人粒细胞巨噬细胞集落刺激因子DNA疫苗预防NOD小鼠的机制探讨
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摘要
目的探讨人粒细胞巨噬细胞集落刺激因子(GM-CSF)DNA疫苗预防非肥胖糖尿病(NOD)小鼠糖尿病的作用机制。方法 4周龄NOD雌鼠随机分为PBS(n=15)、PcDNA(n=15)、GM(n=15)3组,由胫前肌分别注射PBS、质粒PcDNA3.1、人GM-CSF DNA疫苗100μg,1周后重复1次,观察30周龄小鼠的累积糖尿病发病率。各组取12周龄未发病NOD鼠(n=6)胰腺HE染色观察胰岛炎;取PBS组未发病10～12周龄NOD鼠脾脏制成细胞悬液加GM-CSF(2ng/mL)培养72h;ELISA法测定血清、脾细胞培养上清液干扰素-γ(IFN-γ)、白细胞介素-1β(IL-1β)、IL-4和IL-10水平。结果 30周龄时,PBS、PcDNA、GM组糖尿病发病率分别为80.0%、73.3%和46.7%;平均发病时间分别为(143.9±46.1)、(156.9±40.0)、(188.3±30.0)d,与PBS组和PcDNA组比较,GM组发病时间显著延迟(P=0.004和P=0.034)。12周龄时GM组胰岛炎积分低于PBS组(P=0.001)和PcDNA组(P=0.027),GM组血清及脾细胞培养上清液IL-10水平显著高于PBS组和PcDNA组,IL-4水平显著性高于PBS组,而IFN-γ、IL-1β水平则低于PBS组和PcDNA组。GM组脾细胞上清液IL-4和IL-10水平高于PBS组;IFN-γ和IL-1β水平则低于PBS组。结论人GM-CSF DNA疫苗上调了IL-10、IL-4及下调IFN-γ、IL-1β,使免疫平衡偏向Th2,而减轻NOD鼠胰岛炎,预防和延缓糖尿病发生。
Objective To investigate the mechanism of human granulocyte macrophage colony-stimulating factor(GM-CSF)DNA vaccine in preventing diabetes mellitus in(non-obese diabetes)NOD mice.Methods Female NOD mice aged 4 weeks were randomly divided into the PBS(n=15),PcDNA(n=15)and GM(n=15)group.PBS,plasmid PcDNA3.1 and human GM-CSF DNA vaccine were injected into tibialis anterior muscle respectively.The cumulative incidence of diabetes mellitus at 30 weeks was observed.Pancreas was removed from NOD mice at 12-week age in each group(n=6)to score insulitis severity by HE staining.The spleens of 10 to 12-week-old NOD mice in the PBS group were used to prepare cell suspension and cultured with GM-CSF(2 ng/mL)for 72 hours.The levels of IFN-γ,IL-1β,IL-4 and IL-10 in serum and supernatant of spleen cells were determined by ELISA.Results At the age of 30 weeks,the incidence of diabetes mellitus was 80.0%,73.3% and 46.7%in the PBS,PcDNA and GM group,respectively,and the average onset time was(143.9±46.1),(156.9±40.0)and(188.3±30.0)d,respectively.Compared with the PBS and PcDNA groups,the onset time of diabetes mellitus in the GM group was significantly delayed(P=0.004 and P=0.034).At the age of 12 weeks,the score of insulitis in the GM group was lower than that in the PBS group(P=0.001)and the PcDNA group(P=0.027).The levels of IL-10 in serum and spleen cell supernatant of the GM group were significantly higher than those of the PBS group and the PcDNA group,and IL-4 was significantly higher than those of the PBS group,while the levels of IFN-γand IL-1βwere lower than those of the PBS group and the PcDNA group.The levels of IL-4 and IL-10 in spleen cell supernatant of the GM group were higher than those of the PBS group,while the levels of IFN-γand IL-1βwere lower than those of PBS group.Conclusion Human GM-CSF DNA vaccine can up-regulate IL-10,IL-4 and down-regulate IFN-γand IL-1β,so as to make the immune balance incline to Th2 which alleviate insulitis in NOD mice and prevent and delay the occurrence of diabetes.
