Human Lung Alveolar Epithelial Cells Research Articles

Year-long and seasonal differences of PM2.5 chemical characteristics and their role in the viability of human lung epithelial cells (A549)

Fine particulate matters-PM2.5 in the air can have considerable negative effects on human health and the environment. Various human cell-based studies examined the effect of PM2.5 on human health in different cities of the world using various chemical parameters. Unfortunately, limited information is available regarding the relationship between toxicity and chemical characteristics of PM2.5 collected in Istanbul, Türkiye, located in one of the most populated cities in the world. To investigate the chemical characteristics and cytotoxicity of PM2.5 in Istanbul, samples were collected for 12 months, then potentially toxic metals, oxidative potential, and particle indicators (e.g., functional groups and elements) were determined, and the cytotoxicity of PM2.5 on human A549 lung alveolar epithelial cells was examined. The mean PM2.5 mass concentration was 24.0 ± 17.4 µg m−3 and higher in cold months compared to other seasons. Moreover, the results of the metals, elemental, and functional groups indicated that seasonal and monthly characteristics were influenced by the regional anthropogenic sources and photochemistry input. The cytotoxicity results also showed that the viability of A549 cells was reduced with the exposure of PM2.5 (30–53%) and higher cytotoxicity was obtained in summer compared to the other seasons due to the impact of the metals, elements, and oxidative characteristics of PM2.5.

10 Validation of an Aerosol Exposure-Air Liquid Interface (AE-ALI) System to Facilitate Realistic Hazard Identification of Nano-Sized Aerosols

Abstract To facilitate hazard characterisation for inhaled engineered nanomaterials we have established an aerosol exposure air-liquid-interface (AE-ALI) system, incorporating a Cultex RFSTM cell exposure system. While offering potential benefits in terms of more realistic aerosol exposure mode over traditional submerged in vitro models and being in-line with the principles of the 3Rs, (replace, reduce and refine animal use), detailed system characterisation is required to produce reliable and reproducible results. As such, this work looked to fully characterise our AE-ALI system with a view to optimising cell viability of system controls and deposition quantity, uniformity, and reproducibility for engineered nanomaterials. Using a nano-CeO2 aerosol, the effects of exposure conditions and system parameters (e.g. temperature, electrostatic precipitator voltage and airflow) on deposition efficiency, pattern and reproducibility were investigated using ICP-MS. While results showed that appropriate choice of operating parameters can produce broadly uniform deposition, reproducibility was low, and dose monitoring during exposures is recommended. After initial optimisation of the system for deposition, the effect of exposure duration, well size and flowrate on cellular responses (e.g. cytotoxicity (LDH) and selected gene expression (IL-8, CXCL1, HMOX1, SPP1) of system controls (H2O aerosol) was investigated using human lung alveolar (A549) and primary small airway epithelial cells. Results indicate that even after optimisation for cell health, the system itself is “toxic” to cells in comparison to incubator controls, with increasing effects observed with exposure duration meaning exposure durations must be minimised. Results from this study highlight the need for detailed characterisation of AE-ALI systems prior to use.

Lack of ZNF365 Drives Senescence and Exacerbates Experimental Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant activation of the alveolar epithelium, the expansion of the fibroblast population, and the accumulation of extracellular matrix. Global gene expression of human lung fibroblasts stimulated with TGFβ-1, a strong fibrotic mediator revealed the overexpression of ZNF365, a zinc finger protein implicated in cell cycle control and telomere stabilization. We evaluated the expression and localization of ZNF365 in IPF lungs and in the fibrotic response induced by bleomycin in WT and deficient mice of the orthologous gene Zfp365. In IPF, ZNF365 was overexpressed and localized in fibroblasts/myofibroblasts and alveolar epithelium. Bleomycin-induced lung fibrosis showed an upregulation of Zfp365 localized in lung epithelium and stromal cell populations. Zfp365 KO mice developed a significantly higher fibrotic response compared with WT mice by morphology and hydroxyproline content. Silencing ZNF365 in human lung fibroblasts and alveolar epithelial cells induced a significant reduction of growth rate and increased senescence markers, including Senescence Associated β Galactosidase activity, p53, p21, and the histone variant γH2AX. Our findings demonstrate that ZNF365 is upregulated in IPF and experimental lung fibrosis and suggest a protective role since its absence increases experimental lung fibrosis mechanistically associated with the induction of cell senescence.

