雷帕霉素通过上调TGF-β/smad信号通路减轻小鼠的实验性自身免疫性脑脊髓炎
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摘要
目的观察雷帕霉素对实验性自身免疫性脑脊髓炎(EAE)小鼠的治疗作用,并研究相关机制。方法建立C57BL/6小鼠EAE模型,免疫成功后将小鼠分为试验对照组,雷帕霉素小剂量组(0.3 mg/kg/d)和雷帕霉素大剂量组(1 mg/kg/d)。应用Knoz评分观察小鼠的临床评分,免疫组化法观察IL-17浸润情况,流式细胞仪观察外周Treg细胞的分化情况,ElISA法观察小鼠细胞因子的变化情况,Western blot法观察p-smad2和p-smad3表达情况。结果大剂量雷帕霉素可以明显改善EAE小鼠的神经功能缺损评分,在发病初期,高峰期及缓解期大剂量雷帕霉素组评分分别为(0.14±0.38),(0.43±1.13)和(0.14±0.37),而实验对照组分别为(1.14±0.69),(2.14±1.06)和(2.2±0.75);大剂量雷帕霉素组中可IL-17炎性细胞在在中枢神经系统的浸润为(43±1.83),而实验对照组为(153.5±7.02);大剂量雷帕霉素可以抑制IL-12,IFN-γ,IL-17和IL-23炎性细胞因子同时诱导IL-10和TGF-β抗炎性细胞因子;大剂量雷帕霉素组外周Treg细胞为(10.17±0.68),较实验对照组(3.52±0.32)明显增多(P<0.05);雷帕霉素和TGF-β具有协同免疫抑制作用,体外实验发现雷帕霉素和TGF-β共同作用淋巴细胞后Treg细胞为(13.66±1.89),较单用雷帕霉素(6.23±0.80)或单用TGF-β(4.87±0.85)均明显增多(P<0.05);雷帕霉素可以上调p-smad2和p-smad3的表达,并呈剂量依赖关系。结论雷帕霉素通过上调p-smad2和p-smad3表达来促进Treg细胞分化,进而改善EAE的神经功能缺损评分。
Objective To evaluate the efficacy of rapmycin for treatment of experimental autoimmune encephalomyelitis(EAE)in mice and explore the underlying mechanism. Methods An EAE model was established in C57BL/6 mice. After immunization, the mice were divided into model group and rapamycin groups treated daily with low-dose(0.3 mg/kg) or high-dose(1 mg/kg) rapamycin. The clinical scores of the mice were observed using Knoz score, the infiltration of IL-17 cells in the central nervous system(CNS) was determined using immunohistochemistry; the differentiation of peripheral Treg cells was analyzed using flow cytometry, and the changes in the levels of cytokines were detected with ELISA; the changes in the expressions of p-Smad2 and p-smad3 were investigated using Western blotting. Results High-dose rapamycin significantly improved the neurological deficits scores of EAE mice. In high-dose rapamycin group, the scores in the onset stage, peak stage and remission stage were 0.14±0.38, 0.43±1.13 and 0.14±0.37, respectively, as compared with 1.14±0.69, 2.14±1.06 and 2.2±0.75 in the model group. The infiltration of inflammatory IL-17 cells was significantly lower in high-dose rapamycin group than in the model group(43±1.83 vs 153.5±7.02). High-dose rapamycin obviously inhibited the production of IL-12, IFN-γ, IL-17 and IL-23 and induced the anti-inflammatory cytokines IL-10 and TGF-β. The percentage of Treg in CD4+ T cells was significantly higher in high-dose rapamycin group than in the model group(10.17 ± 0.68 vs 3.52 ± 0.32). In the in vitro experiment, combined treatments of the lymphocytes isolated from the mice with rapamycin and TGF-β induced a significant increase in the number of Treg cells(13.66±1.89) compared with the treatment with rapamycin(6.23±0.80) or TGF-β(4.87±0.85) alone. Rapamycin also obviously up-regulated the expression of p-Smad2 and p-Smad3 in the lymphocytes. Conclusion Rapamycin can promote the differentiation of Treg cells by up-regulating the expression of p-Smad2 and p-smad3 to improve neurological deficits in mice with EAE.
