Correspondence: Fengchao Wang, Department of Clinical Laboratory Science, The First Affiliated Hospital of Bengbu Medical College, 287 Chang Huai Road, Bengbu 233004, China. Tel.: +86 552 3086272; fax: +86 552 3086277; e-mail: firstname.lastname@example.org
T-helper (Th) 17 and the more recently identified Th22 cells are of great importance in host defense against pathogens, but can also be responsible for chronic inflammatory disorders. However, the roles of the two cell subsets in syphilis remain elusive. In this study, we show that the frequencies of Th17 and Th22 cells are significantly increased in the peripheral blood of patients with secondary syphilis (SS). A significant positive correlation is observed between Th17 and Th22 cells, whereas a negative correlation exists between Th17 and Th1 cells. Moreover, the frequency of Th17 cells has a significant positive correlation with the plasma interleukin 6 (IL-6) or IL-1β levels, and the frequency of Th22 cells is positively correlated with the IL-6 or IL-23 levels. Finally, the elevated frequencies of Th17 and Th22 cells are positively associated with plasma C-reactive protein levels. Our results suggest that Th17 and Th22 cells may be implicated in the pathogenesis of the SS.
Syphilis is a sexually transmitted disease (STD) caused by the spirochetal bacterium Treponema pallidum (Tp), which is able to disseminate systemically and induce chronic inflammation in diverse tissues and organ systems (LaFond & Lukehart, 2006). Being a multistage illness, syphilis begins with the development of primary lesions or chancre at the spot of infection. In most instances, early syphilis usually goes undiagnosed and untreated for several weeks or several months until it progresses to the secondary stage.
The early immune response against Tp is characteristic of delayed-type hypersensitivity. Tp components preferentially activate Th1 lymphocytes, which contribute to bacterial clearance by cytokine-activated macrophages (Lukehart, 1982; Van Voorhis et al., 1996; Bouis et al., 2001; Leader et al., 2007). In contrast, a strong Th2-mediated humoral immune response is associated with prolonged infection and progression to the late syphilis (Podwinska et al., 2000).
Th17 is a unique CD4+ T-cell subset defined by its characteristic production of interleukin 17A (IL-17A) (Miossec et al., 2009). Th17 cells are involved in the pathogenesis of multiple inflammatory and autoimmune disorders because of the broad distribution of the IL-17 receptors (Da Silva et al., 2008; Ishigame et al., 2009). Mounting evidences have shown that Th17 cells are crucial effector cells in several chronic inflammatory diseases that were considered to be Th1-mediated, such as multiple sclerosis (Kebir et al., 2007), rheumatoid arthritis (Shen et al., 2009), inflammatory bowel disease (Fujino et al., 2003), systemic lupus erythematosus (Wong et al., 2008), and psoriasis (Kagami et al., 2010). Th22 represents a distinct T-cell subset, which is characterized by secretion of IL-22 but not interferon-γ (IFN-γ) or IL-17A (Eyerich et al., 2009; Trifari et al., 2009). By virtue of the specific expression of the CC chemokine receptor 6 (CCR6) as well as the skin-homing receptors CCR4 and CCR10, Th22 cells are allowed for localization to the skin and play important roles in inflammatory skin disorders (Duhen et al., 2009). However, the roles of Th17 and Th22 cells in syphilis are currently unknown.
In this study, peripheral blood IL-17A- and IL-22-expressing cells in patients with secondary syphilis (SS) were measured through FACS intracellular cytokine staining. We further correlated the frequency of IL-17A- or IL-22-expressing cells with that of IFN-γ-producing Th1 cells or IL-4-producing Th2 cells, and with TRUST titer or plasma C-reactive protein (CRP) level to elucidate the proinflammatory roles of Th17 and Th22 cells in SS.
Materials and methods
Patients and control subjects
This study included 32 patients with newly diagnosed SS from April 2010 to June 2011 at the Department of Dermatology & STD, the First Affiliated Hospital of Bengbu Medical College, Anhui, China. The diagnosis was based on a compatible history, the appearance of skin lesions, and the results of nontreponemal and treponemal tests. The clinical and epidemiological features of the 32 patients are listed in Table 1. Twenty healthy volunteers were recruited as health controls (HCs). The two groups were well matched on gender and age. Patients who were HIV positive, pregnant, receiving anti-inflammatory or immunosuppressive medications, treated with antibiotics recently, with a history of a chronic dermatitis (i.e., eczema or psoriasis) and other underlying acute or chronic disease were all excluded from this study. Informed consent was obtained from each participant, and the study protocol was approved by the Ethics Committee of Bengbu Medical College, Anhui, China.
