9688%;\" valign=\"top\" width=\"11.30952380952381%\"\u003e\n \u003cp\u003eCSF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.1445%;\" valign=\"top\" width=\"12.648809523809524%\"\u003e\n \u003cp\u003eCXCL13\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 12.6934%;\" valign=\"top\" width=\"15.476190476190476%\"\u003e\n \u003cp\u003eELISA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 22.1816%;\" valign=\"top\" width=\"26.785714285714285%\"\u003e\n \u003cp\u003eMarra et al. (2010)\u003csup\u003e27\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 16.9688%;\" valign=\"top\" width=\"47.20496894409938%\"\u003e\n \u003cp\u003eSerum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.1445%;\" valign=\"top\" width=\"52.79503105590062%\"\u003e\n \u003cp\u003eCXCL13\u0026uarr;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviations: NIND, non-inflammatory neurological diseases; MB, magnetic beads; HC, healthy controls; ELISA, enzyme-linked immunosorbent assay; CSF, cerebrospinal fluid.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cytokines, Chemokine, Neurosyphilis, Neuro-inflammation, Cerebrospinal fluid.","lastPublishedDoi":"10.21203/rs.3.rs-1562299/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1562299/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eNeurosyphilis remains a chronic infectious disease within central nervous system infected by Treponema pallidum (TP). At present, the diagnosis and efficacy evaluation of neurosyphilis still facing clinical challenges. It has been reported that cytokines, which play multiple roles in neuroinflammation, showed abnormal expression patterns in neurosyphilis. To search for potential biomarkers of neurosyphilis, we analyzed the changes of cytokine profiles in cerebrospinal fluid (CSF) and serum of neurosyphilis. This may provide insight into clinical values for neurosyphilis.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eUsing the quantitative antibody array platform, we measured 80 cytokines expression in the CSF and serum of 16 subjects. 9 patients diagnosed with neurosyphilis, included samples before and after antibiotic treatment. 7 patients with non-inflammatory neurological diseases (NIND) were used as controls. Compared to the differences in cytokine expression between groups and analyzed to the correlations between cytokines and clinical CSF parameters. The ability of valuable biomarkers to diagnosis of neurosyphilis was evaluated by receiver operating characteristic (ROC) curves.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe observed significantly increased of multiple cytokines (26/80) in CSF of neurosyphilis were significantly increased compared to the NIND group. Concentrations of 15 cytokines were significantly decreased after treatment, of which 11 cytokines were down regulated to the levels were similar to the NIND group. Most CSF cytokine concentrations consistently correlated with clinical parameters, positively related to white blood cells (WBCs, 42/80), total protein (36/80), and albumin quotient (Qalb, 37/80) and negatively related to Glucose level (22/80). More than 40 cytokines concentrations correlated with each other in CSF, most of them were positively correlated. But correlations between CSF cytokine and serum cytokine values were scarce (6/80). The area under the ROC curve (AUC) of G-CSF, ICAM-1, CCL4, CCL19 and IL-12p40 in CSF were all greater than 0.9, with high diagnostic values.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur data showed that multiple CSF cytokines were highly expressed of neurosyphilis, and their levels were significantly reduced after antibiotic treatment. Meanwhile, these cytokines were significantly correlated with various CSF parameters. The AUCs of G-CSF, ICAM-1, CCL4, CCL19 and IL-12p40 were all greater than 0.9, suggesting that these cytokines may serve as potential biomarkers for neurosyphilis. Unfortunately, more meaningful results were not observed in the analysis at the serum level. Therefore, along with previous related reports, it is believed that CSF measures are irreplaceable in the current clinical diagnosis, and the combined dynamic detection of multiple cytokines such as G-CSF, ICAM-1, CCL4, CCL19 and IL-12p40 in CSF can serve as a supplementary diagnostic tool for neurosyphilis.\u003c/p\u003e","manuscriptTitle":"Cytokines in cerebrospinal fluid and serum as biomarkers are associated with the diagnosis and efficacy evaluation of neurosyphilis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-04-21 15:49:46","doi":"10.21203/rs.3.rs-1562299/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8fbb74e6-591a-4059-9ba3-508108434fc9","owner":[],"postedDate":"April 21st, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-05-23T21:29:09+00:00","versionOfRecord":[],"versionCreatedAt":"2022-04-21 15:49:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1562299","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1562299","identity":"rs-1562299","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}