Particulate matter air pollution exposure disrupts the Nrf2 pathway in sinonasal epithelium via epigenetic alterations in a murine model

Abstract

Exposure to airborne environmental pollutants including fine particulate matter (PM2.5) has been linked to respiratory disease, including chronic rhinosinusitis (CRS), allergies, and anosmia.1 Recent work has established a link between PM2.5 exposure and the development of nonallergic CRS.1 In addition, CRS symptom severity is influenced by air pollutant exposure, and chronic PM2.5 exposure has been found to induce sinonasal epithelial barrier dysfunction and type 2 eosinophilic rhinosinusitis in a novel mouse model.2 Molecular mechanisms of airway responses to these environmental stimuli, including possible epigenomic reprogramming, remain unknown. PM2.5 contains redox cyclic chemicals which induce oxidative stress upon exposure, causing inflammation.3 One endogenous mechanism that detoxifies these effects is the nuclear erythroid 2-related factor 2 (Nrf2) pathway.4, 5 Upon activation by environmental stressors such as PM2.5, Nrf2 (a transcription factor) translocates to the nucleus and upregulates the expression of several antioxidant cytoprotective genes: heme oxidase (HMOX-1), glutamate cysteine ligase catalytic subunit (GCLC), and glutamate cysteine ligase modulatory subunit (GCLM).5 Through such responses, Nrf2 activation restores in vitro sinonasal epithelial barrier permeability that was restricted by PM2.5 and other toxicants.5 In this study, we utilized next-generation sequencing (NGS) approaches to identify altered expression pathways and epigenetic modifications that arise in response to chronic sinonasal PM2.5 exposure in a mouse model. Adolescent male C57BL/6 mice (with Institutional Animal Care and Use Committee [IACUC] protocol approval) were exposed to either PM2.5 (n = 4, 70–100 μg/m3, comparable to extreme exposures in major urban cities in Asia) or filtered air (n = 2) for 14 weeks as previously described, and RNA from sinonasal tissue samples were isolated as previously described2 and prepared for sequencing (Illumina TrueSeq). We quantified gene expression using a TaRGETII uniform pipeline,6 and raw counts were measured with featureCounts. We identified 64 differentially expressed genes (DEGs) in sinonasal tissue from PM2.5-exposed mice compared with the filtered-air controls (Figure 1, false discovery rate [FDR] < 0.05). Interestingly, nine of the 53 downregulated genes were within the Nrf2 oxidative stress response pathway (Table 1). These genes include hmox-1 (FA 83.34 CPM vs. PM2.5 32.37 CPM), Gclc (Gclc; 561.02 vs. 222.17), Akr1b8 (16.43 vs. 7.44), Gpx2 (111.85 vs. 60.7), catalase (252.98 vs. 137.52), glutatione S-transferase 1 (89.85 vs. 51.48), sulfiredoxin (67.25 vs. 40.47), glutamate-cysteine ligase modifier subunit (Gclm; 237.6 vs. 147.13), and Ppargc1a (62.23 vs. 40.86). No common pathways were identified among the 11 upregulated genes, although two olfactory receptor genes were identified (Olfr140 and Olfr455) of uncertain significance. In order to explore whether chronic PM2.5 exposure created epigenomic modifications, we then applied bisulfite treatment to genomic DNA extracted from the same sinonasal tissues via the Accel-NGS Methyl-Seq DNA library kit (Swift Biosciences). Differentially methylated regions (DMRs) and sites were identified with a minimum of five CpG sites per region and at least > 5% difference in methylation (p-value < 0.01) using the BSmooth package. In this exploratory analysis, we observed global hypomethylation in intergenic/intron sites and hypermethylation in promoters (total DMR = 3.6K) in mice exposed to PM2.5 compared with controls (Figure 1). We queried these locations for known Nrf2 binding sites contained in the Cistrome database and identified 13 DMRs of transcription start sites (TSS) (< 2 kb from TSS) that correspond to Nrf2 target genes (Table 1).7 These data suggest that PM2.5 exposure results in epigenetic reprogramming of Nrf2 antioxidant response genes in the sinonasal mucosa. Here, we report for the first time that the Nrf2 pathway is globally downregulated in the sinonasal mucosa after long-term exposure to airborne PM2.5 in mice. These findings are analogous to the chronic obstructive pulmonary disease (COPD) literature, where Nrf2 levels are also downregulated, making Nrf2 supplementation or enhancement a therapeutic option. We also report the unique findings of epigenetic modifications of the Nrf2 gene in the sinonasal mucosa, demonstrating that chronic PM2.5 exposure may have long lasting, potentially irreversible effects. Few studies have examined the link between PM2.5 stimulation and Nrf2 expression. A study by Piao et al. demonstrated that PM2.5 exposure alone for 5 weeks caused a decrease in Nrf2 signaling with limited sinonasal inflammation.8 These findings are in contrast to previous cell culture studies stimulated with acute exposures to PM2.5 which demonstrated increased Nrf2 expression. Based on our findings, we postulate that chronic PM2.5 exposures are necessary to overcome the acute oxidative stress phase to create downregulation of Nrf2 with robust eosinophilic inflammation. Disruption of key antioxidant responses at the mucosal interface may represent a critical mechanism by which air pollution causes CRS. In prior research, activation of the Nrf2 pathway was shown to strengthen the sinonasal epithelial barrier and mitigate the type 2 inflammatory response and eosinophilia in the nose and sinuses of mice.5, 9 Indeed, Nrf2 activation via Keap1 deletion attenuates sinonasal inflammation and improves epithelial barrier function in mice, while deletion of Nrf2 exacerbates inflammation and creates epithelial barrier dysfunction.10 Thus, our data in this pilot study with a limited sample size, demonstrating that chronic PM2.5 exposure creates eosinophilic inflammation and epigenetic reprogramming of Nrf2 antioxidant response genes in the sinonasal mucosa, suggests that the Nrf2 pathway may be a useful therapeutic target for treatment of PM2.5-induced or exacerbated CRS. Studies of this phenomenon in larger samples and in translational studies in humans could provide greater insight into the molecular mechanisms that underlie environmentally mediated inflammatory disease in the upper airway. We would like to acknowledge Jinhu Yin, PhD and Daifeng Wang, PhD for their technical and analytical expertise. N. London holds stock in Navigen Pharmaceuticals and was a consultant for Cooltech Inc., neither of which are relevant to the study. The other authors declare no relevant conflict of interest.

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