重组甲型流感病毒基质蛋白1和2通过ERK信号因子诱导小鼠气管上皮细胞产生IFN-γ
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摘要
探讨重组甲型流感病毒基质蛋白1和2(rM1和rM2)通过细胞外信号调节蛋白激酶(Extracellular signal regulated kinase,ERK)诱导小鼠气管上皮细胞产生γ-干扰素(Interferon-γ,IFN-γ)作用及机制。以原代小鼠气管上皮细胞为实验模型,实验分为6组(rM1组、rM2组、甲型流感病毒(Influenza A virus,IAV)组、rM1+IAV组、rM2+IAV组和正常对照组)。在各组分干预细胞4h、8h、24h时,采用半定量RT-PCR法检测各组细胞中IFN-γmRNA的表达和免疫印迹法检测各组细胞中IFN-γ、ERK、磷酸化ERK(phospho-ERK,p-ERK)蛋白的表达;用抑制剂阻断ERK信号因子信号传导,观察对各组分诱导小鼠气管上皮细胞产生IFN-γ的影响。各组分干预细胞4h、8h、24h,rM1组、rM2组、IAV组、rM1+IAV组、rM2+IAV组细胞的IFN-γmRNA和IFN-γ蛋白表达水平高于正常对照组(P<0.01或P<0.05);rM1+IAV组、rM2+IAV组细胞的IFN-γmRNA和IFN-γ蛋白表达水平高于IAV组(P<0.01或P<0.05)。在干预细胞4h,仅rM2组细胞中ERK磷酸化水平显著高于正常对照组(P<0.01),在干预细胞8h、24h,rM1组、rM2组、IAV组、rM1+IAV组、rM2+IAV组细胞中ERK磷酸化水平均显著高于正常对照组(P<0.01或P<0.05)。加入ERK抑制剂,rM1组、rM2组、rM1+IAV组、rM2+IAV组细胞的IFN-γmRNA和IFN-γ蛋白表达水平显著低于非抑制剂组。本研究数据表明rM1和rM2可通过上调ERK信号因子的磷酸化水平诱导小鼠气管上皮细胞中产生IFN-γ,该诱导作用在干预4h即显著表现,并维持至少24h。
We wished to investigate the role and mechanism of recombinant influenza A virus matrix protein1 and 2(rM1 and rM2,respectively)in inducing interferon-γ(IFN-γ)expression through extracellular signal regulated kinase(ERK)signaling.The tracheal epithelial cells of mice were used as the experimental model and were divided into six groups according to treatment type:rM1,rM2,influenza A virus(IAV),rM1+IAV,rM2+IAV,and normal control.At 4,8,and 24 hafter treatment,expression of IFN-γmRNA was measured semi-quantitatively by reverse-transcription polymerase chain reaction and expression of IFN-γ,ERK and phospho-ERK(p-ERK)proteins were tested by Western Blotting.Then,an ERK inhibitor was used to study the effect of ERK signaling upon IFN-γexpression.At 4,8,and 24 hafter treatment,expression of IFN-γmRNA and IFN-γprotein in all groups was up-regulated significantly compared with that in the normal control group(P<0.01 or P<0.05),and expression of IFN-γmRNA and IFN-γprotein in the rM1+IAV group and rM2+IAV group was up-regulated significantly compared with that in IAV group(P<0.01 or P<0.05).p-ERK expression was up-regulated only in the rM2 group 4 hafter treatment,but was up-regulated significantly in all groups compared with the normal control group 8 hand24 hafter the treatment(P<0.01 or P<0.05).Expression of IFN-γmRNA and IFN-γprotein was downregulated significantly if an ERK inhibitor was used(P<0.01 or P<0.05).These data suggest that rM1 and rM2 promote IFN-γexpression in the tracheal epithelial cells of mice by enhancing p-ERK expression;their effect is significant 4 hafter treatment,and lasted for at least 24 h.
