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Pulmonary tolerance to ozone is associated with changes in the alveolar macrophage transcriptome

ABSTRACT: Tolerance to the air pollutant ozone refers to the phenomenon by which pre-exposure to a relatively low concentration confers protection against from lung injury and inflammation caused by a subsequent challenge with high concentration ozone. We investigated whether alveolar macrophages play a central role in this process. First, we established a model of ozone tolerance in which inbred C57BL/6NJ mice were pre-exposed to 0.8 ppm ozone for 4 days then challenged with 2 ppm ozone on day 7, followed by sacrifice and phenotyping on Day 8. As expected, pre-exposure significantly reduced ozone-induced airway injury and inflammation on day 8. To investigate the role of alveolar macrophages, we treated mice with clodronate on day 5, which reduced macrophage number by ~5-fold. Following ozone challenge on day 7, clodronate treated mice exhibited increased injury compared to vehicle treated controls. Cytokines involved in neutrophil chemotaxis such as IL-6, MIP-1b, and KC, were also significantly elevated in clodronate treated mice. Collectively, these data indicate an important role of AMs in this model of tolerance. To identify differences in the phenotype of AMs caused by pre-exposure to ozone, we isolated AMs from ozone pre-exposed mice vs. filtered air controls using flow sorting and performed RNA-seq analysis of these samples. We found that a number of genes and pathways were altered by ozone pre-exposure, most importantly downregulation of pathways involved in toll-like receptor cell signaling, proinflammatory gene expression, and NF-kB mediated gene regulation.

ORGANISM(S): Mus musculus

PROVIDER: GSE248291 | GEO | 2025/07/18

REPOSITORIES: GEO

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Project description:Rationale: Previous work demonstrated that pre-exposure to ozone primes innate immunity and increases TLR4-mediated response to subsequent stimulation with lipopolysaccharide (LPS). To further explore the pulmonary innate immune response to ozone exposure, we investigated the effect of ozone in combination with Pam3CYS, a synthetic TLR2/TLR1 agonist. Methods: Bronchoalveolar lavage (BAL) and lungs were harvested from C57Bl/6 mice after exposure to ozone or filtered air followed by saline or Pam3CYS 24 hours later. Cells and cytokines in the BAL, surface expression of TLRs on macrophages, and lung RNA genomic expression profiles were examined. Results: We demonstrate an increased BAL cell influx, increased IL-6 and KC, and decreased MIP-1α and TNF-α in response to Pam3CYS as a result of ozone pre-exposure. We also observed increased cell surface expression of TLR4, TLR2 and TLR1 on macrophages as a result of ozone alone or in combination with Pam3CYS. Gene expression analysis of lung tissue revealed a significant increase in expression of genes related to injury repair and cell cycle as a result of ozone exposure. When comparing Pam3CYS treated animals to saline treated animals with or without ozone, genes associated with inflammation were significantly increased. Potentially novel ozone exposure candidate genes (CCK, RELM-α and β) were identified. Conclusion: Our results extend previous findings with ozone/LPS to other TLRs PAMPs and suggest that ozone priming of innate immunity is a general mechanism. Gene expression profiling of lung tissue identified transcriptional networks and genes that contribute to the priming of innate immunity at the molecular level. Mice were exposed to either 2ppm ozone or filtered air (FA) for 3 hours. 24 hours following ozone exposure, mice from either the ozone or FA group were treated intratracheally with either 100ug of Pam3CYS in saline or saline alone. Animals were euthanized 4 or 24 hours post-Pam3CYS exposure. RNA from whole lung tissue from 4 animals per group was profiled.

Project description:Long-term exposure to particulate air pollutants has been linked to increased incidence of Type 2 Diabetes (T2DM). Recently, we showed that the gaseous pollutant, O3, induced glucose intolerance, and increased serum leptin and epinephrine in Brown Norway rats. In this study, we hypothesized that O3 exposure will cause broad scale changes in metabolic homeostasis involving liver, muscle and adipose tissues, and that serum metabolomic and liver transcriptomic profiling will provide mechanistic insights into pollutant-induced metabolic alterations. In the first experiment, male Wistar Kyoto (WKY) rats were exposed to filtered air (FA) or O3 at 0.25, 0.50, or 1.00 ppm, 6h/day for two consecutive days to establish concentration-related effects on glucose tolerance and lung injury. In a second experiment, rats were exposed to FA or 1.0 ppm O3, 6h/day for either one day or two consecutive days and systemic metabolic responses were determined immediately after each exposure or after an 18h recovery period. In WKY rats, O3 increased serum fasting glucose and leptin on day 1. Glucose intolerance persisted through two days of exposure but reversed 18h post second exposure. O3 exposure increased circulating metabolites of glycolysis, long-chain free fatty acids, branched chain amino acids (BCAA) and cholesterol while 1,5- anhydroglucitol, bile acids and metabolites of TCA cycle were decreased, indicating impaired glycemic control, muscle proteolysis and adipose lipolysis. Liver gene expression profile after O3 exposure reflected a response to the serum metabolite changes, as evidenced by increased expression of genes for glycolysis, TCA cycle and gluconeogenesis, and decreased expression of genes involved in steroid and fat biosynthesis. In conclusion, short-term O3 exposure induced hormonal changes and global metabolic disorder reflective of changes in peripheral glucose, lipid, and amino acid metabolism, representative of a stress-response. It remains to be examined if these metabolic alterations contribute to insulin resistance upon chronic exposure. Rats were exposed to filtered air (FA) or Ozone at 1 ppm, 6h/day for two consecutive days to establish ozone-exposure related effects on transcriptomic profiles. Samples were taken at three time points: 1 day (1day0hrs) - at the end of the 6 hour exposure period, 2 days (2day0hrs) - at the end of the of the second day 6 hour exposure period) and 2 days 18 hours (2day18hrs) - 18 hours after the 6 hour exposure period on the second day. Total liver RNA was isolated from ~20 mg tissue with a commercially available RNeasy mini kit (Qiagen, Valencia, CA) using silica gel membrane purification. Liver RNA was resuspended in 30μl of RNAse- free water. RNAse inhibitor was added and RNA yield was determined spectrophotometrically on a NanoDrop 1000 (Thermo Scientific, Wilmington, DE). RNA integrity was assessed by the RNA 6000 LabChip® kit using a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). We examined global gene expression changes using the Affymetrix platform (RG- 230 PM Array strip). Biotin-labeled cRNA was produced from total RNA using an Affymetrix “IVT-express labeling kit “(cat# 901229).

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Pulmonary tolerance to ozone is associated with changes in the alveolar macrophage transcriptome

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