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Th17 cytokines and up-regulated T regulatory cells
Cellular Immunology 271 (2011) 455–461
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Atorvastatin ameliorates experimental autoimmune neuritis by decreased Th1/Th17 cytokines and up-regulated T regulatory cells Xiao-Li Li a, Ying-Chun Dou b, Ying Liu a, Chang-Wen Shi c, Li-Li Cao c, Xiu-Qing Zhang a, Jie Zhu d, Rui-Sheng Duan a,⇑ a
Department of Neurology, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014, PR China College of Basic Medical Sciences, Shandong University of Traditional Chinese Medicine, Jinan 250014, PR China Central Laboratory, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014, PR China d Department of Neurobiology, Care Sciences and Society, Karolinska Institute, Stockholm SE-141 86, Sweden b c
a r t i c l e
i n f o
Article history: Received 11 April 2011 Accepted 12 August 2011 Available online 23 August 2011 Keywords: Atorvastatin Experimental autoimmune neuritis IL-17 CD80 Treg cells
a b s t r a c t Statins have anti-inflammatory and immune-regulating properties. To investigate the effects of atorvastatin on experimental autoimmune neuritis (EAN), an animal model of Guillain–Barré syndrome (GBS), atorvastatin was administered to Lewis rats immunized with bovine peripheral myelin in complete Freund’s adjuvant. We found that atorvastatin ameliorated the clinical symptoms of EAN, decreased the numbers of inflammatory cells as well as IFN-c+ and IL-17+ cells in sciatic nerves, decreased the CD80 expression and increased the number of CD25+Foxp3+ cells in mononuclear cells (MNC), and decreased the levels of IFN-c in MNC culture supernatants. These data provide strong evidence that atorvastatin can act as an inhibitor in EAN by inhibiting the immune response of Th1 and Th17, decreasing the expression of co-stimulatory molecule, and up-regulating the number of T regulatory cells. These data demonstrated that statins could be used as a therapeutic strategy in human GBS in future. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Experimental autoimmune neuritis (EAN) is a T cell-mediated acute inflammatory disease of the peripheral nervous system (PNS) that serves as a model for the human Guillain–Barré syndrome (GBS). EAN can be induced in susceptible animal species and strains by immunization with autoantigen emulsified in complete Freund’s adjuvant [1]. Accumulation of reactive inflammatory cells, e.g. T cells and macrophages in the PNS is a pathological mark of EAN and human GBS [2,3]. Statins, including atorvastatin, are inhibitors of 3-hydroxy-3methylglutaryl coenzyme A reductase in the mevalonate pathway for cholesterol biosynthesis, and they are widely used in clinical practice for the treatment of hypercholesterolemia [4,5]. In the past few years, accumulated evidence from animal experiments and clinical studies has shown that statins have anti-inflammation and immunomodulatory effects. The effects of statins on the immune system are pleiotropic and include inhibition of T cell activation, proliferation, and migration [6–8]. Reportedly atorvastatin could promote shifting the T cell response from a pro-inflammatory Th1 to an anti-inflammatory Th2 profile in experimental autoimmune encephalomyelitis [EAE, an animal model of multiple
⇑ Corresponding author. Fax: +86 531 82963647. E-mail address: [email protected] (R.-S. Duan). 0008-8749/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2011.08.015
sclerosis mediated by Th1 cell in the central nervous system (CNS)] [9,10]. Furthermore, simvastatin could directly inhibit IL-17 secretion in CD4+ T cells derived from relapsing remitting multiple sclerosis patients [11], which plays a critical role in the development of autoimmune diseases. These evidences suggest that statins can inhibit Th1 and Th17 inflammatory response in autoimmune diseases. In addition, statins can inhibit the maturation and function of antigen presenting cells (APCs), such as dendritic cells and B cells. Administration of atorvastatin could inhibit the expression of costimulatory molecules, such as CD40, CD83, CD86, and human leukocyte antigen-DR (HLA-DR) on dendritic cells in human [12]. Statins also could inhibit major histocompatibility complex (MHC) expression on IFN-c-stimulated human macrophages and endothelial cells, and reduce the ability of these cells to stimulate T cells [13]. Naturally occurring CD4+CD25+ T regulatory cells (Treg cells) play important roles in the prevention of various inflammatory and autoimmune diseases by suppressing immune responses [14,15]. Reportedly atorvastatin could increase the number of CD4+CD25high cells and CD4+CD25+Foxp3+ cells in human peripheral blood mononuclear cells [16]. So far, there is no report about the immunomodulatory properties of atorvastatin in GBS/EAN. Based on previous studies, we hypothesize that atorvastatin could attenuate EAN, in which Th1/ Th17 play essential roles in the pathogenesis. In the present study,
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we administered atorvastatin in the initial phase of EAN to evaluate its possible immunomodulatory effects during the process. We found that atorvastatin administration could ameliorate EAN by immune regulation.
2. Materials and methods 2.1. Reagents Bovine peripheral nerve myelin (BPM) was prepared from fresh cauda equina of bovine according to the described method [17]. Atorvastatin was provided by Beijing Garlin Pharmaceutical Co. Ltd. (Beijing, China) and dissolved in dimethyl sulfoxide (DMSO) (Sigma–Aldrich, St. Louis, MO, USA).
2.2. Induction of EAN and assessment of clinical symptoms Female Lewis rats, 6–8 weeks of age (body weight 140–170 g), were purchased from Chinese Academy of Medical Sciences (Beijing, China). All rats were housed at the local animal house, fed standard rat chow and water ad libitum and maintained on a 10 h/14 h light/dark cycle. All the experimental protocols were approved by the institutional ethics committee. EAN was induced by subcutaneous injection into both hind footpads of the rats with 200 ll inoculum containing 5 mg BPM and 2 mg Mycobacterium tuberculosis (strain H37RA; Difco, Detroit, MI, USA) emulsified in incomplete Freund’s adjuvant (Sigma–Aldrich). The rats were monitored for clinical symptoms of disease by two independent investigators. Clinical scores were assessed immediately before immunization (day 0) and thereafter every day until day 18 post immunization (p.i.). The severity of clinical symptoms was scored as follows: 0 = no illness; 1 = flaccid tail; 2 = dragging both hind limbs; 3 = paralysis of both hind limbs; and 4 = paralysis of four limbs or death, intermediate scores of 0.5 increment were given to rats with intermediate signs.
