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Scand J Rheumatol 2014;43:194–201
Circulating Th17 and Th1 cells expressing CD161 are associated with disease activity in rheumatoid arthritis J Miao1, K Zhang1, M Lv1, Q Li1, Z Zheng1, Q Han1, N Guo2, C Fan1, P Zhu1
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1 Department of Clinical Immunology, Xijing Hospital and 2Cell Engineering Research Center, Fourth Military Medical University, Shaanxi Province, PR China
Objectives: To determine whether the percentages of major CD4þCD161þ T-cell subsets [T-helper (Th)17, Th1, and Th17/Th1] in peripheral blood are correlated with disease activity of rheumatoid arthritis (RA). Method: In 42 RA patients and 15 healthy controls (HCs), the percentages of interleukin (IL)-17- and/or interferon (IFN)-γ-producing CD4þCD161þ T cells and the plasma levels of related cytokines were assessed by flow cytometry and cytometric bead array (CBA) analysis, respectively. Disease activity was evaluated by the 28-joint Disease Activity Score (DAS28). Results: The percentage of circulating CD4þCD161þIL-17þIFN-γ T cells (CD161þ Th17) in RA patients increased significantly and was higher in patients with active disease status (DAS28 > 3.2) compared with those with low disease status (DAS28 3.2), and correlated positively with DAS28, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), IL-17, and IL-6 levels in RA patients. The percentage of circulating CD4þCD161þIL-17IFN-γþ T cells (CD161þ Th1) decreased and correlated negatively with DAS28, CRP, and ESR levels in RA patients, while the percentage of CD4þCD161þIL-17þIFN-γþ T cells (CD161þ Th17/Th1) was unchanged in RA patients and was not correlated with RA disease activity. Conclusions: These data suggest that the percentages of circulating CD161þ Th17 and CD161þ Th1 cells in RA patients reflect the degree of disease activity. They support the hypothesis that Th17 cells are involved in the pathogenesis of RA and that CD161þ Th17/CD161þ Th1-cell imbalance may contribute to the development of RA.
Rheumatoid arthritis (RA) is a systemic inflammatory disease leading to joint damage and functional disability and a common autoimmune disease marked by persistent synovitis, systemic inflammation, and autoantibody production (1). Although the precise immunopathological mechanism of RA is not completely understood, the abnormality of T cells is implicated in the pathogenesis of RA (2). Conventionally, T helper type 1 (Th1) cells have been shown to play an important pathogenetic role in RA (3). More recently, a distinct lineage of CD4þ effector T cells producing interleukin (IL)-17, termed Th17 cells, has been described (4, 5). A study by van Hamburg et al reported that Th17 cells, but not Th1 cells, from patients with RA can drive chronic destruction by cooperating with synovial fibroblasts in a proinflammatory feedback loop (6). There is further clinical evidence that the percentage of circulating Th17 cells of RA patients is significantly elevated and correlates positively with local and systemic disease activity indices (7–9). Ping Zhu, Department of Clinical Immunology, Xijing Hospital, Fourth Military Medical University, No. 127 West Changle Road, Xi’an 710032, Shaanxi Province, PR China. E-mail: [email protected] Accepted 17 September 2013
IL-17, secreted mainly by Th17 cells, is found to have induced massive damage with extensive inflammatory cell migration, bone erosion, and cartilage degradation in both animal RA models and human RA cases (10, 11). Although the exact mechanism of Th17 cells in the pathogenesis of RA is not yet clear, these observations suggest that Th17 cells make an important contribution to the inflammatory pathology of RA. The lectin receptor CD161, a marker of human Th17 cells, has been identified in recent years (12, 13). CD161 is a human homologue of the murine natural killer (NK)-1.1 antigen, which is expressed on the surface of NK T cells and on subsets of CD4þ T cells (14). Several studies have shown that human Th17 cells originate from CD4þCD161þ-naive T-cell precursors (12, 15) and almost all Th17 cells are found to be contained within the CD161þ fraction of circulating CD4þ T cells (12, 13, 15, 16). Besides containing Th17 cells (CD4þCD161þ IL-17þIFN-γ T cells), CD4þCD161þ T-cell subpopulations also contain Th1-cell (CD4þCD161þIL-17IFNγþ T cells) and Th17/Th1-cell subsets (CD4þCD161þ IL-17þIFN-γþ T cells) in human peripheral blood (12, 16). In addition, CD4þCD161þ naive T-cell precursors can differentiate into mature Th17, Th17/Th1, and Th1 cells under the same polarizing conditions (12). It has
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DOI: 10.3109/03009742.2013.846407
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Table 1. Clinical characteristics of rheumatoid arthritis (RA) patients. Characteristics
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Patients (n) Age (years), mean SD Sex (male/female) Disease duration (years), mean SD RF positivity, n (%) Anti-CCP positivity, n (%) ESR (mm/h), mean SD CRP (mg/dL), mean SD DAS28 score, mean SD Treatment, n (%) None Steroid NSAIDs DMARDs TNF-α antagonist
All RA patients
Low disease status
Active disease status
42 41.38 11.71 33/9 3.93 4.54 34 (81.0) 29 (69.0) 34.17 28.96 1.86 3.07 3.85 1.79
18 39.17 13.73 14/4 4.25 3.93 15 (83.3) 12 (66.7) 11.39 7.06 0.38 0.57 2.06 0.47
24 43.04 9.91 19/5 3.69 5.01 19 (79.2) 17 (70.8) 51.25 27.35 2.97 3.69 5.19 1.07
1 (2.4) 6 (14.3) 4 (9.5) 36 (85.7) 5 (11.9)
0 2 (11.1) 1 (5.6) 16 (88.9) 2 (11.1)
1 (5.2) 4 (16.7) 3 (12.5) 20 (83.3) 3 (12.5)
RF, Rheumatoid factor; Anti-CCP, anti-cyclic citrullinated peptide antibody; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; DAS28, 28-joint Disease Activity Score; NSAID, non-steroidal anti-inflammatory drug; DMARD, disease-modifying anti-rheumatic drug; TNF-α, tumour necrosis factor-α; SD, standard deviation.
also been reported that the percentage of CD161þ Th17/ Th1 cells in juvenile idiopathic arthritis (JIA) synovial fluid is positively correlated with erythrocyte sedimentation rate (ESR) and levels of C-reactive protein (CRP) (16). However, there has been little discussion about the percentages of peripheral blood CD4þCD161þ T-cell subpopulations (Th17, Th1, and Th17/Th1 cell subsets) in RA patients and their potential link to disease activity of RA. Therefore, in this study, we measured the percentages of CD4þCD161þ T-cell subsets (Th17, Th1, and Th17/ Th1) in peripheral blood of RA patients, and assayed their correlations with disease activity parameters.
Method
Flow cytometric analysis The following monoclonal antibodies (mAbs) were used: for surface phenotype staining, phycoerythrin (PE)-conjugated CD4 (clone RPA-T4) and phycoerythrin-cyanin 5 (PC5)-conjugated CD161 (DX12) (all from BD Biosciences, San Diego, CA, USA); for intracellular cytokine detection, allophycocyanin (APC)-conjugated IL-17A (eBio64DEC17, eBiosciences, San Diego, CA, USA) and fluorescein isothiocyanate (FITC)-conjugated IFN-γ (25723.11, BD Biosciences). Appropriately conjugated immunoglobulin (Ig)G antibodies were used as isotype controls. Stained cells were analysed on a Fluorescence Activated Cell Sorter (FACS)Calibur flow cytometer, using CellQuest software (BD Bioscience).
Patients Peripheral blood samples were obtained from 42 patients with RA (33 females and nine males; mean age SD ¼ 41.38 11.71 years) and 15 age- and sex-matched healthy controls (HCs: 12 females and three males; mean age SD ¼ 39.07 10.26 years). Diagnosis of RA was established according to the 1987 revised criteria of the American College of Rheumatology (ACR) for RA (17), and disease activity was assessed by the 28-joint Disease Activity Score (DAS28) (18). DAS28 3.2 was considered to reflect low disease status and DAS28 > 3.2 to reflect an active disease status. In this study, 18 patients had DAS28 3.2 and 24 patients had DAS28 > 3.2. Table 1 shows the patients’ demographic and clinical characteristics and current medications. The study conforms to the recommendations of the Declaration of Helsinki. The Ethics Committee of Xijing Hospital approved this study, and informed consent was obtained from all patients and controls.