Hazard identification of nanomaterials: In silico unraveling of descriptors for cytotoxicity and genotoxicity

Hazard identification and safety assessment of the huge variety of nanomaterials (NMs), calls for robust and validated toxicity screening tests in combination with cheminformatics approaches to identify factors that can drive toxicity. Cytotoxicity and genotoxicity of seventeen JRC repository NMs, derived from titanium dioxide, zinc oxide, silver and silica, were tested in vitro using human lung alveolar epithelial cells A549. Cytotoxicity was assessed with the AlamarBlue (AB) and colony forming efficiency (CFE) assays, and genotoxicity by the enzyme-linked version of the comet assay. Nanoparticle tracking analysis (NTA) was used to measure size of the NMs in stock and in cell culture medium at different time points. Categorization and ranking of cytotoxic and genotoxic potential were performed (EU-NanoREG2 project approach). Descriptors for prediction of NMs toxicity were identified by quantitative structure-activity relationship (QSAR) analysis. Our results showed that ZnO NMs (NM-110 and NM-111), and Ag NMs (NM-300K and NM-302) were cytotoxic, while the TiO2 and SiO2 NMs were non-cytotoxic. Regarding genotoxicity, TiO2 NM-100, ZnO NM-110, SiO2 NM-203 and Ag NM-300K were categorized as positive. Cheminformatics modeling identified electron properties and overall chemical reactivity as important descriptors for cytotoxic potential, HOMO-LUMO energy parameter, ionization potential, pristine size for the NMs´ genotoxic potential, and presence of surface coating as descriptor for induction of DNA oxidized base lesions.

Universal lung epithelium DNA methylation markers for detection of lung damage in liquid biopsies

Circulating biomarkers for lung damage are lacking. Lung epithelium-specific DNA methylation patterns can potentially report the presence of lung-derived cell-free DNA (cfDNA) in blood, as an indication of lung cell death. We sorted human lung alveolar and bronchial epithelial cells from surgical specimens, and obtained their methylomes using whole-genome bisulfite sequencing. We developed a PCR sequencing assay determining the methylation status of 17 loci with lung-specific methylation patterns, and used it to assess lung-derived cfDNA in the plasma of healthy volunteers and patients with lung disease. Loci that are uniquely unmethylated in alveolar or bronchial epithelial cells are enriched for enhancers controlling lung-specific genes. Methylation markers extracted from these methylomes revealed that normal lung cell turnover probably releases cfDNA into the air spaces, rather than to blood. People with advanced lung cancer show a massive elevation of lung cfDNA concentration in blood. Among individuals undergoing bronchoscopy, lung-derived cfDNA is observed in the plasma of those later diagnosed with lung cancer, and to a lesser extent in those diagnosed with other lung diseases. Lung cfDNA is also elevated in patients with acute exacerbation of COPD compared with patients with stable disease, and is associated with future exacerbation and mortality in these patients. Universal cfDNA methylation markers of normal lung epithelium allow for mutation-independent, sensitive and specific detection of lung-derived cfDNA, reporting on ongoing lung injury. Such markers can find broad utility in the study of normal and pathologic human lung dynamics.

KAP1 Positively Modulates Influenza A Virus Replication by Interacting with PB2 and NS1 Proteins in Human Lung Epithelial Cells.

