Nanoplastics Penetrate Human Bronchial Smooth Muscle and Small Airway Epithelial Cells and Affect Mitochondrial Metabolism.

Micro- and nanoplastic particles, including common forms like polyethylene and polystyrene, have been identified as relevant pollutants, potentially causing health problems in living organisms. The mechanisms at the cellular level largely remain to be elucidated. This study aims to visualize nanoplastics in bronchial smooth muscle (BSMC) and small airway epithelial cells (SAEC), and to assess the impact on mitochondrial metabolism. Healthy and asthmatic human BSMC and SAEC in vitro cultures were stimulated with polystyrene nanoplastics (PS-NPs) of 25 or 50 nm size, for 1 or 24 h. Live cell, label-free imaging by holotomography microscopy and mitochondrial respiration and glycolysis assessment were performed. Furthermore, 25 and 50 nm NPs were shown to penetrate SAEC, along with healthy and diseased BSMC, and they impaired bioenergetics and induce mitochondrial dysfunction compared to cells not treated with NPs, including changes in oxygen consumption rate and extracellular acidification rate. NPs pose a serious threat to human health by penetrating airway tissues and cells, and affecting both oxidative and glycolytic metabolism.

Airway epithelial CD47 plays a critical role in inducing influenza virus-mediated bacterial super-infection.

Respiratory viral infection increases host susceptibility to secondary bacterial infections, yet the precise dynamics within airway epithelia remain elusive. Here, we elucidate the pivotal role of CD47 in the airway epithelium during bacterial super-infection. We demonstrated that upon influenza virus infection, CD47 expression was upregulated and localized on the apical surface of ciliated cells within primary human nasal or bronchial epithelial cells. This induced CD47 exposure provided attachment sites for Staphylococcus aureus, thereby compromising the epithelial barrier integrity. Through bacterial adhesion assays and in vitro pull-down assays, we identified fibronectin-binding proteins (FnBP) of S. aureus as a key component that binds to CD47. Furthermore, we found that ciliated cell-specific CD47 deficiency or neutralizing antibody-mediated CD47 inactivation enhanced in vivo survival rates. These findings suggest that interfering with the interaction between airway epithelial CD47 and pathogenic bacterial FnBP holds promise for alleviating the adverse effects of super-infection.

Mitigation of acute lung injury by human bronchial epithelial cell-derived extracellular vesicles via ANXA1-mediated FPR signaling.

Acute lung injury (ALI) is characterized by respiratory failure resulting from the disruption of the epithelial and endothelial barriers as well as immune system. In this study, we evaluated the therapeutic potential of airway epithelial cell-derived extracellular vesicles (EVs) in maintaining lung homeostasis. We isolated human bronchial epithelial cell-derived EVs (HBEC-EVs), which endogenously express various immune-related surface markers and investigated their immunomodulatory potential in ALI. In ALI cellular models, HBEC-EVs demonstrated immunosuppressive effects by reducing the secretion of proinflammatory cytokines in both THP-1 macrophages and HBECs. Mechanistically, these effects were partially ascribed to nine of the top 10 miRNAs enriched in HBEC-EVs, governing toll-like receptor-NF-κB signaling pathways. Proteomic analysis revealed the presence of proteins in HBEC-EVs involved in WNT and NF-κB signaling pathways, pivotal in inflammation regulation. ANXA1, a constituent of HBEC-EVs, interacts with formyl peptide receptor (FPR)2, eliciting anti-inflammatory responses by suppressing NF-κB signaling in inflamed epithelium, including type II alveolar epithelial cells. In a mouse model of ALI, intratracheal administration of HBEC-EVs reduced lung injury, inflammatory cell infiltration, and cytokine levels. Collectively, these findings suggest the therapeutic potential of HBEC-EVs, through their miRNAs and ANXA1 cargo, in mitigating lung injury and inflammation in ALI patients.

Coinfection with respiratory syncytial virus and rhinovirus increases IFN-λ1 and CXCL10 expression in human primary bronchial epithelial cells.

