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. Author manuscript; available in PMC: 2024 Nov 1.
Published in final edited form as: Adv Biol (Weinh). 2023 May 19;7(11):e2300045. doi: 10.1002/adbi.202300045

Acute and repeated ozone exposures differentially affect circadian clock gene expression in mice

Isaac Kirubakaran Sundar 1,*, Santhosh Kumar Duraisamy 1, Ishita Choudhary 2, Yogesh Saini 2, Patricia Silveyra 3
PMCID: PMC10657336  NIHMSID: NIHMS1903133  PMID: 37204107

Abstract

Circadian rhythms have an established role in regulating physiological processes such as inflammation, immunity, and metabolism. Ozone (O3), a common environmental pollutant with strong oxidative potential, is implicated in lung inflammation/injury in asthmatics. However, whether O3 exposure affects the gene expression of circadian clock genes in the lung is not known. In this study, we analyzed changes in the expression of core clock genes in the lungs of adult female and male mice exposed to either filtered air (FA) or O3 using qRT-PCR. We confirmed our findings using an existing RNA-sequencing dataset from repeated FA- and O3-exposed mouse lungs and validated them by qRT-PCR. Acute O3 exposure significantly altered the expression of clock genes in the lungs of females (Per1, Cry1, and Rora) and males (Per1). RNA-seq data revealing sex-based differences in clock gene expression in the airway of males (decreased Nr1d1/Rev-erbα) and females (increased Skp1), parenchyma of females and males (decreased Nr1d1 and Fbxl3 and increased Bhlhe40 and Skp1), and alveolar macrophages of males (decreased Arntl/Bmal1, Per1, Per2, Prkab1, and Prkab2) and females (increased Cry2, Per1, Per2, Csnk1d, Csnk1e, Prkab2, and Fbxl3). Our findings suggest that O3 exposure-induced heightened lung inflammation affects circadian clock genes that involve key canonical signaling pathways.

Keywords: Ozone, Circadian rhythms, Lung inflammation, Sex differences, Transcriptomics

Graphical Abstract

Ozone (O3) exposure affects circadian clock gene expression in the lungs. O3-exposed mice showed compartment-specific lung transcriptomics and sex-based difference in clock gene expression. Nr1d1 (Rev-erbα) gene expression in the airways and parenchyma of O3-exposed males was reduced compared to O3-exposed females. Implicating circadian clock disruption may play a major role in regulating immune-inflammatory response and DNA damage/repair pathways.

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1. Introduction

Ground-level ozone (O3) is a reactive oxidant gas and a common urban air pollutant generated by the photochemical reaction of several pollutants, particularly volatile organic compounds, and nitrogen oxides. O3 exposure has been largely associated with severe cardiopulmonary disease-related morbidity and mortality globally (1, 2). Exposure to O3 occurs through the inhalation route, and due to its low water-solubility, inhaled O3 can penetrate deep into the conducting airways of the lung resulting in increased lung inflammation, altered lung function and changes in epithelial barrier function (3–8). Thus, O3 exposure often leads to exacerbations in young and old individuals suffering from chronic inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease (COPD) (9–11). Additionally, chronic exposure to O3 can potentially result in the onset of chronic respiratory conditions such as asthma and COPD in susceptible individuals (10, 12, 13). The constantly evolving regulatory policies and preventive measures worldwide have significantly improved the air quality standards and their related adverse health outcomes (14, 15). Nevertheless, exposure to an unhealthy level of ambient O3 occurs on a daily basis due to climate change and increasing warm temperature conditions (16, 17).

Accumulating evidence from current literature indicates that O3 exposure induces oxidative stress, exaggerated airway inflammation, and enhances the innate immune response in the lungs (18–20). Additionally, epidemiological, and clinical studies have shown that sex (biological) and gender (social) factors can affect the susceptibility to negative effects of O3 exposure (21–25). The airway epithelial cells and resident alveolar macrophages play important roles during the initiation and resolution of O3-induced lung injury (26, 27). In recent years, transcriptomic and metabolomic approaches have been widely used to understand the gene-environment interaction that occurs following O3 exposure in the male and female lung (7, 28–30). Evidence from prior reports show that O3 exposure transcriptionally affects lung tissues and inflammatory cells in the lungs, altering key canonical signaling pathways and molecules such as heat-shock proteins, extracellular matrix remodeling proteins, and proinflammatory signaling pathways (7, 18, 28, 31, 32). The lung is a very complex tissue that consists of a multitude of different cell types and hence bulk transcriptomics data cannot direct us towards cell-type-specific changes in gene expression contributing to the observed phenotypes (33). However, several reports from animal models of O3 exposure have indicated compartment-specific transcriptomic changes in O3-exposed mice compared to controls exposed to filtered air (FA) (28). Importantly, these studies have revealed sex differences in the expression of inflammatory genes and microRNAs that could contribute to lung disease development and exacerbation in males and females (3, 34–37).

