大鼠肝移植免疫耐受过程Th细胞和Treg细胞因子及信号通路蛋白的变化研究
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摘要
目的探讨大鼠肝移植免疫耐受过程中,辅助性T细胞(Th)和调节性T细胞(Treg)细胞因子及相关信号通路蛋白的变化与免疫耐受的关系。方法双袖套法建立原位肝移植模型,将大鼠分为3组:手术对照组(6只),假手术不进行肝移植;短期组(10只),术后存活10 d;耐受组(10只),术后存活100 d,移植肝功能恢复正常。检测各组大鼠肝功能指标如丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST),Th1细胞因子[干扰素(IFN)-γ、白细胞介素(IL)-2、肿瘤坏死因子(TNF)-α],Th2细胞因子(IL-4、IL-5、IL-6、IL-13),Th17细胞因子[粒细胞-巨噬细胞集落刺激因子(GM-CSF)、IL-17A],Treg细胞因子[IL-10、转化生长因子(TGF)-β、IL-12p]表达水平。对各组大鼠血清进行蛋白芯片检测。结果与手术对照组比较,短期组AST明显降低,ALT明显升高(P<0.05)。而耐受组与手术对照组的AST、ALT水平比较,差异均无统计学意义(均为P>0.05)。各组大鼠的肝组织中,与手术对照组比较,短期组Th1细胞因子IFN-γ、IL-2表达水平明显升高(均为P<0.05);耐受组Th2细胞因子IL-4的表达水平明显低于手术对照组(P<0.05);短期组Th2细胞因子IL-5、IL-6、IL-13的表达水平明显低于手术对照组(P<0.05);耐受组IL-17A的表达水平明显高于手术对照组(P<0.05)。耐受组IL-10、IL-12p的表达水平明显高于手术对照组(均为P<0.05),短期组TGF-β的表达水平明显高于手术对照组(P<0.05)。与手术对照组比较,短期组细胞间黏附分子(ICAM)-1、前血小板碱性蛋白(Ppbp)、神经纤毛蛋白(Neuropilin)-2、Notch-2蛋白的表达水平明显升高(均为P<0.05);耐受组趋化因子配基17(CXCL17)、ICAM-1、Neuropilin-2蛋白的表达水平明显升高(均为P<0.05),而B7-1蛋白的表达水平显著减低(P<0.05)。结论在大鼠肝移植自然免疫耐受过程中,Treg类细胞因子(IL-10、TGF-β、IL-12p), IL-6, IL-17以及细胞表面的跨膜信号通路分子(ICAM-1、Neuropilin-2、B7-1蛋白)在其中起到了重要的作用。
Objective To investigate the relationship between immune tolerance and the changes of helper T cell(Th), regulatory T cell(Treg) cytokines, related signaling pathway proteins during immune tolerance process in rat models of liver transplantation. Methods The orthotopic liver transplantation rat models were established by double-cuff technique. All rats were divided into 3 groups. In the operative control group(n=6), sham operation was performed without liver transplantation. In the short-term group(n=10), the rats survived for 10 d after liver transplantation. In the immune tolerance group(n=10), the rats survived for 100 d after operation and the function of the transplanted liver was restored to normal. The expression levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), Th1 cytokines[interferon(IFN)-γ, interleukin(IL)-2 and tumor necrosis factor(TNF)-α], Th2 cytokines(IL-4, IL-5, IL-6 and IL-13),Th17 cytokines [granulocyte-macrophage colony-stimulating factor(GM-CSF) and IL-17 A], Treg cytokines [IL-10,transforming growth factor(TGF)-β and IL-12 p] were quantitatively measured. The serum sample of rats in each group was detected by protein chip analysis. Results Compared with the operative control group, the AST level in the short-term group was significantly down-regulated, whereas the ALT level was significantly up-regulated(both P<0.05). However,the AST and ALT levels did not significantly differ between the immune tolerance group and operative control group(both P>0.05). In the liver tissues of rats in each group, the expression levels of Th1 cytokines IFN-γ and IL-2 in the shortterm group were significantly higher than those in the operative control group(both P<0.05). The expression level of Th2 cytokine IL-4 in the immune tolerance group was significantly lower than that in the operative control group(P<0.05).The expression levels of Th2 cytokines IL-5, IL-6 and IL-13 in the short-term group were significantly lower than those in the operative control group(all P<0.05). The expression level of IL-17 A in the immune tolerance group was significantly higher than that in the operative control group(P<0.05). In the immune tolerance group, the expression levels of IL-10 and IL-12 p were significantly higher than those in the operative control group(both P<0.05). The expression level of TGF-β in the short-term group was significantly higher than that in the operative control group(P<0.05). Compared with the operative control group, the expression levels of intercellular adhesion molecule(ICAM)-1, pro-platelet basic protein(Ppbp), Neuropilin-2, Notch-2 protein in the short-term group were significantly up-regulated(all P<0.05). The expression levels of CXC chemokine ligand 17(CXCL17), ICAM-1 and Neuroleptin-2 protein were markedly up-regulated(all P<0.05), whereas that of B7-1 protein was significantly down-regulated(P<0.05) in the immune tolerance group.Conclusions Treg cytokines(IL-10, TGF-β and IL-12 p), IL-6, IL-17 and trans-membrane signaling pathway molecules(ICAM-1, Neuropilin-2, B7-1 proteins) play a pivotal role in the natural immune tolerance process of rat models of liver transplantation.
