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

In this study, we investigated the hypothesis that regulatory T cells (T reg ) 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. T reg numbers, suppressive function, serum inﬂ ammatory markers, and disease activity were evaluated before and after the therapy. Furthermore, the effects of AT on the frequency and suppressive function of T reg were determined in vitro. Our data revealed that the suppressive function of T reg from RA patients signiﬁ cantly decreased compared with that of control subjects. AT signiﬁ cantly reduced erythrosedi-mentation, C-reactive protein, and disease activity. Con-comitantly, T reg numbers and suppressive functions were signiﬁ cantly improved by AT. Consistent with the in vivo experiments, AT promoted the generation of T reg from primary T cells and enhanced preexisting T reg function in vitro. and signal the Atorvastatin upregulates regulatory T cells and reduces clinical disease activity in patients with arthritis. 28; 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-inﬂ ammatory PBMC, blood mononuclear rheumatoid triglyceride; TNF- (cid:5) M) 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 (cid:5) g) was used for each sample and was separated by SDS-PAGE and then transferred to nitrocellulose membranes. The protein bands were subsequently probed using speciﬁ c 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.


Cell isolation
CD4 + CD25 Ϫ primary T cells and CD4 + CD25 + CD127 low T reg were magnetic-sorted using a CD4 + CD25 + CD127 dim/ Ϫ regulatory T-cell isolation kit (Miltenyi Biotec, Germany) according to the manufacturer's instructions. Briefl y, PBMCs were fi rst incubated with a biotin-labeled cocktail of antibodies, CD4 + CD127 low T cells were isolated using negative selection, and then CD25 Ϫ T cells were removed using positive selection after incubation with anti-CD25 microbeads. A purity of >90% was confi rmed for CD4 + CD25 Ϫ T cells and CD4 + CD25 + CD127 low T reg by fl ow 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 Ϫ T cells from the RA patients were prepared according to the manufacturer's instructions and incubated at a density of 2 × 10 6 cells/ml in RPMI 1640 medium with 100 U/ml penicillin and 100 g/ml streptomycin, 2 mM glutamine, and 10% (v/v) heat-inactivated fetal bovine serum (Gibco BRL) in the presence of 2 g/ml plate-bound anti-CD3 (OKT3; eBioscience), 2 g/ml soluble anti-CD28 (eBioscience) antibodies, and 20 IU/ml IL-2 (Peprotech). AT (1, 5, and 10 M) was added, and the cells were then incubated for 5 days at 37°C in 5% CO 2 . In some experiments, 200 M L -mevalonate and the inhibitors of ERK (U0126, 5 M) and PI3K (LY294002, 5 M) were added to the culture by themselves or with AT.

Suppression assays
To assess the suppressive function, CD4 + CD25 Ϫ T cells (T responder cells [T resp ]) and CD4 + CD25 + CD127 low T reg were cocultured at T resp /T reg ratios of 1:0, 4:1, 2:1, and 1:1 in 200 l of RPMI 1640 medium. Plate-bound anti-CD3 (2 g/ml; eBioscience) and soluble anti-CD28 (5 g/ml; eBioscience) (anti-CD3/28) were added at the beginning of the experiment. After 5 days of culture, 3 H-labeled thymidine (1 Ci/well) was added 16 h before the culture was terminated. The cells were harvested and assayed by scintillation counting. Isolated CD4 low T reg were harvested and washed vigorously with medium after a 24-h incubation period with AT. Then, T reg were cocultured with T resp and tested using the suppression assay. Blood samples were obtained again on the next day, and T resp were freshly isolated when T reg were preincubated with AT.

Cytokines detection
Cultures of 1 × 10 5 magnetic-sorted CD4 + CD25 + CD127 low T reg and 1 × 10 5 CD4 + CD25 Ϫ T cells were cocultured with anti-CD3/28 in U-shaped-bottom 96 well plates to a fi nal volume of 200 l. Three days later, culture supernatants were collected, and tumor necrosis factor-␣ (TNF-␣ ) and IFN-␥ were quantifi ed using ELISA kits (eBioscience) according to the manufacturer's instructions. 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 T reg in RA patients and tested the hypothesis that T reg are involved in the immunomodulatory effects of statins on RA patients.

Subjects
In this study, we enrolled 55 patients with active RA who fulfi lled 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-infl ammatory 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 fi xed and permeabilized according to the manufacturer's instructions before an antibody was added. Isotype controls were used to allow for correct compensation and to confi rm antibody specifi city. The samples were analyzed using fl ow 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 tested ( p < 0.05) ( Fig. 2A ). We also investigated whether T reg from the RA patients were able to suppress TNF-␣ and IFN-␥ production by activated CD4 + CD25 Ϫ T cells. Because CD4 + CD25 Ϫ T cells from the RA patients and the control subjects may differ in their respective abilities to produce TNF-␣ and IFN-␥ upon activation, we fi rst measured the production of cytokines by activated T cells from the two groups. The results suggested an increase in the production of TNF-␣ and IFN-␥ in activated T cells from the RA patients compared with those of the control subjects. Therefore, T reg 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, T reg from the RA patients were less effective at inhibiting the production of the two cytokines than those from the control group ( Fig. 2B ).