Influenza virus only encodes a dozen of viral proteins, which need to use host machinery to complete the viral life cycle. Previously, KAP1 was identified as one host protein that potentially interacts with influenza viral proteins in HEK 293 cells. However, the role of KAP1 in influenza virus replication in human lung alveolar epithelial cells and the underlying mechanism remains unclear. In this study, we first generated KAP1 KO A549 cells by CRISPR/Cas9 gene editing. KAP1 deletion had no significant effect on the cell viability and lack of KAP1 expression significantly reduced the influenza A virus replication. Moreover, we demonstrated that KAP1 is involved in the influenza virus entry, transcription/replication of viral genome, and viral protein synthesis in human lung epithelial cells and confirmed that KAP1 interacted with PB2 and NS1 viral proteins during the virus infection. Further study showed that KAP1 inhibited the production of type I IFN and overexpression of KAP1 significantly reduced the IFN-β production. In addition, influenza virus infection induces the deSUMOylation and enhanced phosphorylation of KAP1. Our results suggested that KAP1 is required for the replication of influenza A virus and mediates the replication of influenza A virus by facilitating viral infectivity and synthesis of viral proteins, enhancing viral polymerase activity, and inhibiting the type I IFN production.

Complete Genome Sequences of Three Staphylococcus haemolyticus Strains Isolated from the Lung of a TGFβ1 Transgenic Mouse with Lung Fibrosis

ABSTRACTWe report here the complete genome sequences of three Staphylococcus haemolyticus strains isolated from a mouse fibrotic lung tissue and exhibiting proapoptotic activity on human lung alveolar epithelial cells. The genomes were obtained from a combination of Illumina MiSeq and Oxford Nanopore MinION sequencing.

Real-time chemical composition of ambient fine aerosols and related cytotoxic effects in human lung epithelial cells in an urban area

Particulate matter with aerodynamic diameters ≤1 μm (PM1) in the atmosphere, especially that which is emitted from anthropogenic sources, can induce considerable negative effects on the cardiopulmonary system. To investigate the chemical emission characteristics and organic sources in Yuen Long (Hong Kong), both offline and online approaches for PM1 samples were applied by filter-based samplers and a Quadrupole Aerosol Chemical Speciation Monitor (Q-ACSM), respectively. The toxicological effects on human A549 lung alveolar epithelial cells were investigated, and associations between cytotoxicity and organic sources and compositions were evaluated. The organics from the Q-ACSM measurement were the largest contributor to submicron aerosols in both seasons of our study, and the mass fraction was higher in winter (60%) than it was in autumn (46%). Regarding organic sources, the mass fraction of hydrocarbon-like organics (HOA) increased from 7% in autumn to 38% in winter, whereas cooking organics (COA) decreased from 30% in autumn to 18% in winter, and oxygenated organics (OOA) decreased from 63% to 45%. Organic compounds contributed more during pollution episodes, and more secondary ions were formed by means of the oxidation process. Oxidative and inflammatory responses in A549 cells were found with PM1 exposures; the differences in chemical compositions resulted in the higher cytotoxicity in winter than autumn. The cooking organic aerosol in residential area was significantly correlated with cell inflammation. Both elemental carbon and specific inorganic ions (SO42− and Mg2+) contributed to the intracellular cytotoxicity. This study demonstrated that specific atmospheric particulate matter chemical properties and sources can trigger distinct cell reactions; the inorganic ions from cooking emissions cannot be disregarded in terms of their pulmonary health risks in residential areas.

Bioreactivity of a novel poly(epsilon-caprolactone) nanocapsule containing atrazine with human lung alveolar epithelial cells

Nanoencapsulation of pesticide atrazine improves efficacy. However, this study discovered adverse bioreactivity in alveolar type 1 epithelial cells (a major recipient of inhaled nano-sized particles), following exposure to nanoencapsulated atrazine.

