Oxidative stress mediated by reactive oxygen species (ROS) is a key process for adverse aerosol health effects. Secondary organic aerosols (SOA) account for a major fraction of fine particulate ...matter, and their inhalation and deposition into the respiratory tract causes the formation of ROS by chemical and cellular processes, but their relative contributions are hardly quantified and their link to oxidative stress remains uncertain. Here, we quantified cellular and chemical superoxide generation by 9,10-phenanthrenequinone (PQN) and isoprene SOA using a chemiluminescence assay combined with electron paramagnetic resonance spectroscopy as well as kinetic modeling. We also applied cellular imaging techniques to study the cellular mechanism of superoxide release and oxidative damage on cell membranes. We show that PQN and isoprene SOA activate NADPH oxidase in macrophages to release massive amounts of superoxide, overwhelming the superoxide formation by aqueous chemical reactions in the epithelial lining fluid. The activation dose for PQN is 2 orders of magnitude lower than that of isoprene SOA, suggesting that quinones are more toxic. While higher exposures trigger cellular antioxidant response elements, the released ROS induce oxidative damage to the cell membrane through lipid peroxidation. Such mechanistic and quantitative understandings provide a basis for further elucidation of adverse health effects and oxidative stress by fine particulate matter.
Successful recovery from lung injury requires the repair and regeneration of alveolar epithelial cells to restore the integrity of gas-exchanging regions within the lung and preserve organ function. ...Improper regeneration of the alveolar epithelium is often associated with severe pulmonary fibrosis, the latter of which involves the recruitment and activation of fibroblasts, as well as matrix accumulation. Type 2 alveolar epithelial cells (AEC2s) are stem cells in the adult lung that contribute to the lung repair process. The mechanisms that regulate AEC2 renewal are incompletely understood. We provide evidence that expression of the innate immune receptor Toll-like receptor 4 (TLR4) and the extracellular matrix glycosaminoglycan hyaluronan (HA) on AEC2s are important for AEC2 renewal, repair of lung injury and limiting the extent of fibrosis. Either deletion of TLR4 or HA synthase 2 in surfactant-protein-C-positive AEC2s leads to impaired renewal capacity, severe fibrosis and mortality. Furthermore, AEC2s from patients with severe pulmonary fibrosis have reduced cell surface HA and impaired renewal capacity, suggesting that HA and TLR4 are key contributors to lung stem cell renewal and that severe pulmonary fibrosis is the result of distal epithelial stem cell failure.
Introduction
Somatic mutations in myeloid growth factor pathway genes, such as JAK2, and genes involved in epigenetic regulation, such as TET2, in hematopoietic stem cells (HSCs) leads to clonal ...hematopoiesis of indeterminate potential (CHIP) which presents a risk factor for hematologic malignancy and cardiovascular disease. Smoking behavior has been repeatedly associated with the occurrence of CHIP but whether smoking is an environmental inflammatory stressor in promoting clonal expansion has not been investigated.
Methods
We performed in vivo smoke exposures in both wildtype (WT) mice and transplanted mice carrying Jak2
V617F
mutant and Tet2 knockout (Tet-/-) cells to determine the impact of cigarette smoke (CS) in the HSC compartment as well as favoring mutant cell expansion.
Results
WT mice exposed to smoke displayed increased oxidative stress in long-term HSCs and suppression of the hematopoietic stem and progenitor compartment but smoke exposure did not translate to impaired hematopoietic reconstitution in primary bone marrow transplants. Gene expression analysis of hematopoietic cells in the bone marrow identified an imbalance between Th17 and Treg immune cells suggesting a local inflammatory environment. We also observed enhanced survival of Jak2V617F cells exposed to CS in vivo and cigarette smoke extract (CSE) in vitro. WT bone marrow hematopoietic cells from WT/Jak2V617F chimeric mice exposed to CS demonstrated an increase in neutrophil abundance and distinct overexpression of bone marrow stromal antigen 2 (Bst2) and retinoic acid early transcript 1 (Raet1) targets. Bst2 and Raet1 are indicative of increased interferon signaling and cellular stress including oxidative stress and DNA damage, respectively. In chimeric mice containing both WT and Tet2-/- cells, we observed an increased percentage of circulating mutant cells in peripheral blood post-cigarette smoke exposure when compared to pre-exposure levels while this difference was absent in air-exposed controls.
