Abstract The association between airborne fine particulate matter (PM[2.5]) concentration and the risk of respiratory diseases has been well documented by epidemiological studies. However, the mechanism underlying the harmful effect of PM[2.5] has not been fully understood. In this study, we exposed the C57BL/6J mice to airborne PM[2.5] for 3 months (mean daily concentration ~50 or ~110 μg/m^3, defined as PM[2.5]–3L or PM[2.5]–3H) or 6 months (mean daily concentration ~50 μg/m^3, defined as PM[2.5]–6L) through a whole-body exposure system. Histological and biochemical analysis revealed that PM[2.5]–3H exposure caused more severe lung injury than did PM[2.5]–3L, and the difference was greater than that of PM[2.5]–6L vs PM[2.5]–3L exposure. With RNA-sequencing technique, we found that the lungs exposed with different concentration of PM[2.5] have distinct transcriptional profiles. PM[2.5]–3H exposure caused more differentially expressed genes (DEGs) in lungs than did PM[2.5]–3L or PM[2.5]–6L. The DEGs induced by PM[2.5]–3L or PM[2.5]–6L exposure were mainly enriched in immune pathways, including Hematopoietic cell lineage and Cytokine-cytokine receptor interaction, while the DEGs induced by PM[2.5]–3H exposure were mainly enriched in cardiovascular disease pathways, including Hypertrophic cardiomyopathy and Dilated cardiomyopathy. In addition, we found that upregulation of Cd5l and reduction of Hspa1 and peroxiredoxin-4 was associated with PM[2.5]-induced pulmonary inflammation and oxidative stress. These results may provide new insight into the cytotoxicity mechanism of PM[2.5] and help to development of new strategies to attenuate air pollution associated respiratory disease. Keywords: PM[2.5], Lung injury, RNA-Sequencing, Inflammation, Oxidative stress Graphical abstract [39]Image 1 [40]Open in a new tab Highlights * • High concentration of PM[2.5] has a profound effect on lung injury and gene expression profile. * • RNA-Seq analysis revealed that PM[2.5] exposure affects immune and cardiovascular pathways. * • PM[2.5] increases Cd5l expression and decreases Hspa1 and Prdx4 expression in a concentration dependently manner. Abbreviations 3′-NT 3-nitrotyrosine 4-HNE 4-hydroxynonenal BALF bronchoalveolar lavage fluid Cd5l CD5 antigen-like COPD chronic obstructive pulmonary disease DCM dilated cardiomyopathy DEGs differentially expressed genes FA filtered air Gpx glutathione peroxidase GSH reduced glutathione GSSG oxidized glutathione H&E hematoxylin and eosin HCM hypertrophic cardiomyopathy Hspa1 heat shock 70 kDa protein 1 IL interleukin ILr interleukin receptor KEGG kyoto encyclopedia of genes and genomes Ndufs NADH dehydrogenase iron-sulfur protein PM particulate matter Prdx4 peroxiredoxin-4 qPCR quantitative real-time polymerase chain reaction RNA-seq RNA-sequencing ROS reactive oxygen species Sod superoxide dismutase TLR Toll-like receptors TNFα Tumor Necrosis Factor TNFrsf TNF receptor superfamily member 14 Trxn thioredoxin Trxr2 thioredoxin reductase 1. Introduction Currently, ambient air pollution has become a large threat to public health. Fine particulate matter (PM[2.5], aerodynamic diameter ≤ 2.5 μm) is one of the most important components of outdoor air pollution. High concentration of PM[2.5] increases the risk of respiratory diseases, including asthma [[41]1], bronchitis [[42]2], chronic obstructive pulmonary disease (COPD) [[43]3] and lung cancer [[44]4,[45]5]. Using an analysis of daily time-series for the 20 largest US cities, the PM-mortality dose-response curves and threshold levels were firstly described in 2000 [[46]6]. Then, multiple epidemiological studies have validated that there is a concentration-response relationship between airborne PM[2.5] and its harmful effects on respiratory system [[47]5,[48][7], [49][8], [50][9]]. In mice models, a recent study found that the severity of lung