Compartment-specific transcriptomics of ozone-exposed murine lungs reveals sex- and cell type-associated perturbations relevant to mucoinflammatory lung diseases

Abstract

Ozone is known to cause lung injury and resident cells of the respiratory tract, i.e., epithelial cells and macrophages, respond to inhaled ozone in a variety of ways that affect their survival, morphology, and functioning. However, a complete understanding of the sex-associated and the cell type-specific gene expression changes in response to ozone exposure is still limited. Through transcriptomics, we aimed to analyze gene expression alterations and associated enrichment of biological pathways enrichment in three distinct cell type-enriched compartments of ozone-exposed murine lungs. We sub-chronically exposed adult males and females to 0.8ppm ozone or filtered air. RNA-Seq was performed on airway epithelium-enriched airways, parenchyma, and purified airspace macrophages. Differential gene expression and biological pathway analyses were performed and supported by cellular and immunohistochemical analyses. While a majority of differentially expressed genes (DEGs) in ozone-exposed versus air-exposed groups were common between both sexes, sex-specific DEGs were also identified in all the three tissue compartments. As compared to ozone-exposed males, ozone-exposed females had significant alterations in gene expression in three compartments. Pathways relevant to cell division and DNA repair were enriched in the ozone-exposed airways indicating ozone-induced airway injury and repair which was further supported by immunohistochemical analyses. In addition to cell division and DNA repair pathways, inflammatory pathways were also enriched within the parenchyma supporting contribution by both epithelial and immune cells. Finally, immune response and cytokine-cytokine receptor interactions were enriched in macrophages, indicating ozone-induced macrophage activation. Lastly, our analyses also revealed ozone-induced upregulation of mucoinflammation- and mucous cell metaplasia-associated pathways.

Vesicular and Extravesicular Protein Signatures From the Airspaces of Ozone-Exposed Mice Reflect Muco-Inflammatory Disturbances.

“…Exposure to high levels of ozone for nearly a decade increases the susceptibility to the development of COPD 77 Previous reports have demonstrated that female mice show exaggerated inflammatory responses to ozone inhalation as compared to their male counterparts. In our recent report 17 , we also reported that, as compared to ozone-exposed males, ozone-exposed females exhibit exaggerated recruitment of inflammatory cells including macrophages, neutrophils, eosinophils, and lymphocytes. Consistent with cellular recruitment, the levels of total protein were also significantly elevated within the lungs of the ozone-exposed females versus ozone-exposed males.…”

“…Tissues were harvested from experimental mice 12-16 hours after the end of the last exposure. Of note, BALF samples and lung tissues were harvested from the same cohort of mice that were exposed to air or ozone for our recently published study 17 .…”

“…In our recent publication 17 Under homeostatic conditions, epithelial cells and macrophages are known to communicate with each other to restrict their proinflammatory characteristics 54,55 . This adaptive phenomenon is critical in effectively curbing the exaggerated immune responses and disruption of the gas exchange function of the respiratory tract.…”

Integrative phenotypic and genomic analyses reveal strain-dependent responses to acute ozone exposure and their associations with airway macrophage transcriptional activity

“…To address this research gap, we exposed a panel of Collaborative Cross (CC) recombinant inbred mouse strains that span a broad phenotypic range of responses to acute O3 exposure and performed genomic analyses of airway macrophages, a key cell type known to mount dynamic responses to O3 (23)(24)(25)(26)(27)(28). The CC is a mouse genetic reference population derived from eight founder strains, whose genomes collectively harbor around 40 million singlenucleotide variants (SNVs) (29,30), thus capturing more than 90% of the genetic variation present in all inbred mouse strains (31).…”

Ozone exposure upregulates the expression of host susceptibility protein TMPRSS2 to SARS-CoV-2

“…To begin to understand the impact of ozone inhalation on the host susceptibility to SARS-CoV-2, we analyzed lung tissues collected from mice that were sub-chronically exposed to filtered-air or 0.8ppm ozone for three weeks (4h/night, 5 nights/week) 21 , and analyzed the expression of gene and protein signatures associated with host susceptibility to SARS-CoV-2. We used western blotting and immunohistochemistry for assessing expression levels of TMPRSS2 protein in three different lung tissue compartments.…”