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...