Molecular Mechanism Of Lenalidomide Ameliorates Multiple Sclerosis By Promoting Macrophage M2 Polarization

Objectives:Multiple Sclerosis(MS)is the most common clinical demyelinating disease in central nervous system,with fast progression,high disability and poor prognosis,seriously affecting patients' life quality.Immunotherapy is main therapeutic strategy of MS,which can't repair damaged myelin despite of alleviating MS development.As a result,detecting a drug for immunosuppression and myelin regeneration has become one of highlights in scientific research.According to the reports,macrophage polarization is closely related to MS development,playing a role in regulating inflammatory response and repairing damaged myelin,which is expected to be one of MS effective treatment strategies.We adopted blood monocytes derived macrophage to screen drugs for regulating macrophage polarization and studied its effect as well as molecular mechanism on the treatment of multiple sclerosis.Methods:1.Drug screening for promoting macophage M2 polarization1)Bone Marrow Derived Macrophages(BMDMs)were treated alone with drugs exerting immunomodultory effects,such as anti-metabolism drugs Pemetrexed and Gemcitabine,anti-histamine drugs Loratadine and Promethazine,anti-5-hydroxytryptamine drugs Azasetron and Palonosetron and immunomodulators Lenalidomide and Pomalidomide,qRT-PCR to test the macrophage M2 type genes Argl and Mrc1 mRNA and M1 type genes iNOS and CXCL10 expression change;2)BMDMs were treated with the above drugs along with IL-13 stimulation or LPS stimulation,qRT-PCR to test the macrophage M2 type genes Argl and Mrcl mRNA and M1 type genes iNOS and CXCL10 expression change;3)BMDMs were treated with lenalidomide of different concentrations alone,qRT-PCR to test M2 macrophage genes Arg1,Yml,Mrc1,Ccl17 and Ccl22 mRNA expression.2.The therapeutic effects of lenalidomide exerted on EAE modelEAE model mimicing MS was induced by MOG35-55,and EAE mice were gagved with lenalidomide of different doses everyday,lasting 23 days.1)Referring to Kono's standards to judge symptoms severity of EAE mice and record the body weight;2)HE staining to detect for inflammatory focal point of spinal cord after lenalidomide adminstration;3)Immunostaining to investigate astrocytes activation in spinal cord;4)Immunostaining to investigate microglia activation in spinal cord;5)qRT-PCR and immunostaining to detect the effects of lenalidomide on oligodendrocytes of EAE mice;6)LFB staining and transmission electron microscopy technique to evaluate myelin repair and regeneration process of EAE mice with lenalidomide administration.3.The molecular mechanism research of lenalidomide promoting macrophage M2 polarization1)Western blot and immunofluorescence to detect expression of M2 macrophage marker Argl in spleen and spinal cord at acute phase and peak phase;2)qRT-PCR to detect to cytokines expression associated with macrophage in EAE mice;3)Immunostaining and western blot to test the expression change of IL-10 protein in the spleen and spinal cord of EAE mice;4)Immunostaining IL-10 with CD4,CD8,CD19 and CD68 respectively to examine its main origin;5)qRT-PCR to detect Arg1 and IL-10 mRNA from BMDMs of wild type mice and IL-10-/-mice;6)Western blot to dectect the activiation of STAT3,STAT6,AKT and p38 in RAW264.7 cell at different time treated with lenalidomide of different concentrations;7)Immunostaining to investigate the expression of p-p38 and p-STAT3(Y705)with M2 macrophage marker Argl.Results:1.Drug screening for promoting macophage M2 polarization1)Effects of Anti-metabolism drugs on macrophage genes expressionPemetrexed alone promoted Arg1 mRNA expressionof BMDMs(p<0.05),but without affecting Mrcl mRNA expression significantly.And there were n o significant increase of Arg1 and Mrcl mRNA of BMDMs when they were treated with Pemetrexed and IL-13 in the meantime.Gemcitabine treated alone or with IL-13 stimulation did not promote BMDMs to express Arg1 and Mrcl mRNA.iNOS and CXCL10 mRNA expression of BMDMs had not changed greatly when Pemetrexed treating alone.But after stimulated with LPS,Pemetrexed increased iNOS