Irf1- and Egr1-activated transcription plays a key role in macrophage polarization: A multiomics sequencing study with partial validation.

BACKGROUND: Macrophage polarization has a causal role in the pathogenesis and resolution of various clinical diseases. DNA-binding transcription factors (TFs) have been identified as essential factors during gene transcription. Better insight into the TFs that regulate macrophage polarization could provide novel therapeutic targets. METHODS: IFN-γ (50 ng/mL) or IL4 (20 ng/mL) was utilized to stimulate bone marrow-derived macrophages from mice for 24 h for M1- and M2-polarized macrophage model construction, respectively. First, ATAC-seq (Assay for Targeting Accessible-Chromatin with high throughout sequencing) and motif analysis were conducted to identify potential transcription factors (TFs) involved in M1 and M2 macrophage polarization. Second, essential TFs were identified through RNA-seq, after which, their expression was compared between M0-polarized and M1/M2-polarized macrophages. Furthermore, a multiomic analysis of RNA-seq (siRNA knock down of the identified TFs), ChIP-seq and ATAC-seq was utilized to explore the TF-regulated molecular network. GO and KEGG analyses were used to expound the main functions of the TF-regulated molecular network. Finally, the top 5 TF-regulated genes were validated through flow cytometry, ELISA and qPCR. The cut-off values for high-throughput sequencing and qPCR were FDR < 0.05 and P < 0.05, respectively. RESULTS: Compared with M0 macrophages, 10,771 and 4,848 peaks were identified by ATAC-seq during M1 and M2 macrophage polarization, respectively (FDR < 0.05). Fifty and 62 TF binding motifs were identified for the TFs that participate in M1 and M2 macrophage polarization, respectively. The most significantly highly expressed TFs in M1 and M2 macrophages were identified by RNA-seq as Irf1 and Egr1, with LogFC values of 3.2 and 2.8, respectively. Multiomic analyses further found that Irf1 regulated the transcription of 90 genes and that Egr1 regulated the transcription of 116 genes. The Irf1-regulated molecular network played a key role in the inflammatory response and viral defence of M1 macrophages, and 116 Egr1-regulated genes included anti-inflammatory and cell proliferation genes. Validation experiments indicated that IFN-γ-induced Gbp5, Nos2, CD86, Cxcl10 and Cxcl5 expression was significantly downregulated in siIrf1-BMDMs, and IL4-induced Itgax, Nipal1, Bhlhe40, CD206 and Ffar4 expression was significantly downregulated in siEgr1-BMDMs (P < 0.05). CONCLUSIONS: Through multiomic analyses of epigenetic sequencing and RNA-seq with partial validation, the current study found that Irf1- and Egr1-induced transcription plays key roles in M1 and M2 macrophage polarization, respectively.

Pirfenidone Inhibits Hypoxic Pulmonary Hypertension through the NADPH/ROS/p38 Pathway in Adventitial Fibroblasts in the Pulmonary Artery.

Hypoxic pulmonary hypertension (HPH) is a devastating disease characterized by progressive vasoconstriction and vascular remodeling. Pirfenidone (PFD) inhibits the progression of HPH, though the molecular mechanisms remain unknown. This study is aimed at determining the role and mechanism of PFD in HPH in human pulmonary artery adventitial fibroblasts (HPAAFs), which were cultured under normal or hypoxic conditions. NOX4 and Rac1 were inhibited or overexpressed by shRNA or pcDNA3.1, respectively. Proliferation of HPAAFs was quantified by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assays to assess cellular metabolic activity, cell counts, and ethynyldeoxyuridine (EdU) assays to detect DNA synthesis. Migration of HPAAFs was assessed by a wound healing assay. The expression levels of smooth muscle alpha-actin (a-SMA) and procollagen I (COL1A1) were assessed by RT-PCR and western blot analysis. PFD suppressed hypoxia-induced proliferation and migration of HPAAFs. Compared with the hypoxic control group, PFD reduced the expression of a-SMA and procollagen I (COL1A1). PFD reduced hypoxia-induced phosphorylation of p38 through the NOX4/reactive oxygen species (ROS) signaling pathway. Moreover, Rac1 also decreased hypoxia-induced phosphorylation of p38, without any cross-interaction with NOX4. These findings demonstrate that PFD is a novel therapeutic agent to prevent cell proliferation, migration, and fibrosis, which might be useful in inhibiting vascular remodeling in patients with HPH.

