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J Lipid Res. May 2011; 52(5): 1023–1032.
PMCID: PMC3073458

Atorvastatin upregulates regulatory T cells and reduces clinical disease activity in patients with rheumatoid arthritis

Abstract

In this study, we investigated the hypothesis that regulatory T cells (Treg) are involved in the immunomodulatory effects of statins on rheumatoid arthritis (RA) patients. The 12-week study cohort consisted of 55 RA patients and 42 control subjects allocated to either a group treated with atorvastatin (AT) (20 mg/day) or a non-AT group. Treg numbers, suppressive function, serum inflammatory markers, and disease activity were evaluated before and after the therapy. Furthermore, the effects of AT on the frequency and suppressive function of Treg were determined in vitro. Our data revealed that the suppressive function of Treg from RA patients significantly decreased compared with that of control subjects. AT significantly reduced erythrosedimentation, C-reactive protein, and disease activity. Concomitantly, Treg numbers and suppressive functions were significantly improved by AT. Consistent with the in vivo experiments, AT promoted the generation of Treg from primary T cells and enhanced preexisting Treg function in vitro. Moreover, we showed that PI3K-Akt-mTOR and ERK signal pathways were involved in the induction of Treg by AT. In conclusion, AT significantly increased Treg numbers and restored their suppressive function in the RA patients, and this may be relevant in the modulation of uncontrolled inflammation in this disorder.

Keywords: HMG-CoA reductase inhibition, immunity, cell signaling, drug therapy/hypolipidemic drugs, inflammation, lymphocytes, statins

Regulatory T cells (Treg), a subset of T cells that constitutively expresses CD4 and CD25, play a crucial role in preventing autoimmune disorders and actively controlling autoimmune responses (1). Foxp3, a member of the fork-head/winged-helix family of the transcriptional factor, has been identified as the best marker of Treg (2). Recently, a downregulation of CD127 has been shown to be closely correlated with Foxp3 (3). Thus, it can be used as a reliable surface marker for Treg. It has been well documented that Treg are involved in the pathogenesis of autoimmune disorders, such as multiple sclerosis (4) and type 1 diabetes (5), and they have certain protective effects.

Rheumatoid arthritis (RA) is a chronic inflammatory arthropathy associated with systemic inflammation and often leads to clinically significant functional impairment. The etiology of RA is unclear. However, it is accepted that autoimmune responses play distinct roles in the pathogenesis of RA. Studies of experimental models of inflammatory arthritis have revealed that Treg are the protective regulators of the disorder (68). However, to date, there is limited information about the role of Treg in RA (914).

Statins, which inhibit 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, have been shown to possess anti-inflammatory and immunomodulatory properties and contribute to cholesterol reduction (15). A number of studies have shown that statin treatment benefits RA patients, which may be partly due to its immunomodulatory properties (16, 17). In this study, we employed both in vivo and in vitro strategies to evaluate the effects of statins on Treg in RA patients and tested the hypothesis that Treg are involved in the immunomodulatory effects of statins on RA patients.

MATERIALS AND METHODS

Subjects

In this study, we enrolled 55 patients with active RA who fulfilled the 1987 American College of Rheumatology criteria for RA (18). A patient was determined to have active RA if he or she met at least two of the following criteria: (1) the patient had six tender joints, (2) 45 min of morning stiffness, and (3) three swollen joints. The inclusion criteria included a history of RA for at least 1 year of the ongoing active disease and the use of stable doses of disease-modifying antirheumatic drugs (DMARD) for at least 3 months before recruitment. Exclusion criteria included inability to give informed consent, pregnancy or lactation, dyslipidemia, use of any lipid-lowering medication, presence of known hepatic disease or elevated liver transaminase levels within the previous 3 months, and hydroxychloroquine treatment in the previous 3 months. We also included 42 control subjects matched by age and gender. The investigation conformed to the principles outlined in the Declaration of Helsinki. The trial was approved by the country's ethics committee, and the patients and controls provided written informed consent.

