| Objective: Multiple sclerosis (MS) is characterized by theintermittent development of inflammatory lesions in the brain and spinalcord, resulting in plaques of demyelination and axonal loss. Lymphocytemigration across the blood–brain barrier is thought to be an importantearly step in the formation of lesions. Experimental autoimmuneencephalomyelitis (EAE) mimics many aspect of multiple sclerosis.Different classes of immunomodulatory agents, such as GA, IFN-β,Mitoxantrone, natalizumab, and fingolimod, with distinct mechanisms ofaction, are approved for multiple sclerosis treatment. However, thecurrent medications are only partially effective. They are associated withside effects and potential toxicities, and there is ongoing debate regardinglong-term efficacy of certain agents. While one strategy to improve MStherapy is to develop novel agents that may have greater efficacy, it isimportant to identify existing or novel classes of drugs that maycomplement one another in combination to provide additive or synergisticbenefit.Oral therapy for multiple sclerosis is welcome news for the2.5million patients who have this chronic, disabling disease. Oralformulations for treating RRMS are becoming more important because ofthe relative ease of administration, which should improve adherence andreduce lifestyle restrictions. Phase3trial results of oral therapies forRRMS have recently been reported. Fingolimod, Cladribine andteriflunomide each have advantages and disadvantages. The benefit ofteriflunomide, for patients with RRMS, with respect to the rate of relapse and disability progression, is inferior to Fingolimod and Cladribine.However, serious adverse events of Cladribine include infection andneoplasm. Adverse events related to fingolimod include bradycardia,atrioventricular conduction block, macular edema, elevated liver-enzymelevels, and mild hypertension. We assume that combination therapy couldreduce adverse events and improve curative effects.Rapamycin is an oral immunomodulatory agent approved for toprevent rejection in human organ transplantation. In mammalian cells,rapamycin forms a complex with the intracellular immunophilinFK506-binding protein-12(FKBP12), which blocks activation of aserine/threonine protein kinase called mammalian target of rapamycin(mTOR). mTOR is crucial for cell cycle progression, protein synthesis,and inhibiting antigen-induced T and B cell proliferation. It isincreasingly evident that promotion of Treg activity contributes totolerance induction by this compound. For example, Battaglia andcolleagues reported that rapamycin selectively expands naturallyoccurring murine Tregs in vitro. Furthermore, Battaglia and Straussreported rapamycin selectively promotes expansion of functional humanTregs, while depleting human CD4+CD25T effector cells. Loenen et alfound that rapamycin permits thymic generation and peripheralpreservation of mouse Tregs. Rapamycin efficacy and safety havepreviously been evaluated in an open-label trial. Patients with clinicallydefinitive relapsing-remitting MS (RRMS) or secondary progressive MS(SPMS) with relapses displayed a significant beneficial effect withrespect to new magnetic resonance imaging lesions and number ofrelapses, with an acceptable risk/benefit profile.Rapamycin is an oral immunosuppressant previously reported toinduce naturally occurring CD4+CD25+Foxp3+regulatory T cells,re-establishing long-term immune self-tolerance in autoimmune disease.Rapamycin suppressed IFN-γ, and IL-17release from antigen-specific Tcells and selectively expanded Treg cells in lymphoid organs affected by EAE. Rapamycin could be combined with1,25-dihydroxyvitamin D3orFTY720, which could find clinical application in reducingtherapeutically efficient doses without causing side effects.Recent studies demonstrate that oral cholesterol-loweringHMG-CoA reductase inhibitors (i.e. statins) have immunomodulatoryproperties that may benefit the treatment of T cell–mediated,organ-specific autoimmune diseases and other inflammatory conditions.Promising results were obtained in initial clinical trials testing simvastatin(Zocor) and atorvastatin (Lipitor) in MS and RA, respectively.Atorvastatin is an oral cholesterol-lowering HMG-CoA reductaseinhibitor with immunomodulatory properties. In EAE models,atorvastatin promotes differentiation and expansion of myelinprotein-reactive regulatory Th2cells and suppresses upregulation ofMHC class II and costimulatory molecules on APCs. This indicates thatthe