2.3. Administration of atorvastatin Atorvastatin administration has been investigated in various autoimmune diseases of animal models, such as experimental autoimmune uveitis (EAU) in mice [18], experimental autoimmune myocarditis in Lewis rats [19,20]. Based on previous studies, it can be concluded that higher bioavailability can be obtained by atorvastatin at doses of 1 and 10 mg/kg/day in EAN rats. To determine whether atorvastatin protected against the development of EAN, atorvastatin (at doses of 1 and 10 mg/kg/day in a volume of 100 ll DMSO) was administered to immunized rats by intraperitoneal injection from day 5 to 18 p.i. Control EAN rats received the same volume injections of vehicle (DMSO). The dose and route were chosen in accordance with previous studies on the effects of atorvastatin on related autoimmune diseases.
2.5. Immunohistochemistry Paraffin tissue sections (5 lm) were deparaffinized and hydrated. The sections were treated with 0.3% hydrogen peroxide for 20 min to block endogenous peroxidase activity. After three washes in phosphate-buffered saline (PBS), the sections were exposed to ethylene diamine tetraacetic acid (EDTA) for antigen retrieval and incubated overnight at 4 °C with rabbit anti-rat IFN-c antibody (1:100; Boster, Wuhan, China) and rabbit anti-rat IL-17 antibody (1:75; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Then sections were stained with HRP-conjugated goat anti-rabbit secondary antibody (Zhongshan Goldenbridge Biotechnology, Beijing, China), followed by development with diaminobenzidine (DAB) substrate (Zhongshan Goldenbridge Biotechnology) to detect the numbers of IFN-c+ cells and IL-17+ cells. As negative controls for immunostaining, the primary antibodies were omitted. The tissue areas were measured by image analysis in five sections per sciatic nerve and the results were expressed as the number of positive cells per mm2 tissue section. 2.6. Preparation of lymph node mononuclear cells Inguinal lymph nodes were removed under aseptic conditions. Mononuclear cells (MNC) suspensions from inguinal lymph nodes of individual rats were obtained by grinding through cell strainers (Becton Dickenson, Franklin Lakes, NJ, USA) in medium. The cells were then washed three times before being suspended to 2 106/ml in RPMI 1640 (containing 2.05 mM glutamine, HyClone, Beijing, China) supplemented with 1% (v/v) minimum essential medium (MEM; Sigma–Aldrich) and 50 lg/ml gentamicin (Shandong Lukang Cisen, Jining, China) and 10% (v/v) fetal bovine serum (FBS; Gibco, Grand Land, NY, USA). 2.7. Flow cytometric analysis of surface molecules in MNC from lymph nodes Cell surface molecules were examined by a FACScan (Beckman Coulter, Los Angeles, CA, USA). Briefly, MNC suspensions in PBS containing 0.5% bovine serum albumin (BSA; Sigma–Aldrich) were incubated with phycoerythrin (PE)-labeled anti-rat CD80, fluorescein isothiocyanate (FITC)-labeled anti-rat CD86 (BioLegend, San Diego, CA, USA) and FITC-labeled anti-rat MHC class II (eBioscience, San Diego, CA, USA) for 30 min at 4 °C in the dark, respectively. After staining, the cells were washed and re-suspended in PBS. Then the cells were analyzed by a FACScan. 2.8. Flow cytometric analysis of Treg cells in MNC from lymph nodes Fixation and permeabilization of MNC from lymph node cells were performed using the eBioscience Foxp3 Staining Buffer Set (eBioscience). FITC-labeled anti-rat CD4, PE-labeled anti-rat CD25, and PE-Cy5-labeled anti-mouse/rat Foxp3 antibodies (all from eBioscience) were used for staining according to the protocol recommended by eBioscience. After staining, the cells were re-suspended in PBS and analyzed by a FACScan.
2.4. Histopathological assessment 2.9. Determination of cytokines in culture supernatants by ELISA Five rats of each group were killed on day 18 p.i. just after the clinical symptoms of EAN peaked. Sciatic nerve segments were excised close to the lumbar spinal cord, fixed in 10% paraformaldehyde and embedded in paraffin. Multiple longitudinal sections (5 lm) of sciatic nerves were stained with hematoxylin and eosin for evaluation of the inflammatory cells by light microscopy. The number of inflammatory cells was counted at 200 magnification. The average results were expressed as cells per mm2 tissue section.
MNC (4 105 cells/well) were cultured in the presence of BPM (10 lg/ml). After 60 h incubation with 5% CO2 at 37 °C, the supernatants were collected and stored at 20 °C for cytokine determination. IFN-c (Bender, Vienna, Austria) and IL-17 (Uscn Life Science Inc. Wuhan, China) were measured by sandwich ELISA kits according to the manufacturer’s instructions. Determinations were performed in duplicate and the results were expressed as qg/ml.
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2.10. Statistical analysis The SPSS 17.0 computer programme (SPSS Inc., Chicago, IL, USA) was used for all calculations and statistical evaluations. Results were expressed as means ± standard deviation (SD). Differences among three groups were tested by one-factor analysis of variance (ANOVA) followed by Least Significant Difference (LSD) test as a post hoc test. The levels of significance was set to p = 0.05. All tests were two-sided. 3. Results 3.1. Atorvastatin suppresses the severity of clinical EAN Female Lewis rats were immunized with BPM and treated with atorvastatin from day 5 to 18 p.i. All the immunized rats exhibited the clinical syndromes of EAN. Atorvastatin, at doses of 10 and 1 mg/kg/day, not only delayed the onset of EAN, but also significantly decreased the neurological severity of EAN, compared to DMSO treatment. The mean day of EAN onset was day 13.4 ± 1.5 in the 10 mg/kg-treated group, 12.2 ± 0.4 in the 1 mg/kg-treated group, and 11.6 ± 0.5 in the DMSO-treated group. Moreover, the 10 mg/kg-treated group and DMSO-treated group differed significantly (p < 0.05). The rats in 10 mg/kg-treated group exhibited significantly lower clinical scores from day 13 to 17 p.i. compared with the rats in DMSO group (p < 0.05 on each time point). Meanwhile, the rats in 1 mg/kg-treated group had lower clinical scores from day 13 to 17 p.i. compared with the rats in DMSO group, but difference was significant only on day 16 p.i. (p < 0.05) (Fig. 1). 3.2. Atorvastatin decreases the numbers of inflammatory cells in the PNS To confirm whether the benefits of atorvastatin against clinical symptoms of EAN were due to the decreased inflammation in the PNS, the sciatic nerves of EAN rats were examined histologically just after the peak of the clinical course of EAN. The rats in the group treated with 10 mg/kg atorvastatin and in the group treated with 1 mg/kg atorvastatin showed fewer inflammatory cells in sciatic nerves than those in the DMSO control group (p < 0.001 and