Objective To investigate the relationship between immune tolerance and the changes of helper T cell(Th), regulatory T cell(Treg) cytokines, related signaling pathway proteins during immune tolerance process in rat models of liver transplantation. Methods The orthotopic liver transplantation rat models were established by double-cuff technique. All rats were divided into 3 groups. In the operative control group(n=6), sham operation was performed without liver transplantation. In the short-term group(n=10), the rats survived for 10 d after liver transplantation. In the immune tolerance group(n=10), the rats survived for 100 d after operation and the function of the transplanted liver was restored to normal. The expression levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), Th1 cytokines[interferon(IFN)-γ, interleukin(IL)-2 and tumor necrosis factor(TNF)-α], Th2 cytokines(IL-4, IL-5, IL-6 and IL-13),Th17 cytokines [granulocyte-macrophage colony-stimulating factor(GM-CSF) and IL-17 A], Treg cytokines [IL-10,transforming growth factor(TGF)-β and IL-12 p] were quantitatively measured. The serum sample of rats in each group was detected by protein chip analysis. Results Compared with the operative control group, the AST level in the short-term group was significantly down-regulated, whereas the ALT level was significantly up-regulated(both P<0.05). However,the AST and ALT levels did not significantly differ between the immune tolerance group and operative control group(both P>0.05). In the liver tissues of rats in each group, the expression levels of Th1 cytokines IFN-γ and IL-2 in the shortterm group were significantly higher than those in the operative control group(both P<0.05). The expression level of Th2 cytokine IL-4 in the immune tolerance group was significantly lower than that in the operative control group(P<0.05).The expression levels of Th2 cytokines IL-5, IL-6 and IL-13 in the short-term group were significantly lower than those in the operative control group(all P<0.05). The expression level of IL-17 A in the immune tolerance group was significantly higher than that in the operative control group(P<0.05). In the immune tolerance group, the expression levels of IL-10 and IL-12 p were significantly higher than those in the operative control group(both P<0.05). The expression level of TGF-β in the short-term group was significantly higher than that in the operative control group(P<0.05). Compared with the operative control group, the expression levels of intercellular adhesion molecule(ICAM)-1, pro-platelet basic protein(Ppbp), Neuropilin-2, Notch-2 protein in the short-term group were significantly up-regulated(all P<0.05). The expression levels of CXC chemokine ligand 17(CXCL17), ICAM-1 and Neuroleptin-2 protein were markedly up-regulated(all P<0.05), whereas that of B7-1 protein was significantly down-regulated(P<0.05) in the immune tolerance group.Conclusions Treg cytokines(IL-10, TGF-β and IL-12 p), IL-6, IL-17 and trans-membrane signaling pathway molecules(ICAM-1, Neuropilin-2, B7-1 proteins) play a pivotal role in the natural immune tolerance process of rat models of liver transplantation.