Conclusion
Altogether, these findings demonstrate that CS results in an inflamed bone marrow environment that provides a selection pressure for existing CHIP mutations such as Jak2V617F and Tet2 loss-of-function.
Severe pulmonary fibrosis such as idiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of extracellular matrix and fibroblast activation. Targeting fibroblast activation has ...contributed to the development of antifibrotic therapeutics for patients with IPF. Mitogen-activated protein kinase-activated protein kinase 2 (MK2), downstream in the transforming growth factor-β/p38 mitogen-activated protein kinase pathway, has been implicated in inflammatory and fibrosing diseases. Increased concentrations of activated MK2 were expressed in IPF lung and in the mouse bleomycin model of lung fibrosis. The aim of the present study was to determine the role and the mechanisms of MK2 in fibroblast invasion and lung fibrosis. Our results showed that an MK2 inhibitor (MMI-0100) was able to inhibit the invasive capacity of lung fibroblasts isolated from patients with IPF, as well as fibroblasts isolated from both wild-type mice and mice with overexpressing hyaluronan synthase 2 (HAS2) in the myofibroblast compartment. We previously showed that hyaluronan and HAS2 regulate fibroblast invasion and lung fibrosis in vivo. The results of the present study showed that MMI-0100 reduced transforming growth factor-β-induced hyaluronan production in human and mouse fibroblasts in vitro and that HAS2 mediated MK2 activation, suggesting a feed-forward loop in fibroblast activation. More importantly, MK2 inhibition attenuated hyaluronan accumulation and reduced collagen content in bleomycin-injured mouse lungs in vivo. Conditional deletion of MK2 in fibroblasts attenuated bleomycin-induced lung fibrosis. These data provide evidence that MK2 has a role in fibroblast invasion and fibrosis and may be a novel therapeutic target in pulmonary fibrosis.
Dysregulated repair of lung injury often results in lung fibrosis characterized by unremitting deposition of matrix components including glycosaminoglycan hyaluronan (HA). HA is mainly produced by ...hyaluronan synthases (HAS) in mesenchymal cells. We previously demonstrated that over-expression of HAS2 in mesenchymal cells in mice regulates the invasiveness of fibroblasts and promotes severe lung fibrosis. The mechanisms that control the resolution of lung fibrosis are unknown. We propose that a critical step in resolving fibrosis is the induction of senescence in fibrotic fibroblasts and hyaluronan synthase 2 may regulate this process. We found that fibrotic fibroblasts developed the characteristics of replicative senescence in culture and that HAS2 expression was dramatically down-regulated. Furthermore, down-regulation of HAS2 initiated and regulated fibroblast senescence through a p27-CDK2-SKP2 pathway. Deletion of HAS2 in mouse mesenchymal cells increased the cellular senescence of fibroblasts in bleomycin-induced mouse lung fibrosis in vivo. These data suggest that HAS2 may be a critical regulator of the fate of pulmonary fibrosis and we propose a model where over-expression of HAS2 promotes an invasive phenotype resulting in severe fibrosis and down-regulation of HAS2 promotes resolution. Targeting HAS2 to induce fibroblast senescence could be an attractive approach to resolve tissue fibrosis.
•IPF fibroblasts show senescence in vitro.•HAS2 expression correlates fibroblast senescence.•Deletion of HAS2 in fibroblasts increases fibroblast senescence in vitro and in vivo.•HAS2 regulates fibroblast senescence through a p27-CDK2-SKP2 pathway.
Electronic cigarettes (eC) may not be entirely benign. There is a lack of data on the effect of a single acute exposure of eC vapor using various heating sources and power settings upon lung injury. ...The purpose of this study was to determine if an acute exposure with eC vapor heated with different heating elements and power levels induced inflammatory changes in the lungs and heart.
Rats were exposed to pure air or received a single, 4-h exposure to eC vapor. The devices used either a stainless steel (SS) or nichrome (NC) heating element randomized to a low or high atomization power (45 versus 70 W). Rats were euthanized within 48 h of exposure.
