Genetic Mechanisms Of Susceptibility To Ozone-induced Pu

臭氧诱导Pu易感性的遗传机制

• 批准号: 7170027
• 负责人: STEVEN R KLEEBERGER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7170027
• 关键词: biological signal transduction; fibronectins; gene environment interaction; gene expression profiling; genetic susceptibility; genetically modified animals; immune response; immunogenetics; inflammation; laboratory mouse; lung injury; microarray technology; nucleic acid sequence; ozone; polymerase chain reaction; quantitative trait loci; respiratory epithelium; tenascin; toll like receptor

We previously identified significant quantitative trait loci (QTLs) for inflammatory and hyperpermeability responses to 0.3 ppm ozone (O3) on chromosomes 17 and 4, respectively. The chromosome 17 QTL includes the candidate gene Tnf that encodes TNF-a. TNF-a is a key proinflammatory cytokine, and pretreatment of susceptible C57BL/6J mice with anti-TNF-a antibody significantly attenuated O3-induced pulmonary injury, providing strong evidence for Tnf as an O3-susceptibility gene. To understand further the mechanisms through which TNF-a modulates O3-induced inflammation and injury, we began to investigate TNF-a cell signaling following O3 exposure. The cellular effects of TNF-a are mediated by two structurally related, but functionally distinct, receptors: TNF receptor 1 (55 kDa, TNFR1) and TNF receptor 2 (75 kDa, TNFR2). To investigate the roles of TNFR1 and TNFR2 in this model, O3-susceptible C57BL/6J mice (B6; wild type, wt) and B6 mice with targeted disruption (knockout) of the genes for p55 TNF receptor (TNFR1-/-), p75 TNF receptor (TNFR2-/-), or both (TNFR1/TNFR2-/-), were exposed to 0.3 ppm O3 for 48 h (sub-acute), and lung responses were determined by bronchoalveolar lavage. All TNFR-deficient mice had significantly less O3-induced inflammation and epithelial damage, but not lung hyperpermeability, than wt mice did. Compared to air controls, O3 elicited up-regulation of lung TNFR1 and TNFR2 mRNA in wt mice, and down-regulated TNFR1 and TNFR2 mRNA in TNFR2-/- and TNFR1-/- mice, respectively. Airway hyperreactivity induced by acute O3 exposure (2 ppm, 3 hr) was diminished in knockout mice compared to wt, though lung inflammation and permeability remained elevated. Results suggested a critical role for TNFR signaling in sub-acute O3-induced pulmonary epithelial injury and inflammation, and in acute O3-induced airway hyperreactivity. We have continued these studies to further investigate the mechanisms through which TNFR modulates O3-induced lung injury. We identified toll like receptor 4 (Tlr4) as a candidate susceptibility gene within the chromosome 4 QTL. Tlr4 is particularly intriguing because of its role in innate immunity in Drosophila and humans, and its importance in modulating responses to endotoxin. TLR4 belongs to a family of Toll-like receptors (TLRs) that activate intracellular signaling which results in the induction of a variety of effector genes. The cytoplasmic domain of TLRs is homologous to the cytoplasmic domain of the interleukin (IL)-1 receptor (IL-1R) family and share signaling components. In particular, the transcription factor NF (nuclear factor)-kB is an important effector of Tlr4 activation, as NF-kB has been shown to have critical involvement in multiple inflammatory processes. Strong supportive evidence for Tlr4 as a candidate gene in O3 susceptibility was provided by two observations. First, O3-induced lung hyperpermeability was significantly different between HeJ and OuJ mice, which differ only at a missense mutation in the third exon of the Tlr4 gene. Second, Tlr4 mRNA levels in lungs of HeJ mice were decreased relative to OuJ after exposure to O3 which suggested that down regulation of Tlr4 gene expression may contribute to O3 resistance in HeJ mice. To begin investigation of the mechanism through which Tlr4 modulates the O3 response, we tested the hypotheses that 1) inducible nitric oxide synthase (iNOS) mediates O3-induced lung hyperpermeability, and 2) mRNA levels of the gene for iNOS (Nos2) are modulated by Tlr4 during O3 exposure. Pre-treatment of O3-susceptible C57BL/6J (B6) mice with a specific inhibitor of total NOS (L-NMMA, NG-methyl-L-arginine) significantly decreased mean lavageable protein concentration (a marker of lung permeability) induced by O3 (0.3 ppm, 72 hr) compared to vehicle controls. Further, lavageable protein in B6 mice with targeted disruption of Nos2 (Nos2-/-) was 50% less than protein in wild type Nos2+/+ mice following O3. Together, results are consistent with an important role for iNOS in O3-induced lung hyperpermeability and suggest that Nos2 mRNA levels are mediated through Tlr4. Our laboratory has also begun to investigate the role innate immunity genes (in particular, TLR4) in lung neoplasia. TLRs transduce exogenous and endogenous signals into production of inflammatory cytokines to shape and coordinate adaptive immune responses. By using models of Tlr4 deletion and mutation, we have determined that TLR4 has a protective role in chronic lung inflammation and carcinogenesis.

