Transcriptional profiling of lung macrophages following ozone exposure in mice identifies signaling pathways regulating immunometabolic activation

Macrophages play a key role in ozone-induced lung injury by regulating both the initiation and resolution of inflammation. These distinct activities are mediated by pro-inflammatory and anti-inflammatory/pro-resolution macrophages which sequentially accumulate in injured tissues. Macrophage activation is dependent, in part, on intracellular metabolism. Herein, we used RNA-sequencing (seq) to identify signaling pathways regulating macrophage immunometabolic activity following exposure of mice to ozone (0.8 ppm, 3 hr) or air control. Analysis of lung macrophages using an Agilent Seahorse showed that inhalation of ozone increased macrophage glycolytic activity and oxidative phosphorylation at 24 and 72 hr post exposure. An increase in the percentage of macrophages in the S phase of the cell cycle was observed 24 hr post ozone. RNA-seq revealed significant enrichment of pathways involved in innate immune signaling and cytokine production among differentially expressed genes at both 24 and 7..., Total RNA was extracted as described above from 3 mice/treatment group. In a pilot study, we found that 3 mice were sufficient to identify a significant difference in Ptgs2 gene expression by qPCR at α = 0.05 and power = 80%. RNA integrity numbers (RINs) were confirmed to be ≥ 8.8 using a 2100 Bioanalyzer Instrument (Agilent, Santa Clara, CA). cDNA libraries were prepared using mouse TruSeq® Stranded Total RNA Library Prep kit (illumina, San Diego, CA) and quantified using a KAPA Library Quantification kit (Roche, Pleasanton, CA). cDNA libraries were sequenced (75 bp single-ended, ~35-44M reads per sample) on an Illumina NextSeq instrument. Raw reads in FastQ files were trimmed using Trimmomatic-0.39 (Bolger et al. 2014) and quality control of trimmed files performed using FastQC. Salmon was used to align reads in mapping-based mode with selective alignment against a decoy-aware transcriptome generated from mouse transcriptome GENCODE Release M23 (GRCm38.p6). Estimated counts per transc..., , # Transcriptional profiling of lung macrophages following ozone exposure in mice identifies signaling pathways regulating immunometabolic activation

[https://doi.org/10.5061/dryad.b8gtht7mq](https://doi.org/10.5061/dryad.b8gtht7mq)

We analyzed gene expression profiles in bronchoalveolar lavage cells (>95% macrophages) isolated from adult female mice exposed to ozone using bulk RNA-sequencing. Mice were sampled 24 and 72 hr after exposure. Specific analysis details are available in the associated manuscript.

## Description of the data and file structure

Counts data were analyzed using DESeq2 which resulted in multiple results files:

* File \"Supplementary File 1_24 hr DEGs\" contains differential expression data generated from DESeq2 comparing counts at 24 hr to counts in air controls.
* File \"Supplementary File 2_72 hr DEGs\" contains differential expression data generated from DESeq2 comparing counts at 72 hr to counts in air controls.
* File \"Supplementary File 3_72 hr_vs_24 hr D...

巨噬细胞通过调控炎症的启动与消退，在臭氧诱导的肺损伤中发挥关键作用。这两种截然不同的功能分别由促炎型与抗炎/促消退型巨噬细胞介导，它们会依次在损伤组织中聚集。巨噬细胞的激活在一定程度上依赖于细胞内代谢过程。本研究采用RNA测序（RNA-seq），旨在探究小鼠暴露于臭氧（0.8 ppm，3小时）或空气对照后，调控肺巨噬细胞免疫代谢活性的信号通路。通过安捷伦海马生物能量代谢分析仪对肺巨噬细胞进行分析，结果显示，臭氧吸入可在暴露后24小时和72小时增强巨噬细胞的糖酵解活性与氧化磷酸化水平。臭氧暴露后24小时，可观察到处于细胞周期S期的巨噬细胞占比升高。RNA-seq结果显示，在24小时和7... 时间点的差异表达基因中，先天免疫信号通路与细胞因子产生相关通路均显著富集。

总RNA提取：每处理组取3只小鼠，按照前述方法提取总RNA。预实验结果显示，当检验水准α=0.05、检验效能为80%时，3只小鼠即可通过实时定量PCR（qPCR）检测到Ptgs2基因表达的显著差异。采用2100生物分析仪（安捷伦，美国加利福尼亚州圣克拉拉市）验证RNA完整性数（RINs）≥8.8。使用小鼠TruSeq® 链特异性总RNA文库制备试剂盒（Illumina，美国加利福尼亚州圣迭戈市）构建cDNA文库，并采用KAPA文库定量试剂盒（罗氏，美国加利福尼亚州普莱森顿市）对文库进行定量。在Illumina NextSeq测序仪上对cDNA文库进行测序（75 bp单端测序，每个样本约35-44M条读段）。使用Trimmomatic-0.39（Bolger等人，2014年）对FastQ文件中的原始读段进行修剪，并通过FastQC对修剪后的读段进行质量控制。采用Salmon软件以映射模式结合选择性比对策略，将读段比对到由小鼠转录组GENCODE发布版M23（GRCm38.p6）构建的诱饵感知转录组。每个转录本的估计计数... , , # 小鼠臭氧暴露后肺巨噬细胞的转录组分析揭示调控免疫代谢激活的信号通路

[https://doi.org/10.5061/dryad.b8gtht7mq](https://doi.org/10.5061/dryad.b8gtht7mq)

我们采用批量RNA测序（bulk RNA-seq）分析了暴露于臭氧的成年雌性小鼠分离得到的支气管肺泡灌洗液细胞（其中巨噬细胞占比＞95%）的基因表达谱。小鼠于暴露后24小时和72小时被取样。具体分析细节见相关研究论文。

## 数据与文件结构说明
采用DESeq2对计数数据进行分析，生成了多份结果文件：
* 文件"Supplementary File 1_24 hr DEGs"包含通过DESeq2生成的、对比24小时暴露组与空气对照组计数数据的差异表达分析结果。
* 文件"Supplementary File 2_72 hr DEGs"包含通过DESeq2生成的、对比72小时暴露组与空气对照组计数数据的差异表达分析结果。
* 文件"Supplementary File 3_72 hr_vs_24 hr D..."