The eC groups showed accumulation of inflammatory cells in bronchial lumen, near the pleura, and within the alveolar spaces. The numbers of inflammatory cells per field in the lung parenchyma were significantly greater in the rats exposed to eC groups vs. the air group. There were significantly higher inflammatory gene expression changes in the lungs of animals assigned to 70 W power. We observed that eC vapor generated using burnt coils were toxic and could cause acute respiratory distress and myocarditis.
In conclusion, one 4-h exposure to eC vapor, in the absence of vitamin E oil or nicotine, significantly increased lung inflammation. Effects were seen after exposures to vapor generated using SS and NC heating elements at either high or low power. Vapor from devices with burnt coils can negatively affect the heart and lung.
Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease. Although the pathogenesis is poorly understood, evidence suggests that genetic and epigenetic alterations, such as DNA methylation, ...may play a key role. Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β (TGF-β) superfamily and are important regulators in IPF. Here we identified BMP endothelial cell precursor-derived regulator (BMPER) as a key regulator of fibroblast activation. BMPER is a secreted glycoprotein that binds directly to BMPs and may regulate TGF-β/BMP signaling, but its role in lung fibrosis is not clear. BMPER is highly expressed in human IPF lung fibroblasts compared to normal lung fibroblasts. Demethylation agent 5'-azacytidine decreased BMPER expression in fibroblasts, and attenuated the invasion and migration of IPF lung fibroblasts. Furthermore, siRNA-mediated reduction of BMPER in the human lung fibroblasts impaired cell migration and invasion. 5'-azacytidine treatment additionally regulated BMPER expression and reduced lung fibrosis in mice in vivo. These findings demonstrate that methylation of specific genes in fibroblasts may offer a new therapeutic strategy for IPF by modulating fibroblast activation.
Introduction: Human exposure to biomass smoke particulate matter has been documented to induce immunomodulatory effects that can promote respiratory and cardiovascular injuries. Despite the prevalent ...use of solid fuels and the potential detrimental impact on global health, the effects of specific solid biomass fuels on innate immune cell function remains poorly understood. Therefore, I aimed to begin to fill this gap by studying the effects of acute exposure to particulate matter emissions from dung and mixtures of dung with brushwood used with chulha and angithi traditional cookstoves commonly used in India, collected from in-field sampling.Methods: Particles collected on to Teflon filters in-field were extracted into aqueous suspension for cell-free and cellular in vitro toxicity assessment. The intrinsic cell-free oxidative capacity of particulate matter samples was compared to urban particulate matter and a single diesel quinone compound using dithiothreitol measures. Biological toxicity was assessed using submerged culture exposure with the macrophage-like RAW 264.7 cell line. Biological dose response changes in viability and innate immune function were assessed following four-hour exposures. Biochemical approaches were used to evaluate respiratory burst, metabolic activity, cell cycling dynamics, and gene expression changes. Associations between combustion emission factors, particle oxidative potential, and biological effects were evaluated using multivariate analysis to explore how particle toxicity is influenced by combustion processes.Results: PM emissions from dung-angithi combustion had higher oxidative potential measures than emissions from solid fuel use with chulha cookstoves. The oxidative potential of emissions from dung-chulha cookstoves were lower compared to all other sources of PM including urban dust and quinone compounds. Acute four-hour exposure to solid biomass particles altered critical innate immune respiratory burst function but did not reduce cell viability. Gene expression changes were observed following acute exposures in macrophages exposed to all the air pollutants studied here, including the downregulation of antioxidant response element transcription factor Nrf2 and antioxidant Gpx1, yet an upregulation of detoxifying enzyme Nqo1. However, differential gene expression patterns were observed for antioxidant Gsr, and metabolic housekeeping gene G6pd. The degree of particle reduction-oxidation (redox) activity, and biological respiratory burst dynamics and gene expression changes were associated with pyrolysis combustion processes that influence the generation of volatile organic compounds.Conclusions: In summary, this dissertation describes differences in the toxicity of air pollution particles from distinct sources. Particles generated by the combustion dung fuel and dung brushwood mixtures combustion commonly used with chulha and angithi cookstoves, induce innate immune cell changes through distinct mechanisms compared to urban dust and quinone. This study provides valuable insight into flaming, smoldering, and pyrolysis combustion processes that contribute to air pollution compositional differences and particle toxicity related to household cooking with solid biomass fuels.
