| ObjectiveMultiple sclerosis(MS)is an immune system-mediated inflammatory demyelinating disease in the central nervous system(CNS)that can lead to multiple dysfunctions including sensory,motor,and neurocognitive effects with progressions possibly leading to paralysis and even death.Researches have found that activated CD4+ T cells could infiltrate the CNS through the blood-brain barrier and release inflammatory cytokines and chemokines,with recruiting T cells,B cells and macrophages to further infiltrate CNS,which breaks the balance between pro-inflammatory and anti-inflammatory factors,resulting in damage to oligodendrocytes and subsequently inducing MS.In previous studies,we have demonstrated that lenalidomide promotes M2 polarization of macrophages through drug screening and in vitro experiments,and could also alleviate the clinical symptoms in EAE mice.However,whether lenalidomide has effects on other immune cells in regulating EAE progression and by which way have not been elucidated.To further validate the mechanism of lenalidomide protecting against EAE disease,this study used different models and experimental methods at the cellular and animal levels to investigate the immune cells types which lenalidomide regulates in ameliorating EAE and its molecular mechanisms.Methods1.The therapeutic effect of lenalidomide on EAE(1)The MOG35-55-induced EAE mouse model was used as the research object.After 20%of model mice showed decreased muscle strength of the tail end,the test drug lenalidomide(30 mg/kg)and positive control dexamethasone(DXM,10mg/kg)were administered by gavage once daily for 18 days.The clinical scores were calculated with Kono's method,in order to determine the therapeutic effect of lenalidomide on EAE.(2)The spleen and draining lymph nodes(DLN)of EAE mice in the acute phase(day 17)were collected,and the ratio of Th1,Th17,B cells and Treg cells in the peripheral immune organs after treated with lenalidomide was measured by flow cytometry.(3)The brain and spinal cord in the CNS of EAE mice on day 17 after the model establishment were collected,and the mononuclear cells(MNCs)were separated by collagenase digestion and non-continuous density gradient centrifugation,and counted by cell-counting methods.Flow cytometry was used to detect the changes in the ratio pro-inflammatory Th1 and Th17 cells in MNCs after lenalidomide treatment.2.The role of macrophages in EAE treated with lenalidomide(1)Isolated splenic CD4+ T cells were differentiated into myelin-specific CD4+ T cells after treated with anti-CD3.The percentage of Th1 and Th17 in myelin-specific CD4+ T cells was determined by flow cytometry.(2)CD4+ T cells were labeled with carboxyfluorescein succinimidyl amino ester(CFSE)and the proportion of CFSE-attenuated CD4+ T cells was detected by flow cytometry.(3)Splenic CD4+ T cells were isolated from MOG-treated wild-type mice,co-cultured with lenalidomide-treated BMDMs at a ratio of 1:4 after CSFE labeling,and the proportion of CFSE-attenuated CD4+ T cells was detected by flow cytometry.(4)50 mg/kg clodronate liposome and its empty liposome control were administered by tail vein injection on days 7,8,11,12,15,and 16 after modeling.By using Kono's method,the clinical scores of EAE were calculated to determine the role of macrophages in lenalidomide treating EAE.(5)Flow cytometry was used to detect the ratio of M2 and M1 macrophages in the spleen and DLN of EAE mice in the model group and the lenalidomide-treated group on day 17 after modeling.(6)Flow cytometry was used to detect the ratio of M2 macrophages in CNS of EAE mice in the model group and the lenalidomide-treated group on day 17 after modeling.(7)Flow cytometry was used to verify the effect of lenalidomide on macrophage polarization.(8)On day 9,14 and 19 after model establishment,BMDMs without or with lenalidomide treatment were intravenously injected into EAE mice,and the clinical scores were obtained by using Kono's method to confirm the role M2 macrophage plays in the therapeutic effect of lenalidomide on EAE.3.M2 macrophages-secreted IL10 mediates the therapeutic effect of lenalidomide(1)Real-time PCR was used to detect the relative expression levels of Il4,Il10,Il13 and Tgf-? in BMDMs after lenalidomide treatment.