Immunofluorescent intracellular staining of cytokines Intracellular cytokines were analysed by flow cytometry to characterize the cytokine-producing cells. In brief, sodium heparinized venous blood was obtained from all participants. After sampling, 500 μL of blood was mixed immediately with an equal volume of RPMI 1640 (Gibco, Paisley, Scotland, UK). To assess the production of intracellular cytokines, diluted blood was stimulated with 50 ng/mL of phorbol myristate acetate (PMA; SigmaAldrich, Steinheim, Germany) and 1 μg/mL of ionomycin (Sigma-Aldrich) in the presence of 10 μg/mL GolgiStop (BD Bioscience) for 5 h at 37˚C in a humidified 5% CO2 incubator. After surface staining of the cells, erythrocytes were lysed by adding FACS lysing solution (BD Biosciences). Then intracellular staining was performed using BD Cytofix/Cytoperm solution and BD Perm/Wash solution following the manufacturer’s instructions (BD Biosciences).
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coefficient was used to calculate the correlations. A two-sided p-value < 0.05 was considered significant. Statistical analyses were performed using SPSS version 17.0 (SPSS Inc, Chicago, IL, USA).
Plasma samples were harvested and stored at –70˚C until cytokine testing was performed. CBA analysis for IL-17, IFN-γ, IL-1β, IL-6, and IL-21 in plasma was performed according to the manufacturer’s instructions (BD Biosciences). In brief, five bead populations with distinct fluorescence intensities coated with capture Abs specific for IL-17, IFN-γ, IL-1β, IL-6, and IL-21 were mixed. Then the beads were incubated in serum enhancement buffer to reduce the possibility of false-positive results. Plasma and PE detection reagent were mixed with a cocktail of capture beads. The mixtures were incubated at room temperature for 3 h in the dark, washed, and resuspended in wash buffer before analysis on the FACSCalibur (BD Biosciences). Data were analysed using CBA analysis software from BD Biosciences. The concentration of each cytokine in plasma was determined by reference to a standard curve.
To investigate whether circulating IL-17þ T cells from HCs and RA patients are contained in the CD161þ fraction, we gated CD4þ T cells into CD4þCD161þ and CD4þCD161 fractions, and each fraction was assessed for their ability to produce IL-17. As shown in Figure 1, the CD4þCD161þ T-cell subsets showed a significantly higher frequency of IL-17-producing cells compared with the CD4þCD161 T cells in both HCs (p < 0.001) and RA patients (p < 0.001).
Statistical analysis
Percentage of CD4þCD161þ T-cell subsets (Th17, Th1, and Th17/Th1) in RA patients
The non-parametric Mann–Whitney U-test was used for statistical analysis of the difference between two groups in all experiments. Spearman’s rank correlation
Expression of the intracellular cytokines IL-17 and/or IFN-γ was assessed in CD4þCD161þ T cells, and representative examples of flow cytometric dot-plots from RA
Percentage of IL-17-producing cells in CD4þCD161þ T cells in HCs and RA patients
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Cytometric bead array (CBA) analysis
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CD
Figure 1. Circulating CD4þCD161þ T cells produce more IL-17 than CD4þCD161 T cells. (A) Flow cytometric dot-plots of intracellular IL-17 production in CD4þCD161 and CD4þCD161þ cells obtained from one representative healthy control (HC) and one patient with rheumatoid arthritis (RA). (B, C) Frequencies of circulating IL-17-producing cells between CD4þCD161 and CD4þCD161þ fractions in (B) HCs and (C) RA patients. The horizontal line indicates the median value. p-values were assessed by the Mann–Whitney U-test.
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significant differences in the frequency of CD161þ Th17/Th1 cells between the two groups (p ¼ 0.959).
As shown in Figure 3, the plasma levels of Th17related cytokines including IL-17 (Figure 3A), IL-1β (Figure 3C), IL-6 (Figure 3D), and IL-21 (Figure 3E) were significantly higher in RA patients than in HCs (all p < 0.05). Among the RA patients with different disease activity, significantly higher IL-17 and IL-6 levels were observed in the patients with DAS28 > 3.2 than in those with DAS28 3.2 (p ¼ 0.008 and p < 0.001, respectively) (Figures 3F and 3I). Although the patients with DAS28 > 3.2 seemed to have higher levels of IL-1β and IL-21 when compared to the levels in the patients with DAS28 3.2, the differences did not reach statistical significance (p ¼ 0.150 and p ¼ 0.055, respectively) (Figures 3H and 3J). In addition, the differences in IFN-γ (Th1-related cytokine) concentration were not significant between RA patients and HCs (p ¼ 0.949) (Figure 3B) or between patients with DAS28 > 3.2 and those with DAS28 3.2 (p ¼ 0.809) (Figure 3G).
A
HC
60 50 40 30 20 10
p < 0.001
–
IL-17+IFN-Y p = 0.198
+
IL-17-IFN-Y
6 4 2 0
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HCs
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HCs
RA
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6.87 0.45 63.03 29.65
102 103
104
4.33 0.48 57.22 37.97
DAS28 > 3.2
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+
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IL-17 101 102 103 100
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Plasma levels of cytokines in RA patients
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IL-17+IFN-Y
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SSC-H 0 200 400 600 800 1000
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patients and HCs are shown in Figure 2A. The percentage of IL-17þIFN-γ cells in CD4þCD161þ T cells (CD161þ Th17 cells) increased significantly in RA patients [median 4.83, interquartile range (IQR) 3.68–6.54%] compared to HCs (median 2.69, IQR 2.15–3.68%; p < 0.001) (Figure 2B). By contrast, the percentage of IL-17IFNγþ cells in CD4þCD161þ T cells (CD161þ Th1 cells) was significantly lower in RA patients (median 37.42, IQR 25.14–42.84%) than in HCs (median 45.50, IQR 40.33– 49.35%; p ¼ 0.004) (Figure 2B). For CD4þCD161þ T cells producing both IL-17 and IFN-γ (CD161þ Th17/ Th1 cells), there was no difference between RA patients and HCs (p ¼ 0.198) (Figure 2B). Then we analysed the percentages of CD4þCD161þ T-cell subpopulations in RA patients with different disease activity (Figure 2C). The percentage of CD161þ Th17 cells was increased in the patients with DAS28 > 3.2 (median 5.79, IQR 4.83–6.82%) compared to those with DAS28 3.2 (median 4.10, IQR 3.20– 4.79%; p ¼ 0.001), while a significantly lower frequency of circulating CD161þ Th1 cells was observed in the patients with DAS28 > 3.2 (median 29.89, IQR 20.80– 42.10%) than in those with DAS28 3.2 (median 39.73, IQR 36.85–44.27%; p ¼ 0.021). However, there were no
percentages of CD4+CD161+ T cells
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Th17 and Th1 cells expressing CD161 in RA
p = 0.001
60 50 40 30 20 10
–
IL-17+IFN-Y p = 0.959
0
+
101 102 103 104 IFN-Y IL-17-IFN-Y p = 0.021
+
8 6 4 2 0
.2 .2 .2 .2 3.2 3.2 8≤3 S28>3 S28≤3 S28>3 28≤ AS28> DA DA DAS D DA
2 DAS
Figure 2. Expression of IL-17 and/or IFN-γ on CD4þCD161þ T cells in RA patients and healthy controls (HCs). (A) Whole blood from RA patients and HCs was stimulated with PMA/ionomycin and then the intracellular cytokine expression of IL-17 and/or IFN-γ among CD4þCD161þ T cells was analysed. (B) Frequencies of IL-17- and/or IFN-γ-producing cells in CD4þCD161þ T cells from RA patients and HCs are shown. (C) Detection of IL-17- and/or IFN-γ-producing cells in CD4þCD161þ T cells from RA patients with low disease status (DAS28 3.2) and active disease status (DAS28 > 3.2). The horizontal line indicates the median value. p-values were assessed by the Mann–Whitney U-test.
