Repeated exposure to eucalyptus wood smoke alters pulmonary gene and metabolic profiles in male Long-Evans rats.

Exposure to wildfire smoke is associated with both acute and chronic cardiopulmonary illnesses, which are of special concern for wildland firefighters who experience repeated exposure to wood smoke. It is necessary to better understand the underlying pathophysiology by which wood smoke exposure increases pulmonary disease burdens in this population. We hypothesize that wood smoke exposure produces pulmonary dysfunction, lung inflammation, and gene expression profiles associated with future pulmonary complications. Male Long-Evans rats were intermittently exposed to smoldering eucalyptus wood smoke at 2 concentrations, low (11.0 ± 1.89 mg/m3) and high (23.7 ± 0.077 mg/m3), over a 2-week period. Whole-body plethysmography was measured intermittently throughout. Lung tissue and lavage fluid were collected 24 h after the final exposure for transcriptomics and metabolomics. Increasing smoke exposure upregulated neutrophils and select cytokines in the bronchoalveolar lavage fluid. In total, 3446 genes were differentially expressed in the lungs of rats in the high smoke exposure and only 1 gene in the low smoke exposure (Cd151). Genes altered in the high smoke group reflected changes to the Eukaryotic Initiation Factor 2 stress and oxidative stress responses, which mirrored metabolomics analyses. xMWAS-integrated analysis revealed that smoke exposure significantly altered pathways associated with oxidative stress, lung morphogenesis, and tumor proliferation pathways. These results indicate that intermittent, 2-week exposure to eucalyptus wood smoke leads to transcriptomic and metabolic changes in the lung that may predict future lung disease development. Collectively, these findings provide insight into cellular signaling pathways that may contribute to the chronic pulmonary conditions observed in wildland firefighters.

Scavenger receptor BI attenuates oxidized phospholipid-induced pulmonary inflammation.

Damage associated molecular patterns (DAMPs) are molecules released from dead/dying cells following toxicant and/or environmental exposures that activate the immune response through binding of pattern recognition receptors (PRRs). Excessive production of DAMPs or failed clearance leads to chronic inflammation and delayed inflammation resolution. One category of DAMPs are oxidized phospholipids (oxPLs) produced upon exposure to high levels of oxidative stress, such as following ozone (O3) induced inflammation. OxPLs are bound by multiple classes of PRRs that include scavenger receptors (SRs) such as SR class B-1 (SR-BI) and toll-like receptors (TLRs). Interactions between oxPLs and PRRs appear to regulate inflammation; however, the role of SR-BI in oxPL-induced lung inflammation has not been defined. Therefore, we hypothesize that SR-BI is critical in protecting the lung from oxPL-induced pulmonary inflammation/injury. To test this hypothesis, C57BL/6J (WT) female mice were dosed with oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (oxPAPC) by oropharyngeal aspiration which increased pulmonary SR-BI expression. Following oxPAPC exposure, SR-BI deficient (SR-BI-/-) mice exhibited increased lung pathology and inflammatory cytokine/chemokine production. Lipidomic analysis revealed that SR-BI-/- mice had an altered pulmonary lipidome prior to and following oxPAPC exposure, which correlated with increased oxidized phosphatidylcholines (PCs). Finally, we characterized TLR4-mediated activation of NF-κB following oxPAPC exposure and discovered that SR-BI-/- mice had increased TLR4 mRNA expression in lung tissue and macrophages, increased nuclear p65, and decreased cytoplasmic IκBα. Overall, we conclude that SR-BI is required for limiting oxPAPC-induced lung pathology by maintaining lipid homeostasis, reducing oxidized PCs, and attenuating TLR4-NF-κB activation, thereby preventing excessive and persistent inflammation.

Ozone impairs endogenous compensatory responses in allergic asthma.

Asthma is a chronic inflammatory airway disease characterized by acute exacerbations triggered by inhaled allergens, respiratory infections, or air pollution. Ozone (O3), a major component of air pollution, can damage the lung epithelium in healthy individuals. Despite this association, little is known about the effects of O3 and its impact on chronic lung disease. Epidemiological data have demonstrated that elevations in ambient O3 are associated with increased asthma exacerbations. To identify mechanisms by which O3 exposure leads to asthma exacerbations, we developed a two-hit mouse model where mice were sensitized and challenged with three common allergens (dust mite, ragweed and Aspergillus fumigates, DRA) to induce allergic inflammation prior to exposure to O3 (DRAO3). Changes in lung physiology, inflammatory cells, and inflammation were measured. Exposure to O3 following DRA significantly increased airway hyperreactivity (AHR), which was independent of TLR4. DRA exposure resulted in increased BAL eosinophilia while O3 exposure resulted in neutrophilia. Additionally, O3 exposure following DRA blunted anti-inflammatory and antioxidant responses. Finally, there were significantly less monocytes and innate lymphoid type 2 cells (ILC2s) in the dual challenged DRA-O3 group suggesting that the lack of these immune cells may influence O3-induced AHR in the setting of allergic inflammation. In summary, we developed a mouse model that mirrors some aspects of the clinical course of asthma exacerbations due to air pollution and identified that O3 exposure in the asthmatic lung leads to impaired endogenous anti-inflammatory and antioxidant responses and alterations inflammatory cell populations.