Table 1. Clinical and epidemiological features of study subjects
No. of patients
36.5 ± 12.8
Disease duration (days)
Type of skin lesions, no./total
TRUST titer, no./total
1 : 8
1 : 16
1 : 32
1 : 64
1 : 128
CRP (mg L−1)
15.92 ± 10.03
Clinical laboratory assays
Toluidine red unheated serum test (TRUST; Rongsheng Biotech, China) was used in combination with treponemal pallidum particle agglutination (TPPA; Flijirebio Inc, Japan) for the serological diagnosis of SS. Plasma CRP levels were measured by immunoturbidimetric method via an automated chemistry analyzer (AU-5400; Olympus Optical Co, Tokyo, Japan). All tests were performed in accordance with the manufacturers' instructions.
Cell stimulation and intracellular cytokine analysis
Fresh heparinized peripheral whole blood (500 μL) was diluted in an equal volume of PBS (pH 7.2) followed by incubation at 37 °C 5% CO2 for 5 h in the presence of 50 ng mL−1 of phorbol myristate acetate (PMA; Alexis Biochemicals, San Diego, CA) and 1 μg mL−1 of ionomycin (Alexis). Monensin (2.0 μΜ Alexis) was added after 2 h of incubation. Cells were harvested and stained with allophycocyanin-conjugated anti-CD3 (Caltag Laboratories, San Francisco, CA) and FITC-conjugated anti-CD8 (Invitrogen) or FITC-conjugated anti-TCRγδ (Invitrogen). After lysis of red blood cells, cells were then washed twice, fixed, and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences). Intracellular staining was performed using PerCPCy5.5-conjugated anti-IL-17A (eBioscience San Diego, CA), PE-conjugated anti-IL-22 (R&D Systems, Minneapolis, MN), PE-conjugated anti-IFN-γ (Ancell Corporation) or PE-conjugated anti-IL-4 (Ancell). In some cases, Biotinylated anti-IFN-γ or anti-IL-4 (Ancell) was also used followed by labeling with Streptavidin-Cy5 (eBioscience). Controls for nonspecific staining were monitored with isotype-matched mAbs (Caltag Laboratories). We defined CD3+CD8− cells as CD4+ T cells because of activation-induced downregulation of surface CD4 molecules. Cells were detected on FACSCalibur (BD Biosciences), and the acquired data were analyzed with cellquest software.
Cytokine measurement by ELISA
Heparinized venous blood samples were collected from 32 patients with SS and 20 healthy volunteers. Plasma was isolated and stored at −80 °C until analysis. The concentrations of IL-1β, IL-6, and IL-23p19 in plasma were determined by ELISA using Quantikine kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. The sensitivity of the assay for IL-1β was 1.0 pg mL−1, for IL-6 was 0.7 pg mL−1, and for IL-23 p19 was 6.8 pg mL−1. All standards and samples were tested in duplicate. The mean values of duplicate measurements were used in the analyses.
Values were expressed as the mean ± standard deviation. Comparison between two groups was performed using the nonparametric Mann–Whitney U test. Correlation analysis was performed with the Spearman's rank order correlation. All statistical analyses were performed using spss 19.0 software. Differences with a value of P <0.05 were considered statistically significant.
Peripheral Th17 cells and Th22 cells are significantly increased in patients with SS
We first identified IL-17A- or IL-22-producing cells in peripheral blood stimulated with PMA and ionomycin. The percentages of CD3+CD8−IL-17A+ and CD3+CD8−IL-22+ cells were 1.78 ± 0.41% and 3.11 ± 0.48% in patients with SS against 0.88 ± 0.23% and 1.29 ± 0.31% in HCs, respectively (P <0.001). In contrast, no significant difference was observed in the percentages of IL-17A- or IL-22-producing CD3+CD8+ T cells and γδ+ T cells between patients with SS and HCs (Fig. 1a–c). Interestingly, the frequency of IL-17A+IL-22+CD3+CD8− T cells was also significantly increased in patients with SS as compared with HCs (0.60 ± 0.13% vs. 0.16 ± 0.05%, P <0.001; Fig. 1d and e). Furthermore, a significant positive correlation was observed between Th22 and Th17 cells (r = 0.548, P =0.001; Fig. 1f) in patients with SS.