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2
s
HC
IFN-Y (pg/mL)
6
IL-17 (pg/mL)
100 80 60 40 20 6 4 2 0
G
p = 0.008
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C p = 0.949 IL-1β (pg/mL)
100 80 60 40 20 6 4 2 0
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IL-17 (pg/mL)
p = 0.004
3.2 3.2 28 > S A D
p < 0.001
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IL-21 (pg/mL)
B
A
8≤
2 DAS
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2000 1500 1000 500 100 80 60 40 20 0 8≤
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RA
p = 0.055
3.2 3.2 28 > S A D
Figure 3. Plasma levels of cytokines in RA patients. Comparisons of the levels of plasma (A) IL-17, (B) IFN-γ, (C) IL-1β, (D) IL-6, and (E) IL-21 are shown between RA patients and HCs. Detection of (F) IL-17, (G) IFN-γ, (H) IL-1β, (I) IL-6, and (J) IL-21 levels from RA patients with low disease status (DAS28 3.2) and those with active disease status (DAS28 > 3.2). The horizontal line indicates the median value. p-values were assessed by the Mann– Whitney U-test.
Correlation of CD4þCD161þ T-cell subsets with disease activity and their related cytokines in RA patients The correlation of circulating CD4þCD161þ T-cell subsets with disease activity parameters in RA is shown in Figure 4. The frequency of circulating CD161þ Th17 cells was positively correlated with DAS28 (r ¼ 0.546, p < 0.001) (Figure 4A), CRP levels (r ¼ 0.520, p < 0.001) (Figure 4B), and ESR levels (r ¼ 0.542, p < 0.001) (Figure 4C). By contrast, the frequency of CD161þ Th1 cells was negatively correlated with DAS28 (r ¼ –0.468, p ¼ 0.002) (Figure 4D), CRP (r ¼ –0.430, p ¼ 0.005) (Figure 4E), and ESR (r ¼ –0.341, p ¼ 0.027) (Figure 4F). However, there was no correlation between the percentages of CD161þ Th17/Th1 cells and RA disease activity parameters (all p > 0.05) (Figures 4G, 4H, and 4I). In RA patients, circulating CD161þ Th17 cell percentages were positively correlated with IL-17 levels (r ¼ 0.315, p ¼ 0.042) and IL-6 levels (r ¼ 0.488, p ¼ 0.001). However, CD161þ Th17 cells failed to show a significant correlation with levels of IL-1β (r ¼ 0.241, p ¼ 0.124) and IL-21 (r ¼ 0.256, p ¼ 0.102). In addition, no correlation was present between percentages of CD161þ Th1 cells and IFN-γ levels (r ¼ –0.125, p ¼ 0.430). Furthermore, a significant negative correlation was found between CD161þ Th17 cells and CD161þ Th1 cells (r ¼ –0.570, p < 0.001) (data not shown).
Discussion Numerous inflammatory cells, proinflammatory cytokines, and chemokines are known to play crucial roles
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in the pathogenesis of inflammatory arthritis. In studies of models of autoimmunity, including experimental allergic encephalomyelitis (EAE) (19) and several murine arthritis models (20), the Th17-cell subset has been identified as the principal driver of inflammation. There is also considerable evidence to suggest that Th17 cells are involved in the development of human autoimmune diseases such as RA (21–23). To further investigate the role of Th17 cells in the pathogenesis of RA, we examined the Th17-cell profile in RA patients. Our data show that IL-17-producing cells were found almost exclusively in the CD4þCD161þ fraction in both RA patients and HCs, as reported previously (12, 16). We also found that the frequencies of circulating CD161þ Th17 cells were significantly increased in RA patients and also elevated in the patients with DAS28 > 3.2 compared to those with DAS28 3.2. Moreover, levels of CD161þ Th17 cells were positively correlated with DAS28 and with ESR and CRP levels in RA patients. These results are in partial agreement with findings reported in other investigations (7, 8), in which an increased percentage of circulating Th17 cells correlated with RA disease activity. Our observations are compatible with the hypothesis that Th17 cells contribute to the pathogenesis of RA and suggest that circulating CD161þ Th17 cells may reflect disease activity status in RA patients. The Th17-cell subset has a specific role in immune function through the secretion of effector cytokines. IL-17, the main cytokine of Th17 cells, is involved in the pathogenesis of arthritis, as has been demonstrated in animal arthritis models. By secreting IL-17, Th17 cells can stimulate monocytes to produce proinflammatory
Th17 and Th1 cells expressing CD161 in RA
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.0
1.0 10.0 20.0 30.0 40.0 50.0 60.0 CD161 + Th1 (%)
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0 10.0 20.0 30.0 40.0 50.0 60.0 CD161 + Th1 (%) H
r = –0.072 p = 0.649
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10.0 20.0 30.0 40.0 50.0 60.0 CD161 + Th1 (%) I
r = 0.092 p = 0.561
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.0 F
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6.0
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40
E r = –0.468 p = 0.002
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80
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r = 0.059 p = 0.712
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5.0 20
2.0 1.0
.0 .00 .20 .40 .60 .80 1.00 1.20 CD161 + Th17/Th1 (%)
0 .00 .20 .40 .60 .80 1.00 1.20 CD161 + Th17/Th1 (%)
.00 .20 .40 .60 .80 1.00 1.20 CD161 + Th17/Th1 (%)
Figure 4. Correlations between the levels of CD4þCD161þ T-cell subsets and RA disease activity indices. (A–C) Correlations of CD161þ Th17-cell percentage with (A) DAS28, (B) CRP, and (C) ESR levels. (D–F) Correlations of CD161þ Th1-cell percentage with (D) DAS28, (E) CRP, and (F) ESR levels. (G–I) Correlations of CD161þ Th17/Th1-cell percentage with (G) DAS28, (H) CRP, and (I) ESR levels. All correlations were calculated using Spearman’s rank correlation coefficient.
