Treponema pallidum, the bacterium that causes syphilis, disseminates to the cerebrospinal fluid (CSF) and meninges very early during the infection.1 It is assumed that inflammatory response associated with early invasion of the central nervous system (CNS) with T. pallidum may be restrained in most individuals, even without therapy targeted to achieve treponemicidal concentrations in the CSF. It is less clear, however, why the clearance of T. pallidum is not effective in some patients, and the disease may progress further (ie, to symptomatic neurosyphilis).2,3
Asymptomatic neurosyphilis may occur during every stage of syphilis and is defined by CSF abnormalities (ie, reactive Venereal Disease Research Laboratory [VDRL], pleocytosis, and elevated protein concentration) in a patient without any neurological signs and symptoms.4 The clinical importance of identifying asymptomatic neurosyphilis is still a matter of debate because it may progress only in some individuals.
It has been shown that T cells are important components of the immune response against T. pallidum, although current understanding of the adaptive immune response in syphilis is based on very few studies.5 It is widely accepted that T. pallidum infection evokes a vigorous immune response in the early stages of infection characterized by Th1 predominance. Moreover, by studying a nonhuman model of syphilis infection, Marra et al.6 demonstrated that CD4+ lymphocytes could play a pivotal role in the clearance of treponemas from the CNS. Little is known, however, about the adaptive immune response in the CSF of patients with neurosyphilis.
The Th1/Th2 concept has recently been extended to include a new population of T-helper cells named Th17, based on their secretion of interleukin (IL)-17A. Th17 lymphocytes participate in host defense against many microorganisms, although they also contribute to immunopathology during infection.7 Only recently, Stary et al.8 identified IL-17A–producing cells in humans with syphilis. Increased frequency of Th17 cells has also been found in the peripheral blood of patients with secondary syphilis.9 There are no data, however, concerning whether Th17 cells and their associated cytokines contribute to inflammatory response in neurosyphilis.
Here, we measured the levels of 3 T-helper cell–associated cytokines (namely, IL-4, IL-17, and IFN-γ both in the sera and in the CSF of patients with secondary and early latent syphilis, to compare cytokine profiles in the neurosyphilis and no-neurosyphilis group.
MATERIALS AND METHODS
Thirty-three patients (all male, aged 19–53 years) with newly diagnosed secondary and early latent syphilis were enrolled in the study that took place in the Department of Dermatology (Jagiellonian University School of Medicine, Cracow, Poland) between November 2011 and August 2012. Exclusion criteria were as follows: contraindication to lumbar puncture, pregnancy, anti-inflammatory or immunosuppressive therapy, treatment with antibiotics within the last 6 months, and other chronic inflammatory disorders (eg, autoimmunity). Syphilis staging was done by a specialist in dermatology and venerology and was based on clinical history, physical examination, and laboratory findings according to the Centers for Disease Control and Prevention classification.10 All patients were serologically positive for serum T. pallidum hemagglutionation assay (TPHA), fluorescent treponemal antibody (FTA), FTA absorbed, and VDRL tests. Eight patients (24%) were HIV positive. Three HIV-positive individuals received antiretroviral therapy resulting in a viral load of less than 50 RNA copies/mL plasma. All study participants were also evaluated by a certified neurologist for the assessment of neurological symptoms.
For the purpose of the study, asymptomatic neurosyphilis was defined as (1) any stage of syphilis and (2) a reactive CSF-VDRL, and (3) no neurological symptoms. Presumptive neurosyphilis was defined as (1) any stage of syphilis and CSF pleocytosis (ie, ≥5 cells/μL). Based on this definition, patients with syphilis were divided into 3 groups: (1) patients with positive CSF-VDRL result (neurosyphilis group, n = 5), (2) patients with presumptive neurosyphilis (presumptive neurosyphilis group, n = 6), and (3) patients with negative CSF-VDRL result without CSF pleocytosis (no-neurosyphilis group, n = 22). The epidemiological and clinical characteristics of the studied groups are shown in Table 1. All patients from the neurosyphilis group and presumptive neurosyphilis group were treated with penicillin regimens appropriate for neurosyphilis (ie, intravenously crystalline penicillin). No-neurosyphilis individuals were treated with intramuscular benzathine penicillin.
