PM2.5 exposure causes alterations in the ADMA/DDAH pathway. To determine the effect of “real-world” PM2.5 exposure on the ADMA/DDAH pathway, we exposed the mice to ambient PM2.5 using the whole-body PM2.5 exposure system for 3 months or 6 months (July-December 2017), and the experimental processes are illustrated in Additional file 1: Figure S1A. As described previously [8, 10], the PM2.5 exposure system was located in the Zhongguancun campus of University of Chinese academy of Science, which is ~ 50 m away from a main traffic artery (Sihuan Road, Beijing, China). During the exposure period, PM2.5 was continuously collected using high-volume sampler particle collectors in the same place. The morphology of PM2.5 were examined by scanning electron microscopy. Dynamic light scattering measurement showed that the size range of collected PM2.5 was about 250–2500 nm and the mean size was 970.96 nm (Additional file 1: Figure S1). The concentrations of metals, soluble inorganic ions, polycyclic aromatic hydrocarbons (PAHs) and carbon in PM2.5 are listed in Additional file 2: Table S1. In addition, ambient air was pumped into the PM2.5 chamber and all PM with an aerodynamic diameter greater than 2.5 μM was removed by the swirler device. In the FA chamber, the pumped air was filtered with a high-efficiency particulate air filter. After PM2.5 exposure, serum ADMA levels were significantly increased. Mice in the PM2.5-6 M group had higher serum ADMA levels than mice in the PM2.5-3 M group (Fig. 1B). PM2.5 exposure decreased pulmonary DDAH1 expression in a time-dependent manner, but had no obvious effect on pulmonary DDAH2 expression. After 3 months of exposure to PM2.5, a significant reduction in pulmonary protein arginine methyltransferase 1 (PRMT1) expression was observed. However, ambient PM2.5 exposure for 6 months had no obvious effect on pulmonary PRMT1 expression (Fig. 1C). There were no significant differences in lung weight and the ratio of lung weight to bodyweight among the FA-6 M, PM2.5-3 M and PM2.5-6 M groups (Additional file 2: Table S2).
To investigate the effect of high concentration of PM2.5 exposure under some extreme conditions on ADMA/DDAH1 pathway, mice were subjected to acute PM2.5 exposure via intratracheal instillation for different time. The experimental protocol is illustrated in Fig. 1D and the dose used here was equal to daily exposure to ~ 1500 μg/m3 of PM2.5 [18]. As shown in Fig. 1E, serum ADMA levels were increased in PM2.5-exposed mice and PM2.5 exposure caused more increases in serum ADMA levels in the PM2.5-4 W group than in the PM2.5-2 W group. However, there was no significant difference in serum ADMA levels between the PM2.5-4 W and PM2.5-8 W groups (Fig. 1E). In addition, DDAH1 expression was increased in the lungs of mice from the PM2.5-2 W and PM2.5-4 W groups. However, the upregulation of pulmonary DDAH1 was diminished in the mice from the PM2.5-8 W group (Fig. 1F). DDAH2 expression was not affected by PM2.5 exposure in any group. In the mice from the PM2.5-4 W group, pulmonary PRMT1 was upregulated but it was dramatically downregulated in the mice from the PM2.5-8 W group (Fig. 1F). The lung weight and ratio of lung weight to bodyweight were significantly increased in acute PM2.5-exposed mice (Additional file 2: Table S3).
Ddah1 deficiency exacerbates long-term PM2.5-induced systemic inflammation, lung vessel remodeling and fibrosis. To determine whether DDAH1 affects PM2.5-induced lung injury, we exposed WT and Ddah1−/− mice to either ambient PM2.5 or FA for 6 months. During PM2.5 exposure, the body weights of the WT and Ddah1−/− mice were recorded weekly. There was no obvious difference in the body weight changes between the WT and Ddah1−/− mice (Additional file 1: Figure S2A). At the end of the experiment, there were still no significant differences in body weight, lung weight or the ratio of lung to body weight between the WT and Ddah1−/− mice (Additional file 2: Table S4). After 6 months of PM2.5 exposure, the levels of tumor necrosis factor alpha (TNFα), interleukin 6 (IL-6) and ADMA in serum, as well as the number of cells in the bronchoalveolar lavage fluid (BALF), were significantly increased in both WT and Ddah1−/− mice. However, these alternations were greater in Ddah1−/− mice than in WT mice (Fig. 2A–D).
