索曲妥林对人T细胞产生细胞因子抑制作用的机制研究
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摘要
目的观察索曲妥林(sotrastaurin或AEB071)对人外周血中单个核细胞(PBMCs)、纯化CD4~+T细胞和CD8~+T细胞分泌细胞因子的抑制作用及其机制。方法采集健康志愿者的静脉血,肝素抗凝,密度梯度离心法分离PBMCs后纯化CD4~+T和CD8~+T细胞,应用抗CD3抗体联合CD28抗体进行刺激,加或不加入AEB071培养后,检测细胞因子的产生、转录因子的表达和细胞的活化与增殖。结果索曲妥林处理不促进外周血T细胞的凋亡,而能抑制T细胞分泌细胞因子IFN-γ、TNF-α和IL-2,且呈时间和剂量依赖性;进一步研究发现,索曲妥林通过下调STAT-1和STAT-4的磷酸化以及T-bet的表达抑制细胞因子的产生。同时经研究发现,索曲妥林处理后抑制T细胞的活化和增殖。结论索曲妥林抑制T细胞转录因子的表达与磷酸化而抑制细胞因子的产生,从而抑制人体免疫功能。为此,进一步研究索曲妥林免疫抑制的机制,对索曲妥林在器官移植、银屑病和溃疡性结肠炎的治疗方面的应用,具有参考价值。
To investigate the inhibitory effect and mechanism of sotrastaurin on the production of cytokines,human PBMCs and purified CD4~+ or CD8~+T cells were stimulated with anti-CD3 plus anti-CD28 or with immobilized anti-CD3 and soluble anti-CD28 in the presence or absence of sotrastaurin. The levels of IFN-γ, TNF-α and IL-2 in the culture supernatants were detected by ELISA; the production of cytokines and transcription factors including T-bet, p-STAT-1 as well as p-STAT-4 were analyzed by FACS. Data showed that sotrastaurin lacked sufficient cytotoxicity toward T cells from healthy volunteers but exhibited significant antiproliferation. In addition, sotrastaurin had significant suppressive effects on the production of IFN-γ, TNF-α and IL-2 in a dosetime-dependent manner. Further study revealed that sotrastaurin down-regulated the phosphorylation of STAT4 and STAT1 and the expression of T-bet. All these results indicated that the main mechanism of sotrastaurin inhibiting the cytokine production is down-regulating the phosphorylation of STAT1 and STAT4 and the expression of T-bet.The further study on the mechanism of sotrastaurin immunosuppression is of great significance for its application in the treatment of organ transplantation, psoriasis and ulcerative colitis.
To investigate the inhibitory effect and mechanism of sotrastaurin on the production of cytokines,human PBMCs and purified CD4~+ or CD8~+T cells were stimulated with anti-CD3 plus anti-CD28 or with immobilized anti-CD3 and soluble anti-CD28 in the presence or absence of sotrastaurin. The levels of IFN-γ, TNF-α and IL-2 in the culture supernatants were detected by ELISA; the production of cytokines and transcription factors including T-bet, p-STAT-1 as well as p-STAT-4 were analyzed by FACS. Data showed that sotrastaurin lacked sufficient cytotoxicity toward T cells from healthy volunteers but exhibited significant antiproliferation. In addition, sotrastaurin had significant suppressive effects on the production of IFN-γ, TNF-α and IL-2 in a dosetime-dependent manner. Further study revealed that sotrastaurin down-regulated the phosphorylation of STAT4 and STAT1 and the expression of T-bet. All these results indicated that the main mechanism of sotrastaurin inhibiting the cytokine production is down-regulating the phosphorylation of STAT1 and STAT4 and the expression of T-bet.The further study on the mechanism of sotrastaurin immunosuppression is of great significance for its application in the treatment of organ transplantation, psoriasis and ulcerative colitis.
引文
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[15]Biswas S,Myszor M,De Silva A.Management of psoriasis in patients treated with anti-TNF-αtherapy for inflammatory bowel disease[J].J Crohns Colitis,2013,7(12):708-709.
[16]Skvara H,Dawid M,Kleyn E,et al.The PKC inhibitor AEB071 may be a therapeutic option for psoriasis[J].J Clin Invest,2008,118(9):3151-3159.477
[17]Zanin Zhorov A,Ding Y,Kumari S.et al.Protein kinase C-theta mediates negative feedback on regulatory T cell function[J].Science,2010,328(5976):372-376.
[18]Zanin Zhorov A,Lin J,Scher J,et al.Scaffold protein Disc large homolog 1 is required for T-cell receptor-induced activation of regulatory T-cell function[J].Proc Natl Acad Sci U S A,2012,109(5):1625-1630.
[19]Mateen S,Zafar A,Moin S,et al.Understanding the role of cytokines in the pathogenesis of rheumatoid arthritis[J].Clin Chim Acta,2016,455:161-171.