All study participants gave their informed written consent, and the study was approved by the Jagiellonian University Bioethical Committee.
Laboratory Investigations and CSF Sampling
Blood samples were taken at the time of entry into the study for routine laboratory testing. For cytokine measurements, aliquots of serum were stored at −80°C until analysis. Lumbar puncture and CSF sampling was performed in all patients with syphilis. Cerebrospinal fluid samples were sent for routine examination (ie, cell count, glucose, and protein concentration) and serology testing (ie, VDRL, TPHA, and FTA). Aliquots of CSF were centrifuged and stored at −80°C until cytokine measurements were executed.
Serum and CSF cytokine levels were measured using a high-sensitive enzyme-linked immunosorbent assay (IFN-γ: R&D Systems, Minneapolis, MN; IL-4 and IL-17A: eBiosciences, San Diego, CA), according to instructions provided by the manufacturers. The lower detection limits for serum and CSF were as follows: IFN-γ, 0.08 pg/mL; IL-4, 0.13 pg/mL; and IL-17A, 0.12 pg/mL. Samples below the threshold of quantification were assigned the value of lower limit of detection.
Statistical analysis was performed with the Statistica 7.1 PL package (StatSoft, Inc, 2005). Data are expressed as median and interquartile range if not otherwise stated. Between-group comparisons were performed with the Mann-Whitney U test or χ 2 test. The associations between the individual parameters were measured using Spearman rank correlation coefficient. A P value less than 0.05 was considered statistically significant.
Characteristics of the Patients and CSF Abnormalities
Thirty-three patients with secondary and early latent syphilis were included in the study. Five individuals (15%) had a positive CSF-VDRL test result. All of them had CSF pleocytosis (≥5 cells/μL) and an increased protein CSF concentration (≥45 mg/dL). Because the patients with positive CSF-VDRL test result had no neurological symptoms, they were diagnosed as having asymptomatic neurosyphilis. Six patients had CSF pleocytosis but a negative CSF-VDRL test result (presumptive neurosyphilis group). Three individuals from the presumptive neurosyphils group had additionally elevated CSF protein concentration (ie, ≥0.45 mg/dL). The CSF-TPHA test result was also positive in all 5 patients with a positive CSF-VDRL test result. The neurosyphilis group was compared with syphilitic patients with a presumptive neurosyphilis (n = 6) and with those with a negative CSF-VDRL test result without CSF pleocytosis (n = 22; no-neurosyphilis group). Cerebrospinal fluid samples from all no-neurosyphilis patients had a negative TPHA assay result. Only 1 patient from the presumptive neurosyphilis group had CSF-TPHA positive test result. The characteristics of the neurosyphilis, presumptive neurosyphilis, and no-neurosyphilis groups are shown in Table 1.
Groups did not differ with respect to age, disease duration, clinical symptoms (except hair loss), baseline titer of serum VDRL and FTA, white blood cell count, lymphocyte count, and serum glucose levels (Table 1). Patients with positive CSF-VDRL test results (n = 5) when compared with no-neurosyphilis group (n = 22) were characterized by a significantly higher level of CSF-WBC (P = 0.00002), higher CSF protein concentration (P = 0.001), and lower CSF glucose concentration (P = 0.02) (Table 1). Cerebrospinal fluid results (pleocytosis and elevated protein concentration) in patients with syphilis classified according to the presence of HIV infection and positive CSF-VDRL test result (confirmed diagnosis of neurosyphilis) are summarized in Table 2.