injury caused by ambient PM exposure is associated with cumulative dose [[51]10]. Acute exposure to low doses of fine PM by intranasal instillation also induced lung inflammation and oxidative stress in a dose-dependent manner [[52]11]. It has been suggested that PM[2.5]-induced inflammatory response was associated with Toll-like receptors (TLR2/TLR4) and PM[2.5] can drive a Th2-biased immune response in mice [[53]12]. Notably, during inflammation induced by PM[2.5], enhanced production of reactive oxygen species (ROS) could result in DNA damage, lipid peroxidation and cell death [[54][13], [55][14], [56][15]]. However, the comprehensive mechanisms by which PM[2.5] causes lung injury have not been full elucidated. RNA-sequencing (RNA-seq) is a precise and sensitive tool for measuring global gene expression profiles expression. Recently, this technique has been used to investigate the mechanism of PM[2.5]-induce cytotoxicity in cell models, including 16HBE [[57]16], BEAS-2B [[58]17], A549 [[59]18] and human non-small-cell lung cancer (H1299) cells [[60]19]. To better understand the harmful effects of PM[2.5] on respiratory system, we exposed mice to either airborne PM[2.5] or filtered air (FA) for 3–6 months through a whole-body exposure system and then obtained global gene expression profiles in lungs of FA or PM[2.5] exposed mice using RNA-seq. 2. Materials methods 2.1. Reagents and antibodies BCA protein assay kit and reduced/oxidized glutathione (GSH and GSSG) kit were purchased from the Beyotime Institute of Biotechnology (#P0012, #S0053, Shanghai, China). Elisa kits for mouse tumor necrosis factor alpha (TNFα), 3′-nitrotyrosine (3′-NT) and 4-hydroxynonenal (4-HNE) were purchased from Sino Biological Inc (#[61]SEK50349, Beijing, China), Abcam PLC (#ab116691, Cambridge, UK) and Donggeboye Biological Technology Co. LTD (#DG30947 M, Beijing, China), respectively. The Masson's trichrome stain kit and superoxide dismutase 3 (SOD3) antibody were obtained from Solarbio Science &Technology Co. LTD (#G1340, #K006598P, Beijing, China). Primary antibodies against β-tubulin, SOD1, SOD2, peroxiredoxin 3 (PRDX3), PRDX4, PRDX5 and thioredoxin reductase 2(TRXR2) were purchased from Signalway Antibody LLC (#38075, #32058, #32265, #38567, #43303, #38828, #32885, College Park, MD, USA). Anti-galectin 3 and neutrophil antibodies were purchased from Bioss Biotechnology Co. LTD (#bs-20700R, #bs-6982R, Beijing, China). 2.2. Animal experiments As described previously [[62]20], male C57BL/6J mice (20–22g, obtained from HFK Bioscience Co., Beijing, China) were exposed to either ambient PM[2.5] or FA in a “real-world” exposure system for 12 h/day, 7 days/week, for 3–6 months (October–December 2015 or July–December 2017). The exposure system contains two chambers and ambient air was inhaled into the chambers by air pump. In the inlet valve of FA chamber, a high efficiency particulate air filter (Shanghai Liancheng Purification Equipment CO., LTD, Shanghai) was installed to remove all the microparticles. In the PM[2.5] exposure chamber, PM with an aerodynamic diameter greater than 2.5 μM was removed by a swirler. The exposure system locates at Zhongguancun campus of the University of Chinese of Academy of Sciences (N39°57′39.83″E116°20′10.97″), which is ~50 m away from a traffic main artery (Sihuan Road). During the whole exposure stage, the mice were fed commercial mouse chow and distilled water ad libitum, and were housed under a controlled temperature (22 ± 2 °C) and relative humidity (40–60%) with a 12 h light/dark cycle. Animal studies were performed in accordance with the principles of laboratory animal care (NIH publication no. 85–23, revised 1985) and with approval by the University Of Chinese Academy of Sciences Animal Care and Use Committee. 