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Atorvastatin Modulates Regulatory T Cells and Attenuates Cerebral Damage in a Model of Transient Middle Cerebral Artery Occlusion in Rats

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An Erratum to this article was published on 14 October 2016

Abstract

Regulatory T cells (Tregs) inhibit the activation of the immune response which could down-regulate the systemic and focal activation observed during ischemic stroke. In fact, in animal models, Tregs infiltrate the infarcted brain and reduce the pro-inflammatory cytokine production and infarct volume, mainly in late stages of ischemia. Recently, an expansion and greater suppressive capacity of circulating Tregs after treatment with statins was observed, in addition to their cardio- and neuroprotective actions demonstrated previously. Thus, to determine whether Treg modulation mediated by statins can also be beneficial during stroke, cerebral ischemia was artificially induced in Wistar rats by transient middle cerebral artery occlusion (tMCAO) during 60 minutes with subsequent reperfusion for 7 days. Six hours after surgery, some animals were treated with atorvastatin (ATV, 10 mg/kg) or carboxymethylcellulose as vehicle at the same concentration every other day during 7 days. Some animals were sham operated as control group of surgery. Interestingly, ATV treatment prevented the development of infarct volume, reduced the neurological deficits, and the circulating and cervical lymph node CD25+FoxP3+ Treg population. Moreover, there was a reduction of glial cell activation, which correlated with decreased circulating Tregs. Remarkably, treatment with ATV induced an increase in the frequency of CD4+CD25+ T cells, in particular of those expressing CTLA-4, in brain samples. Together, these results suggest that ATV can modulate Tregs in peripheral tissue and favor their accumulation in the brain, where they can exert neuroprotective actions maybe by the reduction of glial cell activation.

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References

  • Ajmo CT, Vernon DO, Collier L, Hall AA, Garbuzova-Davis S, Willing A, Pennypacker KR (2008) The spleen contributes to stroke-induced neurodegeneration. J Neurosci Res 86:2227–2234

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Anderson SL, Duke-Novakovski T, Singh B (2014) The immune response to anesthesia: part 1. Vet Anaesth Analg 41:113–126

    Article  CAS  PubMed  Google Scholar 

  • Barakat R, Redzic Z (2015) The role of activated microglia and resident macrophages in the neurovascular unit during cerebral ischemia: is the jury still out? Med Princ Pract 25:3–14

    Article  PubMed  Google Scholar 

  • Callaway JK, Wood C, Jenkins TA, Royse AG, Royse CF (2016) Isoflurane in the presence or absence of surgery increases hippocampal cytokines associated with memory deficits and responses to brain injury in rats. Behav Brain Res 303:44–52

    Article  CAS  PubMed  Google Scholar 

  • Céspedes-Rubio A, Jurado FW, Cardona-Gómez GP (2010) p120 catenin/αN-catenin are molecular targets in the neuroprotection and neuronal plasticity mediated by atorvastatin after focal cerebral ischemia. J Neurosci Res 88:3621–3634

    Article  PubMed  Google Scholar 

  • Chan A, Yan J, Csurhes P, Greer J, McCombe P (2015) Circulating brain derived neurotrophic factor (BDNF) and frequency of BDNF positive T cells in peripheral blood in human ischemic stroke: effect on outcome. J Neuroimmunol 286:42–47

    Article  CAS  PubMed  Google Scholar 

  • Chen J, Zhang ZG, Li Y, Wang Y, Wang L, Jiang H, Zhang C, Lu M, Katakowski M, Feldkamp CS, Chopp M (2003) Statins induce angiogenesis, neurogenesis, and synaptogenesis after stroke. Ann Neurol 53:743–751

    Article  CAS  PubMed  Google Scholar 

  • Corbett D, Nurse S (1998) The problem of assessing effective neuroprotection in experimental cerebral ischemia. Prog Neurobiol 54:531–548

    Article  CAS  PubMed  Google Scholar 

  • Cui X, Chopp M, Zacharek A, Roberts C, Lu M, Savant-Bhonsale S, Chen J (2009) Chemokine, vascular and therapeutic effects of combination simvastatin and BMSC treatment of stroke. Neurobiol Dis 36:35–41

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Culley DJ, Cotran EK, Karlsson E, Palanisamy A, Boyd JD, Xie Z, Crosby G (2013) Isoflurane affects the cytoskeleton but not survival, proliferation, or synaptogenic properties of rat astrocytes in vitro. Br J Anaesth 110(Suppl 1):i19–i28