(2)The effect of lenalidomide on IL10 secretion in serum,spleen and spinal cord of EAE mice was detected by ELISA on day 17 after modeling.(3)The effect of lenalidomide on the ratio of CD206+ IL10+macrophages in spleen and DLN on day 17 after modeling was detected by flow cytometry.(4)BMDMs were isolated from wild-type mice and Il10-/-mice,and western blotting was used to detect the effect of lenalidomide on the protein level of M2 macrophage gene Ym1.(5)The EAE disease model was constructed using wild-type and Il10-/-mice,and the clinical scores were calculated by Kono's method to determine the effect of IL10 deficiency on lenalidomide inhibiting EAE disease progression.(6)Immunohistochemistry of MBP and LFB staining were used to investigate the myelin sheath changes in the wild-type and Il10-/-EAE mice treated with lenalidomide on day 15 after model establishment.(7)Adaptive transfer of untreated wild-type BMDMs,lenalidomide-treated wild-type BMDMs or lenalidomide-treated Il10-/-BMDMs to the tail vein were performed on day 12 after modeling.By using Kono's method,EAE clinical scores were calculated to determine the effect of IL 10-deficient macrophages on lenalidomide treating EAE.4.Lenalidomide regulates M2 macrophages polarization via IL10-STAT3 pathway(1)Western blotting was used to investigate the different activation state of STAT6,AKT and STAT3 by lenalidomide in RAW264.7 and BMDMs.(2)Western blotting was used to investigate different protein levels of IL10 and p-STAT3 in RAW264.7 and BMDMs cells treated with lenalidomide for different duration of time.(3)The different IL10 cytokine secretion level in the supernatant of RAW264.7 and BMDMs cells after lenalidomide treatment was determined by using ELISA method.(4)The effect of lenalidomide on the expression levels of p-STAT3 and Ym1 was investigated after IL10 was neutralized with IL10 antibody.(5)The inhibition of STAT3 by STAT3 inhibitor Cucurbitacin(CuCu)was used to investigate the effect of lenalidomide on the expression levels of p-STAT3 and Ym1.Results1.The therapeutic effect of lenalidomide on EAE(1)On day 8 after the construction of the chronic mouse EAE model,mice were intragastrically administered with 30 mg/kg lenalidomide and 10 mg/kg dexamethasone(DXM)as a positive control.The result showed that both lenalidomide and DXM could downregulate the clinical score of EAE mice,in which in acute phase(day 17),DXM group scored 0.67 ± 0.15 points(p<0.001)and lenalidomide group got 1.17 ± 0.30 points(p<0.001),which is much lower than the control group(about 3 points).(2)On day 8 after the construction of the chronic mouse EAE model,mice were intragastrically administered with 30 mg/kg lenalidomide,and the ratio of IFNy+ CD4+ T(Th1)and IL17+CD4+ T(Th17)cells in spleen and DLN was detected on day 17 in the acute phase.The data revealed that lenalidomide could downregulate the percentage of Thl cells(15.81 ±0.97%to 5.58 ± 0.57%,p<0.001)and Th17 cells(13.03 ± 0.93%to 4.63 ± 0.21%,p<0.001).At the same time,the proportion of Th1 cells in DLN(12.18 ± 0.92%to 5.28 ±0.47%,p<0.001)and the ratio of Th17 cells(15.69 ± 1.11%to 5.53 ± 0.36%,p<0.001)were also downregulated.(3)On day 17,B220+ B cells and CD4+ Foxp3+ Treg cells in the spleen and DLN of EAE mice were detected.It was found that there was no significant change in the percentage of B cells in spleen and DLN after lenalidomide administration(15.62 ± 0.72%to 14.40 ± 0.69%,p= 0.2501;29.29 ± 1.25%to 25.65 ± 1.39%,p =0.0797),and neither in Treg cells in spleen and DLN(14.53 ± 1.04%to 16.52 ± 1.49%,p = 0.3000;14.93 ± 0.96%to 17.71 ± 1.06%,p=0.0806).(4)Mononuclear cells in the CNS of EAE mice were isolated and counted on day 17,and the result showed that the number of MNCs was significantly decreased after lenalidomide treatment(466,700 ±29,600 to 194,700 ± 18,000,p<0.001).(5)The ratio of Thl and Th17 cells in the CNS of EAE mice was detected on day 17.The result showed that the proportion of Th1 and Th 17 cells in the CNS was significantly downregulated after lenalidomide administration(5.77±0.51%to 2.51 ±0.36%,p<0.001;6.01 ±0.69%to 2.83±0.38%,P<0.001).The above results showed that lenalidomide could reduce the number of Th1 and Th1 7 cells in peripheral immune organs and the CNS in EAE mice,suggesting that lenalidomide could inhibit EAE autoimmune response and therefore inhibit CNS inflammation and disease progression.2.The role of macrophages in EAE treated with lenalidomide.