2.3. Bronchoalveolar lavage The mice were anesthetized by pentobarbital sodium after exposure. Then, the whole lungs were lavaged 3 times with 1 ml phosphate buffer solution (PBS, pH = 7.4). The bronchoalveolar lavage fluid (BALF) was collected and centrifuged at 1000 rpm for 5–10 min. Total cell number and the protein content of the BALF were measured respectively. 2.4. Histologic assessment Mouse lungs were harvested quickly, and then washed with ice-cold PBS for three times. After fixation with 4% paraformaldehyde for 48 h, the lungs were embedded in paraffin. Tissue sections (5 μm) were stained with hematoxylin and eosin (H&E) or Masson trichrome staining kits. To identify macrophages and neutrophils, tissue sections were stained with anti-galectin 3 and anti-neutrophil monoclonal antibodies, respectively. 2.5. RNA isolate and RNA-sequencing Total RNA was extracted from the lungs of FA- or PM[2.5]-exposed mice using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA quality was measured by Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA) and samples with an RNA integrity number over than 8 were used for subsequent experiments. The total RNA was further purified by digesting the double-stranded and single-stranded DNA with DNase I and remove of rRNA using Ribo-Zero method (human, mouse, plants) (Illumina, USA). The library construction and RNA sequencing were performed on a BGISEQ500 platform (BGI-Shenzhen, China). 2.6. Read mapping and differentially expressed gene analysis The raw data were firstly counted and cleaned using SOAPnuke (BGI-Shenzhen, China) and trimmomatic [[63]21] software to remove ligation sequence, low quality sequence and repeats. The sequencing data for clean reads generated by this study have been deposited in the NCBI Sequence Read Archive (SRA) database (accession number: PRJNA540011). Then the clean reads were mapped to the reference genome (Mus_musculus, GCF_000001635.25_GRCm38.p5) using HISAT (Hierarchical Indexing for Spliced Alignment of Transcripts) [[64]22] or Bowtie 2 [[65]23] software. The matched reads were calculated and then normalized to RPKM value (reads per kilo base per million mapped reads) using RESM software [[66]24] to obtain the gene expression level. The differential expression of genes (DEGs) between two groups was screened by DEGseq [[67]25] with the thresholds of fold change ≥ 2 and adjusted P value ≤ 0.001. To further understand the biological functions of genes, the identified DEGs in each pair were mapped to terms in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database ([68]http://www.genome.jp/kegg/pathway.html). In addition, we performed enrichment analysis using the phyper function of R software. The p-value was adjusted for false discovery rate (FDR) to get q-value, and q-value ≤ 0.05 was considered as significant enrichment. 2.7. Quantitative real-time PCR analysis and western blot The cDNA was synthesized using a PrimeScript RT Reagent Kit (#RR036B, TaKaRa, Otsu, Japan) and mRNA expression were measured by quantitative real-time polymerase chain reaction (qPCR) with the SYBR^® Premix Ex Taq™ II Kit (#RR820A). The results were normalized to 18S ribosomal RNA. Primers used in this study are listed in [69]Table S1. Proteins were extracted from mouse lung using lysis buffer (150 mM NaCl, 100 μg/ml phenylmethylsulfonyl fluoride, 50 mM Tris-Cl and 1% Triton X-100) with protease and phosphatase inhibitor cocktail (#04693124001, #4906837001, Roche, Basel, Switzerland) on 4 °C for 20–30 min. After centrifugation at 12,000 g and 4 °C for 20 min, the supernatant was used for western blot analysis as reported previously [[70]20]. 2.8. Statistical analysis All data were analyzed by StatView (SAS Institute Inc.) and expressed as mean ± SEM. One-way analysis of variance (ANOVA) with Tukey's correction was used to make multiple comparisons among the groups. p < 0.05 was defined statistical significance. 