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0.05, respectively). In addition, the rats treated with high dose of atorvastatin had fewer inflammatory cells than the rats treated with low dose of atorvastatin (p < 0.05; Fig. 2A and B). These data indicated that the mild EAN clinical scores in the groups of atorvastatin administration were associated with the decreased numbers of inflammatory cells in the PNS. 3.3. Atorvastatin inhibits the production of IFN-c and IL-17 in sciatic nerves of EAN rats Sciatic nerves were taken from atorvastatin-treated and control EAN rats on day 18 p.i. for immunohistochemistry analysis. Atorvastatin at daily doses of 10 and 1 mg/kg decreased the numbers of IFN-c+ and IL-17+ cells in sciatic nerves compared to the control DMSO administration (for IFN-c: p < 0.001 and 0.01, respectively, for IL-17: p < 0.001 for both comparisons). There was no difference for the numbers of IFN-c+ cells in sciatic nerves between the two groups treated with 10 and 1 mg/kg atorvastatin. Whereas, the rats treated with the high dose of atorvastatin had fewer IL-17+ cells in sciatic nerves than the rats treated with the low dose of atorvastatin (p < 0.01). Further analysis showed that the percentages of IFN-c+ and IL-17+ cells among infiltrating inflammatory cells in rats with 10 and 1 mg/kg atorvastatin were lower than those in control rats (for IFN-c: p < 0.01 and 0.001, respectively, for IL-17: p < 0.01 and 0.05, respectively), while the two treated groups did not differ significantly (Figs. 3 and 4). 3.4. Effects of atorvastatin on the expression of co-stimulatory molecules in lymphocytes The CD80, CD86, and MHC class II in lymphocytes were analyzed by FACS. Atorvastatin treatment at doses of 10 and 1 mg/kg clearly inhibited the expression of CD80 in lymphocytes when compared to control group (p < 0.05 for both comparisons). However, the two treated groups did not differ significantly (Fig. 5). 3.5. Atorvastatin up-regulates the number of Treg cells We also examined whether atorvastatin altered the number of Treg cells in lymphocytes by FACS. The results showed that both 10 and 1 mg/kg atorvastatin-treated groups increased the percentages of CD25+Foxp3+ cells among CD4+ cells from lymph node MNC when compared with the control group (p < 0.05 and 0.01). Meanwhile the two treated groups did not differ significantly (Fig. 6A and B). These data indicated that the immunomodulatory effects of atorvastatin on EAN were associated with up-regulated the number of Treg cells. 3.6. Effects of atorvastatin on the levels of IFN-c and IL-17 in the culture supernatants MNC were obtained from inguinal lymph nodes of individual rats and cultured in the presence of BPM. The production of IFNc in the culture supernatants of the 10 and 1 mg/kg-treated group were significantly reduced when compared with the control group (p < 0.05 and 0.001, respectively). There were no significant differences for the levels of IL-17 among the three groups (Fig. 7).
Fig. 1. Atorvastatin ameliorated the clinical scores of EAN. Lewis rats were immunized with BPM in complete Freund’s adjuvant and monitored for initiation and development of EAN. Rats were treated with atorvastatin at daily doses of 10 or 1 mg/kg in a volume of 100 ll from day 5 to 18 p.i. (n = 5 in each group). Control rats were treated with the same volume of DMSO (n = 5). The rats treated with 10 mg/kg atorvastatin showed significantly lower clinical scores from day 13 to 17 p.i. compared with the rats in the DMSO group (⁄p < 0.05 on each time point). The difference was significant only on day 16 p.i. between the group treated with 1 mg/ kg atorvastatin and the DMSO group (#p < 0.05). The results are presented as mean ± SD.
4. Discussion To examine whether atorvastatin has anti-inflammation and immune modulation effects, it was administered to EAN rats from day 5 to 18 p.i. Although the clinical symptoms of the EAN rats were not exhibited on day 5 p.i., the immune response to the myelin in peripheral nerves had been activated in vivo. We found that
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Fig. 2. Histopathological analysis of the sciatic nerves of EAN rats. (A) The number of inflammatory cells in sciatic nerve sections was reduced in EAN rats treated with atorvastatin at daily doses of 10 and 1 mg/kg compared to those in control EAN rats. The arrow referred to typical inflammatory cells. Original magnifications: 200. (B) The number of inflammatory cells per mm2 tissue section was determined. The results are presented as mean ± SD (n = 5 in each group) (⁄p < 0.05 and ⁄⁄⁄p < 0.001).
Fig. 3. Atorvastatin suppressed the production of IFN-c in sciatic nerves of EAN rats. The number of IFN-c+ cells in sciatic nerves of EAN rats was analyzed by immunohistochemistry. Atorvastatin or DMSO was intraperitoneally injected from day 5 to 18 p.i. Rats were sacrificed on day 18 p.i. (A) Representative micrographs show the fewer IFN-c+ cells in sciatic nerves in rats treated with atorvastatin at daily doses of 10 and 1 mg/kg compared to those in control rats, respectively. The arrow referred to typical IFN-c+ cells, which appear to be infiltrating inflammatory cells morphologically and the cytoplasm was filled with brown. Original magnification: 400. (B) The numbers of IFN-c+ cells per mm2 sciatic nerve sections (a) and the percentages of IFN-c+ cells among inflammatory cells (b) are expressed as mean ± SD (n = 5 in each group) (⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001).
atorvastatin markedly ameliorated the clinical symptoms of EAN, decreased the numbers of inflammatory cells as well as IFN-c+ and IL-17+ cells in sciatic nerves, decreased the levels of IFN-c in MNC culture supernatants, decreased the CD80 expression and increased Treg cells in MNC from the lymphoid nodes. These data provide strong evidence that atorvastatin can act as an inhibitor in EAN by inhibiting the immune response of Th1 and Th17, decreasing the expression of co-stimulatory molecule, as well as up-regulating the number of Treg cells. Statins could reduce inflammation in the CNS. Administration of atorvastatin significantly reduced the severity and incidence of EAE, along with a large reduction in the number of inflammatory lesions in brains and spinal cords [10]. In our study, we found that atorvastatin significantly reduced inflammatory cells infiltration in
sciatic nerves of EAN rats. The result indicates that atorvastatin may play a role in anti-inflammation not only in the CNS but also in the PNS. Moreover, atorvastatin can dose-dependently exhibit the protective effects on EAN rats. Th17, which is considered as a subset of CD4+ T cells, has recently been found to play a critical role in the development of autoimmune diseases [21,22]. IL-17, also named IL-17A, is mainly expressed by Th17 and involved in inducing and mediating proinflammatory responses. Reportedly nasal administration of recombinant mouse IL-17 to Lewis rats with ongoing chronic EAN enhanced clinical symptoms and immune cell activity during the initial phase of the disease, which indicated that IL-17 played an important role in EAN [23]. Recently, it was found that accumulation of IL-17+ cells in sciatic nerves was correlated with the pathological progress in
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Fig. 4. Atorvastatin suppressed the production of IL-17 in sciatic nerves of EAN rats. (A) IL-17 immune reactivity in sciatic nerves was shown in 10, 1 mg/kg atorvastatintreated EAN rats, and control rats. The arrow referred to typical IL-17+ cells, which appear to be infiltrating inflammatory cells morphologically and the cytoplasm was filled with brown. Original magnification: 400. (B) The numbers of IL-17+ cells per mm2 sciatic nerve sections (a) and the percentages of IL-17+ cells among inflammatory cells (b) are expressed as mean ± SD (n = 5 in each group) (⁄p < 0.05, ⁄⁄p < 0.01, and ⁄⁄⁄p < 0.001).
