Epigenetic Regulation of Interleukin 6 by Histone Acetylation in Macrophages and Its Role in Paraquat-Induced Pulmonary Fibrosis

Abstract

Overexpression of interleukin 6 (IL-6) has been proposed to contribute to pulmonary fibrosis and other fibrotic diseases. However, the regulatory mechanisms and the role of IL-6 in fibrosis remain poorly understood. Epigenetics refers to alterations of gene expression without changes in the DNA sequence. Alternation of chromatin accessibility by histone acetylation acts as a critical epigenetic mechanism to regulate various gene transcriptions. The goal of this study was to determine the impact of IL-6 in paraquat (PQ)-induced pulmonary fibrosis and to explore whether the epigenetic regulations may play a role in transcriptional regulation of IL-6. In PQ-treated lungs and macrophages, we found that the mRNA and protein expression of IL-6 was robustly increased in a time-dependent and a dose-dependent manner. Our data demonstrated that PQ-induced IL-6 expression in macrophages plays a central role in pulmonary fibrosis through enhanced epithelial-to-mesenchymal transition (EMT). IL-6 expression and its role to enhance PQ-induced pulmonary fibrosis were increased by histone deacetylase (HDAC) inhibition and prevented by histone acetyltransferase (HAT) inhibition. In addition, the ability of CRISPR-ON transcription activation system (CRISPR-ON) to promote transcription of IL-6 was enhanced by HDAC inhibitor and blocked by HAT inhibitor. Chromatin immunoprecipitation experiments revealed that HDAC inhibitor increased histones activation marks H3K4me3 and H3K9ac at IL-6 promoter regions. In conclusion, IL-6 functioning through EMT in PQ-induced pulmonary fibrosis was regulated dynamically by HDAC and HAT both in vitro and in vivo via epigenetically regulating chromatin accessibility.

Keywords: IL-6; epigenetics; forensic toxicology; histone acetylation; paraquat; pulmonary fibrosis.

Figure 1

Paraquat (PQ)-induced acute pulmonary inflammation, pulmonary fibrosis, and increased IL-6 expression. Wild-type C57BL/6 male mice were treated with vehicle (saline) or 10 mg/kg of PQ for 3 days or 1 month. (A) Lung sections from 3-day mice were stained with hematoxylin and eosin staining for visualization of inflammatory cell infiltration. (B) Lung sections from day 30 mice were stained with Masson’s trichrome for visualization of collagen deposition (green). (C) IL-6 was measured in whole-lung RNA of 3-day mice. (D) ELISA measurement of IL-6 in the serum of day 3 mice. (E) Macrophages were treated with increasing concentrations of PQ for 24 h, and IL-6 mRNA expression was determined by real-time PCR. (F) ELISA measurement of IL-6 in lysates of macrophages. (G) Macrophages were treated with PQ (80 µM) for 0, 4, 8, 12, and 24 h, and IL-6 mRNA expression was determined by real-time PCR. Sections are representative of n ≥ 5 mice from each group. Macrophages were plated in six-well plates at a density of 1.5–2 × 10⁶ cells/well, and the data are representative of n > 5 from each group. Data are expressed as means ± SEM, *P < 0.05 versus vehicle.

Figure 2

GP130Fc downregulated the expression of fibrotic markers and ameliorated paraquat (PQ)-induced pulmonary fibrosis. Wild-type mice were injected with saline or PQ 10 mg/kg, respectively, for 33 days. Mice were treated with vehicle (200 µl sterile PBS) or gp130Fc (2 µg/mouse reconstituted in 200 µl sterile PBS) 1 h before and 18 days after PQ injection. (A–D) The mRNA expression of TGF-β, α-smooth muscle actin (α-SMA), GREM1, and FN1 were measured in whole-lung RNA. (E) Western blot analysis of α-SMA expression in whole-lung lysates. (F) Masson’s trichrome for collagen deposition (green). Representative images of n = 4–6 mice from each group are shown. Data are expressed as means ± SEM, *P < 0.05 versus vehicle and ^#P < 0.05 versus PQ alone.

Figure 3

Paraquat (PQ)-induced pulmonary fibrosis through epithelial-to-mesenchymal transition. (A–E) 16HBE cells were treated with vehicle or recombinant IL-6 10 ng/ml for 48 h. STAT-3, TGF-β, α-smooth muscle actin (α-SMA), GREM1, and FN1 mRNA expression were determined by real-time PCR. Bar graphs show the normalized levels of STAT-3, TGF-β, α-SMA, GREM1, and FN1 by GAPDH. (F–J) 16HBE were incubated with or without 50% macrophage lysates that were treated with or without PQ or recombinant gp130Fc. STAT-3, α-SMA, TGF-β, GREM1, and FN1 mRNA expression were determined by real-time PCR. 16HBE cells were plated in six-well plates at a density of 1.5–2 × 10⁶ cells/well, and the data are representative of n > 5 from each group. *P < 0.05 versus vehicle and ^#P < 0.05 versus 50% macrophage lysates (n = 4–6).

