Role of Notch receptors in ozone induced lung injury in mice

Ozone is a highly toxic air pollutant and global health concern. Mechanisms of genetic susceptibility to ozone-induced lung inflammation are not completely understood. We hypothesized Notch3 and Notch4 are important determinants of susceptibility to ozone-induced lung inflammation. Wild type (WT), Notch3 (Notch3-/-) and Notch4 (Notch4-/-) knockout mice were exposed to ozone (0.3 ppm) or filtered air for 6-72 hours. Ozone increased bronchoalveolar lavage fluid (BALF) protein, a marker of lung permeability, in all genotypes, but significantly greater concentrations were found in Notch4-/- compared to WT and Notch3-/-. Significantly greater mean numbers of BALF neutrophils were found in Notch3-/- and Notch4-/- mice compared to WT mice after ozone. Expression of whole lung Tnf was significantly increased after ozone in all genotypes, and was significantly greater in Notch3-/- mice compared to WT. Statistical analyses of the transcriptome identified differentially expressed gene networks between WT and knockout mice basally and after ozone, and included Trim30, a member of the inflammasome pathway, and Traf6, an inflammatory signaling member. These novel findings are consistent with Notch3 and Notch4 as susceptibility genes for ozone-induced lung injury, and suggest that Notch receptors protect against innate immune inflammation. Wild-type, Notch3 knockout, and Notch4 knockout mice at 7-13 weeks of age were exposed continuously to air or 0.3 ppm ozone for 6, 24, or 48 hours. Three biological replicates from individual animals were included in each exposure group from each genotype and samples hybridized to the GeneChip Mouse Genome 430 2.0 array (Affymetrix).臭氧是一种剧毒空气污染物，亦是全球公共健康领域的重点关切问题。臭氧诱导肺部炎症的遗传易感机制尚未完全阐明。本研究推测，Notch3与Notch4是影响臭氧诱导肺部炎症易感性的关键调控因子。实验对象为野生型（Wild type，WT）、Notch3敲除（Notch3-/-）及Notch4敲除（Notch4-/-）小鼠，将其分别暴露于0.3 ppm臭氧或过滤空气中，暴露时长为6至72小时。臭氧暴露可升高所有基因型小鼠的支气管肺泡灌洗液（bronchoalveolar lavage fluid, BALF）蛋白水平——该指标为肺部通透性的标志物，但Notch4-/-小鼠的灌洗液蛋白浓度显著高于WT及Notch3-/-小鼠。臭氧暴露后，Notch3-/-及Notch4-/-小鼠的支气管肺泡灌洗液中性粒细胞平均计数显著高于WT小鼠。所有基因型小鼠的全肺组织肿瘤坏死因子（Tumor necrosis factor, Tnf）转录水平在臭氧暴露后均显著升高，且Notch3-/-小鼠的Tnf表达水平显著高于WT小鼠。转录组（transcriptome）统计分析显示，野生型与敲除小鼠在基础状态及臭氧暴露后均存在差异表达基因网络，其中包含炎性小体通路成员Trim30以及炎症信号通路成员Traf6。上述新发现支持Notch3与Notch4为臭氧诱导肺损伤的易感基因，并提示Notch受体可抵御先天免疫炎症反应。本实验选用7至13周龄的野生型、Notch3敲除及Notch4敲除小鼠，将其持续暴露于空气或0.3 ppm臭氧中，暴露时长分别为6、24或48小时。每个基因型的各暴露组均设置3只独立个体作为生物学重复，提取样本后在Affymetrix GeneChip Mouse Genome 430 2.0芯片上进行杂交检测。