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• W2000135771 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. 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. 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 (1Brusko T.M. Putnam A.L. Bluestone J.A. Human regulatory T cells: role in autoimmune disease and therapeutic opportunities.Immunol. Rev. 2008; 223: 371-390Crossref PubMed Scopus (302) Google Scholar). Foxp3, a member of the fork-head/winged-helix family of the transcriptional factor, has been identified as the best marker of Treg (2Zheng Y. Rudensky A. Foxp3 in the control of the regulatory T cell lineage.Nat. Immunol. 2007; 8: 457-462Crossref PubMed Scopus (566) Google Scholar). Recently, a downregulation of CD127 has been shown to be closely correlated with Foxp3 (3Liu W. Putnam A.L. Xu-Yu Z. Szot G.L. Lee M.R. Zhu S. Gottlieb P.A. Kapranov P. Gingeras T.R. Fazekas de St. Groth B. CD127 expression inversely correlates with FOXP3 and suppressive function of human CD4+ Treg cells.J. Exp. Med. 2006; 203: 1701-1711Crossref PubMed Scopus (2095) Google Scholar). 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 (4Viglietta V. Baecher-Allan C. Weiner H.L. Hafler D.A. Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis.J. Exp. Med. 2004; 199: 971-979Crossref PubMed Scopus (1500) Google Scholar) and type 1 diabetes (5Tang Q. Henriksen K.J. Bi M. Finger E.B. Szot G. Ye J. Masteller E.L. McDevitt H. Bonyhadi M. Bluestone J.A. In vitro-expanded antigen-specific regulatory T cells suppress auto­immune diabetes.J. Exp. Med. 2004; 199: 1455-1465Crossref PubMed Scopus (989) Google Scholar), 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 (6Morgan M.E. Flierman R. van Duivenvoorde L.M. Witteveen H.J. van Ewijk W. van Laar J.M. de Vries R.R. Toes R.E. Effective treatment of collagen-induced arthritis by adoptive transfer of CD25+ regulatory T cells.Arthritis Rheum. 2005; 52: 2212-2221Crossref PubMed Scopus (328) Google Scholar, 7Morgan M.E. Sutmuller R.P. Witteveen H.J. van Duivenvoorde L.M. Zanelli E. Melief C.J. Snijders A. Offringa R. de Vries R.R. Toes R.E. CD25+ cell depletion hastens the onset of severe disease in collagen induced arthritis.Arthritis Rheum. 2003; 48: 1452-1460Crossref PubMed Scopus (262) Google Scholar, 8Nguyen L.T. Jacobs J. Mathis D. Benoist C. Where FoxP3-dependent regulatory T cells impinge on the development of inflammatory arthritis.Arthritis Rheum. 2007; 56: 509-520Crossref PubMed Scopus (115) Google Scholar). However, to date, there is limited information about the role of Treg in RA (9Han G.M. O'Neil-Andersen N.J. Zurier R.B. Lawrence D.A. CD4+CD25high T cell numbers are enriched in the peripheral blood of patients with rheumatoid arthritis.Cell. Immunol. 2008; 253: 92-101Crossref PubMed Scopus (112) Google Scholar, 10van Amelsfort J.M. Jacobs K.M. Bijlsma J.W. Lafeber F.P. Taams L.S. CD4+CD25+ regulatory T cells in rheumatoid arthritis: differences in the presence, phenotype, and function between peripheral blood and synovial fluid.Arthritis Rheum. 2004; 50: 2775-2785Crossref PubMed Scopus (437) Google Scholar, 11Möttönen M. Heikkinen J. Mustonen L. Isomäki P. Luukkainen R. Lassila O. CD4+ CD25+ T cells with the phenotypic and functional characteristics of regulatory T cells are enriched in the synovial fluid of patients with rheumatoid arthritis.Clin. Exp. Immunol. 2005; 140: 360-367Crossref PubMed Scopus (259) Google Scholar, 12de Kleer I.M. Wedderburn L.R. Taams L.S. Patel A. Varsani H. Klein M. de Jager W. Pugayung G. Giannoni F. Rijkers G. CD4+CD25bright regulatory T cells actively regulate inflammation in the joints of patients with the remitting form of juvenile idiopathic arthritis.J. Immunol. 