Therapeutic Potential Of Atorvastatin And Atorvastatin-modrfied Dendritic Cells In Neuroimmunological Diseases

Background:Experimental autoimmune neuritis (EAN) is a CD4+T cell-mediated acute inflammatory demyelinating disease of the peripheral nervous system (PNS) that serves as a model for the human Guillain-Barre syndrome (GBS). Its pathogenesis, clinical symptoms, electrophysiological signs, neuropathological changes and immunological parameters are similar to human GBS. EAN can be induced in susceptible animal species and strains by immunization with autoantigen emulsified in complete Freund’s adjuvant.Statins are inhibitors of3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in the mevalonate pathway for cholesterol biosynthesis, and they are widely used in clinical practice for the treatment of hypercholesterolemia. In the past few years, accumulated evidence has shown that statins have immunomodulatory effects. The effects of statins on the immune system are pleiotropic and include inhibition of T cell activation, proliferation, migration, and so on. Reportedly atorvastatin could promote shifting the T cell response from a pro-inflammatory Th1to an anti-inflammatory Th2profile in experimental autoimmune encephalomyelitis (EAE). It has been recently reported that IL-17plays a critical role in the development of autoimmune diseases. Furthermore, simvastatin could directly inhibit IL-17secretion in CD4+T cells derived from relapsing remitting multiple sclerosis patients.These evidences suggest that statins can inhibit Thl and Th17inflammatory 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 or lovastatin could inhibit the expression of co-stimulatory molecules, such as CD40, CD83, CD86. and human leukocyte antigen-DR (HLA-DR) on dendritic cells in human, then it could significantly reduce the ability of dendritic cells to induce T cell proliferation. Statins also could inhibit major histocompatibility complex (MHC) expression on IFN-y-stimulated human macrophages and endothelial cells, and reduce the ability of these cells to stimulate T cells. Reportedly atorvastatin could increase the number of CD4+CD25high cells and CD4+CD25+Foxp3+cells in human peripheral blood mononuclear cells.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 by immune regulation, in which Thl/Th17play essential roles in the pathogenesis. In the present study, we administered atorvastatin in the initial phase of EAN to evaluate its possible immunomodulatory effects during the process.Objective:The aim of our present study was to analyze the effects of atorvastatin on the induction of immune tolerance in Lewis rats with EAN.Methods:1. Induction of EAN and assessment of clinical symptoms Female Lewis rats were induced by subcutaneous injection into both hind footpads of the rats with200μ1inoculums containing5mg BPM and2mg Mycobacterium tuberculosis emulsified in incomplete Freund’s adjuvant.The rats were monitored for clinical symptoms of disease by two independent investigators. Clinical scores were assessed immediately before immunization (day0) and thereafter every day until day18post immunization (p.i.).2. Administration of atorvastatin To determine whether atorvastatin protected against the development of EAN, atorvastatin (at doses of1and10mg/kg/day in a volume of100μl DMSO) was administered to immunized rats by intraperitoneal injection from day5to18p.i. Control EAN rats received the same volume injections of vehicle (DMSO).3. Histopathological assessment Sciatic nerve segments were excised close to the lumbar spinal cord, fixed in10% paraformaldehyde and embedded in paraffin. Multiple longitudinal sections (5μm) of sciatic nerves were stained with hematoxylinandeosin for evaluation of the inflammatory cells by light microscopy. The number of inflammatory cells was counted and the average results were expressed as cells per mm2tissue section.4. Immunohistochemistry Paraffin tissue sections (5μm) were deparaffinized and hydrated. The sections were treated with0.3%hydrogen peroxide to block endogenous peroxidase activiry. Then the sections were exposed to ethylene diamine tetraacetic acid (EDTA) for antigen retrieval and incubated overnight at4℃with rabbit anti-rat IFN-y antibody and rabbit anti-rat IL-17antibody. Then sections were stained with HRP-conjugated goat anti-rabbit secondary