Acute respiratory tract infection (ARTI) is common in all age groups, especially in children and the elderly. About 85% of children who present with bronchiolitis are infected with respiratory syncytial virus (RSV); however, nearly one-third are coinfected with another respiratory virus, such as human rhinovirus (HRV). Therefore, it is necessary to explore the immune response to coinfection to better understand the molecular and cellular pathways involving virus-virus interactions that might be modulated by innate immunity and additional host cell response mechanisms. This study aims to investigate the host innate immune response against RSV-HRV coinfection compared with monoinfection. Human primary bronchial/tracheal epithelial cells (HPECs) were infected with RSV, HRV, or coinfected with both viruses, and the infected cells were collected at 48 and 72 hours. Gene expression profiles of IL-6, CCL5, TNF-α, IFN-ß, IFN-λ1, CXCL10, IL-10, IL-13, IRF3, and IRF7 were investigated using real-time quantitative PCR, which revealed that RSV-infected cells exhibited increased expression of IL-10, whereas HRV infection increased the expression of CXCL10, IL-10, and CCL5. IFN-λ1 and CXCL10 expression was significantly different between the coinfection and monoinfection groups. In conclusion, our study revealed that two important cytokines, IFN-λ1 and CXCL10, exhibited increased expression during coinfection.

Biochemical changes in lipid and protein metabolism caused by mannose-Raman spectroscopy studies.

Biochemical analysis of human normal bronchial cells (BEpiC) and human cancer lung cells (A549) has been performed by using Raman spectroscopy and Raman imaging. Our approach provides a biochemical compositional mapping of the main cell components: nucleus, mitochondria, lipid droplets, endoplasmic reticulum, cytoplasm and cell membrane. We proved that Raman spectroscopy and Raman imaging can distinguish successfully BEpiC and A549 cells. In this study, we have focused on the role of mannose in cancer development. It has been shown that changes in the concentration of mannose can regulate some metabolic processes in cells. Presented results suggest lipids and proteins can be considered as Raman biomarkers during lung cancer progression. Analysis obtained for bands 1444 cm-1, and 2854 cm-1 characteristic for lipids and derivatives proved that the addition of mannose reduced levels of these compounds. Results obtained for protein compounds based on bands 858 cm-1, 1004 cm-1 and 1584 cm-1 proved that the addition of mannose increases the values of protein in BEpiC cells and blocks protein glycolisation in A549 cells. Noticing Raman spectral changes in BEpiC and A549 cells supplemented with mannose can help to understand the mechanism of sugar metabolism during cancer development and could play in the future an important role in clinical treatment.

Deficiency of the BKCa potassium channel displayed significant implications for the physiology of the human bronchial epithelium.

Plasma membrane large-conductance calcium-activated potassium (BKCa) channels are important players in various physiological processes, including those mediated by epithelia. Like other cell types, human bronchial epithelial (HBE) cells also express BKCa in the inner mitochondrial membrane (mitoBKCa). The genetic relationships between these mitochondrial and plasma membrane channels and the precise role of mitoBKCa in epithelium physiology are still unclear. Here, we tested the hypothesis that the mitoBKCa channel is encoded by the same gene as the plasma membrane BKCa channel in HBE cells. We also examined the impact of channel loss on the basic function of HBE cells, which is to create a tight barrier. For this purpose, we used CRISPR/Cas9 technology in 16HBE14o- cells to disrupt the KCNMA1 gene, which encodes the α-subunit responsible for forming the pore of the plasma membrane BKCa channel. Electrophysiological experiments demonstrated that the disruption of the KCNMA1 gene resulted in the loss of BKCa-type channels in the plasma membrane and mitochondria. We have also shown that HBE ΔαBKCa cells exhibited a significant decrease in transepithelial electrical resistance which indicates a loss of tightness of the barrier created by these cells. We have also observed a decrease in mitochondrial respiration, which indicates a significant impairment of these organelles. In conclusion, our findings indicate that a single gene encodes both populations of the channel in HBE cells. Furthermore, this channel is critical for maintaining the proper function of epithelial cells as a cellular barrier.

Modeling PAH Mixture Interactions in a Human In Vitro Organotypic Respiratory Model.

