SYPHILIS IS A SEXUALLY TRANSMITTED infection caused by the spirochete Treponema pallidum. A number of recent outbreaks have been reported.1 Of particular concern is an increase in rates of syphilis among HIV–1-infected men who have sex with other men because coinfection facilitates the transmission of either infection. Further, we and others have shown that coinfection is associated with peripheral CD4 T cell loss and plasma HIV RNA increase.2–4
Infection with T. pallidum activates leukocytes and macrophages through phagocytosis of T. pallidum, which leads to antigen presentation and secretion of cytokines such as interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12, and tumor necrosis factor (TNF)-α.5,6 The subsequent adaptive response to T. pallidum is believed to be of the Th1 type because primary and secondary syphilitic lesions express the Th1 cytokines interferon (IFN)-γ and IL-2.7,8 Experimental work suggest that some Th2-associated cytokines such as IL-10 also are expressed, whereas others, such as IL-4, are absent.8,9 In fact, the increase in IL-10 in an animal model correlated to a high treponemal burden.8 Although, cytokines exert a number of important immunomodulatory actions during disease and also regulate the expression of HIV-1, little is known about cytokine responses during coinfection with T. pallidum and HIV-1 or the mechanisms accounting for CD4 T cell decline and plasma HIV RNA increase.
In the current study, we investigated the impact of syphilis infection on plasma concentrations of the cytokines IL-2, IL-4, IL-6, IL-8, IL-10, IFN-γ, and TNF-α. Additionally, we examined the interplay between T. pallidum-induced cytokine secretion and correlates of HIV-1 disease progression.
MATERIALS AND METHODS
A retrospective cohort study of consecutive HIV-1 and syphilis-coinfected patients, attending the outpatient clinic at the Department of Infectious Diseases at Copenhagen University Hospital, Hvidovre. The clinic follows ∼1200 HIV-infected patients. Between early 2003 and late 2006, 46 patients infected with HIV-1 were diagnosed with syphilis either by screening or by self-approach.
HIV-positive patients attending the clinic have plasma samples drawn and frozen quarterly, thus plasma samples drawn 3 months before, during, and 3 months after the diagnosis of syphilis were available from the majority of patients.
Syphilis staging was done according to the Centers for Disease Control classification as described previously.2,10 Patient characteristics, choice of treatment, combination antiretroviral therapy, plasma HIV RNA viral load, peripheral CD4 T cell counts, and syphilis titres were recorded from patient records. The Scientific Ethical Committee of Copenhagen and Frederiksberg approved the study (01-2006-2921).
Plasma HIV RNA was determined by the Ultra Sensitive method (Roche Molecular Systems, Branchburg, NJ). The analyses had a lower limit of detection of 20 copies/mL. We assigned the value 19 copies/mL to samples recorded below. Viral loads of more than 150,000 copies/mL were not diluted further and assigned the value 150,000 copies/mL.
Blood was drawn into EDTA-coated Vacutainer tubes (Becton Dickinson, Plymouth, UK), separated by centrifuge, and then plasma was frozen at −80°C.
IL-2, IL-4, IL-6, IL-8, IL-10, IFN-γ, and TNF-α were measured using a high sensitive Luminex multiplex assay (Millipore, LINCO Research Inc, St. Charles, MO).
All measurements were performed in duplicates masked from clinical data. The coefficients of variation of the measurements were: IL-2 18.2%, IL-4 16.8%, IL-6 14.1%, IL-8 18.3%, IL-10 15.5%, TNF-α 17.6%, and IFN-γ 17.2%. In our laboratory, the lower limit of quantification was 2 pg/mL for all cytokines. Samples below the limit of quantification were assigned the value 2 pg/mL.
Values are presented as median with interquartile ranges (IQR). Comparison of the interindividual data between the primary and secondary, and latent syphilis groups was performed using the Mann-Whitney U-test or Fisher exact test, as appropriate. Intraindividual changes from before to during and during to after syphilis infection were analyzed using Wilcoxon’s signed rank test, whereas comparison of data for the group of patients having both before, during, and after samples was performed using the Friedman test. Correlation analysis was done by the Spearman test. Analyses were performed using Graphpad Prism (GraphPad Software, San Diego, CA) and SPSS version 13 (SSPS Inc., Chicago, IL).
Of the 46 patients identified, 10 were excluded due to missing or failing cytokine measurements, or participation in an IL-2 treatment trial.