Circadian rhythms are intrinsic biological oscillations governed by an autoregulatory feedback loop controlled by the core clock genes that are virtually expressed in all cells. The central clock in the suprachiasmatic nucleus of the brain entrains the peripheral tissues (lung, heart, liver, spleen, kidney, gut, etc.) via neuronal and hormonal signaling. CLOCK:BMAL1 forms the core clock complex that heterodimerizes to mediate the transcription of core clock genes such as Period (PER 1–3), Cryptochrome (CRY 1–2), and REV-ERBα/β that form the two inhibitory feedback loops thereby blocking CLOCK:BMAL1 heterodimer-mediated transactivation function (38). The circadian clock plays an important role in mediating several physiological processes such as innate and adaptive immune response, inflammation, and metabolism (39–42). In the lung, a role of the circadian clock in immune function during respiratory diseases and systemic inflammation has also been proposed (43). We have previously reported that circadian clock disruption in the lungs occurs following acute and chronic exposure to environmental tobacco smoke (ETS)/cigarette smoke (CS) and in ETS-exposed mice infected with influenza A virus (IAV) (44–46). Rev-erbα (Nr1d1: Nuclear receptor subfamily 1 group D member 1), a key core circadian clock regulatory gene has been shown to play an important role in CS, allergen, lipopolysaccharide (LPS), and bleomycin-induced lung inflammation and injury (46–51). Prior studies support that Nr1d1-based targeted therapy (e.g., Rev-erbα agonists: GSK4112, SR9009, and SR9011) may be beneficial in chronic lung diseases such as COPD, asthma, sepsis, and pulmonary fibrosis highlighting the importance of circadian clock regulation in pulmonary diseases (46–53). Currently, there are no studies available that demonstrate circadian clock disruption/dysregulation in the lung following O3 exposure.

In the present study, we first aimed to determine whether O3 exposure differentially affects the gene expression of core clock genes in the lungs of female and male mice. We chose females along with males due to their increased susceptibility to O3-induced inflammation, based on our prior studies (35). We next validated our findings using one previously available gene expression omnibus (GEO) dataset, an RNA-sequencing (RNA-seq) dataset from FA and O3-exposed mice. We focused on 25 selected circadian rhythms associated genes to compare the results of the RNA-seq dataset and support the findings from our study (acute O3 exposure model). Here, for the first time, we provide evidence to support that acute and repeated O3 exposure alters the lung transcript levels of genes involved in circadian rhythms in a sex- and time-dependent manner.

2. Materials and Methods

2.1. Experimental Animals

Adult female (10 weeks of age, n=4–7) and male (9 weeks of age n=3–4) C57BL/6J mice for acute and repeated ozone exposure (female and male ~7 weeks of age followed by 3 weeks acclimatization) were purchased from The Jackson Laboratories (Bar Harbor, ME) and maintained in a 12:12-hr light-dark cycle, with ad libitum access to food and water. These mice were chosen based on their strain-dependent susceptibility to O3-induced lung injury as described previously (5, 6). The Pennsylvania State University College of Medicine Institutional Animal Care and Use Committee (for acute O3 exposure) and Louisiana State University College of Medicine Institutional Animal Care and Use Committee (for repeated O3 exposure) approved all animal procedures. All animal experiments were conducted as per the ARRIVE guidelines.

2.2. Acute Ozone Exposure

Adult mice were exposed to 2 ppm of ozone (O3) or filtered air (FA: control); (between 11:00 am and 2:00 pm [Zeitgeber time ZT5–8] in females and between 12:00 pm and 3:00 pm [ZT6–9] in males) for 3 h in chambers as described previously (35). All mice were euthanized at 21 h post-exposure (11:00 am and 2:00 pm [ZT5–8] in females and 12:00 pm and 3:00 pm [ZT6–9] in males) on the following day (see acute O3 exposure model schematic Fig. 1A). Lung tissue was harvested for gene expression analysis of selected 10 circadian clock genes including Clock, Arntl/Bmal1, Nr1d1/Rev-erbα, Nr1d2/Rev-erbβ, Nfil3, Per1, Per2, Cry1, Cry2, and Rora by quantitative real-time PCR (qRT-PCR).

Fig. 1. Schematic representation of acute and repeated O3 exposure models used in this study.

Fig. 1.

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WT (C57BL/6J) female and male mice were exposed to FA or O3 (2 ppm; 3 hrs during the light cycle between Zeitgeber time [ZT5–8 in females and ZT6–9 in males] for acute O3 exposure and euthanized 21 hrs post-exposure) and (0.8 ppm; 4 hrs/day during the dark cycle between ZT12–16 [for a total of 14 days] for repeated O3 exposure and euthanized 12–16 hrs post-exposure). Lung tissues were harvested to determine gene expression of circadian clock genes from acute O3 and repeated O3 exposures.

2.3. Repeated Ozone Exposure

Repeated O3 (0.8 ppm; 4 h/day for 14 days) or FA control exposure was conducted between 6:00 pm and 10:00 pm [ZT12–16] in female and male C57BL/6J mice (~7 weeks of age + 3 weeks acclimatization [~10 weeks of age]; 5 days/week with no exposure during the weekend for the first 2 weeks followed by 4 consecutive days of O3 or FA exposure) and euthanized within 12–16 hr post-exposure [between ZT4–8] (see repeated O3 exposure model schematic Fig. 1B) (28). Compartment-specific isolation of airways, parenchyma, and alveolar macrophages for qRT-PCR validation of RNA-sequencing data was performed as described (28). In brief, the extra-pulmonary airway sample includes the trachea (devoid of the first three cartilaginous rings) along with the first and second-generation extralobular airways were dissected using the dissection microscope. For parenchyma samples, 1 mm margins of the left lung lobes were trimmed. Finally, for alveolar macrophages, CD11b- microbeads were used to isolate alveolar macrophages from bronchoalveolar lavage (BAL) fluid of FA vs. O3-exposed female and male mice.