One of the most significant challenges in human health risk assessment is to evaluate hazards from exposure to environmental chemical mixtures. Polycyclic aromatic hydrocarbons (PAHs) are a class of ubiquitous contaminants typically found as mixtures in gaseous and particulate phases in ambient air pollution associated with petrochemicals from Superfund sites and the burning of fossil fuels. However, little is understood about how PAHs in mixtures contribute to toxicity in lung cells. To investigate mixture interactions and component additivity from environmentally relevant PAHs, two synthetic mixtures were created from PAHs identified in passive air samplers at a legacy creosote site impacted by wildfires. The primary human bronchial epithelial cells differentiated at the air-liquid interface were treated with PAH mixtures at environmentally relevant proportions and evaluated for the differential expression of transcriptional biomarkers related to xenobiotic metabolism, oxidative stress response, barrier integrity, and DNA damage response. Component additivity was evaluated across all endpoints using two independent action (IA) models with and without the scaling of components by toxic equivalence factors. Both IA models exhibited trends that were unlike the observed mixture response and generally underestimated the toxicity across dose suggesting the potential for non-additive interactions of components. Overall, this study provides an example of the usefulness of mixture toxicity assessment with the currently available methods while demonstrating the need for more complex yet interpretable mixture response evaluation methods for environmental samples.

mTORC1 - TFEB pathway was involved in sodium arsenite induced lysosomal alteration, oxidative stress and genetic damage in BEAS-2B cells.

The mechanistic target of rapamycin (RAPA) complex 1 (mTORC1) - transcription factor EB (TFEB) pathway plays a crucial role in response to nutritional status, energy and environmental stress for maintaining cellular homeostasis. But there is few reports on its role in the toxic effects of arsenic exposure and the related mechanisms. Here, we show that the exposure of bronchial epithelial cells (BEAS-2B) to sodium arsenite promoted the activation of mTORC1 (p-mTORC1) and the inactivation of TFEB (p-TFEB), the number and activity of lysosomes decreased, the content of reduced glutathione (GSH) and superoxide dismutase (SOD) decreased, the content of malondialdehyde (MDA) increased, the DNA and chromosome damage elevated. Further, when mTORC1 was inhibited with RAPA, p-mTORC1 and p-TFEB down-regulated, GSH and SOD increased, MDA decreased, the DNA and chromosome damage reduced significantly, as compared with the control group. Our data revealed for the first time that mTORC1 - TFEB pathway was involved in sodium arsenite induced lysosomal alteration, oxidative stress and genetic damage in BEAS-2B cells, and it may be a potential intervention target for the toxic effects of arsenic.

Metabolic alterations and mitochondrial dysfunction in human airway BEAS-2B cells exposed to vanadium pentoxide.

Vanadium pentoxide (V+5) is a hazardous material that has drawn considerable attention due to its wide use in industrial sectors and increased release into environment from human activities. It poses potential adverse effects on animals and human health, with pronounced impact on lung physiology and functions. In this study, we investigated the metabolic response of human bronchial epithelial BEAS-2B cells to low-level V+5 exposure (0.01, 0.1, and 1 ppm) using liquid chromatography-high resolution mass spectrometry (LC-HRMS). Exposure to V+5 caused extensive changes to cellular metabolism in BEAS-2B cells, including TCA cycle, glycolysis, fatty acids, amino acids, amino sugars, nucleotide sugar, sialic acid, vitamin D3, and drug metabolism, without causing cell death. Altered mitochondrial structure and function were observed with as low as 0.01 ppm (0.2 µM) V+5 exposure. In addition, decreased level of E-cadherin, the prototypical epithelial marker of epithelial-mesenchymal transition (EMT), was observed following V+5 treatment, supporting potential toxicity of V+5 at low levels. Taken together, the present study shows that V+5 has adverse effects on mitochondria and the metabolome which may result in EMT activation in the absence of cell death. Furthermore, results suggest that high-resolution metabolomics could serve as a powerful tool to investigate metal toxicity at levels which do not cause cell death.

Anti-inflammatory actions of aspirin-triggered resolvin D1 (AT-RvD1) in bronchial epithelial cells stimulated by cigarette smoke extract.

Smoking causes several diseases such as chronic obstructive pulmonary disease (COPD). Aspirin-triggered-resolvin D1 (AT-RvD1) is a lipid mediator produced during the resolution of inflammation and demonstrates anti-inflammatory and pro-resolution effects in several inflammatory experimental models including in the airways. Here we evaluated the role of AT-RvD1 (100 nM) in bronchial epithelial cells (BEAS-2B) stimulated by cigarette smoke extract (CSE; 1%; 1 cigarette) for 24 h. CSE induced the productions of IL-1ß, TNF-α, IL-10, IL-4 and IFN-γ as well as the activations of NF-κB and STAT3 and the expression of ALX/FPR2 receptor. AT-RvD1 reduced the IL-1ß and TNF-α production and increased the production of IFN-γ. These effects were reversed BOC2, an antagonist of ALX/FPR2 receptor for AT-RvD1. The production of IL-4 and IL-10 were not altered by AT-RvD1. In addition, AT-RvD1 reduced the phosphorylation of NF-κB and STAT3 when compared to CSE-stimulated BEAS-2B cells. No alteration of ALX/FPR2 expression was observed by AT-RvD1 when compared to CSE group. In the human monocytic leukemia cell line, the relative number of copies of IL-1ß and IL-4 was significantly higher in CSE + AT-RvD1 group compared CSE group, however, the expression of M1 cytokine was more pronounced than M2 profile. AT-RvD1 could be an important target for the reduction of inflammation in the airways associated with smoking.