Cytokine Concentrations in CSF and Serum
Serum levels of IFN-γ, IL-4, and IL-17A were similar in all studied groups (data not shown). Patients with neurosyphilis had significantly higher levels of CSF IL-17A (2.31 pg/mL vs. 0.29 pg/mL; P = 0.005) and CSF IFN-γ (1.02 pg/mL vs. 0.13 pg/mL; P = 0.02) when compared with the no-neurosyphilis group. Mean CSF IL-17A and CSF IFN-γ levels in the presumptive neurosyphilis group were similarly high to those found in the neurosyphilis group (1.55 and 1.04 pg/mL, respectively; Fig. 1). All measurements of CSF IL-4 were below the detection limit of the assay.
In all patients studied, CSF concentrations of IFN-γ correlated with serum IFN-γ (r = 0.42, P = 0.01), CSF IL-17A (r = 0.4, P = 0.01), and CSF pleocytosis (r = 0.61, P = 0.0001). Cerebrospinal fluid IL-17A levels correlated with CSF pleocytosis (r = 0.6, P = 0.0001) and CSF protein concentration (r = 0.47, P = 0.004). Interestingly enough, there was no correlation between CSF and serum IL-17 levels. In addition, serum IFN-γ levels inversely correlated with the duration of T. pallidum infection (r = −0.48, P = 0.03).
Analyzing all patients, we observed a strong correlation between CSF pleocytosis, CSF protein concentration (r = 0.54, P = 0.0008), and CSF glucose concentration (r = −0.38, P = 0.04).
Finally, we compared CSF pleocytosis, CSF protein concentration, and CSF levels of analyzed cytokines in HIV-infected and HIV-uninfected patients with CSF pleocytosis. There were no significant differences found between both groups (data not shown).
Studies from the preantibiotic era demonstrate that approximately 25% of infected patients did not clear T. pallidum organisms from the CNS.11 Moreover, the extent of abnormalities in the CSF (such as pleocytosis, elevated CSF protein concentration, and positive CSF Wasserman test result) correlated with the probability of developing neurological complications. In the penicillin era, the incidence of neurosyphilis has decreased. However, still a few individuals fail to clear CSF abnormalities after penicillin treatment.12 The mechanisms of this phenomenon still remain unclear.
The diagnosis of neurosyphilis, especially the asymptomatic form (ie, only CSF abnormalities without any neurological symptoms), is a widely debated issue. Currently, it is acknowledged that a positive CSF-VDRL test result is specific and establishes the diagnosis of neurosyphilis. However, the sensitivity of a CSF-VDRL test in patients with a clinical diagnosis ranges between 30% and 70%.13–15 Therefore, some authorities suggest that the diagnosis of neurosyphilis in cases with a negative CSF-VDRL test result may rely on another CSF abnormalities (ie, pleocytosis, CSF FTA absorbed test, CSF protein concentration).10 Altogether, in the current study, 16 patients (48%) had at least 1 CSF abnormality, with elevated CSF protein concentration being the most common. However, only 5 individuals (15%) had a confirmed diagnosis of neurosyphilis according to Centers of Disease Control and Prevention criteria (ie, positive CSF-VDRL test result). There were 6 remaining individuals with a negative CSF-VDRL test result but with CSF pleocytosis (3 of them had additionally elevated CSF protein concentration). In those patients, the diagnosis of neurosyphilis was presumptive. Our results are consistent with previous studies that estimated the frequency of neurosyphilis in early syphilis between 13.5% and 20%.2
Cell-mediated immunity is thought to be of critical importance in antitreponemal host defense. The infiltration of skin lesions with T cells and macrophages in primary and secondary syphilis, together with the presence of IL-2 and IFN-γ, suggests the dominance of a classic Th1 immune response.16,17 T cells are believed to mediate the eradication of treponemas primarily through the production of cytokines such as IFN-γ, therefore activating macrophages, which further kill opsonized spirochetes. Indeed, the numbers of IFN-γ–producing T cells in skin lesions peak roughly at the same time as T. pallidum. Early responses were followed by macrophage infiltration a few days later, which coincided with a sharp decline in lesional spirochetes.18 Similar mechanisms were observed during the clearance of T. pallidum from the CNS in the nonhuman model of syphilis.6