Objective To investigate the mechanism of human granulocyte macrophage colony-stimulating factor(GM-CSF)DNA vaccine in preventing diabetes mellitus in(non-obese diabetes)NOD mice.Methods Female NOD mice aged 4 weeks were randomly divided into the PBS(n=15),PcDNA(n=15)and GM(n=15)group.PBS,plasmid PcDNA3.1 and human GM-CSF DNA vaccine were injected into tibialis anterior muscle respectively.The cumulative incidence of diabetes mellitus at 30 weeks was observed.Pancreas was removed from NOD mice at 12-week age in each group(n=6)to score insulitis severity by HE staining.The spleens of 10 to 12-week-old NOD mice in the PBS group were used to prepare cell suspension and cultured with GM-CSF(2 ng/mL)for 72 hours.The levels of IFN-γ,IL-1β,IL-4 and IL-10 in serum and supernatant of spleen cells were determined by ELISA.Results At the age of 30 weeks,the incidence of diabetes mellitus was 80.0%,73.3% and 46.7%in the PBS,PcDNA and GM group,respectively,and the average onset time was(143.9±46.1),(156.9±40.0)and(188.3±30.0)d,respectively.Compared with the PBS and PcDNA groups,the onset time of diabetes mellitus in the GM group was significantly delayed(P=0.004 and P=0.034).At the age of 12 weeks,the score of insulitis in the GM group was lower than that in the PBS group(P=0.001)and the PcDNA group(P=0.027).The levels of IL-10 in serum and spleen cell supernatant of the GM group were significantly higher than those of the PBS group and the PcDNA group,and IL-4 was significantly higher than those of the PBS group,while the levels of IFN-γand IL-1βwere lower than those of the PBS group and the PcDNA group.The levels of IL-4 and IL-10 in spleen cell supernatant of the GM group were higher than those of the PBS group,while the levels of IFN-γand IL-1βwere lower than those of PBS group.Conclusion Human GM-CSF DNA vaccine can up-regulate IL-10,IL-4 and down-regulate IFN-γand IL-1β,so as to make the immune balance incline to Th2 which alleviate insulitis in NOD mice and prevent and delay the occurrence of diabetes.
引文
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[8]GOGESCH P,SCHLKE S,SCHEURER S,et al.Modular MLV-VLPs co-displaying ovalbumin peptides and GM-CSF effectively induce expansion of CD11b+APCand antigen-specific T cell responses in vitro[J].Mol Immunol,2018,101:19-28.
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[12]BOUDALY S,MORIN J,BERTHIER R,et al.Altered dendritic cells(DC)might be responsible for regulatory T cell imbalance and autoimmunity in nonobese diabetic(NOD)mice[J].Eur Cytokine Netw,2002,13(1):29-37.
[13]GANGI E,VASU C,CHEATEM D,et al.IL-10-producing CD4+CD25+regulatory T cells play a critical role in granulocyte-macrophage colony-stimulating factor-induced suppression of experimental autoimmune thyroiditis[J].J Immunol,2005,174(11):7006-7013.
[14]BHATTACHARYA P,THIRUPPATHI M,ELSHABRAWYH A,et al.GM-CSF:An immune modulatory cytokine that can suppress autoimmunity[J].Cytokine,2015,75(2):261-271.
[15]CURTI A,FOGLI M,RATTA M,et al.Stem cell factor and FLT3-ligand are strictly required to sustain the longterm expansion of primitive CD34+DR-dendritic cell precursors[J].J Immunol,2001,166(2):848-854.
[16]VASU C,HOLTERMAN M J,PRABHAKAR B S.Modulation of dendritic cell function and cytokine production to prevent thyroid autoimmunity[J].Autoimmunity,2003,36(6/7):389-396.
[17]SATO K,UTO T,FUKAYA T,et al.Regulatory dendritic cells[J].Curr Top Microbiol Immunol,2017,410:47-71.
[18]MORRIS G P,KONG Y C.Interference with CD4+CD25+T-cell-mediated tolerance to experimental autoimmune thyroiditis by glucocorticoid-induced tumor necrosis factor receptor monoclonal antibody[J].J Autoimmun,2006,26(1):24-31.
[19]O′SULLIVAN B J,THOMAS H E,PAI S,et al.IL-1beta breaks tolerance through expansion of CD25+effector T cells[J].J Immunol,2006,176(12):7278-7287.