In addition, H&E staining of the lung sections revealed that chronic PM2.5 exposure resulted in obvious interstitial lung pathologic changes, as indicated by the widening of alveolar spaces (blue arrows), inflammatory cell infiltration (green arrows), and alveolar structure collapse (red arrows). Lung fibrosis is characterized by increased collagen deposition. As indicated by blue color area in Masson staining (deep red arrows), PM2.5 exposure caused lung fibrosis in both WT and Ddah1−/− mice. Compared with the lungs of WT mice, lungs from Ddah1−/− mice developed more severe morphological changes and fibrosis in response to PM2.5 (Fig. 2E, F). Vascular remodeling is a dynamic process that occurs in response to a variety of stimuli. In this study, vascular remodeling was determined histologically by staining for α-smooth muscle actin (αSMA) and CD31. The vessels in which > 75% of the vessel ring was encircled by smooth muscle cells are defined as fully muscularized vessels and vessels with 25% to 75% of the vessel ring encircled by smooth muscle cells were defined as partially muscularized vessels. As shown in Fig. 2E, PM2.5 exposure produced a greater number of muscularized vessels (including both fully and total muscularized vessels) in the Ddah1−/− lungs than in WT lungs (Fig. 2E, G–H). PM2.5 exposure significantly increased TGF-β mRNA levels in the lungs of mice of both genotypes; however, this change was significantly greater in the lungs of Ddah1−/− mice (Fig. 2I).
Ddah1 deficiency aggravates the pulmonary inflammatory response, cell death and oxidative stress in PM2.5-exposed mice. The results of the immunohistochemical staining using antibodies against neutrophils and F4/80 (a macrophage-specific marker) showed that PM2.5 exposure resulted in serious infiltration of neutrophils and macrophages in Ddah1−/− lungs compared to WT lungs (Fig. 3A–C). Vascular cell adhesion molecule 1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) contribute to inflammatory cells infiltration under various inflammatory conditions [19, 20]. Here, the pulmonary VCAM-1 and ICAM-1 distribution and expression in each experimental group were further examined by immune staining. Ddah1 deficiency did not affect lung VCAM-1 and ICAM-1 levels in FA-exposed mice. PM2.5 exposure caused significant increases in lung VCAM-1 and ICAM-1 expression in both genotypes; however, these increases were significantly greater in the lung of Ddah1−/− mice (Fig. 3A, D, E). Ddah1 deficiency increased lung ADMA levels in both FA- and PM2.5-exposed mice (Fig. 3F). To determine whether PM2.5 exposure differentially stimulates pulmonary inflammatory responses in the lungs of WT and Ddah1−/− mice, we performed qPCR to examine the mRNA levels of TNFα, IL-6 and IL-1β. PM2.5 exposure significantly increased the TNFα, IL-6 and IL-1β mRNA levels in the lungs of Ddah1−/− mice compared to the lungs of WT mice (Fig. 3G–I).
To determine whether Ddah1 deficiency affects PM2.5-induced pulmonary oxidative stress, the changes in oxidative stress markers were measured. In the lungs of FA-exposed mice, Ddah1 deficiency resulted in significant increase in 3′-nitrotyrosine (3′-NT), 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) levels (Fig. 4A–C). PM2.5 exposure increased the levels of 3′-NT, 4-HNE, and MDA in the lungs of WT and Ddah1−/− mice; however, these changes were significantly greater in the lungs of Ddah1−/− mice (Fig. 4A–C). Moreover, PM2.5 exposure also increased the pulmonary nitrate/nitrite (NOx) levels in WT mice and such increase was attenuated by Ddah1 deficiency (Fig. 4D).
