The synergistic effect of circRNA methylation promotes pulmonary fibrosis

Rationale: N6-Methyladenosine (m6A) is the most common type of RNA methylation modification, mainly occurring on mRNA. Whether m6A-modified circRNAs are involved in different settings of pulmonary fibrosis remains unclear. Methods and Results: Using an m6A-circRNA epitranscriptomic chip, candidate circRNAs were selected, in which hsa_circ_0000672 and hsa_circ_0005654 were specifically involved in SiO2-induced pulmonary fibrosis by targeting the same protein, eIF4A3, indicating that the m6A modification of these two circRNAs has a synergistic effect on fibroblast dysfunction induced by SiO2. A mechanistic study revealed that the m6A modification of circRNAs was mainly mediated by the methyltransferase METTL3. Furthermore, METTL3 promoted the activation, migration and activity of pulmonary fibroblasts and participated in SiO2-induced pulmonary fibrosis via circRNA m6A modification. Conclusion: m6A methylation of circRNAs mediates silica-induced fibrosis via synergistic effects, enriching the understanding of circRNAs and uncovering a potential new target to treat fibrosis-related diseases.

Extracellular CIRP Induces an Inflammatory Phenotype in Pulmonary Fibroblasts via TLR4

Extracellular cold-inducible RNA-binding protein (eCIRP), a new damage-associated molecular pattern (DAMP), has been recently shown to play a critical role in promoting the development of bleomycin-induced pulmonary fibrosis. Although fibroblast activation is a critical component of the fibrotic process, the direct effects of eCIRP on fibroblasts have never been examined. We studied eCIRP’s role in the induction of inflammatory phenotype in pulmonary fibroblasts and its connection to bleomycin-induced pulmonary fibrosis in mice. We found that eCIRP causes the induction of proinflammatory cytokines and differentially expression-related pathways in a TLR4-dependent manner in pulmonary fibroblasts. Our analysis further showed that the accessory pathways MD2 and Myd88 are involved in the induction of inflammatory phenotype. In order to study the connection of the enrichment of these pathways in priming the microenvironment for pulmonary fibrosis, we investigated the gene expression profile of lung tissues from mice subjected to bleomycin-induced pulmonary fibrosis collected at various time points. We found that at day 14, which corresponds to the inflammatory-to-fibrotic transition phase after bleomycin injection, TLR4, MD2, and Myd88 were induced, and the transcriptome was differentially enriched for genes in those pathways. Furthermore, we also found that inflammatory cytokines gene expressions were induced, and the cellular responses to these inflammatory cytokines were differentially enriched on day 14. Overall, our results show that eCIRP induces inflammatory phenotype in pulmonary fibroblasts in a TLR4 dependent manner. This study sheds light on the mechanism by which eCIRP induced inflammatory fibroblasts, contributing to pulmonary fibrosis.

Asthmatic Eosinophils Promote Contractility and Migration of Airway Smooth Muscle Cells and Pulmonary Fibroblasts In Vitro

Enhanced contractility and migration of airway smooth muscle cells (ASMC) and pulmonary fibroblasts (PF) are part of airway remodeling in asthma. Eosinophils are the central inflammatory cells that participate in airway inflammation. However, the role of asthmatic eosinophils in ASMC and PF contractility, migration, and differentiation to contractile phenotype has not yet been precisely described. A total of 38 individuals were included in this study: 13 steroid-free non-severe allergic asthma (AA) patients, 11 severe non-allergic eosinophilic asthma (SNEA) patients, and 14 healthy subjects (HS). For AA patients and HS groups, a bronchial allergen challenge with D. pteronyssinus was performed. Individual combined cell cultures were prepared from isolated peripheral blood eosinophils and immortalized ASMC or commercial PF cell lines separately. The migration of ASMC and PF was evaluated using wound healing assay and contractility using collagen gel assay. Gene expression of contractile apparatus proteins, COL1A1, COL5A1, and FN, in ASMC and PF was evaluated using qRT-PCR. We found that contractility and migration of ASMC and PF significantly increased after incubation with asthmatic eosinophils compared to HS eosinophils, p < 0.05, and SNEA eosinophils demonstrated the highest effect on contractility of ASMC and migration of both cell lines, p < 0.05. AA and SNEA eosinophils significantly increased gene expression of contractile apparatus proteins, COL1A1 and FN, in both cell lines, p < 0.05. Furthermore, the allergen-activated AA eosinophils significantly increased the contractility of ASMC, and migration and gene expression in ASMC and PF, p < 0.05. Thus, asthmatic eosinophils change ASMC and PF behavior by increasing their contractility and migration, contributing to airway remodeling.