我们先前鉴定出明显的定量性状基因座（QTL），分别在染色体17和4上分别针对0.3 ppm臭氧（O3）的炎症和高度过度响应。染色体17 QTL包括编码TNF-A的候选基因TNF。 TNF-A是一种关键的促炎细胞因子，并且具有抗TNF-A抗体的易感C57BL/6J小鼠的预处理显着减弱了O3诱导的肺损伤，为TNF作为O3-敏感性基因提供了有力的TNF证据。为了进一步了解TNF-A调节O3诱导的炎症和损伤的机制，我们开始研究O3暴露后TNF-A细胞信号传导。 TNF-A的细胞作用是由两个结构相关但功能上不同的受体介导的：TNF受体1（55 kDa，TNFR1）和TNF受体2（75 kDa，TNFR2）。为了研究该模型中TNFR1和TNFR2的作用，具有p55 TNF受体（TNFR1-/ - ），p75 TNF受体（p75 tnf受体（TNF）的基因的O3敏感性C57BL/6J小鼠（B6;野生型，WT）和B6小鼠的作用暴露于0.3 ppm O3持续48小时（亚急性），并通过支气管肺泡灌洗确定肺反应。与WT小鼠相比，所有TNFR缺陷型小鼠的炎症和上皮损伤的明显少得多，但没有肺过敏性。与空气对照相比，O3分别在WT小鼠中引起了肺TNFR1和TNFR2 mRNA的上调，分别在TNFR2 - / - 和TNFR1 - / - 小鼠中被调节的TNFR1和TNFR2 mRNA下调。与WT相比，急性O3暴露（2 ppm，3小时）诱导的气道高反应性降低，尽管肺部炎症和渗透性仍然升高。结果表明，TNFR信号在亚急性O3诱导的肺上皮损伤和炎症以及急性O3诱导的气道高反应性中的关键作用。我们继续进行这些研究，以进一步研究TNFR调节O3诱导的肺损伤的机制。 我们将Toll像受体4（TLR4）一样确定为染色体4 QTL中的候选敏感性基因。 TLR4由于其在果蝇和人类的先天免疫中的作用而引人入胜，及其在调节内毒素反应中的重要性。 TLR4属于一系列Toll样受体（TLR），该家族激活细胞内信号传导，从而导致各种效应基因诱导。 TLR的细胞质结构域与白介素（IL）-1受体（IL-1R）家族的细胞质结构域同源，并共享信号传导成分。特别是，转录因子NF（核因子）-Kb是TLR4激活的重要效应因子，因为NF-KB已显示出在多种炎症过程中至关重要。两种观察结果提供了TLR4作为O3敏感性的候选基因的强大支持证据。首先，HEJ和OUJ小鼠之间O3诱导的肺过敏性显着差异，这仅在TLR4基因的第三个外显子中的错义突变中有所不同。其次，暴露于O3后，相对于OUJ，HEJ小鼠肺中的TLR4 mRNA水平降低，这表明对TLR4基因表达的下降调节可能有助于HEJ小鼠的O3耐药性。 为了开始研究TLR4调节O3响应的机制，我们测试了以下假设：1）诱导的一氧化氧化物合酶（INOS）介导O3诱导的肺过敏性和2）INOS基因（NOS2）的mRNA水平由TLR4在O3曝光过程中调节。对O3敏感的C57BL/6J（B6）小鼠的预处理具有特定的NOS（L-NMMA，NG-甲基-L-精氨酸）的特异性抑制剂，可显着降低平均液平均可厕所蛋白浓度（肺通透性标记），诱导的是O3（0.3 ppm，72 HR）。此外，在O3之后，在野生型NOS2+/+小鼠中，NOS2靶向破坏（NOS2 - / - ）的B6小鼠中的可溶剂蛋白比蛋白低50％。总之，结果与INOS在O3诱导的肺过敏性中的重要作用一致，并表明NOS2 mRNA水平是通过TLR4介导的。 我们的实验室还开始研究肺部肿瘤中先天免疫基因（尤其是TLR4）的作用。 TLRS将外源和内源性信号转换为炎症细胞因子的产生，以塑造和协调适应性免疫反应。通过使用TLR4缺失和突变的模型，我们确定TLR4在慢性肺部炎症和致癌作用中具有保护作用。