Study design

The patients continued to take stable doses of prestudy DMARD, nonsteroidal anti-inflammatory drugs (NSAID), and prednisone during the study. The subjects were randomly allocated to either a group receiving AT treatment (20 mg daily, RA-A and control [Con]-A) or a group that did not receive AT (RA-C and Con-C) for 12 weeks. RA disease activity was evaluated using the Disease Activity Score 28 (DAS28) instrument at the baseline and after the therapy. DAS28 was calculated as previously described (19). Blood samples were collected at the baseline (RA-AB, RA-CB, Con-AB, and Con-CB) and at 12 weeks after the therapy (RA-A1, RA-C1, Con-A1, and Con-C1). After centrifugation, serum was obtained for the assessment of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), highly sensitive C-reactive protein (hs-CRP), interleukin 6 (IL-6), and intercellular adhesion molecule-1 (ICAM-1). Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Hypaque (Sigma-Aldrich) and were then used for serial analyses.

Flow cytometry analysis

The following antibodies were used: anti-CD4-PerCP, anti-CD25-FITC, anti-CD127-Alexa Fluor® 647, anti-CD45RO-APC, anti-CD62L-APC, anti-HLA-DR-APC, and anti-Foxp3-PE (all from eBioscience). For the surface staining, cells were incubated with antibodies for 20 min at 4°C. For the intracellular staining of Foxp3, cells were fixed and permeabilized according to the manufacturer's instructions before an antibody was added. Isotype controls were used to allow for correct compensation and to confirm antibody specificity. The samples were analyzed using flow cytometry on a FACSCalibur (BD Biosciences).

Foxp3 expression by real-time PCR

Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized using random hexamer primers and RNase H-reverse transcriptase (Invitrogen). The sequences of the primers were as follows: Foxp3 forward, 5′-GAAACAGCACATTCCCAGAGTTC-3′, and reverse, 5′-ATGGCCCAGCGGATGAG-3′; and GAPDH, forward, 5′-CCA­CATCGCTCAGACACCAT-3′, and reverse, 5′-GGCAACAATATC­CACTTTACCAGAGT-3′. The samples were analyzed with an ABI Prism 7900 sequence detection system (Applied BioSystems) using SYBR Green Master Mix (Takara, Japan). The relative expression level of Foxp3 was normalized with GAPDH as a housekeeping gene and was calculated by the 2−ΔΔCt method.

Cell isolation

CD4+CD25 primary T cells and CD4+CD25+CD127low Treg were magnetic-sorted using a CD4+CD25+CD127dim/− regulatory T-cell isolation kit (Miltenyi Biotec, Germany) according to the manufacturer's instructions. Briefly, PBMCs were first incubated with a biotin-labeled cocktail of antibodies, CD4+CD127low T cells were isolated using negative selection, and then CD25 T cells were removed using positive selection after incubation with anti-CD25 microbeads. A purity of >90% was confirmed for CD4+CD25 T cells and CD4+CD25+CD127low Treg by flow cytometry.

Cell culture

Pure AT (Honghui medicine Co., Ltd., Beijing, China) was dissolved in 2% DMSO-ethanol (the carrier was tested as a vehicle control). l-Mevalonic acid lactone (Sigma-Aldrich) was activated in 1 N NaOH and then neutralized with 1 N HCl to pH 7.2.

magnetic-sorted CD4+CD25 T cells from the RA patients were prepared according to the manufacturer's instructions and incubated at a density of 2 × 106 cells/ml in RPMI 1640 medium with 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM glutamine, and 10% (v/v) heat-inactivated fetal bovine serum (Gibco BRL) in the presence of 2 μg/ml plate-bound anti-CD3 (OKT3; eBioscience), 2 μg/ml soluble anti-CD28 (eBioscience) antibodies, and 20 IU/ml IL-2 (Peprotech). AT (1, 5, and 10 μM) was added, and the cells were then incubated for 5 days at 37°C in 5% CO2. In some experiments, 200 μM l-mevalonate and the inhibitors of ERK (U0126, 5 μM) and PI3K (LY294002, 5 μM) were added to the culture by themselves or with AT.

