Inflammopharmacol DOI 10.1007/s10787-015-0252-1
Inflammopharmacology
RESEARCH ARTICLE
Simvastatin ameliorates experimental autoimmune encephalomyelitis by inhibiting Th1/Th17 response and cellular infiltration Daniel May de Oliveira1,5 • Enedina Maria Lobato de Oliveira4 • Merari de Fa´tima Ramires Ferrari2 • Patrı´cia Semedo6 • Meire Ioshie Hiyane5 • Marcos Antoˆnio Cenedeze3 • Alvaro Pacheco-Silva3 • Niels Olsen Saraiva Caˆmara3,5 Jean Pierre Schatzmann Peron1
•
Received: 26 May 2015 / Accepted: 20 October 2015 Springer Basel 2015
Abstract Aim Experimental autoimmune encephalomyelitis (EAE) is a CD4?-mediated autoimmune pathology of the central nervous system (CNS) that is used as a model for the study of the human neuroinflammatory disease, multiple sclerosis. During the development of EAE, auto-reactive Th1 and Th17 CD4? T cells infiltrate the CNS promoting inflammatory cells recruitment, focal inflammation and tissue destruction. In this sense, statins, agents used to lower lipid levels, have recently shown to exert interesting immunomodulatory function. In fact, statins promote a bias towards a Th2 response, which ameliorates the clinical Niels Olsen Saraiva Caˆmara and Jean Pierre Schatzmann Peron have contributed equally to this manuscript. & Daniel May de Oliveira [email protected] 1
Neuroimmune Interactions Laboratory, Department of Immunology, University of Sa˜o Paulo, Sa˜o Paulo, SP 05508-900, Brazil
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Department of Genetics and Evolutive Biology, Biosciences Institute, University of Sa˜o Paulo (USP), Sa˜o Paulo, SP 05508-090, Brazil
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Laboratory of Clinical and Experimental Immunology, Nephrology Division, Medicine Department, Federal University of Sa˜o Paulo UNIFESP, Sa˜o Paulo, SP 04023-900, Brazil
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Discipline of Clinical Neurology, Federal University of Sa˜o Paulo UNIFESP, Sa˜o Paulo, SP 04023-900, Brazil
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Laboratory for Transplantation Immunobiology, Department of Immunology, Institute of Biomedical Science IV, University of Sa˜o Paulo (USP), Sa˜o Paulo, SP 05508-090, Brazil
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Biosciences Institute, University of Sa˜o Paulo, Sa˜o Paulo, SP 05508-900, Brazil
outcome of EAE. Additionally, simvastatin can inhibit Th17 differentiation. However, many other effects exerted on the immune system by statins have yet to be clarified, in particular during neuroinflammation. Thus, the aim of this study was to investigate the effects of simvastatin on the development of experimental autoimmune encephalomyelitis. Methods Mice were immunized with MOG35–55 and EAE severity was assessed daily and scored using a clinical scale. Cytokine secretion by mononuclear cells infiltrating the CNS was evaluated by flow cytometry. Results Simvastatin (5 mg/kg/day) improved clinical outcome, induced an increase in TGF-b mRNA expression and inhibited IL-6, IL-12p40, IL-12p70, RANTES and MIP-1b secretion (p \ 0.05). This was accompanied by a significant decrease in CNS inflammatory mononuclear cell infiltration, with reduced frequencies of both Th1 and Th17 cells. Simvastatin inhibited the proliferation of T lymphocytes co-cultured with primary microglial cells. Conclusions Simvastatin treatment promotes EAE clinical amelioration by inhibiting T cell proliferation and CNS infiltration by pathogenic Th1 and Th17 cells. Keywords Simvastatin Experimental autoimmune encephalomyelitis Th1 Th17 Chemokine Proliferation
Introduction Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) whose hallmark is the presence of multifocal plaques consisting of inflammatory infiltrate, demyelination, and secondary axonal loss
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(Compston and Coles 2002). Typically, MS lesions are found in white matter, and active lesions contain reactive astrocytes, activated microglia, phagocytic macrophages, lymphocytes (CD4?, CD8? and B cells) and plasma cells (Hu and Lucchinetti 2009). Myelin-derived auto-reactive CD4? and CD8? T cells become activated after encountering the antigens in the CNS, leading to the initiation and perpetuation of the inflammatory processes (Frohman et al. 2006). Experimental autoimmune encephalomyelitis (EAE), the model for multiple sclerosis (MS) (Kuchroo et al. 2002; Peron et al. 2012), is also characterized by the activation of auto-reactive T lymphocytes, which infiltrate the CNS and initiate local inflammatory processes with pro-inflammatory cytokines, chemokines, matrix metalloproteinases (MMPs) and nitric oxide (NO), also resulting in demyelination and secondary axonal injury (Lavi and Constantinescu 2005). Loss of blood–brain barrier integrity and oxidative stress greatly contribute to these processes (Lavi and Constantinescu 2005). IFN-c-secreting Th1 lymphocytes were first described as the main T cell type responsible for EAE pathology (Voskuhl et al. 1993; Ando et al. 1989). However, further researches have demonstrated that IL-17-secreting Th17 cells, dependent on IL-6 ? TGF-b and IL-23, are essential to EAE development and maintenance (Cua et al. 2003; Langrish et al. 2005). Both IFN-c and IL-17 seem to act directly on CNS resident cells, inducing astrocytes proliferation, cytokine secretion, antigen-presentation related molecules expression and many others. Corroborating this, MS patients have higher levels of IL-6 as well as IL-17 mRNA both in their blood and spinal fluid during relapses (Lock et al. 2002; Matusevicius et al. 1998). Moreover, patients with optic spinal MS, a more aggressive type of MS, demonstrate increased levels of IL-17 compared to patients with less aggressive relapsing–remitting multiple sclerosis (RR MS) or control patients (Ishizu et al. 2005). However, an attempt to treat MS with recombinant IFN-c resulted in disease flare-ups in several patients (Panitch et al. 1987). Therefore, the view of MS as a mixed Th1/Th17 disorder is more likely to be correct. Statins are 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) inhibitors widely used in clinical practice to lower cholesterol levels (Weber et al. 2006). In 1995, a clinical study showed that cardiac transplantation patients treated with pravastatin had better clinical outcomes. Interestingly, this result was independent on the ability of pravastatin to lower lipid levels (Kobashigawa et al. 1995). This brought attention to the possible effects exerted by these drugs on the immune system. Since then, several different regulatory actions of statins have been described: inhibition of nitric oxide synthase and cytokine production (Pahan et al. 1997), decreased expression and secretion of MMP-9
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(Bellosta et al. 1998; Ganne et al. 2000), decreased secretion of chemokines and expression of chemokine receptors (Rallidis et al. 2008; Vieira et al. 2005; Neuhaus et al. 2002) and class II MHC molecule expression (Yilmaz et al. 2004; Youssef et al. 2002). Furthermore, it has already been demonstrated that statins inhibit T cell proliferation (Waiczies et al. 2005) and immune cell migration to inflammatory sites in EAE and other models (Bustos et al. 1998; Kim et al. 1995; Zhang et al. 2008, 2011; Paintlia et al. 2004). Statins are able to prevent or even reverse disease progression in murine EAE models, inducing a bias towards Th2 differentiation with decreased secretion of Th1 cytokines such as IL-2, IL-12 and IFN-c (Youssef et al. 2002). These drugs can also inhibit human Th17 cell proliferation, suggesting a role for statins in preventing Th17 development in vivo (Zhang et al. 2008). However, many questions are still to be addressed concerning the effects of statins on Th17 commitment during EAE (Paintlia et al. 2012; Li et al. 2012). Statins have already been tested in clinical trials regarding their capacity to treat MS. However, most of these studies have addressed atorvastatin and simvastatin as add-on therapies to interferon-b. Unfortunately, most of them showed disappointing results (Togha et al. 2010; Sorensen et al. 2011; Kamm et al. 2012, 2014; Lanzillo et al. 2010; Birnbaum et al. 2008). Since IFN-b also has pleiotropic actions like statins, the lack of an additive effect in these studies can actually represent an overlap of drugs effect (Neuhaus et al. 2005). It is worth mentioning that animal studies have demonstrated an additive effect when statins are associated with other drugs like glatiramer acetate (Stuve et al. 2006), minocycline (Luccarini et al. 2008) and rapamycin (Li et al. 2012), but not IFN-b. On the other hand, studies using simvastatin alone have shown encouraging results in MS patients. For instance, simvastatin 80 mg/day improved visual parameters in MS patients with acute optic neuritis (Tsakiri et al. 2012). Recently, a study addressed simvastatin as a treatment to secondary progressive MS. The results indicate that simvastatin as single therapy was able to decrease the rate of annualized whole-brain atrophy progression by 43 % (Chataway et al. 2014). These findings bring attention for possible actions of simvastatin directly inside the CNS. Regarding this possibility, among nine statins tested, simvastatin was the one with better BBB penetration profile even though atorvastatin was more lipophilic (Sierra et al. 2011). Here we show that simvastatin is able to reduce the absolute numbers of Th1 and Th17 cells in the CNS of EAE mice. Moreover, treatment of co-cultured lymphocytes and microglia resulted in decreased numbers of T cells, suggesting that this effect can in part be the result of an anti-proliferative action exerted on T cells.
Simvastatin ameliorates experimental autoimmune encephalomyelitis by inhibiting Th1/Th17…
Materials and methods Animals Eight- to twelve-week-old female C57BL/6 mice from the University of Sa˜o Paulo were housed in a facility with a controlled light cycle and were given commercial food pellets and water ad libitum. All experiments were approved by the ethical committee for animal research of this institution (Comissa˜o de E´tica no Uso de Animais—CEUA), protocol number 800-42-2. A minimum number of animals was use in order to provide statistically significant differences. EAE induction and clinical evaluation Mice were immunized with subcutaneous s.c. injections of MOG35–55 (150 lg) emulsified in complete Freund’s adjuvant (Sigma-Aldrich, St. Louis, USA) containing 5 mg/ml of BCG (Butantan Institute, Sa˜o Paulo, Brazil) in a total volume of 150 ll. Pertussis toxin (Sigma-Aldrich) was given i.p. (200 ng) on days 0 and 2 post-immunization. Disease severity was assessed and scored using a scale ranging from 0 to 5, as follows: 0 = normal, 1 = limp tail, 2 = hind limb weakness, 3 = hind limb paralysis, 4 = hind limb paralysis and fore limb weakness, 5 = moribund or death. In order to alleviate the distress of animals effort was made to induce a disease that was not severe in its clinical evolution which was achieved with the dose of MOG35–55 (150 lg). Animals were assessed daily. If an animal reached the score of 4, it was assessed again after 12 h and, if still on score 4 or worse, animals were euthanized. Animals at score 5 were immediately euthanized. However, no animal have reached score 4 or 5 during the experiments. The experiments were finished and animals were euthanized in CO2 chambers once mice have reached stable disease (at day 21st). Simvastatin treatment Simvastatin (Zocor, Merk Sharp & Dohme) was brought into suspension in PBS and administered orally at a dose of 5 mg/kg/day in a total volume of 0.2 ml daily using 20-mm feeding needles. PBS was administered in the same volume to control animals. Treatment was started at day seven after EAE induction through the end of the experiment. Real-time quantitative polymerase chain reaction Whole spinal cord samples were taken from animals and immediately snap-frozen in liquid nitrogen. For total RNA extraction, samples were transferred to a tube with 1 ml of Trizol reagent (Invitrogen, Karlsruhe, Germany) and homogenized. Chloroform (Synth, Diadema, Brazil)
(200 ll) was added and samples centrifuged at 12,000g for 15 min at 4–8 C for phase separation. The aqueous phase on the top was carefully taken and transferred to a fresh tube. Further, 500 ll of isopropanol (Synth, Diadema, Brazil) was added and the columns were centrifuged again at 12,000g for 15 min at 4–8 C. Following centrifugation, the supernatant was removed and the pellet washed with 75 % ethanol (Synth, Diadema, Brazil). After another centrifugation at 12,000g for 15 min at 4–8 C, the supernatant was removed and the pellet dissolved in 50 ll of DEPC-treated water. For cDNA synthesis, an M-MLV Reverse Transcriptase reaction kit (Promega, Madison, USA) was performed following the manufacturer’s recommendations. A quantity of 2.5 lg of total RNA was treated with DNase I (Promega, Madison, USA) (1U/ml) and incubated for 15 min at 25 C. The mRNA was separated from the obtained total RNA by using 2 ll of oligo (dT) 12–18 primer (Invitrogen, Carlsbad, CA, USA) (0.1 mg/ml) which was incubated at 65 C for 10 min. The samples were kept at 4 C for 5–10 min and then reverse transcribed using a solution with 1 ll acetylated BSA (20 mg/ml) (Promega, Madison, USA); 10 ll of 59 First Strand Buffer (Invitrogen, Carlsbad, CA, USA); 10 ll of deoxynucleotide triphosphate (dNTP) 10 mM (Promega, Madison, USA), and 2 ll MMLV Reverse Transcriptase (200 U/ml). This solution was incubated at 37 C for 1 h, with subsequent incubation at 65 C for 10 min. Finally, the samples were left on ice for 5 min and diluted at a ratio of 1: 3 with DEPC-treated water. RT-PCR was performed on a 7300 Real Time-PCR system (Applied Biosystems, Foster City, USA) using PCR master Mix (Applied Biosystems, Warrington, UK) and gene-specific primers. For normalization, we determined the distribution of HPRT. The sequences of the primer sets are as follows (50 –30 ): HPRT: 50 -CTCATGGACTGATTA TGGACAGGA-30 (sense), 50 -GCAGGTCAGCAAAGAA CTTATAGCC-30 (anti-sense); TGF-b: 50 -AACTATTG CTTCAGCTCCACAGAGA-30 (sense), 50 -AGTTGGATG GTAGCCCTTG-30 (anti-sense); IL-6: 50 -AGGATACCAC TCCCAACAGACCT-30 (sense), 50 -TTTCTCATTTCCAC GATTTCCC-30 (anti-sense). Quantification was performed using 7500 System SDS software (Applied Biosystems). Cytokine determination by Bio-Plex assay A Bio-Plex mouse cytokine assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to test samples for the presence of the following cytokines: interleukin (IL)1b, IL-6, IL-12 (p40), IL-12 (p70), IL-17, interferon (IFN)c, macrophage chemoattractant protein 1 (MCP-1/CCL2), macrophage inflammatory protein 1a (MIP-1a/CCL3), macrophage inflammatory protein 1b (MIP-1b/CCL4) and RANTES/CCL5. In brief, premixed spinal cord samples were reconstituted in 0.5 ml of a Bio-Plex mouse serum
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standard diluent, generating a stock concentration of 50,000 pg/ml for each cytokine. The standard stock was serially diluted in the Bio-Plex human serum diluent to generate 8 points for the standard curve. The assay was performed in a 96-well filtration plate supplied with the assay kit. The samples were diluted 1:4 in the Bio-Plex serum sample diluent. Homogenates of CNS tissue samples were used in this assay. The amounts of cytokines measured were corrected for the amount of protein in each sample. Protein content was measured with a Bradford assay (Bio-Rad). Premixed standards or diluted samples (50 ll) were added to each well containing washed beads. The plate was shaken for 30 s at high speed (1100 rpm) and then incubated at room temperature for 2 h at low speed (300 rpm). After incubation and washing, premixed detection antibodies (25 ll) were added to each well. Then, the plate was incubated for 1 h on a shaker at low speed (300 rpm). After incubation and washing, streptavidin-PE was added to each well. The incubation was terminated after shaking for 30 min at room temperature. After washing, the beads were re-suspended in 125 ll of BioPlex assay buffer. 250 beads were acquired for each sample and the data were analyzed using Bio-Plex Manager software version 4.0. The lowest limit of concentration for the detection of each cytokine was as follows (pg/ml): IL-1b, 3.01 pg/ml; IL-6, 0.81 pg/ml; IL-12 (p40),1.36 pg/ml; IL12 (p70), 1.08 pg/ml; IL-17, 2.04 pg/ml; IFN-c, 1.93 pg/ ml; MCP-1, 2.0 pg/ml; MIP-1a, 4.63 pg/ml; MIP-1b, 1.3 pg/ml and RANTES, 0.42 pg/ml. CNS-infiltrating cell separation All mice were euthanized in CO2 chambers and perfused with 10 ml of cold PBS. The whole CNS (brain, cerebellum and spinal cords) was excised, macerated and maintained in 4 mL of DMEM supplemented with 2.5 % collagenase D (Roche) at 37 C in a 5 % CO2 incubator. Forty-five minutes later, suspensions were washed in DMEM and centrifuged at 450g for 5 min at 4 C. After that, cells were re-suspended in 37 % Percoll and gently layered over 70 % Percoll in 15-ml tubes. The tubes were centrifuged at 950g for 20 min with the centrifuge brake turned off. After centrifugation, the ring containing mononuclear cells was collected, washed in DMEM and centrifuged at 450g for 5 min. Cells were then counted and suspended in complete DMEM medium. Cells were treated with brefeldin A at 1 lg/ml and stimulated with medium (control) or phorbol myristate acetate (PMA) (50 ng/ ml) plus ionomycin (1 lg/ml) for 12 h before analysis. Primary microglial cultures Primary microglia was isolated from mixed glial cultures. Glial cultures were isolated from 2-day-old C57Bl/6 mice.