antibody, followed by development with diaminobenzidine (DAB) substrate to detect the numbers of IFN-y+cells and IL-17+cells. As negative controls for immunostaining, the primary antibodies were omitted.5. Preparation of lymph node MNC On day18p.i., rats were sacrificed and inguinal lymph nodes were removed under aseptic conditions. MNC suspensions were obtained by grinding the organs through cell strainers in serum-free medium. Then cells were re-suspended to2×106/ml for the following experiments.6. Flow cytometric analysis of surface molecules on MNC from lymph nodes MNC suspensions in PBS containing0.5%bovine serum albumin (BSA) were incubated with phycoerythrin (PE)-labeled anti-rat CD80, fluorescein isothiocyanate (FITC)-labeled anti-rat CD86and FITC-labeled anti-rat MHC class II antibodies in the dark, respectively. After staining, the cells were analyzed by flow cytometry.7. Flow cytometric analysis of Treg cells on lymph node MNC FITC-conjugated anti-rat CD4, PE-conjugated anti-rat CD25and PE-Cy5-conjugated anti-mouse/rat Foxp3antibodies were used for staining according to the protocol recommended by eBioscience. Then the cells were re-suspended in PBS and analyzed by flow cytometry.8. Determination of cytokines by ELISA MNC were cultured in the presence of BPM peptide. After60h mcubarion, the supernatants were collected and measured for IFN-γ and IL-17by ELISA kits following the manufacturer’s instructions.9. Statistical analysis The SPSS17.0computer program was used for all calculations and statistical evaluations. Differences among different groups were tested by one-factor analysis of variance (ANOVA). Results were presented as means±SD and a level ofp<0.05was considered significant.Results:1. Atorvastatin suppresses the severity of clinical EAN Atorvastatin, at doses of10and1mg/kg/day, not only delayed the onset of EAN, but also significantly decreased the neurological severity of EAN, compared to DMSO treatment. Moreover, the10mg/kg-treated group and DMSO-treated group differed significantly. The rats in10mg/kg-treated group exhibited significantly lower clinical scores from day13to17p.i. compared with the rats in DMSO group. Meanwhile, the rats in1mg/kg-treated group had lower clinical scores from day13to17p.i. compared with the rats in DMSO group, but difference was significant only on day16p.i.2. Atorvastatin decreases the numbers of inflammatory cells in the PNS The rats in the group treated with10mg/kg atorvastatin and in the group treated with1mg/kg atorvastatin showed fewer inflammatory cells in sciatic nerves than those in the DMSO control group. In addition, the rats treated with high dose of atorvastatin had fewer inflammatory cells than the rats treated with low dose of atorvastatin.3. Atorvastatin inhibits the production of IFN-γ and IL-17in sciatic nerves of EAN rats Atorvastatin at daily doses of10and1mg/kg decreased the numbers of IFN-γ+and IL-17+cells in sciatic nerves compared to the control DMSO administration. There was no difference for the numbers of IFN-γ+cells in sciatic nerves between the two groups treated with10and1mg/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. Further analysis showed that the percentages of IFN-γ+and IL-17+cells among infiltrating inflammatory cells in rats with10and1mg/kg atorvastatin were lower than those in control rats, while the two treated groups did not differ significantly. 4. Effects of atorvastatin on the expression of co-stimulatory molecules in lymphocytes Atorvastatin treatment at doses of10and1mg/kg clearly inhibited the expression of CD80in lymphocytes when compared to control group. However, the two treated groups did not differ significantly.5. Atorvastatin up-regulates the number of CD25+Foxp3+Treg cells Both10and1mg/kg atorvastatin-treated groups increased the percentages of CD25+Foxp3+cells among CD4+cells from lymph node MNC when compared with the control group. Meanwhile the two treated groups did not differ significantly.6. Effects of atorvastatin on the levels of IFN-y and IL-17in the culture supematants The production of IFN-y in the culture supernatants of the10and1mg/kg-treated group were significantly reduced when compared with the control group. There were no significant differences for the levels of IL-17among the three groups.Conclusions:1. Atorvastatin can induce the immune tolerance of EAN.2. The