Suppression assays

To assess the suppressive function, CD4+CD25 T cells (T responder cells [Tresp]) and CD4+CD25+CD127low Treg were cocultured at Tresp/Treg ratios of 1:0, 4:1, 2:1, and 1:1 in 200 μl of RPMI 1640 medium. Plate-bound anti-CD3 (2 μg/ml; eBioscience) and soluble anti-CD28 (5 μg/ml; eBioscience) (anti-CD3/28) were added at the beginning of the experiment. After 5 days of culture, 3H-labeled thymidine (1 μCi/well) was added 16 h before the culture was terminated. The cells were harvested and assayed by scintillation counting. Isolated CD4+CD25+CD127low Treg were harvested and washed vigorously with medium after a 24-h incubation period with AT. Then, Treg were cocultured with Tresp and tested using the suppression assay. Blood samples were obtained again on the next day, and Tresp were freshly isolated when Treg were preincubated with AT.

Cytokines detection

Cultures of 1 × 105 magnetic-sorted CD4+CD25+CD127low Treg and 1 × 105 CD4+CD25 T cells were cocultured with anti-CD3/28 in U-shaped-bottom 96 well plates to a final volume of 200 μl. Three days later, culture supernatants were collected, and tumor necrosis factor-α (TNF-α) and IFN-γ were quantified using ELISA kits (eBioscience) according to the manufacturer's instructions.

Western blotting

The CD4+CD25 T cells from the RA patients were preincubated with AT (5 μM) for 2 h and then stimulated with anti-CD3/28. At different times, cell lysates were prepared using a 1% NP-40 lysis buffer, and the protein concentration in the lysates was determined using a BCA protein kit (Pierce). Protein extract (25 μg) was used for each sample and was separated by SDS-PAGE and then transferred to nitrocellulose membranes. The protein bands were subsequently probed using specific primary antibodies, phosphorylated ERK1/2, ERK1/2, phosphorylated p38, p38, phosphorylated JNK, JNK, phosphorylated Akt, Akt, phosphorylated mTOR, mTOR, phosphorylated STAT5, STAT5, phosphorylated Smad3, and Smad3 (all at 1/1,000 dilution; Cell Signaling Technology), and then by anti-rabbit-IgG-horseradish peroxidase (1/20,000 dilution). Proteins were detected using an ECL detection kit (Pierce). A comparative analysis was performed using quantitative densitometry.

Statistical analyses

Values are expressed as means ± standard deviations (SD) or percentages in text and figures. For variables with normal distribution and homogeneity of variance, an independent t test or one-way ANOVA was used to test differences among two or more groups. For skewed variables, a nonparametric Kruskal-Wallis-(H) test or Mann-Whitney U test was used for analyses. The effect of AT in vivo was assessed using a paired-sample t test or Wilcoxon signed-rank test within groups, depending on normality. For the ranked data, Pearson's chi-square test or Fisher's exact test was used for the comparison among multiple groups. In all cases, a two-tailed p value of <0.05 was considered significant.

RESULTS

Treg display compromised function in patients with RA

Table 1 summarizes the demographic and clinical characteristics of the study population. The PBMCs of RA patients (n = 55) and control (n = 42) donors were stained with fluorescent-labeled antibodies against CD4, CD25, and Foxp3. The gating strategy for the CD4+CD25+Foxp3+ Treg analysis is shown in Fig. 1A. Further phenotypic analysis revealed that these CD4+CD25+Foxp3+ cells showed a low expression of CD127 and a high expression of CD45RO, CD62L, and HLA-DR, demonstrating their regulatory phenotype (Fig. 1B). CD4+CD25+Foxp3+ Treg did not differ significantly between the RA patients and the control subjects (RA vs. Con, 5.2% ± 1.3% vs. 5.0% ± 1.5% of CD4+ T cells, respectively; p = 0.542 (Fig. 1C). In line with this observation, Foxp3 mRNA expression in the PBMCs appeared to be similar between the RA patients and the control subjects (p = 0.432) (Fig. 1D).