2.4. Total RNA Isolation and qRT-PCR Analysis

Lung tissue was homogenized in TRIzol reagent (LifeTechnologies, Carlsbad CA) and total RNA was extracted using the Direct-zol RNA MiniPrep kit (Zymo Research, Irvine, CA) according to the manufacturer’s instructions. RNA concentration and purity were determined by a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE). Mouse circadian gene-specific primers (Clock, Arntl, Nr1d1, Nr1d2, Nfil3, Per1, Per2, Cry1, Cry2, Rora, Npas2, Bhlhe40, Fbxl3, and Skp1) were synthesized by IDT (www.idtdna.com) (Table 1). One microgram of total RNA was used for cDNA synthesis by the RT2 first strand kit (Qiagen, Valencia, CA) and qPCR reactions were performed using a CFX Opus 96 Real-Time PCR System (Bio-Rad). Relative expression of target genes was determined by the 2−ΔΔCt method with 18s rRNA (Rn18s) as housekeeping gene as described previously (54, 55).

Table 1.

qRT-PCR primer pairs for circadian clock and housekeeping genes used in this study.

Genes Primer Sequence
Clock (F) 5’-GGAGTCTCCAACACCCACAG-3’
(R) 5’-GGCACGTGAAAGAAAAGCAC-3’
Arntl (F) 5’-AAGGGCCACTGTAGTTGCTG-3’
(R) 5’-CTGCAGTGAATGCTTTTGGA-3’
Nr1d1 (F) 5’-GAGTCAGGGACTGGAAGCTG-3’
(R) 5’-AAGACATGACGACCCTGGAC-3’
Nr1d2 (F) 5’-TGGAGGCAGAGCTAGAGGAA-3’
(R) 5’-ACCCGGTGCTCATGATGT- 3’
Nfil3 (F) 5’-GAACTCTGCCTTAGCTGAGGT-3’
(R) 5’-ATTCCCGTTTTCTCCGACACG-3’
Per1 (F) 5’-AACGCTTTGCTTTAGATCGG-3’
(R) 5’-TCCTCAACCGCTTCAGAGAT-3’;
Per2 (F) 5’-CTTGGGGAGAAGTCCACGTA-3’
(R) 5’-TACTGGGACTAGCGGCTCC-3’
Cry1 (F) 5’-GTCCCCGAATCACAAACAGA-3’
(R) 5’-TGCGTCTATATCCTCGACCC-3’
Cry2 (F) 5’-TCCCCGGACTACAAACAGAC-3’
(R) 5’-GTCTACATCCTCGACCCGTG-3
Rora (F) 5’-TTGCAGCCTTCACACGTAAT-3’
(R) 5’-AGGCAGAGCTATGCGAGC-3’
Bhlhe40 (F) 5’-ACGGAGACCTGTCAGGGATG-3’
(R) 5’-GGCAGTTTGTAAGTTTCCTTGC-3’
Fbxl3 (F) 5’-CCTGACTTGTGGCGATGTTTT-3’
(R) 5’-ACTGTAGGTGGTTTGAGTGCC-3’
Skp1 (F) 5’-ATGCCTACGATAAAGTTGCAGAG-3’
(R) 5’-TCCATTCCCAAATCTTCCAGC-3’
Rn18s (F) 5’-GTAACCCGTTGAACCCCATT-3’
(R) 5’-CCATCCAATCGGTAGTAGCG-3’
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2.5. Repeated Ozone Exposure Dataset Analyses

We utilized a dataset available from the Gene Expression Omnibus (GEO), from previously published report, to determine the differential expression of core clock genes: Compartment-specific transcriptomics of O3-exposed murine lungs reveal sex- and cell type-associated perturbations relevant to mucoinflammatory lung diseases GSE156799 (28). In this study, C57BL/6J female and male mice (7 weeks of age) were exposed to filtered air (FA) or repeated O3 exposure (0.8 ppm; 4 h/night, 5 nights/week, for 3 weeks; exposures were performed between 6:00 pm and 10:00 pm) and mice were euthanized within 12–16 hr post-exposure (28). To determine differential expression of selected circadian clock genes, we extracted the RNA-seq fragment per kilobase per million mapped reads (FPKM) values from the respective GEO dataset and compared values between FA vs. O3-exposed mice. For detailed materials and methods, please refer to the original article (28).

2.6. Gene Expression Analyses

The dataset with an accession number GSE156799 was downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). This dataset GSE156799 contains compartment-specific transcriptomic analyses of O3-exposed murine lungs that showed sex- and cell type-specific changes to mucoinflammatory lung diseases. Adult mice (females and males) were repeatedly exposed to 0.8 ppm O3 or FA between 6:00 pm and 10:00 pm, and RNA-seq was performed from airway epithelium enriched airways, parenchyma, and isolated alveolar macrophages on a platform of the Illumina NovaSeq 6000 as described previously (28).