We wished to investigate the role and mechanism of recombinant influenza A virus matrix protein1 and 2(rM1 and rM2,respectively)in inducing interferon-γ(IFN-γ)expression through extracellular signal regulated kinase(ERK)signaling.The tracheal epithelial cells of mice were used as the experimental model and were divided into six groups according to treatment type:rM1,rM2,influenza A virus(IAV),rM1+IAV,rM2+IAV,and normal control.At 4,8,and 24 hafter treatment,expression of IFN-γmRNA was measured semi-quantitatively by reverse-transcription polymerase chain reaction and expression of IFN-γ,ERK and phospho-ERK(p-ERK)proteins were tested by Western Blotting.Then,an ERK inhibitor was used to study the effect of ERK signaling upon IFN-γexpression.At 4,8,and 24 hafter treatment,expression of IFN-γmRNA and IFN-γprotein in all groups was up-regulated significantly compared with that in the normal control group(P<0.01 or P<0.05),and expression of IFN-γmRNA and IFN-γprotein in the rM1+IAV group and rM2+IAV group was up-regulated significantly compared with that in IAV group(P<0.01 or P<0.05).p-ERK expression was up-regulated only in the rM2 group 4 hafter treatment,but was up-regulated significantly in all groups compared with the normal control group 8 hand24 hafter the treatment(P<0.01 or P<0.05).Expression of IFN-γmRNA and IFN-γprotein was downregulated significantly if an ERK inhibitor was used(P<0.01 or P<0.05).These data suggest that rM1 and rM2 promote IFN-γexpression in the tracheal epithelial cells of mice by enhancing p-ERK expression;their effect is significant 4 hafter treatment,and lasted for at least 24 h.
引文
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[19]朱颜鑫,牟秋菊,赵兵兵,罗红,智妍,兰露莎,江滟.重组甲型流感病毒NS2蛋白抑制感染小鼠肺组织干扰素的产生[J].细胞与分子免疫学杂志,2015,31(12):1615-1619.
[20]Hayashi T,Macdonald L A,Takimoto T.Influenza A virus protein PA-X contributes to viral growth and suppression of the host antiviral and immune responses[J].J Virol,2015,89(12):6442-6452.
[21]Xia C,Vijayan M,Pritzl C J,Fuchs S Y,McDermott A B,Hahm B.Hemagglutinin of influenza A virus antagonizes type I IFN responses by inducing degradation of type I IFN receptor 1[J].J Virol,2015,90(5):2403-2417.
[22]Dong Q,Sugiura T,Toyohira Y,Yoshida Y,Yanagihara N,Karasaki Y.Stimulation of IFN-γproduction by garlic lectin in mouse spleen cells:involvement of IL-12via activation of p38 MAPK and ERK in macrophages[J].Phytomedicine,2011,18(4):309-316.
[2]Hause B M,Collin E A,Liu R,Huang B,Sheng Z,Lu W,Wang D,Nelson EA,Li F.Characterization of a novel influenza virus in cattle and Swine:proposal for a new genus in the Orthomyxoviridae family[J/OL].MBio,2014,5(2):e00031-14.
[3]Lee V J,Ho Z J M,Goh E H,Campbell H,Cohen C,Cozza V,Fitzner J,Jara J,Krishnan A,Bresee J.Advances in measuring influenza burden of disease[J].Influenza Other Respi Viruses,2018,12(1):3-9.
[4]Boulo S,Akarsu H R,Baudin F.Nuclear traffic of influenza virus proteins and ribonucleoprotein complexes[J].Virus Res,2007,124(1):12-21.
[5]Nayak D P,Hui E K,Barman S.Assembly and budding of influenza virus.[J].Virus Res,2004,106(2):147-165.
[6]Hale B G,Barclay W S,Randall R E,Russell R J.Structure of an avian influenza A virus NS1protein effector domain[J].Virology,2008,378(1):1-5.
[7]Hai R,Schmolke M,Varga Z T,Manicassamy B,Wang T T,Belser J A,Pearce M B,García-Sastre A,Tumpey T M,Palese P.PB1-F2expression by the 2009pandemic H1N1influenza virus has minimal impact on virulence in animal models[J].J Virol,2010,84(9):4442-4450.