mRNA level(p<0.05).Gemcitabine alone promoted BMDMs to express CXCL10 mRNA(p<0.05),but without affecting iNOS mRNA expression significantly.iNOS and CXCL10 mRNA had no observable significant reduction when BMDMs were treated with Gemcitabine and LPS in the meantime.The above results showed that Gemcitabine had no observable effects on promoting M2 macrophaege genes to express.Pemetrexed only increased Arg1 mRNA expression when treating BMDMs alone,but the promoting effect of it was limited with only upregulated Arg1 mRNA expression for 1.65 times compared with control.2)Effects of Anti-histamine drugs on macrophage genes expressionArgl,Mrcl and iNOS mRNA of BMDMs had not significant change when treated with Loratadine alone.Loratadine alone only promoted BMDMs to express CXCL10 mRNA(p<0.05).There were no observable significant change of Argl and Mrcl mRNA expression when BMDMs were treated with Loratadine and IL-13 in the meantime.And there were also no observable significant reduction of iNOS and CXCL10 mRNA expression when BMDMs were treated with Loratadine and LPS in the meantime.Promethazine had on observable effects on Argl,Mrcl,iNOS and CXCL10 mRNA expression of BMDMs whenever treated alone or stimulated with inducible factor.The above results showed that Loratadine and Promethazine had no observable effects on promoting M2 macrophaege genes to express.3)Effects of Anti-5-hydroxytryptamine drugs on macrophage genes expressionAzasetron upregulated Argl mRNA but had no effect on Mrcl mRNA expression when treating on BMDMs alone.And Azasetron even decreased Argl and Mrcl mRNA expression of BMDMs with IL-13 stimulation.There were no significant reduction of iNOS and CXCL10 mRNA when BMDMs were treated with Azasetron and LPS in the meantime.Only when Azasetron were treated alone on BMDMs could it decrease CXCL10 mRNA level(p<0.05).However,Palonosetron promoted BMDMs to increase Argl mRNA expression with significant difference and IL-13 amplifyed its upregulation(p<0.05).Palonosetron did not affect Mrcl mRNA expression.Also there were no observable reduction of iNOS and CXCL10 mRNA expression no matter Palonosetron treated alone or with LPS stimulation.The above results showed that Azasetron had on visible significant effects to promote M2 macrophage genes Argl and Mrc1 mRNAexpression.Palonosetron selectivly promoted Arg1 and iNOS mRN A expression significantly.4)Effects of immunomodulator on macrophage genes expressionBoth Lenalidomide and Pomalidomide had no significant promotion of Argl and Mrcl mRNA expression of BMDMs.But there were significant increase of Argl and Mrcl mRNA when BMDMs were treated with Lenalidomide and IL-13 meanwhile(p<0.05).And lenalidomide had no observable sinificant effect on decreasing iNOS and CXCL10 mRNA level when treated BMDMs alone or with LPS stimulation.Pomalidomide decrease iNOS mRNA expression when treating alone on BMDMs.The above results showed that lenalidomide selectivly promoted Argl and Mrcl mRNA expression with great significance.But Pomalidomide had on observable effects on promoting M2 macrophage genes expression.5)Effects of lenalidomide on M2 macrrophage genes expressionLenalidomide(25 nM)alone could not promote M2 macrophage genes Arg?Ym1?Mrc1?Ccl17 and Ccl22 mRNA expression after treating BMDMs alone for 24 hour(p>0.05).Lenalidomide(100 nM)increased Ym1 mRNA expression only after treating BMDMs alone for 24 hours(p<0.05).Lenalidomide(400 nM)increased Arg1?Ym1?Mrc1 and Ccl17 mRNA expression except Ccl22 mRNA after treating BMDMs alone for 24 hours(p<0.05).The above results showed that Lenalidomide alone increased M2 macrophage genes expression with concentrations dependence.The effects of Lenalidomide(400 nM)upregulating M2 macrophage genes expression were most remarkable.2.The therapeutic effects of lenalidomide exerted on EAE model1)Lenalidomide could slow and alleviate the progression of EAE diseaseEight days after EAE model was constructed,there 20 percent of EAE mice appeared a bit weak tail.Twelve days of post-immunization,the mean clinical EAE score upped to 1.94 points.EAE mice showed low response and its tail were fully