2.7. Online Tools for Hierarchical cluster analysis

Hierarchical clustering was performed using the FPKM values for 25 circadian clock genes obtained from GSE156799 dataset with the help of the Morpheus tool (https://software.broadinstitute.org/morpheus/) by selecting the option metric: one minus Pearson correlation, linkage method: average; cluster: rows and columns.

2.8. Statistical Analysis

Statistical significance was calculated between the two groups FA vs. O3 using unpaired Student’s t-test. The probability of significance compared with FA control was based on a two-tail t-test. Statistical differences in more than two groups (FA vs. O3 in Females and Males) were analyzed by two-way ANOVA using Tukey’s multiple-comparison test with the GraphPad Prism 9 (La Jolla, CA). For analysis of the dataset from the GEO database, differences among the experimental groups were analyzed using the appropriate statistical test described above based on the FPKM values (RNA-seq) obtained from the GEO dataset. The results are shown as means ± SEM with a P < 0.05 considered as statistically significant.

3. Results

3.1. Acute ozone exposure differentially affects gene expression of circadian clock genes in the mouse lung.

Acute 2 ppm O3 exposure in females (between 11:00 am and 2:00 pm) showed altered gene expression of core clock genes in the mouse lung compared to FA-exposed control as confirmed by qRT-PCR. qRT-PCR analysis of 10 different clock genes (Clock, Arntl, Nr1d1, Nr1d2, Nfil3, Per1, Per2, Cry1, Cry2, and Rora) revealed that acute O3-exposed mice had a significant increase in the transcript levels of Per1, Cry1 and Rora in the lungs compared to FA-exposed controls. However, we observed an increasing trend in the gene expression of remaining circadian genes such as Clock, Arntl, Nr1d2, Per2 and Cry2 in O3-exposed female mice compared to FA control (Fig. 2A). Acute 2 ppm O3 exposure in males (between 1:00–3:00 pm and 4:00–6:00 pm) showed altered gene expression of clock genes in lung compared to FA-exposed control as confirmed by qRT-PCR. Selected clock genes Bmal1/Arntl (Aryl hydrocarbon receptor nuclear translocator like) and Nfil3 (Nuclear factor, interleukin 3 regulated) showed a trend toward reduced expression and Per1 was significantly reduced compared to FA-exposed control (Fig. 2B). We did not compare expression of core clock genes between sexes because the FA and O3 (2 ppm) exposure and tissue harvest time points were different in females and males.

Fig. 2. Acute ozone exposure differentially affects the gene expression of circadian clock genes in the lungs analyzed by qRT-PCR.

Fig. 2.

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(A-B) Gene expression of circadian clock genes (Clock, Arntl, Nr1d1, Nr1d2, Nfil3, Per1, Per2, Cry1, Cry2, and Rora) in females and males exposed to FA vs. O3 (2 ppm) for 3 h. Data are mean ± SEM (n=3–4 in FA group and n=4–7 in O3 group) and significance determined using Student’s t-test. * P < 0.05, FA vs. O3.

3.2. Repeated ozone exposure differentially affects circadian clock genes in lung airways, parenchyma and resident alveolar macrophages confirmed by RNA-seq analysis from existing dataset.

We utilized the existing gene expression RNA-sequencing dataset from GEO (GSE156799) to analyze the sex difference in the expression of core clock genes in three different compartments of the lung (airways, parenchyma, and alveolar macrophages) in female and male C57BL/6J mice exposed to either FA or O3. Hierarchical cluster analysis of 25 selected circadian clock target genes in three different compartments of the lung: airways, parenchyma, and alveolar macrophages revealed sex-based differences in clock gene expression in different compartments of the lung in response to O3 vs. FA exposure (Fig 3A–C, Fig. 4A–C and Fig. 5A–C). We found the males exposed to O3 showed significantly reduced expression of Nr1d1 (Rev-erbα) transcript levels in the airways compared to respective FA control (P < 0.01). In contrast, O3-exposed females did not show any significant change in Nr1d1 expression in the airways compared to respective FA control (Fig 3C). Additionally, Skp1 (S-phase kinase associated protein 1) gene expression was significantly increased in the airways of O3-exposed females compared to respective FA control (P < 0.01). The males did not show any difference in the gene expression of Skp1 in the airways of FA- and O3-exposed groups (Fig. 3C). The basal transcript levels of Skp1 were significantly higher in the airway of males vs. females FA group (P < 0.01) (Fig. 3C).

Fig. 3. Repeated ozone exposure differentially affects the expression of circadian clock genes in the airways.

Fig. 3.

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Hierarchical cluster analysis and Heatmap showing gene expression of circadian clock genes in the airways from FA vs. O3-exposed (A) female and (B) male mice. (C) RNA-seq analysis from GSE156799 showed differential expression of Nr1d1 and Skp1 genes based on their FPKM values compared among FA vs. O3-exposed female and male mice. Data are mean ± SEM (n=4/group) and significance determined using 2-way ANOVA. ** P < 0.01, FA vs. O3 or FA females vs. FA males.