beneficial immunomodulatory effects of statins may involve bothAPC and T cell functions. Other studies report that statins enhancedifferentiation of Foxp3+Tregs and inhibit differentiation of Th17cellsin vitro.Data from previous studies indicate that atorvastatin mediatesimmunomodulatory effects via T cells and APCs primarily by inhibitingsynthesis of isoprenoid compounds in the mevalonate pathway.Isoprenylation of small GTP-binding proteins is necessary for theirintracellular trafficking and subcellular localization to the plasmamembrane. Ras proteins, members of a large family of lowmolecular-weight GTP/GDP binding proteins, serve as a 'molecularswitch' that regulate cell fate by cycling between active GTP and inactiveGDP conformations. Statins may facilitate Th2differentiation, possiblyby reducing available Ras, which has an important role in ERK activationand Th1differentiation.Ras inhibition is associated with upregulation of Foxp3and hence inthe frequency and suppressive properties of Treg. Other studies suggest that ERK/MAPK plays a critical role in activating innate production ofIL-23and IL-1-β, which directs induction and expansion of Th17cells.Simvastatin inhibited differentiation of Th17cells but enhanceddifferentiation of Treg cells.Rapamycin mediated disruption of kinase/mitogen-activated proteinkinase (ERK/MAPK) via mTOR may render cells resistant to mTORinhibition. Pharmacological inhibition of MAPK enhances theantitumoral action of rapamycin associated with abrogation of thefeedback response. Since both the AKT/mTOR and ERK/MAPKsignaling pathways mediate differentiation of CD4+FoxP3+T cells, thecombined inhibition of mTOR and MAPK may hold greater promise thana single agent in treating multiple sclerosis. Thus, the mechanism ofaction of these two drugs is distinct, and complements each other. Havingmet all of the criteria described above, we reasoned that both drugs wouldbe excellent candidates for combination therapy.Interestingly, abrogation of a newly identified mTOR-mediatednegative feedback regulation of extracellular signal-regulatedkinase/mitogen-activated protein kinase (ERK/MAPK) signaling, and onthe well-documented RTK-PI3K-AKT signaling cascade, could limit theefficacy of rapamycin. Therefore, we speculated that dual inhibition ofmTOR and ERK/MAPK pathways may overcome the disadvantage ofsingle agent therapies and boost the efficacy of mTOR targeted therapiesfor MS patients. The majority of atorvastatin mediatedimmunomodulatory effects may be related to the inhibition ofisoprenylation of GTPases such as Ras and Rho, which have importantroles in multiple cellular differentiation and proliferation signalingpathways, including those in immune cells. Because atorvastatincomplements rapamycin, prompts development of protectivemyelin-reactive Treg cells and inhibits development of destructive Th17cells, we tested whether atorvastatin could augment the therapeutic andimmunomodulatory effects of rapamycin on myelin-reactive T cells in EAE.One must consider that FDA-approved therapies and several noveltherapies, administered individually, potently suppress EAE. As we havedescribed, one strategy to evaluate potential complementary therapeuticbenefit is to administer candidate drugs using suboptimal doses. In thisregard, atorvastatin and rapamycin were synergistic when administered atdoses that had no detectable clinical or immunomodulatory effects aloneMethods:MiceFemale C57BL/6mice were purchased from vital river laboratories(Beijing, China). Mice were housed in specific pathogen free conditions.Induction and assessment of EAEEAE was induced in C57BL/6mice by immunization with250μg ofMOGp35–55(lysine Bio-system, XiAn,China). All peptides weredissolved in complete Freund's adjuvant (Sigma, St Louis, MO, USA)containing4mg/ml of heat-killed mycobacterium tuberculosis H37Ra(Difco Laboratories, Detroit, MI, USA). At day0and48h postimmunization, C57BL/6mice were injected with500ng of pertussis toxin(Alexis, San Diego, CA, USA) in PBS, intraperitoneally (i.p.). Mice wereexamined daily for clinical signs of EAE and scored as follows:0, noparalysis;1, loss of tail tone;2, hindlimb weakness;3, hindlimb paralysis;4, hindlimb and forelimb paralysis;5, moribund or dead.Atorvastatin and Rapamycin treatmentAtorvastatin (prescription formulation; Pfizer) was dissolved in PBS.Atorvastatin was administered orally in0.5ml PBS, once daily, using a20-mm feeding needle. Rapamycin (Sigma) was suspend in