Objective To evaluate the efficacy of rapmycin for treatment of experimental autoimmune encephalomyelitis(EAE)in mice and explore the underlying mechanism. Methods An EAE model was established in C57BL/6 mice. After immunization, the mice were divided into model group and rapamycin groups treated daily with low-dose(0.3 mg/kg) or high-dose(1 mg/kg) rapamycin. The clinical scores of the mice were observed using Knoz score, the infiltration of IL-17 cells in the central nervous system(CNS) was determined using immunohistochemistry; the differentiation of peripheral Treg cells was analyzed using flow cytometry, and the changes in the levels of cytokines were detected with ELISA; the changes in the expressions of p-Smad2 and p-smad3 were investigated using Western blotting. Results High-dose rapamycin significantly improved the neurological deficits scores of EAE mice. In high-dose rapamycin group, the scores in the onset stage, peak stage and remission stage were 0.14±0.38, 0.43±1.13 and 0.14±0.37, respectively, as compared with 1.14±0.69, 2.14±1.06 and 2.2±0.75 in the model group. The infiltration of inflammatory IL-17 cells was significantly lower in high-dose rapamycin group than in the model group(43±1.83 vs 153.5±7.02). High-dose rapamycin obviously inhibited the production of IL-12, IFN-γ, IL-17 and IL-23 and induced the anti-inflammatory cytokines IL-10 and TGF-β. The percentage of Treg in CD4+ T cells was significantly higher in high-dose rapamycin group than in the model group(10.17 ± 0.68 vs 3.52 ± 0.32). In the in vitro experiment, combined treatments of the lymphocytes isolated from the mice with rapamycin and TGF-β induced a significant increase in the number of Treg cells(13.66±1.89) compared with the treatment with rapamycin(6.23±0.80) or TGF-β(4.87±0.85) alone. Rapamycin also obviously up-regulated the expression of p-Smad2 and p-Smad3 in the lymphocytes. Conclusion Rapamycin can promote the differentiation of Treg cells by up-regulating the expression of p-Smad2 and p-smad3 to improve neurological deficits in mice with EAE.
引文
[1]Schloeder J,Berges C,Luessi F,et al.Dimethyl fumarate therapy significantly improves the responsiveness of T cells in multiple sclerosis patients for immunoregulation by regulatory T cells[J].Int J Mol Sci,2017,18(2):271.
[2]Sakaguchi S.Naturally arising CD4+regulatory t cells for immunologic self-tolerance and negative control of immune responses[J].Annu Rev Immunol,2004,22:531-62.
[3]Mcpherson RC,Turner DG,Iris M,et al.T-bet expression by Foxp3+T regulatory cells is not essential for their suppressive function in CNS autoimmune disease or colitis[J].Frontiers Immunol,2015,6:69.
[4]Zozulya AL,Wiendl H.The role of regulatory T cells in multiple sclerosis[J].Nat Clin Pract Neurol,2008,4(7):384-98.
[5]Zhao YG,Wang Y,Guo Z,et al.Dihydroartemisinin ameliorates inflammatory disease by its reciprocal effects on Th and regulatory T cell function via modulating the mammalian target of rapamycin pathway[J].J Immunol,2012,189(9):4417-25.
[6]Koga T,Hedrich CM,Mizui MA,et al.CaMK4-dependent activation of AKT/mTOR and CREM-alpha underlies autoimmunity-associated Th17 imbalance[J].J Clini Invest,2014,124(5):2234-45.
[7]Chapman NM,Chi HB.mTOR signaling,tregs and immune modulation[J].Immunotherapy,2014,6(12):1295-311.
[8]Kim KW,Chung BH,Kim BM,et al.The effect of mammalian target of rapamycin inhibition on T helper type 17 and regulatory T cell differentiation in vitro and in vivo in kidney transplant recipients[J].Immunology,2015,144(1):68-78.