Fig. 5. Expression of CD80, CD86 and MHC class II on the surface of lymph node MNC. MNC were prepared on day 18 p.i. from lymph nodes of rats treated with atorvastatin (at daily doses of 10 and 1 mg/kg) or DMSO and stained with PElabeled anti-rat CD80, FITC-labeled anti-rat CD86, and anti-rat MHC class II. The percentage of CD80, CD86, and MHC class II positive cells among all lymph node MNC are expressed as mean ± SD (⁄p < 0.05).
EAN rats and it was suggested that the Sphingosine-1-phosphate (S1P) signal pathway is involved in regulating IL-17+ cell trafficking [24]. Furthermore, AUY954, an S1P receptor modulator, effectively suppressed local inflammation in EAN, which might partly be due to a reduced peripheral proportion of IL-17 expressing cells and consequently attenuated infiltration of IL-17+ cells into inflammatory areas [25]. However, there has been no report about the roles of statins on Th17 in EAN. In autoimmune and inflammatory diseases of animals and human, including rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus, an increased level of IL17 expression has been observed [26–28]. Reportedly simvastatin could directly suppress IL-17 gene expression, as well as IL-17 secretion from the CD4+ T cells in relapsing remitting multiple sclerosis patients [11]. Our data showed that atorvastatin dose-dependently inhibited not only the numbers of IL-17+ cells but also the percentages of IL-17+ cells among inflammatory cells in sciatic nerves of EAN rats compared to the control DMSO administration, although
it did not significantly decrease IL-17 level in culture MNC supernatants. IFN-c is a pro-inflammatory cytokine, which can be produced by variety of cells, including Th1 cells, activated macrophages and so on, and it plays an important pathogenic role in EAN. In the present study, we found that atorvastatin could decrease not only the numbers of IFN-c+ cells and the percentages of IFN-c+ cells among inflammatory cells in sciatic nerves, but also the production of IFN-c in culture MNC supernatants in EAN rats. In accordance with our findings, it has recently been reported that simvastatin could inhibit the production of IFN-c in collagen induced arthritis (CIA, the experimental model for rheumatoid arthritis) [29]. Moreover, lovastatin could ameliorate EAU (a Th1-mediated autoimmune disease model), which was associated with inhibiting the production of pro-inflammatory cytokines, such as IFN-c, but did not require the induction of Th2 cytokine profile [30]. Our data showed that atorvastatin decreased the percentages of both IFN-c+ and IL-17+ cells among the inflammatory cells in sciatic nerves, which indirectly indicated that atorvastatin can ameliorate EAN by inhibiting Th1 and Th17 cell differentiation rather than by inhibiting the migration of inflammatory cells. Clearly in our study, atorvastatin can decrease IFN-c and IL-17 production in EAN. As known, MHC class II and co-stimulatory molecules such as CD80 and CD86 are expressed on APC. They are thought to be engaged in delivering signals to T cells and then determine the fate of naïve T cell. Up to now, three signals are thought to be involved in this process [31]. MHC class II is thought to be engaged in signal 1 which can be delivered to the T cell receptor. Signal 1 alone is considered as a factor to promote naïve T cell inactivation by anergy, deletion, or generation of regulatory cell, and thereby leading to tolerance. Co-stimulatory molecules are taken as an accessory factor of signal 1 in inducing immunity and considered as signal 2. Besides, signal 3 is referred to deliver signals from APCs to T cells that determine their differentiation into effector cells (e.g., Th1 cells, Th2 cells, or cytotoxic T lymphocytes (CTLs)) [32]. It has been reported that IL-12 is a mediator that delivers a signal 3 and promotes Th1 cells or CTLs development [33]. Statins could affect autoreactive leukocyte proliferation by altering the expression of MHC class II or co-stimulatory molecules on APC. Atorvastatin could suppress IFN-c inducible expression of CD40, CD80, and
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Fig. 6. Effects of atorvastatin on Treg cells in EAN rats. A representative FACS analysis of the expression of CD25+Foxp3+ cells among CD4+ cells from lymph node MNC in different groups are shown in (A). The percentages of CD25+Foxp3+ cells among CD4+ cells from lymph node MNC in rats treated with atorvastatin (at daily doses of 10 and 1 mg/kg) or DMSO are shown in (B). The results are expressed as mean ± SD (n = 5 in each group) (⁄p < 0.05 and ⁄⁄p < 0.01).
atorvastatin-treated groups increased the number of CD25+Foxp3+ cells among CD4+ cells from lymph node MNC. These indicate that atorvastatin may inhibit EAN by increasing the Treg cells. In summary, we provide strong evidence that atorvastatin can act as an inhibitor in EAN by inhibiting the immune response of Th1 and Th17, decreasing the expression of co-stimulatory molecule, as well as up-regulating Treg cells. All our data suggest that it is a new challenge for statins, which may be used as a new medicine in human GBS. Acknowledgments
Fig. 7. Cytokine production. MNC from lymph nodes of three group rats were cultured with BPM. The supernatants were collected for measuring the levels of IFN-c and IL-17. The results are expressed as mean ± SD (n = 5 in each group) (⁄p < 0.05 and ⁄⁄⁄p < 0.001).
