Identification of immunostimulatory CpG DNA and its activation mechanism in human immune-conmpetent cells

人免疫活性细胞中免疫刺激CpG DNA的鉴定及其激活机制

• 批准号: 13670266
• 负责人: IHO Sumiko
• 金额: $ 2.3万
• 依托单位: 福井医科大学
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670266/
• 关键词: CpG DNA; PDC; IFN-α; IP-10; MIP-1α; p38 MAPK; STAT1; NF-κB; CpG-DNA; Plasmacytoid Dendritic Cells; IFN-alpha; MIP-1alpha; endosomal maturation; PI3K; p38MAPK; Bacterial DNA; INF-α; IRF-7; ISGF3

CpG DNA induces Th1-dominant immune responses in vertebrate. The activity of CpG DNA is dependent on both the base sequences and species in mammalian. In this study, we identify the base sequence of CpG DNA that activates human plasmacytoid cells (PDC), and examine its activation mechanisms.Poly G-flanked palindrome CpG DNA, GGGGGGGGGG-GACGATCGTC-GGGGGGGGGG(G10GACGA), was identified as an active form of CpG DNA for PDC to induce IFN-α/IP-10/MIP-1α. The activity of G10GACGA is dependent on the sequence of GACGATCGTC, and endosomal maturation is required for the induction of IFN-α/IP-10/MIP-1α. No activity is observed in TLR9-KO mice. In G10GACGA-treated PDC, phosphorylation of p38 MAPK is induced in a manner independent of autocrine IFN-α/β signaling, and this pathway is essential for the induction of IFN-α/IP-10/MIP-1α. PDC constitutively express both IRF-3 and IRF-7, and G10GACGA enhances the expression of IRF-7 but not of IRF-3. Prior to the increase of IRF-7, STAT1 is phosphorylated … More dependently on p38 MAPK, and ISGF3 component (STAT1, STAT2, and IRF-9) translocates to the nuclei. PDC also constitutively express NF-κB p65 and p50 in the nuclei. The activities increase by the treatment with G10GACGA, but not in the presence of p38 MAPK inhibitor. The NF-κB inhibitors, PDTC, MG-132, Dexamethasone, and CAPE, suppress G10GACGA-induced IRF-7/IFN-α/IP-10/MIP-1α expression. In late phase, the autocrine activation via IFNAR takes part in PDC.From these results, we propose that poly-G-flanked palindrome CpG DNA up-taken in PDC activates both STAT1 and NF-κB through the processes of endosomal maturation and p38 MAPK activation. These pathways would cause, respectively or cooperatively, transcription of the genes, in the promoters of which ISRE and/or κB site exists, such as IRF-7/IP-10 and IRF-7/IP-10/MIP-1α, thereby inducing IFN-α/IP-10/MIP-1α in a manner independent of autocrine activation of IFNAR. The consecutive activation of IFNAR-signaling loop produces a large amount of IFN-α/IP-10/MIP-1α in PDC. Less

CpG DNA诱导脊椎动物Th1显性免疫反应。在哺乳动物中，CpG DNA的活性依赖于碱基序列和物种。在本研究中，我们鉴定了激活人浆细胞样细胞的CpGDNA的碱基序列，并研究了其激活机制。多G侧翼回文CpGDNA，GGGGGGGGGGG-GACGATCGTC-GGGGGGGGGGG(G10GACGA)被鉴定为激活人浆细胞样细胞的CpGDNA的活性形式，可诱导干扰素-α/IP-10/MIP-1α。G10GACGA的活性依赖于GACGATCGTC的序列，诱导干扰素-α/IP-10/MIP-1α需要内体成熟。在TLR9-KO小鼠中未观察到任何活性。在G10GACGA处理的pDC中，p38MAPK的磷酸化不依赖于自分泌的干扰素-α/β信号，这一途径是诱导干扰素-α/IP-10/MIP-1α所必需的。PDC同时表达IRF-3和IRF-7，G10GACGA增强IRF-7的表达，但不增强IRF-3的表达。在IRF-7增加之前，STAT1是磷酸化的…更依赖于p38 MAPK，ISGF3组分(STAT1、STAT2和IRF-9)移位到细胞核。PDC还在细胞核中结构性表达NF-κB p65和p50。G10GACGA处理后活性增加，但p38 MAPK抑制剂的作用不明显。NF-κB抑制剂PDTC、MG-132、地塞米松和CAPE可抑制G10GACGA诱导的IRF-7/干扰素-α/IP-10/MIP-1α的表达。在晚期，通过IFNAR的自分泌激活参与了PDC。根据这些结果，我们认为PDC中的多G侧翼回文CpGDNA通过内体成熟和p38MAPK激活过程激活STAT1和NF-κB。这些途径将分别或协同地引起存在ISRE和/或κB位点的启动子的基因的转录，如iRF-7/IP-10和iRF-7/IP-10/MIP-1α，从而以一种不依赖于IFNAR自分泌激活的方式诱导α-α/IP-10/MIP-1。IFNAR信号转导环的连续激活可在DC中产生大量的干扰素-α/IP-10/MIP-1α。较少

项目成果

Takauji, R., et al.: "Lecture : Activation of plasmacytoid dendritic cells by CpG DNA (In Japanese)."Rinsho Meneki. 41. 107-112 (2004)

Takauji, R. 等人：“讲座：CpG DNA 激活浆细胞样树突状细胞（日语）。”Rinsho Meneki。

Yamamoto, S., et al.: "Activation of NK cell by immunostimulatory oligo-DNA in mouse and human."Microbial DNA and Host Immunity, Edited by Raz, E.. 81-89 (2002)

Yamamoto, S. 等人：“小鼠和人类中通过免疫刺激性寡 DNA 激活 NK 细胞。”微生物 DNA 和宿主免疫，Raz, E.. 编辑 81-89 (2002)

Yamamoto, S., et al.: "Activation of NK cell by immunostimulatory oligo-DNA in mouse and human. In Microbial DNA and Host Immunity, Edited by Raz, E."Humana Press, Totowa, NJ, USA. 9 (2002)

Yamamoto, S. 等人：“小鼠和人类中免疫刺激寡 DNA 激活 NK 细胞。微生物 DNA 和宿主免疫，由 Raz, E 编辑。”Humana Press，Totowa，新泽西州，美国。

Takauji, R., et al.: "Lecture : Mechanism of CpG DNA-induced IFN-α production in pDC (In Japanese)."Rinsho Meneki. 41(in press). (2004)

Takauji, R., et al.：“讲座：pDC 中 CpG DNA 诱导的 IFN-α 产生的机制（日语）。”Rinsho Meneki 41（印刷中）。

Iho, S.: "Type I IFN synthesis in plasmacytoid dendritic cells"J.Immunol.. 171(6). 2767 (2003)

Iho, S.：“浆细胞样树突状细胞中的 I 型 IFN 合成”J.Immunol.. 171(6)。