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dirnagl U, Klehmet J, Braun JS, Harms H, Meisel C, Ziemssen T, Prass K, Meisel A (2007) Stroke-induced immunodepression: experimental evidence and clinical relevance. Stroke; J Cereb Circ 38:770–773

    Article  Google Scholar 

  • García-Bonilla L, Campos M, Giralt D, Salat D, Chacón P, Hernández-Guillamon M, Rosell A, Montaner J (2012) Evidence for the efficacy of statins in animal stroke models: a meta-analysis. J Neurochem 122:233–243

    Article  PubMed  Google Scholar 

  • Gelderblom M, Leypoldt F, Steinbach K, Behrens D, Choe CU, Siler DA, Arumugam TV, Orthey E, Gerloff C, Tolosa E, Magnus T (2009) Temporal and spatial dynamics of cerebral immune cell accumulation in stroke. Stroke; J Cereb Circ 40:1849–1857

    Article  Google Scholar 

  • Gelderblom M, Weymar A, Bernreuther C, Velden J, Arunachalam P, Steinbach K, Orthey E, Arumugam TV, Leypoldt F, Simova O, Thom V, Friese MA, Prinz I, Hölscher C, Glatzel M, Korn T, Gerloff C, Tolosa E, Magnus T (2012) Neutralization of the IL-17 axis diminishes neutrophil invasion and protects from ischemic stroke. Blood 120:3793–3802

    Article  CAS  PubMed  Google Scholar 

  • Graca L, Cobbold SP, Waldmann H (2002) Identification of regulatory T cells in tolerated allografts. J Exp Med 195:1641–1646

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Greenwood J, Steinman L, Zamvil SS (2006) Statin therapy and autoimmune disease: from protein prenylation to immunomodulation. Nat Rev Immunol 6:358–370

    Article  CAS  PubMed  Google Scholar 

  • Gu L, Xiong X, Wei D, Gao X, Krams S, Zhao H (2013) T cells contribute to stroke-induced lymphopenia in rats. PLoS One 8:e59602

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gutierrez-Vargas JA, Muñoz-Manco JI, Garcia-Segura LM, Cardona-Gómez GP (2014) GluN2B N-methyl-D-aspartic acid receptor subunit mediates atorvastatin-induced neuroprotection after focal cerebral ischemia. J Neurosci Res 92:1529–1548

    Article  CAS  PubMed  Google Scholar 

  • Gutiérrez-Vargas JA, Moreno H, Cardona-Gómez GP (2016) Targeting CDK5 post-stroke provides long-term neuroprotection and rescues synaptic plasticity. J Cereb Blood Flow Metab. doi:10.1177/0271678X16662476

  • Hewett SJ, Jackman NA, Claycomb RJ (2012) Interleukin-1β in central nervous system injury and repair. Eur J Neurodegener Dis 1:195–211

    PubMed  PubMed Central  Google Scholar 

  • Hu Y, Zheng Y, Wu Y, Ni B, Shi S (2014) Imbalance between IL-17 A-producing cells and regulatory T cells during ischemic stroke. Mediat Inflamm 2014:813045

    Google Scholar 

  • Huehn J, Siegmund K, Lehmann JC, Siewert C, Haubold U, Feuerer M, Debes GF, Lauber J, Frey O, Przybylski GK, Niesner U, de la Rosa M, Schmidt CA, Bräuer R, Buer J, Scheffold A, Hamann A (2004) Developmental stage, phenotype, and migration distinguish naive- and effector/memory-like CD4+ regulatory T cells. J Exp Med 199:303–313

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kawai H, Deguchi S, Deguchi K, Yamashita T, Ohta Y, Omote Y, Kurata T, Ikeda Y, Matsuura T, Abe K (2011) Protection against ischemic stroke damage by synergistic treatment with amlodipine plus atorvastatin in Zucker metabolic rat. Brain Res 1382:308–314

    Article  CAS  PubMed  Google Scholar 

  • Ke D, Fang J, Fan L, Chen Z, Chen L (2013) Regulatory T cells contribute to rosuvastatin-induced cardioprotection against ischemia-reperfusion injury. Coron Artery Dis 24:334–341