Abstract only Background: A new form of lung injury was described in electronic cigarette users beginning July 2019 with patients presenting with respiratory distress. The condition is known as ...E-cigarette (eC) or vaping product use associated lung injury or EVALI and was thought to be related to Vit E oils used to dilute cannabis. Whether a single acute exposure of eC smoke can induce lung injury has not been reported. The purpose of this study was to determine if such an acute exposure with eC smoke heated with different heating elements and power levels induced inflammatory changes in the lungs and heart. Methods: Rats were exposed to pure air or received a single, 4-hour exposure to eC vapor (50% propylene glycol, 50% vegetable glycerin; tobacco flavoring, n=16 in each group). The devices used either a stainless steel (SS) or nichrome (NC) heating element randomized to a low or high atomization power (45 versus 70 Watts). Rats were euthanized within 48 hours of exposure and hematoxylin and eosin stained lung and cardiac histology sections were assessed. Inflammatory cells were counted from 12 random areas per section at 10 х magnification. Results: The lung alveoli appeared normal in the air group (Figure, panel A); the eC groups using the SS or NC heating element with high or low power showed accumulation of inflammatory cells (mainly macrophages) in bronchial lumen (B), near the pleura (C), and within the alveolar spaces (D). The numbers of inflammatory cells per field in the lung parenchyma were significantly greater in the rats exposed to eC using SS or NC heating element compared to the air control group (P < 0.05, One way ANOVA, Tukey multiple comparison test; panel E). Cardiac tissue appeared normal with no evidence of inflammation. Conclusion: One 4-hour exposure to eC vapor, in the absence of vitamin E oil or nicotine, significantly increased the number of inflammatory cells in the lung. Effects were seen after exposures to vapor generated using SS and NC heating elements at either high or low power.
Abstract only Introduction: lectronic cigarette (eC) or Vaping induced Lung Injury (EVALI) was first described in the summer of 2019 in patients presenting with acute respiratory distress. Several ...factors contributing to EVALI are suspected, including use of oil carriers, and heating and power elements. This study examined whether a single acute exposure to eC smoke using two popular heating elements on high or low power levels would induce inflammatory gene expression and proteins, and changes in alveolar macrophage function. Hypothesis: We hypothesize that nichrome, but not stainless-steel atomizers and high power wattage used in the eC device contribute to the extent of changes in pulmonary inflammatory response and alveolar macrophage activity. Methods: Adult Sprague Dawley rats were exposed to clean air (n=8) or eC vapor (50% propylene glycol, 50% vegetable glycerin, tobacco flavoring, no nicotine, n=8) for a single four hour period using devices equipped with stainless steel (SS) or nichrome (NC) heating elements at 45 or 70 wattage power (5 exposure conditions per sex group). Rats were euthanized and bronchoalveolar lavage was performed to collect alveolar macrophages for ex-vivo cell studies. The remaining lung tissue was homogenized for inflammatory protein and gene expression analyses. Results: Significant changes were observed in animals assigned to 70 watts. Increases ranging from 1.5-fold to 2-fold change in expression of the inflammation related genes IL-6, TNF-α and CRP occurred in rats assigned to NC atomizers (p ≤ 0.05), predominantly in males. While the concentration of CRP protein was significantly increased in females, CRP gene expression was decreased at the time point examined. Other proteins were not significantly elevated. Significant (p ≤ 0.05) changes in phagocytic activity were only observed in males exposed to 70 watts NC vapor, which generated exaggerated superoxide production by 4-fold during ex-vivo incubation. Evidence of depressed phagocytic activity was observed in other exposure groups, although not statistically significant. Conclusions: These results demonstrate that a single 4-hr exposure to e-cigarettes can initiate increased gene expression of inflammatory markers IL-6, TNF-α and CRP and oxidative changes in the lung.
PDF