weak.As days post-immunization went on,symptoms of EAE mice aggravated more and more.Eighteen days after model construction,EAE mice showed hemiplegia and its mean clinical EAE score was 3 points.Dexamethasone administration reduced the mean clinical EAE score(1 points)compared with the EAE mice(2.56 points)at day sixteen post-immunization.Lenalidomide(30 mg/kg,100 mg/kg)significantly reduced EAE mean clinical score after administrated for one week,which was equal to the positive control dexametahasone's effect.Lenalidomide continued to alleviate clinical symptoms of EAE mice,expanding the scores gap between Lenalidomide-treated mice and EAE mice.With aspect of body weight,EAE vehicle mice presented weight decrease,but lenalidomide maintained body weight.When the experiment ended,body weight of lenalidomide-treated mice were consistent with the those at initial phase.The above results suggested lenalidomide ameliorated EAE progression,improving movement dysfunction,and maintain body weight stability of EAE mice.2)Lenalidomide suppressed inflammatory responses in EAE miceHE staining showed there were a large number of inflammatory cells infiltrating in spinal cord white matter of EAE mice.Lenalidomide-treated mice had no observable inflammatory cell infiltration occurring in spinal cord.Immunostaining results showed that at EAE early phase,astrocytes activation were found in the EAE mice in the spinal cord.Astrocytes increased in the number,with ridges growing thick and interwoven with stellate.While microglia had not been fully activated.During peak phase and plateau of EAE,both astrocytes and microglia were significantly activated.Lenalidomide continued to suppress the activation of astrocytes and microglia.The above results suggested lenalidomide inhibited the activation of astrocytes and microglia,reducing inflammatory reaction.3)Lenalidomide protected oligodendrocytes and myelinAccording to immunostaining,there were sharp decrease of Olig2 and MBP in spinal cord white matte of EAE mice at EAE acute phase,while lenalidomde administration reversed the reduction.qRT-PCR results showed oligodendrocytes related genes Olig2,Mbp,Cnp and Sox10 mRNA in EAE vehicle group decreased at different extent while lenalidomide reversed the reduction,promoting those genes expression.Immunostaining results showed that EAE mice presented sharp decline of Olig2 and CC1 at EAE peak phase equally.While lenalidomide administration significantly promoted Olig2 and CC1 expression and increased the rate of CC1+/Olig2.The above results suggested that lenalidomide protected oligodendrocytes from inflammatory attack.LFB staining showed EAE mice had serious myelin lost,with vacuolization.After treated with lenalidomide,EAE mice maintained pretty complete regional myelination,reducing myelin damage.Moreover,transmission electron microscope results showed that the spinal cord of EAE mice showed severe myelin sheaths,loosely organized,deformation and breakage.Lenalidomide therapeutic administraton alleviated myelin damage.Using Image J software to calculate the inner and outer radius of axons.Data shows g-ratio values of EAE mice was significantly raised(0.78 ± 0.01 ?m),higher than those of normal mice(0.66±0.009 ?m).Lenalidomide administration decreased g-ratio values(0.67 ± 0.007?m).The above results suggested lenalidomide relieved myelin damage.3.The molecular mechanism research of lenalidomide promoting M2 macrophage polarization1)Lenalidomide promoted M2 macrophage expression in vivoImmunostaining showed that at peack phase,M2 macrophage marker Argl in spleen of EAE mice decreased,while lenalidomide administration increaseed Argl protein expression.Western blot also proved that lenalidomide treatment increased Argl expression in spinal cord.The above results suggested lenalidomide promoted the expression of M2 macrophage.2)Lenalidomide regulated inflammation related cytokines expression in vivoqRT-PCR data showed at early phase of EAE,IL-6 and IFN-y mRNA in EAE mice increased greatly but TNF-a expressed equally to normal mice.Lenalidomide administration reduced IL-6 and IFN-y mRNA expression(p<0.05).And