Fig. 4. Repeated ozone exposure differentially affects the expression of circadian clock genes in the parenchyma.

Fig. 4.

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Hierarchical cluster analysis and Heatmap showing gene expression of circadian clock genes in the parenchyma from FA vs. O3-exposed (A) female and (B) male mice. (C) RNA-seq analysis from GSE156799 showed differential expression of Nr1d1, Bhlhe40, Fbxl3, and Skp1 circadian target genes based on their FPKM values compared among FA vs. O3-exposed female and male mice. Data are mean ± SEM (n=4/group) and significance determined using 2-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, FA vs. O3-exposed females or males.

Fig. 5. Repeated ozone exposure differentially affects the expression of circadian clock genes in alveolar macrophages.

Fig. 5.

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Hierarchical cluster analysis and Heatmap showing gene expression of circadian clock genes in the alveolar macrophages from FA vs. O3-exposed (A) female and (B) male mice. (C) RNA-seq analysis from GSE156799 showed differential expression of Arntl (Bmal1), Cry2, Per1, and Per2 circadian target genes based on their FPKM values compared among FA vs. O3-exposed female and male mice. Data are mean ± SEM (n=4/group) and significance determined using 2-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, FA vs. O3-exposed females or males; FA females vs. FA males; O3 females vs. O3 males.

Next, we analyzed the FPKM values from lung parenchyma of FA vs. O3-exposed female and male mice. Hierarchical cluster analysis revealed a difference in the pattern of core clock gene expression among FA vs. O3-exposed female and male mice (Fig. 4A–B; Supplementary Fig. 1). Four of the clock genes Nr1d1, Bhlhe40 (Basic helix-loop-helix family member E40), Fbxl3 (F-box and leucine rich repeat protein 3) and Skp1 showed altered transcript levels among the FA vs. O3-exposed female and male mice (Fig. 4A–C). Similar to the findings of the lung airway, males exposed to O3 showed significantly reduced expression of Nr1d1 and females exposed to O3 showed significantly increased expression of Skp1 in the parenchyma compared to FA control (P < 0.01 and P < 0.05 in males and females respectively) (Fig. 4C). We observed a trend in reduced expression of Nr1d1 in females and increased Skp1 in males exposed to O3 compared to FA control although results were not significantly different. Additionally, female, and male mice exposed to O3 showed increased transcript levels of Bhlhe40 in the parenchyma compared to FA control (P < 0.001 in females and P < 0.05 in males) (Fig. 4C). Both female and male O3-exposed mice show reduced expression of Fbxl3 in the parenchyma compared to FA control (P < 0.05 and P < 0.01 in females and males respectively) (Fig. 4C). The transcript levels of other clock genes were highly variable among O3 vs. FA-exposed females and males did not reach statistical significance.

Additionally, we analyzed the FPKM values from alveolar macrophages (AMs) of FA vs. O3-exposed female and male mice. Hierarchical cluster analysis revealed a distinct difference in the transcript levels of core clock genes between FA vs. O3-exposed female and male mice (Fig. 5A–B; Supplementary Fig. 1). Among the 25 clock genes evaluated, Bmal1/Arntl, Cry2, Per1 and Per2 showed altered expression in the AMs of both females and males exposed to O3 compared to FA control (Fig. 5C). Male mice exposed to O3 showed reduced expression of Arntl in the AMs (P < 0.01). The basal transcript levels of Arntl in the FA control group were significantly higher in males compared to females in the AMs (P < 0.01). Additionally, AMs from females exposed to O3 showed a significant increase in the expression of Cry2, Per1 and Per2 compared to those from FA control (Fig. 5C), whereas AMs from males exposed to O3 showed significantly reduced expression of Per1 and Per2 than those from FA control mice (P < 0.05 and P < 0.001 for Per1 and Per2, respectively). The transcript levels of circadian clock genes Arntl, Cry2, Per1, and Per2 were significantly higher in the AMs from FA-exposed male vs. FA-exposed female mice (Fig. 5C). Furthermore, transcript levels of a few other circadian genes were altered among females and males exposed to O3 compared to FA control. These include protein kinases (Csnk1d, Csnk1e, Prkab1, and Prkab2), ligases (Fbxl3) and other interacting targets (Bhlhe41) (Supplementary Fig. 2). Among these, females exposed to O3 showed a significant increase in the expression of protein kinase genes Csnk1d, Csnk1e and Prkab2 in the AMs compared to FA control (Supplementary Fig. 2), while males exposed to O3 show reduced expression of Prkab1 and Prkab2 in the AMs compared to FA control (P < 0.01) (Supplementary Fig. 2). Interestingly, O3-exposed females show reduced, and males show increased transcript level of Bhlhe41 in AMs compared to FA control. Female mice exposed to O3 also showed increased Fbxl3 expression in the AMs compared to FA control (Supplementary Fig. 2). Finally, transcript levels of protein kinase genes Csnk1d, Csnk1e, and Prkab2 and ligase gene Fbxl3 were significantly reduced in O3-exposed males vs. females demonstrating a sex-based difference in AMs among the O3-exposed female vs. male mice (Supplementary Fig. 2).