Exosome-encapsulated lncRNA HOTAIRM1 contributes to PM2.5-aggravated COPD airway remodeling by enhancing myofibroblast differentiation.

Emphysema, myofibroblast accumulation and airway remodeling can occur in the lungs due to exposure to atmospheric pollution, especially fine particulate matter (PM2.5), leading to chronic obstructive pulmonary disease (COPD). Specifically, bronchial epithelium-fibroblast communication participates in airway remodeling, which results in COPD. An increasing number of studies are now being conducted on the role of exosome-mediated cell-cell communication in disease pathogenesis. Here, we investigated whether exosomes generated from bronchial epithelial cells could deliver information to normal stromal fibroblasts and provoke cellular responses, resulting in airway obstruction in COPD. We studied the mechanism of exosome-mediated intercellular communication between human bronchial epithelial (HBE) cells and primary lung fibroblasts (pLFs). We found that PM2.5-induced HBE-derived exosomes promoted myofibroblast differentiation in pLFs. Then, the exosomal lncRNA expression profiles derived from PM2.5-treated HBE cells and nontreated HBE cells were investigated using an Agilent Human LncRNA Array. Combining coculture assays and direct exosome treatment, we found that HBE cell-derived exosomal HOTAIRM1 facilitated the myofibroblast differentiation of pLFs. Surprisingly, we discovered that exosomal HOTAIRM1 enhanced pLF proliferation to secrete excessive collagen secretion, leading to airway obstruction by stimulating the TGF-ß/SMAD3 signaling pathway. Significantly, PM2.5 reduced FEV1/FVC and FEV1 and increased the level of serum exosomal HOTAIRM1 in healthy people; moreover, serum exosomal HOTAIRM1 was associated with PM2.5-related reductions in FEV1/FVC and FVC. These findings show that PM2.5 triggers alterations in exosome components and clarify that one of the paracrine mediators of myofibroblast differentiation is bronchial epithelial cell-derived HOTAIRM1, which has the potential to be an effective prevention and therapeutic target for PM2.5-induced COPD.

TMPRSS2 is a functional receptor for human coronavirus HKU1.

Four endemic seasonal human coronaviruses causing common colds circulate worldwide: HKU1, 229E, NL63 and OC43 (ref. 1). After binding to cellular receptors, coronavirus spike proteins are primed for fusion by transmembrane serine protease 2 (TMPRSS2) or endosomal cathepsins2-9. NL63 uses angiotensin-converting enzyme 2 as a receptor10, whereas 229E uses human aminopeptidase-N11. HKU1 and OC43 spikes bind cells through 9-O-acetylated sialic acid, but their protein receptors remain unknown12. Here we show that TMPRSS2 is a functional receptor for HKU1. TMPRSS2 triggers HKU1 spike-mediated cell-cell fusion and pseudovirus infection. Catalytically inactive TMPRSS2 mutants do not cleave HKU1 spike but allow pseudovirus infection. Furthermore, TMPRSS2 binds with high affinity to the HKU1 receptor binding domain (Kd 334 and 137 nM for HKU1A and HKU1B genotypes) but not to SARS-CoV-2. Conserved amino acids in the HKU1 receptor binding domain are essential for binding to TMPRSS2 and pseudovirus infection. Newly designed anti-TMPRSS2 nanobodies potently inhibit HKU1 spike attachment to TMPRSS2, fusion and pseudovirus infection. The nanobodies also reduce infection of primary human bronchial cells by an authentic HKU1 virus. Our findings illustrate the various evolution strategies of coronaviruses, which use TMPRSS2 to either directly bind to target cells or prime their spike for membrane fusion and entry.