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Euthanasia was performed by decapitation by a properly trained participant. The brains were taken and separated from the meninges and minced into small pieces, filtered through a 70-lm strainer and separated by trypsinization (0.025 % trypsin, 37 C, 20 min). Fetal bovine serum (FBS) was added at a 1:10 proportion to inactivate the trypsin. The cells were centrifuged (200g for 5 min), the pellets were re-suspended in media and the cells were transferred to a 75-mm2 flask (one brain per flask). Cells were cultured at 37 C in a 5 % CO2 atmosphere in DMEM media supplemented with 10 % heatinactivated FBS (Gibco), 100 U/ml penicillin and 100 lg/ml streptomycin. Medium was changed every 4 days until day 14, when cells reached confluence. Primary microglial cells were isolated from mixed glial cultures as they matured on top of astrocyte monolayers. Flasks were shaken for 5 min in an orbital shaker. The floating microglia were harvested and transferred to 96-well dishes, and, after 2 h, the medium was changed to remove non-adherent cells. The purity of the cultures was confirmed by immunofluorescence using an antiIba1 antibody (Santa Cruz Biotechnology, Santa Cruz, USA). Magnetic activated cell sorting (MACS) of CD41 cells from spleen MACS separation was undertaken using a CD4 (L3T4) MicroBeads, mouse kit (Miltenyi Biotec, Auburn, USA) according to the manufacturer’s instructions. Initially, splenocytes were collected from C57BL/6 mice. The cells were re-suspended in 90 ll of MACS buffer, 10 ll of CD4 (L3T4) magnetic beads were added per every 107 cells, and incubated at 4 C for 15 min. Then the cells were washed and re-suspended in 1 ml MACS buffer. For magnetic separation, the cell suspension was applied to a magnetic column and washed three times with MACS buffer. The collected material containing unlabeled cells was discarded. The column was then removed from the magnetic separator, placed in a suitable collection tube and a plunger was used to flush out fraction with magnetically labeled cells. The effluent containing the labeled cells was collected and the purity of the cells evaluated by fluorescence activated cell sorting (FACS). Co-culture of microglia and CD41 T Cells After magnetic separation, the CD4? T cells were centrifuged and re-suspended 1 9 106 cells per ml in a total volume of 2 ml. CFSE (5-(6)-carboxyfluorescein N-succinimidyl ester diacetate) (Sigma-Aldrich, Saint Louis, USA) (5 mM) was added to a final concentration of 2.5 lM. The cells were incubated at 37 C, protected from light, for 20 min. The CFSE was inactivated with 8 ml of medium supplemented with 10 % FBS by incubating at 37 C for 5 min. Finally, the cells were centrifuged and re-
Simvastatin ameliorates experimental autoimmune encephalomyelitis by inhibiting Th1/Th17…
suspended in supplemented medium (1 9 106 cells per mL) for use in co-culture experiments. For co-culture experiments, 25 9 104 microglial cells were seeded per well in a 96-well flat bottom dish (Corning Incorporated, Corning, NY, USA). After 3 days, 5 9 105 CD4? T cells labeled with CFSE were added. The cultures were treated with simvastatin (5 lM) or vehicle. Treatment with LPS (lipopolysaccharide from Salmonella enterica serotype abortus equi, Sigma-Aldrich, Saint Louis, USA) was given simultaneously to simvastatin. Five days after treatment, CD4? T cells were collected and labeled with anti-CD4APC antibody (BioLegend, San Diego, USA) (clone: RM45) for analysis by flow cytometry in a BD Accuri C6 (BD Biosciences, Singapore). Simvastatin preparation to cell culture Five milligrams of simvastatin (Sigma-Aldrich) was dissolved in 125 ll of ethanol, and then 187.5 ll of 0.1 N NaOH was added and the solution incubated at 50 C for 2 h. The pH was brought to 7.0, and the final concentration adjusted to 5 mg/ml. Statistical analysis Data are presented as mean ± SEM. For clinical scores, the significance between each two groups was examined using the Mann–Whitney test. A one-tailed unpaired Student’s t test was employed to analyze gene expression, protein production and flow cytometry results. A value of p \ 0.05 was considered significant.
Results Clinical disease development
Fig. 1 EAE clinical development. Animals were immunized with MOG35-55 peptide in CFA ? BCG. Pertussis toxin was given i.p. at the time of immunization and 48 h later. Oral simvastatin at 5 mg/ kg/day or vehicle (control) was given daily beginning on day 7. Mice were followed to assess disease clinical scores. N = 5 per group. Error bar represents mean ± SEM. *p \ 0.05 for control versus simvastatin 5 mg/kg/day
Cytokine and chemokine synthesis To further corroborate the findings obtained with real-time PCR, we assessed whether simvastatin treatment promoted changes in protein expression. Mice treated with simvastatin at 5 mg/kg/day exhibited decreased synthesis of the pro-inflammatory cytokines IL-6 (p \ 0.05), IL-12p70 (p \ 0.05) and IL-12p40 (p \ 0.01) (Fig. 3). The effects exerted on IL-1b, IL-17 and IFN-c expression were not statistically significant (p [ 0.05) (Fig. 3). Furthermore, simvastatin treatment was able to modulate chemokine synthesis. Simvastatin decreased RANTES and MIP-1b expression (p \ 0.05) (Fig. 4), whereas changes in MCP-1 and MIP-1a were not significant (p [ 0.05) (Fig. 4). CNS inflammatory mononuclear cell infiltration To further investigate the effects of simvastatin on CNS inflammatory cell recruitment, we undertook a cell
Mice received simvastatin at 5 mg/kg/day starting on day 7 after immunization until the end of follow-up on day 21. Mice receiving simvastatin treatment demonstrated decreased mean clinical scores (p \ 0.05) and overall better disease outcomes (Fig. 1). Cytokine gene expression analysis To clarify the mechanisms by which simvastatin changes EAE clinical outcome, we investigated the expression of different genes implicated in EAE inflammatory lesions. We explored the possibility that simvastatin affects the gene expression of TGF-b and IL-6 in situ. Indeed, there was an increase in TGF-b mRNA expression (Fig. 2). However, the effect on IL-6 expression was not significant (Fig. 2).
Fig. 2 The effect of simvastatin treatment on TGF-b and IL-6 mRNA expression. Real-time PCR analysis of TGF-b and IL-6 mRNA expression in the CNS during EAE development. Oral simvastatin at 5 mg/kg/day or vehicle (control) were given daily beginning on day 7 through the end of the experiment. Samples of CNS tissue were collected on day 21 after immunization. Data are shown as mean ± SEM. Each group contained 4 animals
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D. M. Oliveira et al. Fig. 3 CNS cytokine expression. CNS samples were taken from mice with EAE at day 21 after disease induction, and cytokine expression was analyzed with a Bio-Plex assay. Oral simvastatin at 5 mg/kg/day or vehicle (control) were given daily beginning on day 7 through the end of the experiment. The cytokines IL1b, IL-6, IL-17, IL-12p40, IL12p70, IFN-c were analyzed. Each bar represents mean ± SEM (3 animals per group)
Fig. 4 CNS chemokine expression. CNS samples were taken from mice with EAE on day 21 after disease induction, and cytokine expression was analyzed with a Bio-Plex assay. Oral simvastatin at 5 mg/kg/day or vehicle (control) were given daily beginning on day 7 through the end of the experiment. The chemokines MCP-1, RANTES, MIP-1a, MIP-1b were analyzed. Each bar represents mean ± SEM (3 animals per group)
separation assay to obtain mononuclear inflammatory cells from CNS samples. Total absolute numbers of infiltrating mononuclear cells were lower in mice receiving simvastatin (control, 3.1 ± 0.36; simvastatin at 5 mg/kg/day, 1.31 ± 0.37) (Fig. 5). For cytokine analysis, these cells were seeded on 96-well round-bottomed plates and stimulated with PMA ? ionomycin or remained unstimulated. After 12 h of culture, cells were analyzed by flow cytometry. Simvastatin decreased both relative and absolute
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numbers of IFN-c secreting Th1 cells infiltrating the CNS of the experimental groups (Fig. 6b, c). With regard to Th17 cells, no differences were detected in terms of relative numbers (Fig. 7b). However, the absolute number of CD4? IL-17? cells was significantly lower in the CNS of mice under simvastatin treatment (Fig. 7c). These results indicate that in vivo simvastatin treatment may modulate both Th1 and Th17 biology, either by impairing cell differentiation in the periphery or by
Simvastatin ameliorates experimental autoimmune encephalomyelitis by inhibiting Th1/Th17…
reducing cellular ability to infiltrate the CNS. We can also speculate that simvastatin exerts a more prominent effect on Th1 cells than on Th17 cells, as results demonstrated decreased Th1 cell infiltration in terms of percent and absolute numbers of cells, while Th17 analysis indicated that differences were present only in terms of absolute numbers of cells. Lymphocyte proliferation and interaction with glial cells
Fig. 5 The effect of simvastatin on CNS mononuclear cell infiltration. Animals were immunized with MOG35-55 peptide in CFA ? BCG. Pertussis toxin was given i.p. at the time of immunization and 48 h later. Oral simvastatin at 5 mg/kg/day or vehicle were given daily beginning on day 7 until the peak of disease (day 15), when CNS samples were taken for analysis. CNS-infiltrating mononuclear cells were separated from CNS tissue in a cell separation assay, and total numbers of inflammatory cells were counted in each animal sample. Each bar represents mean ± SEM (4 animals per group)
Fig. 6 IFN-c expression in CNS-infiltrating mononuclear cells. Animals were immunized with MOG35-55 peptide in CFA ? BCG. Pertussis toxin was given i.p. at the time of immunization and 48 h later. Oral simvastatin at 5 mg/kg/day or vehicle were given daily beginning on day 7 through the end of the experiment (day 15), when CNS samples were taken for analysis. CNS-infiltrating mononuclear cells were separated from CNS tissue in a cell separation assay. Collected cells were stimulated with medium (control) or
We asked whether the anti-proliferative effects of simvastatin contributed to the results of CNS mononuclear cell infiltrate analysis. The anti-proliferative effects of statins have previously been demonstrated in different cell types and lineages (Yu et al. 2013; Neuhaus et al. 2002; Saito et al. 2008). If simvastatin is able to inhibit lymphocyte proliferation in the CNS, then decreased infiltrating mononuclear cell numbers could be attributed, at least in part, to the direct effects of simvastatin inside the CNS. To analyze this hypothesis, CD4? T cells were purified from spleen samples, labeled with CFSE and added to primary microbial cultures
PMA ? ionomycin. After 12 h, cells were analyzed by flow cytometry. a Gates from medium (control) and from PMA ? ionomycin stimulated cells, b percentage numbers of IFN-c? cells, c absolute numbers of IFN-c? cells. Each bar represents mean ± SEM. Numbers of mice per group: medium, N = 3 (control), N = 4 (simvastatin); PMA ? ionomycin, N = 4 (control), N = 4 (simvastatin)
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Fig. 7 IL-17 expression in CNS-infiltrating mononuclear cells. Animals were immunized with MOG35-55 peptide in CFA ? BCG. Pertussis toxin was given i.p. at the time of immunization and 48 h later. Oral simvastatin at 5 mg/kg/day or vehicle were given daily beginning on day 7 through the end of the experiment (day 15), when CNS samples were taken for analysis. CNS-infiltrating mononuclear cells were separated from CNS tissue in a cell separation assay. Collected cells
were stimulated with medium (control) or PMA ? ionomycin. After 12 h, cells were analyzed by flow cytometry. a Gates from medium (control) and from PMA ? ionomycin stimulated cells, b percentage numbers of IL-17? cells, c absolute numbers of IL-17? cells. Each bar represents mean ± SEM. Numbers of mice per group: medium, N = 4 (control), N = 4 (simvastatin); PMA ? ionomycin, N = 3 (control), N = 4 (simvastatin)
stimulated with LPS at 0.25 lg/ml or vehicle. After 5 days, cells were collected and analyzed by flow cytometry. Simvastatin at 5 lM decreased CD4? T cell proliferation in unstimulated (Fig. 8a, b) and LPS-stimulated cultures (Fig. 8c, d). In this context, the anti-proliferative effects of simvastatin can be attributed to its inhibition of inflammation, as simvastatin can inhibit the expression of several inflammatory molecules such as CD80, CD86, MHC, and other cytokines (Neuhaus et al. 2002).