引文
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[19]REN J,ZHOU T,PILLI VSS,et al.Novel paracrine functions of smooth muscle cells in supporting endothelial regeneration following arterial injury[J].Circ Res,2019,124(8):1253-1265.DOI:10.1161/CIRCRESAHA.118.314567.
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[23]WATANABE M,FUJIHARA C,RADTKE AJ,et al.Co-stimulatory function in primary germinal center responses:CD40 and B7 are required on distinct antigenpresenting cells[J].J Exp Med,2017,214(9):2795-2810.DOI:10.1084/jem.20161955.
[24]KANG S,ZHANG C,OHNO T,et al.Unique B7-H1expression on masticatory mucosae in the oral cavity and trans-coinhibition by B7-H1-expressing keratinocytes regulating CD4+T cell-mediated mucosal tissue inflammation[J].Mucosal Immunol,2017,10(3):650-660.DOI:10.1038/mi.2016.89.
[2]叶林森,张英才,唐晖,等.长期生存的肝移植受者外周血免疫细胞分析[J].中华普通外科杂志,2017,32(6):508-511.DOI:10.3760/cma.j.issn.1007-631X.2017.06.014.YE LS,ZHANG YC,TANG H,et al.Peripheral blood immune cells in long-term survival patients after liver transplantation[J].Chin J Gen Surg,2017,32(6):508-511.DOI:10.3760/cma.j.issn.1007-631X.2017.06.014.
[3]HWANG W,LEE J.Pathophysiologic implications of cytokines secretion during liver transplantation surgery[J].Int J Med Sci,2018,15(14):1737-1745.DOI:10.7150/ijms.28382.
[4]LóPEZ-ABENTE J,BERNALDO-DE-QUIRóS E,CAMINO M,et al.Immune dysregulation and Th2polarization are associated with atopic dermatitis in heart-transplant children:a delicate balance between risk of rejection or atopic symptoms[J].Am J Transplant,2019,19(5):1536-1544.DOI:10.1111/ajt.15245.
[5]ETEGHADI A,PAK F,AHMADPOOR P,et al.Th1,Th2,Th17 cell subsets in two different immunosuppressive protocols in renal allograft recipients(sirolimus vs mycophenolate mofetil):a cohort study[J].Int Immunopharmacol,2019,67:319-325.DOI:10.1016/j.intimp.2018.12.033.
[6]LIU M,LI S,LI MO.TGF-βcontrol of adaptive immune tolerance:a break from Treg cells[J].Bioessays,2018,40(11):e1800063.DOI:10.1002/bies.201800063.
[7]HOE E,ANDERSON J,NATHANIELSZ J,et al.The contrasting roles of Th17 immunity in human health and disease[J].Microbiol Immunol,2017,61(2):49-56.DOI:10.1111/1348-0421.12471.
[8]NOACK M,MIOSSEC P.Th17 and regulatory T cell balance in autoimmune and inflammatory diseases[J].Autoimmun Rev,2014,13(6):668-677.DOI:10.1016/j.autrev.2013.12.004.
[9]ZHENG L,LI Z,LING W,et al.Exosomes derived from dendritic cells attenuate liver injury by modulating the balance of Treg and Th17 cells after ischemia reperfusion[J].Cell Physiol Biochem,2018,46(2):740-756.DOI:10.1159/000488733.
