Inhibition of EZH2 prevents acute respiratory distress syndrome (ARDS)-associated pulmonary fibrosis by regulating the macrophage polarization phenotype

Abstract

Background: We recently reported histone methyltransferase enhancer of zeste homolog 2 (EZH2) as a key epigenetic regulator that contributes to the dysfunction of innate immune responses to sepsis and subsequent lung injury by mediating the imbalance of macrophage polarization. However, the role of EZH2 in acute respiratory distress syndrome (ARDS)-associated fibrosis remains poorly understood. Methods: In this study, we investigated the role and mechanisms of EZH2 in pulmonary fibrosis in a murine model of LPS-induced ARDS and in ex-vivo cultured alveolar macrophages (MH-S) and mouse lung epithelial cell line (MLE-12) by using 3-deazaneplanocin A (3-DZNeP) and EZH2 the small interfering (si) RNA. Results: We found that treatment with 3-DZNeP significantly ameliorated the LPS-induced direct lung injury and fibroproliferation by blocking the epithelial to mesenchymal transition (EMT) through transforming growth factor-β1 (TGF-β1)/Smad signaling pathway and regulating shift of macrophage phenotypes. In the ex-vivo polarized alveolar macrophages cells, treatment with EZH2 siRNA or 3-DZNeP suppressed the M1 while promoted the M2 macrophage differentiation through modulating the STAT/SOCS signaling pathway and activating PPAR-γ. Moreover, we identified that blockade of EZH2 with 3-DZNeP suppressed EMT in co-cultured bronchoalveolar lavage fluid (BALF) and mouse lung epithelial cell line through down-regulation of TGF-β1, TGF-βR1, Smad2 while up-regulation of Smad7 expression. Conclusions: These results indicate that EZH2 is involved in the pathological process of ARDS-associated pulmonary fibrosis. Targeting EZH2 may be a potential therapeutic strategy to prevent and treat pulmonary fibrosis post ARDS.

CircN4bp1 Facilitates Sepsis-Induced Acute Respiratory Distress Syndrome through Mediating Macrophage Polarization via the miR-138-5p/EZH2 Axis

“…Analysis of BALF was conducted and wet/ dry weight ratio was calculated as we previously described [13]. Macrophages were isolated from BALF and lung tissue homogenates according to our previously reported methods [13,16]. All samples and isolated cells are stored at −20 °C for determinations.…”

“…Therefore, EZH2 might be the targeted gene of miR-138-5p. As we had reported that EZH2 was involved in the activation of M1 macrophage and inhibition of M2 differentiation [16], we hypothesized that circN4bp1 regulates macrophage differentiation through preventing EZH2 downregulation by miR-138-5p. To test this hypothesis, we overexpressed miR-138-5p mimics in the aforementioned two macrophage cell lines and found that the expression level of EZH2 was increased in the presence of the circN4bp1 overexpression (Figure 6(c)), whereas miR-138-5pmimics significantly inhibited the upregulation of EZH2 after the circN4bp1 overexpression (Figures 6(d) and 6(e)).…”

“…MH-S cells, the SV40 transformed mouse alveolar macrophage cell line (CRI-2019, ATCC, Baltimore, Md, USA), and Raw264.7 cells were cultured in RPMI-1640 medium (Gibco, CA, USA) containing 10% fetal bovine serum (Gibco, Australia Origin) and 1% penicillin/streptomycin in an atmosphere of 5% CO 2 and 95% air at 37 °C. According to the previous report [16], MH-S and Raw264.7 cells were polarized as M1 or M2 macrophages with the indicated stimulants: 50 ng/ml LPS (L3024, Sigma, Mo) 24 h for M1 polarization and 10 ng/ml IL-4 (404-ML, R&D Systems) 24 h for M2 polarization. CircN4bp1 overexpressed vector (pc-circN4bp1, 2 μg), circN4bp1 silence vectors (sh-circN4bp1, 2 μg), miR-138-5p mimics (50 pmol), miR-138-5p inhibitor (50 pmol), and the corresponding control vectors were constructed by GenePharma (Shanghai, China).…”

“…Immunoblotting Analysis. Immunoblotting analysis was conducted as described previously [13,16]. Densitometry analysis of immunoblot results was conducted by using Ima-geJ software.…”

“…To determine the functional influence ofcircN4bp1on macrophage differentiation, we first specifically knocked down the expression of cir-cN4bp1(circN4bp1-KD) in RAW264.7 and MH-S cells with small interfering RNAs (siRNAs) targeting the circN4bp1 junction site and overexpressed of circN4bp1with a circN4bp1 lentivirus plasmids (circN4bp1-OE) transfected into the two macrophage cell lines in contrast to the NC (scrambled control) group (Figure 3(a)). Next, we cultured RAW264.7and MH-S cells in the presence or absence of LPS (M1 polarization) or IL-4 (M2 phenotype) as previously reported [16] and treated them with Si-circN4bp1 or circN4bp1 lentivirus plasmids for24 h. We found that genetic knockdown of circN4bp1 significantly inhibited M1 polarization as evidenced by a downregulation of M1 marker INOS (Figures 3(c As we had previously documented the involvement of STAT1 pathway in M1 polarization and PPAR-γ in M2 differentiation [16], we further investigated the effects of cir-cN4bp1on these pathways. Intriguingly, western blotting showed that the overexpression of circN4bp1 did indeed 4(g) and 4(h)) in contrast to the miR-138-5p-mimic and miR-NC group.…”