Synergistic activation of PTGS2 and CXCL8 by Ni and microbial stimuli

描述（由申请人提供）：拟议项目的总体目标是获得有关颗粒物（PM）空气污染和微生物刺激物的成分如何协同相互作用以促进肺部炎症样过程的新信息，从而有助于呼吸系统疾病的发作。许多流行病学研究将PM暴露与慢性呼吸道和心血管疾病联系起来。研究表明，镍是PM的常见成分，在体外和体内介导PM的一些生物学效应。慢性炎性疾病也与感染因子（包括发酵支原体）的定殖有关。炎症的标志之一是通过诱导环氧合酶-2酶（加兰他汀-内过氧化物酶合酶2，PTGS 2）增加加兰他汀（PG）的加工。初步数据显示，暴露于NiS 〇 4和M.发酵多糖衍生的脂肽MALP-2协同增强培养的人肺成纤维细胞（HLF）中PTGS 2和免疫调节趋化因子CXCL 8（IL-8）的表达。在诱导PTGS 2和CXCL 8的同时，Ni和MALP-2协同刺激PGE 2和CXCL 8蛋白从HLF释放。拟议的研究将探讨PTGS 2衍生的PGE 2有助于在混合暴露于Ni和微生物刺激期间从HLF扩增CXCL 8生产的假设。具体目的是1）确定响应于同时的Ni和MALP-2暴露而协同诱导HLF中PTGS 2表达的细胞和分子机制，和2）确定在混合暴露于Ni和MALP-2之后PGE 2的产生与随后的CXCL 8的产生之间的机制关系。将使用由人PTGS 2和CXCL 8启动子沿着驱动的荧光素酶报告质粒以及电泳迁移率变动测定（EMSA）来确定Ni和MALP-2如何协同增强PTGS 2和CXCL 8的活化。相关的顺式调控DNA结合元件和反式激活蛋白因子负责将使用全长，截短和突变体版本的PTGS 2和CXCL 8启动子映射。将使用siRNA沿着药理学激动剂和拮抗剂来探测PTGS 2、PGE 2和PGE 2受体亚型的特异性贡献，所述PTGS 2、PGE 2和PGE 2受体亚型参与转导Ni和MALP-2诱导的CXCL 8表达和随后从HLF释放的蛋白质。现实生活中的环境暴露可能涉及有毒物质和微生物刺激的混合物。这项研究的结果将增加我们对镍等金属如何使细胞对肺部微生物驱动的炎症敏感，以及毒物和微生物刺激如何相互作用以促进人类呼吸道疾病发作的理解。

DESCRIPTION (provided by applicant): The overall objective of the proposed project is to gain novel information regarding how components of particulate matter (PM) air pollution and microbial stimuli synergistically interact to promote inflammatory-like processes in the lung, and thereby contribute to the onset of respiratory disease. Numerous epidemiological studies have linked exposure to PM with chronic respiratory and cardiovascular diseases. Studies have shown nickel, a common component of PM, mediates some of the biological effects of PM both in vitro and in vivo. Chronic inflammatory diseases are also associated with colonization by infectious agents, including Mycoplasma fermentans. One of the hallmarks of inflammation is increased elaboration of prostaglandins (PG) through the induction of the cyclooxygenase-2 enzyme (prostaglandin-endoperoxidase synthase 2, PTGS2). Preliminary data show that exposure to NiS04 and the M. fermentans-derived lipopeptide MALP-2 synergistically enhances expression of PTGS2 and the immune-modulating chemokine CXCL8(IL-8) in cultured human lung fibroblast cells (HLF). Concurrent with the induction of PTGS2 and CXCL8, Ni and MALP-2 synergistically stimulate PGE2 and CXCL8 protein release from HLF. The proposed study will explore the hypothesis that PTGS2-derived PGE2 contributes to the amplification of CXCL8 production from HLF during mixed exposures to Ni and microbial stimuli. The specfic aims are to 1) determine the cellular and molecular mechanisms underlying the synergistic induction of PTGS2 expression in HLF in response to concurrent Ni and MALP-2 exposure and 2) determine the mechanistic relationship between the production of PGE2 following mixed exposures to Ni and MALP-2 and subsequent production of CXCL8. Luciferase reporter plasmids driven by human PTGS2 and CXCL8 promoters along with electrophoretic mobility shift assays (EMSA) will be employed to determine how Ni and MALP-2 synergistically enhance activation of PTGS2 and CXCL8. Relevant cis-regulatory DNA binding elements and transactivating protein factors responsible will be mapped using full-length, truncated and mutant versions of the PTGS2 and CXCL8 promoters. siRNA along with pharmacological agonists and antagonists will be used to probe the specific contribution of PTGS2, PGE2 and PGE2 receptor subtypes involved in transducing Ni and MALP-2-induced CXCL8 expression and subsequent protein release from HLF. Real-life environmental exposures are likely to involve a mixture of toxicants and microbial stimuli. Results from this study will increase our understanding of how metals such as Ni can sensitize cells to microbial-driven inflammation in the lung, and how toxicant and microbial stimuli interact to promote the onset of respiratory disease in human populations.