2004; 172: 6435-6443Crossref PubMed Scopus (333) Google Scholar, 13Valencia X. Stephens G. Goldbach-Mansky R. Wilson M. Shevach E.M. Lipsky P.E. TNF downmodulates the function of human CD4+CD25hi T-regulatory cells.Blood. 2006; 108: 253-261Crossref PubMed Scopus (662) Google Scholar, 14Ehrenstein M.R. Evans J.G. Singh A. Moore S. Warnes G. Isenberg D.A. Mauri C. Compromised function of regulatory T cells in rheumatoid arthritis and reversal by anti-TNF-α therapy.J. Exp. Med. 2004; 200: 277-285Crossref PubMed Scopus (1056) Google Scholar). 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 (15Schönbeck U. Libby P. Inflammation, immunity, and HMG-CoA reductase inhibitors statins as antiinflammatory agents?.Circulation. 2004; 109: II18-II26Crossref PubMed Google Scholar). A number of studies have shown that statin treatment benefits RA patients, which may be partly due to its immunomodulatory properties (16Mäki-Petäjä K.M. Booth A.D. Hall F.C. Wallace S.M. Brown J. McEniery C.M. Wilkinson I.B. Ezetimibe and simvastatin reduce inflammation, disease activity, and aortic stiffness and improve endothelial function in rheumatoid arthritis.J. Am. Coll. Cardiol. 2007; 50: 852-858Crossref PubMed Scopus (229) Google Scholar, 17Kanda H. Yokota K. Kohno C. Sawada T. Sato K. Masao Y. Komagata Y. Shimada K. Yamamoto K. Mimura T. Effects of low-dosage simvastatin on rheumatoid arthritis through reduction of Th1/Th2 and CD4/CD8 ratios.Mod. Rheumatol. 2007; 17: 364-368Crossref PubMed Google Scholar). 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. In this study, we enrolled 55 patients with active RA who fulfilled the 1987 American College of Rheumatology criteria for RA (18Arnett F.C. Edworthy S.M. Bloch D.A. McShane D.J. Fries J.F. Cooper N.S. Healey L.A. Kaplan S.R. Liang M.H. Luthra H.S. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis.Arthritis Rheum. 1988; 31: 315-324Crossref PubMed Scopus (18641) Google Scholar). 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. 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 (19Prevoo M.L. van't Hof M.A. Kuper H.H. van Leeuwen M.A. van de Putte L.B. van Riel P.L. 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: 44-48Crossref PubMed Scopus (5105) Google Scholar). 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. 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). 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. 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. 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. 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. 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. 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. 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. 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 1Demographic and clinical characteristics of the study populationCharacteristicsControl group (n = 42)RA group (n = 55)Age (years)50 ± 1251 ± 13Male/female, %5/37, 12/886/49, 11/89Rheumatoid factor-positive, no., %—39, 71Disease duration (years)—12.03 ± 8.38hs-CRP (mg/l)—14.32 ± 7.84ESR (mm/h)—20.75 ± 8.05DAS28—5.67 ± 0.85IL-6 (pg/ml)—17.57 ± 12.37ICAM-1 (ng/ml)—245.61 ± 79.67Total cholesterol (mmol/l)4.54 ± 0.434.50 ± 0.42HDL cholesterol (mmol/l)1.21 ± 0.321.25 ± 0.29LDL cholesterol (mmol/l)2.61 ± 0.342.51 ± 0.40Triglycerides (mmol/l)1.30 ± 0.321.31 ± 0.35Mediations, no. (%)Methotrexate—36 (65)Leflunomide—4 (7)Sulfasalazine—6 (11)TNF inhibitor—16 (29)NSAIDs—42 (76)Prednisone—7 (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-inflammatory drugs. Open table in a new tab 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-inflammatory drugs. 