This study was supported by Grants from the Foundation for 1020 Project Talent, Health Department of Shandong Province, partly supported by the National Natural Science Foundation of China (30771989), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. We like to thank Beijing Garlin Pharmaceutical Co. Ltd., for assistance in providing atorvastatin. References
CD86 co-stimulatory molecules in EAE [10]. Moreover, lovastatin could significantly reduce the expression of both MHC class II and CD80 co-stimulatory molecule in splenocytes of EAN rats [34]. Consistent with these findings, our results demonstrate that atorvastatin plays an important role in EAN rats by decreasing the expression of CD80, which was unable to provide efficient signals to activate T cells. Treg cells exert their immune suppressive functions by inhibitory cytokines, cytolysis, and modulation of dendritic cells maturation or function [35]. Foxp3 is expressed only on Treg cells and is thought to be correlated with an increased regulatory potential of Treg cells [36]. Statins could induce high levels of Foxp3, which is considered to be the main transcriptional regulator of the development and function of Treg cells. We found that both 10 and 1 mg/kg
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Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Atorvastatin ameliorates experimental autoimmune neuritis by decreased Th1/Th17 cytokines and up-regulated T regulatory cells Xiao-Li Li a, Ying-Chun Dou b, Ying Liu a, Chang-Wen Shi c, Li-Li Cao c, Xiu-Qing Zhang a, Jie Zhu d, Rui-Sheng Duan a,⇑ a
Department of Neurology, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014, PR China College of Basic Medical Sciences, Shandong University of Traditional Chinese Medicine, Jinan 250014, PR China Central Laboratory, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014, PR China d Department of Neurobiology, Care Sciences and Society, Karolinska Institute, Stockholm SE-141 86, Sweden b c
a r t i c l e
i n f o
Article history: Received 11 April 2011 Accepted 12 August 2011 Available online 23 August 2011 Keywords: Atorvastatin Experimental autoimmune neuritis IL-17 CD80 Treg cells
a b s t r a c t Statins have anti-inflammatory and immune-regulating properties. To investigate the effects of atorvastatin on experimental autoimmune neuritis (EAN), an animal model of Guillain–Barré syndrome (GBS), atorvastatin was administered to Lewis rats immunized with bovine peripheral myelin in complete Freund’s adjuvant. We found that atorvastatin ameliorated the clinical symptoms of EAN, decreased the numbers of inflammatory cells as well as IFN-c+ and IL-17+ cells in sciatic nerves, decreased the CD80 expression and increased the number of CD25+Foxp3+ cells in mononuclear cells (MNC), and decreased the levels of IFN-c in MNC culture supernatants. These data provide strong evidence that atorvastatin can act as an inhibitor in EAN by inhibiting the immune response of Th1 and Th17, decreasing the expression of co-stimulatory molecule, and up-regulating the number of T regulatory cells. These data demonstrated that statins could be used as a therapeutic strategy in human GBS in future. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Experimental autoimmune neuritis (EAN) is a T cell-mediated acute inflammatory disease of the peripheral nervous system (PNS) that serves as a model for the human Guillain–Barré syndrome (GBS). EAN can be induced in susceptible animal species and strains by immunization with autoantigen emulsified in complete Freund’s adjuvant [1]. Accumulation of reactive inflammatory cells, e.g. T cells and macrophages in the PNS is a pathological mark of EAN and human GBS [2,3]. Statins, including atorvastatin, are inhibitors of 3-hydroxy-3methylglutaryl coenzyme A reductase in the mevalonate pathway for cholesterol biosynthesis, and they are widely used in clinical practice for the treatment of hypercholesterolemia [4,5]. In the past few years, accumulated evidence from animal experiments and clinical studies has shown that statins have anti-inflammation and immunomodulatory effects. The effects of statins on the immune system are pleiotropic and include inhibition of T cell activation, proliferation, and migration [6–8]. Reportedly atorvastatin could promote shifting the T cell response from a pro-inflammatory Th1 to an anti-inflammatory Th2 profile in experimental autoimmune encephalomyelitis [EAE, an animal model of multiple
⇑ Corresponding author. Fax: +86 531 82963647. E-mail address: [email protected] (R.-S. Duan). 0008-8749/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2011.08.015
sclerosis mediated by Th1 cell in the central nervous system (CNS)] [9,10]. Furthermore, simvastatin could directly inhibit IL-17 secretion in CD4+ T cells derived from relapsing remitting multiple sclerosis patients [11], which plays a critical role in the development of autoimmune diseases. These evidences suggest that statins can inhibit Th1 and Th17 inflammatory response in autoimmune diseases. In addition, statins can inhibit the maturation and function of antigen presenting cells (APCs), such as dendritic cells and B cells. Administration of atorvastatin could inhibit the expression of costimulatory molecules, such as CD40, CD83, CD86, and human leukocyte antigen-DR (HLA-DR) on dendritic cells in human [12]. Statins also could inhibit major histocompatibility complex (MHC) expression on IFN-c-stimulated human macrophages and endothelial cells, and reduce the ability of these cells to stimulate T cells [13]. Naturally occurring CD4+CD25+ T regulatory cells (Treg cells) play important roles in the prevention of various inflammatory and autoimmune diseases by suppressing immune responses [14,15]. Reportedly atorvastatin could increase the number of CD4+CD25high cells and CD4+CD25+Foxp3+ cells in human peripheral blood mononuclear cells [16]. So far, there is no report about the immunomodulatory properties of atorvastatin in GBS/EAN. Based on previous studies, we hypothesize that atorvastatin could attenuate EAN, in which Th1/ Th17 play essential roles in the pathogenesis. In the present study,
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we administered atorvastatin in the initial phase of EAN to evaluate its possible immunomodulatory effects during the process. We found that atorvastatin administration could ameliorate EAN by immune regulation.
2. Materials and methods 2.1. Reagents Bovine peripheral nerve myelin (BPM) was prepared from fresh cauda equina of bovine according to the described method [17]. Atorvastatin was provided by Beijing Garlin Pharmaceutical Co. Ltd. (Beijing, China) and dissolved in dimethyl sulfoxide (DMSO) (Sigma–Aldrich, St. Louis, MO, USA).
2.2. Induction of EAN and assessment of clinical symptoms Female Lewis rats, 6–8 weeks of age (body weight 140–170 g), were purchased from Chinese Academy of Medical Sciences (Beijing, China). All rats were housed at the local animal house, fed standard rat chow and water ad libitum and maintained on a 10 h/14 h light/dark cycle. All the experimental protocols were approved by the institutional ethics committee. EAN was induced by subcutaneous injection into both hind footpads of the rats with 200 ll inoculum containing 5 mg BPM and 2 mg Mycobacterium tuberculosis (strain H37RA; Difco, Detroit, MI, USA) emulsified in incomplete Freund’s adjuvant (Sigma–Aldrich). The rats were monitored for clinical symptoms of disease by two independent investigators. Clinical scores were assessed immediately before immunization (day 0) and thereafter every day until day 18 post immunization (p.i.). The severity of clinical symptoms was scored as follows: 0 = no illness; 1 = flaccid tail; 2 = dragging both hind limbs; 3 = paralysis of both hind limbs; and 4 = paralysis of four limbs or death, intermediate scores of 0.5 increment were given to rats with intermediate signs.
2.3. Administration of atorvastatin Atorvastatin administration has been investigated in various autoimmune diseases of animal models, such as experimental autoimmune uveitis (EAU) in mice [18], experimental autoimmune myocarditis in Lewis rats [19,20]. Based on previous studies, it can be concluded that higher bioavailability can be obtained by atorvastatin at doses of 1 and 10 mg/kg/day in EAN rats. To determine whether atorvastatin protected against the development of EAN, atorvastatin (at doses of 1 and 10 mg/kg/day in a volume of 100 ll DMSO) was administered to immunized rats by intraperitoneal injection from day 5 to 18 p.i. Control EAN rats received the same volume injections of vehicle (DMSO). The dose and route were chosen in accordance with previous studies on the effects of atorvastatin on related autoimmune diseases.