Klebsiella pneumoniae outer membrane vesicles induce strong IL-8 expression via NF-κB activation in normal pulmonary bronchial cells.

BACKGROUND: Outer membrane vesicles (OMVs) derived from bacteria are known to play a crucial role in the interactions between bacteria and their environment, as well as bacteria-bacteria and bacteria-host interactions.Specifically, OMVs derived from Klebsiella pneumoniae have been implicated in contributing to the pathogenesis of this bacterium.Hypervirulent Klebsiella pneumoniae (hvKp) has emerged as a global pathogen of great concern due to its heightened virulence compared to classical K. pneumoniae (cKp), and its ability to cause community-acquired infections, even in healthy individuals.The objective of this study was to investigate potential differences between hvKp-derived OMVs and cKp-derived OMVs in their interactions with microorganisms and host cells. METHODS: Four strains of K. pneumoniae were used to produce OMVs: hvKp strain NTUH-K2044 (K1, ST23), hvKp clinical strain AP8555, and two cKP clinical strains C19 and C250. To examine the morphology and size of the bacterial OMVs, transmission electron microscopy (TEM) was utilized. Additionally, dynamic light scattering (DLS) was used to analyze the size characterization of the OMVs.The normal pulmonary bronchial cell line HBE was exposed to OMVs derived from hvKp and cKP. Interleukin 8 (IL-8) messenger RNA (mRNA) expression was assessed using reverse transcription-polymerase chain reaction (RT-PCR), while IL-8 secretion was analyzed using enzyme-linked immunosorbent assay (ELISA).Furthermore, the activation of nuclear factor kappa B (NF-κB) was evaluated using both Western blotting and confocal microscopy. RESULTS: After purification, OMVs appeared as electron-dense particles with a uniform spherical morphology when observed through TEM.DLS analysis indicated that hvKp-derived OMVs from K2044 and AP8555 measured an average size of 116.87 ± 4.95 nm and 96.23 ± 2.16 nm, respectively, while cKP-derived OMVs from C19 and C250 measured an average size of 297.67 ± 26.3 nm and 325 ± 6.06 nm, respectively. The average diameter of hvKp-derived OMVs was smaller than that of cKP-derived OMVs.A total vesicular protein amount of 47.35 mg, 41.90 mg, 16.44 mg, and 12.65 mg was generated by hvKp-K2044, hvKp-AP8555, cKP-C19, and cKP-C250, respectively, obtained from 750 mL of culture supernatant. Both hvKp-derived OMVs and cKP-derived OMVs induced similar expression levels of IL-8 mRNA and protein. However, IL-8 expression was reduced when cells were exposed to BAY11-7028, an inhibitor of the NF-κB pathway.Western blotting and confocal microscopy revealed increased phosphorylation of p65 in cells exposed to OMVs. CONCLUSIONS: Klebsiella pneumoniae produces outer membrane vesicles (OMVs) that play a key role in microorganism-host interactions. HvKp, a hypervirulent strain of K. pneumoniae, generates more OMVs than cKP.The average size of OMVs derived from hvKp is smaller than that of cKP-derived OMVs.Despite these differences, both hvKp-derived and cKP-derived OMVs induce a similar level of expression of IL-8 mRNA and protein.OMVs secreted by K. pneumoniae stimulate the secretion of interleukin 8 by activating the nuclear factor NF-κB.

Lactobacillus brevis BGZLS10-17 and Lb. plantarum BGPKM22 Exhibit Anti-Inflammatory Effect by Attenuation of NF-κB and MAPK Signaling in Human Bronchial Epithelial Cells.

Bronchial epithelial cells are exposed to environmental influences, microbiota, and pathogens and also serve as a powerful effector that initiate and propagate inflammation by the release of pro-inflammatory mediators. Recent studies suggested that lung microbiota differ between inflammatory lung diseases and healthy lungs implicating their contribution in the modulation of lung immunity. Lactic acid bacteria (LAB) are natural inhabitants of healthy human lungs and also possess immunomodulatory effects, but so far, there are no studies investigating their anti-inflammatory potential in respiratory cells. In this study, we investigated immunomodulatory features of 21 natural LAB strains in lipopolysaccharide (LPS)-stimulated human bronchial epithelial cells (BEAS-2B). Our results show that several LAB strains reduced the expression of pro-inflammatory cytokine and chemokine genes. We also demonstrated that two LAB strains, Lactobacillus brevis BGZLS10-17 and Lb. plantarum BGPKM22, effectively attenuated LPS-induced nuclear factor-κB (NF-κB) nuclear translocation. Moreover, BGZLS10-17 and BGPKM22 reduced the activation of p38, extracellular signal-related kinase (ERK), and c-Jun amino-terminal kinase (JNK) signaling cascade resulting in a reduction of pro-inflammatory mediator expressions in BEAS-2B cells. Collectively, the LAB strains BGZLS10-17 and BGPKM22 exhibited anti-inflammatory effects in BEAS-2B cells and could be employed to balance immune response in lungs and replenish diminished lung microbiota in chronic lung diseases.

