Common Transcriptomic Changes Regulated by Innate Immune Receptors and Nuclear Factor NF-kappa-B in Mice Exposed to Ozone

Abstract

Ozone (O3) is a predominant oxidant in air pollution and has been linked to diminished lung function, respiratory inflammation, and worsening of preexisting airway diseases. Tumor necrosis factor (TNF)-α and toll-like receptor 4 (TLR4) play critical roles in innate immune responses. Signaling through TNF receptor (TNFR) and TLR4 activates nuclear factor NF-kappa-B (NF-κB), leading to the transcriptional regulation of immunity genes. The injurious roles of TNF-α, TLR4, and NF-κB in O3-induced lung pathogenesis have been determined in mice. The current study was designed to examine lung transcriptome changes and downstream mechanisms regulated by these innate immune response factors. Mice lacking Tnfr (Tnfr-/-) or Nfkb (Nfkb-/-), Tlr4 mutant mice (C3H/HeJ), and their wild-types (Tnfr+/+, Nfkb+/+, C3H/HeOuJ) were exposed to air or 0.3 ppm O3. Lung RNAs were isolated for cDNA microarray analysis. Significantly varied genes between the wild-type and gene knockout mice after O3 exposure were determined (2-way ANOVA) to elucidate commonly changed genes between the TNFR-NF-κB, TLR4-NF-κB, and TNFR-TLR4 pathways (Venn Diagram analysis). Downstream mechanisms affected by the common differentially regulated genes and chemicals/drugs that may act on O3-responsive genes were predicted by bioinformatics analysis tools. qPCR, ELISA, Western blotting, and chromatin immunoprecipitation assay validated the results. Tnfr- and Nfkb-dependently changed genes by O3, including matrix metalloproteinase 9 (Mmp9) and endothelin 1 (Edn1), were involved in cell death, connective tissue injury, and inflammation. Interleukin 12 (IL-12) and transforming growth factor (TGF)-β1 were suggested as upstream regulators. Genes changed in common by Tlr4 and Nfkb during O3 exposure (e.g. D-box binding PAR BZIP transcription factor, Dbp; lipocalin 2, Lcn2; serum amyloid A 3, Saa3) were related to lipid derangement, cell morphology, and lymphoid tissue hyperplasia. Common gene transcripts regulated by TNFR and TLR4 such as fatty acid binding protein 1 (Fabp1) and tissue inhibitor of metalloproteinase 1 (Timp1) contributed to the chemotaxis and fatty acid metabolism. In conclusion, the TNFR-NF-κB and TLR4-NF-κB pathways modulated distinct transcriptomes during the development of O3-induced lung injury in mice. Results will provide significant insights into the pulmonary O3 pathogenesis and therapeutic interventions.