    Article  PubMed  Google Scholar 

  • Kim E, Yang J, Beltran CD, Cho S (2014) Role of spleen-derived monocytes/macrophages in acute ischemic brain injury. J Cereb Blood Flow Metab 34:1411–1419

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kingsley CI, Karim M, Bushell AR, Wood KJ (2002) CD25 + CD4+ regulatory T cells prevent graft rejection: CTLA-4- and IL-10-dependent immunoregulation of alloresponses. J Immunol 168:1080–1086

    Article  CAS  PubMed  Google Scholar 

  • Kinsey GR, Sharma R, Huang L, Li L, Vergis AL, Ye H, Ju ST, Okusa MD (2009) Regulatory T cells suppress innate immunity in kidney ischemia-reperfusion injury. J Am Soc Nephrol 20:1744–1753

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kleinschnitz C, Kraft P, Dreykluft A, Hagedorn I, Göbel K, Schuhmann MK, Langhauser F, Helluy X, Schwarz T, Bittner S, Mayer CT, Brede M, Varallyay C, Pham M, Bendszus M, Jakob P, Magnus T, Meuth SG, Iwakura Y, Zernecke A, Sparwasser T, Nieswandt B, Stoll G, Wiendl H (2013) Regulatory T cells are strong promoters of acute ischemic stroke in mice by inducing dysfunction of the cerebral microvasculature. Blood 121:679–691

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Klingenberg R, Gerdes N, Badeau RM, Gisterå A, Strodthoff D, Ketelhuth DF, Lundberg AM, Rudling M, Nilsson SK, Olivecrona G, Zoller S, Lohmann C, Lüscher TF, Jauhiainen M, Sparwasser T, Hansson GK (2013) Depletion of FOXP3+ regulatory T cells promotes hypercholesterolemia and atherosclerosis. J Clin Invest 123:1323–1334

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lai LW, Yong KC, Lien YH (2012) Pharmacologic recruitment of regulatory T cells as a therapy for ischemic acute kidney injury. Kidney Int 81:983–992

    Article  CAS  PubMed  Google Scholar 

  • Li P, Gan Y, Sun BL, Zhang F, Lu B, Gao Y, Liang W, Thomson AW, Chen J, Hu X (2013) Adoptive regulatory T-cell therapy protects against cerebral ischemia. Ann Neurol 74:458–471

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li P, Mao L, Liu X, Gan Y, Zheng J, Thomson AW, Gao Y, Chen J, Hu X (2014) Essential role of program death 1-ligand 1 in regulatory T-cell-afforded protection against blood-brain barrier damage after stroke. Stroke; J Cereb Circ 45:857–864

    Article  CAS  Google Scholar 

  • Liesz A, Suri-Payer E, Veltkamp C, Doerr H, Sommer C, Rivest S, Giese T, Veltkamp R (2009) Regulatory T cells are key cerebroprotective immunomodulators in acute experimental stroke. Nat Med 15:192–199

    Article  CAS  PubMed  Google Scholar 

  • Ling EM, Smith T, Nguyen XD, Pridgeon C, Dallman M, Arbery J, Carr VA, Robinson DS (2004) Relation of CD4 + CD25+ regulatory T-cell suppression of allergen-driven T-cell activation to atopic status and expression of allergic disease. Lancet 363:608–615

    Article  CAS  PubMed  Google Scholar 

  • Mausner-Fainberg K, Luboshits G, Mor A, Maysel-Auslender S, Rubinstein A, Keren G, George J (2008) The effect of HMG-CoA reductase inhibitors on naturally occurring CD4 + CD25+ T cells. Atherosclerosis 197:829–839

    Article  CAS  PubMed  Google Scholar 

  • Mira E, Leon B, Barber DF, Jimenez-Baranda S, Goya I, Almonacid L, Marquez G, Zaballos A, Martinez AC, Stein JV, Ardavin C, Manes S (2008) Statins induce regulatory T cell recruitment via a CCL1 dependent pathway. J Immunol 181:3524–3534

    Article  CAS  PubMed  Google Scholar 

  • Mor A, Planer D, Luboshits G, Afek A, Metzger S, Chajek-Shaul T, Keren G, George J (2007) Role of naturally occurring CD4+ CD25+ regulatory T cells in experimental atherosclerosis. Arterioscler Thromb Vasc Biol 27:893–900

    Article  CAS  PubMed  Google Scholar 

  • Mottet C, Uhlig HH, Powrie F (2003) Cutting edge: cure of colitis by CD4 + CD25+ regulatory T cells. J Immunol 170:3939–3943