A Senescence Bystander Effect in Human Lung Fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterised by a dense fibrosing of the lung parenchyma. An association between IPF and cellular senescence is well established and several studies now describe a higher abundance of senescent fibroblasts and epithelial cells in the lungs of IPF patients compared with age-matched controls. The cause of this abnormal accumulation of senescent cells is unknown but evidence suggests that, once established, senescence can be transferred from senescent to non-senescent cells. In this study, we investigated whether senescent human lung fibroblasts (LFs) and alveolar epithelial cells (AECs) could induce a senescent-like phenotype in “naïve” non-senescent LFs in vitro. Primary cultures of LFs from adult control donors (Ctrl-LFs) with a low baseline of senescence were exposed to conditioned medium (CM) from: (i) Ctrl-LFs induced to become senescent using H2O2 or etoposide; (ii) LFs derived from IPF patients (IPF-LFs) with a high baseline of senescence; or (iii) senescence-induced A549 cells, an AEC line. Additionally, ratios of non-senescent Ctrl-LFs and senescence-induced Ctrl-LFs (100:0, 0:100, 50:50, 90:10, 99:1) were co-cultured and their effect on induction of senescence measured. We demonstrated that exposure of naïve non-senescent Ctrl-LFs to CM from senescence-induced Ctrl-LFs and AECs and IPF-LFs increased the markers of senescence including nuclear localisation of phosphorylated-H2A histone family member X (H2AXγ) and expression of p21, IL-6 and IL-8 in Ctrl-LFs. Additionally, co-cultures of non-senescent and senescence-induced Ctrl-LFs induced a senescent-like phenotype in the non-senescent cells. These data suggest that the phenomenon of “senescence-induced senescence” can occur in vitro in primary cultures of human LFs, and provides a possible explanation for the abnormal abundance of senescent cells in the lungs of IPF patients.

Salidroside Suppresses the Proliferation and Migration of Human Lung Cancer Cells through AMPK-Dependent NLRP3 Inflammasome Regulation.

Inflammatory reactions mediated by the NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome contributes to non-small-cell lung cancer (NSCLC) progression, particularly in patients with bacterial infections. Salidroside (SAL) has recently been shown to suppress lipopolysaccharide- (LPS-) induced NSCLC proliferation and migration, but its mechanism of action remains unclear. It has been shown that SAL improves metabolic inflammation in diabetic rodents through AMP-activated protein kinase- (AMPK-) dependent inhibition of the NLRP3 inflammasome. However, whether the NLRP3 inflammasome is regulated by SAL in NSCLC cells and how its underlying mechanism(s) can be determined require clarification. In this study, human lung alveolar basal carcinoma epithelial (A549) cells were treated with LPS, and the effects of SAL on cell proliferation, migration, AMPK activity, reactive oxygen species (ROS) production, and NLRP3 inflammasome activation were investigated. We found that LPS induction increases the proliferation and migration of A549 cells which was suppressed by SAL. Moreover, SAL protected A549 cells against LPS-induced AMPK inhibition, ROS production, and NLRP3 inflammasome activation. Blocking AMPK using Compound C almost completely suppressed the beneficial effects of SAL. In summary, these results indicate that SAL suppresses the proliferation and migration of human lung cancer cells through AMPK-dependent NLRP3 inflammasome regulation.

Pseudomonas aeruginosa induces spatio-temporal secretion of IL-1β, TNFα, proMMP-9, and reduction of epithelial E-cadherin in human alveolar epithelial type II (A549) cells.

Pseudomonas aeruginosa, is an opportunistic bacterium with a high prevalence in diverse pulmonary infections. Although several genes are involved in the system of resistance and evasion of the immunological response of the host, little is known about the inflammatory, degradative, and cell-binding response induced by P. aeruginosa in human lung alveolar epithelial cells. The purpose of this study was to determine the cytokine expression (IL-1β and TNFα), pro matrix metalloproteinases activation (proMMP-2 and proMMP-9), and the effects on the cell-binding adhesion protein (E-cadherin) in an in vitro model of human lung alveolar epithelial cells. A549 cells were stimulated with a different number of colony-forming units of P. aeruginosa for 3, 6, and 24 hours. Subsequently, the culture medium was collected, IL-1β and TNFα levels were evaluated by ELISA; proMMP-2 and -9 levels were determined by substrate gel zymography, and the MMP-9 and E-cadherin assessed by immunostaining of A549 cells. Our results demonstrated that P. aeruginosa induces mainly the secretion of TNFα, increases actMMP-9 level, and significantly reduces the level of E-cadherin in the A549 cells. In summary, the inflammatory/degradative process induced by P. aeruginosa modulates the expression of the E-cadherin protein. The probable clinical implications of this study suggest the use of inhibitors that reduce the degradative activity of proMMP-9 which will be further explored in the next phase of this study.