[9]Dello Russo C,Lisi L,Feinstein DL.mTOR kinase,a key player in the regulation of glial functions:relevance for the therapy of multiple sclerosis[J].Glia,2013,61(3):301-11.
[10]Lisi L,Navarra P,Cirocchi R,et al.Rapamycin reduces clinical signs and neuropathic pain in a chronic model of experimental autoimmune encephalomyelitis[J].J Neuroimmunol,2012,243(1/2):43-51.
[11]Yang HJ,Rudge DG,Koos JD,et al.mTOR kinase structure,mechanism and regulation[J].Nature,2013,497(7448):217.
[12]Schmidt A,Eriksson M,Shang MM,et al.Comparative analysis of protocols to induce human CD4+Foxp3+regulatory T cells by combinations of IL-2,TGF-beta,retinoic acid,rapamycin and butyrate[J].PLoS One,2016,11(2):e0148474.
[13]Biswas M,Sarkar D,Kumar SR,et al.Synergy between rapamycin and FLT3 ligand enhances plasmacytoid dendritic cell-dependent induction of CD4+CD25+FoxP3+Treg[J].Blood,2015,125(19):2937-47.
[14]Sun IH,Oh MH,Zhao L,et al.mTOR complex 1 signaling regulates the Generation and function of central and effector Foxp3(+)regulatory T cells[J].J Immunol,2018,201(2):481-92.
[15]Zheng SG,Gray JD,Ohtsuka K,et al.Generation ex vivo of TGF-beta-producing regulatory T cells from CD4+CD25-precursors[J].JImmunol,2002,169(8):4183-9.
[16]Benson MJ,Pino-Lagos K,Rosemblatt MA.All-trans retinoic acid mediates enhanced T reg cell growth,differentiation,and gut homing in the face of high levels of co-stimulation[J].J Exp Med,2007,204(8):1765-74.
[17]Mucida D,Park Y,Kim G,et al.Reciprocal TH17 and regulatory Tcell differentiation mediated by retinoic acid[J].Science,2007,317(5835):256-60.
[18]Nolting J,Daniel C,Reuter S,et al.Retinoic acid can enhance conversion of naive into regulatory T cells independently of secreted cytokines[J].J Exp Med,2009,206(10):2131-9.
[19]Candia E,Reyes P,Covian C,et al.Single and combined effect of retinoic acid and rapamycin modulate the Generation,activity and homing potential of induced human regulatory T cells[J].PLoS One,2017,12(7):e0182009.
[20]Zorn E,Nelson EA,Mohseni M,et al.IL-2 regulates FOXP3expression in human CD4+CD25+regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo[J].Blood,2006,108(5):1571-9.
[21]Wu Y,Wang W,Peng XM,et al.Rapamycin upregulates connective tissue growth factor expression in hepatic progenitor cells through TGF-beta-Smad2 dependent signaling[J].Front Pharmacol,2018,9:877.
[22]Tone Y,Furuuchi K,Kojima Y,et al.Smad3 and NFAT cooperate to induce Foxp3 expression through its enhancer[J].Nat Immunol,2008,9(2):194-202.
[23]Wang YY,Jiang H,Wang YC,et al.Deletion of Smad3 improves cardiac allograft rejection in mice[J].Oncotarget,2015,6(19):17016-30.
[24]Xu LL,Kitani A,Stuelten C,et al.Positive and negative transcriptional regulation of the Foxp3 gene is mediated by access and binding of the Smad3 protein to enhancer I[J].Immunity,2010,33(3):313-25.
[25]Zheug SG,Lu L,Wang JL,et al.Role of Smad and non-Smad signals in the development of Th17 and regulatory T cells[J].J Immunol,2010,184(1):4295-306.
