Genetic Mechanisms Of Susceptibility To Ozone-induced Pulmonary Inflammation

我们之前分别在17号和4号染色体上发现了0.3 ppm臭氧（O3）对炎症和高渗透性反应的显著数量性状位点（qtl）。17号染色体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，抗Tnf -a抗体预处理易感C57BL/6J小鼠可显著减轻o3诱导的肺损伤，为Tnf作为o3易感基因提供了有力证据。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TNF-a在O3暴露后的细胞信号传导。TNF-a的细胞效应由两种结构相关但功能不同的受体介导：TNF受体1 （55 kDa, TNFR1）和TNF受体2 （75 kDa, TNFR2）。为了研究TNFR1和TNFR2在该模型中的作用，将p55 TNF受体（TNFR1-/-）、p75 TNF受体（TNFR2-/-）或两者（TNFR1/TNFR2-/-）基因靶向破坏（敲除）的O3易感C57BL/6J小鼠（B6；野生型，wt）和B6小鼠（B6）暴露于0.3 ppm O3中48小时（亚急性），并通过支气管肺泡灌洗测定肺反应。所有tnfr缺陷小鼠与wt小鼠相比，o3诱导的炎症和上皮损伤明显减少，但肺通透性不高。与空气对照组相比，O3诱导wt小鼠肺TNFR1和TNFR2 mRNA表达上调，TNFR2-/-和TNFR1-/-小鼠肺TNFR1和TNFR2 mRNA表达下调。与wt相比，基因敲除小鼠急性暴露于O3 （2ppm， 3小时）诱导的气道高反应性减弱，但肺部炎症和通透性仍然升高。结果表明，TNFR信号在亚急性臭氧诱导的肺上皮损伤和炎症以及急性臭氧诱导的气道高反应性中起关键作用。我们继续进行这些研究，以进一步研究TNFR调节臭氧诱导的肺损伤的机制。一项研究旨在确定TNF受体（TNF- r）介导的细胞信号传导和O3诱导的肺损伤的分子机制。我们发现，在肺损伤发生前，O3显著激活了Tnfr wt小鼠的肺NF-kappaB。在tnf（-/-）小鼠中，基础和o3诱导的NF-kappaB活性被抑制。与tnf（+/+）小鼠相比，tnf（-/-）小鼠的MAPKs和激活蛋白(AP)-1基本和O3后均较低。此外，炎症细胞因子，包括巨噬细胞炎症蛋白-2，在O3后在Tnfr（-/-）和Tnfr wt小鼠中表达差异。与相应的对照动物相比，Nfkb1（-/-）和Jnk1（-/-）小鼠o3诱导的肺损伤明显减轻。我们的研究结果表明，NF-kappaB和MAPK/AP-1信号通路在O3诱导的tnf - r介导的肺毒性中是必不可少的。

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. One investigation was designed to determine the molecular mechanisms of TNF receptor (TNF-R)-mediated cell signaling and lung injury induced by O3. We found that O3 significantly activated lung NF-kappaB in Tnfr wt mice before the development of lung injury. Basal and O3-induced NF-kappaB activity was suppressed in Tnfr(-/-) mice. Compared with Tnfr(+/+) mice, MAPKs and activator protein (AP)-1 were lower in Tnfr(-/-) mice basally and after O3. Furthermore, inflammatory cytokines, including macrophage inflammatory protein-2, were differentially expressed in Tnfr(-/-) and Tnfr wt mice after O3. O3-induced lung injury was significantly reduced in Nfkb1(-/-) and Jnk1(-/-) mice relative to respective control animals. Our results suggest that NF-kappaB and MAPK/AP-1 signaling pathways are essential in TNF-R-mediated pulmonary toxicity induced by O3. To further investigate the genes within the chromosome 17 QTL that control susceptibility to O3-induced inflammation, we have used additional genetic tools to restrict the QTL and focus on additional candidate genes within the restricted QTL. Using congenic mice, differential gene expression, and informative sequence variation we have identified MHC Class II genes as novel important genes that contribute to differential O3 susceptibility. As early inflammatory response by inhaled O3 is characterized primarily by release of inflammatory mediators such as cytokines, chemokines, and airway neutrophil accumulation. Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of oxidative lung disorders including acute lung injury, asthma and chronic obstructive pulmonary disease. We have therefore begun to characterize the role of these mediators and innate immune molecules in the acute phase of the inflammatory response induced by O3 exposure. In collaboration with Les Kobzik at Harvard, we have also begun to investigate the mechanisms through which TLR4 modulates O3-induced inflammation. We used microarray analyses to find that macrophage receptor with collagenous structure (MARCO) was upregulated after O3 exposure in mice with a mutation in TLR4 relative to mice with normal TLR4. We then found that after ozone exposure, MARCO-/- mice showed greater lung injury than did MARCO+/+ mice. Further, intratracheal instillation of oxidized lipids known to be created by ozone exposure caused substantial neutrophil influx in MARCO-/- mice, but had no effect in MARCO+/+ mice. These results led us to conclude that MARCO has an important role in decreasing pulmonary inflammation after oxidant inhalation by scavenging proinflammatory oxidized lipids from lung lining fluids. Our laboratory has also begun to investigate the role innate immunity genes (in particular, TLR4) and inflammation 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.