unrestrained polarized Th17 response that is more aggressive than a normal Th1/Th17 disease in WT animals. In fact, experimental data suggest that Th1- and Th17-polarized disease can exhibit features of different types of MS: Th1 is more similar to relapsing–remitting MS, and Th17 is similar to the more aggressive opticospinal MS or Devic’s disease (Kroenke et al. 2008). There is evidence for many different relevant roles for both subtypes in human disease. In a study attempting to treat MS with IFN-c, many patients experienced disease flare-ups (Panitch et al. 1987). Corroborating this, in humans, IL-6 and IL-17 transcripts can be found in cerebrospinal fluid samples from MS patients, with higher levels present during disease flare-ups (Wen et al. 2012), and higher expression of IL-17F has been associated with a poor response to treatment (Axtell et al. 2010). Statins have been studied in different contexts, such as auto-immunity and inflammation (Neuhaus et al. 2005). Statins have the ability to ameliorate EAE by inducing a transition from a Th1 to a Th2 phenotype (Youssef et al. 2002), and in fact, corroborating our findings, their potential to inhibit Th17 development has already been addressed (Zhang et al. 2008). However, the role for statins
Discussion MS is an organ-specific autoimmune disease mediated by CD4? T cells. Data from its comparable animal model, EAE, have revealed that IFN-c?-producing Th1 lymphocytes are pivotal in disease development. However, studies have demonstrated that mice deficient for INF-c and INF-c receptor (IFN-cR) were, in fact, more susceptible to EAE induction (Bettelli et al. 2004; Ferber et al. 1996; Willenborg et al. 1996). These drawbacks were clarified with the discovery of the Th17 T cell subset. The absence of Th1 cells in IFN-c KO mice permits the development of an
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Simvastatin ameliorates experimental autoimmune encephalomyelitis by inhibiting Th1/Th17…
Fig. 8 Percentages of CFSE-low lymphocytes. CFSE-labeled T cells were added to primary microbial cultures. Vehicle or simvastatin at a concentration of 5 lM were given starting on the first day. In cultures treated with LPS, LPS was given simultaneously with simvastatin. After 5 days, cells were collected and analyzed by flow cytometry. a Histograms representative of control and simvastatin (5 lM) cells.
b Bar graphs represent mean ± SEM from gates of CD4? CFSE-low cells. c Histograms representative of control plus LPS (0.25 lg/ml) and simvastatin (5 lM) plus LPS (0.25 lg/ml) cells. d Bar graphs represent mean ± SEM from gates of CD4? CFSE-low cells from LPS-treated groups
acting on infiltrating inflammatory cells inside the CNS has still to be addressed. Therefore, we focused our experiments on CNS samples taken during disease development. In order to evaluate CNS inflammatory cell recruitment, we performed a cell separation assay by gradient centrifugation, which permits quantification and analysis of inflammatory infiltrates and microglia in CNS tissue. Simvastatin treatment reduced the numbers of mononuclear cells in brain and spinal cord samples. This suggests that the effects exerted by simvastatin on EAE are, at least in part, the result of decreased inflammatory cell infiltration. During the process of EAE development, auto-reactive T cells are activated in the periphery and up-regulate CCR5, the receptor for MIP-1b and RANTES, due to the effects of IL-12 (Bagaeva et al. 2003). Auto-reactive T cells expressing CCR5 are able to infiltrate CNS perivascular sites, where they interact with microglia, leading to release of inflammatory mediators and inflammatory cell
recruitment. This secondary influx of inflammatory cells greatly correlates with clinical disease development (Brocke et al. 1996). In accordance with these findings, CNS samples from animals under simvastatin treatment exhibited decreased amounts of RANTES and MIP-1b. Therefore, the reduced numbers of inflammatory mononuclear cells can be partly explained by the decreased expression of chemokines. The role of chemokines in EAE has already been addressed in several studies. It has been suggested that they enhance rather than initiate cell infiltration (Glabinski et al. 1995). RANTES, MIP-1a, MIP-1b, and MCP-1 among others were induced 1–2 days before clinical disease development in murine EAE (Godiska et al. 1995). During relapses, increased expression of MCP-1, IP-10/CXCL10, MIP-1a, RANTES and GRO-a/CXCL1 was observed (Glabinski et al. 1997). Astrocytes are the source of MCP-1 and IP-10, whereas RANTES and MIP-1a expression was associated with inflammatory cells (Tani et al. 1996;
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Glabinski et al. 1997). In our samples, there was a tendency to observe decreased expression of MCP-1 and MIP-1a with no significant differences. These results can be explained by the time of sample collection on the 21st day after disease induction, when chemokine release was most likely decreasing. Another possibility is that these results are merely attributable to the reduced presence of Th17 cells that may boost the inflammation acting on glial resident cells. We also analyzed the types of T cells present in the inflammatory infiltrates and their response to stimulation with PMA ? ionomycin. Simvastatin could decrease the percentages and absolute numbers of CD4?IFN-c? T cells, whereas CD4?IL-17? T cells were reduced only in terms of absolute numbers with similar percentages in both groups. These results are in accordance with previous data showing that statins inhibit Th1 (Youssef et al. 2002) and Th17 responses (Imamura et al. 2009; Murphy et al. 2008; Eller et al. 2010). Our results suggest that simvastatin exerts a more prominent effect on the Th1 response, as there were decreased percentages of CD4?IFN-c? T cells, while CD4?IL-17? T cells were decreased only in terms of absolute numbers, with similar percentages in the control and simvastatin groups. Zhang et al. (2008) have demonstrated that simvastatin directly inhibits Th17 differentiation. Thus, it was not expected that simvastatin treatment would fail to induce decreased percentages of CD4?IL-17? T cells. This apparent contradiction may be explained by the day chosen for the beginning of the treatment. T lymphocytes migrate into the CNS in two waves. In the first wave, CD4?IL-17? T cells migrate into the CNS through the choroid plexus. Once inside the CNS, these cells activate epithelial cells to initiate a second wave of T cell migration (Reboldi et al. 2009). As the treatment was initiated at day 7 after immunization, it was ineffective to inhibit initial T cell differentiation and it was probably of little efficiency against the first migration wave. As a consequence, simvastatin was more effective against Th1 because Th17 cells migrated early into the CNS. Therefore, most of the Th17 cells were already there when treatment began. In mice, Th17 development is a consequence of the simultaneous action of IL-6 and TGF-b on T lymphocytes (Bettelli et al. 2006) with IL-23 being a survival factor for effector Th17 cells, whereas Th1 differentiation is driven by IL-12. This cytokine is composed of a p35 and a p40 subunit. Likewise, IL-23 is composed of the same p40 subunit with a p19 subunit. Our tissue analysis of cytokine expression indicated reduced levels of IL-6 as well as IL12 p40 and IL-12 p70, which together represent the whole IL-12 molecule. This may indicate effects exerted by simvastatin on macrophage/microglia cytokine production, which in turn reduced Th1 commitment and CNS infiltration.
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It has been demonstrated that statins have anti-proliferative effects, inhibiting T cell proliferation as well as tumor growth. Thus, clinical improvement and reduced inflammatory infiltrates would be consequences of these effects at the periphery but also for cells already inside the inflammatory sites in the CNS tissue. In order to address these questions, we co-cultured CFSE-labeled lymphocytes with microglial cells during 5 days. Simvastatin at a concentration of 5 lM reduced the percentage of CFSE-low lymphocytes, suggesting that an anti-proliferative effect can occur not only at the periphery during clonal expansion, but also inside inflammatory sites. As simvastatin is able to cross the BBB (Sierra et al. 2011), an anti-proliferative effect directly inside the CNS may have contributed to the decreased number of inflammatory cells in the CNS which can partially explain the decreased levels of cytokines and chemokines found at tissue analysis. Moreover, it is suitable to think that, as simvastatin is able to transpose the BBB, it is dampening T cell activation in situ and for example reducing epitope spreading. Collectively, our data show that simvastatin is able to ameliorate EAE by reducing inflammatory cell recruitment to the CNS and by inhibiting both Th1 and Th17 development. Our in vitro results also suggest that simvastatin decreases T lymphocyte proliferation during co-culture with microglia. In summary, our data corroborates previous findings showing that simvastatin has immunomodulatory properties, through which inflammatory immune responses may be blunted. Moreover, besides its peripheral effect, this drug may also exert some effect in situ, dampening resident cells inflammatory cytokines secretion and thus T cell activation. Acknowledgments We thank Claudia da Silva Cunha and Paulo Albe for technical support. Financial Support: CNPq, Complex Fluids INCT and FAPESP (07/01771-0, 07/07139-3, 12/02270-2 and 2011/18703-2). Compliance with ethical standards Conflict of interest
The authors declare no conflict of interest.