3.3. Repeated ozone exposure differentially affects circadian clock genes in lung airways, parenchyma, and resident alveolar macrophages validated by qRT-PCR analysis.

Total RNA from the airways, parenchyma, and alveolar macrophages of FA and repeated O3-exposed females and males were used for qRT-PCR validation. Results revealed a sex-based difference in increased expression of Nfil3, Per1, Per2, Cry1, Cry2, and Fbxl3 in the airway of O3-exposed females compared to respective FA control (Fig. 6). Additionally, we found that O3-exposed females showed increased expression of the above-mentioned clock genes compared to O3-exposed males. However, only two clock genes Nfil3 and Bhlhe40 were significantly altered in the lung parenchyma of O3-exposed males and O3-exposed females compared to respective FA controls (Fig. 7). Finally, the expression of clock genes in alveolar macrophages from O3-exposed females revealed increased expression of Arntl and Nfil3 compared to FA control. Additionally, O3-exposed males showed reduced expression of Arntl, Nfil3, Cry1, Per1, and Fbxl3 compared to O3-exposed females (Supplementary Fig. 3). Together, these findings support the hypothesis that acute as well as repeated O3 exposures affect the transcript levels of circadian clock genes in the lung. Altered expression of these genes in response to air pollution exposure may play an essential role in altered immune responses in males and females, as well as cytokine/chemokine signaling both in the structural and immune cell compartments of the lung that may contribute to inflammation and injury.

Fig. 6. Repeated ozone exposure differentially affects the expression of circadian clock genes in the airways validated by qRT-PCR.

Fig. 6.

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Gene expression of circadian clock genes (Clock, Arntl, Nr1d1, Nr1d2, Nfil3, Per1, Per2, Cry1, Cry2, Rora, Npas2, Bhlhe40, Fbxl3 and Skp1) in females and males exposed to FA vs. O3 (0.8 ppm) for 4 h/night (5 days/week for 3 weeks). Data are mean ± SEM (n=4 in FA group and n=3–4 in O3 group) and significance determined using 2-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, FA vs. O3-exposed females; O3 females vs. O3 males.

Fig. 7. Repeated ozone exposure differentially affects the expression of circadian clock genes in the parenchyma validated by qRT-PCR.

Fig. 7.

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Gene expression of core circadian clock genes (Clock, Arntl, Nr1d1, Nr1d2, Nfil3, Per1, Per2, Cry1, Cry2, Rora, Npas2, Bhlhe40, Fbxl3 and Skp1) in females and males exposed to FA vs. O3 (0.8 ppm) for 4 h/night (5 days/week for 3 weeks). Data are mean ± SEM (n=4 in FA group and n=4 in O3 group) and significance determined using 2-way ANOVA. * P < 0.05, ** P < 0.01, FA vs. O3-exposed females and males.

4. Discussion

Ozone is among the major air pollutants that contribute to chronic inflammatory lung diseases such as asthma and COPD (1, 2, 9–15). The present study is focused on determining mRNA expression of core circadian clock genes and provide experimental evidence to show that sex-specific differences exist in three different compartments of female and male mouse lungs exposed to O3. Prior studies have observed sex-based differences in animal models of O3-induced lung inflammation and injury (4, 28, 34–36, 56, 57). A recent study showed increased total cells and neutrophils in BAL fluid of female mice associated with an increase in acute phase cytokines/chemokines, but different miRNA expression compared to male mice (34). A more recent report validated some of these findings and identified increased expression of proinflammatory eicosanoids and resolvins in female mice (56).

In this study, we found female and male mice showing a difference in the transcript levels of clock genes in the airways vs. parenchyma vs. AMs may have influenced the mucoinflammation and mucous cell metaplasia-associated pathways affected differentially in repeated O3-exposed female and male mice. Compartment-specific transcriptomic analysis of O3-exposed mouse lungs revealed sex differences (O3-exposed females showed more changes compared to males) in gene expression and mucoinflammatory phenotypes (28). Pathway analysis of differentially expressed genes (DEGs) revealed cell division and DNA repair among the most enriched in O3-exposed airways that is evidenced by immunohistochemical analyses. Similarly, in O3-exposed parenchymal tissues, cell division and DNA repair pathways along with inflammatory pathways were enriched that support the direct role of epithelial and immune cells. Furthermore, in O3-exposed alveolar macrophages immune response and cytokine-cytokine receptor interactions were enriched (28). Previously, adult C57BL/6J female mice when exposed to FA, 1 or 2 ppm O3 showed DEGs analyzed by RNA-seq specifically in the conducting airway (CA) and airway macrophages (AM) (7). These studied revealed DEGs in O3-exposed CA that were associated with epithelial barrier function (Gjb3–5, Cldn2/4, Adam12, Tgm1), detoxification processes (Mt1, Ugt1a6a/b, Gstm1/2/6) and cellular proliferation (Pcna, Cdh3), whereas O3-exposed AM showed genes involved in innate immune signaling, cytokine production (Ccl17, Slpi, Ccl22) and extracellular matrix remodeling (Krt7, Krt8, Krt18) (7). Our analysis in GEO data from repeated O3 exposure showed sex differences in the expression of clock genes in a different compartment of FA vs. O3-exposed mouse lung. Interestingly, Nr1d1 (Rev-erbα), a nuclear receptor and transcription factor that plays an essential role in the circadian timing system, was significantly affected in the O3-exposed airway of males and parenchyma of both males and females. Recently, we and others have shown Nr1d1 gene regulates pulmonary inflammation and epithelial-mesenchymal transition during cigarette smoke exposure (46), and allergen- and LPS-induced lung injury models (47, 52). Hence, the Nr1d1 gene may have a profound cell type-specific role in O3-induced lung injury models which need to be further explored.