Transcriptomic-Metabolomic Profiling in Mouse Lung Tissues Reveals Sex- and Strain-Based Differences.

Omics analyses are commonly used for identifying pathways and genes responsible for physiologic and pathologic processes. Though sex is considered a biological variable in rigorous assessments of pulmonary responses to oxidant exposures, the contribution of the murine strain is largely ignored. This study utilized an unbiased integrated assessment of high-resolution metabolomic profiling and RNA-sequencing to explore sex- and strain-dependent pathways in adult mouse lungs. The results indicated that strain exhibited a greater influence than sex on pathways associated with inflammatory and oxidant/antioxidant responses and that interaction metabolites more closely resembled those identified as differentially expressed by strain. Metabolite analyses revealed that the components of the glutathione antioxidant pathway were different between strains, specifically in the formation of mixed disulfides. Additionally, selenium metabolites such as selenohomocystiene and selenocystathionine were similarly differentially expressed. Transcriptomic analysis revealed similar findings, as evidenced by differences in glutathione peroxidase, peroxiredoxin, and the inflammatory transcription factors RelA and Jun. In summary, an multi-omics integrated approach identified that murine strain disproportionately impacts baseline expression of antioxidant systems in lung tissues. We speculate that strain-dependent differences contribute to discrepant pulmonary responses in preclincal mouse models, with deleterious effects on clinical translation.

Characterization of subunit interactions in the hetero-oligomeric retinoid oxidoreductase complex.

The hetero-oligomeric retinoid oxidoreductase complex (ROC) catalyzes the interconversion of all-trans-retinol and all-trans-retinaldehyde to maintain the steady-state output of retinaldehyde, the precursor of all-trans-retinoic acid that regulates the transcription of numerous genes. The interconversion is catalyzed by two distinct components of the ROC: the NAD(H)-dependent retinol dehydrogenase 10 (RDH10) and the NADP(H)-dependent dehydrogenase reductase 3 (DHRS3). The binding between RDH10 and DHRS3 subunits in the ROC results in mutual activation of the subunits. The molecular basis for their activation is currently unknown. Here, we applied site-directed mutagenesis to investigate the roles of amino acid residues previously implied in subunit interactions in other SDRs to obtain the first insight into the subunit interactions in the ROC. The results of these studies suggest that the cofactor binding to RDH10 subunit is critical for the activation of DHRS3 subunit and vice versa. The C-terminal residues 317-331 of RDH10 are critical for the activity of RDH10 homo-oligomers but not for the binding to DHRS3. The C-terminal residues 291-295 are required for DHRS3 subunit activity of the ROC. The highly conserved C-terminal cysteines appear to be involved in inter-subunit communications, affecting the affinity of the cofactor binding site in RDH10 homo-oligomers as well as in the ROC. Modeling of the ROC quaternary structure based on other known structures of SDRs suggests that its integral membrane-associated subunits may be inserted in adjacent membranes of the endoplasmic reticulum (ER), making the formation and function of the ROC dependent on the dynamic nature of the tubular ER network.

Sex Differences in Pulmonary Eicosanoids and Specialized Pro-Resolving Mediators in Response to Ozone Exposure.

Ozone (O3) is a criteria air pollutant known to increase the morbidity and mortality of cardiopulmonary diseases. This occurs through a pulmonary inflammatory response characterized by increased recruitment of immune cells into the airspace, pro-inflammatory cytokines, and pro-inflammatory lipid mediators. Recent evidence has demonstrated sex-dependent differences in the O3-induced pulmonary inflammatory response. However, it is unknown if this dimorphic response is evident in pulmonary lipid mediator metabolism. We hypothesized that there are sex-dependent differences in lipid mediator production following acute O3 exposure. Male and female C57BL/6J mice were exposed to 1 part per million O3 for 3 h and were necropsied at 6 or 24 h following exposure. Lung lavage was collected for cell differential and total protein analysis, and lung tissue was collected for mRNA analysis, metabololipidomics, and immunohistochemistry. Compared with males, O3-exposed female mice had increases in airspace neutrophilia, neutrophil chemokine mRNA, pro-inflammatory eicosanoids such as prostaglandin E2, and specialized pro-resolving mediators (SPMs), such as resolvin D5 in lung tissue. Likewise, precursor fatty acids (arachidonic and docosahexaenoic acid; DHA) were increased in female lung tissue following O3 exposure compared with males. Experiments with ovariectomized females revealed that loss of ovarian hormones exacerbates pulmonary inflammation and injury. However, eicosanoid and SPM production were not altered by ovariectomy despite depleted pulmonary DHA concentrations. Taken together, these data indicate that O3 drives an increased pulmonary inflammatory and bioactive lipid mediator response in females. Furthermore, ovariectomy increases susceptibility to O3-induced pulmonary inflammation and injury, as well as decreases pulmonary DHA concentrations.

Scavenger Receptor BI Attenuates IL-17A-Dependent Neutrophilic Inflammation in Asthma.