References Ando DG, Clayton J, Kono D, Urban JL, Sercarz EE (1989) Encephalitogenic T cells in the B10.PL model of experimental allergic encephalomyelitis (EAE) are of the Th-1 lymphokine subtype. Cell Immunol 124:132–143 Axtell RC, de Jong BA, Boniface K, van der Voort LF, Bhat R, de Sarno P, Naves R, Han M, Zhong F, Castellanos JG, Mair R, Christakos A, Kolkowitz I, Katz L, Killestein J, Polman CH, de Waal Malefyt R, Steinman L, Raman C (2010) T helper type 1 and 17 cells determine efficacy of interferon-beta in multiple sclerosis and experimental encephalomyelitis. Nat Med 16:406–412 Bagaeva LV, Williams LP, Segal BM (2003) IL-12 dependent/IFN gamma independent expression of CCR5 by myelin-reactive T cells correlates with encephalitogenicity. J Neuroimmunol 137:109–116
Simvastatin ameliorates experimental autoimmune encephalomyelitis by inhibiting Th1/Th17… Bellosta S, Via D, Canavesi M, Pfister P, Fumagalli R, Paoletti R, Bernini F (1998) HMG-CoA reductase inhibitors reduce MMP-9 secretion by macrophages. Arterioscler Thromb Vasc Biol 18:1671–1678 Bettelli E, Sullivan B, Szabo SJ, Sobel RA, Glimcher LH, Kuchroo VK (2004) Loss of T-bet, but not STAT1, prevents the development of experimental autoimmune encephalomyelitis. J Exp Med 200:79–87 Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK (2006) Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441:235–238 Birnbaum G, Cree B, Altafullah I, Zinser M, Reder AT (2008) Combining beta interferon and atorvastatin may increase disease activity in multiple sclerosis. Neurology 71:1390–1395 Brocke S, Gijbels K, Allegretta M, Ferber I, Piercy C, Blankenstein T, Martin R, Utz U, Karin N, Mitchell D, Veromaa T, Waisman A, Gaur A, Conlon P, Ling N, Fairchild PJ, Wraith DC, O’Garra A, Fathman CG, Steinman L (1996) Treatment of experimental encephalomyelitis with a peptide analogue of myelin basic protein. Nature 379:343–346 Bustos C, Hernandez-Presa MA, Ortego M, Tunon J, Ortega L, Perez F, Diaz C, Hernandez G, Egido J (1998) HMG-CoA reductase inhibition by atorvastatin reduces neointimal inflammation in a rabbit model of atherosclerosis. J Am Coll Cardiol 32:2057–2064 Chataway J, Schuerer N, Alsanousi A, Chan D, Macmanus D, Hunter K, Anderson V, Bangham CR, Clegg S, Nielsen C, Fox NC, Wilkie D, Nicholas JM, Calder VL, Greenwood J, Frost C, Nicholas R (2014) Effect of high-dose simvastatin on brain atrophy and disability in secondary progressive multiple sclerosis (MS-STAT): a randomised, placebo-controlled, phase 2 trial. Lancet 383:2213–2221 Compston A, Coles A (2002) Multiple sclerosis. Lancet 359:1221–1231 Cua DJ, Sherlock J, Chen Y, Murphy CA, Joyce B, Seymour B, Lucian L, To W, Kwan S, Churakova T, Zurawski S, Wiekowski M, Lira SA, Gorman D, Kastelein RA, Sedgwick JD (2003) Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421:744–748 Eller P, Eller K, Wolf AM, Reinstadler SJ, Tagwerker A, Patsch JR, Mayer G, Rosenkranz AR (2010) Atorvastatin attenuates murine anti-glomerular basement membrane glomerulonephritis. Kidney Int 77:428–435 Ferber IA, Brocke S, Taylor-Edwards C, Ridgway W, Dinisco C, Steinman L, Dalton D, Fathman CG (1996) Mice with a disrupted IFN-gamma gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE). J Immunol 156:5–7 Frohman EM, Racke MK, Raine CS (2006) Multiple sclerosis—the plaque and its pathogenesis. N Engl J Med 354:942–955 Ganne F, Vasse M, Beaudeux JL, Peynet J, Francois A, Mishal Z, Chartier A, Tobelem G, Vannier JP, Soria J, Soria C (2000) Cerivastatin, an inhibitor of HMG-CoA reductase, inhibits urokinase/urokinase-receptor expression and MMP-9 secretion by peripheral blood monocytes–a possible protective mechanism against atherothrombosis. Thromb Haemost 84:680–688 Glabinski AR, Tani M, Tuohy VK, Tuthill RJ, Ransohoff RM (1995) Central nervous system chemokine mRNA accumulation follows initial leukocyte entry at the onset of acute murine experimental autoimmune encephalomyelitis. Brain Behav Immun 9:315–330 Glabinski AR, Tani M, Strieter RM, Tuohy VK, Ransohoff RM (1997) Synchronous synthesis of alpha- and beta-chemokines by cells of diverse lineage in the central nervous system of mice with relapses of chronic experimental autoimmune encephalomyelitis. Am J Pathol 150:617–630 Godiska R, Chantry D, Dietsch GN, Gray PW (1995) Chemokine expression in murine experimental allergic encephalomyelitis. J Neuroimmunol 58:167–176
Hu W, Lucchinetti CF (2009) The pathological spectrum of CNS inflammatory demyelinating diseases. Semin Immunopathol 31:439–453 Imamura M, Okunishi K, Ohtsu H, Nakagome K, Harada H, Tanaka R, Yamamoto K, Dohi M (2009) Pravastatin attenuates allergic airway inflammation by suppressing antigen sensitisation, interleukin 17 production and antigen presentation in the lung. Thorax 64:44–49 Ishizu T, Osoegawa M, Mei FJ, Kikuchi H, Tanaka M, Takakura Y, Minohara M, Murai H, Mihara F, Taniwaki T, Kira J (2005) Intrathecal activation of the IL-17/IL-8 axis in opticospinal multiple sclerosis. Brain 128:988–1002 Kamm CP, El-Koussy M, Humpert S, Findling O, von Bredow F, Burren Y, Schwegler G, Schott D, Donati F, Muller M, Goebels N, Muller F, Slotboom J, Tettenborn B, Kappos L, Naegelin Y, Mattle HP (2012) Atorvastatin added to interferon beta for relapsing multiple sclerosis: a randomized controlled trial. J Neurol 259:2401–2413 Kamm CP, El-Koussy M, Humpert S, Findling O, Burren Y, Schwegler G, Donati F, Muller M, Muller F, Slotboom J, Kappos L, Naegelin Y, Mattle HP (2014) Atorvastatin added to interferon beta for relapsing multiple sclerosis: 12-month treatment extension of the randomized multicenter SWABIMS trial. PLoS ONE 9:e86663 Kim SY, Guijarro C, O’Donnell MP, Kasiske BL, Kim Y, Keane WF (1995) Human mesangial cell production of monocyte chemoattractant protein-1: modulation by lovastatin. Kidney Int 48:363–371 Kobashigawa JA, Katznelson S, Laks H, Johnson JA, Yeatman L, Wang XM, Chia D, Terasaki PI, Sabad A, Cogert GA et al (1995) Effect of pravastatin on outcomes after cardiac transplantation. N Engl J Med 333:621–627 Kroenke MA, Carlson TJ, Andjelkovic AV, Segal BM (2008) IL-12and IL-23-modulated T cells induce distinct types of EAE based on histology, CNS chemokine profile, and response to cytokine inhibition. J Exp Med 205:1535–1541 Kuchroo VK, Anderson AC, Waldner H, Munder M, Bettelli E, Nicholson LB (2002) T cell response in experimental autoimmune encephalomyelitis (EAE): role of self and cross-reactive antigens in shaping, tuning, and regulating the autopathogenic T cell repertoire. Annu Rev Immunol 20:101–123 Langrish CL, Chen Y, Blumenschein WM, Mattson J, Basham B, Sedgwick JD, McClanahan T, Kastelein RA, Cua DJ (2005) IL23 drives a pathogenic T cell population that induces autoimmune inflammation. J Exp Med 201:233–240 Lanzillo R, Orefice G, Quarantelli M, Rinaldi C, Prinster A, Ventrella G, Spitaleri D, Lus G, Vacca G, Carotenuto B, Salvatore E, Brunetti A, Tedeschi G, Brescia Morra V (2010) Atorvastatin combined to interferon to verify the efficacy (ACTIVE) in relapsing-remitting active multiple sclerosis patients: a longitudinal controlled trial of combination therapy. Mult Scler 16:450–454 Lavi E, Constantinescu CS (2005) Experimental models of multiple sclerosis. Springer, New York Li Z, Chen L, Niu X, Liu J, Ping M, Li R, Xie X, Guo L (2012) Immunomodulatory synergy by combining atorvastatin and rapamycin in the treatment of experimental autoimmune encephalomyelitis (EAE). J Neuroimmunol 250:9–17 Lock C, Hermans G, Pedotti R, Brendolan A, Schadt E, Garren H, Langer-Gould A, Strober S, Cannella B, Allard J, Klonowski P, Austin A, Lad N, Kaminski N, Galli SJ, Oksenberg JR, Raine CS, Heller R, Steinman L (2002) Gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis. Nat Med 8:500–508 Luccarini I, Ballerini C, Biagioli T, Biamonte F, Bellucci A, Rosi MC, Grossi C, Massacesi L, Casamenti F (2008) Combined treatment with atorvastatin and minocycline suppresses severity of EAE. Exp Neurol 211:214–226
123
D. M. Oliveira et al. Matusevicius D, Kivisakk P, Navikas VV, Tian W, Soderstrom M, Fredrikson S, Link H (1998) Influence of IFN-beta1b (Betaferon) on cytokine mRNA profiles in blood mononuclear cells and plasma levels of soluble VCAM-1 in multiple sclerosis. Eur J Neurol 5:265–275 Murphy DM, Forrest IA, Corris PA, Johnson GE, Small T, Jones D, Fisher AJ, Egan JJ, Cawston TE, Ward C, Lordan JL (2008) Simvastatin attenuates release of neutrophilic and remodeling factors from primary bronchial epithelial cells derived from stable lung transplant recipients. Am J Physiol Lung Cell Mol Physiol 294:L592–L599 Neuhaus O, Strasser-Fuchs S, Fazekas F, Kieseier BC, Niederwieser G, Hartung HP, Archelos JJ (2002) Statins as immunomodulators: comparison with interferon-beta 1b in MS. Neurology 59:990–997 Neuhaus O, Stuve O, Archelos JJ, Hartung HP (2005) Putative mechanisms of action of statins in multiple sclerosis—comparison to interferon-beta and glatiramer acetate. J Neurol Sci 233:173–177 Pahan K, Sheikh FG, Namboodiri AM, Singh I (1997) Lovastatin and phenylacetate inhibit the induction of nitric oxide synthase and cytokines in rat primary astrocytes, microglia, and macrophages. J Clin Invest 100:2671–2679 Paintlia AS, Paintlia MK, Singh AK, Stanislaus R, Gilg AG, Barbosa E, Singh I (2004) Regulation of gene expression associated with acute experimental autoimmune encephalomyelitis by Lovastatin. J Neurosci Res 77:63–81 Paintlia AS, Paintlia MK, Hollis BW, Singh AK, Singh I (2012) Interference with RhoA-ROCK signaling mechanism in autoreactive CD4? T cells enhances the bioavailability of 1,25dihydroxyvitamin D3 in experimental autoimmune encephalomyelitis. Am J Pathol 181:993–1006 Panitch HS, Hirsch RL, Haley AS, Johnson KP (1987) Exacerbations of multiple sclerosis in patients treated with gamma interferon. Lancet 1:893–895 Peron JPS, Oliveira D, Branda˜o WN, Fickinger A, Ligeiro De Oliveira AP, Rizzo LV, Caˆmara NOS (2012) Central nervous system resident cells in neuroinflammation: a brave new world. In: Chan J (ed) Autoimmune diseases, contributing factors, specific cases of autoimmune diseases, and stem cell and other therapies. InTech, Rijeka Rallidis LS, Hamodraka ES, Fountoulaki K, Moustogiannis G, Zolindaki MG, Kremastinos DT (2008) Simvastatin exerts its anti-inflammatory effect in hypercholesterolaemic patients by decreasing the serum levels of monocyte chemoattractant protein-1. Int J Cardiol 124:271–272 Reboldi A, Coisne C, Baumjohann D, Benvenuto F, Bottinelli D, Lira S, Uccelli A, Lanzavecchia A, Engelhardt B, Sallusto F (2009) C–C chemokine receptor 6-regulated entry of TH-17 cells into the CNS through the choroid plexus is required for the initiation of EAE. Nat Immunol 10:514–523 Saito A, Saito N, Mol W, Furukawa H, Tsutsumida A, Oyama A, Sekido M, Sasaki S, Yamamoto Y (2008) Simvastatin inhibits growth via apoptosis and the induction of cell cycle arrest in human melanoma cells. Melanoma Res 18:85–94 Sierra S, Ramos MC, Molina P, Esteo C, Vazquez JA, Burgos JS (2011) Statins as neuroprotectants: a comparative in vitro study of lipophilicity, blood–brain-barrier penetration, lowering of brain cholesterol, and decrease of neuron cell death. J Alzheimers Dis 23:307–318 Sorensen PS, Lycke J, Eralinna JP, Edland A, Wu X, Frederiksen JL, Oturai A, Malmestrom C, Stenager E, Sellebjerg F, Sondergaard HB (2011) Simvastatin as add-on therapy to interferon beta-1a for relapsing-remitting multiple sclerosis (SIMCOMBIN study): a placebo-controlled randomised phase 4 trial. Lancet Neurol 10:691–701 Stuve O, Youssef S, Weber MS, Nessler S, von Budingen HC, Hemmer B, Prod’Homme T, Sobel RA, Steinman L, Zamvil SS
123
(2006) Immunomodulatory synergy by combination of atorvastatin and glatiramer acetate in treatment of CNS autoimmunity. J Clin Invest 116:1037–1044 Tani M, Glabinski AR, Tuohy VK, Stoler MH, Estes ML, Ransohoff RM (1996) In situ hybridization analysis of glial fibrillary acidic protein mRNA reveals evidence of biphasic astrocyte activation during acute experimental autoimmune encephalomyelitis. Am J Pathol 148:889–896 Togha M, Karvigh SA, Nabavi M, Moghadam NB, Harirchian MH, Sahraian MA, Enzevaei A, Nourian A, Ghanaati H, Firouznia K, Jannati A, Shekiba M (2010) Simvastatin treatment in patients with relapsing-remitting multiple sclerosis receiving interferon beta 1a: a double-blind randomized controlled trial. Mult Scler 16:848–854 Tsakiri A, Kallenbach K, Fuglo D, Wanscher B, Larsson H, Frederiksen J (2012) Simvastatin improves final visual outcome in acute optic neuritis: a randomized study. Mult Scler 18:72–81 Vieira JM Jr, Mantovani E, Rodrigues LT, Delle H, Noronha IL, Fujihara CK, Zatz R (2005) Simvastatin attenuates renal inflammation, tubular transdifferentiation and interstitial fibrosis in rats with unilateral ureteral obstruction. Nephrol Dial Transpl 20:1582–1591 Voskuhl RR, Martin R, Bergman C, Dalal M, Ruddle NH, McFarland HF (1993) T helper 1 (Th1) functional phenotype of human myelin basic protein-specific T lymphocytes. Autoimmunity 15:137–143 Waiczies S, Prozorovski T, Infante-Duarte C, Hahner A, Aktas O, Ullrich O, Zipp F (2005) Atorvastatin induces T cell anergy via phosphorylation of ERK1. J Immunol 174:5630–5635 Weber MS, Youssef S, Dunn SE, Prod’Homme T, Neuhaus O, Stuve O, Greenwood J, Steinman L, Zamvil SS (2006) Statins in the treatment of central nervous system autoimmune disease. J Neuroimmunol 178:140–148 Wen SR, Liu GJ, Feng RN, Gong FC, Zhong H, Duan SR, Bi S (2012) Increased levels of IL-23 and osteopontin in serum and cerebrospinal fluid of multiple sclerosis patients. J Neuroimmunol 244:94–96 Willenborg DO, Fordham S, Bernard CC, Cowden WB, Ramshaw IA (1996) IFN-gamma plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis. J Immunol 157:3223–3227 Yilmaz A, Reiss C, Tantawi O, Weng A, Stumpf C, Raaz D, Ludwig J, Berger T, Steinkasserer A, Daniel WG, Garlichs CD (2004) HMG-CoA reductase inhibitors suppress maturation of human dendritic cells: new implications for atherosclerosis. Atherosclerosis 172:85–93 Youssef S, Stuve O, Patarroyo JC, Ruiz PJ, Radosevich JL, Hur EM, Bravo M, Mitchell DJ, Sobel RA, Steinman L, Zamvil SS (2002) The HMG-CoA reductase inhibitor, atorvastatin, promotes a Th2 bias and reverses paralysis in central nervous system autoimmune disease. Nature 420:78–84 Yu X, Pan Y, Ma H, Li W (2013) Simvastatin inhibits proliferation and induces apoptosis in human lung cancer cells. Oncol Res 20:351–357 Zhang X, Jin J, Peng X, Ramgolam VS, Markovic-Plese S (2008) Simvastatin inhibits IL-17 secretion by targeting multiple IL-17regulatory cytokines and by inhibiting the expression of IL-17 transcription factor RORC in CD4? lymphocytes. J Immunol 180:6988–6996 Zhang S, Rahman M, Qi Z, Herwald H, Thorlacius H (2011) Simvastatin regulates CXC chemokine formation in streptococcal M1 protein-induced neutrophil infiltration in the lung. Am J Physiol Lung Cell Mol Physiol 300:L930–L939
Inflammopharmacology
RESEARCH ARTICLE
Simvastatin ameliorates experimental autoimmune encephalomyelitis by inhibiting Th1/Th17 response and cellular infiltration Daniel May de Oliveira1,5 • Enedina Maria Lobato de Oliveira4 • Merari de Fa´tima Ramires Ferrari2 • Patrı´cia Semedo6 • Meire Ioshie Hiyane5 • Marcos Antoˆnio Cenedeze3 • Alvaro Pacheco-Silva3 • Niels Olsen Saraiva Caˆmara3,5 Jean Pierre Schatzmann Peron1
•
Received: 26 May 2015 / Accepted: 20 October 2015 Springer Basel 2015
Abstract Aim Experimental autoimmune encephalomyelitis (EAE) is a CD4?-mediated autoimmune pathology of the central nervous system (CNS) that is used as a model for the study of the human neuroinflammatory disease, multiple sclerosis. During the development of EAE, auto-reactive Th1 and Th17 CD4? T cells infiltrate the CNS promoting inflammatory cells recruitment, focal inflammation and tissue destruction. In this sense, statins, agents used to lower lipid levels, have recently shown to exert interesting immunomodulatory function. In fact, statins promote a bias towards a Th2 response, which ameliorates the clinical Niels Olsen Saraiva Caˆmara and Jean Pierre Schatzmann Peron have contributed equally to this manuscript. & Daniel May de Oliveira [email protected] 1