AT reduces RA clinical disease activity and enhances the frequency and suppressive function of circulating T reg 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 T reg 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 infl ammatory markers of RA

Western blotting
The CD4 + CD25 Ϫ T cells from the RA patients were preincubated with AT (5 M) 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 g) was used for each sample and was separated by SDS-PAGE and then transferred to nitrocellulose membranes. The protein bands were subsequently probed using specifi c 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 fi gures. 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 signifi cant. 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 fl uorescent-labeled antibodies against CD4, CD25, and Foxp3. The gating strategy for the CD4 + CD25 + Foxp3 + T reg 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 + T reg did not differ signifi cantly 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 ).

T reg display compromised function in patients with RA
Next, CD4 + CD25 + CD127 low T reg and CD4 + CD25 Ϫ T resp were purifi ed by magnetic sorting and tested in the suppression assay. CD4 + CD25 Ϫ T resp 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 + CD127 low T reg function was performed by coculturing them with autologous CD4 + CD25 Ϫ T resp at different ratios (T reg /T resp ratios of 1:1, 1:2, and 1:4). The data indicated that the RA patients showed signifi cantly reduced T reg suppression function compared with that of the control subjects for all T reg /T resp ratios  (13) Values are presented as means ± SD or percentages. RA, rheumatoid arthritis; hs-CRP, high-sensitivity C-reactive protein; ESR, erythrocyte sedimentation rate; DAS28, disease activity 28; IL-6, interleukin-6; ICAM-1, intercellular adhesion molecule-1; HDL, high density lipoprotein; LDL, low density lipoprotein; TNF, tumor necrosis factor; NSAID, nonsteroidal anti-infl ammatory drugs. signifi cant reduction in TC and LDL was also observed in the control subjects receiving AT after 12 weeks ( p < 0.05, data not shown).

AT induces the conversion of T reg from primary T cells and enhanced the function of preexisting T reg in vitro
Next, we performed experiments to investigate the origin of the newly formed T reg . The CD4 + CD25 Ϫ 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 Ϫ T cells resulted in a dosedependent 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 T reg 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 T reg , 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 + CD127 low T cells were isolated and mixed with freshly autologous T resp . Our data indicated that only newly formed CD4 + CD25 + CD127 low T cells in the presence of AT were able to suppress the proliferation of T resp ( Fig. 4B ). 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 T resp , 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 T reg after AT therapy by using a 1:2 T reg /T resp ratio. As expected, T reg 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 signifi cant increase in the suppressive function of T reg 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.
In summary, the above-described results indicate that statins are promising drugs for RA and that they increase the frequency of T reg and restore the suppressive function of these cells in RA patients.  been reported to be related to the conversion of T reg 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 T reg , we next tested whether treatment with an ERK inhibitor or a PI3K inhibitor during T-cell activation could directly induce CD4 + CD25 + Foxp3 + T reg . As shown in Fig. 5B , primary CD4 + CD25 Ϫ T cells activated in the presence of U0126 or LY294002 showed high expression of both CD25 and Foxp3, as was the case for AT.

DISCUSSION
RA is a systemic autoimmune disease characterized by a chronic relapsing-remitting joint infl ammation. Accumulating evidences suggests that T reg defects are involved in the suppression of immune activation in several human diseases ( 21,22 ). Therefore, it is possible that T reg are involved in the physiopathogenesis of rheumatoid arthritis.
To determine whether AT targeted the preexisting T reg , T reg 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 purifi ed T reg of the RA patients ( Fig. 4C ). Consistent with this observation, enhanced suppressive function was observed in T reg that were preincubated with AT ( Fig. 4D ). Similar effects of AT on T reg were observed in the control subjects (data not shown).