[8]Wang S,Zhao Z,Bi Y,Sun L,Liu X,Liu W.Tyrosine132phosphorylation of influenza A virus M1protein is crucial for virus replication by controlling the nuclear import of M1[J].J Virol,2013,87(11):6182-6191.
[9]Kumar B,Rajput R,Pati D R,Khanna M.Potent intracellular knock-down of influenza A virus M2 gene transcript by DNAzymes considerably reduces viral replication in host cells[J].Mol Biotechnol,2015,57(9):836-845.
[10]Lohia N,Baranwal M.Immune responses to highly conserved influenza A virus matrix 1peptides[J].Microbiol Immunol,2017,61(6):225-231.
[11]Kim M C,Lee Y N,Hwang H S,Lee Y T,Ko E J,Jung Y J,Cho M K,Kim Y J,Lee J S,Ha S H,Kang S M.Influenza M2virus-like particles confer a broader range of cross protection to the strain-specific pre-existing immunity[J].Vaccine,2014,32(44):5824-5831.
[12]Platanias L C.Mechanisms of type-I-and type-Ⅱ-interferon-mediated signalling[J].Nat Rev Immunol,2005,5(5):375-386.
[13]Hu X P,Shao M M,Song X,Wu X L,Qi L,Zheng K,Fan L,Liao C H,Li C Y,He J,Hu Y J,Wu H Q,Li S H,Zhang J,Zhang F X,He Z D.Anti-influenza virus effects of crude phenylethanoid glycosides isolated from ligustrum purpurascens via inducing endogenous interferon-γ[J].J Ethnopharmacol,2016,179:128-136.
[14]Joly S,Organ C C,Johnson G K,McCray P B Jr,Guthmiller J M.Correlation between beta-defensin expression and induction profiles in gingival keratinocytes[J].Mol Immunol,2005,42(9):1073-1084.
[15]Randall R E,Goodbourn S.Interferons and viruses:an interplay between induction,signalling,antiviral responses and virus countermeasures[J].J Gen Virol,2008,89(1):1-47.
[16]Bezniakow N,Gos M,Obersztyn E.The RASopathies as an example of RAS/MAPK pathway disturbancesclinical presentation and molecular pathogenesis of selected syndromes[J].Dev Period Med,2014,18(3):285-296.
[17]Lv S,Yi P F,Shen H Q,Zhang L Y,Dong H B,Wu S C,Xia F,Guo X,Wei X B,Fu B D.Ginsenoside Rh2-B1stimulates cell proliferation and IFN-γproduction by activating the p38 MAPK and ERK-dependent signaling pathways in CTLL-2 cells[J].Immunopharmacol Immunotoxicol,2013,36(1):43-51.
[18]Chen L,Zanker D,Xiao K,Wu C,Zou Q,Chen W.Immunodominant CD4+T-cell responses to influenza A virus in healthy individuals focus on matrix 1and nucleoprotein[J].J Virol,2014,88(20):11760-11773.
[19]朱颜鑫,牟秋菊,赵兵兵,罗红,智妍,兰露莎,江滟.重组甲型流感病毒NS2蛋白抑制感染小鼠肺组织干扰素的产生[J].细胞与分子免疫学杂志,2015,31(12):1615-1619.
[20]Hayashi T,Macdonald L A,Takimoto T.Influenza A virus protein PA-X contributes to viral growth and suppression of the host antiviral and immune responses[J].J Virol,2015,89(12):6442-6452.
[21]Xia C,Vijayan M,Pritzl C J,Fuchs S Y,McDermott A B,Hahm B.Hemagglutinin of influenza A virus antagonizes type I IFN responses by inducing degradation of type I IFN receptor 1[J].J Virol,2015,90(5):2403-2417.
[22]Dong Q,Sugiura T,Toyohira Y,Yoshida Y,Yanagihara N,Karasaki Y.Stimulation of IFN-γproduction by garlic lectin in mouse spleen cells:involvement of IL-12via activation of p38 MAPK and ERK in macrophages[J].Phytomedicine,2011,18(4):309-316.