HIF-1α Stabilization in Flagellin-Stimulated Human Bronchial Cells Impairs Barrier Function.

The respiratory epithelium provides a first line of defense against pathogens. Hypoxia-inducible factor (HIF)1α is a transcription factor which is stabilized in hypoxic conditions through the inhibition of prolyl-hydroxylase (PHD)2, the enzyme that marks HIF1α for degradation. Here, we studied the impact of HIF1α stabilization on the response of primary human bronchial epithelial (HBE) cells to the bacterial component, flagellin. The treatment of flagellin-stimulated HBE cells with the PHD2 inhibitor IOX2 resulted in strongly increased HIF1α expression. IOX2 enhanced the flagellin-induced expression of the genes encoding the enzymes involved in glycolysis, which was associated with the intracellular accumulation of pyruvate. An untargeted pathway analysis of RNA sequencing data demonstrated the strong inhibitory effects of IOX2 toward key innate immune pathways related to cytokine and mitogen-activated kinase signaling cascades in flagellin-stimulated HBE cells. Likewise, the cell-cell junction organization pathway was amongst the top pathways downregulated by IOX2 in flagellin-stimulated HBE cells, which included the genes encoding claudins and cadherins. This IOX2 effect was corroborated by an impaired barrier function, as measured by dextran permeability. These results provide a first insight into the effects associated with HIF1α stabilization in the respiratory epithelium, suggesting that HIF1α impacts properties that are key to maintaining homeostasis upon stimulation with a relevant bacterial agonist.

Ferrostatin-1 and 3-Methyladenine Ameliorate Ferroptosis in OVA-Induced Asthma Model and in IL-13-Challenged BEAS-2B Cells.

Ferroptosis was reported to be involved in the occurrence and development of asthma. However, the potential mechanism underlying the role of ferroptosis in asthma remains unclear. In this study, we established the mouse asthma model following the ovalbumin (OVA) method in C57BL/6 mice and the cell model with IL-13 induction in bronchial epithelial cells (BEAS-2B cells). Treatment of ferrostatin-1 (Ferr-1) and 3-methyladenine (3-MA) decreased iron deposition in IL-13-induced BEAS-2B cells and lung tissues of asthma mice, opposite to that in bronchoalveolar lavage fluid (BALF). Meanwhile, excessive lipid peroxidation asthma model in vivo and in vitro was alleviated by Ferr-1 or 3-MA treatment. In addition, Ferr-1 and 3-MA inhibited the expression of LC-3 in these cells and lung tissues of mice. Moreover, Ferr-1 and 3-MA also suppressed the production of inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and oxidative stress factors (ROS and MDA), while promoting the level of SOD, in vivo and in vitro. Furthermore, application of Ferr-1 exhibited a greater inhibitory effect on iron release and lipid peroxidation in IL-13-induced BEAS-2B cells and asthma mice than 3-MA, accompanied with a weaker effect on ferritinophagy than 3-MA. Collectively, Ferr-1 and 3-MA ameliorated asthma in vivo and in vitro through inhibiting ferroptosis, providing a new strategy for the clinical treatment of asthma.

Methylprednisolone up-regulates annexin A1 (ANXA1) to inhibit the inflammation, apoptosis and oxidative stress of cigarette smoke extract (CSE)-induced bronchial epithelial cells, a chronic obstructive pulmonary disease in vitro model, through the formyl peptide receptor 2 (FPR2) receptors and the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway.