Coexpression of IFN-γ or IL-4 by Th17 cells or Th22 cells in SS
We then examined whether Th17 and Th22 cells coexpressed IFN-γ or IL-4 in peripheral blood of patients with SS. As shown in Fig. 2, the frequencies of IL-17A+IFN-γ+ and IL-22+IFN-γ+ double-positive cells in total CD3+CD8− T cells were significantly increased in patients with SS when compared with HCs (0.66 ± 0.18% vs. 0.17 ± 0.10%, P < 0.001; 1.17 ± 0.29% vs. 0.31 ± 0.10%, P <0.001; respectively). In contrast, either patients with SS or HCs, only a few Th17 or Th22 cells coexpressed IL-4 in response to PMA/ionomycin stimulation. Moveover, the percentage of IFN-γ-producing CD3+CD8− T cells was slightly decreased (45.73 ± 6.03% vs. 50.05 ± 7.20%, P <0.05), while the percentage of IL-4-producing CD3+CD8− T cells was significantly increased in patients with SS as compared with HCs (6.11 ± 1.87% vs. 2.42 ± 0.92%, P <0.001).
High levels of plasma IL-1β, IL-6, and IL-23 are correlated with the frequencies of Th17 or Th22 cells in SS
We next determined the plasma IL-1β, IL-6, and IL-23 levels in patients with SS as these proinflammatory cytokines are involved in the tight regulation of Th17 and Th22 differentiation. As shown in Fig. 3, IL-1β, IL-6, and IL-23 levels were significantly higher in patients with SS than that in healthy controls (IL-1β: 814.9 ± 276.2.5 pg mL−1 vs. 186.5 ± 81.4 pg mL−1, P < 0.001; IL-6: 215.2 ± 92.9 pg mL−1 vs. 95.1 ± 37.2 pg mL−1, P < 0.001; IL-23: 165.1 ± 92.7 pg mL−1 vs. 37.6 ± 15.9 pg mL−1, P < 0.001). Moreover, the increased frequency of Th17 cells has a significant positive correlation with IL-1β (r =0.415, P =0.018) and IL-6 levels (r =0.371, P =0.037). However, the frequency of Th22 cells were observed to be linked with the elevated levels of IL-6 (r =0.472, P =0.006) and IL-23 levels (r =0.392, P = 0.026). Of note, no correlation was observed between Th17 cells and IL-23 levels (r =0.095, P = 0.603) as well as Th22 cells and IL-1β (r =0.077, P =0.675).
Negative correlation exists between Th17 and Th1 cells in SS
We also evaluated the correlation between the frequencies of Th17, Th22 cells and Th1, Th2 cells in SS. Interestingly, a significant negative correlation existed between Th17 and Th1 cells in patients with SS (r = −0.443, P =0.011, Fig. 4a). However, no correlation was observed between Th17 and Th2 cells (r =0.190, P =0.296, Fig. 4b), Th22 and Th1 cells (r = −0.237, P =0.191, Fig. 4c) as well as Th22 and Th2 cells (r =0.186, P = 0.308, Fig. 4d).
Positive correlation between Th17, Th22 cells, and CRP levels in SS
Finally, we explored the relationships between Th17 or Th22 cells and TRUST titers or plasma CRP levels in SS. There was no statistical dependence between TRUST titers and the frequencies of Th17 cells (r =0.024, P =0.894; Fig. 5a) or Th22 cells (r =0.012, P =0.947; Fig. 5b) in patients with SS. In contrast, a significant positive association was observed between plasma CRP levels and the proportion of Th17 cells (r =0.460, P =0.008; Fig. 5c) or Th22 cells (r =0.422, P =0.016; Fig. 5d).
A wealth of information has been gathered concerning the role of cellular immunity in the development of syphilis (Van Voorhis et al., 1996; Bouis et al., 2001). Besides, proinflammatory cytokines are also involved in syphilis-associated lesions (Brightbill et al., 1999; Babolin et al., 2011). However, whether the newly identified Th17 and Th22 subtypes are involved in the pathogenesis of syphilis are not fully understood. Here, we show clear evidence of significant elevations in peripheral IL-17A+ and IL-22+ T cells in patients with SS. Both IL-17A- and IL-22-producing cells are CD3+CD8− and CD3+γδ− T cells, implicating that CD4+ T cells comprise predominant source of IL-17A-producing Th17 cells and IL-22-producing Th22 cells in peripheral blood of patients with SS.