cytokines, thus amplifying the inflammatory cascade (24, 25). In the current study, there was a significant positive correlation between plasma IL-17 levels and the frequency of CD161þ Th17 cells. In line with previous findings (26), we found that the levels of plasma IL-17 were elevated in RA patients compared with HCs. Furthermore, we have shown an increase in plasma IL-17 levels with higher DAS28. Our observations and the data of previous studies indicate that IL-17 may play a crucial role in the pathogenesis of RA and support the therapeutic benefits of IL-17 inhibitors in RA patients (27). The mechanism for increased CD161þ Th17-cell response in patients with RA has not been fully explored. Two previous studies reported that the development of
Th17 cells is dependent on the cytokine milieu, with IL-6, IL-1β, and IL-21 promoting Th17-cell differentiation and expansion (28, 29). Thus, we explored the potential role of these cytokines in promoting IL-17 secretion in RA. In our study, we noted that patients with RA had a trend for increased plasma levels of IL-1β, IL-6, and IL-21, and the high levels of IL-6 were positively correlated with circulating CD161þ Th17-cell frequencies in RA patients. Moreover, a significant increase in plasma IL-6 levels, in parallel with the increase in the frequencies of CD161þ Th17 cells, was observed in RA patients with DAS28 > 3.2. These data suggest the possible involvement of these cytokines, particularly IL-6, in increasing the CD161þ
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Th17-cell response we observed, and support the crucial role of IL-6 in Th17-dependent autoimmune diseases (30). It was previously reported that Th17 cells in culture may lose IL-17 expression, possibly through a Th17/Th1 intermediate, to obtain a Th1 phenotype, and CD161 appears to track this conversion (31, 32). Therefore, levels of CD161þ Th1 cells and CD161þ Th17/Th1 cells were also examined in this study. Contrasting with the results of CD161þ Th17 cells, we observed that the percentage of circulating CD161þ Th1 cells decreased significantly and correlated negatively with DAS28, ESR and CRP levels in RA patients. A possible explanation for the decreased CD161þ Th1-cell levels is that the Th17 cells, characterized by production of the cytokine IL-17, have an inhibitory effect on Th1-cell development (33). Another explanation may be a shift in CD4þCD161þ T cells from the Th17 to the Th1 phenotype, which is rare in peripheral blood of RA, although it can occur in the synovial fluid of children with JIA (16, 32). Further studies are clearly needed to determine the mechanism for decreased CD161þ Th1 cells in peripheral blood and their potential link to RA. There was also a significant negative correlation between the frequency of CD161þ Th17 cells and CD161þ Th1 cells. These observations indicate that the balance of CD161þ Th17- and CD161þ Th1-cell responses is dysregulated in RA patients with different disease activity. The immunocyte balance has long been hypothesized as one of the important factors in the development of autoimmune diseases (34, 35), and the CD161þ Th17/CD161þ Th1 imbalance maybe play an important role in RA. As the hallmark cytokine of Th1 cells, IFN-γ exerts an important role as a mediator of chronic autoimmune inflammation (36). However, in this study, we found no influence of disease activity on the concentration of IFN-γ in RA, and there was no correlation between plasma IFNγ levels and CD161þ Th1-cell levels. This is probably because IFN-γ could also be secreted from CD161 Th1 cells, the other Th1-cell subsets (12), and some cells including CD8þ T cells, NK cells, and macrophages (37). Consistent with our data, previous studies also detected a relatively small but substantial percentage of CD4þ CD161þ cells producing both IL-17 and IFN-γ (12, 16). Subanalysis of the percentage of CD161þ Th17/Th1 cells revealed no difference between RA patients with different disease activity. Furthermore, there was no correlation between CD161þ Th17/Th1-cell frequency and the disease activity indices of RA. This could be due to the relatively limited numbers of patients included, but an intrinsic abnormality is also possible. In conclusion, this study provides evidence for the role of Th17 cells in the pathogenesis of RA, with the demonstration of an elevated percentage of Th17 cells expressing CD161 in RA patients, and correlations of this cell subset with disease activity indices. Although the mechanism underlying our data is yet to be determined,
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it appears that the balance of CD161þ Th17-cell and CD161þ Th1-cell responses is dysregulated in RA, which may play an important role in the development of RA. Further investigations are needed to determine the involvement of CD4þCD161þ T-cell subsets in the joints of RA patients and to clarify the pathogenetic role of the CD161þ Th17/CD161þ Th1 balance in RA. Acknowledgements This study was supported by grants from the Key Program of the National Natural Science Foundation of China (No. 81030058) and the National Science and Technology Major Project of China (No. 2012ZX09103301-026, 2013ZX09301-301).
References 1. Scott DL, Wolfe F, Huizinga TW. Rheumatoid arthritis. Lancet 2010;376:1094–108. 2. McInnes IB, Schett G. The pathogenesis of rheumatoid arthritis. N Engl J Med 2011;365:2205–19. 3. Dolhain RJ, van der Heiden AN, ter Haar NT, Breedveld FC, Miltenburg AM. Shift toward T lymphocytes with a T helper 1 cytokine-secretion profile in the joints of patients with rheumatoid arthritis. Arthritis Rheum 1996;39:1961–9. 4. Park H, Li Z, Yang XO, Chang SH, Nurieva R, Wang YH, et al. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol 2005;6:1133–41. 5. Bettelli E, Korn T, Oukka M, Kuchroo VK. Induction and effector functions of T(H)17 cells. Nature 2008;453:1051–7. 6. van Hamburg JP, Asmawidjaja PS, Davelaar N, Mus AM, Colin EM, Hazes JM, et al. Th17 cells, but not Th1 cells, from patients with early rheumatoid arthritis are potent inducers of matrix metalloproteinases and proinflammatory cytokines upon synovial fibroblast interaction, including autocrine interleukin-17A production. Arthritis Rheum 2011;63:73–83. 7. Shen H, Goodall JC, Hill GJ. Frequency and phenotype of peripheral blood Th17 cells in ankylosing spondylitis and rheumatoid arthritis. Arthritis Rheum 2009;60:1647–56. 8. Zhang L, Li JM, Liu XG, Ma DX, Hu NW, Li YG, et al. Elevated Th22 cells correlated with Th17 cells in patients with rheumatoid arthritis. J Clin Immunol 2011;31:606–14. 9. Jiao Z, Hua S, Wang W, Wang H, Gao J, Wang X. Increased circulating myeloid-derived suppressor cells correlated negatively with Th17 cells in patients with rheumatoid arthritis. Scand J Rheumatol 2013;42:85–90. 10. Lubberts E, Koenders MI, van den Berg WB. The role of T-cell interleukin-17 in conducting destructive arthritis: lessons from animal models. Arthritis Res Ther 2005;7:29–37. 11. Waldburger JM, Firestein GS. Garden of therapeutic delights: new targets in rheumatic diseases. Arthritis Res Ther 2009;11:206. 12. Cosmi L, De Palma R, Santarlasci V, Maggi L, Capone M, Frosali F, et al. Human interleukin 17-producing cells originate from a CD161þCD4þ T cell precursor. J Exp Med 2008;205:1903–16. 13. Kleinschek MA, Boniface K, Sadekova S, Grein J, Murphy EE, Turner SP, et al. Circulating and gut-resident human Th17 cells express CD161 and promote intestinal inflammation. J Exp Med 2009;206:525–34. 14. Lanier LL, Chang C, Phillips JH. Human NKR-P1A. A disulfidelinked homodimer of the C-type lectin superfamily expressed by a subset of NK and T lymphocytes. J Immunol 1994; 153:2417–28. 15. Maggi L, Santarlasci V, Capone M, Peired A, Frosali F, Crome SQ, et al. CD161 is a marker of all human IL-17-producing T-cell subsets and is induced by RORC. Eur J Immunol 2010;40:2174–81.
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Th17 and Th1 cells expressing CD161 in RA 16. Cosmi L, Cimaz R, Maggi L, Santarlasci V, Capone M, Borriello F, et al. Evidence of the transient nature of the Th17 phenotype of CD4þCD161þ T cells in the synovial fluid of patients with juvenile idiopathic arthritis. Arthritis Rheum 2011;63:2504–15. 17. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24. 18. Prevoo ML, van ‘t Hof, Kuper HH, van Leeuwen MA, van de Putte LB, van Riel PL. Modified disease activity scores that include twenty-eight-joint counts. Development and validation in a prospective longitudinal study of patients with rheumatoid arthritis. Arthritis Rheum 1995;38:44–8. 19. Cua DJ, Sherlock J, Chen Y, Murphy CA, Joyce B, Seymour B, et al. Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 2003;421:744–8. 20. Hirota K, Hashimoto M, Yoshitomi H, Tanaka S, Nomura T, Yamaguchi T, et al. T cell self-reactivity forms a cytokine milieu for spontaneous development of IL-17þ Th cells that cause autoimmune arthritis. J Exp Med 2007;204:41–7. 21. Shahrara S, Huang Q, Mandelin AN, Pope RM. TH-17 cells in rheumatoid arthritis. Arthritis Res Ther 2008;10:R93. 22. Bettelli E, Oukka M, Kuchroo VK. T(H)-17 cells in the circle of immunity and autoimmunity. Nat Immunol 2007;8:345–50. 23. Dong W, Zhu P. Functional niche of inflamed synovium for Th17-cell expansion and activation in rheumatoid arthritis: implication to clinical therapeutics. Autoimmun Rev 2012; 11:844–51. 24. Jovanovic DV, Di Battista JA, Martel-Pelletier J, Jolicoeur FC, He Y, Zhang M, et al. IL-17 stimulates the production and expression of proinflammatory cytokines, IL-beta and TNF-alpha, by human macrophages. J Immunol 1998;160:3513–21. 25. Van Bezooijen RL, Van Der Wee-Pals L, Papapoulos SE, Lowik CW. Interleukin 17 synergises with tumour necrosis factor alpha to induce cartilage destruction in vitro. Ann Rheum Dis 2002;61:870–6. 26. Metawi SA, Abbas D, Kamal MM, Ibrahim MK. Serum and synovial fluid levels of interleukin-17 in correlation with disease activity in patients with RA. Clin Rheumatol 2011; 30:1201–7.