Asthma is a common respiratory disease currently affecting more than 300 million worldwide and is characterized by airway inflammation, hyperreactivity, and remodeling. It is a heterogeneous disease consisting of corticosteroid-sensitive T-helper cell type 2-driven eosinophilic and corticosteroid-resistant, T-helper cell type 17-driven neutrophilic phenotypes. One pathway recently described to regulate asthma pathogenesis is cholesterol trafficking. Scavenger receptors, in particular SR-BI (scavenger receptor class B type I), are known to direct cellular cholesterol uptake and efflux. We recently defined SR-BI functions in pulmonary host defense; however, the function of SR-BI in asthma pathogenesis is unknown. To elucidate the role of SR-BI in allergic asthma, SR-BI-sufficient (SR-BI+/+) and SR-BI-deficient (SR-BI-/-) mice were sensitized (Days 0 and 7) and then challenged (Days 14, 15, and 16) with a house dust mite (HDM) preparation administered through oropharyngeal aspiration. Airway inflammation and cytokine production were quantified on Day 17. When compared with SR-BI+/+ mice, the HDM-challenged SR-BI-/- mice had increased neutrophils and pulmonary IL-17A production in BAL fluid. This augmented IL-17A production in SR-BI-/- mice originated from a non-T-cell source that included neutrophils and alveolar macrophages. Given that SR-BI regulates adrenal steroid hormone production, we tested whether the changes in SR-BI-/- mice were glucocorticoid dependent. Indeed, SR-BI-/- mice were adrenally insufficient during the HDM challenge, and corticosterone replacement decreased pulmonary neutrophilia and IL-17A production in SR-BI-/- mice. Taken together, these data indicate that SR-BI dampens pulmonary neutrophilic inflammation and IL-17A production in allergic asthma at least in part by maintaining adrenal function.

Club Cell Heme Oxygenase-1 Deletion: Effects in Hyperoxia-Exposed Adult Mice.

Thioredoxin reductase-1 (TXNRD1) inhibition activates nuclear factor (erythroid-derived 2)-like 2 (Nrf2) responses and prevents acute lung injury (ALI). Heme oxygenase-1 (HO-1) induction following TXNRD1 inhibition is Nrf2-dependent in airway epithelial (club) cells in vitro. The influence of club cell HO-1 on lung development and lung injury responses is poorly understood. The present studies characterized the effects of hyperoxia on club cell-specific HO-1 knockout (KO) mice. These mice were generated by crossing Hmox1 flox mice with transgenic mice expressing cre recombinase under control of the club cell-specific Scgb1a1 promoter. Baseline analyses of lung architecture and function performed in age-matched adult wild-type and KO mice indicated an increased alveolar size and airway resistance in HO-1 KO mice. In subsequent experiments, adult wild-type and HO-1 KO mice were either continuously exposed to >95% hyperoxia or room air for 72 h or exposed to >95 hyperoxia for 48 h followed by recovery in room air for 48 h. Injury was quantitatively assessed by calculating right lung/body weight ratios (g/kg). Analyses indicated an independent effect of hyperoxia but not genotype on right lung/body weight ratios in both wild-type and HO-1 KO mice. The magnitude of increases in right lung/body weight ratios was similar in mice of both genotypes. In the recovery model, an independent effect of hyperoxia but not genotype was also detected. In contrast to the continuous exposure model, right lung/body weight ratio mice were significantly elevated in HO-1 KO but not wild-type mice. Though club cell HO-1 does not alter hyperoxic sensitivity in adult mice, it significantly influences lung development and resolution of lung injury following acute hyperoxic exposure.

Aurothioglucose enhances proangiogenic pathway activation in lungs from room air and hyperoxia-exposed newborn mice.

Bronchopulmonary dysplasia (BPD), a long-term respiratory morbidity of prematurity, is characterized by attenuated alveolar and vascular development. Supplemental oxygen and immature antioxidant defenses contribute to BPD development. Our group identified thioredoxin reductase-1 (TXNRD1) as a therapeutic target to prevent BPD. The present studies evaluated the impact of the TXNRD1 inhibitor aurothioglucose (ATG) on pulmonary responses and gene expression in newborn C57BL/6 pups treated with saline or ATG (25 mg/kg ip) within 12 h of birth and exposed to room air (21% O2) or hyperoxia (>95% O2) for 72 h. Purified RNA from lung tissues was sequenced, and differential expression was evaluated. Hyperoxic exposure altered ~2,000 genes, including pathways involved in glutathione metabolism, intrinsic apoptosis signaling, and cell cycle regulation. The isolated effect of ATG treatment was limited primarily to genes that regulate angiogenesis and vascularization. In separate studies, pups were treated as described above and returned to room air until 14 days. Vascular density analyses were performed, and ANOVA indicated an independent effect of hyperoxia on vascular density and alveolar architecture at 14 days. Consistent with RNA-seq analyses, ATG significantly increased vascular density in room air, but not in hyperoxia-exposed pups. These findings provide insights into the mechanisms by which TXNRD1 inhibitors may enhance lung development.