American Journal of Respiratory Cell and Molecular Biology最新文献

Gap junction protein β2 (GJB2) has been associated with glycolysis and immunosuppression in human tumors. This research aims to explore the roles of GJB2 in these aspects in the context of lung adenocarcinoma (LUAD). GJB2 expression in LUAD was analyzed using bioinformatics tools and verified in human LUAD cells. RNA binding proteins that target GJB2 were predicted using bioinformatics and verified using RNA immunoprecipitation assays. Gain- or loss-of-function assays of GJB2 and high-density lipoprotein binding protein (HDLBP) were performed in LUAD cells to investigate their roles in glycolysis. These LUAD cells underwent coculture with activated CD8⁺ T cells to examine the effect of gene interference on the exhaustion and activity of T cells. A mouse model of allograft tumor was established for in vivo validation. GJB2 exhibited aberrantly heightened expression in LUAD cells. Further overexpression of GJB2 in cancer cells increased glucose uptake, lactate production, and extracellular acidification rate; augmented aggressive phenotype of cancer cells; and increased exhaustion of the cocultured CD8⁺ T cells. HDLBP, an RNA binding protein that binds to GJB2 RNA, was found to be highly expressed in LUAD as well, which enhanced GJB2 expression by stabilizing the GJB2 mRNA. Overexpression of HDLBP similarly rendered glycolysis and T cell inactivity, with these effects negated by GJB2 knockdown. In parallel, GJB2 silencing in mouse 3LL cells suppressed tumorigenesis, glycolysis, and T cell exhaustion in mice promoted by HDLBP. This research suggests that HDLBP-mediated GJB2 RNA stabilization augments glycolysis and CD8⁺ T cell exhaustion in LUAD progression.

The quest for innovative pharmacologic interventions in idiopathic pulmonary fibrosis (IPF) is a challenging journey. The complexity of the disease demands a comprehensive approach that targets multiple cell types and pathways. This study examined the antifibrotic properties of nerandomilast, a preferential phosphodiesterase 4B inhibitor, focusing on its effects on myofibroblasts (MFs) and endothelial cells. Using cytokine-stimulated human IPF lung fibroblasts and RNA sequencing, we assessed the effects of nerandomilast on MF contractility, MF markers, and differentiation mechanisms. In addition, using human microvascular endothelial cells, we assessed endothelial barrier integrity and monocyte adhesion in a three-dimensional microfluidic chip. Our results show that nerandomilast significantly inhibited MF contractility and marker expression in cytokine-stimulated human IPF lung fibroblast cells. Treatment with nerandomilast significantly activated cAMP-associated pathways and G-protein-coupled receptor signaling events while inhibiting mitogen-activated protein kinase signaling pathways and transforming growth factor β signaling. Nerandomilast also significantly reduced microvascular permeability in cytokine-stimulated human lung microvascular endothelial cells. Finally, in an adeno-associated virus-human diphtheria toxin receptor/diphtheria toxin mouse model of acute lung injury, nerandomilast significantly inhibited total protein in lavage, total macrophages, neutrophils, cell count, and VCAM-1 expression. In summary, our results demonstrate that nerandomilast induces the dedifferentiation of human IPF lung MFs and diminishes their contractility in vitro by interfering with transforming growth factor β, mitogen-activated protein kinase phosphatase-1, and G-protein-coupled receptor signaling pathways. It also mitigates vascular dysfunction by strengthening endothelial junctions and inhibiting adhesion protein expression. These findings highlight nerandomilast's potential therapeutic use in IPF by providing insights into its cellular and molecular actions.

Approximately 50% of people living with Human Immunodeficiency Virus (HIV) in the United States misuse alcohol, and they are at increased risk of chronic lung inflammation despite antiretroviral therapy. Acetaldehyde, a metabolite of alcohol, circulates systemically and directly impacts alveolar macrophages (AMs), the primary reservoir of HIV in the lungs. Acetaldehyde promotes AM HIV replication and triggers IL-1β release. We explored the mechanisms by which alcohol-derived acetaldehyde drives HIV replication and IL-1β release in AMs. Furthermore, we tested if the transcription factor peroxisome proliferator-activated receptor (PPAR)γ agonist, pioglitazone, attenuates AM HIV replication and IL-1β release. Primary mouse AMs, MH-S cells (an AM cell line), and THP-1 (human monocyte cell line)-derived macrophages were treated with alcohol-derived acetaldehyde (acetaldehyde-generating system [AGS]), HIV 1_ADA, and EcoHIV, a chimeric HIV that infects murine cells. HIV expression was confirmed by HIV gag RNA (qRT-PCR) and p24 release (ELISA). IL-1β was measured by qRT-PCR and ELISA. Extracellular hydrogen peroxide (H₂O₂) release was quantified by Amplex Red assay. Furthermore, immunoblot analysis of ERK1/2, PPARγ, and NF-ĸB/p65 (p65) was used to identify how acetaldehyde potentiates HIV replication and IL-1β activation in AMs. AGS increased H₂O₂, leading to ERK1/2 phosphorylation, which deactivated PPARγ. AGS drove nuclear p65 translocation in HIV-infected cells, which enhanced HIV replication and IL-1β release. Treatment with pioglitazone decreased nuclear p65, attenuating AGS-induced HIV replication and IL-1β activation in AMs. We identified mechanisms underlying acetaldehyde-induced inflammatory activation and potentiation of HIV replication in AMs, which could be therapeutically targeted with pioglitazone to decrease HIV-related respiratory comorbidities among people living with HIV who misuse alcohol.