Additionally, two important genes Skp1 and Fbxl3 that are involved in the E3 ubiquitin ligase protein complex activity and subsequent degradation of cryptochromes (CRYs) were differentially affected in the airways of female and parenchyma of females and males. This Skp1-Cul1-Fbxl3 (SCF[Fbxl3]) protein complex is well characterized for its ubiquitin ligase E3 activity. Fxbl3 is an F-box protein that exclusively mediates ubiquitination followed by degradation of CRYs and hence regulates circadian oscillation in target tissues and cells (58). In our analysis, Skp1 transcript levels were increased in O3-exposed females in the airways and the parenchyma of females and males compared to FA control. Surprisingly, the levels of Fbxl3 were only affected in the O3-exposed parenchymal compartment of the lung in females and males but not in the airways. Altered expression of these genes that contribute to the E3 ubiquitin ligase complex formation and regulation might contribute to the altered expression of Cry1 and Cry2 observed in O3-exposed mice compared to FA control. We observed sex differences in the expression of circadian clock genes in alveolar macrophages of females and males exposed to O3 compared to FA controls. O3-exposed male mice showed decreased transcript levels of clock genes Bmal1, Per1, and Per2 and protein kinase genes Prkab1, and Prkab2 in alveolar macrophages. However, O3-exposed female mice showed increased transcript levels of clock genes Cry2, Per1, and Per2 and other protein kinases and ligase genes such as Csnk1d, Csnk1e, Prkab2 and Fbxl3 in AMs. Our data analysis indicate that sex differences could contribute to the altered directionality of clock gene expression in the structural vs. immune compartment of the lungs. Future studies determining the status of circadian clock gene expression in the lung myeloid cell types during acute vs. chronic O3 exposure will provide better insights into the molecular mechanisms that lead to lung inflammation and injury.

Our experiments using acute O3 exposure in female mice showed increased expression of Per1, Cry1, and Rora genes but males showed a decreasing trend in Bmal1 and Nfil3, but significantly reduced Per1 expression in the lungs analyzed by qRT-PCR. qRT-PCR validation using samples from FA and repeated O3-exposed airways revealed a significant increase in the expression of clock genes in the O3-exposed females compared to FA control and O3-exposed males (Nfil3, Per1, Per2, Cry2, and Fbxl3). Additionally, Nfil3 was significantly increased in O3 males vs. FA control and Bhlhe40 was significantly increased in O3 females vs. FA control in the parenchyma. Several clock genes such as Nfil3, Cry1, Cry2, Per1, and Per2 showed an increasing trend in O3 females compared to FA control. Similar results (increased expression of Arntl, and Nfil3) were observed in the AMs compartment from O3 females compared to FA control and O3 females vs. O3 males (reduced expression of Arntl, Nfil3, Cry1, Per1, and Fbxl3) validated by qRT-PCR analysis. Microarray data from wild-type and Tlr4-KO mice exposed to O3 at the different time points (6 hr, 24 hr and 48hr) showed Heat-shock protein 70 (HSP70) as an effector molecule downstream of TLR4 that regulates O3-induced lung inflammation (31). Prior evidence suggests that acute TLR4-mediated cytokine response following the LPS challenge affects the degradation of repressor protein REV-ERBα (NR1D1) in the lung epithelial cells and mouse lungs (47).

Evidence from the literature indicates that glucocorticoids (GCs: steroid hormones) produced by the adrenal cortex are under the control of circadian rhythm and stress. GCs have been shown to regulate rhythms of innate and adaptive immune responses (59). Circadian rhythms of tissue inflammation are regulated by GC-mediated inhibition of inflammatory cytokine and chemokine expression at the active phase in mice (dark cycle/night). In mice, T cells accumulate in secondary lymphoid organs (e.g., spleen, lymph nodes, and Peyer’s patches) during the active phase. In contrast, T cells in blood accumulate at the resting phase (light cycle/day). The observed diurnal oscillation is lost in T cell-specific glucocorticoid receptor (GR)-deficient and glucocorticoid response element (GRE) mutant mice (60). CXCR4 expression on T cells show diurnal oscillation similar to IL-7R in mice. This was abolished in T cell-specific CXCR4-deficient mice, suggesting that IL-7R induced by GCs is possibly involved in the diurnal oscillation of the T cell distribution (60). Earlier reports showed the effect of ozone exposure on lymphoid cells at different times post-exposure (61, 62). They found that lymphocyte number in mediastinal lymph node changes dose-dependently at different times during the 1–28 days period (acute phase: hyperplastic increase in cell mass [days 1–7] and subacute phase: functional changes in lymphocyte reactivity [days 8–28] (61). We speculate that observed lung compartment and sex-specific differences in the gene expression of core clock genes following acute and repeated O3 exposure conditions may be due to GC-mediated change in innate and adaptive immune response that differentially affects both the structural and immune cells in the lung which has to be systematically explored.