Chronic obstructive pulmonary disease (COPD) is a progressive degenerative disease, of which smoking is the main causer. We carried out this study with the aim of exploring the underlying mechanism of methylprednisolone (MP) treating the COPD. To stimulate COPD in vitro, cigarette smoke extract (CSE)was employed to induce human bronchial epithelial cells BEAS-2B. With the help of MTT and Tunel assays, the viability and apoptosis of BEAS-2B cells after indicated treatment were assessed. The levels of inflammatory response and oxidative stress were determined by the changes of markers basing on their commercial kits. Additionally, annexin A1 (ANXA1) expressions at both protein and mRNA levels were assessed with Western blot and Reverse transcription­quantitative PCR (RT-qPCR). Moreover, the expressions of apoptosis- and formyl peptide receptor 2 (FPR2) receptors and the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway-related proteins were determined with Western blot., related proteins and proteins. As a result, MP up-regulated the ANXA1 expression in CSE-induced BEAS-2B cells. MP enhanced the viability but suppressed the apoptosis, inflammatory response and oxidative stress of CSE-induced BEAS-2B cells via regulating FPR2/AMPK pathway, while ANXA1 knockdown exhibited oppositive effects on them. In conclusion, MP up-regulated ANXA1 to inhibit the inflammation, apoptosis and oxidative stress of BEAS-2B cells induced by CSE, alleviating COPD through suppressing the FPR2/AMPK pathway.

Nicotine promotes chronic obstructive pulmonary disease via inducing pyroptosis activation in bronchial epithelial cells.

Nicotine is one of the primary components in cigarettes, which is responsible for addiction. Numerous studies have investigated the effects of nicotine on pulmonary disease. The health of epithelial cells is important in the development of chronic obstructive pulmonary disease (COPD). Accumulating evidence has suggested that epithelial cell death may initiate or contribute to the progression of a number of lung diseases via airway remodeling. Pyroptosis is a unique form of inflammatory cell death mediated by the activation of caspase­1 and the NOD­like receptor protein­3 (NLRP3) inflammasome. The present study aimed to evaluate whether pyroptosis of epithelial cells was involved in the progression of COPD. The normal human bronchial epithelial cell line 16HBE was treated with 0.1 or 1 µM nicotine. Then the proliferation ability of 16HBE cells was detected by CCK­8. Cell death was detected by flow cytometry analysis and TUNEL assay. Subsequently, the levels of pro­caspase 1, caspase 1, IL­1ß, IL­18, NLRP3, ASC and cleaved GSDMD were examined by western blotting. It was revealed that nicotine treatment significantly induced cell death and suppressed proliferation of 16HBE cells. Furthermore, nicotine exposure increased the expression levels of caspase­1, IL­1ß, IL­18, NLRP3, apoptosis­associated speck­like protein and gasdermin D in 16HBE cells. Therefore, the present study concluded that nicotine treatment induced pyroptosis in 16HBE cells, which may be associated with the progression of COPD.

The impact of extractable organic matter from gasoline and alternative fuel emissions on bronchial cell models (BEAS-2B, MucilAir™).

Air pollution caused by road traffic has an unfavorable impact on the environment and also on human health. It has previously been shown, that complete gasoline emissions lead to toxic effects in cell models originating from human airways. Here we focused on extractable organic matter (EOM) from particulate matter, collected from gasoline emissions from fuels with different ethanol content. We performed cytotoxicity evaluation, quantification of mucin and extracellular reactive oxygen species (ROS) production, DNA breaks detection, and selected gene deregulation analysis, after one and five days of exposure of human bronchial epithelial model (BEAS-2B) and a 3D model of the human airway (MucilAir™). Our data suggest that the longer exposure had more pronounced effects on the parameters of cytotoxicity and mucin production, while the impacts on ROS generation and DNA integrity were limited. In both cell models the expression of CYP1A1 was induced, regardless of the exposure period or EOM tested. Several other genes, including FMO2, IL1A, or TNF, were deregulated depending on the exposure time. In conclusion, ethanol content in the fuels did not significantly impact the toxicity of EOM. Biological effects were mostly linked to xenobiotics metabolism and inflammatory response. BEAS-2B cells were more sensitive to the treatment.

Zero-preserving imputation of single-cell RNA-seq data.

A key challenge in analyzing single cell RNA-sequencing data is the large number of false zeros, where genes actually expressed in a given cell are incorrectly measured as unexpressed. We present a method based on low-rank matrix approximation which imputes these values while preserving biologically non-expressed genes (true biological zeros) at zero expression levels. We provide theoretical justification for this denoising approach and demonstrate its advantages relative to other methods on simulated and biological datasets.