201 27. Genovese MC, Van den Bosch F, Roberson SA, Bojin S, Biagini IM, Ryan P, et al. LY2439821, a humanized anti-interleukin-17 monoclonal antibody, in the treatment of patients with rheumatoid arthritis: a phase I randomized, double-blind, placebo-controlled, proof-of-concept study. Arthritis Rheum 2010;62:929–39. 28. Acosta-Rodriguez EV, Napolitani G, Lanzavecchia A, Sallusto F. Interleukins 1beta and 6 but not transforming growth factor-beta are essential for the differentiation of interleukin 17-producing human T helper cells. Nat Immunol 2007;8:942–9. 29. Wilson NJ, Boniface K, Chan JR, McKenzie BS, Blumenschein WM, Mattson JD, et al. Development, cytokine profile and function of human interleukin 17-producing helper T cells. Nat Immunol 2007;8:950–7. 30. Zhou L, Ivanov II, Spolski R, Min R, Shenderov K, Egawa T, et al. IL-6 programs T(H)-17 cell differentiation by promoting sequential engagement of the IL-21 and IL-23 pathways. Nat Immunol 2007;8:967–74. 31. Lee YK, Turner H, Maynard CL, Oliver JR, Chen D, Elson CO, et al. Late developmental plasticity in the T helper 17 lineage. Immunity 2009;30:92–107. 32. Nistala K, Adams S, Cambrook H, Ursu S, Olivito B, de Jager W, et al. Th17 plasticity in human autoimmune arthritis is driven by the inflammatory environment. Proc Natl Acad Sci USA 2010; 107:14751–6. 33. Toh ML, Kawashima M, Hot A, Miossec P, Miossec P. Role of IL-17 in the Th1 systemic defects in rheumatoid arthritis through selective IL-12Rbeta2 inhibition. Ann Rheum Dis 2010;69:1562–7. 34. Lina C, Conghua W, Nan L, Ping Z. Combined treatment of etanercept and MTX reverses Th1/Th2, Th17/Treg imbalance in patients with rheumatoid arthritis. J Clin Immunol 2011;31: 596–605. 35. E XQ, Meng HX, Cao Y, Zhang SQ, Bi ZG, Yamakawa M. Distribution of regulatory T cells and interaction with dendritic cells in the synovium of rheumatoid arthritis. Scand J Rheumatol 2012;41:413–20. 36. Schulze-Koops H, Kalden JR. The balance of Th1/Th2 cytokines in rheumatoid arthritis. Best Pract Res Clin Rheumatol 2001;15: 677–91. 37. Shah K, Lee WW, Lee SH, Kim SH, Kang SW, Craft J, et al. Dysregulated balance of Th17 and Th1 cells in systemic lupus erythematosus. Arthritis Res Ther 2010;12:R53.
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Scand J Rheumatol 2014;43:194–201
Circulating Th17 and Th1 cells expressing CD161 are associated with disease activity in rheumatoid arthritis J Miao1, K Zhang1, M Lv1, Q Li1, Z Zheng1, Q Han1, N Guo2, C Fan1, P Zhu1
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1 Department of Clinical Immunology, Xijing Hospital and 2Cell Engineering Research Center, Fourth Military Medical University, Shaanxi Province, PR China
Objectives: To determine whether the percentages of major CD4þCD161þ T-cell subsets [T-helper (Th)17, Th1, and Th17/Th1] in peripheral blood are correlated with disease activity of rheumatoid arthritis (RA). Method: In 42 RA patients and 15 healthy controls (HCs), the percentages of interleukin (IL)-17- and/or interferon (IFN)-γ-producing CD4þCD161þ T cells and the plasma levels of related cytokines were assessed by flow cytometry and cytometric bead array (CBA) analysis, respectively. Disease activity was evaluated by the 28-joint Disease Activity Score (DAS28). Results: The percentage of circulating CD4þCD161þIL-17þIFN-γ T cells (CD161þ Th17) in RA patients increased significantly and was higher in patients with active disease status (DAS28 > 3.2) compared with those with low disease status (DAS28 3.2), and correlated positively with DAS28, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), IL-17, and IL-6 levels in RA patients. The percentage of circulating CD4þCD161þIL-17IFN-γþ T cells (CD161þ Th1) decreased and correlated negatively with DAS28, CRP, and ESR levels in RA patients, while the percentage of CD4þCD161þIL-17þIFN-γþ T cells (CD161þ Th17/Th1) was unchanged in RA patients and was not correlated with RA disease activity. Conclusions: These data suggest that the percentages of circulating CD161þ Th17 and CD161þ Th1 cells in RA patients reflect the degree of disease activity. They support the hypothesis that Th17 cells are involved in the pathogenesis of RA and that CD161þ Th17/CD161þ Th1-cell imbalance may contribute to the development of RA.
Rheumatoid arthritis (RA) is a systemic inflammatory disease leading to joint damage and functional disability and a common autoimmune disease marked by persistent synovitis, systemic inflammation, and autoantibody production (1). Although the precise immunopathological mechanism of RA is not completely understood, the abnormality of T cells is implicated in the pathogenesis of RA (2). Conventionally, T helper type 1 (Th1) cells have been shown to play an important pathogenetic role in RA (3). More recently, a distinct lineage of CD4þ effector T cells producing interleukin (IL)-17, termed Th17 cells, has been described (4, 5). A study by van Hamburg et al reported that Th17 cells, but not Th1 cells, from patients with RA can drive chronic destruction by cooperating with synovial fibroblasts in a proinflammatory feedback loop (6). There is further clinical evidence that the percentage of circulating Th17 cells of RA patients is significantly elevated and correlates positively with local and systemic disease activity indices (7–9). Ping Zhu, Department of Clinical Immunology, Xijing Hospital, Fourth Military Medical University, No. 127 West Changle Road, Xi’an 710032, Shaanxi Province, PR China. E-mail: [email protected] Accepted 17 September 2013
IL-17, secreted mainly by Th17 cells, is found to have induced massive damage with extensive inflammatory cell migration, bone erosion, and cartilage degradation in both animal RA models and human RA cases (10, 11). Although the exact mechanism of Th17 cells in the pathogenesis of RA is not yet clear, these observations suggest that Th17 cells make an important contribution to the inflammatory pathology of RA. The lectin receptor CD161, a marker of human Th17 cells, has been identified in recent years (12, 13). CD161 is a human homologue of the murine natural killer (NK)-1.1 antigen, which is expressed on the surface of NK T cells and on subsets of CD4þ T cells (14). Several studies have shown that human Th17 cells originate from CD4þCD161þ-naive T-cell precursors (12, 15) and almost all Th17 cells are found to be contained within the CD161þ fraction of circulating CD4þ T cells (12, 13, 15, 16). Besides containing Th17 cells (CD4þCD161þ IL-17þIFN-γ T cells), CD4þCD161þ T-cell subpopulations also contain Th1-cell (CD4þCD161þIL-17IFNγþ T cells) and Th17/Th1-cell subsets (CD4þCD161þ IL-17þIFN-γþ T cells) in human peripheral blood (12, 16). In addition, CD4þCD161þ naive T-cell precursors can differentiate into mature Th17, Th17/Th1, and Th1 cells under the same polarizing conditions (12). It has
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DOI: 10.3109/03009742.2013.846407
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Table 1. Clinical characteristics of rheumatoid arthritis (RA) patients. Characteristics
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Patients (n) Age (years), mean SD Sex (male/female) Disease duration (years), mean SD RF positivity, n (%) Anti-CCP positivity, n (%) ESR (mm/h), mean SD CRP (mg/dL), mean SD DAS28 score, mean SD Treatment, n (%) None Steroid NSAIDs DMARDs TNF-α antagonist
All RA patients
Low disease status
Active disease status
42 41.38 11.71 33/9 3.93 4.54 34 (81.0) 29 (69.0) 34.17 28.96 1.86 3.07 3.85 1.79
18 39.17 13.73 14/4 4.25 3.93 15 (83.3) 12 (66.7) 11.39 7.06 0.38 0.57 2.06 0.47
24 43.04 9.91 19/5 3.69 5.01 19 (79.2) 17 (70.8) 51.25 27.35 2.97 3.69 5.19 1.07
1 (2.4) 6 (14.3) 4 (9.5) 36 (85.7) 5 (11.9)
0 2 (11.1) 1 (5.6) 16 (88.9) 2 (11.1)
1 (5.2) 4 (16.7) 3 (12.5) 20 (83.3) 3 (12.5)
RF, Rheumatoid factor; Anti-CCP, anti-cyclic citrullinated peptide antibody; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein; DAS28, 28-joint Disease Activity Score; NSAID, non-steroidal anti-inflammatory drug; DMARD, disease-modifying anti-rheumatic drug; TNF-α, tumour necrosis factor-α; SD, standard deviation.