[26]Tomohito T,Yu W,Takashi S,et al.Smad2 and Smad3 are redundantly essential for the TGF-b-mediated regulation of regulatory T plasticity and Th1 development[J].J Immunol,2010,185:842-55
[27]Zhang W,Dong Z,Shen M,et al.Combined administration of a novel mutant TGF-β1/Fc and rapamycin promotes induction of regulatory T cells and islet allograft tolerance[J].J Immunol,2010,185(8):4750-9.
[28]Kawamoto K,Pahuja A,Hering BJ.Transforming growth factor beta1(TGF-beta 1)and rapamycin synergize to effectively suppress human T cell responses via upregulation of FoxP3(+)Tregs[J].Transpl Immunol,2010,23(1/2):28-33.
[29]Kappos L,Barkhof F,Desmet A,et al.The effect of oral temsirolimus on new magneticresonance imaging scan lesions,brain atrophy,and the number of relapses inmultiplesclerosis:results from a randomized,controlled,clinical trial[J].J Neurol,2005,252:S46.
[30]Neuhaus O,Kieseier BC,Hartung,HP,et al.Immunosupperssive agents in multiple sclerosis[J].Am Soc Exp Neurotherap,2007,4:654-60.
[31]Hou H,Cao R,Quan M et al.Rapamycin and fingolimod modulate Treg/Th17 cells in experimental autoimmune encephalomyelitis by regulating the Akt-mTOR and MAPK/ERK pathways[J].JNeuroimmunol,2018,324:26-34.
[2]Sakaguchi S.Naturally arising CD4+regulatory t cells for immunologic self-tolerance and negative control of immune responses[J].Annu Rev Immunol,2004,22:531-62.
[3]Mcpherson RC,Turner DG,Iris M,et al.T-bet expression by Foxp3+T regulatory cells is not essential for their suppressive function in CNS autoimmune disease or colitis[J].Frontiers Immunol,2015,6:69.
[4]Zozulya AL,Wiendl H.The role of regulatory T cells in multiple sclerosis[J].Nat Clin Pract Neurol,2008,4(7):384-98.
[5]Zhao YG,Wang Y,Guo Z,et al.Dihydroartemisinin ameliorates inflammatory disease by its reciprocal effects on Th and regulatory T cell function via modulating the mammalian target of rapamycin pathway[J].J Immunol,2012,189(9):4417-25.
[6]Koga T,Hedrich CM,Mizui MA,et al.CaMK4-dependent activation of AKT/mTOR and CREM-alpha underlies autoimmunity-associated Th17 imbalance[J].J Clini Invest,2014,124(5):2234-45.
[7]Chapman NM,Chi HB.mTOR signaling,tregs and immune modulation[J].Immunotherapy,2014,6(12):1295-311.
[8]Kim KW,Chung BH,Kim BM,et al.The effect of mammalian target of rapamycin inhibition on T helper type 17 and regulatory T cell differentiation in vitro and in vivo in kidney transplant recipients[J].Immunology,2015,144(1):68-78.
[9]Dello Russo C,Lisi L,Feinstein DL.mTOR kinase,a key player in the regulation of glial functions:relevance for the therapy of multiple sclerosis[J].Glia,2013,61(3):301-11.
[10]Lisi L,Navarra P,Cirocchi R,et al.Rapamycin reduces clinical signs and neuropathic pain in a chronic model of experimental autoimmune encephalomyelitis[J].J Neuroimmunol,2012,243(1/2):43-51.
[11]Yang HJ,Rudge DG,Koos JD,et al.mTOR kinase structure,mechanism and regulation[J].Nature,2013,497(7448):217.
[12]Schmidt A,Eriksson M,Shang MM,et al.Comparative analysis of protocols to induce human CD4+Foxp3+regulatory T cells by combinations of IL-2,TGF-beta,retinoic acid,rapamycin and butyrate[J].PLoS One,2016,11(2):e0148474.
[13]Biswas M,Sarkar D,Kumar SR,et al.Synergy between rapamycin and FLT3 ligand enhances plasmacytoid dendritic cell-dependent induction of CD4+CD25+FoxP3+Treg[J].Blood,2015,125(19):2937-47.