There were 18 cases of primary and secondary and 18 cases of latent (early, late, or unknown) syphilis infection. The median age was 40 years (range, 20–61). Patients with primary and secondary syphilis were younger than patients with latent infection [39 years (20–50) vs. 43 years, (33–60) P = 0.04]. Thirty-five were men and all 36 reported men as their sexual partners. Patients received treatment with either penicillin G benzathine (n = 26) or oral doxycycline (n = 9). One case was treated at another hospital. Six individuals had a coincident diagnosis of syphilis and HIV-1. Of these, 4 had primary or secondary stage syphilis. Of individuals with primary and secondary stage syphilis, 50% (n = 9) received combination antiretroviral therapy (cART), whereas 67% (n = 12) of individuals with latent syphilis received cART (P = 0.69). Two patients commenced cART at the time of the diagnosis of syphilis. Samples taken before the diagnosis of syphilis were available from 25 (69%) patients and were taken a median of 119 days (range, 63–204) before the diagnosis of syphilis. After syphilis samples were available from 30 (83%) patients, these were taken a median of 98 days (range, 36–176) after the diagnosis of syphilis infection.
In primary and secondary stage syphilis, IL-10 levels were significantly increased [46.7 pg/mL (IQR, 28.4–78.9)] at the time of diagnosis of syphilis compared to the before levels [12.8 pg/mL (IQR, 11.0–27.8)] and returned to the before levels after treatment [13.0 pg/mL (IQR; 6.2–19.4); P = 0.008, Friedman test, Table 1 and Fig. 1]. TNF-α was increased at diagnosis of syphilis and returned to before levels after treatment [3.9 pg/mL (IQR, 3.3–9.6) vs. 9.0 pg/mL (IQR, 5.4–12.6) vs. 4.2 pg/mL (IQR 2.7–6.8); P = 0.008, Friedman test, Table 1 and Fig. 1]; however, only the decrease from during to after syphilis infection was found to be significant when doing pair-wise comparisons (P <0.001). IL-6 and IL-8 decreased after treatment (P = 0.042 for both comparisons), but no changes were found from before to during syphilis infection or when comparing all 3 time points. Levels of IL-2 and IL-4 were unchanged in primary and secondary syphilis infection. CD8 and CD4 T cell counts decreased significantly at the time of diagnosis and returned to before levels after treatment of syphilis (P = 0.008 and P = 0.021, respectively, Friedman test). Although the median plasma HIV RNA viral load increased from 1.30 log10 copies/mL (IQR; 1.28–3.17) before syphilis infection to 2.58 log10 copies/mL (IQR; 1.30–5.07) during syphilis infection and decreased to 1.71 log10 copies/mL (IQR; 1.29–4.91) after syphilis, these changes were not significant (P >0.05) as apparent from the wide interquartile ranges of the measurements.
In latent syphilis cytokines and CD4 and CD8 T cell counts were unchanged (Table 1). No changes were observed when comparing before versus during syphilis plasma HIV RNA viral load or when comparing all 3 measurements of plasma HIV RNA; however, plasma HIV RNA values decreased from 1.30 (IQR, 1.28–4.11) log10 copies/mL during to 1.28 (IQR, 1.28–2.53) log10 copies/mL (P = 0.03) after syphilis.
Patients with primary and secondary stage syphilis had significantly higher levels of TNF-α, IL-6, and IL-10 compared to patients with latent syphilis patients at the time of diagnosis of syphilis infection (P = 0.007, 0.033, and 0.002, respectively). There were no significant differences in IL-2, IL-4, IL-8, IFN-γ, CD4 T cell counts, or plasma HIV RNA between primary and secondary stage and latent syphilis.
At diagnosis, IL-10, TNF-α, and HIV RNA correlated inversely with CD4 T cell counts (r = −0.35, P = 0.036; r = −0.34, P = 0.042; and r = −0.38, P = 0.022; respectively). There was no correlation between IL-2, IL-4, IL-6, IL-8, IFN-γ, or CD4 T cell counts.
There were positive correlations between both IL-10 and TNF-α levels and plasma HIV RNA viral load at the time of diagnosis (r = 0.38, P = 0.023 and r = 0.64, P <0.0001). IL-4 correlated inversely with HIV RNA (r = −0.38; P = 0.022). Neither IL-2, IL-6, IL-8 nor IFN-γ correlated with plasma HIV RNA viral load. Rapid plasma reagin values and levels of cytokines did not correlate (IL-6, r = 0.13, P = 0.46; IL-10, r = 0.02, P = 0.91; and TNF-α, r = 0.06, P = 0.74).
All analyses were repeated separately for patients receiving and not receiving cART. The results were unaffected by treatment status.
To our knowledge, this is the first time that plasma cytokine responses have been investigated during coinfection with syphilis and HIV-1. We found that the concentrations of the proinflammatory cytokine TNF-α and the anti-inflammatory cytokine IL-10 were increased during primary and secondary syphilis infection. Cytokine levels during latent infection were unchanged.