TABLE 1.
Demographic and clinical characteristics of the study population
Fig. 1.
Determination of Treg in RA patients and control subjects is shown. (A) PBMCs from 55 RA patients and 42 control subjects were stained for CD4, CD25, and Foxp3 and analyzed by flow cytometry. Dot plots are representative fluorescence-activated cell sorting ...

Next, CD4+CD25+CD127low Treg and CD4+CD25 Tresp were purified by magnetic sorting and tested in the suppression assay. CD4+CD25 Tresp from the RA patients and the control subjects exhibited similar proliferation in the presence of anti-CD3/28 (p = 0.177) (Fig. 2A). Quantitative analysis of CD4+CD25+CD127low Treg function was performed by coculturing them with autologous CD4+CD25 Tresp at different ratios (Treg/Tresp ratios of 1:1, 1:2, and 1:4). The data indicated that the RA patients showed significantly reduced Treg suppression function compared with that of the control subjects for all Treg/Tresp ratios tested (p <0.05) (Fig. 2A). We also investigated whether Treg from the RA patients were able to suppress TNF-α and IFN-γ production by activated CD4+CD25 T cells. Because CD4+CD25 T cells from the RA patients and the control subjects may differ in their respective abilities to produce TNF-α and IFN-γ upon activation, we first measured the production of cytokines by activated T cells from the two groups. The results suggested an increase in the production of TNF-α and IFN-γ in activated T cells from the RA patients compared with those of the control subjects. Therefore, Treg from the two groups were cocultured with activated T cells from the RA patients to determine their effects on cytokines production. In agreement with the results of the suppression assay, Treg from the RA patients were less effective at inhibiting the production of the two cytokines than those from the control group (Fig. 2B).

Fig. 2.
Compromised Treg suppressive function in RA patients is shown. (A) Magnetic-sorted CD4+ CD25+CD127low Treg and CD4+CD25 Tresp from RA patients and control subjects were mixed at the indicated ratios and tested in the proliferation assay. (B) ...

AT reduces RA clinical disease activity and enhances the frequency and suppressive function of circulating Treg in vivo

The potential of statins to modulate immune responses has led to considerable interest in their use for the treatment of RA. Therefore, we set out to investigate whether the clinical response to statin therapy results in changes in the Treg population. RA patients and control subjects in our study population were allocated to either a group that received AT (20 mg daily, RA-A group and Con-A group) or one that did not (RA-C group and Con-C group) for 12 weeks. Table 2 shows that the RA patients and the control subjects with or without AT treatment were generally comparable at baseline. Table 3 shows the effects of statins on lipids, disease activity, and inflammatory markers of RA patients after 12 weeks. AT produced a significant reduction in both TC and LDL (p <0.001). Disease activity, as assessed by DAS28, decreased significantly by AT (p < 0.001). hs-CRP and erythrocyte sedimentation rate (ESR) were also reduced by AT (p < 0.05). Neither IL-6 nor ICAM-1 was significantly affected by the drug (p > 0.05). A significant reduction in TC and LDL was also observed in the control subjects receiving AT after 12 weeks (p < 0.05, data not shown).

TABLE 2.
Baseline clinical characteristics of Con-A, Con-C, RA-A and RA-C groups
TABLE 3.
Difference in serum lipids, DAS28, and inflammatory factors in RA patients after 12 weeks of therapy