2.5. Immunohistochemistry Paraffin tissue sections (5 lm) were deparaffinized and hydrated. The sections were treated with 0.3% hydrogen peroxide for 20 min to block endogenous peroxidase activity. After three washes in phosphate-buffered saline (PBS), the sections were exposed to ethylene diamine tetraacetic acid (EDTA) for antigen retrieval and incubated overnight at 4 °C with rabbit anti-rat IFN-c antibody (1:100; Boster, Wuhan, China) and rabbit anti-rat IL-17 antibody (1:75; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Then sections were stained with HRP-conjugated goat anti-rabbit secondary antibody (Zhongshan Goldenbridge Biotechnology, Beijing, China), followed by development with diaminobenzidine (DAB) substrate (Zhongshan Goldenbridge Biotechnology) to detect the numbers of IFN-c+ cells and IL-17+ cells. As negative controls for immunostaining, the primary antibodies were omitted. The tissue areas were measured by image analysis in five sections per sciatic nerve and the results were expressed as the number of positive cells per mm2 tissue section. 2.6. Preparation of lymph node mononuclear cells Inguinal lymph nodes were removed under aseptic conditions. Mononuclear cells (MNC) suspensions from inguinal lymph nodes of individual rats were obtained by grinding through cell strainers (Becton Dickenson, Franklin Lakes, NJ, USA) in medium. The cells were then washed three times before being suspended to 2 106/ml in RPMI 1640 (containing 2.05 mM glutamine, HyClone, Beijing, China) supplemented with 1% (v/v) minimum essential medium (MEM; Sigma–Aldrich) and 50 lg/ml gentamicin (Shandong Lukang Cisen, Jining, China) and 10% (v/v) fetal bovine serum (FBS; Gibco, Grand Land, NY, USA). 2.7. Flow cytometric analysis of surface molecules in MNC from lymph nodes Cell surface molecules were examined by a FACScan (Beckman Coulter, Los Angeles, CA, USA). Briefly, MNC suspensions in PBS containing 0.5% bovine serum albumin (BSA; Sigma–Aldrich) were incubated with phycoerythrin (PE)-labeled anti-rat CD80, fluorescein isothiocyanate (FITC)-labeled anti-rat CD86 (BioLegend, San Diego, CA, USA) and FITC-labeled anti-rat MHC class II (eBioscience, San Diego, CA, USA) for 30 min at 4 °C in the dark, respectively. After staining, the cells were washed and re-suspended in PBS. Then the cells were analyzed by a FACScan. 2.8. Flow cytometric analysis of Treg cells in MNC from lymph nodes Fixation and permeabilization of MNC from lymph node cells were performed using the eBioscience Foxp3 Staining Buffer Set (eBioscience). FITC-labeled anti-rat CD4, PE-labeled anti-rat CD25, and PE-Cy5-labeled anti-mouse/rat Foxp3 antibodies (all from eBioscience) were used for staining according to the protocol recommended by eBioscience. After staining, the cells were re-suspended in PBS and analyzed by a FACScan.
2.4. Histopathological assessment 2.9. Determination of cytokines in culture supernatants by ELISA Five rats of each group were killed on day 18 p.i. just after the clinical symptoms of EAN peaked. Sciatic nerve segments were excised close to the lumbar spinal cord, fixed in 10% paraformaldehyde and embedded in paraffin. Multiple longitudinal sections (5 lm) of sciatic nerves were stained with hematoxylin and eosin for evaluation of the inflammatory cells by light microscopy. The number of inflammatory cells was counted at 200 magnification. The average results were expressed as cells per mm2 tissue section.
MNC (4 105 cells/well) were cultured in the presence of BPM (10 lg/ml). After 60 h incubation with 5% CO2 at 37 °C, the supernatants were collected and stored at 20 °C for cytokine determination. IFN-c (Bender, Vienna, Austria) and IL-17 (Uscn Life Science Inc. Wuhan, China) were measured by sandwich ELISA kits according to the manufacturer’s instructions. Determinations were performed in duplicate and the results were expressed as qg/ml.
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2.10. Statistical analysis The SPSS 17.0 computer programme (SPSS Inc., Chicago, IL, USA) was used for all calculations and statistical evaluations. Results were expressed as means ± standard deviation (SD). Differences among three groups were tested by one-factor analysis of variance (ANOVA) followed by Least Significant Difference (LSD) test as a post hoc test. The levels of significance was set to p = 0.05. All tests were two-sided. 3. Results 3.1. Atorvastatin suppresses the severity of clinical EAN Female Lewis rats were immunized with BPM and treated with atorvastatin from day 5 to 18 p.i. All the immunized rats exhibited the clinical syndromes of EAN. Atorvastatin, at doses of 10 and 1 mg/kg/day, not only delayed the onset of EAN, but also significantly decreased the neurological severity of EAN, compared to DMSO treatment. The mean day of EAN onset was day 13.4 ± 1.5 in the 10 mg/kg-treated group, 12.2 ± 0.4 in the 1 mg/kg-treated group, and 11.6 ± 0.5 in the DMSO-treated group. Moreover, the 10 mg/kg-treated group and DMSO-treated group differed significantly (p < 0.05). The rats in 10 mg/kg-treated group exhibited significantly lower clinical scores from day 13 to 17 p.i. compared with the rats in DMSO group (p < 0.05 on each time point). Meanwhile, the rats in 1 mg/kg-treated group had lower clinical scores from day 13 to 17 p.i. compared with the rats in DMSO group, but difference was significant only on day 16 p.i. (p < 0.05) (Fig. 1). 3.2. Atorvastatin decreases the numbers of inflammatory cells in the PNS To confirm whether the benefits of atorvastatin against clinical symptoms of EAN were due to the decreased inflammation in the PNS, the sciatic nerves of EAN rats were examined histologically just after the peak of the clinical course of EAN. The rats in the group treated with 10 mg/kg atorvastatin and in the group treated with 1 mg/kg atorvastatin showed fewer inflammatory cells in sciatic nerves than those in the DMSO control group (p < 0.001 and