The Impact of Angiotensin Converting Enzyme Inhibition and/or Angiotensin Receptor Blockade on Tissue Expression of the SARS‐CoV‐2 Receptor ACE2 in Mice

The angiotensin‐converting enzyme 2 (ACE2) is an established receptor and entry point for the novel SARS‐CoV‐2 coronavirus, as the spike protein on the viral envelope binds the ACE2 receptor. Published histological profiling revealed ACE2 to be highly expressed on human lung alveolar epithelial cells and on enterocytes of the small intestine, as well as on arterial and venous endothelial cells. Given the widespread abundance of ACE2 across tissues, there has been much speculation regarding whether ACE inhibitors (ACEi) and/or Angiotensin Receptor Blockers (ARB) might alter tissue ACE2 expression and thereby change the risk of transmission or development of severe complications. Therefore, the aim of this ongoing study is to measure the impact of ACEi and ARB singly and in combination versus placebo control on tissue distribution of ACE2 in male and female mice.A factorial design was employed with the following exposures: ACEi (lisinopril, 10 mg/kg/day), ARB (losartan, 10 mg/kg/day), both drugs, or water control. Drugs were delivered via drinking water. C57BL/6J Mice were 8 weeks of age at the start of the experiment. After 21 days of treatment, four mice (two males and two females) from each treatment group were euthanized, followed by perfusion and tissue collection. 12 more untreated mice were combined with the placebo group to increase the sample size of this reference group to 16 (8 males, 8 females). ACE2 protein abundance in brain, kidney, liver, lung, and small intestine was measured by ELISA (Abcam) and normalized to BCA‐measured total protein. Two‐way ANOVA was performed to test for differences in ACE2 levels by organ or sex among untreated mice; Mann‐Whitney tests were performed to compare each drug treatment group to the control group.Among untreated mice, ACE2 levels differed by organ (p < 0.01), but there were no differences between males and females for a given organ (p = 0.20). Males and females were combined for subsequent analyses. After 21 days of treatment, ACEi, ARB, and the combination therapy lowered ACE2 tissue abundance compared to untreated mice in the brain (ACEi p = 0.04; ARB p = 0.04; ACEi+ARB p = 0.01), kidney (ACEi p = 0.03; ARB p = 0.02; ACEi+ARB p = 0.02), and lung (ACEi p = 0.01; ARB p = 0.01; ACEi+ARB p = 0.01). ACEi, but not ARB or combination therapy, lowered ACE2 tissue abundance in the liver (p = 0.02). None of the treatments changed ACE2 tissue abundance in the small intestine.These results indicate that ACEi, ARB, or combination therapy reduces levels of ACE2, the SARS‐CoV‐2 receptor, in key tissues affected by the virus, including lung, kidney, and brain. An ongoing validation study will evaluate ACE2 protein and gene expression at earlier time points, as well as after discontinuation of treatment. These results will provide valuable insight into the risk or potential benefit of starting or stopping ACEi, ARB, or combination therapy during the COVID‐19 pandemic.

Regulation of ACE-2 enzyme by hyperoxia in lung epithelial cells by post-translational modification