Macrophage-mediated inflammation drives various lung diseases, including chronic obstructive pulmonary disease (COPD). COPD macrophages have dysfunctional mitochondrial metabolism and function, which lead to a chronic inflammatory lung environment. However, the factors regulating this altered metabolism have not been elucidated. ANT1 (adenine nucleotide translocase 1) is a mitochondrial ATP transporter critical to mitochondrial metabolism. We demonstrate that human alveolar macrophages from patients with moderate COPD (Global Initiative for Chronic Obstructive Lung Disease [GOLD] stage 2) have reduced ANT1 expression, whereas macrophages from very severe COPD (GOLD stage 4) have elevated ANT1 compared with normal control subjects. Ant1-deficient mice were protected against cigarette smoke (CS)-induced emphysema, with failure of recruited immune cells to migrate into alveoli. Ant1-null alveolar macrophages had reduced ATP production and mitochondrial respiration, upregulated fewer inflammatory pathways after CS, and reduced migratory capacity. Conditional Ant1 knockout in Cx3cr1-positive monocytes and adoptive transfer of Ant1-deficient bone marrow into CS-treated mice phenocopied the migratory defect in the lung. Our data indicate that ANT1 is a critical regulator of lung macrophage inflammatory signaling and CS-triggered cell migration in the lung, suggesting that metabolic modulation may be a promising therapeutic avenue for COPD.

Cannabidiol (CBD) vaping products pose a significant public health risk because of their high cannabinoid concentrations, additives and contaminants, unsubstantiated claims of health benefits, and their implication in e-cigarette, or vaping, product use-associated lung injury. However, research on the respiratory health effects of vaping CBD is limited. Here we show that the reactive electrophile CBD quinone (CBDQ) is present in significant quantities in commercial CBD vaping products. The effect of vaping on CBDQ concentration was variable across products, indicating that the additives and contaminants we detected in commercial products, including plasticizers, flavorings, and solvents, may play a role in catalyzing or inhibiting vaping-induced CBD oxidation. Using the University of North Carolina Vaping Product Exposure System and click chemistry methodologies, we demonstrate that, in human airway epithelial cells, CBDQ and commercial CBD liquids form covalent adducts with TOP2A, a key protein in DNA replication and cell division. CBDQ downregulated cell cycle genes in an airway epithelial cell line, a finding that was replicated in differentiated human bronchial epithelial cells exposed to commercial CBD vaping products. In addition, CBDQ and vaped CBD products inhibited cell proliferation. We also show that CBDQ is ubiquitous in commercial CBD vaping products, may increase after vaping, and significantly alters the respiratory transcriptome, most notably inhibiting cell cycle genes. Together, these data suggest that CBD vaping products have significant effects on normal airway function and, with chronic use, could pose health risks, including impaired wound healing and increased susceptibility to infections, diseases, and other environmental exposures.

The human airway epithelium is a primary site of toxicant exposure and crucial in the pathogenesis of acute and chronic lung disease. In chronic lung disease (CLD), the airway epithelium is frequently altered and distorted, and its restoration is desirable. The mechanisms underlying human aberrant epithelial regeneration, however, are poorly understood. Importantly, our knowledge about airway epithelial injury and regeneration stems largely from mouse models; yet, airways differ considerably between mice and humans. We hypothesized that treatment of differentiated primary human bronchial epithelial cells (phBECs) with polidocanol (PDOC) or naphthalene would allow study of mechanisms of human airway epithelial injury and regeneration. Injury of differentiated phBECs with 0.04% but not 0.1% PDOC resulted in full restoration of a functional epithelium and epithelial barrier integrity as monitored by qRT-PCR analysis, immunofluorescence staining, and transepithelial electrical resistance measurements. Regeneration was associated with a transient but not parallel increase of p21⁺ and KRT17⁺ cells. Providing proof of concept, DAPT, an inhibitor of Notch signaling, blunted the restoration of secretory cell types after 0.04% PDOC injury. Differentiation of phBECs in the presence of cigarette smoke extract or ethanol as a first hit significantly impaired the regeneration capacity of phBECs. Although naphthalene is known to specifically induce club cell depletion in mouse airways, it failed to do so in phBECs. In conclusion, using fully differentiated phBECs treated with PDOC, we successfully established and thoroughly characterized a human in vitro system that will facilitate studies of mechanisms involved in susceptibility to injury as well as human airway repair and regeneration.