Neuroimmune Interactions Laboratory, Department of Immunology, University of Sa˜o Paulo, Sa˜o Paulo, SP 05508-900, Brazil
2
Department of Genetics and Evolutive Biology, Biosciences Institute, University of Sa˜o Paulo (USP), Sa˜o Paulo, SP 05508-090, Brazil
3
Laboratory of Clinical and Experimental Immunology, Nephrology Division, Medicine Department, Federal University of Sa˜o Paulo UNIFESP, Sa˜o Paulo, SP 04023-900, Brazil
4
Discipline of Clinical Neurology, Federal University of Sa˜o Paulo UNIFESP, Sa˜o Paulo, SP 04023-900, Brazil
5
Laboratory for Transplantation Immunobiology, Department of Immunology, Institute of Biomedical Science IV, University of Sa˜o Paulo (USP), Sa˜o Paulo, SP 05508-090, Brazil
6
Biosciences Institute, University of Sa˜o Paulo, Sa˜o Paulo, SP 05508-900, Brazil
outcome of EAE. Additionally, simvastatin can inhibit Th17 differentiation. However, many other effects exerted on the immune system by statins have yet to be clarified, in particular during neuroinflammation. Thus, the aim of this study was to investigate the effects of simvastatin on the development of experimental autoimmune encephalomyelitis. Methods Mice were immunized with MOG35–55 and EAE severity was assessed daily and scored using a clinical scale. Cytokine secretion by mononuclear cells infiltrating the CNS was evaluated by flow cytometry. Results Simvastatin (5 mg/kg/day) improved clinical outcome, induced an increase in TGF-b mRNA expression and inhibited IL-6, IL-12p40, IL-12p70, RANTES and MIP-1b secretion (p \ 0.05). This was accompanied by a significant decrease in CNS inflammatory mononuclear cell infiltration, with reduced frequencies of both Th1 and Th17 cells. Simvastatin inhibited the proliferation of T lymphocytes co-cultured with primary microglial cells. Conclusions Simvastatin treatment promotes EAE clinical amelioration by inhibiting T cell proliferation and CNS infiltration by pathogenic Th1 and Th17 cells. Keywords Simvastatin Experimental autoimmune encephalomyelitis Th1 Th17 Chemokine Proliferation
Introduction Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) whose hallmark is the presence of multifocal plaques consisting of inflammatory infiltrate, demyelination, and secondary axonal loss
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(Compston and Coles 2002). Typically, MS lesions are found in white matter, and active lesions contain reactive astrocytes, activated microglia, phagocytic macrophages, lymphocytes (CD4?, CD8? and B cells) and plasma cells (Hu and Lucchinetti 2009). Myelin-derived auto-reactive CD4? and CD8? T cells become activated after encountering the antigens in the CNS, leading to the initiation and perpetuation of the inflammatory processes (Frohman et al. 2006). Experimental autoimmune encephalomyelitis (EAE), the model for multiple sclerosis (MS) (Kuchroo et al. 2002; Peron et al. 2012), is also characterized by the activation of auto-reactive T lymphocytes, which infiltrate the CNS and initiate local inflammatory processes with pro-inflammatory cytokines, chemokines, matrix metalloproteinases (MMPs) and nitric oxide (NO), also resulting in demyelination and secondary axonal injury (Lavi and Constantinescu 2005). Loss of blood–brain barrier integrity and oxidative stress greatly contribute to these processes (Lavi and Constantinescu 2005). IFN-c-secreting Th1 lymphocytes were first described as the main T cell type responsible for EAE pathology (Voskuhl et al. 1993; Ando et al. 1989). However, further researches have demonstrated that IL-17-secreting Th17 cells, dependent on IL-6 ? TGF-b and IL-23, are essential to EAE development and maintenance (Cua et al. 2003; Langrish et al. 2005). Both IFN-c and IL-17 seem to act directly on CNS resident cells, inducing astrocytes proliferation, cytokine secretion, antigen-presentation related molecules expression and many others. Corroborating this, MS patients have higher levels of IL-6 as well as IL-17 mRNA both in their blood and spinal fluid during relapses (Lock et al. 2002; Matusevicius et al. 1998). Moreover, patients with optic spinal MS, a more aggressive type of MS, demonstrate increased levels of IL-17 compared to patients with less aggressive relapsing–remitting multiple sclerosis (RR MS) or control patients (Ishizu et al. 2005). However, an attempt to treat MS with recombinant IFN-c resulted in disease flare-ups in several patients (Panitch et al. 1987). Therefore, the view of MS as a mixed Th1/Th17 disorder is more likely to be correct. Statins are 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) inhibitors widely used in clinical practice to lower cholesterol levels (Weber et al. 2006). In 1995, a clinical study showed that cardiac transplantation patients treated with pravastatin had better clinical outcomes. Interestingly, this result was independent on the ability of pravastatin to lower lipid levels (Kobashigawa et al. 1995). This brought attention to the possible effects exerted by these drugs on the immune system. Since then, several different regulatory actions of statins have been described: inhibition of nitric oxide synthase and cytokine production (Pahan et al. 1997), decreased expression and secretion of MMP-9
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(Bellosta et al. 1998; Ganne et al. 2000), decreased secretion of chemokines and expression of chemokine receptors (Rallidis et al. 2008; Vieira et al. 2005; Neuhaus et al. 2002) and class II MHC molecule expression (Yilmaz et al. 2004; Youssef et al. 2002). Furthermore, it has already been demonstrated that statins inhibit T cell proliferation (Waiczies et al. 2005) and immune cell migration to inflammatory sites in EAE and other models (Bustos et al. 1998; Kim et al. 1995; Zhang et al. 2008, 2011; Paintlia et al. 2004). Statins are able to prevent or even reverse disease progression in murine EAE models, inducing a bias towards Th2 differentiation with decreased secretion of Th1 cytokines such as IL-2, IL-12 and IFN-c (Youssef et al. 2002). These drugs can also inhibit human Th17 cell proliferation, suggesting a role for statins in preventing Th17 development in vivo (Zhang et al. 2008). However, many questions are still to be addressed concerning the effects of statins on Th17 commitment during EAE (Paintlia et al. 2012; Li et al. 2012). Statins have already been tested in clinical trials regarding their capacity to treat MS. However, most of these studies have addressed atorvastatin and simvastatin as add-on therapies to interferon-b. Unfortunately, most of them showed disappointing results (Togha et al. 2010; Sorensen et al. 2011; Kamm et al. 2012, 2014; Lanzillo et al. 2010; Birnbaum et al. 2008). Since IFN-b also has pleiotropic actions like statins, the lack of an additive effect in these studies can actually represent an overlap of drugs effect (Neuhaus et al. 2005). It is worth mentioning that animal studies have demonstrated an additive effect when statins are associated with other drugs like glatiramer acetate (Stuve et al. 2006), minocycline (Luccarini et al. 2008) and rapamycin (Li et al. 2012), but not IFN-b. On the other hand, studies using simvastatin alone have shown encouraging results in MS patients. For instance, simvastatin 80 mg/day improved visual parameters in MS patients with acute optic neuritis (Tsakiri et al. 2012). Recently, a study addressed simvastatin as a treatment to secondary progressive MS. The results indicate that simvastatin as single therapy was able to decrease the rate of annualized whole-brain atrophy progression by 43 % (Chataway et al. 2014). These findings bring attention for possible actions of simvastatin directly inside the CNS. Regarding this possibility, among nine statins tested, simvastatin was the one with better BBB penetration profile even though atorvastatin was more lipophilic (Sierra et al. 2011). Here we show that simvastatin is able to reduce the absolute numbers of Th1 and Th17 cells in the CNS of EAE mice. Moreover, treatment of co-cultured lymphocytes and microglia resulted in decreased numbers of T cells, suggesting that this effect can in part be the result of an anti-proliferative action exerted on T cells.
Simvastatin ameliorates experimental autoimmune encephalomyelitis by inhibiting Th1/Th17…
Materials and methods Animals Eight- to twelve-week-old female C57BL/6 mice from the University of Sa˜o Paulo were housed in a facility with a controlled light cycle and were given commercial food pellets and water ad libitum. All experiments were approved by the ethical committee for animal research of this institution (Comissa˜o de E´tica no Uso de Animais—CEUA), protocol number 800-42-2. A minimum number of animals was use in order to provide statistically significant differences. EAE induction and clinical evaluation Mice were immunized with subcutaneous s.c. injections of MOG35–55 (150 lg) emulsified in complete Freund’s adjuvant (Sigma-Aldrich, St. Louis, USA) containing 5 mg/ml of BCG (Butantan Institute, Sa˜o Paulo, Brazil) in a total volume of 150 ll. Pertussis toxin (Sigma-Aldrich) was given i.p. (200 ng) on days 0 and 2 post-immunization. Disease severity was assessed and scored using a scale ranging from 0 to 5, as follows: 0 = normal, 1 = limp tail, 2 = hind limb weakness, 3 = hind limb paralysis, 4 = hind limb paralysis and fore limb weakness, 5 = moribund or death. In order to alleviate the distress of animals effort was made to induce a disease that was not severe in its clinical evolution which was achieved with the dose of MOG35–55 (150 lg). Animals were assessed daily. If an animal reached the score of 4, it was assessed again after 12 h and, if still on score 4 or worse, animals were euthanized. Animals at score 5 were immediately euthanized. However, no animal have reached score 4 or 5 during the experiments. The experiments were finished and animals were euthanized in CO2 chambers once mice have reached stable disease (at day 21st). Simvastatin treatment Simvastatin (Zocor, Merk Sharp & Dohme) was brought into suspension in PBS and administered orally at a dose of 5 mg/kg/day in a total volume of 0.2 ml daily using 20-mm feeding needles. PBS was administered in the same volume to control animals. Treatment was started at day seven after EAE induction through the end of the experiment. Real-time quantitative polymerase chain reaction Whole spinal cord samples were taken from animals and immediately snap-frozen in liquid nitrogen. For total RNA extraction, samples were transferred to a tube with 1 ml of Trizol reagent (Invitrogen, Karlsruhe, Germany) and homogenized. Chloroform (Synth, Diadema, Brazil)
(200 ll) was added and samples centrifuged at 12,000g for 15 min at 4–8 C for phase separation. The aqueous phase on the top was carefully taken and transferred to a fresh tube. Further, 500 ll of isopropanol (Synth, Diadema, Brazil) was added and the columns were centrifuged again at 12,000g for 15 min at 4–8 C. Following centrifugation, the supernatant was removed and the pellet washed with 75 % ethanol (Synth, Diadema, Brazil). After another centrifugation at 12,000g for 15 min at 4–8 C, the supernatant was removed and the pellet dissolved in 50 ll of DEPC-treated water. For cDNA synthesis, an M-MLV Reverse Transcriptase reaction kit (Promega, Madison, USA) was performed following the manufacturer’s recommendations. A quantity of 2.5 lg of total RNA was treated with DNase I (Promega, Madison, USA) (1U/ml) and incubated for 15 min at 25 C. The mRNA was separated from the obtained total RNA by using 2 ll of oligo (dT) 12–18 primer (Invitrogen, Carlsbad, CA, USA) (0.1 mg/ml) which was incubated at 65 C for 10 min. The samples were kept at 4 C for 5–10 min and then reverse transcribed using a solution with 1 ll acetylated BSA (20 mg/ml) (Promega, Madison, USA); 10 ll of 59 First Strand Buffer (Invitrogen, Carlsbad, CA, USA); 10 ll of deoxynucleotide triphosphate (dNTP) 10 mM (Promega, Madison, USA), and 2 ll MMLV Reverse Transcriptase (200 U/ml). This solution was incubated at 37 C for 1 h, with subsequent incubation at 65 C for 10 min. Finally, the samples were left on ice for 5 min and diluted at a ratio of 1: 3 with DEPC-treated water. RT-PCR was performed on a 7300 Real Time-PCR system (Applied Biosystems, Foster City, USA) using PCR master Mix (Applied Biosystems, Warrington, UK) and gene-specific primers. For normalization, we determined the distribution of HPRT. The sequences of the primer sets are as follows (50 –30 ): HPRT: 50 -CTCATGGACTGATTA TGGACAGGA-30 (sense), 50 -GCAGGTCAGCAAAGAA CTTATAGCC-30 (anti-sense); TGF-b: 50 -AACTATTG CTTCAGCTCCACAGAGA-30 (sense), 50 -AGTTGGATG GTAGCCCTTG-30 (anti-sense); IL-6: 50 -AGGATACCAC TCCCAACAGACCT-30 (sense), 50 -TTTCTCATTTCCAC GATTTCCC-30 (anti-sense). Quantification was performed using 7500 System SDS software (Applied Biosystems). Cytokine determination by Bio-Plex assay A Bio-Plex mouse cytokine assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to test samples for the presence of the following cytokines: interleukin (IL)1b, IL-6, IL-12 (p40), IL-12 (p70), IL-17, interferon (IFN)c, macrophage chemoattractant protein 1 (MCP-1/CCL2), macrophage inflammatory protein 1a (MIP-1a/CCL3), macrophage inflammatory protein 1b (MIP-1b/CCL4) and RANTES/CCL5. In brief, premixed spinal cord samples were reconstituted in 0.5 ml of a Bio-Plex mouse serum
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standard diluent, generating a stock concentration of 50,000 pg/ml for each cytokine. The standard stock was serially diluted in the Bio-Plex human serum diluent to generate 8 points for the standard curve. The assay was performed in a 96-well filtration plate supplied with the assay kit. The samples were diluted 1:4 in the Bio-Plex serum sample diluent. Homogenates of CNS tissue samples were used in this assay. The amounts of cytokines measured were corrected for the amount of protein in each sample. Protein content was measured with a Bradford assay (Bio-Rad). Premixed standards or diluted samples (50 ll) were added to each well containing washed beads. The plate was shaken for 30 s at high speed (1100 rpm) and then incubated at room temperature for 2 h at low speed (300 rpm). After incubation and washing, premixed detection antibodies (25 ll) were added to each well. Then, the plate was incubated for 1 h on a shaker at low speed (300 rpm). After incubation and washing, streptavidin-PE was added to each well. The incubation was terminated after shaking for 30 min at room temperature. After washing, the beads were re-suspended in 125 ll of BioPlex assay buffer. 250 beads were acquired for each sample and the data were analyzed using Bio-Plex Manager software version 4.0. The lowest limit of concentration for the detection of each cytokine was as follows (pg/ml): IL-1b, 3.01 pg/ml; IL-6, 0.81 pg/ml; IL-12 (p40),1.36 pg/ml; IL12 (p70), 1.08 pg/ml; IL-17, 2.04 pg/ml; IFN-c, 1.93 pg/ ml; MCP-1, 2.0 pg/ml; MIP-1a, 4.63 pg/ml; MIP-1b, 1.3 pg/ml and RANTES, 0.42 pg/ml. CNS-infiltrating cell separation All mice were euthanized in CO2 chambers and perfused with 10 ml of cold PBS. The whole CNS (brain, cerebellum and spinal cords) was excised, macerated and maintained in 4 mL of DMEM supplemented with 2.5 % collagenase D (Roche) at 37 C in a 5 % CO2 incubator. Forty-five minutes later, suspensions were washed in DMEM and centrifuged at 450g for 5 min at 4 C. After that, cells were re-suspended in 37 % Percoll and gently layered over 70 % Percoll in 15-ml tubes. The tubes were centrifuged at 950g for 20 min with the centrifuge brake turned off. After centrifugation, the ring containing mononuclear cells was collected, washed in DMEM and centrifuged at 450g for 5 min. Cells were then counted and suspended in complete DMEM medium. Cells were treated with brefeldin A at 1 lg/ml and stimulated with medium (control) or phorbol myristate acetate (PMA) (50 ng/ ml) plus ionomycin (1 lg/ml) for 12 h before analysis. Primary microglial cultures Primary microglia was isolated from mixed glial cultures. Glial cultures were isolated from 2-day-old C57Bl/6 mice.