    Article  CAS  PubMed  Google Scholar 

  • Offner H, Subramanian S, Parker SM, Wang C, Afentoulis ME, Lewis A, Vandenbark AA, Hurn PD (2006) Splenic atrophy in experimental stroke is accompanied by increased regulatory T cells and circulating macrophages. J Immunol 176:6523–6531

    Article  CAS  PubMed  Google Scholar 

  • Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Popp A, Jaenisch N, Witte OW, Frahm C (2009) Identification of ischemic regions in a rat model of stroke. PLoS One 4:e4764

    Article  PubMed  PubMed Central  Google Scholar 

  • Posada-Duque RA, Velasquez-Carvajal D, Eckert GP, Cardona-Gomez GP (2013) Atorvastatin requires geranylgeranyl transferase-I and Rac1 activation to exert neuronal protection and induce plasticity. Neurochem Int 62:433–445

    Article  CAS  PubMed  Google Scholar 

  • Potey C, Ouk T, Petrault O, Petrault M, Berezowski V, Salleron J, Bordet R, Gautier S (2015) Early treatment with atorvastatin exerts parenchymal and vascular protective effects in experimental cerebral ischaemia. Br J Pharmacol 172:5188–5198

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Qiu R, Cai A, Dong Y, Zhou Y, Yu D, Huang Y, Zheng D, Rao S, Feng Y, Mai W (2012) SDF-1α upregulation by atorvastatin in rats with acute myocardial infarction via nitric oxide production confers anti-inflammatory and anti-apoptotic effects. J Biomed Sci 19:99

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Reynolds AD, Banerjee R, Liu J, Gendelman HE, Mosley RL (2007) Neuroprotective activities of CD4 + CD25+ regulatory T cells in an animal model of Parkinson's disease. J Leukoc Biol 82:1083–1094

    Article  CAS  PubMed  Google Scholar 

  • Rodríguez-Perea AL, Montoya CJ, Olek S, Chougnet CA, Velilla PA (2015) Statins increase the frequency of circulating CD4+ FOXP3+ regulatory T cells in healthy individuals. J Immunol Res 2015:762506

    Article  PubMed  PubMed Central  Google Scholar 

  • Saito T, Nito C, Ueda M, Inaba T, Kamiya F, Muraga K, Katsura K, Katayama Y (2014) Continuous oral administration of atorvastatin ameliorates brain damage after transient focal ischemia in rats. Life Sci 94:106–114

    Article  CAS  PubMed  Google Scholar 

  • Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M (1995) Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 155:1151–1164

    CAS  PubMed  Google Scholar 

  • Seifert HA, Hall AA, Chapman CB, Collier LA, Willing AE, Pennypacker KR (2012) A transient decrease in spleen size following stroke corresponds to splenocyte release into systemic circulation. J NeuroImmune Pharmacol 7:1017–1024

    Article  PubMed  PubMed Central  Google Scholar 

  • Shim R, Wong CH (2016) Ischemia, immunosuppression and infection--tackling the predicaments of post-stroke complications. Int J Mol Sci 17

  • Skinner R, Georgiou R, Thornton P, Rothwell N (2009) Psychoneuroimmunology of stroke. Immunol Allergy Clin N Am 29:359–379

    Article  Google Scholar 

  • Stubbe T, Ebner F, Richter D, Engel O, Randolf Engel O, Klehmet J, Royl G, Meisel A, Nitsch R, Meisel C, Brandt C (2013) Regulatory T cells accumulate and proliferate in the ischemic hemisphere for up to 30 days after MCAO. J Cereb Blood Flow Metab 33:37–47

    Article  CAS  PubMed  Google Scholar 

  • Tai X, Van Laethem F, Pobezinsky L, Guinter T, Sharrow SO, Adams A, Granger L, Kruhlak M, Lindsten T, Thompson CB, Feigenbaum L, Singer A (2012) Basis of CTLA-4 function in regulatory and conventional CD4(+) T cells. Blood 119:5155–5163

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tang TT, Song Y, Ding YJ, Liao YH, Yu X, Du R, Xiao H, Yuan J, Zhou ZH, Liao MY, Yao R, Jevallee H, Shi GP, Cheng X (2011) Atorvastatin upregulates regulatory T cells and reduces clinical disease activity in patients with rheumatoid arthritis. J Lipid Res 52:1023–1032