Next, we performed dihydroethidium (DHE) and TUNEL staining to assess the effect of Ddah1 deficiency on superoxide generation and cell death. As shown in Fig. 4E, PM2.5 exposure resulted in higher superoxide levels and more TUNEL-positive cells in the lungs of Ddah1−/− mice than in the lungs of WT mice (Fig. 4E–G). To explore the underlying mechanism by which DDAH1 affects PM2.5-induced lung injury, inflammation and cell death, we performed Western blotting to evaluate the expression levels of related proteins. Consistent with the TUNEL staining results, the expression of the pulmonary anti-apoptotic protein Bcl-2 was decreased, whereas the expression of the pro-apoptotic protein Bax was increased in PM2.5-exposed mice. These changes were more pronounced in Ddah1−/− mice. In addition, significant increases in iNOS expression and the ratio of p-p65 (Ser536) to total p65 in the lungs of PM2.5-exposed mice were observed, and these increases were further exacerbated by Ddah1 deficiency (Fig. 4H). Also, PM2.5 exposure significantly decreased the protein expression of SOD1 and PRDX4 in the lungs of WT mice, and these reductions were further exacerbated by Ddah1 deletion (Fig. 4H).
DDAH1 overexpression attenuates long-term PM2.5-induced lung injury, fibrosis and oxidative stress. During the exposed period, the body weight of WT and DDAH1-TG mice was almost identical (Additional file 1: Figure S2B), and there were also no significant differences in body weight, lung weight and the ratio of lung to body weight at the end of the exposure period (Additional file 2: Table S5). After PM2.5 exposure, DDAH1-TG mice exhibited significantly less serum ADMA levels and cell influx in the BALF than WT mice (Fig. 5A, B). H&E and Masson’s staining revealed that the lungs of DDAH1-TG mice exhibited less morphological changes (indicated by blue and red arrows) in alveoli structure and collagen deposition (deep red arrows) than the lungs of the WT mice after PM2.5 exposure (Fig. 5C, D). In addition, TUNEL staining showed that PM2.5 exposure caused less TUNEL-positive cells in the lungs of DDAH1-TG mice than in the lungs of the WT mice (Fig. 5C, E). PM2.5 exposure caused significant increases in ADMA, 3′-NT and 4-HNE in the lungs of WT and DDAH1-TG mice; however, the increases in pulmonary 3′-NT, 4-HNE and MDA levels were significantly attenuated by DDAH1 overexpression (Fig. 5F–H). As shown in Fig. 5I, pulmonary DDAH1 expression in DDAH1-TG mice was ~ twofold higher than in WT mice, and was significantly decreased after PM2.5 exposure. DDAH2 expression was not affected by DDAH1 overexpression or PM2.5 exposure. In the lungs of PM2.5-exposed WT mice, Bcl-2, PRDX4 and SOD1 expressions were decreased whereas Bax and iNOS expression, as well as the p-p65(ser536) to t-p65 ratio were increased. However, downregulation of Bcl-2, PRDX4 and SOD1 and upregulation of iNOS, Bax and p-p65 ser536 were significantly attenuated by DDAH1 overexpression (Fig. 5I).
ADMA treatment exacerbates PM2.5-induced systemic inflammation, lung vessel remodeling, fibrosis and cell death. Next, we treated acute PM2.5-exposed mice with exogenous ADMA (2 mg/kg, every other day) for 2 weeks via intratracheal instillation to determine whether ADMA affects PM2.5-induced lung injury. PM2.5 exposure resulted in significant increases in the serum TNFα and IL-6 levels, and these increases were further enhanced by ADMA administration (Fig. 6A, B). Interestingly, ADMA treatment did not further increase serum ADMA levels in PM2.5-exposed mice (Fig. 6C). ADMA treatment significantly exacerbated the PM2.5-induced upregulation of TGF-β and collagen I and III (Fig. 6D). H&E staining of the lung sections showed that PM2.5-induced alveolar collapse (red arrows), inflammatory cell infiltration (green arrows) and airway epithelial thickening (blue arrows) were exacerbated by ADMA treatment (Fig. 6E). As shown by Masson’s trichrome staining, pulmonary collagen accumulation (black arrows) in PM2.5-exposed mice was further aggravated after ADMA treatment (Fig. 6E, F), indicating that ADMA could promote PM2.5-induced lung fibrosis.