(1)By directly treating CD4+ T cells with lenalidomide,lenalidomide showed no significant effect on the proportion of proliferating CD4+ T cells(6.19 ± 1.29%to 7.19 ±0.91%,p = 0.1238).(2)By in vitro co-culture of lenalidomide-treated BMDMs with CD4+T cells,it showed that lenalidomide-treated BMDMs significantly downregulated the proportion of proliferating CD4+ T cells(7.53 ± 0.35%to 4.36 ± 0.34%,p<0.01).(3)After constructing the chronic mouse EAE model,mice were administered with clodronate liposomes to eliminate macrophages in vivo.The data showed that the therapeutic effect of lenalidomide on EAE was nearly completely inhibited after macrophages were eliminated,indicated by similar clinical scores between the lenalidomide-clodronate liposomes and the clodronate liposomes or empty liposomes group(p>0.05).(4)On day 17,the ratio of M2(F4/80+ CD206+)and M1(F4/80+ CD86+)macrophages in the spleen and DLN of EAE mice was detected,and it was found that the lenalidomide significantly upregulated the proportion of M2 macrophages in the spleen(15.99 ± 1.14%to 30.81 ± 2.48%,p<0.001),but had no effect on the proportion of M1 macrophages in the spleen(32.72 ± 1.54%to 28.47±1.20%,p = 0.0544).The data was similar in DLN(M2,14.47 ± 1.02%to 26.82 ± 2.49%,p<0.01;M1,19.85 ± 1.83%to 18.76 ± 1.49%,p = 0.6529).(5)On day 17,the proportion of M2(CD11b+ CD45hi CD206+)macrophages in the CNS of EAE mice was detected.The result showed that lenalidomide significantly upregulated the proportion of M2 macrophages in the CNS(3.94 ± 0.31%to 8.36 ± 0.61%,p<0.001).(6)After the isolation of mouse BMDMs,the pro-M2 effect of lenalidomide was verified.The data revealed that 4h-treatment of lenalidomide could significantly increase the proportion of M2 cells in BMDMs(20.22 ±2.06%to 48.96 ± 5.54%,p<0.01),but did not have a significant effect on M1 proportion(18.45 ± 1.73%to 21.22 ± 2.15%,p = 0.3241).(7)After constructing the chronic mouse EAE model,the lenalidomide-treated BMDMs were then adoptively transferred into mice.The result showed that the average clinical score of the lenalidomide-BMDMs group(1.44 ± 0.13 points)was significantly lower than that of the control-BMDMs group(2.18± 0.22 points,p<0.01).On day 18 in acute phase,the scores are 1.93 ± 0.31 points and 2.90 ± 0.08 points,respectively(p<0.001).The above results showed that M2 macrophages polarization plays a key role in the therapeutic effect of lenalidomide on EAE.However,lenalidomide does not affect T cells directly.Lenalidomide is likely to regulate M2 macrophages/T cells crosstalk and subsequently inhibit CNS autoimmunity,suppress CNS inflammation and protect myelin.3.M2 macrophages-secreted IL10 mediates the therapeutic effect of lenalidomide.(1)BMDMs were treated with 25 nM for 4 h,then the expression level of 114,1110,1113 and Tgf-? was detected.The result showed that lenalidomide significantly upregulated the mRNA level of Il4,Il13 and Tgf-?,with 114 upregulated 7.41 ± 0.80 times(p<0.01),Il13 5.63 ± 0.51 times(p<0.01)and Tgf-? 13.96 ± 1.30 times(p<0.01).Especially for IL10,it was upregulated 27.70 ± 1.32 times(p<0.001).(2)The secretion of IL10 in serum,spleen and spinal cord was measured by ELISA on day 17 after the construction of the EAE model.The result showed that consisteent with the upregulation of mRNA level,the IL10 secretion in serum was upregulated from 65.00 ± 4.36 pg/mL to 125.00 ±4.36 pg/mL(p<0.001),3.18 ± 0.27 pg/mg protein to 7.25 ± 0.65 pg/mg protein in spleen(p<0.01)and 18.86 ± 1.15 pg/mg protein to 47.06 ± 7.57 pg/mg protein in spinal cord(p<0.01).(3)The ratio of CD206+ IL10+ macrophages in the spleen and DLN was measured on day 17 after the construction of the EAE model.The data showed that lenalidomide significantly upregulated the proportion of CD206+ IL10+ macrophages in the spleen and DLN(5.59 ± 0.91%to 20.30 ± 1.82%,p<0.001;10.70 ± 1.07%to 25.00± 1.44%,p<0.001).(4)BMDMs were isolated from wild-type mice and Il10-/-mice,and the expression of Yml was detected after treatment with 25 nM lenalidomide for 4 h.The result showed that the Ym1 protein level in IL10-deficient BMDMs was significantly downregulated.