therapeutic effects of atorvastatin on EAN rats were associated with inhibited the immune response of Thl and Th17, decreased the expression of co-stimulatory molecule, as well as up-regulated the number of CD25+Foxp3+Treg cells.3. Atorvastatin can dose-dependently exhibit the protective effects on EAN rats. Background:Myasthenia gravis (MG) is an antibody-mediated, T cell-dependent, complements involved autoimmune disease, caused by an immunological response against the acetylcholine receptor (AChR) on the postsynaptic membrane of neuromuscular junction and characterized by fatigability and fluctuating weakness of the skeletal muscles. Experimental autoimmune myasthenia gravis (EAMG) can be induced in Lewis rats by immunization with Torpedo AChR, which is a reliable animal model for human MG and suitable for investigating the mechanisms and the therapeutic strategies. Recently, it was found that EAMG also could be induced by a synthetic peptide corresponding to region97-116of the rat AChR a subunit (R97-116peptide).R97-116peptide can break the tolerance to the antigen and induce autoreactive T cells and specific antibodies to rat AChR. Its clinical symptoms, electrophysiological signs, anti-AChR antibodies and immunological parameters are similar to EAMG induced by TAChR. Therefore, immunization-induced EAMG models are usually employed for the analysis of antigen-specific immune responses and helping to investigate their modulation in order to improve disease progression. Current treatments for MG based on immunomodulation. are usually continued lifelong to maintain disease control,and associated with significant adverse effects arising from prolonged immune suppression. Therefore, it promotes us to explore new therapies to treat MG, which will specifically suppress the immune response to the AChR.AChR antibodies can produce defects in neuromuscular transmission via different mechanisms as follows:(1) binding to AChR and affecting their function;(2) increased degradation of AChR; and (3) complement-mediated damage to the postsynaptic endplate. In addition, the autoantibody response is T cell dependent, which provides help for B cells to produce anti-AChR antibodies. Dendritic cells (DCs) are specialized antigen presenting cells (APCs) with the ability to initiate a primary immune response by activating naive T cells or induce immune tolerance. The immune or tolerance properties of DCs depend on their maturation status. The up-regulation of co-stimulatory molecules (such as CD83, CD40, CD80and CD86) and MHC class II on DCs is essential to activate T cells. Immature DCs with lower levels of co-stimulatory molecules CD80, CD86and MHC class II, are potent inducers of immune tolerance or delay the immune response. Increasing evidence has suggested that immature DCs have potent ability to tolerize T cells. The maturation and function of DCs can be regulated in different pathways in vitro, such as DCs pulsed with AChR, DCs exposed to various cytokines, DCs incubated with Re1B specific small interfering RNA sequences, and so on. Up to now, studies have found that the DCs vaccine can induce tolerance and protect from some autoimmune diseases, such as EAMG. AChR-immunized rats injected intraperitoneally with spleen-derived DCs exposed to IL-10in vitro, showed amelioration of clinical symptoms, which due to the ability of these DCs in modulating T and B cell responses. It indicates that DCs engineered to present AChR epitopes can specifically target AChR-specific T cells, resulting in the reduction of both T cell responses to AChR and anti-AChR antibodies. Recent data also demonstrated that the administration of Re1B-silenced bone marrow DCs was able to suppress EAMG progression in mice, by inducing a shift from Th17/Thl to Th2and regulatory T cell responses, which indicated that DCs had become an attractive cell type for therapeutic manipulation of the immune system.Statins are inhibitors of3-hydroxy-3-methylglutaryl coenzyme A reductase in the mevalonate pathway for cholesterol biosynthesis, and could reduce cardiovascular and cerebrovascular related morbidity and mortality in patients. Increasing evidences from animal experiments and clinical studies have shown that statins have immunomodulatory activity. The effects of statins on immunomodulation are mainly due to inhibiting the isoprenylation