Flow cytometric analyses showed a significant increase in Treg in RA patients (RA-A1 vs. RA-AB or RA-C1: 6.8% ± 1.9% vs. 5.1% ± 1.4% or 5.2% ± 1.1% of CD4+ T cells, respectively, p < 0.05) and the control subjects receiving AT (Con-A1 vs. Con-AB or Con-C1: 6.5% ± 1.2% vs. 5.0% ± 1.6% or 5.3% ± 1.4% of CD4+ T cells, respectively, p < 0.05) compared with the baseline and subjects not receiving AT (Fig. 3A, B). AT showed no effect on the proliferation of Tresp, as a similar extent of proliferation was observed in subjects who received AT or did not receive AT both at baseline and after 12 weeks (Fig. 3C, left panel). Next, we compared the suppressive function of Treg after AT therapy by using a 1:2 Treg/Tresp ratio. As expected, Treg suppressive function remained compromised in patients who did not receive AT 12 weeks later, as it was at baseline (baseline RA-CB vs. Con-CB, p < 0.05; 12 weeks later, RA-C1 vs. Con-C1, p < 0.05) (Fig. 3C, right panel). In contrast, there was a significant increase in the suppressive function of Treg in subjects receiving AT (RA-A1 vs. RA-AB or RA-C1, p < 0.05; Con-A1 vs. Con-AB or Con-C1, respectively, p < 0.05) that was not seen in patients who did not receive AT.

Fig. 3.
Influence of AT on circulating Treg in vivo is shown. (A) Dot plots show representative fluorescence-activated cell sorting pictures of CD4+ CD25+ Foxp3+ Treg from a single person in four groups. (B) The frequency of Treg was compared among four groups ...

In summary, the above-described results indicate that statins are promising drugs for RA and that they increase the frequency of Treg and restore the suppressive function of these cells in RA patients.

AT induces the conversion of Treg from primary T cells and enhanced the function of preexisting Treg in vitro

Next, we performed experiments to investigate the origin of the newly formed Treg. The CD4+CD25 T cells of the RA patients were sorted to >90% purity by magnetic beads separation. As shown in Fig. 4A, the addition of AT to activated RA CD4+CD25 T cells resulted in a dose-dependent increase in the percentage of CD4+CD25+Foxp3+ cells. The increased number of CD4+CD25+Foxp3+ cells was prevented by the product of HMG-CoA reduction, l-mevalonate, indicating that the effects of AT on Treg depended on HMG-CoA reduction. However, a suboptimal increase could also be observed in the absence of AT. Although Foxp3 is the best marker of Treg, previous studies (20) have reported a transient expression of Foxp3 in conventional T cells, triggered by activation in vitro. Therefore, we then tested whether CD4+CD25+Foxp3+ cells differentiated in the presence of AT were functionally suppressive. Five days after the culture, CD4+CD25+CD127low T cells were isolated and mixed with freshly autologous Tresp. Our data indicated that only newly formed CD4+CD25+CD127low T cells in the presence of AT were able to suppress the proliferation of Tresp (Fig. 4B).

Fig. 4.
Effects of AT on Treg in vitro are shown. (A) AT induced CD4+CD25+Foxp3+ from primary CD4+CD25 T cells in a dose-dependent manner and l-mevalonate reversed the induction of these cells by AT. (B) CD4+CD25+Foxp3+ induced only in the presence of ...

To determine whether AT targeted the preexisting Treg, Treg were isolated from the RA patients and incubated with AT for 24 h and then tested in the suppression assay. The results showed that AT increased the expression of Foxp3 in the purified Treg of the RA patients (Fig. 4C). Consistent with this observation, enhanced suppressive function was observed in Treg that were preincubated with AT (Fig. 4D). Similar effects of AT on Treg were observed in the control subjects (data not shown).

AT induces the generation of Treg accompanied by the inhibition of PI3K-Akt-mTOR and ERK signal transduction pathways

To gain insight into the molecular mechanisms, we preincubated the CD4+CD25 T cells from the RA patients with AT, induced activation in these cells using anti-CD3/28, and then probed the cell lysates for the phosphor­ylation of several signaling pathways. Figure 5A shows that AT-treated cells displayed significantly reduced phosphor­ylation levels of Akt, mTOR, and ERK compared with those of untreated cells. Other signaling pathways that have been reported to be related to the conversion of Treg including p38, JNK, STAT5, and smad3 were also tested; however, no alteration was observed (data not shown). To ensure that the inhibition of ERK and the PI3K-Akt-mTOR signaling pathway was downstream of the induction effect of AT on Treg, we next tested whether treatment with an ERK inhibitor or a PI3K inhibitor during T-cell activation could directly induce CD4+CD25+Foxp3+ Treg. As shown in Fig. 5B, primary CD4+CD25 T cells activated in the presence of U0126 or LY294002 showed high expression of both CD25 and Foxp3, as was the case for AT.