We and others have recently shown that peripheral blood CD4 T cell counts decline during syphilis and HIV-1 coinfection.2–4 This decline is most likely due to increased cell turn-over, apoptosis, and alternations in T cell homeostasis.11,12 However, it is notable that increased IL-10 concentrations have been associated with progression of disease and CD4 T cell loss in chronic HIV-1 infection.13 Indeed, higher levels of IL-10 were associated with lower CD4 T cell counts and higher plasma HIV RNA viral loads in the current study.
Similarly, it has been shown that primary and secondary stage syphilis increases plasma HIV RNA viral load.2–4 We found an increase in TNF-α concentrations in this group of patients and a correlation between TNF-α and plasma HIV RNA. This correlation is well-established,14 and it is known that TNF-α increases HIV-1 replication via activation of nuclear factor-κB.15 However, we were not able to show a significant increase in plasma HIV RNA during syphilis infection in the current study. Interestingly, in vitro experiments indicate that IL-10 cooperates with TNF-α and IL-6 to activate HIV-1 from infected cells.16–18
IL-10 is an important anti-inflammatory regulator of innate and adaptive immunity. IL-10 acts in conjunction with other regulatory proteins to prevent the protective immune response from leading to the development of excessive immunopathologic lesions.19–21 However, IL-10 is as well involved in persistence of bacteria and viruses by interfering with innate and adaptive protective immunity. Moreover, pathogens have been shown to hijack and exploit IL-10 to dampen the host immune response and stall their elimination.19–22 Certainly, in a related spirochetal infection, IL-10 knock-out mice infected with Borrelia burgdorferi rapidly cleared infection compared to wild-type mice.23 In a rabbit model of syphilis, increase in IL-10 correlated to a high treponemal burden.8 In accordance, we found that IL-10 was elevated in primary and secondary stage syphilis when the treponemal burden is believed to peak but not in latent syphilis when the burden is low. It is tempting to speculate that elevated levels of IL-10 may help T. pallidum to evade the immune system and thus establish a latent infection.
IL-4 has been shown to down-regulate HIV-1 expression in vitro.24 We did find an inverse correlation between IL-4 and plasma HIV RNA during syphilis infection. However, we did not see significant changes in IL-4 concentrations during infection. Experimental findings support this observation because only negligible amounts of IL-4 mRNA were detected throughout early infection with syphilis in a rabbit model.8
We were unable to show an increase in either IL-2 or IFN-γ as may be expected in a cellular adaptive (Th1) response to a pathogen. We hypothesize that IL-2 and IFN-γ may be secreted locally or alternatively that the high levels of IL-10 inhibit IL-2 and INF-γ secretion from CD4 T lymphocytes.7 Alternatively, IFN-γ may be secreted in high levels in the early stages of the infection, where it is involved in controlling the infection and lesion resolution.8 The samples from patients with primary and secondary stage syphilis infection in our study may have been obtained at a time where the IFN-γ concentrations have already decreased.
Possible limitations include the retrospective cohort study design. The before, during, and after time ranges vary from patient to patient, and for some of the patients with latent syphilis, the before values were measured at a time when the patients were already infected with syphilis; this weakens the before to during changes in this group. Therefore and because of the small sample size and low statistical power, we might have failed to detect some significant changes, whereas other significant changes might have arisen by chance alone. Our study was conducted in patients who were enrolled in an HIV care program in Copenhagen and only included 1 female. Consequently, the results may not be applicable for all HIV- and syphilis-coinfected patients. No samples from non-HIV infected were available thus it is impossible from the current study to conclude which effect the HIV infection and cART had on the observed changes in the cytokine concentrations and T-cell counts. It is of course well known that the initiation of cART has profound effects on the immune system. However, the fact that we included both primary and secondary and latent stages of syphilis infection makes it possible to compare the effects on the immune system of different stages of syphilis. One of the strengths of this study is that we were able to obtain samples from approximately 3 months before the diagnosis of syphilis from the majority of patients. Finally, several factors may influence the cytokine measurements, such as the presence of interfering heterophilic antibodies and other plasma proteins that might skew results,25 and our results may not be directly comparable to results obtained using other cytokine assays.25
In conclusion, we show for the first time in humans that IL-10 and TNF-α increased at the time of diagnosis of primary and secondary but not latent syphilis infection during concomitant HIV-1 infection. Further, TNF-α and IL-10 correlated with low CD4 T cell counts and high plasma HIV RNA viral load. Our findings emphasize the importance of early detection and treatment of syphilis in HIV-infected patients.
1. Centers for Disease Control and Prevention. Trends in primary and secondary syphilis and HIV infections in men who have sex with men—San Francisco and Los Angeles, California, 1998–2002. MMWR Morb Mortal Wkly Rep 2004; 53:575–578.
2. Kofoed K, Gerstoft J, Mathiesen LR, et al. Syphilis and human immunodeficiency virus (HIV)-1 coinfection: Influence on CD4 T-cell count, HIV-1 viral load, and treatment response. Sex Transm Dis 2006; 33:143–148.