of small GTP-binding proteins, which largely independent of its lipid-lowering effects. The effects of statins on immune system are pleiotropic and include inhibiting the maturation and function of APCs, such as DCs and B cells. Simvastatin or atorvastatin pre-incubated DCs exhibited an immature phenotype and a significantly lower expression of the maturation-associated markers CD83, CD40, CD86and human leukocyte antigen-DR in human. Furthermore, it significantly reduced the ability of DCs to induce T cell proliferation. Lovastatin could inhibit MHC class II and CD40expression on bone marrow-derived DCs of mice in a dose-dependent manner. Our studies in vitro confirmed that atorvastatin suppressed the expression of co-stimulatory molecules CD80and CD86on the surface of the spleen-derived DCs from ongoing EAMG rats, which indicated that atorvastatin could successfully induce tolergenic DCs.The aim of the present study was to analyze the effects of DCs modified with atorvastatin (statin-DCs) on the immune tolerance induction in Lewis rats with EAMG. Atorvastatin was co-cultured in vitro with spleen-derived DCs from ongoing EAMG rats to induce tolergenic DCs and reinfusion into the EAMG rats. Our data showed that statin-DCs treatment suppressed clinical symptoms of EAMG, which was associated with up-regulated number and function of Treg cells, inhibited lymphocyte proliferation, shifted cytokine profile from Thl/Th17to Th2type cytokines, and decreased level of anti-R97-116IgG antibody in serum. Importantly, these tolerogenic DCs played their immunomodulatory effects in the immune organs mainly by decreased expression of CD86and MHC class II on endogenous DCs. All these data suggest that statin-DCs could be as a new strategy for future therapy of MG in human.Objective:The aim of our present study was to analyze the effects of DCs modified with atorvastatin (statin-DCs) on the induction of immune tolerance in Lewis rats with EAMG.Methods:1. Induction of EAMG and assessment of clinical symptoms Female Lewis rats,6-8weeks old (body weight155-175g). EAMG were induced by subcutaneous injection into both hind footpads with200μl inoculum containing50μg R97-116peptide,1mg Mycobacterium tuberculosis in incomplete Freund’s adjuvant on day0and were boosted with the same dose along the back on day8after the first immunization. Rats were weighed from the beginning of the experiment every day or every second day until day43post immunization (pi.).The severity of the disease was scored by measuring muscular weakness in a blinded fashion.2. Tolerogenic DCs preparation, modification and injection Spleens were removed under aseptic conditions from EAMG rats on day16p.i., and mononuclear cells (MNC) suspensions from individual rats were obtained by grinding the organs through cell strainers. Then erythrocytes were osmotically lysed. DCs were further enriched by differential adherence. After2h, non-adherent cells were gently removed. New complete medium were added to the flasks. After18h incubation, atorvastatin dissolved in dimethylsulfoxide (DMSO) was added to flasks (final concentration10μM, statin-DCs). The same volume of DMSO was added to the other flasks (untreated-DCs). After48h incubation, floating cells were collected as a DC-enriched fraction. For phenotypic analysis, statin-DCs and untreated-DCs were stained with PE-conjugated anti-rat CD80, FITC-conjugated anti-rat CD86and FITC-conjugated anti-rat MHC class II monoclonal antibodies for30min at4℃. For intracellular cytokine analysis, statin-DCs and untreated-DCs were stained with PE-conjugated anti-rat IL-10antibody and rabbit anti-rat TGF-β antibody. Staining with anti-rat TGF-β was followed by FITC-conjugated anti rabbit IgG antibody for30min at4℃. After staining, the cells were analyzed by FACS. Then DCs were intraperitoneally transferred into EAMG rats at dose of2×106cells/rat on days5and13p.i. The control rats received the same volume of medium.3. Preparation of lymph node MNC On day43p.i., rats were sacrificed and inguinal lymph nodes were removed under aseptic conditions. MNC suspensions were obtained by grinding the organs through cell strainers in serum-free medium. Then cells were re-suspended to2×106/ml for the following experiments.4. Flow cytometric analysis of Treg cells on lymph node MNC Fixation and permeabilization of lymph node MNC