More specifically, we found in patients with SS a significant positive correlation between Th17 and Th22 cells, consistent with previous observations in patients with rheumatoid arthritis (Zhang et al., 2011). We therefore believe that differentiation of the two subsets may be driven in an isotropic manner. Being the major proinflammatory agonists, Tp lipoproteins stimulate antigen presenting cells to secrete proinflammatory cytokines, such as IL-1β, IL-6, and IL-23 (Radolf et al., 1995; Knauer et al., 2007). This cytokine milieu not only drives Th17 differentiation but effectively induces IL-22 expression (Ghoreschi et al., 2010). Our results showed that plasma IL-1β, IL-6, and IL-23 levels were significantly elevated in patients with SS. Moreover, we provided further evidences that the increased frequency of Th17 cells had a significant positive correlation with the serum IL-6 and IL-1β levels, suggesting that IL-6 synergizes with IL-1β enhance the differentiation and the generation of Th17 cells in patients with SS. However, plasma IL-23 was not observed to be correlated with Th17, although previous reports showed that the generation of Ag-specific Th17 effectors was severely curtailed in the absence of IL-23 (Veldhoen et al., 2006; Thakker et al., 2007; Stritesky et al., 2008). We also showed that the frequency of Th22 cells was positively correlated with the IL-6 and IL-23 levels, indicating that IL-6 and IL-23 enhance the differentiation of Th22 cells in patients with SS. Moreover, IL-23 was essential for the sustained preferential production of IL-22 in Th17 cells (Liang et al., 2006), consistent with our results that the frequency of IL-17A+IL-22+ T cells was also significantly increased in patients with SS. It is to be noted that increased peripheral Th17 and Th22 cells were also occured in other inflammatory and autoimmune diseases, such as psoriasis (Kagami et al., 2010), interstitial lung disease (Truchetet et al., 2011) and rhermatoid arthritis (Zhang et al., 2011). This, in combination with the comparable levels of IL-1β, IL-6, and IL-23, indicating that Th17 and Th22 differentiation and expansion are driven by proinflammatory stimuli, and therefore the increased frequencies of both Th17 and Th22 cells in patients with SS may represent a Tp nonspecific immune responses.
It is unknown whether there is a reciprocal relationship between Th17 or Th22 cells and Th1 or Th2 cells, which are altered in patients with SS. Our data showed that there was a negative correlation between Th17 and Th1 cells, implying a developmental association exists between the two subsets. In contrast, no significant correlation was observed between Th17 and Th2 cells, Th22 and Th1 or Th2 cells in patients with SS. Furthermore, consistent with previous report (Podwinska et al., 2000), our results indicated that IFN-γ-producing Th1 cells were slightly decreased in patients with SS while IL-4-producing Th2 cells had a significant increment. Thus, the altered balance between Th17 and Th1 cells in patients with SS predicts that Th-17-mediated responses may be protective in the Tp clearance and conducive to proinflammatory effects induction. It should be noted that both the percentages of IL-17A+IFN-γ+ and IL22+IFN-γ+ cells T cells are significantly increased in patients with SS, whereas few or no Th17 or Th22 cells coexpressed IL-4. A substantial proportion of Th17 and Th22 cells that secreted IFN-γ indicated that the plasticity of Th1 and Th17 or Th22 was increased in SS. Taken together, these findings indicate that boosting Th17 and Th22 responses occur in conjunction with Th1/Th2 imbalance in patients with SS.
TRUST is a nontreponemal test for syphilis for many years. Here, we showed that there was no statistically significant association between TRUST titers and the frequencies of Th17 or Th22 cells. However, plasma CRP level, as a systemic marker of focal inflammation and infection (Pasceri et al., 2000), was observed to have a significant positive correlation with the proportion of Th17 or Th22 cells in patients with SS. This might be attributed to the proinflammatory trigger of IL-17 which stimulates the synthesis of CRP (Patel et al., 2007).
In conclusion, our study shows that peripheral Th17 and Th22 subsets are concurrently increased in patients with SS. A significant positive correlation is observed between Th17 and Th22 cells. Moreover, the increment of the two subsets is accompanied by elevated plasma CRP levels. These results raise the possibility that Th17 and Th22 cells are involved in immune response against Tp infection.
This work was supported by grants from the National Natural Science Foundation of China (No. 81171465) and the Natural Science Research Foundation of Education Bureau of Anhui Province (no. kj2007B063).