Fig. 5.
AT induced the generation of Treg accompanied by the inhibition of PI3K-Akt-mTOR and ERK signal transduction pathways. (A, upper panel) Representative Western blots show the indicated protein in primary CD4+CD25 T cells treated with AT; (lower ...

DISCUSSION

RA is a systemic autoimmune disease characterized by a chronic relapsing–remitting joint inflammation. Accumulating evidences suggests that Treg defects are involved in the suppression of immune activation in several human diseases (21, 22). Therefore, it is possible that Treg are involved in the physiopathogenesis of rheumatoid arthritis. In animal models, Treg depletion was shown to aggravate the progression of arthritis, whereas the adoptive transfer of Treg could suppress the development of arthritis in the mouse model of collagen-induced arthritis, suggesting that there is therapeutic potential for restoring Treg activity in arthritis (68). Until now, there has been no consensus on Treg defects in RA patients. The frequency of circulating Treg in RA patients was reported to be either elevated (9) or unchanged (10, 11) by different researchers. The discrepancy might be due to the use of different strategies to identify Treg. In the present study, we considered T cells with the phenotypic characteristics of CD4+CD25+Foxp3+ to be Treg and confirmed that these cells shared a phenotype identical to that in healthy subjects. Our results showed that the RA patients had numbers of circulating Treg similar to that of the control donors. Consistent with a previous report (13), we observed a reduced capacity of Treg to inhibit proliferation and cytokine production by Teff cells. However, another group of researchers demonstrated that Treg cells from RA patients showed a similar ability to suppress Teff cell proliferation but were not effective for suppressing cytokine production by Teff cells (14). These discrepancies in findings between our study and others may be due to differences in the study populations, the medication, the methods used to identify and purify Treg, or how the suppression assays were performed. In summary, our findings suggested a potential role for Treg in the disease, and we hypothesized that the compromised function of Treg may have contributed to the inappropriate activation of the immune system in patients with RA.

Statins act as immunomodulatory agents that block the adhesion of T cells to antigen-presenting cells, inhibiting MHC-II expression and T-cell activation, suppressing T-cell proliferation, and downregulating Th1 cell function (23). Our previous clinical and experimental studies have demonstrated that statins regulate immune responses and can be used to treat inflammatory diseases (2427). Although statins are not a routine treatment agent for RA patients, mounting evidence shows that statins have beneficial effects against several aspects of the disease including disease activity, inflammatory status, and endothelial function (28). The findings of the Trial of Atorvastatin in Rheumatoid Arthritis, the largest, double-blind, placebo-controlled trial of statin therapy, suggested that AT is a potentially excellent candidate agent for RA patients for reducing both RA disease activity and systemic inflammatory activity, which were assessed by DAS28 and hs-CRP, respectively (29). However, a recent cohort study showed that statins do not have any beneficial effects for reducing disease inflammation, which was evaluated by the use of oral steroids in RA patients (30). The beneficial effects of statins in RA patients were confirmed in the present study, as evidenced by reduced DAS28, ESR, and hs-CRP. Therefore, understanding the immunomodulatory effects of statins on the Treg cells in vivo highlights the role of these cells in RA and might partially explain the immunomodulatory effects of AT. In the present study, we analyzed the effects of AT on the amount and function of Treg. Our results demonstrated that AT significantly increased the frequency of Treg in RA patients. A similar increase of Treg was observed in the control subjects after AT treatment, which is consistent with the research of Karin and colleagues (31). We further confirmed the stability of “newly formed” Treg because they sustained their classic phenotype and suppressive function, even after remaining in vitro for 2 weeks (data not shown). Our data also showed a restored suppressive function of Treg in the RA patients undergoing AT therapy. Considering the protective role of Treg on experimental inflammatory arthritis (32), our results suggested that the upregulation of Treg is associated with the beneficial effects of AT in RA. Ehrenstein et al. (33) found that anti-TNF-α therapy could increase the number of Treg by generating a newly formed population of Treg that lacked CD62L expression. In our study, we did not detect the presence of a population of Treg lacking CD62L in RA patients after AT therapy (data not shown). It seems that anti-TNF-α therapy and AT therapy act through different mechanisms.