[10]ZHANG A,WANG K,ZHOU C,et al.Knockout of microRNA-155 ameliorates the Th1/Th17 immune response and tissue injury in chronic rejection[J].J Heart Lung Transplant,2017,36(2):175-184.DOI:10.1016/j.healun.2016.04.018.
[11]LU L,LI G,RAO J,et al.In vitro induced CD4(+)CD25(+)Foxp3(+)Tregs attenuate hepatic ischemiareperfusion injury[J].Int Immunopharmacol,2009,9(5):549-552.DOI:10.1016/j.intimp.2009.01.020.
[12]PERUCHA E,MELCHIOTTI R,BIBBY JA,et al.The cholesterol biosynthesis pathway regulates IL-10expression in human Th1 cells[J].Nat Commun,2019,10(1):498.DOI:10.1038/s41467-019-08332-9.
[13]PILAT N,MAHR B,GATTRINGER M,et al.CTLA4Ig improves murine iTreg induction via TGFβand suppressor function in vitro[J].J Immunol Res,2018:2484825.DOI:10.1155/2018/2484825.
[14]TEIJEIRA A,HUNTER MC,RUSSO E,et al.T cell migration from inflamed skin to draining lymph nodes requires intralymphatic crawling supported by ICAM-1/LFA-1 interactions[J].Cell Rep,2017,18(4):857-865.DOI:10.1016/j.celrep.2016.12.078.
[15]SILVA PDE M,BIER J,PAIATTO LN,et al.Tolerogenic dendritic cells on transplantation:immunotherapy based on second signal blockage[J].J Immunol Res,2015:856707.DOI:10.1155/2015/856707.
[16]SCHELLENBURG S,SCHULZ A,POITZ DM,et al.Role of neuropilin-2 in the immune system[J].Mol Immunol,2017,90:239-244.DOI:10.1016/j.molimm.2017.08.010.
[17]BURGHARDT S,CLAASS B,ERHARDT A,et al.Hepatocytes induce Foxp3+regulatory T cells by notch signaling[J].J Leukoc Biol,2014,96(4):571-577.DOI:10.1189/jlb.2AB0613-342RR.
[18]FINSTERBUSCH M,NORMAN MU,HALL P,et al.Platelet retention in inflamed glomeruli occurs via selective prolongation of interactions with immune cells[J].Kidney Int,2019,95(2):363-374.DOI:10.1016/j.kint.2018.08.042.
[19]REN J,ZHOU T,PILLI VSS,et al.Novel paracrine functions of smooth muscle cells in supporting endothelial regeneration following arterial injury[J].Circ Res,2019,124(8):1253-1265.DOI:10.1161/CIRCRESAHA.118.314567.
[20]SHARPE AH,FREEMAN GJ.The B7-CD28superfamily[J].Nat Rev Immunol,2002,2(2):116-126.
[21]SHEN H,WU N,NANAYAKKARA G,et al.Cosignaling receptors regulate T-cell plasticity and immune tolerance[J].Front Biosci(Landmark Ed),2019,24:96-132.
[22]LI K,CHENG X,TILEVIK A,et al.In situ and in silico kinetic analyses of programmed cell death-1(PD-1)receptor,programmed cell death ligands,and B7-1protein interaction network[J].J Biol Chem,2017,292(16):6799-6809.DOI:10.1074/jbc.M116.763888.
[23]WATANABE M,FUJIHARA C,RADTKE AJ,et al.Co-stimulatory function in primary germinal center responses:CD40 and B7 are required on distinct antigenpresenting cells[J].J Exp Med,2017,214(9):2795-2810.DOI:10.1084/jem.20161955.
[24]KANG S,ZHANG C,OHNO T,et al.Unique B7-H1expression on masticatory mucosae in the oral cavity and trans-coinhibition by B7-H1-expressing keratinocytes regulating CD4+T cell-mediated mucosal tissue inflammation[J].Mucosal Immunol,2017,10(3):650-660.DOI:10.1038/mi.2016.89.