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0.05, respectively). In addition, the rats treated with high dose of atorvastatin had fewer inflammatory cells than the rats treated with low dose of atorvastatin (p < 0.05; Fig. 2A and B). These data indicated that the mild EAN clinical scores in the groups of atorvastatin administration were associated with the decreased numbers of inflammatory cells in the PNS. 3.3. Atorvastatin inhibits the production of IFN-c and IL-17 in sciatic nerves of EAN rats Sciatic nerves were taken from atorvastatin-treated and control EAN rats on day 18 p.i. for immunohistochemistry analysis. Atorvastatin at daily doses of 10 and 1 mg/kg decreased the numbers of IFN-c+ and IL-17+ cells in sciatic nerves compared to the control DMSO administration (for IFN-c: p < 0.001 and 0.01, respectively, for IL-17: p < 0.001 for both comparisons). There was no difference for the numbers of IFN-c+ cells in sciatic nerves between the two groups treated with 10 and 1 mg/kg atorvastatin. Whereas, the rats treated with the high dose of atorvastatin had fewer IL-17+ cells in sciatic nerves than the rats treated with the low dose of atorvastatin (p < 0.01). Further analysis showed that the percentages of IFN-c+ and IL-17+ cells among infiltrating inflammatory cells in rats with 10 and 1 mg/kg atorvastatin were lower than those in control rats (for IFN-c: p < 0.01 and 0.001, respectively, for IL-17: p < 0.01 and 0.05, respectively), while the two treated groups did not differ significantly (Figs. 3 and 4). 3.4. Effects of atorvastatin on the expression of co-stimulatory molecules in lymphocytes The CD80, CD86, and MHC class II in lymphocytes were analyzed by FACS. Atorvastatin treatment at doses of 10 and 1 mg/kg clearly inhibited the expression of CD80 in lymphocytes when compared to control group (p < 0.05 for both comparisons). However, the two treated groups did not differ significantly (Fig. 5). 3.5. Atorvastatin up-regulates the number of Treg cells We also examined whether atorvastatin altered the number of Treg cells in lymphocytes by FACS. The results showed that both 10 and 1 mg/kg atorvastatin-treated groups increased the percentages of CD25+Foxp3+ cells among CD4+ cells from lymph node MNC when compared with the control group (p < 0.05 and 0.01). Meanwhile the two treated groups did not differ significantly (Fig. 6A and B). These data indicated that the immunomodulatory effects of atorvastatin on EAN were associated with up-regulated the number of Treg cells. 3.6. Effects of atorvastatin on the levels of IFN-c and IL-17 in the culture supernatants MNC were obtained from inguinal lymph nodes of individual rats and cultured in the presence of BPM. The production of IFNc in the culture supernatants of the 10 and 1 mg/kg-treated group were significantly reduced when compared with the control group (p < 0.05 and 0.001, respectively). There were no significant differences for the levels of IL-17 among the three groups (Fig. 7).
Fig. 1. Atorvastatin ameliorated the clinical scores of EAN. Lewis rats were immunized with BPM in complete Freund’s adjuvant and monitored for initiation and development of EAN. Rats were treated with atorvastatin at daily doses of 10 or 1 mg/kg in a volume of 100 ll from day 5 to 18 p.i. (n = 5 in each group). Control rats were treated with the same volume of DMSO (n = 5). The rats treated with 10 mg/kg atorvastatin showed significantly lower clinical scores from day 13 to 17 p.i. compared with the rats in the DMSO group (⁄p < 0.05 on each time point). The difference was significant only on day 16 p.i. between the group treated with 1 mg/ kg atorvastatin and the DMSO group (#p < 0.05). The results are presented as mean ± SD.
4. Discussion To examine whether atorvastatin has anti-inflammation and immune modulation effects, it was administered to EAN rats from day 5 to 18 p.i. Although the clinical symptoms of the EAN rats were not exhibited on day 5 p.i., the immune response to the myelin in peripheral nerves had been activated in vivo. We found that
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Fig. 2. Histopathological analysis of the sciatic nerves of EAN rats. (A) The number of inflammatory cells in sciatic nerve sections was reduced in EAN rats treated with atorvastatin at daily doses of 10 and 1 mg/kg compared to those in control EAN rats. The arrow referred to typical inflammatory cells. Original magnifications: 200. (B) The number of inflammatory cells per mm2 tissue section was determined. The results are presented as mean ± SD (n = 5 in each group) (⁄p < 0.05 and ⁄⁄⁄p < 0.001).
Fig. 3. Atorvastatin suppressed the production of IFN-c in sciatic nerves of EAN rats. The number of IFN-c+ cells in sciatic nerves of EAN rats was analyzed by immunohistochemistry. Atorvastatin or DMSO was intraperitoneally injected from day 5 to 18 p.i. Rats were sacrificed on day 18 p.i. (A) Representative micrographs show the fewer IFN-c+ cells in sciatic nerves in rats treated with atorvastatin at daily doses of 10 and 1 mg/kg compared to those in control rats, respectively. The arrow referred to typical IFN-c+ cells, which appear to be infiltrating inflammatory cells morphologically and the cytoplasm was filled with brown. Original magnification: 400. (B) The numbers of IFN-c+ cells per mm2 sciatic nerve sections (a) and the percentages of IFN-c+ cells among inflammatory cells (b) are expressed as mean ± SD (n = 5 in each group) (⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001).
atorvastatin markedly ameliorated the clinical symptoms of EAN, decreased the numbers of inflammatory cells as well as IFN-c+ and IL-17+ cells in sciatic nerves, decreased the levels of IFN-c in MNC culture supernatants, decreased the CD80 expression and increased Treg cells in MNC from the lymphoid nodes. These data provide strong evidence that atorvastatin can act as an inhibitor in EAN by inhibiting the immune response of Th1 and Th17, decreasing the expression of co-stimulatory molecule, as well as up-regulating the number of Treg cells. Statins could reduce inflammation in the CNS. Administration of atorvastatin significantly reduced the severity and incidence of EAE, along with a large reduction in the number of inflammatory lesions in brains and spinal cords [10]. In our study, we found that atorvastatin significantly reduced inflammatory cells infiltration in
sciatic nerves of EAN rats. The result indicates that atorvastatin may play a role in anti-inflammation not only in the CNS but also in the PNS. Moreover, atorvastatin can dose-dependently exhibit the protective effects on EAN rats. Th17, which is considered as a subset of CD4+ T cells, has recently been found to play a critical role in the development of autoimmune diseases [21,22]. IL-17, also named IL-17A, is mainly expressed by Th17 and involved in inducing and mediating proinflammatory responses. Reportedly nasal administration of recombinant mouse IL-17 to Lewis rats with ongoing chronic EAN enhanced clinical symptoms and immune cell activity during the initial phase of the disease, which indicated that IL-17 played an important role in EAN [23]. Recently, it was found that accumulation of IL-17+ cells in sciatic nerves was correlated with the pathological progress in
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Fig. 4. Atorvastatin suppressed the production of IL-17 in sciatic nerves of EAN rats. (A) IL-17 immune reactivity in sciatic nerves was shown in 10, 1 mg/kg atorvastatintreated EAN rats, and control rats. The arrow referred to typical IL-17+ cells, which appear to be infiltrating inflammatory cells morphologically and the cytoplasm was filled with brown. Original magnification: 400. (B) The numbers of IL-17+ cells per mm2 sciatic nerve sections (a) and the percentages of IL-17+ cells among inflammatory cells (b) are expressed as mean ± SD (n = 5 in each group) (⁄p < 0.05, ⁄⁄p < 0.01, and ⁄⁄⁄p < 0.001).