Butyrate Prevents TGF-β1-Induced Alveolar Myofibroblast Differentiation and Modulates Energy Metabolism

Idiopathic pulmonary fibrosis (IPF) is a serious lung disease characterized by excessive collagen matrix deposition and extracellular remodeling. Signaling pathways mediated by fibrotic cytokine transforming growth factor β1 (TGF-β1) make important contributions to pulmonary fibrosis, but it remains unclear how TGF-β1 alters metabolism and modulates the activation and differentiation of pulmonary fibroblasts. We found that TGF-β1 lowers NADH and NADH/NAD levels, possibly due to changes in the TCA cycle, resulting in reductions in the ATP level and oxidative phosphorylation in pulmonary fibroblasts. In addition, we showed that butyrate (C4), a short chain fatty acid (SCFA), exhibits potent antifibrotic activity by inhibiting expression of fibrosis markers. Butyrate treatment inhibited mitochondrial elongation in TGF-β1-treated lung fibroblasts and increased the mitochondrial membrane potential (MMP). Consistent with the mitochondrial observations, butyrate significantly increased ADP, ATP, NADH, and NADH/NAD levels in TGF-β1-treated pulmonary fibroblasts. Collectively, our findings indicate that TGF-β1 induces changes in mitochondrial dynamics and energy metabolism during myofibroblast differentiation, and that these changes can be modulated by butyrate, which enhances mitochondrial function.

Chemical Elements in Electronic Cigarette Solvents and Aerosols Inhibit Mitochondrial Reductases and Induce Oxidative Stress

Introduction Chemical elements and their toxicity were evaluated in electronic cigarette (EC) solvents, fluids, and aerosols. Aims and Methods Element identification and quantification in propylene glycol (PG), glycerin (G), refill fluids before and after use, and aerosols was done using inductively coupled plasma optical emission spectrometry. Cytotoxicity and oxidative stress were evaluated using in vitro assays. Results Seven elements were present in PG, G, and popular refill fluids, and they transferred to aerosols made with ECs. Selenium was in all products (0.125–0.292 mg/L), while arsenic, aluminum, and tin were frequently in solvent and refill fluid samples at lower concentrations. Iron, chromium, copper, nickel, zinc, and lead were only detected in fluid after EC use, indicating they came from heated atomizers. Elements transferred most efficiently to aerosols made with second-/third-generation ECs. Of the elements in fluid, selenium and arsenic were the most cytotoxic to human bronchial epithelial cells (BEAS-2B) and pulmonary fibroblasts in the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. Selenium increased superoxide production in mitochondria and nucleoli and elevated selenoprotein H in nucleoli of BEAS-2B cells at concentrations found in EC aerosols (10 nM or 0.002 mg/L). Conclusions Elements in EC aerosols came from both e-fluids and atomizing units. Within second-/third-generation products, transfer became more efficient as power increased. In vitro responses occurred at concentrations of selenium found in some EC aerosols. Human exposure to chemical elements in ECs could be reduced by regulating (decreasing) allowable EC power and by improving the purity of PG and G. Implications PG, G, refill fluids, and e-fluids contained potentially toxic chemical elements that transferred to aerosols. Transfer was more efficient in second- and third-generation EC products and increased as power increased. Selenium and arsenic were the most cytotoxic of the elements tested in the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. Selenium tetrachloride-induced oxidative stress in BEAS-2B cells, but not in human pulmonary fibroblasts. All fluids contained selenium above the concentration that induced oxidative stress in human bronchial epithelial cells. Selenium increased superoxide in mitochondria and nucleoli and increased selenoprotein H, a redox responsive DNA-binding protein that is upregulated by superoxide and an indicator of nucleolar stress. EC users are exposed to elements in aerosols, which may with chronic exposure contribute to diseases associated with oxidative stress.

CXCR3A promotes the secretion of the antifibrotic decoy receptor sIL-13Rα2 by pulmonary fibroblasts

CXC chemokine receptor 3 (CXCR3) A and its IFN-inducible ligands CXCL9 and CXCL10 regulate vascular remodeling and fibroblast motility. IL-13 is a profibrotic cytokine implicated in the pathogenesis of inflammatory and fibroproliferative conditions. Previous work from our laboratory has shown that CXCR3A is negatively regulated by IL-13 and is necessary for the basal regulation of the IL-13 receptor subunit IL-13Rα2. This study investigates the regulation of fibroblast phenotype, function, and downstream IL-13 signaling by CXCR3A in vitro. CXCR3A was overexpressed via transient transfection. CXCR3A−/− lung fibroblasts were isolated for functional analysis. Additionally, the contribution of CXCR3A to tissue remodeling following acute lung injury was assessed in vivo with wild-type (WT) and CXCR3−/− mice challenged with IL-13. CXCR3 and IL-13Rα2 displayed a reciprocal relationship after stimulation with either IL-13 or CXCR3 ligands. CXCR3A reduced expression of fibroblast activation makers, soluble collagen production, and proliferation. CXCR3A enhanced the basal expression of pERK1/2 while inducing IL-13-mediated downregulation of NF-κB-p65. CXCR3A−/− pulmonary fibroblasts were increasingly proliferative and displayed reduced contractility and α-smooth muscle actin expression. IL-13 challenge regulated expression of the CXCR3 ligands and soluble IL-13Rα2 levels in lungs and bronchoalveolar lavage fluid (BALF) of WT mice; this response was absent in CXCR3−/− mice. Alveolar macrophage accumulation and expression of genes involved in lung remodeling was increased in CXCR3−/− mice. We conclude that CXCR3A is a central antifibrotic factor in pulmonary fibroblasts, limiting fibroblast activation and reducing extracellular matrix (ECM) production. Therefore, targeting of CXCR3A may be a novel approach to regulating fibroblast activity in lung fibrosis and remodeling.