Genetic Mechanisms Of Susceptibility To Ozone-induced Pu

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。

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.