also been reported that the percentage of CD161þ Th17/ Th1 cells in juvenile idiopathic arthritis (JIA) synovial fluid is positively correlated with erythrocyte sedimentation rate (ESR) and levels of C-reactive protein (CRP) (16). However, there has been little discussion about the percentages of peripheral blood CD4þCD161þ T-cell subpopulations (Th17, Th1, and Th17/Th1 cell subsets) in RA patients and their potential link to disease activity of RA. Therefore, in this study, we measured the percentages of CD4þCD161þ T-cell subsets (Th17, Th1, and Th17/ Th1) in peripheral blood of RA patients, and assayed their correlations with disease activity parameters.
Method
Flow cytometric analysis The following monoclonal antibodies (mAbs) were used: for surface phenotype staining, phycoerythrin (PE)-conjugated CD4 (clone RPA-T4) and phycoerythrin-cyanin 5 (PC5)-conjugated CD161 (DX12) (all from BD Biosciences, San Diego, CA, USA); for intracellular cytokine detection, allophycocyanin (APC)-conjugated IL-17A (eBio64DEC17, eBiosciences, San Diego, CA, USA) and fluorescein isothiocyanate (FITC)-conjugated IFN-γ (25723.11, BD Biosciences). Appropriately conjugated immunoglobulin (Ig)G antibodies were used as isotype controls. Stained cells were analysed on a Fluorescence Activated Cell Sorter (FACS)Calibur flow cytometer, using CellQuest software (BD Bioscience).
Patients Peripheral blood samples were obtained from 42 patients with RA (33 females and nine males; mean age SD ¼ 41.38 11.71 years) and 15 age- and sex-matched healthy controls (HCs: 12 females and three males; mean age SD ¼ 39.07 10.26 years). Diagnosis of RA was established according to the 1987 revised criteria of the American College of Rheumatology (ACR) for RA (17), and disease activity was assessed by the 28-joint Disease Activity Score (DAS28) (18). DAS28 3.2 was considered to reflect low disease status and DAS28 > 3.2 to reflect an active disease status. In this study, 18 patients had DAS28 3.2 and 24 patients had DAS28 > 3.2. Table 1 shows the patients’ demographic and clinical characteristics and current medications. The study conforms to the recommendations of the Declaration of Helsinki. The Ethics Committee of Xijing Hospital approved this study, and informed consent was obtained from all patients and controls.
Immunofluorescent intracellular staining of cytokines Intracellular cytokines were analysed by flow cytometry to characterize the cytokine-producing cells. In brief, sodium heparinized venous blood was obtained from all participants. After sampling, 500 μL of blood was mixed immediately with an equal volume of RPMI 1640 (Gibco, Paisley, Scotland, UK). To assess the production of intracellular cytokines, diluted blood was stimulated with 50 ng/mL of phorbol myristate acetate (PMA; SigmaAldrich, Steinheim, Germany) and 1 μg/mL of ionomycin (Sigma-Aldrich) in the presence of 10 μg/mL GolgiStop (BD Bioscience) for 5 h at 37˚C in a humidified 5% CO2 incubator. After surface staining of the cells, erythrocytes were lysed by adding FACS lysing solution (BD Biosciences). Then intracellular staining was performed using BD Cytofix/Cytoperm solution and BD Perm/Wash solution following the manufacturer’s instructions (BD Biosciences).
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coefficient was used to calculate the correlations. A two-sided p-value < 0.05 was considered significant. Statistical analyses were performed using SPSS version 17.0 (SPSS Inc, Chicago, IL, USA).
Plasma samples were harvested and stored at –70˚C until cytokine testing was performed. CBA analysis for IL-17, IFN-γ, IL-1β, IL-6, and IL-21 in plasma was performed according to the manufacturer’s instructions (BD Biosciences). In brief, five bead populations with distinct fluorescence intensities coated with capture Abs specific for IL-17, IFN-γ, IL-1β, IL-6, and IL-21 were mixed. Then the beads were incubated in serum enhancement buffer to reduce the possibility of false-positive results. Plasma and PE detection reagent were mixed with a cocktail of capture beads. The mixtures were incubated at room temperature for 3 h in the dark, washed, and resuspended in wash buffer before analysis on the FACSCalibur (BD Biosciences). Data were analysed using CBA analysis software from BD Biosciences. The concentration of each cytokine in plasma was determined by reference to a standard curve.
To investigate whether circulating IL-17þ T cells from HCs and RA patients are contained in the CD161þ fraction, we gated CD4þ T cells into CD4þCD161þ and CD4þCD161 fractions, and each fraction was assessed for their ability to produce IL-17. As shown in Figure 1, the CD4þCD161þ T-cell subsets showed a significantly higher frequency of IL-17-producing cells compared with the CD4þCD161 T cells in both HCs (p < 0.001) and RA patients (p < 0.001).
Statistical analysis
Percentage of CD4þCD161þ T-cell subsets (Th17, Th1, and Th17/Th1) in RA patients
The non-parametric Mann–Whitney U-test was used for statistical analysis of the difference between two groups in all experiments. Spearman’s rank correlation
Expression of the intracellular cytokines IL-17 and/or IFN-γ was assessed in CD4þCD161þ T cells, and representative examples of flow cytometric dot-plots from RA
Percentage of IL-17-producing cells in CD4þCD161þ T cells in HCs and RA patients
B
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0
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104 100
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Cytometric bead array (CBA) analysis
p < 0.001
12
8
4
0 0
200
400
600
800 1000 FCS
1–
D16
CD
C 4+
1+
D16
C 4+
CD
Figure 1. Circulating CD4þCD161þ T cells produce more IL-17 than CD4þCD161 T cells. (A) Flow cytometric dot-plots of intracellular IL-17 production in CD4þCD161 and CD4þCD161þ cells obtained from one representative healthy control (HC) and one patient with rheumatoid arthritis (RA). (B, C) Frequencies of circulating IL-17-producing cells between CD4þCD161 and CD4þCD161þ fractions in (B) HCs and (C) RA patients. The horizontal line indicates the median value. p-values were assessed by the Mann–Whitney U-test.
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significant differences in the frequency of CD161þ Th17/Th1 cells between the two groups (p ¼ 0.959).
As shown in Figure 3, the plasma levels of Th17related cytokines including IL-17 (Figure 3A), IL-1β (Figure 3C), IL-6 (Figure 3D), and IL-21 (Figure 3E) were significantly higher in RA patients than in HCs (all p < 0.05). Among the RA patients with different disease activity, significantly higher IL-17 and IL-6 levels were observed in the patients with DAS28 > 3.2 than in those with DAS28 3.2 (p ¼ 0.008 and p < 0.001, respectively) (Figures 3F and 3I). Although the patients with DAS28 > 3.2 seemed to have higher levels of IL-1β and IL-21 when compared to the levels in the patients with DAS28 3.2, the differences did not reach statistical significance (p ¼ 0.150 and p ¼ 0.055, respectively) (Figures 3H and 3J). In addition, the differences in IFN-γ (Th1-related cytokine) concentration were not significant between RA patients and HCs (p ¼ 0.949) (Figure 3B) or between patients with DAS28 > 3.2 and those with DAS28 3.2 (p ¼ 0.809) (Figure 3G).