Arginase inhibitor attenuates pulmonary artery hypertension induced by hypoxia.

Hypoxia-induced pulmonary arterial hypertension (HPAH) is a refractory disease characterized by increased proliferation of pulmonary vascular smooth cells and progressive pulmonary vascular remodeling. The level of nitric oxide (NO), a potential therapeutic vasodilator, is low in PAH patients. L-arginine can be converted to either beneficial NO by nitric oxide synthases or to harmful urea by arginase. In the present study, we aimed to investigate whether an arginase inhibitor, S-(2-boronoethyl)-L-cysteine ameliorates HPAH in vivo and vitro. In a HPAH mouse model, we assessed right ventricle systolic pressure (RVSP) by an invasive method, and found that RSVP was elevated under hypoxia, but was attenuated upon arginase inhibition. Human pulmonary artery smooth muscle cells (HPASMCs) were cultured under hypoxic conditions, and their proliferative capacity was determined by cell counting and flow cytometry. The levels of cyclin D1, p27, p-Akt, and p-ERK were detected by RT-PCR or Western blot analysis. Compared to hypoxia group, arginase inhibitor inhibited HPASMCs proliferation and reduced the levels of cyclin D1, p-Akt, p-ERK, while increasing p27 level. Moreover, in mouse models, compared to control group, hypoxia increased cyclin D1 expression but reduced p27 expression, while arginase inhibitor reversed the effects of hypoxia. Taken together, these results suggest that arginase plays an important role in increased proliferation of HPASMCs induced by hypoxia and it is a potential therapeutic target for the treatment of pulmonary hypertensive disorders.

Regulatory T cells protect against hypoxia-induced pulmonary arterial hypertension in mice.

Pulmonary arterial hypertension (PAH) is a life­threatening disease characterized by the complex proliferation of the pulmonary vascular endothelium and progressive pulmonary vascular remodeling. CD4+CD25+ regulatory T cells (Tregs) have been the focus of numerous studies into PAH. The present study aimed to investigate the role and mechanisms of Tregs in hypoxia­induced PAH. A total of 60 male mice were divided at random into three groups: Normoxia group, hypoxia control group and Tregs group. Measurements were obtained of the right ventricle systolic pressure (RVSP) and the Fulton's index; in addition, the mRNA and protein expression of pro­inflammatory cytokines including monocyte chemotactic protein 1 (MCP­1), interleukin (IL)­1ß and IL­6, as well as the anti­inflammatory cytokine IL­10 in the lungs were determined by reverse transcription quantitative polymerase chain reaction and western blot analysis in vivo. Human pulmonary artery smooth muscle cells (HPASMCs) were cultured under hypoxic condition with or without Tregs for 48 h, and the proliferation rate and cell cycle of HPASMCs were determined. In addition, the protein levels of phosphorylated (p)­Akt and p­extracellular signal­regulated kinase (ERK) were measured in HPASMCs in vitro. The results showed that Treg treatment significantly reduced the increased the hypoxia­induced RVSP and Fulton's index, decreased pro­inflammatory cytokine expression as well as enhanced IL­10 levels in vivo. Furthermore, Treg treatment significantly reduced HPASMCs proliferation and the expression of cyclin D1, cyclin­dependent kinase (CDK)4, p­Akt and p­ERK, as well as increased p27 expression in vitro. In conclusion, the results of the present study indicated that Tregs protected against hypoxia­induced PAH in mice; the mechanisms of which may proceed via the suppression of the inflammatory response, as Tregs were found to enhance anti­inflammatory cytokine levels, inhibit HPASMCs proliferation and regulate the cell cycle. These results therefore indicated that Tregs may be a potential novel target for the treatment of PAH.