Figure 4

Histone acetylation regulates IL-6 expression. (A,B) Wild-type C57BL/6 male mice were treated with vehicle (saline) or 10 mg/kg of paraquat (PQ) for 3 days, and mice received VPA (3.5 mg/kg) or anacardic acid (5 mg/kg) starting 24 and 1 h before PQ injection. IL-6 mRNA expression was determined by real-time PCR. (C,D) Macrophages were treated with vehicle or PQ (80 µM) for 24 h, and cells received VPA (1 mM) or anacardic acid (25 µM) starting 1 h before PQ treatment. IL-6 mRNA expression was determined by real-time PCR. (E–H) ELISA measurement of IL-6 in serum of day 3 mice and in lysates of macrophages in the above mentioned experimental groups. Data are expressed as means ± SEM, *P < 0.05 versus vehicle and ^#P < 0.05 versus PQ (n = 4–6).

Figure 5

Changes in pulmonary acute inflammation, pulmonary fibrosis following paraquat (PQ) exposure in mice treated with histone deacetylase or histone acetyltransferase inhibitors. Wild-type C57BL/6 male mice were treated with vehicle (saline) or 10 mg/kg of PQ for 3 days, and mice received VPA (3.5 mg/kg) or anacardic acid (5 mg/kg) starting 24 and 1 h before PQ injection. (A,B) Lung sections from day 3 mice were stained with hematoxylin and eosin staining for visualization of inflammatory cell infiltration. Wild-type C57BL/6 male mice were treated with vehicle (saline) or 10 mg/kg of PQ for1 month, and mice received anacardic acid (5 mg/kg) starting 1 h before PQ injection, 24 h and 15 days after PQ injection. (C) Lung sections from day 30 mice were stained with Masson’s trichrome for visualization of collagen deposition (green). (D) COL1-α immunofluorescence (red) in lung sections. Sections are representative of n = 4–6 mice from each group.

Figure 5

Changes in pulmonary acute inflammation, pulmonary fibrosis following paraquat (PQ) exposure in mice treated with histone deacetylase or histone acetyltransferase inhibitors. Wild-type C57BL/6 male mice were treated with vehicle (saline) or 10 mg/kg of PQ for 3 days, and mice received VPA (3.5 mg/kg) or anacardic acid (5 mg/kg) starting 24 and 1 h before PQ injection. (A,B) Lung sections from day 3 mice were stained with hematoxylin and eosin staining for visualization of inflammatory cell infiltration. Wild-type C57BL/6 male mice were treated with vehicle (saline) or 10 mg/kg of PQ for1 month, and mice received anacardic acid (5 mg/kg) starting 1 h before PQ injection, 24 h and 15 days after PQ injection. (C) Lung sections from day 30 mice were stained with Masson’s trichrome for visualization of collagen deposition (green). (D) COL1-α immunofluorescence (red) in lung sections. Sections are representative of n = 4–6 mice from each group.

Figure 6

Influence of histone acetyltransferase inhibition on gene expression of fibrotic markers. Wild-type C57BL/6 male mice were treated with vehicle (saline) or 10 mg/kg of paraquat (PQ) for 1 month, and mice received anacardic acid (5 mg/kg) starting 1 h before PQ injection, 24 h and 15 days after PQ injection. (A–D) TGF-β, α-smooth muscle actin (α-SMA), GREM1, and FN1 expression were determined by real-time PCR. (E) Western blot analysis of α-SMA expression in whole-lung lysates. Data are expressed as means ± SEM, *P < 0.05 versus vehicle and ^#P < 0.05 versus PQ (n = 4–6).

Figure 7

Modulation of endogenous IL-6 expression using CRISPR-ON, and the effects of histone deacetylase (HDAC) or histone acetyltransferase inhibition and epigenetic regulation of IL-6 transcription by HDAC inhibition. (A) HEK 293 T cells cotransfected with Firefly luciferase (LUC) reporter plasmids consisting of human IL-6 promoter and Renilla luciferase as a transfection control were treated with vehicle (DMSO), paraquat (20, 40, 60, and 80 μM), anacardic acid (25 µM), or VPA (1mM) for 24 h, and luciferase activity determined using a Polarstar Omega (BMG Labtech, Durham, NC, USA) luminometer. (B) Schematic of the CRISPR-ON strategy to activate IL-6 gene expression. (C) Guide RNA (gRNA) sequences and binding regions on the IL-6 promoters. HEK-293 cells were transfected with either a control gRNA or gRNA targeting the proximal IL-6 promoter regions in the presence or absence of VPA (1 mM) or anacardic acid (25 µM) for 24 h. (D) HEK-293 cells were co-transfected with IL-6 gRNA and dCas9-p300 or dCas9-p300 (D1399Y) plasmids. IL-6 mRNA levels were determined by real-time PCR relative to GAPDH. (E,F) Macrophages treated with vehicle (DMSO) or scriptaid (3 µg/ml) were fixed with 1% formaldehyde for 10 min and chromatin prepared by enzymatic shearing for 10 min. ChIP was performed on isolated sheared chromatin using a negative control IgG and ChIP grade antibodies to H3K4 trimethylation (H3K4me3) or H3K9 acetylation (H3K9ac). Real-time PCR was performed on DNA purified from each of the ChIP reactions using primers specific for the IL-6 promoter regions located upstream of IL-6 transcriptional start site. HEK 293 T cells were plated in 12-well plates at a density of 0.5–1 × 10⁶ cells/well, and the data are representative of n > 5 from each group. Macrophages were plated in 100 mm dish at a density of 1–1.5 × 10⁷ cells/dish, and the data are representative of n > 5 from each group. Data are expressed as means ± SEM, *P < 0.05 (n = 4–6).