3. Buchacz K, Patel P, Taylor M, et al. Syphilis increases HIV viral load and decreases CD4 cell counts in HIV-infected patients with new syphilis infections. AIDS 2004; 18:2075–2079.
4. Palacios R, Jimenez-Onate F, Aguilar M, et al. Impact of syphilis infection on HIV viral load and CD4 cell counts in HIV-infected patients. J Acquir Immun Defic Syndr 2007; 44:356–359.
5. Baker-Zander SA, Lukehart SA. Macrophage-mediated killing of opsonized Treponema pallidum
. J Infect Dis 1992; 165:69–74.
6. Radolf JD, Arndt LL, Akins DR, et al. Treponema pallidum
and Borrelia burgdorferi
lipoproteins and synthetic lipopeptides activate monocytes/macrophages. J Immunol 1995; 154:2866–2877.
7. Van Voorhis WC, Barrett LK, Koelle DM, et al. Primary and secondary syphilis lesions contain mRNA for Th1 cytokines. J Infect Dis 1996; 173:491–495.
8. Leader BT, Godornes C, VanVoorhis WC, et al. CD4+ lymphocytes and gamma interferon predominate in local immune responses in early experimental syphilis. Infect Immun 2007; 75:3021–3026.
9. Podwinska J, Lusiak M, Zaba R, et al. The pattern and level of cytokines secreted by Th1 and Th2 lymphocytes of syphilitic patients correlate to the progression of the disease. FEMS Immunol Med Microbiol 2000; 28:1–14.
10. Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines 2002. MMWR Morb Mortal Wkly Rep 2002; 51:1–78.
11. Mellors JW, Munoz A, Giorgi JV, et al. Plasma viral load and CD4+ lymphocytes as prognostic markers of HIV-1 infection. Ann Intern Med 1997; 126:946–954.
12. McCune JM. The dynamics of CD4+ T-cell depletion in HIV disease. Nature 2001; 410:974–979.
13. Stylianou E, Aukrust P, Kvale D, et al. IL-10 in HIV infection: Increasing serum IL-10 levels with disease progression-down-regulatory effect of potent anti-retroviral therapy. Clin Exp Immunol 1999; 116:115–120.
14. Aukrust P, Müller F, Lien E, et al. Tumor necrosis factor (TNF) system levels in human immunodeficiency virus-infected patients during highly active antiretroviral therapy: persistent TNF activation is associated with virologic and immunologic treatment failure. J Infect Dis 1999; 179:74–82.
15. Poli G, Kinter A, Justement JS, et al. Tumor necrosis factor α functions in an autocrine manner in the induction of human immunodeficiency virus expression. Proc Natl Acad Sci U S A 1990; 87:782–785.
16. Weissman D, Poli G, Fauci AS. IL-10 synergizes with multiple cytokines in enhancing HIV production in cells of monocytic lineage. J Acquir Immune Defic Syndr Hum Retrovirol 1995; 9:442–449.
17. Finnegan A, Roebuck KA, Nakai BE, et al. IL-10 cooperates with TNF-alpha to activate HIV-1 from latently and acutely infected cells of monocyte/macrophage lineage. J Immunol 1996; 156:841–851.
18. Rabbi MF, Finnegan A, Al-Harthi L, et al. Interleukin-10 enhances tumor necrosis factor-alpha activation of HIV-1 transcription in latently infected T cells. J Acquir Immune Defic Syndr Hum Retrovirol 1998; 19:321–331.
19. Couper KN, Blount DG, Riley EM. IL-10: The master regulator of immunity to infection. J Immunol 2008; 180:5771–5777.
20. Redpath S, Ghazal P, Gascoigne NR. Hijacking and exploitation of IL-10 by intracellular pathogens. Trends Microbiol 2001; 9:86–92.
21. Mege JL, Meghari S, Honstettre A, et al. The two faces of interleukin 10 in human infectious diseases. Lancet Infect Dis 2006; 6:557–569.
22. Brooks DG, Trifilo MJ, Edelmann KH, et al. Interleukin-10 determines viral clearance or persistence in vivo. Nat Med 2006; 12:1301–1309.
23. Lazarus JJ, Meadows MJ, Lintner RE, et al. IL-10 deficiency promotes increased Borrelia burgdorferi
clearance predominantly through enhanced innate immune responses. J Immunol 2006; 177:7076–7085.
24. Schuitemaker H, Kootstra NA, Koppelman MH, et al. Proliferation-dependent HIV-1 infection of monocytes occurs during differentiation into macrophages. J Clin Invest 1992; 89:1154–1160.
25. Kofoed K, Schneider UV, Scheel T, et al. Development and validation of a multiplex add-on assay for sepsis biomarkers using xMAP technology. Clin Chem 2006; 52:1284–1293.