Fig. 5. Expression of CD80, CD86 and MHC class II on the surface of lymph node MNC. MNC were prepared on day 18 p.i. from lymph nodes of rats treated with atorvastatin (at daily doses of 10 and 1 mg/kg) or DMSO and stained with PElabeled anti-rat CD80, FITC-labeled anti-rat CD86, and anti-rat MHC class II. The percentage of CD80, CD86, and MHC class II positive cells among all lymph node MNC are expressed as mean ± SD (⁄p < 0.05).
EAN rats and it was suggested that the Sphingosine-1-phosphate (S1P) signal pathway is involved in regulating IL-17+ cell trafficking [24]. Furthermore, AUY954, an S1P receptor modulator, effectively suppressed local inflammation in EAN, which might partly be due to a reduced peripheral proportion of IL-17 expressing cells and consequently attenuated infiltration of IL-17+ cells into inflammatory areas [25]. However, there has been no report about the roles of statins on Th17 in EAN. In autoimmune and inflammatory diseases of animals and human, including rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus, an increased level of IL17 expression has been observed [26–28]. Reportedly simvastatin could directly suppress IL-17 gene expression, as well as IL-17 secretion from the CD4+ T cells in relapsing remitting multiple sclerosis patients [11]. Our data showed that atorvastatin dose-dependently inhibited not only the numbers of IL-17+ cells but also the percentages of IL-17+ cells among inflammatory cells in sciatic nerves of EAN rats compared to the control DMSO administration, although
it did not significantly decrease IL-17 level in culture MNC supernatants. IFN-c is a pro-inflammatory cytokine, which can be produced by variety of cells, including Th1 cells, activated macrophages and so on, and it plays an important pathogenic role in EAN. In the present study, we found that atorvastatin could decrease not only the numbers of IFN-c+ cells and the percentages of IFN-c+ cells among inflammatory cells in sciatic nerves, but also the production of IFN-c in culture MNC supernatants in EAN rats. In accordance with our findings, it has recently been reported that simvastatin could inhibit the production of IFN-c in collagen induced arthritis (CIA, the experimental model for rheumatoid arthritis) [29]. Moreover, lovastatin could ameliorate EAU (a Th1-mediated autoimmune disease model), which was associated with inhibiting the production of pro-inflammatory cytokines, such as IFN-c, but did not require the induction of Th2 cytokine profile [30]. Our data showed that atorvastatin decreased the percentages of both IFN-c+ and IL-17+ cells among the inflammatory cells in sciatic nerves, which indirectly indicated that atorvastatin can ameliorate EAN by inhibiting Th1 and Th17 cell differentiation rather than by inhibiting the migration of inflammatory cells. Clearly in our study, atorvastatin can decrease IFN-c and IL-17 production in EAN. As known, MHC class II and co-stimulatory molecules such as CD80 and CD86 are expressed on APC. They are thought to be engaged in delivering signals to T cells and then determine the fate of naïve T cell. Up to now, three signals are thought to be involved in this process [31]. MHC class II is thought to be engaged in signal 1 which can be delivered to the T cell receptor. Signal 1 alone is considered as a factor to promote naïve T cell inactivation by anergy, deletion, or generation of regulatory cell, and thereby leading to tolerance. Co-stimulatory molecules are taken as an accessory factor of signal 1 in inducing immunity and considered as signal 2. Besides, signal 3 is referred to deliver signals from APCs to T cells that determine their differentiation into effector cells (e.g., Th1 cells, Th2 cells, or cytotoxic T lymphocytes (CTLs)) [32]. It has been reported that IL-12 is a mediator that delivers a signal 3 and promotes Th1 cells or CTLs development [33]. Statins could affect autoreactive leukocyte proliferation by altering the expression of MHC class II or co-stimulatory molecules on APC. Atorvastatin could suppress IFN-c inducible expression of CD40, CD80, and
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Fig. 6. Effects of atorvastatin on Treg cells in EAN rats. A representative FACS analysis of the expression of CD25+Foxp3+ cells among CD4+ cells from lymph node MNC in different groups are shown in (A). The percentages of CD25+Foxp3+ cells among CD4+ cells from lymph node MNC in rats treated with atorvastatin (at daily doses of 10 and 1 mg/kg) or DMSO are shown in (B). The results are expressed as mean ± SD (n = 5 in each group) (⁄p < 0.05 and ⁄⁄p < 0.01).
atorvastatin-treated groups increased the number of CD25+Foxp3+ cells among CD4+ cells from lymph node MNC. These indicate that atorvastatin may inhibit EAN by increasing the Treg cells. In summary, we provide strong evidence that atorvastatin can act as an inhibitor in EAN by inhibiting the immune response of Th1 and Th17, decreasing the expression of co-stimulatory molecule, as well as up-regulating Treg cells. All our data suggest that it is a new challenge for statins, which may be used as a new medicine in human GBS. Acknowledgments
Fig. 7. Cytokine production. MNC from lymph nodes of three group rats were cultured with BPM. The supernatants were collected for measuring the levels of IFN-c and IL-17. The results are expressed as mean ± SD (n = 5 in each group) (⁄p < 0.05 and ⁄⁄⁄p < 0.001).
This study was supported by Grants from the Foundation for 1020 Project Talent, Health Department of Shandong Province, partly supported by the National Natural Science Foundation of China (30771989), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. We like to thank Beijing Garlin Pharmaceutical Co. Ltd., for assistance in providing atorvastatin. References
CD86 co-stimulatory molecules in EAE [10]. Moreover, lovastatin could significantly reduce the expression of both MHC class II and CD80 co-stimulatory molecule in splenocytes of EAN rats [34]. Consistent with these findings, our results demonstrate that atorvastatin plays an important role in EAN rats by decreasing the expression of CD80, which was unable to provide efficient signals to activate T cells. Treg cells exert their immune suppressive functions by inhibitory cytokines, cytolysis, and modulation of dendritic cells maturation or function [35]. Foxp3 is expressed only on Treg cells and is thought to be correlated with an increased regulatory potential of Treg cells [36]. Statins could induce high levels of Foxp3, which is considered to be the main transcriptional regulator of the development and function of Treg cells. We found that both 10 and 1 mg/kg
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