Mechanism of Activation of Innate Immunity by ISS-DNA

ISS-DNA 激活先天免疫的机制

• 批准号: 6744001
• 负责人: Wen-Ming Chu
• 金额: $ 27.48万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-15 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6744001
• 关键词: bacterial DNA; biological signal transduction; immunoregulation; laboratory mouse; leukocyte activation /transformation; macrophage; microorganism immunology; protein kinase; toll like receptor

DESCRIPTION (provided by applicant): Innate immunity enables an organism to respond rapidly to invading microorganisms. To do this, innate immune system uses a variety of receptors or intracellular proteins that can recognize a microbial pathogen by the motifs pathogen-associated molecular patterns (PAMPs). Pathogen-associated motifs include mannans in the yeast cell wall, formylated peptides and various bacterial cell-wall components such as lipopolysaccharide (LPS), lipopeptides, peptidoglycans and teichoic acids. Bacterial DNA is also a potent inducer of innate immunity. Bacterial DNA contains increased frequency of CpG dinucleotide motifs in particular sequence contexts (PuPuCpGPyPy), which are greatly reduced in frequency in the mammalian genome, and where it occurs, the cytosine commonly is methylated. DNA containing unmethylated CpG motifs is immunostimulatory and able to activate cellular component of innate immunity, including macrophages and dendritic cells (DC) as well as B cells. Thus, DNA containing unmethylated CpG motif in sequences of PuPuCpGPyPy is also called ISS-DNA. Activation of immune cells by ISS-DNA appears to require uptake into the cells by receptor- or pinocytosis-mediated endocytosis and endosome acidification, suggesting that the recognition structure is either endosome-associated or cytoplasmic. Recent studies suggested that Toll-like receptor (TLR) 9 and adapter protein MyD88 are required for ISS-DNA to induce immune cells to produce interlukin-12, and for activation of NF-kB by ISS-ODN. The studies from our group suggested that catalytic subunit of DNA-dependent protein kinase (DNAPKcs) and IkB kinase (IKK) are required for the activation of innate immunity by ISS-DNA. ISS-DNA activates DNA-PKcs, which in turn activates IKK, leading to NF-kB activation. Null mutations in either the DNA-PKcs- or IKKb gene were found to selectively impair the ability of mice or their macrophages, to respond to ISS-DNA with inducible cytokine gene expression. TLR9 is on cell membrane while DNA-PKcs is in cytoplasm and nucleus. The connection between TLR9 and DNA-PKcs is not apparent. To further understand the molecular mechanisms of activation of innate immunity by ISS-DNA, we will investigate the relationship between TLR9-signaling pathway and DNA-PK by using biochemical and genetic means. Moreover, we will investigate if the regulatory subunits of DNA-PK, Ku70 and Ku80, and their associated factors are required for the innate response to ISS-DNA. Furthermore, we will identify new molecules that are required for activation of innate immunity by ISS-DNA.

描述（由申请人提供）：先天免疫使生物体能够 对入侵的微生物做出快速反应。为此，先天免疫系统 使用多种可以识别的受体或细胞内蛋白质 微生物病原体的图案 病原体相关的分子模式 （PAMP）。病原体相关基序包括酵母细胞壁中的甘露聚糖， 甲酰化肽和各种细菌细胞壁成分，例如 脂多糖（LPS）、脂肽、肽聚糖和磷壁酸。 细菌 DNA 也是先天免疫的有效诱导剂。 细菌 DNA 中 CpG 二核苷酸基序的频率增加 特定的序列上下文（PuPuCpGPyPy），在 哺乳动物基因组中的频率及其出现的位置通常是胞嘧啶 被甲基化。含有未甲基化 CpG 基序的 DNA 具有免疫刺激作用 能够激活先天免疫的细胞成分，包括巨噬细胞 和树突状细胞 (DC) 以及 B 细胞。因此，含有未甲基化的DNA PuPuCpGPyPy 序列中的 CpG 基序也称为 ISS-DNA。激活 ISS-DNA 的免疫细胞似乎需要通过受体摄取到细胞中 或胞饮作用介导的内吞作用和内体酸化，表明 识别结构是内体相关的或细胞质的。最近的 研究表明 Toll 样受体 (TLR) 9 和衔接蛋白 MyD88 ISS-DNA 诱导免疫细胞产生白细胞介素 12 所需，并且 ISS-ODN 激活 NF-kB。我们小组的研究表明 DNA 依赖性蛋白激酶 (DNAPKcs) 和 IkB 激酶的催化亚基 (IKK) 是 ISS-DNA 激活先天免疫所必需的。国际空间站DNA 激活 DNA-PKcs，进而激活 IKK，从而导致 NF-kB 激活。 发现 DNA-PKcs- 或 IKKb 基因中的无效突变选择性地 损害小鼠或其巨噬细胞对 ISS-DNA 做出反应的能力 诱导细胞因子基因表达。 TLR9位于细胞膜上，而DNA-PKcs位于细胞质和细胞核中。这 TLR9 和 DNA-PKcs 之间的联系并不明显。为了进一步了解 ISS-DNA 激活先天免疫的分子机制，我们将 使用以下方法研究 TLR9 信号通路与 DNA-PK 之间的关系 生物化学和遗传手段。此外，我们将调查监管部门是否 需要 DNA-PK、Ku70 和 Ku80 亚基及其相关因子 对 ISS-DNA 的先天反应。此外，我们将识别新分子 这是 ISS-DNA 激活先天免疫所必需的。