were performed using the eBioscience Foxp3Staining Buffer Set. FITC-conjugated anti-rat CD4, PE-conjugated anti-rat CD25and PE-Cy5-conjugated anti-mouse/rat Foxp3antibodies were used for staining according to the protocol recommended by eBioscience. Then the cells were re-suspended in PBS and analyzed by flow cytometry.5. Cell proliferation assay MNC suspended in200μl aliquots containing4×105cells were cultured in flat-bottomed96-well microtitre plates in the absence or presence of R97-116peptide. After40h of incubation, the cells were incubated with CCK-8for4h at37℃. Then the absorbance was read at450nm on a microplate reader. Data are expressed as mean absorbance value (optical density, OD) and stimulation index (SI=OD(R97-116)/OD(o antigen)) of samples±standard deviation (SD).6. Cell cycle analysis MNC were harvested after40h and64h co-culture in the absence or presence of R97-116peptide. Then the cells were fixed in70%ice-cold ethanol at-20℃overnight. After washed in PBS, the cells were stained with propidium iodide (PI) solution (containing20μg/ml RNase A) for30min at4℃in the dark. Then the cells were analyzed by flow cytometry.7. Determination of cytokines by ELISA MNC were cultured in the presence of R97-116peptide. After40h incubation, the supernatants were collected and measured for IFN-y, IL-4and IL-17by ELIS A kits following the manufacturer’s instructions.8. Detection of serum anti-R97-116IgG antibody On day43p.i., the rats serums were collected to detect anti-R97-116IgG antibody by ELISA.9. DCs tracking Both statin-DCs and untreated-DCs were labeled with PKH26(red florescent cell linker) according to the manufacturer’s protocol. Then the labeled DCs were intraperitoneally transferred into EAMG rats at dose of2×106cells/rat on day5p.i. Rats were killed on days1and3after injection, and tissues (spleen, liver, thymus, popliteal and inguinal lymph nodes) were harvested and frozen for histological analysis. Cryostat sections were made from these samples and examined by fluorescence microscopy.10.Detection of the effect of statin-DCs injection on endogenous DCs Spleen-derived DCs from EAMG rats were prepared as previous methods on days1and3after injection of PKH26-labeled DCs respectively. Thereafter, we labeled these spleen-derived DCs (including exogenous DCs and endogenous DCs) with FITC-conjugated anti-rat CD80, FITC-conjugated anti-rat CD86and FITC-conjugated anti-rat MHC class II antibodies. Then the PKH26negative endogenous DCs were separated from PKH26positive exogenous DCs by FACS and the expression of CD80, CD86and MHC class II on endogenous DCs were examined.11. Statistical analysis The SPSS17.0computer programme was used for all calculations and statistical evaluations. Differences among different groups were tested by two-tailed Studen t test and one-factor analysis of variance (ANOVA) followed by Least Significant Difference (LSD) test as a post-hoc test. Results were presented as means±SD and a level of p<0.05was considered significant.Results:1. Atorvastatin could successfully induce tolerogenic DCs in vitro The phenotypic analysis of DCs showed that the expression of CD80and CD86were inhibited on statin-DCs when compared with untreated-DCs. Meanwhile, the results showed that there was no statistical difference for intracellular IL-10and TGF-β production between statin-DCs and untreated-DCs.2. Statin-DCs treatment suppress the development of EAMG The rats in statin-DCs group exhibited lower clinical scores when compared with rats in untreated-DCs group and control group. Moreover, the clinical symptoms between untreated-DCs group and control group did not differ significantly. Furthermore, the rats in untreated-DCs group and control group exhibited more loss of body weight when compared with the rats in statin-DCs group. Although the body weight between untreated-DCs group and statin-DCs group was not statistically different, there was significant difference between control group and statin-DCs group (p<0.05, from day29to43p.i.).3. Statin-DCs treatment increase the number and function of Treg cells The results showed that statin-DCs treatment increased the percentages of CD4+D25+T cells and the percentages of CD4+Foxp3+T cells among lymph node MNC when compared with untreated-DCs and control. There were no difference in the percentages of CD4+D25+T cells and the