After PM2.5 exposure, the fully muscularized vessels and total muscularized vessels (including both fully and partially muscularized vessels) were significantly increased in the lungs of PM2.5-exposed mice. ADMA treatment further promoted lung vessel muscularization in PM2.5-exposed mice, as demonstrated by the increased number of muscularized vessels (Fig. 6E, G). Moreover, TUNEL staining revealed that ADMA significantly increased the number of apoptotic cells in the PM2.5-exposed lungs (Fig. 6E, H). Western blot analysis showed that the upregulation of pulmonary DDAH1 in PM2.5-exposed mice was attenuated by ADMA treatment, whereas DDAH2 expression was unaffected after PM2.5 exposure and/or ADMA treatment (Fig. 6I). Consistent with the TUNEL results, the upregulation of the proapoptotic protein Bax caused by PM2.5 exposure was exacerbated by ADMA treatment (Fg. 6I).
ADMA treatment exacerbates pulmonary inflammation and oxidative stress in PM2.5-exposed mice. Immunohistochemical staining showed that PM2.5 exposure increased infiltration of neutrophils and macrophages and expression of VCAM-1 and ICAM-1 in the lungs. Moreover, inflammatory cell infiltration and upregulation of VCAM-1 and ICAM-1 in the lungs of PM2.5-exposed mice were exacerbated by ADMA treatment (Fig. 7A–E). DHE staining demonstrated that pulmonary superoxide levels were increased in PM2.5-mice, while the increase was further enhanced by ADMA treatment (Fig. 7A, F). As expected, ADMA administration further increased ADMA levels but decreased NOx levels in PM2.5-exposed lungs (Fig. 7G, H). ADMA treatment also significantly increased pulmonary 3’-NT and 4-HNE levels in PM2.5-exposed mice (Fig. 7I, J). Western blot analysis revealed that PM2.5 exposure resulted in significant increases in phosphorylation of NF-κB p65 (Ser536) and iNOS expression, and these increases were enhanced by ADMA administration (Fig. 7K). ADMA treatment also aggravated the reduction in SOD1 and PRDX4 protein expression in PM2.5-exposed lungs (Fig. 7K).
ADMA exacerbates the inflammatory response and ROS generation in PM2.5-exposed macrophages. The induction of proinflammatory mediators by alveolar macrophages is a key factor in PM2.5-induced lung inflammation [21]. To determine whether ADMA affects the inflammatory response in PM2.5-exposed macrophages, RAW264.7 cells were exposed to 50 μg/ml PM2.5 in the presence or absence of ADMA for 6 h. Compared to the untreated control cells, treatment with 50 μM ADMA significantly increased the mRNA levels of IL-1β but had no obvious effect on the mRNA levels of IL-6 and TNFα. In PM2.5-exposed cells, ADMA treatment significantly increased the mRNA levels of IL-6, IL-1β and TNFα (Fig. 8A–C). Moreover, ADMA treatment also triggered more intracellular ROS generation in both control and PM2.5-exposed cells (Fig. 8D). PM2.5 exposure significantly increased intracellular NO levels, while the increase of NO was blocked by ADMA treatment (Fig. 8E). Western blot results showed that PM2.5 exposure increased iNOS and p-p65 expression and decreased PRDX4 expression in RAW264.7 cells, while these changes were further exacerbated by ADMA treatment (Fig. 8F). A previous report showed that ADMA can uncouple purified iNOS and cause superoxide generation [22]. To determine whether ADMA exacerbated PM2.5-induced the inflammatory response and oxidative stress by upregulating and/or uncoupling iNOS, cells were treated with the iNOS-specific inhibitor 1400 W, which caused significant decreases in the mRNA levels of IL-6, IL-1β and TNFα and intracellular ROS levels in PM2.5 plus ADMA-treated cells (Fig. 8G–J). We also transfected cells with PQCXIN–iNOS expression vector, which increased iNOS expression approximately threefold (Fig. 8K). Interestingly, ADMA caused more ROS generation in iNOS-overexpressing cells than in control cells, and this effect was diminished by 1400 W (Fig. 8L).