Bronchopulmonary Dysplasia (BPD) occurs in premature neonates with respiratory distress who require supplemental oxygen in the first days after birth. BPD involves uniform arrest of alveolar development and variable interstitial cellularity and/or fibroproliferation. Previous studies by our lab showed that the enzyme, angiotensin converting enzyme-2 (ACE-2) and its product Ang1-7 exerting action on the receptor Mas oncogene in what is known as ACE-2/Mas axis is protective to lung cells. We also showed that ACE-2 is expressed in fetal human lung fibroblasts but is significantly decreased by hyperoxic gas lung injury, an effect caused by ACE-2 enzyme shedding mediated by TNF-alpha-converting enzyme (TACE/ADAM17). However, no reports yet exist about the regulation of ACE-2 in the alveolar epithelia in hyperoxic lung injury. In this study we aim to define the effects of hyperoxic lung injury on the protective ACE-2 enzyme in the human lung alveolar epithelial cell line A549. Cultured A549 cells were exposed to hyperoxia (95% O2) or normoxia (21% O2) for 3 or 7 days in serum-free nutrient media. Cells were lysed and culture media were collected to test for cellular ACE-2 enzymatic activity and for ACE-2, Mas receptor, TACE/ADAM17, and ubiquitin proteins abundance by immunoblotting. Cells were harvested in Trizol for RNA extraction and ACE-2 qRT-PCR. Whole cell extracts of A549 cell line was used for ACE-2 immunoprecipitation and subsequent ubiquitin immunoblotting. Total ubiquitinated proteins were increased by hyperoxia treatment, while ACE-2 and Mas receptor proteins abundance and ACE-2 enzymatic activity were decreased significantly in A549 cells exposed to hyperoxia relative to the normoxia controls. The percent decrease in ACE-2 activity corresponded with increased time of hyperoxic gas exposure. However, in contrast to our data from lung fibroblasts, no significant change was noted in ACE-2 protein released into the media or in ACE-2 mRNA levels by the hyperoxic treatment. Ubiquitin immunoreactive bands were detectable in the ACE-2 immunoprecipitate. These data suggest that hyperoxic exposure of the lung epithelial cells decreases the protective enzyme ACE-2 by cell type specific mechanisms independent of shedding by TACE/ADAM17. The data also suggest a regulatory level of ACE-2 downstream of transcription may involve ACE-2 ubiquitination and targeting for degradation.

Akt-independent effects of triciribine on ACE2 expression in human lung epithelial cells: Potential benefits in restricting SARS-CoV2 infection.

The severe acute respiratory syndrome coronavirus 2 that causes coronavirus disease 2019 (COVID‐19) binds to the angiotensin‐converting enzyme 2 (ACE2) to gain cellular entry. Akt inhibitor triciribine (TCBN) has demonstrated promising results in promoting recovery from advanced‐stage acute lung injury in preclinical studies. In the current study, we tested the direct effect of TCBN on ACE2 expression in human bronchial (H441) and lung alveolar (A549) epithelial cells. Treatment with TCBN resulted in the downregulation of both messenger RNA and protein levels of ACE2 in A549 cells. Since HMGB1 plays a vital role in the inflammatory response in COVID‐19, and because hyperglycemia has been linked to increased COVID‐19 infections, we determined if HMGB1 and hyperglycemia have any effect on ACE2 expression in lung epithelial cells and whether TCBN has any effect on reversing HMGB1‐ and hyperglycemia‐induced ACE2 expression. We observed increased ACE2 expression with both HMGB1 and hyperglycemia treatment in A549 as well as H441 cells, which were blunted by TCBN treatment. Our findings from this study, combined with our previous reports on the potential benefits of TCBN in the treatment of acute lung injury, generate reasonable optimism on the potential utility of TCBN in the therapeutic management of patients with COVID‐19.

Inhibition of inflammation-induced injury and cell migration by coelonin and militarine in PM2.5-exposed human lung alveolar epithelial A549 cells

Accumulating studies suggest that fine particulate matter (PM2.5) pollutants in the air are easily enter into alveoli and even the bloodstream, resulting in an inflammatory response that not only triggers respiratory disorders but also causes permanent damage to various organs. Recent findings suggest that coelonin and militarine enriched in orchids can inhibit inflammation-induced injury against respiratory diseases. Here, we evaluated the anti-inflammatory properties of coelonin and militarine and examined their underlying molecular mechanisms in A549 cells exposed to PM2.5. PM2.5 induced significant intracellular reactive oxidative stress accumulation at a concentration of 250 μg/ml, as determined using the dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence assay. Cell viability was assessed via the MTS assay to determine the concentrations of compounds appropriate for use in subsequent experiments. Data from the enzyme-linked immunosorbent assay (ELISA) showed that both coelonin (10 and 20 μg/ml) and militarine (5 and 10 μg/ml) mitigated PM2.5-induced inflammation by reducing the generation of inflammatory factors, including interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Quantitative real-time PCR (qRT-PCR) analysis revealed a remarkable decrease in IL-6, TNF-α, cyclooxygenase-2 (COX-2) and interleukin-1β (IL-1β) mRNA levels in the coelonin and militarine-pretreated groups. In Western blot analysis, expression of inhibitor of NF-κB (IκBα) protein in the coelonin and militarine pretreatment groups was significantly increased compared with the PM2.5 (only) treatment group (P < 0.05), concomitant with a significant decrease in phospho-IκB kinase β/IκB kinase β (p-IKKβ/IKKβ), phospho-nuclear factor of kappa B p65/nuclear factor of kappa B p65 (p–NF–κBp65/NF-κBp65) and COX-2 proteins (P < 0.05). Both coelonin and militarine inhibited migration and inflammation by suppressing PM2.5-induced IKK phosphorylation, and followed by IκBα protein degradation and NF-κB activation. Our collective data strongly supported the utility of coelonin and militarine as novel sources for development of treatments for PM2.5-induced lung diseases.