distilledwater, stored at4°C, and protected from light according to themanufacturer's instructions. Rapamycin i.p. injections were given oncedaily. Control mice received PBS (vehicle) orally or a daily injection (i.p.)of distilled water (vehicle).For EAE reversal, daily treatment with rapamycin and atorvastatin began when a clinical score of≥2.0was reached. Rapamycin (0.3mg/kg/d)was administered i.p. in0.1ml distilled water, and atorvastatin(1mg/kg/d)was administered by oral gavage.Isolation of MOG p35–55-primed T cellsAfter10d of immunization, spleens were collected from mice, andsingle-cell suspensions were prepared in RPMI1640media containing10%FBS,2mM L-glutamine,50mM2-ME,100U/ml penicillin, and100mg/ml streptomycin. Splenocytes cultured at a concentration of0.5–1.0×106cells/ml in24-well plates were incubated with20mg/mlMOGp35–55for48or96h. Nonadherent splenic T cells were collectedand used for RNA isolation and FACS analysis.Cytokine analysis.Splenocytes were isolated from atorvastatin, rapamycin, vehicletreated, or combination treated mice and cultured in vitro with thespecific encephalitogenic peptide (MOGp35–55) used for theimmunization. Cell culture supernatants were collected at48h (IL-12andIL-23),72h (IFN-γ, TGF-β and IL-17), and120h (IL-4and IL-10)incubation for cytokine analysis. Quantitative ELISA was performedusing paired monoclonal Abs specific for corresponding cytokines, perthe manufacturer's recommendations (eBioscience, San Diego, CA,USA). ELISA results are expressed as the mean of triplicate wells±SD.Flow cytometryIntracellular staining of Foxp3and surface staining for CD25andCD4on splenic T cells was performed according to the manufacturer'sprotocol (eBioscience). Approximately1×106cells suspended in flowstaining buffer were incubated at4°C with appropriately dilutedFITC-labeled anti-CD4and APC-labeled Abs to CD25for30min,washed, and resuspended in fixation and permeabilization solution.Following incubation in the dark for30min, cells were washed, blockedwith test Fc block (anti-mouse CD16/32) in permeabilization buffer, andsubsequently incubated with appropriately diluted PE-labeled anti-Foxp3 at4°C in the dark. After incubation, the cell suspension was centrifuged,washed three times, and resuspended in an appropriate volume of flowstaining buffer. A minimum of10,000cells was accepted for FACS (BDBiosciences, San Jose, CA, USA) analysis. Cells were gated based onmorphological characteristics. Apoptotic and necrotic cells were notaccepted for FACS analysis.Total RNA isolationSplenocytes were isolated from MOG-immunized female C57BL/6mice. Briefly, splenocyte cultures were washed once with PBS, and RNAwas isolated using Trizol (Life Technologies, Foster City, CA, USA)reagent according to the manufacturer's protocol. RNA concentration wasmeasured by spectrophotometry at260nm.Real-time PCR analysisTo remove any contaminating genomic DNA, total RNA wasdigested with DNase. cDNA was obtained using RevertAid First StrandcDNA Synthesis Kit (Fermentas, Glen Burnie, MD, USA). Briefly,1μgtotal RNA was reverse-transcribed using oligo(dT)12–18as a primer andMoloney murine leukemia virus reverse transcriptase in a20ml reactionmixture. cDNA was amplified using Titanium Taq DNA polymerase andthe following primers: Foxp3sense5-CCC AGG AAA GAC AGC AACCTT-3, anti-sense5-TTC TCA CAA CCA GGC CAC TTG-3; RORCsense5-CCG CTG AGA GGG CTT CAC-3, anti-sense5-TGT AATGTG GCC TAC TCC TGC A-3; ERK sense5-AGC CTT CCA ATCTGC TTA TCA-3, anti-sense5-AGA TGT CGA TGG ATT TGG TGT-3;and S6K sense5-CAA CAT CAT CCT CAA AGA T-3, anti-sense5-TTC CCA GAC TCA TCC ACA TAG A-3. Real-time PCR analysiswas performed using the Stratagene MX3005P sequence detection system(Agilent technologies, Santa Clara, CA, USA). qRT–PCR parameters:initial incubation10min at95C, followed by40cycles: denature10s at95C, annealing20s at55C, extension30s at72C. β-actin wasamplified from all samples as a housekeeping gene to which expression was normalized. A control (no template) was included for each primer set.Data were analyzed by Sequence Detection Systems software (LifeTechnologies). Ct values were compared with those obtained on astandard curve.Western blot analysisSplenocytes were harvested in lysis buffer (2%SDS,0.1mol/L DTT,10%glycerol, and60mmol/L Tris, pH6.8) and heated at98°C for10min. The protein lysates were separated by4–12%Bis-Tris SDS-PAGEgel (Life Technologies) and transferred onto PVDF membranes (PierceChemical, Rockford, IL, USA). Nonspecific reactivity