there were a sharp decrease of anti-inflamatory cytokines TGF-?,IL-4 and IL-10 mRNA in EAE mice.On the contrary,lenalidomide-treated mice exerted increase of TGF-?,IL-4 and IL-10 mRNA expression(p<0.05).Among those,IL-10 mRNA of leanlidomide-treated mice was 7 times higer than EAE mice and 5 times higher than nornal mice.The above results showed lenalidomide reduced pro-inflammatory cytokines IL-6 and IFN-y mRNA expression but increased anti-inflammatory cytokines TGF-?,IL-4 and IL-10 mRNA expression in EAE model.3)Lenalidomide increased IL-10 expression in vivoImmunostaining and western blot results showed a sharp decrease of IL-10 of EAE mice both in spinal cord and spleen at acute and peak phase.Lenalidomide treatment reversed IL-10 protein decrease and upregulated IL-10 expression greatly at peak phase.The above results suggested that lenalidomide promoted IL-10 protein expression despite of EAE development.4)Lenalidomide increased IL-10 mainly expressed in M2 macrophagesImmunofluorescence positioning technology to dye CD4,CD8,CD 19 and CD68 with IL-10 respectively.Results showed that IL-10 was most confocaled with CD19 and CD68.But lenalidomide only upregulated the percentage of IL-10+cells/CD68+ macrophages,the percentages of IL-10+ cells/CD4+ T cells and IL-10+ cells/CD8+ T cells and IL-10+/CD19+ B cells were no different between EAE vehicle group and lenalidomide-treated group(p>0.05).The above results suggested lenalidomide increased IL-10 mainly expressed in M2 macrophages.5)Lenalidomide had no significant upregulation on Argl mRNA in BMDMs of IL-10-/-miceqRT-PCR showed IL-10 mRNA expression in IL-10-/-mice BMDMs was much lower than which in wild type(WT)mice BMDMs(p<0.05).While Argl mRNA expressed eqully in BMDMs from both IL-10-/-mice and WT mice.Lenalidomide(100 nM)promoted Argl mRNA epression in BMDMs from WT mice(p=0.061).However,when IL-10 was knock out,the upregulation of Argl mRNA in BMDMs was diminished.The above results suggested lenalidomide promoting M2 macrophage polarization may relate to that lenalidomide increased IL-10 expression.6)Lenalidomide did not affect STAT6 and AKT phosphorylationWestern blot showed that there was on observable p-AKT(Y473)and p-STAT6(Y641)expression in RAW246,7 cell after treating with lenalidomide for 1 hour.The aboved results showed lenalidomide did not activiate STAT6 and AKT phosphorylation in vitro.7)Lenalidomide activiated p38 and STAT3 phosphorylation in vitroWestern blot showed that p-p38 increased significantly with concentration dependence 2 hour later for lenlidomide treatment.Lenalidomide increased p-STAT3(Y705)expression with concentration dependence.The aboved results showed lenalidomide activiated p38 and STAT3 phosphorylation in vitro.8)Lenalidomide mainly activiated p38 and STAT3 phosphorylation in M2 macrophagesImmunostaining showed there were an increase of p-p38 and p-STAT3(Y705)expression in EAE mice at acute phase of EAE,but there were little merge of p-p38,p-STAT3(Y705)with Arg1.However,lenalidomide administration not only promoted p-p38 and p-STAT3(Y705)expression,but also most p-p38 and p-STAT3(Y705)could merge with Arg1.The above results suggested that the activiated p38 and STAT3 was related with increased M2 macrophage.Conclusion:This topic found lenalidomide promoted M2 macrophages polarization selectively both in vivo and in vitro and exerted great therapeutic effect on multiple sclerosis with suppressing inflammation response as well as protecting oligodendrocytes.Involved molecular mechanism of lenalidomide alleviating EAE was that lenalidomide increasing IL-10 production,which led p38 and STAT3 activation,promoting macrophage M2 polarization.As a result,lenalidomide relieved inflammatory symptoms and protected oligodendrocytes and damaged myelin of EAE mice.In conclusion,this topic set forth the function of lenalidomide promoted macrophage M2 polarization and exerted therapeutic effect in MS via activiating IL-10/p38/STAT3 signaling axis.It enriched the molecular mechanism of M2 macrophage polarization in multiple sclerosis therapy as well as broadened the new idea for lenalidomide clinical use.