AT induces the generation of T reg 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 Ϫ 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 signifi cantly reduced phosphorylation levels of Akt, mTOR, and ERK compared with those of untreated cells. Other signaling pathways that have The frequency of T reg was compared among four groups before (RA-AB, RA-CB, Con-AB, and Con-CB) and after 12 weeks (RA-A1, RA-C1, Con-A1, and Con-C1) of treatment. (C) The proliferation of T resp when they were cultured alone or together with T reg at a 2:1 ratio were compared among four groups before and after 12 weeks of treatment. *, p < 0.05 versus the indicated group.
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 infl ammatory diseases (24)(25)(26)(27). Although statins are not a routine treatment agent for RA patients, mounting evidence shows that statins have benefi cial effects against several aspects of the disease including disease activity, infl ammatory status, and endothelial function ( 28 ). The fi ndings of the Trial of Atorvastatin in Rheumatoid Arthritis, the largest, double-blind, placebocontrolled trial of statin therapy, suggested that AT is a potentially excellent candidate agent for RA patients for reducing both RA disease activity and systemic infl ammatory activity, which were assessed by DAS28 and hs-CRP, respectively ( 29 ). However, a recent cohort study showed that statins do not have any benefi cial effects for reducing disease infl ammation, which was evaluated by the use of oral steroids in RA patients ( 30 ). The benefi cial effects of statins in RA patients were confi rmed in the present study, as evidenced by reduced DAS28, ESR, and hs-CRP. Therefore, understanding the immunomodulatory effects of statins on the T reg 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 T reg . Our results demonstrated that AT signifi cantly increased the frequency of T reg in RA patients. A similar increase of T reg was observed in the control subjects after AT treatment, which is consistent with the research of Karin and colleagues ( 31 ). We further confi rmed the stability of "newly formed" T reg 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 T reg in the RA patients undergoing In animal models, T reg depletion was shown to aggravate the progression of arthritis, whereas the adoptive transfer of T reg could suppress the development of arthritis in the mouse model of collagen-induced arthritis, suggesting that there is therapeutic potential for restoring T reg activity in arthritis (6)(7)(8). Until now, there has been no consensus on T reg defects in RA patients. The frequency of circulating T reg 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 T reg . In the present study, we considered T cells with the phenotypic characteristics of CD4 + CD25 + Foxp3 + to be T reg and confi rmed that these cells shared a phenotype identical to that in healthy subjects. Our results showed that the RA patients had numbers of circulating T reg similar to that of the control donors. Consistent with a previous report ( 13 ), we observed a reduced capacity of T reg to inhibit proliferation and cytokine production by Teff cells. However, another group of researchers demonstrated that T reg 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 fi ndings between our study and others may be due to differences in the study populations, the medication, the methods used to identify and purify T reg , or how the suppression assays were performed. In summary, our fi ndings suggested a potential role for T reg in the disease, and we hypothesized that the compromised function of T reg 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 sient expression of Foxp3 in conventional T cells triggered by activation ( 20 ). Furthermore, the inhibitory function was confi rmed in the presence of AT, supporting the existence of a unique ability of AT to convert T reg . It has been demonstrated that AT could confer its anti-infl ammatory effects both depending on ( 34 ) and independently of ( 35 ) the inhibition of HMG-CoA reductase. To clarify whether the conversion of T reg is mediated by the HMG-CoA reductase pathway, we added mevalonate along with AT and found that this combination could reverse the induction of T reg by AT. These data suggested that the anti-infl ammatory 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 AT therapy. Considering the protective role of T reg on experimental infl ammatory arthritis ( 32 ), our results suggested that the upregulation of T reg is associated with the benefi cial effects of AT in RA. Ehrenstein et al. ( 33 ) found that anti-TNF-␣ therapy could increase the number of T reg by generating a newly formed population of T reg that lacked CD62L expression. In our study, we did not detect the presence of a population of T reg lacking CD62L in RA patients after AT therapy (data not shown). It seems that anti-TNF-␣ therapy and AT therapy act through different mechanisms.
In the in vitro study, CD4 + CD25 Ϫ T primary cells showed a classic phenotype of T reg , as evidenced by the high expression of CD25 and Foxp3 in the presence of AT following activation. However, acquisition of the T reg phenotype does not exclusively mean the conversion of T reg from human T cells because we also observed a population of nonsuppressive T cells with the phenotype of T reg in the absence of AT, and previous research has reported a tran- 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 T reg . Therefore, we propose that the inhibition of ERK and PI3K-Akt-mTOR could be an intermediate step in the induction of T reg by AT. Several other signal pathways, such as p38, JNK, STAT5, and smad3, have been also reported to be involved in the conversion of T reg ( 41,42 ). However, we did not observe any effects of AT on these pathways. As for the preexisting T reg cells, AT enhanced their suppressive function in vitro.
In conclusion, we have demonstrated the systemic infl ammatory response in RA patients assessed by decreased T reg suppressive function. AT signifi cantly upregulates the frequency and impaired function of T reg and reduces clinical disease activity in patients with RA. This is the fi rst study to prospectively analyze the effects of statin therapy on T reg in RA patients. Analysis of the impact of statins on T reg 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 profi le, statin treatment is a promising approach for treating RA and other chronic infl ammatory diseases.