was blocked by5%non-fat dry milk in TBST buffer (20mmol/L Tris-HCl,150mmol/LNaCl,0.05%Tween20, and pH7.5) at room temperature for1h,followed by incubation with primary antibodies for phospho-ERK(1:1,000, Cell Signaling Technology, Danvers, MA, USA), ERK (1:500,Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-actin (1:8,000,Santa Cruz Biotechnology) at4°C for12h, and subsequently incubatedwith HRP-conjugated secondary antibody (Santa Cruz Biotechnology) at1:5,000dilution, at room temperature for2h.Histological evaluationAt least5mice per group were perfused through the left cardiacventricle with saline, plus0.5M EDTA for5–10min followed by fixationwith cold4%paraformaldehyde (PFA)(Sigma) in0.1M phosphate buffer(pH7.4). Subsequently, spinal cords and brains were carefully dissectedout and post-fixed in4%PFA for3–4h and processed for paraffinembedding.Quantification of neurological damage in EAE mice was performedvia histological analysis of5μm paraffin CNS sections of control,atorvastatin, rapamycin, and combination treated EAE mice. Twodifferent stains were used to detect inflammatory infiltrates (H&E) anddemyelination (Luxol fast blue). Immunohistochemistry for IL-17(SantaCruz Biotechnology) was performed to assess IL-17positive cells in the infiltrates. Antibodies were revealed with biotin-labeled secondaryantibodies and developed with the ABC kit (Haoyang BiologicalTechnology Ltd., Tianjin, China), followed by liquid DAB SubstrateChromogen System (Zhongshan Goldenbridge, Beijing, China).Neuropathological findings were quantified on an average of10completespinal cord cross-sections per mouse taken at8different levels from thespinal cord. The number of perivascular inflammatory infiltrates werecalculated and expressed as the numbers of inflammatory infiltrates permm2. Demyelinated areas were expressed as the percentage of damagedarea per mm2. The number of IL-17positive cells lining the subarachnoidspace or infiltrating the CNS parenchyma was calculated and expressedas the number of cells per mm2. Micrographs were taken with anOlympus microscope.Statistical Analysis.Unless otherwise indicated, data are presented as mean±SD. Forclinical scores, significance between groups was examined byMann-Whitney U test. All other statistical comparisons between groupswere examined using1-way multiple range ANOVA adjusted for multiplecomparisons. P values less than0.05were considered statisticallysignificant.Results:Combination therapy using suboptimal doses of rapamycin andatorvastatin prevents clinical EAEAtorvastatin(10mg/kg) and rapamycin(1mg/kg) could preventclinical signs of EAE. In dose-response experiments we consistentlyobserved that0.3mg/kg rapamycin administered prior to EAE onset wasno more effective than control (vehicle only) treatment. Similarly, weobserved that daily oral treatment of atorvastatin (1mg/kg) beginning at3days post immunization had no detectable effect.To evaluate combinationtherapy in the EAE model using medications that are effectiveindividually, it is necessary to test the drugs in combination at suboptimal doses. We therefore combined1mg/kg atorvastatin with0.3mg/kgrapamycin. Combination therapy at individually suboptimal dosesprevented clinical signs of EAE. In fact, this combination was as effectiveas treatment with each individual agent at its optimal dose (atorvastatin,10mg/kg; rapamycin,1mg/kg).Combination therapy using suboptimal doses of rapamycin andatorvastatin inhibit IL-17T cell CNS infiltration in EAETh17is a recently identified subset of CD4T cells critical for thedevelopment of autoimmune diseases. Th17cells produce IL-17A (alsoknown as IL-17). IL-17is expressed in target organs of patients withother autoimmune diseases, including rheumatoid arthritis, psoriasis andautoimmune uveitis. It may play a role in human autoimmune processesand represents a target for the treatment of select autoimmune diseases.We evaluated Th17T cells infiltrating the CNS byimmunohistochemistry (IHC). Atorvastatin(10mg/kg) and rapamycin(1mg/kg) inhibited Th17infiltration of SC white matter in EAEmice,compared with vehicle-treated. Combination therapy of rapamycinand atorvastatin significantly inhibited Th17infiltration of SC whitematter in EAE mice, compared with vehicle-treated. Th17infiltration wasreduced compared with those treated with atorvastatin (1mg) orrapamycin (0.3mg). rorc mRNA relative expression followingcombination therapy (2.92±0.35) was lower than for vehicle-treated(79.67±3.47)(Fig.5).Combination