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Euthanasia was performed by decapitation by a properly trained participant. The brains were taken and separated from the meninges and minced into small pieces, filtered through a 70-lm strainer and separated by trypsinization (0.025 % trypsin, 37 C, 20 min). Fetal bovine serum (FBS) was added at a 1:10 proportion to inactivate the trypsin. The cells were centrifuged (200g for 5 min), the pellets were re-suspended in media and the cells were transferred to a 75-mm2 flask (one brain per flask). Cells were cultured at 37 C in a 5 % CO2 atmosphere in DMEM media supplemented with 10 % heatinactivated FBS (Gibco), 100 U/ml penicillin and 100 lg/ml streptomycin. Medium was changed every 4 days until day 14, when cells reached confluence. Primary microglial cells were isolated from mixed glial cultures as they matured on top of astrocyte monolayers. Flasks were shaken for 5 min in an orbital shaker. The floating microglia were harvested and transferred to 96-well dishes, and, after 2 h, the medium was changed to remove non-adherent cells. The purity of the cultures was confirmed by immunofluorescence using an antiIba1 antibody (Santa Cruz Biotechnology, Santa Cruz, USA). Magnetic activated cell sorting (MACS) of CD41 cells from spleen MACS separation was undertaken using a CD4 (L3T4) MicroBeads, mouse kit (Miltenyi Biotec, Auburn, USA) according to the manufacturer’s instructions. Initially, splenocytes were collected from C57BL/6 mice. The cells were re-suspended in 90 ll of MACS buffer, 10 ll of CD4 (L3T4) magnetic beads were added per every 107 cells, and incubated at 4 C for 15 min. Then the cells were washed and re-suspended in 1 ml MACS buffer. For magnetic separation, the cell suspension was applied to a magnetic column and washed three times with MACS buffer. The collected material containing unlabeled cells was discarded. The column was then removed from the magnetic separator, placed in a suitable collection tube and a plunger was used to flush out fraction with magnetically labeled cells. The effluent containing the labeled cells was collected and the purity of the cells evaluated by fluorescence activated cell sorting (FACS). Co-culture of microglia and CD41 T Cells After magnetic separation, the CD4? T cells were centrifuged and re-suspended 1 9 106 cells per ml in a total volume of 2 ml. CFSE (5-(6)-carboxyfluorescein N-succinimidyl ester diacetate) (Sigma-Aldrich, Saint Louis, USA) (5 mM) was added to a final concentration of 2.5 lM. The cells were incubated at 37 C, protected from light, for 20 min. The CFSE was inactivated with 8 ml of medium supplemented with 10 % FBS by incubating at 37 C for 5 min. Finally, the cells were centrifuged and re-
Simvastatin ameliorates experimental autoimmune encephalomyelitis by inhibiting Th1/Th17…
suspended in supplemented medium (1 9 106 cells per mL) for use in co-culture experiments. For co-culture experiments, 25 9 104 microglial cells were seeded per well in a 96-well flat bottom dish (Corning Incorporated, Corning, NY, USA). After 3 days, 5 9 105 CD4? T cells labeled with CFSE were added. The cultures were treated with simvastatin (5 lM) or vehicle. Treatment with LPS (lipopolysaccharide from Salmonella enterica serotype abortus equi, Sigma-Aldrich, Saint Louis, USA) was given simultaneously to simvastatin. Five days after treatment, CD4? T cells were collected and labeled with anti-CD4APC antibody (BioLegend, San Diego, USA) (clone: RM45) for analysis by flow cytometry in a BD Accuri C6 (BD Biosciences, Singapore). Simvastatin preparation to cell culture Five milligrams of simvastatin (Sigma-Aldrich) was dissolved in 125 ll of ethanol, and then 187.5 ll of 0.1 N NaOH was added and the solution incubated at 50 C for 2 h. The pH was brought to 7.0, and the final concentration adjusted to 5 mg/ml. Statistical analysis Data are presented as mean ± SEM. For clinical scores, the significance between each two groups was examined using the Mann–Whitney test. A one-tailed unpaired Student’s t test was employed to analyze gene expression, protein production and flow cytometry results. A value of p \ 0.05 was considered significant.
Results Clinical disease development
Fig. 1 EAE clinical development. Animals were immunized with MOG35-55 peptide in CFA ? BCG. Pertussis toxin was given i.p. at the time of immunization and 48 h later. Oral simvastatin at 5 mg/ kg/day or vehicle (control) was given daily beginning on day 7. Mice were followed to assess disease clinical scores. N = 5 per group. Error bar represents mean ± SEM. *p \ 0.05 for control versus simvastatin 5 mg/kg/day
Cytokine and chemokine synthesis To further corroborate the findings obtained with real-time PCR, we assessed whether simvastatin treatment promoted changes in protein expression. Mice treated with simvastatin at 5 mg/kg/day exhibited decreased synthesis of the pro-inflammatory cytokines IL-6 (p \ 0.05), IL-12p70 (p \ 0.05) and IL-12p40 (p \ 0.01) (Fig. 3). The effects exerted on IL-1b, IL-17 and IFN-c expression were not statistically significant (p [ 0.05) (Fig. 3). Furthermore, simvastatin treatment was able to modulate chemokine synthesis. Simvastatin decreased RANTES and MIP-1b expression (p \ 0.05) (Fig. 4), whereas changes in MCP-1 and MIP-1a were not significant (p [ 0.05) (Fig. 4). CNS inflammatory mononuclear cell infiltration To further investigate the effects of simvastatin on CNS inflammatory cell recruitment, we undertook a cell
Mice received simvastatin at 5 mg/kg/day starting on day 7 after immunization until the end of follow-up on day 21. Mice receiving simvastatin treatment demonstrated decreased mean clinical scores (p \ 0.05) and overall better disease outcomes (Fig. 1). Cytokine gene expression analysis To clarify the mechanisms by which simvastatin changes EAE clinical outcome, we investigated the expression of different genes implicated in EAE inflammatory lesions. We explored the possibility that simvastatin affects the gene expression of TGF-b and IL-6 in situ. Indeed, there was an increase in TGF-b mRNA expression (Fig. 2). However, the effect on IL-6 expression was not significant (Fig. 2).
Fig. 2 The effect of simvastatin treatment on TGF-b and IL-6 mRNA expression. Real-time PCR analysis of TGF-b and IL-6 mRNA expression in the CNS during EAE development. Oral simvastatin at 5 mg/kg/day or vehicle (control) were given daily beginning on day 7 through the end of the experiment. Samples of CNS tissue were collected on day 21 after immunization. Data are shown as mean ± SEM. Each group contained 4 animals
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D. M. Oliveira et al. Fig. 3 CNS cytokine expression. CNS samples were taken from mice with EAE at day 21 after disease induction, and cytokine expression was analyzed with a Bio-Plex assay. Oral simvastatin at 5 mg/kg/day or vehicle (control) were given daily beginning on day 7 through the end of the experiment. The cytokines IL1b, IL-6, IL-17, IL-12p40, IL12p70, IFN-c were analyzed. Each bar represents mean ± SEM (3 animals per group)
Fig. 4 CNS chemokine expression. CNS samples were taken from mice with EAE on day 21 after disease induction, and cytokine expression was analyzed with a Bio-Plex assay. Oral simvastatin at 5 mg/kg/day or vehicle (control) were given daily beginning on day 7 through the end of the experiment. The chemokines MCP-1, RANTES, MIP-1a, MIP-1b were analyzed. Each bar represents mean ± SEM (3 animals per group)
separation assay to obtain mononuclear inflammatory cells from CNS samples. Total absolute numbers of infiltrating mononuclear cells were lower in mice receiving simvastatin (control, 3.1 ± 0.36; simvastatin at 5 mg/kg/day, 1.31 ± 0.37) (Fig. 5). For cytokine analysis, these cells were seeded on 96-well round-bottomed plates and stimulated with PMA ? ionomycin or remained unstimulated. After 12 h of culture, cells were analyzed by flow cytometry. Simvastatin decreased both relative and absolute
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numbers of IFN-c secreting Th1 cells infiltrating the CNS of the experimental groups (Fig. 6b, c). With regard to Th17 cells, no differences were detected in terms of relative numbers (Fig. 7b). However, the absolute number of CD4? IL-17? cells was significantly lower in the CNS of mice under simvastatin treatment (Fig. 7c). These results indicate that in vivo simvastatin treatment may modulate both Th1 and Th17 biology, either by impairing cell differentiation in the periphery or by
Simvastatin ameliorates experimental autoimmune encephalomyelitis by inhibiting Th1/Th17…
reducing cellular ability to infiltrate the CNS. We can also speculate that simvastatin exerts a more prominent effect on Th1 cells than on Th17 cells, as results demonstrated decreased Th1 cell infiltration in terms of percent and absolute numbers of cells, while Th17 analysis indicated that differences were present only in terms of absolute numbers of cells. Lymphocyte proliferation and interaction with glial cells
Fig. 5 The effect of simvastatin on CNS mononuclear cell infiltration. Animals were immunized with MOG35-55 peptide in CFA ? BCG. Pertussis toxin was given i.p. at the time of immunization and 48 h later. Oral simvastatin at 5 mg/kg/day or vehicle were given daily beginning on day 7 until the peak of disease (day 15), when CNS samples were taken for analysis. CNS-infiltrating mononuclear cells were separated from CNS tissue in a cell separation assay, and total numbers of inflammatory cells were counted in each animal sample. Each bar represents mean ± SEM (4 animals per group)
Fig. 6 IFN-c expression in CNS-infiltrating mononuclear cells. Animals were immunized with MOG35-55 peptide in CFA ? BCG. Pertussis toxin was given i.p. at the time of immunization and 48 h later. Oral simvastatin at 5 mg/kg/day or vehicle were given daily beginning on day 7 through the end of the experiment (day 15), when CNS samples were taken for analysis. CNS-infiltrating mononuclear cells were separated from CNS tissue in a cell separation assay. Collected cells were stimulated with medium (control) or
We asked whether the anti-proliferative effects of simvastatin contributed to the results of CNS mononuclear cell infiltrate analysis. The anti-proliferative effects of statins have previously been demonstrated in different cell types and lineages (Yu et al. 2013; Neuhaus et al. 2002; Saito et al. 2008). If simvastatin is able to inhibit lymphocyte proliferation in the CNS, then decreased infiltrating mononuclear cell numbers could be attributed, at least in part, to the direct effects of simvastatin inside the CNS. To analyze this hypothesis, CD4? T cells were purified from spleen samples, labeled with CFSE and added to primary microbial cultures
PMA ? ionomycin. After 12 h, cells were analyzed by flow cytometry. a Gates from medium (control) and from PMA ? ionomycin stimulated cells, b percentage numbers of IFN-c? cells, c absolute numbers of IFN-c? cells. Each bar represents mean ± SEM. Numbers of mice per group: medium, N = 3 (control), N = 4 (simvastatin); PMA ? ionomycin, N = 4 (control), N = 4 (simvastatin)
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Fig. 7 IL-17 expression in CNS-infiltrating mononuclear cells. Animals were immunized with MOG35-55 peptide in CFA ? BCG. Pertussis toxin was given i.p. at the time of immunization and 48 h later. Oral simvastatin at 5 mg/kg/day or vehicle were given daily beginning on day 7 through the end of the experiment (day 15), when CNS samples were taken for analysis. CNS-infiltrating mononuclear cells were separated from CNS tissue in a cell separation assay. Collected cells
were stimulated with medium (control) or PMA ? ionomycin. After 12 h, cells were analyzed by flow cytometry. a Gates from medium (control) and from PMA ? ionomycin stimulated cells, b percentage numbers of IL-17? cells, c absolute numbers of IL-17? cells. Each bar represents mean ± SEM. Numbers of mice per group: medium, N = 4 (control), N = 4 (simvastatin); PMA ? ionomycin, N = 3 (control), N = 4 (simvastatin)
stimulated with LPS at 0.25 lg/ml or vehicle. After 5 days, cells were collected and analyzed by flow cytometry. Simvastatin at 5 lM decreased CD4? T cell proliferation in unstimulated (Fig. 8a, b) and LPS-stimulated cultures (Fig. 8c, d). In this context, the anti-proliferative effects of simvastatin can be attributed to its inhibition of inflammation, as simvastatin can inhibit the expression of several inflammatory molecules such as CD80, CD86, MHC, and other cytokines (Neuhaus et al. 2002).