3. Results 3.1. Effect of exposure time and concentration on PM[2.5]-induced lung inflammation & fibrosis The average monthly concentration of PM[2.5] during the exposed period was calculated based on the daily data from [71]http://datacenter.mep.gov.cn/. The experiment groups were defined as PM[2.5]–3L group (exposed from July to September in 2017, average concentration ~50 μg/m^3), PM[2.5]–6L group (exposed from July to December in 2017, average concentration ~50 μg/m^3) and PM[2.5]–3H group (exposed from October to December in 2015, average concentration ~115 μg/m^3) ([72]Fig. 1A). As to FA-exposed mice, 3 months or 6 months exposure had no obvious difference in lung morphology, BALF cell flux and serum tumor necrosis factor (TNFα) levels. Therefore, the mice exposed to FA from July to September in 2017 were used as the control group. Fig. 1. [73]Fig. 1 [74]Open in a new tab Effect of exposure time and concentration on PM[2.5]-induced systematic inflammation and lung injury. PM[2.5] concentration of Wanliu monitoring station was recorded during the exposure period. Mean monthly PM[2.5] concentration of July, August, September, October, November and December in 2017, as well as October, November and December in 2015 were shown (A). After exposure to filtered air (FA), low concentration of PM[2.5] for 3–6 months (PM[2.5]–3L, PM[2.5]–6L) or high concentration of PM[2.5] (PM[2.5]–3H) for 3 months, total cell number (B) and protein content (C) in bronchoalveolar lavage fluid (BALF), and serum TNFα level was measured (D). Data are presented as the means ± SEM. N = 3–4. *p < 0.05; **p < 0.01; NS, not significant. To determine whether PM[2.5] concentration or exposure time affects pulmonary alveoli injury, the cell flux and protein content in BALF were measured. As shown in [75]Fig. 1B–C, PM[2.5] exposure significantly increased the cell number and protein concentration of BALF. In addition, BALF from PM[2.5]–3H mice had significantly more cell number and higher protein concentration than that of PM[2.5]–3L mice, while the differences in cell number and protein concentration of BALF between PM[2.5]–3L and PM[2.5]–6L groups were not significant ([76]Fig. 1B–C). Although serum TNFα levels were elevated in PM[2.5]-exposed mice, there was no obvious difference among PM[2.5]–3L, PM[2.5]–6L and PM[2.5]–3H groups ([77]Fig. 1D). As revealed by H&E and Masson staining, PM[2.5] exposure resulted in obvious lung injury & fibrosis, as indicated by the collapse of alveoli, airway epithelial thickening and collagen deposition. Histopathological analysis of lung sections further demonstrated that lungs from PM[2.5]–6L or PM[2.5]–3H group developed significantly more severe injury and fibrosis than lungs from FA or PM[2.5]–3L group. Immunohistochemical staining by antibodies specific for macrophage marker (galectin-3) and neutrophil also revealed that lungs from PM[2.5]–3H group exhibited more infiltration of macrophages and neutrophils than lungs from FA or PM[2.5]–3L group ([78]Fig. 2). Together, these results indicated that the degree of lung injury was associated with the PM[2.5] concentration and exposure time. Fig. 2. [79]Fig. 2 [80]Open in a new tab Effect of exposure time and concentration on PM[2.5]-induced pulmonary fibrosis and inflammation. (A) Representative lung sections from FA, PM[2.5]–3L, PM[2.5]–6L and PM[2.5]–3H exposed mice were stained with hematoxylin and eosin (H&E, Scale bar = 200 μm), Masson trichrome (Scale bar = 200 μm), and antibodies specific for macrophages (galetin-3, Gal-3) and neutrophils (brown staining). Scale bar = 100 μm. The relative collagenous fiber area (B), Gal-3 (C) or neutrophil (D) positive cell number were quantified by Image J software. Data were shown as means ± SEM. N = 4, *p < 0.05; ** indicates p < 0.01; NS, not significant. (For interpretation of the references to color in this figure legend, the reader is referred to