Characterization of organic aerosols in PM1 and their cytotoxicity in an urban roadside area in Hong Kong

Organic compounds in fine particles play major roles in cardiopulmonary diseases. A study was conducted to determine the characteristics and cytotoxicity of organic aerosols (OA) in an urban roadside area in Hong Kong. Chemical components in nonrefractory submicron aerosol (NR-PM1) were observed using a Quadrupole Aerosol Chemical Speciation Monitor (Q-ACSM), and the chemical profile of organic compounds in NR-PM1 was examined with filter-based approach. Associations between cytotoxicity and organic sources and compositions were evaluated. NR-PM1 contributed to 84% of the PM1 concentrations. The NR-PM1 was composed of organics (55 ± 15%), followed by sulfate (21 ± 9%), ammonium (13 ± 6%), nitrate (10 ± 6%) and chloride (1 ± 1%). Three major organic sources were identified using positive matrix factorization, namely primary organic aerosol (POA, 40 ± 19%), more-oxidized oxygenated OA (MO-OOA, 32 ± 22%) and less-oxidized oxygenated OA (LO-OOA, 28 ± 19%). Variations in organic groups, including alkanes, hopanes, steranes, polycyclic aromatic hydrocarbons (PAHs), oxy-PAHs (OPAHs), and fatty acids, demonstrated that traffic and cooking emissions were dominant pollution sources in this roadside station. Human lung alveolar epithelial (A549) cells were exposed to PM1, revealing increases in lactate dehydrogenase (LDH), reactive oxygen species (ROS), and interlukin-6 (IL-6), which indicated the occurrence of inflammatory and oxidative responses. POA was significantly associated with ROS and IL-6, and alkanes, hopanes, steranes, PAHs and OPAHs, and fatty acids presented medium to high correlations with LDH and IL-6, demonstrating the importance of primary emissions and organic compounds in cytotoxicity. This study demonstrated that organic compounds emitted from traffic and cooking play critical roles in PM-induced oxidative stress and inflammation in urban areas.

Assessment of transporter-mediated efflux of nintedanib using in vitro cell line models of idiopathic pulmonary fibrosis.

Objective: We aimed to investigate the involvement of efflux transporters, including multidrug resistant protein 1 (MDR1), multidrug resistance-associated protein 1 (MRP1), MRP2, and breast cancer resistance protein (BCRP), in the intracellular accumulation of the antifibrotic agent nintedanib in fibrotic lung cells. Methods: We used transforming growth factor-β1 (TGF-β1)-treated human lung fibroblasts (WI-38) and alveolar epithelial cells (A549) as in vitro models. The expression and activities of efflux transporters in TGF-β1-treated WI-38 and A549 cells were evaluated using immunoblotting and flow cytometry. Cells were treated with nintedanib and then incubated with inhibitors of these transporters. The intracellular concentration of nintedanib was determined. Results: MDR1, MRP1, MRP2, and BCRP were found to be expressed in WI-38 and A549 cells with or without TGF-β1 stimulation, with the exception of MRP2 in WI-38 cells. The efflux activities of these transporters were observed in these cells. MDR1 inhibitors significantly increased the intracellular accumulation of nintedanib, whereas MRP inhibitors did not show an effect. The BCRP inhibitor significantly increased the transporter activity in A549 cells but not in WI-38 cells. Conclusion: This study suggests that the efflux via MDR1 and BCRP is involved in the intracellular accumulation of nintedanib in fibrotic lung cells.