percentages of CD4+Foxp3+T cells between untreated-DCs group and control group.4. Statin-DCs treatment suppress lymphocyte proliferation In our study, lymphocyte proliferation was measured after40h of culture in the absence or presence of R97-116peptide by using the CCK-8assay. There was no difference among three groups when MNC cultured without R97-116antigen. In the presence of R97-116antigen, lymphocyte proliferation was significantly decreased in statin-DCs group compared with control group. Meanwhile, we did not find statistical difference between statin-DCs group and untreated-DCs group. However, SI analysis showed that R97-116-specific T cells proliferation was suppressed by statin-DCs treatment when compared with untreated-DCs and control treatments.5. Statin-DCs treatment mediate cell cycle changes After co-cultured with PBS for40h and64h, the percentages of S phase in cell cycle of statin-DCs group were reduced compared with those in control group. However, there was no statistical difference between statin-DCs group and untreated-DCs group. After co-cultured with R97-116peptide for40h and64h, the percentages of S phase in cell cycle of statin-DCs group were reduced compared with those in control group and untreated-DCs group, meanwhile there was no difference between control group and untreated-DCs group. After co-cultured with R97-116peptide for40h, the percentage of cells of S phase plus G2/M phase in statin-DCs group was lower than those in control group and untreated-DCs group, while no statistical difference was found between control group and untreated-DCs group.6. Effects of statin-DCs treatment on the secretion of cytokines Statin-DCs treatment resulted in a higher level of IL-4and lower levels of IFN-y as well as IL-17when compared with those in untreated-DCs group and control group. There were no differences between untreated-DCs group and control group for the secretion of IL-4, IFN-y and IL-17.7. Detection of the level of anti-R97-116IgG antibody in serum The rats in statin-DCs group had a lower level of anti-rat R97-116IgG antibody compared with those in untreated-DCs group and control group, while there was no statistical difference between untreated-DCs group and control group. 8. DCs tracking in vivo Both the labeled statin-DCs and untreated-DCs were detected in spleen, thymus, popliteal and inguinal lymph nodes, but were absent in liver. Moreover, less labeled cells were found on day3when compared with those on day1after injection. These labeled statin-DCs and untreated-DCs were mainly located in the red pulp of spleen and the cortex of thymus, while the labeled DCs could be found to be distributed diffusely within the lymph nodes. Meanwhile, the distribution of statin-DCs and untreated-DCs is almost consistent with each other.9. The effect of injected exogenous statin-DCs on endogenous DCs The results showed that both the endogenous and exogenous DCs from EAMG rats of statin-DCs group expressed lower levels of CD86and MHC class Ⅱ on day3after injection when compared with those from untreated-DCs group. There were no significant difference for the levels of CD80, CD86and MHC class II expressed on endogenous and exogenous DCs from EAMG rats on day1after injection between untreated-DCs group and statin-DCs group. Moreover, the percentage of PKH26-labeled exogenous DCs (both statin-DCs and untreated-DCs) on day3after injection (1.93±0.86%) was lower than those on day1after injection (6.68±3.63%).Conclusions:1. Atorvastatin could successfully induce tolerogenic DCs in vitro.2. These tolergenic statin-DCs could induce the immune tolerance of EAMG rats in vivo.3. The therapeutic effects of statin-DCs on EAMG rats were associated with up-regulated number and function of Treg cells, inhibited lymphocyte proliferation, shifted cytokine profile from Thl/Thl7to Th2type cytokines, and decreased level of anti-R97-116IgG antibody in serum.4. These tolerogenic statin-DCs can migrate to spleen, thymus, popliteal and inguinal lymph nodes after they were injected into the EAMG rats intraperitoneally. The characteristics of distribution indicated that these DCs could play their immunomodulatory effects in the immune organs.5. These exogenous statin-DCs played their immunomodulatory effects in the immune organs mainly by decreased expression of CD86and MHC class Ⅱ on endogenous DCs.