Role of Notch receptors in ozone induced lung injury in mice. Mus musculus

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. Overall design: 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，亦是全球范围内广受关注的公共健康问题。臭氧诱导性肺部炎症的遗传易感机制尚未完全阐明。本研究提出假说：Notch3与Notch4是臭氧诱导性肺部炎症易感性的关键决定因素。将野生型（Wild type, WT）、Notch3基因敲除（Notch3-/-）及Notch4基因敲除（Notch4-/-）小鼠暴露于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——该指标可反映肺部通透性，但Notch4-/-小鼠的BALF蛋白浓度显著高于WT及Notch3-/-小鼠。臭氧暴露后，Notch3-/-与Notch4-/-小鼠的BALF中性粒细胞平均计数均显著高于WT小鼠。所有基因型小鼠的全肺Tnf表达水平均因臭氧暴露显著上调，且Notch3-/-小鼠的Tnf表达水平显著高于WT小鼠。对转录组（transcriptome）的统计分析显示，在基线状态及臭氧暴露后，WT小鼠与基因敲除小鼠间存在差异表达基因网络，其中包括炎症小体通路（inflammasome pathway）成员Trim30以及炎症信号通路成员Traf6。上述新发现支持Notch3与Notch4可作为臭氧诱导性肺损伤的易感基因，同时提示Notch受体可对抗先天免疫炎症（innate immune inflammation）。总体实验设计：选取7至13周龄的野生型、Notch3基因敲除及Notch4基因敲除小鼠，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及48小时。每个基因型的每个暴露组均设置3只来自不同个体的生物学重复（biological replicates），样本均与GeneChip Mouse Genome 430 2.0基因芯片（Affymetrix）进行杂交。