Time-resolved mRNA and miRNA expression profiling reveals crucial coregulation of molecular pathways involved in epithelial-pneumococcal interactions.

Streptococcus pneumoniae is a major causative agent of pneumonia worldwide and its complex interaction with the lung epithelium has not been thoroughly characterized. In this study, we exploited both RNA‐sequencing and microRNA (miRNA)‐sequencing approaches to monitor the transcriptional changes in human lung alveolar epithelial cells infected by S. pneumoniae in a time‐resolved manner. A total of 1330 differentially expressed (DE) genes and 45 DE miRNAs were identified in all comparisons during the infection process. Clustering analysis showed that all DE genes were grouped into six clusters, several of which were primarily involved in inflammatory or immune responses. In addition, target gene enrichment analyses identified 11 transcription factors that were predicted to link at least one of four clusters, revealing transcriptional coregulation of multiple processes or pathways by common transcription factors. Notably, pharmacological treatment suggested that phosphorylation of p65 is important for optimal transcriptional regulation of target genes in epithelial cells exposed to pathogens. Furthermore, network‐based clustering analysis separated the DE genes negatively regulated by DE miRNAs into two functional modules (M1 and M2), with an enrichment in immune responses and apoptotic signaling pathways for M1. Integrated network analyses of potential regulatory interactions in M1 revealed that multiple DE genes related to immunity and apoptosis were regulated by multiple miRNAs, indicating the coordinated regulation of multiple genes by multiple miRNAs. In conclusion, time‐series expression profiling of messenger RNA and miRNA provides a wealth of information for global transcriptional changes, and offers comprehensive insight into the molecular mechanisms underlying host–pathogen interactions.

ZNF365 is overexpressed in Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease of unknown etiology characterized by the expansion of fibroblast population and excessive extracellular matrix (ECM) deposition. Transforming growth factor‐beta 1 (TGF‐β1) is one of the most potent mediators of the fibrotic response and induces fibroblast proliferation, and their differentiation to myofibroblasts, cells characterized by increased contractility, apoptosis resistance and the production of multiple ECM components (mainly collagens).Global gene expression of human lung fibroblasts stimulated with TGFβ performed in our laboratory demonstrated the overexpression of ZNF365, a zinc finger protein that belongs to the C2H2 domain family that has been poorly described in the literature. The purpose of this study is to evaluate ZNF365 expression in IPF lung tissue, the expression of its ortholog Zfp365 in mice lung tissue and its functional effects on human lung fibroblasts and epithelial cells in vitro.Expression of ZNF365 and Zfp365 were evaluated by qPCR and WB in human and mice samples, as well as in fibroblasts and epithelial cells stimulated with TGFβ. Localization of the protein was performed by immunohistochemistry in IPF lungs and in bleomycin‐induced lung fibrosis in mice. For silencing experiments, specific siRNAs and scrambled controls designed for ZNF365 were used. For overexpression, cells were stably transfected with ZNF365. Growth rate and proliferation were analyzed using WST‐1 and CyQUANT reagent, and apoptosis was measured by flow cytometry.We found that ZNF365 is overexpressed in IPF lung tissues and localized in alveolar and bronchial epithelial cells and in fibroblasts/myofibroblasts. The orthologous Zfp365 is overexpressed in lung tissue from bleomycin instilled mice, and the immunoreactive protein was observed in alveolar and airway epithelium. In vitro studies confirmed that ZNF365 is overexpressed in control and IPF fibroblasts stimulated with TGFβ. Likewise, TGFβ induced the upregulation of ZNF365 in a human alveolar epithelial cell line. Functional assays revealed that ZNF365 silencing in human lung fibroblasts and alveolar epithelial cells induced a significant reduction of growth rate and proliferation compared with the scrambled and WT controls. Preliminary data show that silencing of this protein increases senescence‐asociated β‐galactosidase activity and increases expression of the histone variant γH2A.X, another senescence marker. These results demonstrate that ZNF365 is upregulated in IPF and experimental lung fibrosis and may be involved in the regulation of proliferation and senescence of human lung cells.Support or Funding InformationSupported by PAPIIT 201520