A
HC
60 50 40 30 20 10
p < 0.001
–
IL-17+IFN-Y p = 0.198
+
IL-17-IFN-Y
6 4 2 0
RA
HCs
RA
HCs
RA
104
6.87 0.45 63.03 29.65
102 103
104
4.33 0.48 57.22 37.97
DAS28 > 3.2
101 102 103 104100 101 102 103 104 10
+
IL-17+IFN-Y
C
p = 0.004
8
HCs
100
2.69 0.46 51.28 45.57
100 101
100 101 102 103 104 CD4
RA DAS28 ≤ 3.2
100 101 102 103
104
IL-17 101 102 103 100
R2
Plasma levels of cytokines in RA patients
percentages of CD4+CD161+ T cells
IL-17+IFN-Y
B
101 R1 0 200 400 600 8001000 FSC-H
100
CD161 102 103
SSC-H 0 200 400 600 800 1000
104
patients and HCs are shown in Figure 2A. The percentage of IL-17þIFN-γ cells in CD4þCD161þ T cells (CD161þ Th17 cells) increased significantly in RA patients [median 4.83, interquartile range (IQR) 3.68–6.54%] compared to HCs (median 2.69, IQR 2.15–3.68%; p < 0.001) (Figure 2B). By contrast, the percentage of IL-17IFNγþ cells in CD4þCD161þ T cells (CD161þ Th1 cells) was significantly lower in RA patients (median 37.42, IQR 25.14–42.84%) than in HCs (median 45.50, IQR 40.33– 49.35%; p ¼ 0.004) (Figure 2B). For CD4þCD161þ T cells producing both IL-17 and IFN-γ (CD161þ Th17/ Th1 cells), there was no difference between RA patients and HCs (p ¼ 0.198) (Figure 2B). Then we analysed the percentages of CD4þCD161þ T-cell subpopulations in RA patients with different disease activity (Figure 2C). The percentage of CD161þ Th17 cells was increased in the patients with DAS28 > 3.2 (median 5.79, IQR 4.83–6.82%) compared to those with DAS28 3.2 (median 4.10, IQR 3.20– 4.79%; p ¼ 0.001), while a significantly lower frequency of circulating CD161þ Th1 cells was observed in the patients with DAS28 > 3.2 (median 29.89, IQR 20.80– 42.10%) than in those with DAS28 3.2 (median 39.73, IQR 36.85–44.27%; p ¼ 0.021). However, there were no
percentages of CD4+CD161+ T cells
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Th17 and Th1 cells expressing CD161 in RA
p = 0.001
60 50 40 30 20 10
–
IL-17+IFN-Y p = 0.959
0
+
101 102 103 104 IFN-Y IL-17-IFN-Y p = 0.021
+
8 6 4 2 0
.2 .2 .2 .2 3.2 3.2 8≤3 S28>3 S28≤3 S28>3 28≤ AS28> DA DA DAS D DA
2 DAS
Figure 2. Expression of IL-17 and/or IFN-γ on CD4þCD161þ T cells in RA patients and healthy controls (HCs). (A) Whole blood from RA patients and HCs was stimulated with PMA/ionomycin and then the intracellular cytokine expression of IL-17 and/or IFN-γ among CD4þCD161þ T cells was analysed. (B) Frequencies of IL-17- and/or IFN-γ-producing cells in CD4þCD161þ T cells from RA patients and HCs are shown. (C) Detection of IL-17- and/or IFN-γ-producing cells in CD4þCD161þ T cells from RA patients with low disease status (DAS28 3.2) and active disease status (DAS28 > 3.2). The horizontal line indicates the median value. p-values were assessed by the Mann–Whitney U-test.
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2
s
HC
IFN-Y (pg/mL)
6
IL-17 (pg/mL)
100 80 60 40 20 6 4 2 0
G
p = 0.008
3.2 3.2 28 ≤ AS28 > S A D D
RA
2 0 8≤
3.2 3.2 28 > DAS
s
HC
RA
p = 0.150
300 200 100 10 8 6 4 2 0 8≤
2 DAS
p < 0.001
2000 1500 1000 500 100 80 60 40 20 0
s
RA
HC
J
I
H p = 0.809
4
2 DAS
s
HC
400 300 200 100 50 40 30 20 10 0
IL-6 (pg/mL)
F
10 8 6 4 2 0
IL-1β (pg/mL)
RA
300 200 100
E
p < 0.001 IL-21 (pg/mL)
4
D
p = 0.001 IL-6 (pg/mL)
6
0 s HC
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C p = 0.949 IL-1β (pg/mL)
100 80 60 40 20 6 4 2 0
IFN-Y (pg/mL)
IL-17 (pg/mL)
p = 0.004
3.2 3.2 28 > S A D
p < 0.001
400 300 200 100 50 40 30 20 10 0
IL-21 (pg/mL)
B
A
8≤
2 DAS
3.2 3.2 28 > S A D
2000 1500 1000 500 100 80 60 40 20 0 8≤
2 DAS
RA
p = 0.055
3.2 3.2 28 > S A D
Figure 3. Plasma levels of cytokines in RA patients. Comparisons of the levels of plasma (A) IL-17, (B) IFN-γ, (C) IL-1β, (D) IL-6, and (E) IL-21 are shown between RA patients and HCs. Detection of (F) IL-17, (G) IFN-γ, (H) IL-1β, (I) IL-6, and (J) IL-21 levels from RA patients with low disease status (DAS28 3.2) and those with active disease status (DAS28 > 3.2). The horizontal line indicates the median value. p-values were assessed by the Mann– Whitney U-test.
Correlation of CD4þCD161þ T-cell subsets with disease activity and their related cytokines in RA patients The correlation of circulating CD4þCD161þ T-cell subsets with disease activity parameters in RA is shown in Figure 4. The frequency of circulating CD161þ Th17 cells was positively correlated with DAS28 (r ¼ 0.546, p < 0.001) (Figure 4A), CRP levels (r ¼ 0.520, p < 0.001) (Figure 4B), and ESR levels (r ¼ 0.542, p < 0.001) (Figure 4C). By contrast, the frequency of CD161þ Th1 cells was negatively correlated with DAS28 (r ¼ –0.468, p ¼ 0.002) (Figure 4D), CRP (r ¼ –0.430, p ¼ 0.005) (Figure 4E), and ESR (r ¼ –0.341, p ¼ 0.027) (Figure 4F). However, there was no correlation between the percentages of CD161þ Th17/Th1 cells and RA disease activity parameters (all p > 0.05) (Figures 4G, 4H, and 4I). In RA patients, circulating CD161þ Th17 cell percentages were positively correlated with IL-17 levels (r ¼ 0.315, p ¼ 0.042) and IL-6 levels (r ¼ 0.488, p ¼ 0.001). However, CD161þ Th17 cells failed to show a significant correlation with levels of IL-1β (r ¼ 0.241, p ¼ 0.124) and IL-21 (r ¼ 0.256, p ¼ 0.102). In addition, no correlation was present between percentages of CD161þ Th1 cells and IFN-γ levels (r ¼ –0.125, p ¼ 0.430). Furthermore, a significant negative correlation was found between CD161þ Th17 cells and CD161þ Th1 cells (r ¼ –0.570, p < 0.001) (data not shown).