(5)30 mg/kg lenalidomide was given on day 8 after the chronic EAE model was constructed using wild-type and Il10-/-mice.The data showed that the IL 10-deficient EAE mice scored significantly higher than the wild-type group(p<0.001 since day 10).On day 17 in the acute phase,wild-type mice scored 3.33 ± 0.25 points,while wild-type lenalidomide mice got 1.10 ± 0.39(p<0.001),and Il10-/-model and Il10-/-lenalidomide mice scored 2.50 ± 1.00 points and 3.50 ± 0.00 points,respectively(p>0,05,both).(6)On day 17,the severity of myelin sheath damage in the spinal cord of EAE mice was detected.The result showed that the MBP expression in the white matter region of the Il10-/-EAE mice was significantly reduced after administered with lenalidomide,and LFB staining appeared to be much lighter in the white matter region and even vacuolated in Il10-/-group.(7)Wild-type mice were successfully transferred with lenalidomide-treated wild-type or Il10-/-mice BMDMs.On day 14,only lenalidomide-treated wild-type BMDMs(0.70 ± 0.10 points)had therapeutic effect on EAE(vs.vehicle-treated wild-type BMDMs,1.67 ± 0.17 points,p<0.01).However,the lenalidomide-treated Il10-/-BMDMs group showed similar scores with vehicle-treated wild-type BMDMs group(p>0.05,all).The above results showed that the therapeutic effect of lenalidomide on EAE is dependent on macrophages-derived IL10.4.Lenalidomide regulates M2 macrophages polarization via IL10-STAT3 pathway(1)RAW264.7 and BMDMs cells were treated with 0,6.25,25 nM lenalidomide for 4 h,and cells were collected to test the activation of STAT6,AKT and STAT3.The result showed that lenalidomide did not change the expression level of p-STAT6 and p-AKT,but upregulated the expression level of p-STAT3 in a dose-dependent manner.(2)RAW264.7 and BMDMs cells were treated with 25 nM lenalidomide,and cells were collected at different time points to detect the protein level of IL10 and p-STAT3.The data showed that both IL10 and p-STAT3 upregulated with the prolongation of time and the rise of IL10 is earlier than p-STAT3.(3)RAW264.7 and BMDMs cells were treated with 25 nM lenalidomide,and media was collected at different time points to detect the secretion of IL10.The result showed that IL10 secretion was significantly upregulated under lenalidomide stimulation.In RAW264.7,the IL10 level was upregulated from 83.44 ± 3.30 pg/mL to 104.36 ± 2.68 pg/mL in 4 h(p<0.05)and reached 125.58 ± 3.32 pg/mL in 12 h(p<0.001).In BMDMs,IL10 was upregulated from 13.12 ± 1.33 pg/mL to 32.12 ± 2.57 pg/mL in 4 h(p<0.001)and to 59.18 ± 3.17 pg/mL in 12 h(p<0.001).(4)RAW264.7 and BMDMs cells were administered with 2 ?M anti-IL10 antibody for 12 h and 25 nM lenalidomide for another 4 h to measure the variation of p-STAT3 and Ym1 levels.The result indicated that lenalidomide-induced p-STAT3 and Yml upregulation was almost reversed by anti-IL10.(5)RAW264.7 and BMDMs cells were administered with 2 ?M STAT3 inhibitor Cucurbitacin(CuCu)for 2 h and 25 nM lenalidomide for another 4 h to test the change of p-STAT3 and Yml levels.The data demonstrated that CuCu caused a significant inhibition of p-STAT3 levels,and the upregulation of Yml expression by lenalidomide was partially reversed.The above results showed that lenalidomide mainly promotes M2 macrophages polarization by upregulating IL 10 and subsequently activating STAT3 phosphorylation to form IL10-STAT3-IL10 positive feedback loop.ConclusionOur study found that lenalidomide targets macrophages and promotes M2 macrophages polarization through the IL10-STAT3 signaling pathway.Lenalidomide can promote IL10 expression and subsequently phosphorylate STAT3,which then translocates into the nucleus to promote transcriptional expression of M2 macrophages polarization-related genes,such as Argl,Yml and CD206.The anti-inflammatory cytokines secreted by M2 macrophages could promote M2 macrophage/T cell crosstalk and protect against EAE by reducing peripherally-derived pro-inflammatory Thl and Thl7 cells infiltrating CNS and inhibiting demyelination.Therefore,this study not only reveals the significant role of lenalidomide in the treatment of EAE,but also enriches the theories of macrophages regulating MS.These findings will help to expand the clinical indications of lenalidomide for MS,as well as provide a new therapeutic strategy for the design and discovery of novel MS agents. |
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