[20]Zhu TZ,Li XM,Luo LH,et al.beta-Elemene inhibits proliferation through crosstalk between glia maturation factor beta and extracellular signalregulated kinase 1/2 and impairs drug resistance to temozolomide in glioblastoma cells[J].Mol Med Rep,2014,10(2):1122-1128.
[21]Bermejo M,López Huertas MR,Hedgpeth J,et al.Analysis of protein kinase C theta inhibitors for the control of HIV-1replication in human CD4+T cells reveals an effect on retrotranscription in addition to viral transcription[J].Biochem Pharmacol,2015,94(4):241-256.
[2]周俐斐,赵家文,侯桂兰,等.PKCθ小分子抑制剂及其免疫抑制作用的研究进展[J].中国现代应用药学,2017,1(34):122-129.
[3]Chen YM,Chuang HC,Lin WC,et al.Germinal center kinase-like kinase overexpression in T cells as a novel biomarker in rheumatoid arthritis[J].Arthritis Rheum,2013,65(10):2573-2582.
[4]Vedran B,Juan TW,Nabila S.PKC-theta in regulatory and effector T-cell functions[J].Front Immunol,2015,6:530.
[5]Igumenova T.Dynamics and membrane interactions of protein kinase C[J].Biochemistry,2015,54(32):4953-4968.
[6]Vincenti F,Kirk AD.What’s next in the pipeline[J].Am JTransplant,2008,8(10):1972-1981.
[7]He X,Koenen HJ,Smeets RL,et al.Targeting PKC in human T cells using sotrastaurin(AEB071)preserves regulatory T cells and prevents IL-17 production[J].JInvest Dermatol,2014,134(4):975-983.
[8]Wagner J,Von Mattp,Faller B,et al.Structure-activity relationship and pharmacokinetic studies of sotrastaurin(AEB071),a promising novel medicinefor prevention of graft rejection and treatment of psoriasis[J].J Med Chem,2011,54(17):6028-6039.
[9]Russ GR,Tedesco Silva H,Kuypers DR,et al.Efficacy of sotrastaurin plus tacrolimus after de novo kidney transplantation:randomized,phase II trial results[J].Am JTransplant,2013,13(7):1746-1756.
[10]赵珺,吴琼丽,沈娟,等.人外周血中γδT细胞亚群、表型和细胞因子产生的研究[J].免疫学杂志,2018,34(1):1-9.
[11]谢淑华,沈娟,张燕楠.IL-12恢复化疗药物对人细胞免疫功能的抑制作用[J].免疫学杂志,2018,6(34):461-475.
[12]吴昆仑,赵珺,吴琼丽,等.托法替尼抑制外周血中T细胞细胞因子的分泌及机制[J].细胞与分子免疫学杂志,2017,33(11):1448-1455.
[13]El Gamal D,Williams K,La Follette TD,et al.PKC-βas a therapeutic target in CLL:PKC inhibitor AEB071demonstrates preclinical activity in CLL[J].Blood,2014,124(9):1481-1491.
[14]丁晓岚,王婷琳,沈佚葳,等.中国六省市银屑病流行病学调查[J].中国皮肤性病学杂志,2010,24(7):598-601.
[15]Biswas S,Myszor M,De Silva A.Management of psoriasis in patients treated with anti-TNF-αtherapy for inflammatory bowel disease[J].J Crohns Colitis,2013,7(12):708-709.
[16]Skvara H,Dawid M,Kleyn E,et al.The PKC inhibitor AEB071 may be a therapeutic option for psoriasis[J].J Clin Invest,2008,118(9):3151-3159.477
[17]Zanin Zhorov A,Ding Y,Kumari S.et al.Protein kinase C-theta mediates negative feedback on regulatory T cell function[J].Science,2010,328(5976):372-376.
[18]Zanin Zhorov A,Lin J,Scher J,et al.Scaffold protein Disc large homolog 1 is required for T-cell receptor-induced activation of regulatory T-cell function[J].Proc Natl Acad Sci U S A,2012,109(5):1625-1630.
[19]Mateen S,Zafar A,Moin S,et al.Understanding the role of cytokines in the pathogenesis of rheumatoid arthritis[J].Clin Chim Acta,2016,455:161-171.
[20]Zhu TZ,Li XM,Luo LH,et al.beta-Elemene inhibits proliferation through crosstalk between glia maturation factor beta and extracellular signalregulated kinase 1/2 and impairs drug resistance to temozolomide in glioblastoma cells[J].Mol Med Rep,2014,10(2):1122-1128.
[21]Bermejo M,López Huertas MR,Hedgpeth J,et al.Analysis of protein kinase C theta inhibitors for the control of HIV-1replication in human CD4+T cells reveals an effect on retrotranscription in addition to viral transcription[J].Biochem Pharmacol,2015,94(4):241-256.