Discussion Numerous inflammatory cells, proinflammatory cytokines, and chemokines are known to play crucial roles
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in the pathogenesis of inflammatory arthritis. In studies of models of autoimmunity, including experimental allergic encephalomyelitis (EAE) (19) and several murine arthritis models (20), the Th17-cell subset has been identified as the principal driver of inflammation. There is also considerable evidence to suggest that Th17 cells are involved in the development of human autoimmune diseases such as RA (21–23). To further investigate the role of Th17 cells in the pathogenesis of RA, we examined the Th17-cell profile in RA patients. Our data show that IL-17-producing cells were found almost exclusively in the CD4þCD161þ fraction in both RA patients and HCs, as reported previously (12, 16). We also found that the frequencies of circulating CD161þ Th17 cells were significantly increased in RA patients and also elevated in the patients with DAS28 > 3.2 compared to those with DAS28 3.2. Moreover, levels of CD161þ Th17 cells were positively correlated with DAS28 and with ESR and CRP levels in RA patients. These results are in partial agreement with findings reported in other investigations (7, 8), in which an increased percentage of circulating Th17 cells correlated with RA disease activity. Our observations are compatible with the hypothesis that Th17 cells contribute to the pathogenesis of RA and suggest that circulating CD161þ Th17 cells may reflect disease activity status in RA patients. The Th17-cell subset has a specific role in immune function through the secretion of effector cytokines. IL-17, the main cytokine of Th17 cells, is involved in the pathogenesis of arthritis, as has been demonstrated in animal arthritis models. By secreting IL-17, Th17 cells can stimulate monocytes to produce proinflammatory
Th17 and Th1 cells expressing CD161 in RA
A 8.0
199
B
r = 0.546 p < 0.001
C
r = 0.520 p < 0.001
20.0
120
DAS28
6.0 5.0 4.0 3.0
100
15.0
ESR (mm/h)
CRP (mg/dL)
7.0
10.0 5.0
1.0
.0 .0
2.0 4.0 6.0 8.0 CD161 + Th17 (%)
10.0
10.0
120
4.0 3.0
15.0
ESR (mm/h)
5.0
10.0 5.0
2.0 4.0 6.0 8.0 CD161 + Th17 (%)
10.0
r = –0.341 p = 0.027
.0
1.0 10.0 20.0 30.0 40.0 50.0 60.0 CD161 + Th1 (%)
60 40
0 10.0 20.0 30.0 40.0 50.0 60.0 CD161 + Th1 (%) H
r = –0.072 p = 0.649
15.0 10.0
10.0 20.0 30.0 40.0 50.0 60.0 CD161 + Th1 (%) I
r = 0.092 p = 0.561
120 ESR (mm/h)
20.0 CRP (mg/dL)
G
80
20
2.0
4.0
.0 F
r = –0.430 p = 0.005
20.0 CRP (mg/dL)
DAS28
2.0 4.0 6.0 8.0 CD161 + Th17 (%)
100
6.0
DAS28
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7.0
5.0
40
E r = –0.468 p = 0.002
8.0
6.0
60
0 .0
D
7.0
80
20
2.0
8.0
r = 0.542 p < 0.001
r = 0.059 p = 0.712
100 80 60 40
3.0
5.0 20
2.0 1.0
.0 .00 .20 .40 .60 .80 1.00 1.20 CD161 + Th17/Th1 (%)
0 .00 .20 .40 .60 .80 1.00 1.20 CD161 + Th17/Th1 (%)
.00 .20 .40 .60 .80 1.00 1.20 CD161 + Th17/Th1 (%)
Figure 4. Correlations between the levels of CD4þCD161þ T-cell subsets and RA disease activity indices. (A–C) Correlations of CD161þ Th17-cell percentage with (A) DAS28, (B) CRP, and (C) ESR levels. (D–F) Correlations of CD161þ Th1-cell percentage with (D) DAS28, (E) CRP, and (F) ESR levels. (G–I) Correlations of CD161þ Th17/Th1-cell percentage with (G) DAS28, (H) CRP, and (I) ESR levels. All correlations were calculated using Spearman’s rank correlation coefficient.
cytokines, thus amplifying the inflammatory cascade (24, 25). In the current study, there was a significant positive correlation between plasma IL-17 levels and the frequency of CD161þ Th17 cells. In line with previous findings (26), we found that the levels of plasma IL-17 were elevated in RA patients compared with HCs. Furthermore, we have shown an increase in plasma IL-17 levels with higher DAS28. Our observations and the data of previous studies indicate that IL-17 may play a crucial role in the pathogenesis of RA and support the therapeutic benefits of IL-17 inhibitors in RA patients (27). The mechanism for increased CD161þ Th17-cell response in patients with RA has not been fully explored. Two previous studies reported that the development of
Th17 cells is dependent on the cytokine milieu, with IL-6, IL-1β, and IL-21 promoting Th17-cell differentiation and expansion (28, 29). Thus, we explored the potential role of these cytokines in promoting IL-17 secretion in RA. In our study, we noted that patients with RA had a trend for increased plasma levels of IL-1β, IL-6, and IL-21, and the high levels of IL-6 were positively correlated with circulating CD161þ Th17-cell frequencies in RA patients. Moreover, a significant increase in plasma IL-6 levels, in parallel with the increase in the frequencies of CD161þ Th17 cells, was observed in RA patients with DAS28 > 3.2. These data suggest the possible involvement of these cytokines, particularly IL-6, in increasing the CD161þ
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200
Th17-cell response we observed, and support the crucial role of IL-6 in Th17-dependent autoimmune diseases (30). It was previously reported that Th17 cells in culture may lose IL-17 expression, possibly through a Th17/Th1 intermediate, to obtain a Th1 phenotype, and CD161 appears to track this conversion (31, 32). Therefore, levels of CD161þ Th1 cells and CD161þ Th17/Th1 cells were also examined in this study. Contrasting with the results of CD161þ Th17 cells, we observed that the percentage of circulating CD161þ Th1 cells decreased significantly and correlated negatively with DAS28, ESR and CRP levels in RA patients. A possible explanation for the decreased CD161þ Th1-cell levels is that the Th17 cells, characterized by production of the cytokine IL-17, have an inhibitory effect on Th1-cell development (33). Another explanation may be a shift in CD4þCD161þ T cells from the Th17 to the Th1 phenotype, which is rare in peripheral blood of RA, although it can occur in the synovial fluid of children with JIA (16, 32). Further studies are clearly needed to determine the mechanism for decreased CD161þ Th1 cells in peripheral blood and their potential link to RA. There was also a significant negative correlation between the frequency of CD161þ Th17 cells and CD161þ Th1 cells. These observations indicate that the balance of CD161þ Th17- and CD161þ Th1-cell responses is dysregulated in RA patients with different disease activity. The immunocyte balance has long been hypothesized as one of the important factors in the development of autoimmune diseases (34, 35), and the CD161þ Th17/CD161þ Th1 imbalance maybe play an important role in RA. As the hallmark cytokine of Th1 cells, IFN-γ exerts an important role as a mediator of chronic autoimmune inflammation (36). However, in this study, we found no influence of disease activity on the concentration of IFN-γ in RA, and there was no correlation between plasma IFNγ levels and CD161þ Th1-cell levels. This is probably because IFN-γ could also be secreted from CD161 Th1 cells, the other Th1-cell subsets (12), and some cells including CD8þ T cells, NK cells, and macrophages (37). Consistent with our data, previous studies also detected a relatively small but substantial percentage of CD4þ CD161þ cells producing both IL-17 and IFN-γ (12, 16). Subanalysis of the percentage of CD161þ Th17/Th1 cells revealed no difference between RA patients with different disease activity. Furthermore, there was no correlation between CD161þ Th17/Th1-cell frequency and the disease activity indices of RA. This could be due to the relatively limited numbers of patients included, but an intrinsic abnormality is also possible. In conclusion, this study provides evidence for the role of Th17 cells in the pathogenesis of RA, with the demonstration of an elevated percentage of Th17 cells expressing CD161 in RA patients, and correlations of this cell subset with disease activity indices. Although the mechanism underlying our data is yet to be determined,
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J Miao et al
it appears that the balance of CD161þ Th17-cell and CD161þ Th1-cell responses is dysregulated in RA, which may play an important role in the development of RA. Further investigations are needed to determine the involvement of CD4þCD161þ T-cell subsets in the joints of RA patients and to clarify the pathogenetic role of the CD161þ Th17/CD161þ Th1 balance in RA. Acknowledgements This study was supported by grants from the Key Program of the National Natural Science Foundation of China (No. 81030058) and the National Science and Technology Major Project of China (No. 2012ZX09103301-026, 2013ZX09301-301).
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