[14]Sun IH,Oh MH,Zhao L,et al.mTOR complex 1 signaling regulates the Generation and function of central and effector Foxp3(+)regulatory T cells[J].J Immunol,2018,201(2):481-92.
[15]Zheng SG,Gray JD,Ohtsuka K,et al.Generation ex vivo of TGF-beta-producing regulatory T cells from CD4+CD25-precursors[J].JImmunol,2002,169(8):4183-9.
[16]Benson MJ,Pino-Lagos K,Rosemblatt MA.All-trans retinoic acid mediates enhanced T reg cell growth,differentiation,and gut homing in the face of high levels of co-stimulation[J].J Exp Med,2007,204(8):1765-74.
[17]Mucida D,Park Y,Kim G,et al.Reciprocal TH17 and regulatory Tcell differentiation mediated by retinoic acid[J].Science,2007,317(5835):256-60.
[18]Nolting J,Daniel C,Reuter S,et al.Retinoic acid can enhance conversion of naive into regulatory T cells independently of secreted cytokines[J].J Exp Med,2009,206(10):2131-9.
[19]Candia E,Reyes P,Covian C,et al.Single and combined effect of retinoic acid and rapamycin modulate the Generation,activity and homing potential of induced human regulatory T cells[J].PLoS One,2017,12(7):e0182009.
[20]Zorn E,Nelson EA,Mohseni M,et al.IL-2 regulates FOXP3expression in human CD4+CD25+regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo[J].Blood,2006,108(5):1571-9.
[21]Wu Y,Wang W,Peng XM,et al.Rapamycin upregulates connective tissue growth factor expression in hepatic progenitor cells through TGF-beta-Smad2 dependent signaling[J].Front Pharmacol,2018,9:877.
[22]Tone Y,Furuuchi K,Kojima Y,et al.Smad3 and NFAT cooperate to induce Foxp3 expression through its enhancer[J].Nat Immunol,2008,9(2):194-202.
[23]Wang YY,Jiang H,Wang YC,et al.Deletion of Smad3 improves cardiac allograft rejection in mice[J].Oncotarget,2015,6(19):17016-30.
[24]Xu LL,Kitani A,Stuelten C,et al.Positive and negative transcriptional regulation of the Foxp3 gene is mediated by access and binding of the Smad3 protein to enhancer I[J].Immunity,2010,33(3):313-25.
[25]Zheug SG,Lu L,Wang JL,et al.Role of Smad and non-Smad signals in the development of Th17 and regulatory T cells[J].J Immunol,2010,184(1):4295-306.
[26]Tomohito T,Yu W,Takashi S,et al.Smad2 and Smad3 are redundantly essential for the TGF-b-mediated regulation of regulatory T plasticity and Th1 development[J].J Immunol,2010,185:842-55
[27]Zhang W,Dong Z,Shen M,et al.Combined administration of a novel mutant TGF-β1/Fc and rapamycin promotes induction of regulatory T cells and islet allograft tolerance[J].J Immunol,2010,185(8):4750-9.
[28]Kawamoto K,Pahuja A,Hering BJ.Transforming growth factor beta1(TGF-beta 1)and rapamycin synergize to effectively suppress human T cell responses via upregulation of FoxP3(+)Tregs[J].Transpl Immunol,2010,23(1/2):28-33.
[29]Kappos L,Barkhof F,Desmet A,et al.The effect of oral temsirolimus on new magneticresonance imaging scan lesions,brain atrophy,and the number of relapses inmultiplesclerosis:results from a randomized,controlled,clinical trial[J].J Neurol,2005,252:S46.
[30]Neuhaus O,Kieseier BC,Hartung,HP,et al.Immunosupperssive agents in multiple sclerosis[J].Am Soc Exp Neurotherap,2007,4:654-60.
[31]Hou H,Cao R,Quan M et al.Rapamycin and fingolimod modulate Treg/Th17 cells in experimental autoimmune encephalomyelitis by regulating the Akt-mTOR and MAPK/ERK pathways[J].JNeuroimmunol,2018,324:26-34.