[20]SUN S C,CHANG J H,JIN J.Regulation of nuclear factor-κB in autoimmunity[J].Trends Immunol,2013,34(6):282-289.
[2]COQUERELLE C,MOSER M.Are dendritic cells central to regulatory T cell function?[J].Immunol Lett,2008,119(1/2):12-16.
[3]DONG Y,ARIF A A,POON G F,et al.Generation and I-dentification of GM-CSF derived alveolar-like macrophages and dendritic cells from mouse bone marrow[J].J Vis Exp,2016,25112:.
[4]BHATTACHARYA P,THIRUPPATHI M,ELSHABRAWYH A,et al.GM-CSF:an immune modulatory cytokine that can suppress autoimmunity[J].Cytokine,2015,75(2):261-271.
[5]罗建华,周智广,蒋铁建,等.人GAD65DNA疫苗预防NOD鼠糖尿病的机制探讨[J].中华医学杂志,2004,84(21):1791-1795.
[6]裴剑浩,周智广,罗建华,等.吡格列酮对NOD鼠糖尿病的预防作用及机制探讨[J].中华医学杂志,2004,84(5):411-415.
[7]PUGLIESE A.Autoreactive T cells in type 1diabetes[J].J Clin Invest,2017,127(8):2881-2891.
[8]GOGESCH P,SCHLKE S,SCHEURER S,et al.Modular MLV-VLPs co-displaying ovalbumin peptides and GM-CSF effectively induce expansion of CD11b+APCand antigen-specific T cell responses in vitro[J].Mol Immunol,2018,101:19-28.
[9]MBONGUE J C,NIEVES H,TORREZ T W,et al.The role of dendritic cell maturation in the induction of Insulin-Dependent diabetes mellitus[J].Front Immunol,2017,8:327.
[10]RAKER V K,DOMOGALLA M P,STEINBRINK K.Tolerogenic dendritic cells for regulatory T cell induction in man[J]Front Immunol,2015,6:569.
[11]MAHNKE K,RING S,ENK A H.Antibody targeting of“Steady-State”dendritic cells induces tolerance mediated by regulatory T cells[J].Front Immunol,2016,7:63.
[12]BOUDALY S,MORIN J,BERTHIER R,et al.Altered dendritic cells(DC)might be responsible for regulatory T cell imbalance and autoimmunity in nonobese diabetic(NOD)mice[J].Eur Cytokine Netw,2002,13(1):29-37.
[13]GANGI E,VASU C,CHEATEM D,et al.IL-10-producing CD4+CD25+regulatory T cells play a critical role in granulocyte-macrophage colony-stimulating factor-induced suppression of experimental autoimmune thyroiditis[J].J Immunol,2005,174(11):7006-7013.
[14]BHATTACHARYA P,THIRUPPATHI M,ELSHABRAWYH A,et al.GM-CSF:An immune modulatory cytokine that can suppress autoimmunity[J].Cytokine,2015,75(2):261-271.
[15]CURTI A,FOGLI M,RATTA M,et al.Stem cell factor and FLT3-ligand are strictly required to sustain the longterm expansion of primitive CD34+DR-dendritic cell precursors[J].J Immunol,2001,166(2):848-854.
[16]VASU C,HOLTERMAN M J,PRABHAKAR B S.Modulation of dendritic cell function and cytokine production to prevent thyroid autoimmunity[J].Autoimmunity,2003,36(6/7):389-396.
[17]SATO K,UTO T,FUKAYA T,et al.Regulatory dendritic cells[J].Curr Top Microbiol Immunol,2017,410:47-71.
[18]MORRIS G P,KONG Y C.Interference with CD4+CD25+T-cell-mediated tolerance to experimental autoimmune thyroiditis by glucocorticoid-induced tumor necrosis factor receptor monoclonal antibody[J].J Autoimmun,2006,26(1):24-31.
[19]O′SULLIVAN B J,THOMAS H E,PAI S,et al.IL-1beta breaks tolerance through expansion of CD25+effector T cells[J].J Immunol,2006,176(12):7278-7287.
[20]SUN S C,CHANG J H,JIN J.Regulation of nuclear factor-κB in autoimmunity[J].Trends Immunol,2013,34(6):282-289.