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). 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 2Baseline clinical characteristics of Con-A, Con-C, RA-A and RA-C groupsCharacteristicsCon-A(n = 21)Con-C(n = 21)pRA-A(n = 28)RA-C(n = 27)pAge (years)50 ± 1150 ± 130.92051 ± 1451 ± 130.914Male/female, %3/18, 14/862/19, 10/900.6344/24,14/862/24,7/930.413Rheumatoid factor-positive, no. (%)———18 (64)21 (78)0.271Disease duration (years)———10.95 ± 8.5213.15 ± 8.240.217hs-CRP (mg/l)———14.00 ± 7.2914.65 ± 8.500.822ESR (mm/h)———20.86 ± 8.9320.63 ± 7.200.930DAS28———5.74 ± 0.865.60 ± 0.860.577IL-6 (pg/ml)———16.53 ± 12.5618.7 ± 12.30.348ICAM-1 (ng/ml)———222.50 ± 88.45269.6 ± 62.30.081Total cholesterol (mmol/l)4.51 ± 0.404.57 ± 0.460.6874.51 ± 0.384.48 ± 0.470.738HDL cholesterol (mmol/l)1.18 ± 0.241.24 ± 0.380.9701.23 ± 0.291.27 ± 0.290.606LDL cholesterol (mmol/l)2.60 ± 0.372.61 ± 0.330.9702.50 ± 0.382.51 ± 0.430.754Triglycerides (mmol/l)1.24 ± 0.391.35 ± 0.250.0991.37 ± 0.371.25 ± 0.310.105Medication, no. (%)Methotrexate———19 (68)17 (63)0.703Leflunomide———3 (11)1 (4)0.317Sulfasalazine———2 (7)4 (15)0.362TNF inhibitor———6 (21)10 (37)0.203NSAIDs———24 (86)18 (66)0.096Prednisone———5 (18)2 (7)0.245Values are presented as means ± SD. Open table in a new tab TABLE 3Difference in serum lipids, DAS28, and inflammatory factors in RA patients after 12 weeks of therapyParameterRA-A (n = 28)RA-C (n = 27)pTotal cholesterol (mmol/l)−1.03 (−1.16, −0.89)0.03 (−0.15, 0.20)<0.001HDL cholesterol (mmol/l)0.05 (0, 0.09)0.03 (−0.05, 0.12)0.797LDL cholesterol (mmol/l)−0.99 (−1.13, −0.86)0.06 (−0.11, 0.23)<0.001Triglycerides (mmol/l)−0.10 (−0.21, 0.01)−0.03 (−0.11, 0.06)0.304DAS28−0.41 (−0.52, −0.30)−0.03 (−0.15, 0.10)<0.001hs-CRP (mg/l)−5.41 (−6.48, −4.34)−0.06 (−1.77, 1.65)<0.001ESR (mm/h)−5.46 (−7.31, −3.62)0.03 (−0.15, 0.2)0.005IL-6 (pg/ml)−2.12 (−5.56, 1.32)1.04 (−0.80, 2.87)0.104ICAM-1 (ng/ml)−21.32 (−43.11, 0.48)0.76 (−23.99, 25.51)0.174Values are presented as means (95% confidence interval). Open table in a new tab Values are presented as means ± SD. Values are presented as means (95% confidence interval). 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. 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. 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 (20Wang J. Ioan-Facsinay A. van der Voort E.I. Huizinga T.W. Toes R.E. Transient expression of FOXP3 in human activated nonregulatory CD4+ T cells.Eur. J. Immunol. 2007; 37: 129-138Crossref PubMed Scopus (835) Google Scholar) 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). 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). 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. 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 (21Crispin J.C. Martinez A. Alcocer-Varela J. Quantification of regulatory T cells in patients with systemic lupus erythematosus.J. Autoimmun. 2003; 21: 273-276Crossref PubMed Scopus (363) Google Scholar, 22Kukreja A. Cost G. Marker J. Zhang C. Sun Z. Lin-Su K. Ten S. Sanz M. Exley M. Wilson B. Multiple immuno-regulatory defects in type-1 diabetes.J. Clin. Invest. 2002; 109: 131-140Crossref PubMed Scopus (623) Google Scholar). 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 (6Morga" @default.
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• W2000135771 date "2011-05-01" @default.
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• W2000135771 title "Atorvastatin upregulates regulatory T cells and reduces clinical disease activity in patients with rheumatoid arthritis" @default.
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