In the in vitro study, CD4+CD25 T primary cells showed a classic phenotype of Treg, as evidenced by the high expression of CD25 and Foxp3 in the presence of AT following activation. However, acquisition of the Treg phenotype does not exclusively mean the conversion of Treg from human T cells because we also observed a population of nonsuppressive T cells with the phenotype of Treg in the absence of AT, and previous research has reported a transient expression of Foxp3 in conventional T cells triggered by activation (20). Furthermore, the inhibitory function was confirmed in the presence of AT, supporting the existence of a unique ability of AT to convert Treg. It has been demonstrated that AT could confer its anti-inflammatory effects both depending on (34) and independently of (35) the inhibition of HMG-CoA reductase. To clarify whether the conversion of Treg is mediated by the HMG-CoA reductase pathway, we added mevalonate along with AT and found that this combination could reverse the induction of Treg by AT. These data suggested that the anti-inflammatory effects of AT are mediated by the HMG-CoA reductase pathway.

Statins not only prevent the synthesis of cholesterol but also reduce the synthesis of isoprenoid intermediates, such as farnesyl pyrophosphate or geranylgeranyl pyrophosphate, thus affecting the activation of the small GTPases, including Ras and Rho-GTPases (36). Two important substrates of GTPases, PI3K-Akt-mTOR and ERK signaling pathways, which regulate FoxP3 expression in activated naïve T cells (37, 38), have been demonstrated to be regulated by AT in some systems (39, 40). In our study, we observed that AT inhibited the phosphorylation of ERK and PI3K-Akt-mTOR and U0126 (ERK inhibitor) or LY294002 (PI3K-Akt inhibitor) treatment of activated primary T cells mimicked the effects of AT on Treg. Therefore, we propose that the inhibition of ERK and PI3K-Akt-mTOR could be an intermediate step in the induction of Treg by AT. Several other signal pathways, such as p38, JNK, STAT5, and smad3, have been also reported to be involved in the conversion of Treg (41, 42). However, we did not observe any effects of AT on these pathways. As for the preexisting Treg cells, AT enhanced their suppressive function in vitro.

In conclusion, we have demonstrated the systemic inflammatory response in RA patients assessed by decreased Treg suppressive function. AT significantly upregulates the frequency and impaired function of Treg and reduces clinical disease activity in patients with RA. This is the first study to prospectively analyze the effects of statin therapy on Treg in RA patients. Analysis of the impact of statins on Treg is essential not only to elucidate its role in the pathogenesis of the disorder but also to clarify its mechanism of action. Considering the above-described immunomodulatory effects, its oral bioavailability, and its good safety profile, statin treatment is a promising approach for treating RA and other chronic inflammatory diseases.

Footnotes

Abbreviations:

Con
control
DAS28
Disease Activity Score 28
DMARD
disease-modifying antirheumatic drugs
ESR
erythrocyte sedimentation rate
hs-CRP
highly sensitive C-reactive protein
ICAM-1
intercellular adhesion molecule-1
IL
interleukin
NSAID
nonsteroidal anti-inflammatory drug
PBMC
peripheral blood mononuclear cell
RA
rheumatoid arthritis
TC
total cholesterol
TG
triglyceride
TNF-α
tumor necrosis factor-α
Treg
regulatory T cell
Tresp
T responder cell

This study was supported by the National Natural Science Foundation of China (grants 30600234 and 30871067), the Program for New Century Excellent Talents in the University of China (NCET-09-0380), and the National Basic Research Program of China (973 program grants 2007CB512000 and 2007CB512005).

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