unrestrained polarized Th17 response that is more aggressive than a normal Th1/Th17 disease in WT animals. In fact, experimental data suggest that Th1- and Th17-polarized disease can exhibit features of different types of MS: Th1 is more similar to relapsing–remitting MS, and Th17 is similar to the more aggressive opticospinal MS or Devic’s disease (Kroenke et al. 2008). There is evidence for many different relevant roles for both subtypes in human disease. In a study attempting to treat MS with IFN-c, many patients experienced disease flare-ups (Panitch et al. 1987). Corroborating this, in humans, IL-6 and IL-17 transcripts can be found in cerebrospinal fluid samples from MS patients, with higher levels present during disease flare-ups (Wen et al. 2012), and higher expression of IL-17F has been associated with a poor response to treatment (Axtell et al. 2010). Statins have been studied in different contexts, such as auto-immunity and inflammation (Neuhaus et al. 2005). Statins have the ability to ameliorate EAE by inducing a transition from a Th1 to a Th2 phenotype (Youssef et al. 2002), and in fact, corroborating our findings, their potential to inhibit Th17 development has already been addressed (Zhang et al. 2008). However, the role for statins
Discussion MS is an organ-specific autoimmune disease mediated by CD4? T cells. Data from its comparable animal model, EAE, have revealed that IFN-c?-producing Th1 lymphocytes are pivotal in disease development. However, studies have demonstrated that mice deficient for INF-c and INF-c receptor (IFN-cR) were, in fact, more susceptible to EAE induction (Bettelli et al. 2004; Ferber et al. 1996; Willenborg et al. 1996). These drawbacks were clarified with the discovery of the Th17 T cell subset. The absence of Th1 cells in IFN-c KO mice permits the development of an
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Simvastatin ameliorates experimental autoimmune encephalomyelitis by inhibiting Th1/Th17…
Fig. 8 Percentages of CFSE-low lymphocytes. CFSE-labeled T cells were added to primary microbial cultures. Vehicle or simvastatin at a concentration of 5 lM were given starting on the first day. In cultures treated with LPS, LPS was given simultaneously with simvastatin. After 5 days, cells were collected and analyzed by flow cytometry. a Histograms representative of control and simvastatin (5 lM) cells.
b Bar graphs represent mean ± SEM from gates of CD4? CFSE-low cells. c Histograms representative of control plus LPS (0.25 lg/ml) and simvastatin (5 lM) plus LPS (0.25 lg/ml) cells. d Bar graphs represent mean ± SEM from gates of CD4? CFSE-low cells from LPS-treated groups
acting on infiltrating inflammatory cells inside the CNS has still to be addressed. Therefore, we focused our experiments on CNS samples taken during disease development. In order to evaluate CNS inflammatory cell recruitment, we performed a cell separation assay by gradient centrifugation, which permits quantification and analysis of inflammatory infiltrates and microglia in CNS tissue. Simvastatin treatment reduced the numbers of mononuclear cells in brain and spinal cord samples. This suggests that the effects exerted by simvastatin on EAE are, at least in part, the result of decreased inflammatory cell infiltration. During the process of EAE development, auto-reactive T cells are activated in the periphery and up-regulate CCR5, the receptor for MIP-1b and RANTES, due to the effects of IL-12 (Bagaeva et al. 2003). Auto-reactive T cells expressing CCR5 are able to infiltrate CNS perivascular sites, where they interact with microglia, leading to release of inflammatory mediators and inflammatory cell
recruitment. This secondary influx of inflammatory cells greatly correlates with clinical disease development (Brocke et al. 1996). In accordance with these findings, CNS samples from animals under simvastatin treatment exhibited decreased amounts of RANTES and MIP-1b. Therefore, the reduced numbers of inflammatory mononuclear cells can be partly explained by the decreased expression of chemokines. The role of chemokines in EAE has already been addressed in several studies. It has been suggested that they enhance rather than initiate cell infiltration (Glabinski et al. 1995). RANTES, MIP-1a, MIP-1b, and MCP-1 among others were induced 1–2 days before clinical disease development in murine EAE (Godiska et al. 1995). During relapses, increased expression of MCP-1, IP-10/CXCL10, MIP-1a, RANTES and GRO-a/CXCL1 was observed (Glabinski et al. 1997). Astrocytes are the source of MCP-1 and IP-10, whereas RANTES and MIP-1a expression was associated with inflammatory cells (Tani et al. 1996;
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Glabinski et al. 1997). In our samples, there was a tendency to observe decreased expression of MCP-1 and MIP-1a with no significant differences. These results can be explained by the time of sample collection on the 21st day after disease induction, when chemokine release was most likely decreasing. Another possibility is that these results are merely attributable to the reduced presence of Th17 cells that may boost the inflammation acting on glial resident cells. We also analyzed the types of T cells present in the inflammatory infiltrates and their response to stimulation with PMA ? ionomycin. Simvastatin could decrease the percentages and absolute numbers of CD4?IFN-c? T cells, whereas CD4?IL-17? T cells were reduced only in terms of absolute numbers with similar percentages in both groups. These results are in accordance with previous data showing that statins inhibit Th1 (Youssef et al. 2002) and Th17 responses (Imamura et al. 2009; Murphy et al. 2008; Eller et al. 2010). Our results suggest that simvastatin exerts a more prominent effect on the Th1 response, as there were decreased percentages of CD4?IFN-c? T cells, while CD4?IL-17? T cells were decreased only in terms of absolute numbers, with similar percentages in the control and simvastatin groups. Zhang et al. (2008) have demonstrated that simvastatin directly inhibits Th17 differentiation. Thus, it was not expected that simvastatin treatment would fail to induce decreased percentages of CD4?IL-17? T cells. This apparent contradiction may be explained by the day chosen for the beginning of the treatment. T lymphocytes migrate into the CNS in two waves. In the first wave, CD4?IL-17? T cells migrate into the CNS through the choroid plexus. Once inside the CNS, these cells activate epithelial cells to initiate a second wave of T cell migration (Reboldi et al. 2009). As the treatment was initiated at day 7 after immunization, it was ineffective to inhibit initial T cell differentiation and it was probably of little efficiency against the first migration wave. As a consequence, simvastatin was more effective against Th1 because Th17 cells migrated early into the CNS. Therefore, most of the Th17 cells were already there when treatment began. In mice, Th17 development is a consequence of the simultaneous action of IL-6 and TGF-b on T lymphocytes (Bettelli et al. 2006) with IL-23 being a survival factor for effector Th17 cells, whereas Th1 differentiation is driven by IL-12. This cytokine is composed of a p35 and a p40 subunit. Likewise, IL-23 is composed of the same p40 subunit with a p19 subunit. Our tissue analysis of cytokine expression indicated reduced levels of IL-6 as well as IL12 p40 and IL-12 p70, which together represent the whole IL-12 molecule. This may indicate effects exerted by simvastatin on macrophage/microglia cytokine production, which in turn reduced Th1 commitment and CNS infiltration.
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It has been demonstrated that statins have anti-proliferative effects, inhibiting T cell proliferation as well as tumor growth. Thus, clinical improvement and reduced inflammatory infiltrates would be consequences of these effects at the periphery but also for cells already inside the inflammatory sites in the CNS tissue. In order to address these questions, we co-cultured CFSE-labeled lymphocytes with microglial cells during 5 days. Simvastatin at a concentration of 5 lM reduced the percentage of CFSE-low lymphocytes, suggesting that an anti-proliferative effect can occur not only at the periphery during clonal expansion, but also inside inflammatory sites. As simvastatin is able to cross the BBB (Sierra et al. 2011), an anti-proliferative effect directly inside the CNS may have contributed to the decreased number of inflammatory cells in the CNS which can partially explain the decreased levels of cytokines and chemokines found at tissue analysis. Moreover, it is suitable to think that, as simvastatin is able to transpose the BBB, it is dampening T cell activation in situ and for example reducing epitope spreading. Collectively, our data show that simvastatin is able to ameliorate EAE by reducing inflammatory cell recruitment to the CNS and by inhibiting both Th1 and Th17 development. Our in vitro results also suggest that simvastatin decreases T lymphocyte proliferation during co-culture with microglia. In summary, our data corroborates previous findings showing that simvastatin has immunomodulatory properties, through which inflammatory immune responses may be blunted. Moreover, besides its peripheral effect, this drug may also exert some effect in situ, dampening resident cells inflammatory cytokines secretion and thus T cell activation. Acknowledgments We thank Claudia da Silva Cunha and Paulo Albe for technical support. Financial Support: CNPq, Complex Fluids INCT and FAPESP (07/01771-0, 07/07139-3, 12/02270-2 and 2011/18703-2). Compliance with ethical standards Conflict of interest
The authors declare no conflict of interest.