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Urra X, Cervera A, Villamor N, Planas AM, Chamorro A (2009) Harms and benefits of lymphocyte subpopulations in patients with acute stroke. Neuroscience 158:1174–1183

    Article  CAS  PubMed  Google Scholar 

  • Veenhof AA, Vlug MS, van der Pas MH, Sietses C, van der Peet DL, de Lange-de Klerk ES, Bonjer HJ, Bemelman WA, Cuesta MA (2012) Surgical stress response and postoperative immune function after laparoscopy or open surgery with fast track or standard perioperative care: a randomized trial. Ann Surg 255:216–221

    Article  CAS  PubMed  Google Scholar 

  • Villamil-Ortiz JG, Cardona-Gomez GP (2015) Comparative analysis of autophagy and tauopathy related markers in cerebral ischemia and Alzheimer's disease animal models. Front Aging Neurosci 7:84

    Article  PubMed  PubMed Central  Google Scholar 

  • Wang J, Xie L, Yang C, Ren C, Zhou K, Wang B, Zhang Z, Wang Y, Jin K, Yang GY (2015) Activated regulatory T cell regulates neural stem cell proliferation in the subventricular zone of normal and ischemic mouse brain through interleukin 10. Front Cell Neurosci 9:361

    CAS  PubMed  PubMed Central  Google Scholar 

  • Xie L, Choudhury GR, Winters A, Yang SH, Jin K (2015) Cerebral regulatory T cells restrain microglia/macrophage-mediated inflammatory responses via IL-10. Eur J Immunol 45:180–191

    Article  CAS  PubMed  Google Scholar 

  • Yan J, Read SJ, Henderson RD, Hull R, O'Sullivan JD, McCombe PA, Greer JM (2012) Frequency and function of regulatory T cells after ischaemic stroke in humans. J Neuroimmunol 243:89–94

    Article  CAS  PubMed  Google Scholar 

  • Yang S, Liu J, Zhang X, Tian J, Zuo Z, Yue X (2016) Anesthetic isoflurane attenuates activated microglial cytokine-induced VSC4.1 motoneuronal apoptosis. Am J Transl Res 8:1437–1446

    PubMed  PubMed Central  Google Scholar 

  • Yasuno F, Taguchi A, Yamamoto A, Kajimoto K, Kazui H, Kudo T, Kikuchi-Taura A, Sekiyama A, Kishimoto T, Iida H, Nagatsuka K (2014) Microstructural abnormality in white matter, regulatory T lymphocytes, and depressive symptoms after stroke. Psychogeriatrics 14:213–221

    Article  PubMed  Google Scholar 

  • Yilmaz G, Arumugam TV, Stokes KY, Granger DN (2006) Role of T lymphocytes and interferon-gamma in ischemic stroke. Circulation 113:2105–2112

    Article  PubMed  Google Scholar 

  • Zhou W, Liesz A, Bauer H, Sommer C, Lahrmann B, Valous N, Grabe N, Veltkamp R (2013) Postischemic brain infiltration of leukocyte subpopulations differs among murine permanent and transient focal cerebral ischemia models. Brain Pathol 23:34–44

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The authors wish to thank Anne-Lise Haenni for her constructive comments. Ana Lucia Rodriguez is recipient of a doctoral scholarship from COLCIENCIAS.

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Authors and Affiliations

  1. Grupo Inmunovirología, Facultad de Medicina, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia

    Ana Lucía Rodríguez-Perea, Carlos Julio Montoya Guarin & Paula Andrea Velilla Hernández

  2. Area de Neurobiología Celular y Molecualr, Grupo de Neurociencias de Antioquia, Facultad de Medicina, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia

    Johanna Gutierrez-Vargas & Gloria Patricia Cardona-Gómez

  3. Grupo de Inmunología Celular e Inmunogenética (GICIG), Instituto de Investigaciones Médicas, Facultad de Medicina, Universidad de Antioquia UdeA, Calle 70 No 52-21, Medellín, Colombia

    Mauricio Rojas

  4. Unidad de Citometría, Facultad de Medicina, Sede de Investigación Universitaria, Universidad de Antioquia UdeA, Calle 70-52-21, Medellín, Colombia

    Mauricio Rojas

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  1. Ana Lucía Rodríguez-Perea
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Correspondence to Ana Lucía Rodríguez-Perea.