of atorvastatin and rapamycin promotes induction ofTh2and Treg myelin reactive T cells and inhibits induction of Th1andTh17As EAE is mediated by Th2and Treg cells, we tested whether thesynergistic beneficial effects observed using the combination ofatorvastatin and rapamycin was associated with secretion of Th2and Tregcytokines. Compared with vehicle-treated mice, treatment withatorvastatin and rapamycin suppressed recall responses to encephalitogenic myelin peptide. Proliferation inhibition appeared doserelated. Culture supernatants were examined for Th1cytokines (IL-12,IFN-γ), Th17cytokines (IL-17, IL-23), Th2cytokines (IL-4, IL-10), andTreg cytokines (TGF-β). As shown in Fig.3, atorvastatin and rapamycincombination therapy was associated with reduced secretion of IL-12,IFN-γ, IL-17, and IL-23. In contrast, secretion of IL-4, IL-10, and TGF-βincreased. Thus, combined atorvastatin and rapamycin treatment inducesa Th2and Treg bias in murine EAE.Combination of atorvastatin and rapamycin protects against EAE viaan expansion of peripheral regulatory T-cell populations.The therapeutic effect of rapamycin in autoimmunity is associatedwith the increase of Foxp3+Treg cells. The mechanism of statinprotection also involves Treg. Therefore, we examined changes inCD4+CD25+Foxp3+T cells in atorvastatin-treated, rapamycin-treated,combination treated, and untreated mice by flow cytometry. Thepercentage of CD4+CD25+Foxp3+T cells in the atorvastatin-treatedgroup did not increase relative to the untreated group. The percentage ofCD4+CD25+Foxp3+T cells in the suboptimal combination-treated groupwas greater than for untreated, atorvastatin-treated and rapamycin-treatedgroups. Foxp3mRNA expression in rapamycin and atorvastatincombination therapy (18.28±1.24) was increased relative tovehicle-treated (0.26±0.05).Atorvastatin impaired rapamycin induced ERK/MAPK activation.Rapamycin disrupts mTOR-mediated suppression of theERK-PI3K-AKT signaling cascade, so we hypothesized that reactivationof ERK/MAPK might compromise the effectiveness of rapamycin inEAE treatment. Thus, to prevent rapamycin mediated hyperactivation ofERK/MAPK, atorvastatin was used to inhibit ERK/MAPK signaling.After treatment with rapamycin alone or in combination with atorvastatin,splenocytes of EAE mice were harvested for western blot analysis. Thisanalysis suggested that mTOR inhibition by rapamycin up-regulated ERK1/2phosphorylation (Fig.5B). Furthermore, it appeared thatrapamycin-reactivation of ERK1/2was blunted by atorvastatin. Thus, wepropose that combining rapamycin and atorvastatin may be a promisingregimen for the treatment of EAE.Combination therapy using suboptimal doses of atorvastatin andrapamycin reverses clinicalIn general, immunomodulatory therapy in MS is initiated afterpatients have developed clinical signs or symptoms of CNSdemyelinating disease. Therefore, it is important to test whether a noveltreatment regimen, which is effective in EAE prevention, can also reverseestablished disease. We evaluated whether suboptimal doses ofatorvastatin in combination with rapamycin could reverse establishedEAE. Treatment was initiated when individual mice developed a clinicalscore>2(see Methods). We consistently observed that treatment using acombination of suboptimal doses of atorvastatin and rapamycin reversedclinical severity of EAE.Conclusion: The EAE model is useful for initial testing of potentialcombination therapies for multiple sclerosis. Rapamycin (0.3mg/kg) oratorvastatin (1mg/kg) administered prior to EAE onset was no moreeffective than control (vehicle only) treatment. Combination therapyusing suboptimal doses of rapamycin and atorvastatin prevents clinicalEAE.Combination therapy using suboptimal doses of rapamycin andatorvastatin inhibit IL-17T cell CNS infiltration in EAE.Combination ofatorvastatin and rapamycin protects against EAE via an expansion ofperipheral regulatory T-cell populations. Combination of atorvastatin andrapamycin promotes induction of Th2and Treg myelin reactive T cellsand inhibits induction of Th1and Th17Atorvastatin impaired rapamycin induced ERK/MAPK activation.Atorvastatin didn't disturb path of PI3K/AKT/mTOR which rapamycinblock. One potential mechanism that could account for the observed clinical and immunological synergistic effects of the combinationtherapy.Our results clearly support testing the combination of atorvastatinand rapamycin in multiple sclerosis clinical trials. |
PDF Full Text Request