This study has some limitations. First, lung tissues from female and male C57BL/6J mice (9–10 weeks of age) exposed to acute O3 or FA for 3 h during the light cycle/resting phase and analyzed 24 h post exposure at different times were used for the qRT-PCR analysis of circadian clock genes from a prior study (35). Second, we utilized a different repetitive O3 exposure model (FA or O3 exposure for 4 h/night, 5 nights/week, for a total of 3 weeks during the dark cycle/active phase) from the available GEO dataset to demonstrate the proof of concept that circadian clock genes were altered following repetitive O3 exposure in mouse lungs (28). Third, we were unable to perform a comparative analysis of sex-based differences in the expression of clock genes in acute O3-exposed female and male mice since the experiments were not conducted in parallel (time of exposure and tissue harvest were not the same). In the FA- and repeated O3-exposed group validated by qRT-PCR, we had limited samples available in alveolar macrophages from females (n=2/group) which limited the statistical analysis of the data presented in Fig. S3, although the trends in FPKM and qRT-PCR data were similar for specific clock genes (e.g., Cry2, Per1, Per2, and Fbxl3 variably between females and males). Additional data to evaluate whether single vs. repeated O3 exposure develops tolerance in vivo and if there is a correlation in mRNA vs. protein levels of altered clock targets in different lung compartments which is beyond the focus of the current study. We strongly suggest based on prior evidence from other lung injury models (e.g., air pollution, cigarette smoke, viral and bacterial infection) that the circadian clock has a profound role in regulating lung inflammation and immune response following O3 exposure in a cell-type dependent manner. Future studies will investigate on the acute vs. chronic O3 exposure-induced inflammatory response and associated lung pathophysiological changes using transcriptomics and proteomics approaches.

This study for the first time provides evidence that circadian clock genes were altered during O3-induced lung inflammation and injury in the lung. Our findings suggest that selected core clock genes such as Bmal1, Nr1d 1–2, Per 1–3 and Cry 1–2 may have a profound cell type-specific role in modulating inflammation, immune response and DNA repair following acute vs. repeated O3 exposure which need to be further investigated. It is possible that both stress hormones as well as sex hormones, may directly or indirectly contribute to the sex difference in circadian clock gene expression observed among female and male mice exposed to O3 compared to FA control. Additionally, there could be a time-of-day effect in the measured outcomes that may lead to increased expression of certain clock genes whereas reduced expression of other clock genes. Utilizing targeted circadian clock deletion global and lung cell-type specific transgenic mice for acute and chronic studies will provide a better understanding of the novel role of the molecular clock in O3-induced lung inflammation and injury.

Supplementary Material

Sup apendix

Acknowledgements

This work was supported in part by the National Institute of Health NIH R01HL142543 (I.K.S), R01ES030125 (Y.S.) and the University of Kansas Medical Center, School of Medicine, Internal Medicine Start-Up Funds (I.K.S.). We acknowledge Mr. Allan Giri, MS for his help in editing the final version of this manuscript.

List of Abbreviations:

AMs

Alveolar Macrophages

ARs

Adrenergic Receptors

CA

Conducting Airways

COPD

Chronic Obstructive Pulmonary Disease

DEGs

Differentially Expressed Genes

ETS

Environmental Tobacco Smoke

FA

Filtered Air

FPKM

Fragment Per Kilobase Per Million

GEO

Gene Expression Omnibus

GRs

Glucocorticoid Receptors

Hr

Hours

IAV

Influenza A Virus

miRNA

microRNA

O3

Ozone

qRT-PCR

Quantitative Real-Time Polymerase Chain Reaction

RNA-seq

RNA-sequencing

Tlr4

Toll-like receptor 4

WT

Wild Type

Footnotes

Conflict of Interest

The authors declare that they have no conflict of interest.

Credit authorship contribution statement

IKS, SKD, IC, YS, and PS: designed the study and conducted the experiments; IKS, YS and PS: primarily responsible for the experimental design, critical interpretation of the data, preparation of figures, and writing of the entire manuscript. PS and YS: provided FA and O3-exposed mouse lungs from acute and repetitive exposures analyzed in this study. All the authors checked the content and approved the final version of the revised manuscript.

Appendix A. Supplementary data

The Supplementary Figures 1–3 is presented as Appendix A.

Availability of data and materials

The data generated and/or analyzed during this study are available from the corresponding author upon reasonable request. The GEO dataset (https://www.ncbi.nlm.nih.gov/geo/) used in this study can be accessed using the GEO accession number GSE156799.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Sup apendix
NIHMS1903133-supplement-Sup_apendix.docx (988.4KB, docx)

Data Availability Statement

The data generated and/or analyzed during this study are available from the corresponding author upon reasonable request. The GEO dataset (https://www.ncbi.nlm.nih.gov/geo/) used in this study can be accessed using the GEO accession number GSE156799.

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