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An erratum to this article is available at http://dx.doi.org/10.1007/s11481-016-9714-5.

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Supplemental Figure 1

mRNA expression of IL-1β (a), IFN-γ (b), IL-6 (c), TNF-α (d), MPO (e), MMP-9 (f), NOX-2 (g) and TjP-1 (h) from tissue obtained of penumbra zone in sham-operated rats, tMCAO and tMCAO ATV. Graphs represent median and IQR; n = 1–5 rats/group. (TIFF 110 kb) (GIF 110 kb)

High Resolution Image (TIFF 106 kb)

Supplemental Figure 2

The percentage of CD25+FoxP3+ Treg cells was determined by flow cytometry in peripheral blood (a) spleen (b) and cervical lymph nodes (c) after 60 min of tMCAO at day 7 and 15 postreperfusion. The sham-operated rats and tMCAO rats were treated with vehicle (carboxymethylcellulose) administered at the same dose and frequency as the treatment. Another group of tMCAO rats was treated with ATV (10 mg/Kg). First doses of ATV or vehicle began 6 h after surgical procedure; the rats sacrificed at day 3 received each day a dose of ATV or vehicle, whereas animals sacrificed at day 15 received doses every other day. Graphs represent median and IQR. Mann–Whitney test shows no significant differences between the groups studied. n = 2–5 rats/group. (GIF 67 kb)

High Resolution Image (TIFF 69 kb)

Supplemental Figure 3

The percentage of CD3+CD4+FoxP3−CTLA-4+ cells was determined by flow cytometry in peripheral blood, spleen and lymphoid nodules in sham operated rats, tMCAO-Veh and tMCAO-ATV at day 7 post reperfusion. Graphs represent median and IQR. The Mann–Whitney test showed no significant differences between the groups studied. n = 8–9 rats/group. (GIF 24 kb)

High Resolution Image (TIFF 26 kb)

Supplemental Figure 4

mRNA expression of FOXP3 (a), CTLA-4 (b), IDO (c), IL-10 (d) and TGF-β (e) from splenic CD4+ T cells in sham operated rats, tMCAO-Veh and tMCAO-ATV at day 7 post reperfusion. Graphs represent median and IQR. The Mann–Whitney test showed no significant differences between the groups studied. n = 4–5 rats/group. (GIF 60 kb)

High Resolution Image (TIFF 58 kb)

Supplemental Figure 5

mRNA expression of IL-1β (a), IFN-γ (b), IL-6 (c), TNF-α (d) and IL-17 (e) from splenic CD4+ T cells in sham operated rats, tMCAO-Veh and tMCAO-ATV at day 7 post reperfusion. Graphs represent median and IQR. The Mann–Whitney test showed no significant differences between the groups studied. n = 4–5 rats/group. (GIF 65 kb)

High Resolution Image (TIFF 55 kb)

Supplemental Figure 6

Representative histograms of CFSE labelling of CD3+CD4+ and CD3+CD4− cells showing proliferation of both cell types. Mononuclear cells from spleen of sham operated rats, tMCAO-Veh and tMCAO-ATV at day 7 post reperfusion were thawed and stained with Carboxyfluorescein succinimidyl ester (CFSE) (1.25 μM, Molecular Probes/Invitrogen, Eugene, OR, USA) and seeded in 96-well culture plates and stimulation or not with concanavalin A (1.5 μg/ml, Sigma) at 37 °C and 5 % CO2 for 72 h. Proliferation was measured in both T cell types. Data were collected on a FACSDiVa (BD Biosciences) flow cytometer and analyzed using FlowJo software (Tree Star, San Carlos, CA, USA). Proliferation index (PI) is shown in each histogram. The Mann–Whitney test showed no significant differences between the groups studied. n = 2–4 rats/group. (GIF 112 kb)

High Resolution Image (TIFF 295 kb)

Supplemental Table 1

(GIF 153 kb)

High Resolution Image (TIFF 147 kb)

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Rodríguez-Perea, A.L., Gutierrez-Vargas, J., Cardona-Gómez, G.P. et al. Atorvastatin Modulates Regulatory T Cells and Attenuates Cerebral Damage in a Model of Transient Middle Cerebral Artery Occlusion in Rats. J Neuroimmune Pharmacol 12, 152–162 (2017). https://doi.org/10.1007/s11481-016-9706-5

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