The paradigm of the Th1/Th2 immune response has recently been expanded to include a population of T cells called Th17, based on their secretion of a potent proinflammatory cytokine IL-17. Although IL-17–producing cells were considered to play a key role in the induction and development of tissue injury in certain autoimmune diseases, growing evidence indicates that they may also play a key role in host defense against microorganisms. It has also been suggested that IL-17 may bridge the innate and adaptive immune responses by supporting the generation of Th1 adaptive immunity.7
To determine both local and systemic immune responses, we have collected serum and CSF samples from 33 patients with syphilis and measured the concentration of cytokines (IFN-γ, IL-4, and IL-17A) representing the 3 major T-helper cell subsets. Although we used very sensitive enzyme-linked immunosorbent assays, serum levels of measured cytokines in both studied groups were very low and did not differ from the healthy control group (data not shown). However, CSF analysis revealed a very significant, approximately 8-fold increase in the concentration of IL-17A and IFN-γ in patients with neurosyphilis. Interestingly enough, there was no correlation between CSF and serum levels of IL-17A, and only a weak correlation regarding INFγ levels. Nevertheless, CSF levels of both IL-17A and IFN-γ were substantially higher when compared with serum levels (5-fold and 12-fold, respectively). This observation indicates that in asymptomatic neurosyphilis, both cytokines are produced intrathecally. In addition, we have found that also patients with CSF pleocytosis but a negative CSF-VDRL test result (presumptive neurosyphilis group) had mean CSF levels of IFN-γ and IL-17A similar to those found in the neurosyphilis group. This may suggest an important role of IL-17A and IFN-γ in the inflammatory response in neurosyphilis. Further studies on larger groups are needed to determine the usefulness of CSF IL-17A and CSF IFN-γ as laboratory markers of neurosyphilis.
HIV-related immunosuppression may be the single factor that influences T-cell response against T. pallidum infection. Of all participants studied, 8 (24%) were infected with HIV, but their CD4+ cell count, viral load, and clinical picture did not indicate a significant degree of immunosuppression. In the current study, 4 patients from the presumptive neurosyphilis group were HIV infected. Thus, one may argue that in this group, CSF pleocytosis and, consequently, elevated CSF IFN-γ and CSF IL-17A levels are more likely caused by HIV than by syphilis. On the other hand, when comparing 2 groups—HIV-infected and HIV-uninfected patients with CSF pleocytosis, we did not find differences in CSF cytokines levels, CSF pleocytosis, and CSF protein concentration.
This study has some limitations. First, the sample size is limited. Despite increasing number of cases worldwide, syphilis remains a relatively rare disease in developed countries (up to 2.5 cases/100-000 per year in Poland). Second, we do not use the “gold standard” for diagnosis of neurosyphilis (ie, rabbit infectivity test). Finally, we did not evaluate clinical end points to answer the question whether higher CSF levels of IL-17A and INFγ were related to a worse clinical outcome (eg, progression to symptomatic neurosyphilis, poor serological response to treatment, and relapse of symptoms)
In summary, we showed for the first time that patients with syphilis who had asymptomatic neurosyphilis had higher levels of IL-17A and INF-γ in the CSF compared with those without neurosyphilis. Cerebrospinal fluid IL-17A and CSF IFN-γ levels in patients with CSF pleocytosis but negative CSF-VDRL test result were similarly high to those found in the neurosyphilis group. However, it remains to be evaluated (1) which cells are the main source of IL-17A and IFN-γ in the CSF of patients with neurosyphilis and (2) if there are any differences in CSF cytokine levels between patients with asymptomatic and symptomatic disease.
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