References Ando DG, Clayton J, Kono D, Urban JL, Sercarz EE (1989) Encephalitogenic T cells in the B10.PL model of experimental allergic encephalomyelitis (EAE) are of the Th-1 lymphokine subtype. Cell Immunol 124:132–143 Axtell RC, de Jong BA, Boniface K, van der Voort LF, Bhat R, de Sarno P, Naves R, Han M, Zhong F, Castellanos JG, Mair R, Christakos A, Kolkowitz I, Katz L, Killestein J, Polman CH, de Waal Malefyt R, Steinman L, Raman C (2010) T helper type 1 and 17 cells determine efficacy of interferon-beta in multiple sclerosis and experimental encephalomyelitis. Nat Med 16:406–412 Bagaeva LV, Williams LP, Segal BM (2003) IL-12 dependent/IFN gamma independent expression of CCR5 by myelin-reactive T cells correlates with encephalitogenicity. J Neuroimmunol 137:109–116
Simvastatin ameliorates experimental autoimmune encephalomyelitis by inhibiting Th1/Th17… Bellosta S, Via D, Canavesi M, Pfister P, Fumagalli R, Paoletti R, Bernini F (1998) HMG-CoA reductase inhibitors reduce MMP-9 secretion by macrophages. Arterioscler Thromb Vasc Biol 18:1671–1678 Bettelli E, Sullivan B, Szabo SJ, Sobel RA, Glimcher LH, Kuchroo VK (2004) Loss of T-bet, but not STAT1, prevents the development of experimental autoimmune encephalomyelitis. J Exp Med 200:79–87 Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, Weiner HL, Kuchroo VK (2006) Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441:235–238 Birnbaum G, Cree B, Altafullah I, Zinser M, Reder AT (2008) Combining beta interferon and atorvastatin may increase disease activity in multiple sclerosis. Neurology 71:1390–1395 Brocke S, Gijbels K, Allegretta M, Ferber I, Piercy C, Blankenstein T, Martin R, Utz U, Karin N, Mitchell D, Veromaa T, Waisman A, Gaur A, Conlon P, Ling N, Fairchild PJ, Wraith DC, O’Garra A, Fathman CG, Steinman L (1996) Treatment of experimental encephalomyelitis with a peptide analogue of myelin basic protein. Nature 379:343–346 Bustos C, Hernandez-Presa MA, Ortego M, Tunon J, Ortega L, Perez F, Diaz C, Hernandez G, Egido J (1998) HMG-CoA reductase inhibition by atorvastatin reduces neointimal inflammation in a rabbit model of atherosclerosis. J Am Coll Cardiol 32:2057–2064 Chataway J, Schuerer N, Alsanousi A, Chan D, Macmanus D, Hunter K, Anderson V, Bangham CR, Clegg S, Nielsen C, Fox NC, Wilkie D, Nicholas JM, Calder VL, Greenwood J, Frost C, Nicholas R (2014) Effect of high-dose simvastatin on brain atrophy and disability in secondary progressive multiple sclerosis (MS-STAT): a randomised, placebo-controlled, phase 2 trial. Lancet 383:2213–2221 Compston A, Coles A (2002) Multiple sclerosis. Lancet 359:1221–1231 Cua DJ, Sherlock J, Chen Y, Murphy CA, Joyce B, Seymour B, Lucian L, To W, Kwan S, Churakova T, Zurawski S, Wiekowski M, Lira SA, Gorman D, Kastelein RA, Sedgwick JD (2003) Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421:744–748 Eller P, Eller K, Wolf AM, Reinstadler SJ, Tagwerker A, Patsch JR, Mayer G, Rosenkranz AR (2010) Atorvastatin attenuates murine anti-glomerular basement membrane glomerulonephritis. Kidney Int 77:428–435 Ferber IA, Brocke S, Taylor-Edwards C, Ridgway W, Dinisco C, Steinman L, Dalton D, Fathman CG (1996) Mice with a disrupted IFN-gamma gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE). J Immunol 156:5–7 Frohman EM, Racke MK, Raine CS (2006) Multiple sclerosis—the plaque and its pathogenesis. N Engl J Med 354:942–955 Ganne F, Vasse M, Beaudeux JL, Peynet J, Francois A, Mishal Z, Chartier A, Tobelem G, Vannier JP, Soria J, Soria C (2000) Cerivastatin, an inhibitor of HMG-CoA reductase, inhibits urokinase/urokinase-receptor expression and MMP-9 secretion by peripheral blood monocytes–a possible protective mechanism against atherothrombosis. Thromb Haemost 84:680–688 Glabinski AR, Tani M, Tuohy VK, Tuthill RJ, Ransohoff RM (1995) Central nervous system chemokine mRNA accumulation follows initial leukocyte entry at the onset of acute murine experimental autoimmune encephalomyelitis. Brain Behav Immun 9:315–330 Glabinski AR, Tani M, Strieter RM, Tuohy VK, Ransohoff RM (1997) Synchronous synthesis of alpha- and beta-chemokines by cells of diverse lineage in the central nervous system of mice with relapses of chronic experimental autoimmune encephalomyelitis. Am J Pathol 150:617–630 Godiska R, Chantry D, Dietsch GN, Gray PW (1995) Chemokine expression in murine experimental allergic encephalomyelitis. J Neuroimmunol 58:167–176
Hu W, Lucchinetti CF (2009) The pathological spectrum of CNS inflammatory demyelinating diseases. Semin Immunopathol 31:439–453 Imamura M, Okunishi K, Ohtsu H, Nakagome K, Harada H, Tanaka R, Yamamoto K, Dohi M (2009) Pravastatin attenuates allergic airway inflammation by suppressing antigen sensitisation, interleukin 17 production and antigen presentation in the lung. Thorax 64:44–49 Ishizu T, Osoegawa M, Mei FJ, Kikuchi H, Tanaka M, Takakura Y, Minohara M, Murai H, Mihara F, Taniwaki T, Kira J (2005) Intrathecal activation of the IL-17/IL-8 axis in opticospinal multiple sclerosis. Brain 128:988–1002 Kamm CP, El-Koussy M, Humpert S, Findling O, von Bredow F, Burren Y, Schwegler G, Schott D, Donati F, Muller M, Goebels N, Muller F, Slotboom J, Tettenborn B, Kappos L, Naegelin Y, Mattle HP (2012) Atorvastatin added to interferon beta for relapsing multiple sclerosis: a randomized controlled trial. J Neurol 259:2401–2413 Kamm CP, El-Koussy M, Humpert S, Findling O, Burren Y, Schwegler G, Donati F, Muller M, Muller F, Slotboom J, Kappos L, Naegelin Y, Mattle HP (2014) Atorvastatin added to interferon beta for relapsing multiple sclerosis: 12-month treatment extension of the randomized multicenter SWABIMS trial. PLoS ONE 9:e86663 Kim SY, Guijarro C, O’Donnell MP, Kasiske BL, Kim Y, Keane WF (1995) Human mesangial cell production of monocyte chemoattractant protein-1: modulation by lovastatin. Kidney Int 48:363–371 Kobashigawa JA, Katznelson S, Laks H, Johnson JA, Yeatman L, Wang XM, Chia D, Terasaki PI, Sabad A, Cogert GA et al (1995) Effect of pravastatin on outcomes after cardiac transplantation. N Engl J Med 333:621–627 Kroenke MA, Carlson TJ, Andjelkovic AV, Segal BM (2008) IL-12and IL-23-modulated T cells induce distinct types of EAE based on histology, CNS chemokine profile, and response to cytokine inhibition. J Exp Med 205:1535–1541 Kuchroo VK, Anderson AC, Waldner H, Munder M, Bettelli E, Nicholson LB (2002) T cell response in experimental autoimmune encephalomyelitis (EAE): role of self and cross-reactive antigens in shaping, tuning, and regulating the autopathogenic T cell repertoire. Annu Rev Immunol 20:101–123 Langrish CL, Chen Y, Blumenschein WM, Mattson J, Basham B, Sedgwick JD, McClanahan T, Kastelein RA, Cua DJ (2005) IL23 drives a pathogenic T cell population that induces autoimmune inflammation. J Exp Med 201:233–240 Lanzillo R, Orefice G, Quarantelli M, Rinaldi C, Prinster A, Ventrella G, Spitaleri D, Lus G, Vacca G, Carotenuto B, Salvatore E, Brunetti A, Tedeschi G, Brescia Morra V (2010) Atorvastatin combined to interferon to verify the efficacy (ACTIVE) in relapsing-remitting active multiple sclerosis patients: a longitudinal controlled trial of combination therapy. Mult Scler 16:450–454 Lavi E, Constantinescu CS (2005) Experimental models of multiple sclerosis. Springer, New York Li Z, Chen L, Niu X, Liu J, Ping M, Li R, Xie X, Guo L (2012) Immunomodulatory synergy by combining atorvastatin and rapamycin in the treatment of experimental autoimmune encephalomyelitis (EAE). J Neuroimmunol 250:9–17 Lock C, Hermans G, Pedotti R, Brendolan A, Schadt E, Garren H, Langer-Gould A, Strober S, Cannella B, Allard J, Klonowski P, Austin A, Lad N, Kaminski N, Galli SJ, Oksenberg JR, Raine CS, Heller R, Steinman L (2002) Gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis. Nat Med 8:500–508 Luccarini I, Ballerini C, Biagioli T, Biamonte F, Bellucci A, Rosi MC, Grossi C, Massacesi L, Casamenti F (2008) Combined treatment with atorvastatin and minocycline suppresses severity of EAE. Exp Neurol 211:214–226
123
D. M. Oliveira et al. Matusevicius D, Kivisakk P, Navikas VV, Tian W, Soderstrom M, Fredrikson S, Link H (1998) Influence of IFN-beta1b (Betaferon) on cytokine mRNA profiles in blood mononuclear cells and plasma levels of soluble VCAM-1 in multiple sclerosis. Eur J Neurol 5:265–275 Murphy DM, Forrest IA, Corris PA, Johnson GE, Small T, Jones D, Fisher AJ, Egan JJ, Cawston TE, Ward C, Lordan JL (2008) Simvastatin attenuates release of neutrophilic and remodeling factors from primary bronchial epithelial cells derived from stable lung transplant recipients. Am J Physiol Lung Cell Mol Physiol 294:L592–L599 Neuhaus O, Strasser-Fuchs S, Fazekas F, Kieseier BC, Niederwieser G, Hartung HP, Archelos JJ (2002) Statins as immunomodulators: comparison with interferon-beta 1b in MS. Neurology 59:990–997 Neuhaus O, Stuve O, Archelos JJ, Hartung HP (2005) Putative mechanisms of action of statins in multiple sclerosis—comparison to interferon-beta and glatiramer acetate. J Neurol Sci 233:173–177 Pahan K, Sheikh FG, Namboodiri AM, Singh I (1997) Lovastatin and phenylacetate inhibit the induction of nitric oxide synthase and cytokines in rat primary astrocytes, microglia, and macrophages. J Clin Invest 100:2671–2679 Paintlia AS, Paintlia MK, Singh AK, Stanislaus R, Gilg AG, Barbosa E, Singh I (2004) Regulation of gene expression associated with acute experimental autoimmune encephalomyelitis by Lovastatin. J Neurosci Res 77:63–81 Paintlia AS, Paintlia MK, Hollis BW, Singh AK, Singh I (2012) Interference with RhoA-ROCK signaling mechanism in autoreactive CD4? T cells enhances the bioavailability of 1,25dihydroxyvitamin D3 in experimental autoimmune encephalomyelitis. Am J Pathol 181:993–1006 Panitch HS, Hirsch RL, Haley AS, Johnson KP (1987) Exacerbations of multiple sclerosis in patients treated with gamma interferon. Lancet 1:893–895 Peron JPS, Oliveira D, Branda˜o WN, Fickinger A, Ligeiro De Oliveira AP, Rizzo LV, Caˆmara NOS (2012) Central nervous system resident cells in neuroinflammation: a brave new world. In: Chan J (ed) Autoimmune diseases, contributing factors, specific cases of autoimmune diseases, and stem cell and other therapies. InTech, Rijeka Rallidis LS, Hamodraka ES, Fountoulaki K, Moustogiannis G, Zolindaki MG, Kremastinos DT (2008) Simvastatin exerts its anti-inflammatory effect in hypercholesterolaemic patients by decreasing the serum levels of monocyte chemoattractant protein-1. Int J Cardiol 124:271–272 Reboldi A, Coisne C, Baumjohann D, Benvenuto F, Bottinelli D, Lira S, Uccelli A, Lanzavecchia A, Engelhardt B, Sallusto F (2009) C–C chemokine receptor 6-regulated entry of TH-17 cells into the CNS through the choroid plexus is required for the initiation of EAE. Nat Immunol 10:514–523 Saito A, Saito N, Mol W, Furukawa H, Tsutsumida A, Oyama A, Sekido M, Sasaki S, Yamamoto Y (2008) Simvastatin inhibits growth via apoptosis and the induction of cell cycle arrest in human melanoma cells. Melanoma Res 18:85–94 Sierra S, Ramos MC, Molina P, Esteo C, Vazquez JA, Burgos JS (2011) Statins as neuroprotectants: a comparative in vitro study of lipophilicity, blood–brain-barrier penetration, lowering of brain cholesterol, and decrease of neuron cell death. J Alzheimers Dis 23:307–318 Sorensen PS, Lycke J, Eralinna JP, Edland A, Wu X, Frederiksen JL, Oturai A, Malmestrom C, Stenager E, Sellebjerg F, Sondergaard HB (2011) Simvastatin as add-on therapy to interferon beta-1a for relapsing-remitting multiple sclerosis (SIMCOMBIN study): a placebo-controlled randomised phase 4 trial. Lancet Neurol 10:691–701 Stuve O, Youssef S, Weber MS, Nessler S, von Budingen HC, Hemmer B, Prod’Homme T, Sobel RA, Steinman L, Zamvil SS
123
(2006) Immunomodulatory synergy by combination of atorvastatin and glatiramer acetate in treatment of CNS autoimmunity. J Clin Invest 116:1037–1044 Tani M, Glabinski AR, Tuohy VK, Stoler MH, Estes ML, Ransohoff RM (1996) In situ hybridization analysis of glial fibrillary acidic protein mRNA reveals evidence of biphasic astrocyte activation during acute experimental autoimmune encephalomyelitis. Am J Pathol 148:889–896 Togha M, Karvigh SA, Nabavi M, Moghadam NB, Harirchian MH, Sahraian MA, Enzevaei A, Nourian A, Ghanaati H, Firouznia K, Jannati A, Shekiba M (2010) Simvastatin treatment in patients with relapsing-remitting multiple sclerosis receiving interferon beta 1a: a double-blind randomized controlled trial. Mult Scler 16:848–854 Tsakiri A, Kallenbach K, Fuglo D, Wanscher B, Larsson H, Frederiksen J (2012) Simvastatin improves final visual outcome in acute optic neuritis: a randomized study. Mult Scler 18:72–81 Vieira JM Jr, Mantovani E, Rodrigues LT, Delle H, Noronha IL, Fujihara CK, Zatz R (2005) Simvastatin attenuates renal inflammation, tubular transdifferentiation and interstitial fibrosis in rats with unilateral ureteral obstruction. Nephrol Dial Transpl 20:1582–1591 Voskuhl RR, Martin R, Bergman C, Dalal M, Ruddle NH, McFarland HF (1993) T helper 1 (Th1) functional phenotype of human myelin basic protein-specific T lymphocytes. Autoimmunity 15:137–143 Waiczies S, Prozorovski T, Infante-Duarte C, Hahner A, Aktas O, Ullrich O, Zipp F (2005) Atorvastatin induces T cell anergy via phosphorylation of ERK1. J Immunol 174:5630–5635 Weber MS, Youssef S, Dunn SE, Prod’Homme T, Neuhaus O, Stuve O, Greenwood J, Steinman L, Zamvil SS (2006) Statins in the treatment of central nervous system autoimmune disease. J Neuroimmunol 178:140–148 Wen SR, Liu GJ, Feng RN, Gong FC, Zhong H, Duan SR, Bi S (2012) Increased levels of IL-23 and osteopontin in serum and cerebrospinal fluid of multiple sclerosis patients. J Neuroimmunol 244:94–96 Willenborg DO, Fordham S, Bernard CC, Cowden WB, Ramshaw IA (1996) IFN-gamma plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis. J Immunol 157:3223–3227 Yilmaz A, Reiss C, Tantawi O, Weng A, Stumpf C, Raaz D, Ludwig J, Berger T, Steinkasserer A, Daniel WG, Garlichs CD (2004) HMG-CoA reductase inhibitors suppress maturation of human dendritic cells: new implications for atherosclerosis. Atherosclerosis 172:85–93 Youssef S, Stuve O, Patarroyo JC, Ruiz PJ, Radosevich JL, Hur EM, Bravo M, Mitchell DJ, Sobel RA, Steinman L, Zamvil SS (2002) The HMG-CoA reductase inhibitor, atorvastatin, promotes a Th2 bias and reverses paralysis in central nervous system autoimmune disease. Nature 420:78–84 Yu X, Pan Y, Ma H, Li W (2013) Simvastatin inhibits proliferation and induces apoptosis in human lung cancer cells. Oncol Res 20:351–357 Zhang X, Jin J, Peng X, Ramgolam VS, Markovic-Plese S (2008) Simvastatin inhibits IL-17 secretion by targeting multiple IL-17regulatory cytokines and by inhibiting the expression of IL-17 transcription factor RORC in CD4? lymphocytes. J Immunol 180:6988–6996 Zhang S, Rahman M, Qi Z, Herwald H, Thorlacius H (2011) Simvastatin regulates CXC chemokine formation in streptococcal M1 protein-induced neutrophil infiltration in the lung. Am J Physiol Lung Cell Mol Physiol 300:L930–L939
