Genetic Mechanisms Of Susceptibility To Inflammation

我们以前确定了显着的数量性状位点（QTL）的炎症和高通透性反应0.3 ppm臭氧（O3）的染色体17和4，分别。染色体17 QTL包括编码TNF-α的候选基因Tnf。TNF-α是一种关键的促炎细胞因子，用抗TNF-α抗体预处理易感的C57 BL/6 J小鼠显著减弱了O3诱导的肺损伤，为TNF作为O3易感基因提供了强有力的证据。为了进一步了解TNF-α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-α细胞信号转导。TNF-α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，TNFR 1）和TNF受体2（75 kDa，TNFR 2）。为了研究TNFR 1和TNFR 2在该模型中的作用，O3易感的C57 BL/6 J小鼠（B6;野生型，wt）和B6小鼠p55 TNF受体基因的（敲除）在48小时（亚急性）内，将肿瘤坏死因子受体（TNFR 1-/-）、p75肿瘤坏死因子受体（TNFR 2-/-）或两者（TNFR 1/TNFR 2-/-）暴露于0.3 ppm O3，通过支气管肺泡灌洗测定肺反应。与野生型小鼠相比，所有TNFR缺陷小鼠的O3诱导的炎症和上皮损伤显着减少，但肺渗透性增高却没有减少。与空气对照组相比，O3引起野生型小鼠肺TNFR 1和TNFR 2 mRNA的上调，并分别下调TNFR 2-/-和TNFR 1-/-小鼠的TNFR 1和TNFR 2 mRNA。急性O3暴露（2 ppm，3小时）诱导的气道高反应性基因敲除小鼠相比，野生型减少，但肺部炎症和通透性仍然升高。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起关键作用。 我们确定了Toll样受体4（Tlr 4）作为4号染色体QTL内的候选易感基因。Tlr 4是特别有趣的，因为它在果蝇和人类的先天免疫中的作用，以及它在调节对内毒素的反应中的重要性。TLR 4属于Toll样受体（TLR）家族，其激活导致诱导多种效应基因的细胞内信号传导。TLR的胞质结构域与白细胞介素（IL）-1受体（IL-1 R）家族的胞质结构域同源，并共享信号传导组分。特别地，转录因子NF（核因子）-kB是Tlr 4活化的重要效应物，因为已经显示NF-kB在多种炎症过程中具有关键参与。两项观察结果为Tlr 4作为O3易感性的候选基因提供了强有力的支持证据。首先，O3诱导的肺高通透性是显着不同的HeJ和OuJ小鼠，不同的只是在Tlr 4基因的第三外显子的错义突变。第二，HeJ小鼠肺中Tlr 4 mRNA水平在暴露于O3后相对于OuJ降低，这表明Tlr 4基因表达的下调可能有助于HeJ小鼠的O3抗性。 为了开始研究Tlr 4调节O3反应的机制，我们测试了以下假设：1）诱导型一氧化氮合酶（iNOS）介导O3诱导的肺通透性增高，2）在O3暴露期间，Tlr 4调节iNOS（Nos 2）基因的mRNA水平。与溶剂对照组相比，用总NOS的特异性抑制剂（L-NMMA，NG-甲基-L-精氨酸）预处理O3-敏感C57 BL/6 J（B6）小鼠显著降低了O3（0.3 ppm，72小时）诱导的平均可灌洗蛋白浓度（肺渗透性的标志物）。此外，在O3后，具有Nos 2靶向破坏（Nos 2-/-）的B6小鼠中的可灌洗蛋白质比野生型Nos 2 +/+小鼠中的蛋白质少50%。总之，结果与iNOS在O3诱导的肺高通透性中的重要作用一致，并表明Nos 2 mRNA水平通过Tlr 4介导。

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 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.