Human Tracheal Epithelial Cells Research Articles

Translational 3D-Cell Culture Model to Assess Hyperoxia Effects on Human Neonatal Airway Epithelial Cells.

The preterm neonatal airway epithelium is constantly exposed to environmental stressors. One of these stressors in neonates with lung disease includes oxygen (O2) tension higher than the ambient atmosphere - termed hyperoxia (>21% O2). The effect of hyperoxia on the airway depends on various factors, including the developmental stage of the airway, the degree of hyperoxia, and the duration of exposure, with variable exposures potentially leading to unique phenotypes. While there has been extensive research on the effect of hyperoxia on neonatal lung alveolarization and airway hyperreactivity, little is known about the short and long-term underlying effect of hyperoxia on human neonatal airway epithelial cells. A major reason for this is the scarcity of an effective in vitro model to study human neonatal airway epithelial development and function. Here, we describe a method for isolating and expanding human neonatal tracheal airway epithelial cells (nTAECs) utilizing human neonatal tracheal aspirates and culturing these cells in air-liquid interface (ALI) culture. We demonstrate that nTAECs form a mature polarized cell-monolayer in ALI culture and undergo mucociliary differentiation. We also present a method for moderate hyperoxia exposure of the cell monolayer in ALI culture using a specialized incubator. Additionally, we describe an assay to measure cellular oxidative stress following hyperoxia exposure in ALI culture using fluorescent quantification, which confirms that moderate hyperoxia exposure induces cellular oxidative stress but does not cause significant cell membrane damage or apoptosis. This model can potentially be used to simulate clinically relevant hyperoxia exposure encountered by neonatal airways in the Neonatal Intensive Care Unit (NICU) and used to study the short and long-lasting effects of O2 on neonatal airway epithelial programming. Studies using this model could be utilized to explore ways to mitigate early-life oxidative injury to developing airways, which is implicated in the development of long-term airway diseases in former premature infants.

Extracellular and intracellular antiviral effects of ultraviolet A against severe acute respiratory syndrome coronavirus-2 are variant-independent

Under controlled settings, narrow-band ultraviolet A (UVA) exposure exerts antiviral effects both in vivo and in vitro. The effect is thought to be mediated via direct effect on viral particles and indirectly, by modulation of metabolic pathways of host cells. We aimed to explore the extracellular and intracellular antiviral effects of UVA exposure against Alpha, Beta, and Delta variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). MethodsVero E6 kidney normal epithelial cells and human tracheal epithelial cells were infected with Alpha, Beta, and Delta variants in a BSL-3 laboratory. To assess extracellular effects, SARS-CoV-2 variants were directly exposed to a single dose of UVA prior to infection of the host cells (Vero E6 kidney normal epithelial cells and human tracheal epithelial cells) The intracellular effects of UVA were assessed by first infecting the cells with SARS-CoV-2 variants followed by UVA treatment of infected cell monolayers. Efficacy was quantified by both plaque reduction assay and quantitative real-time polymerase chain reaction. Additionally, transcriptomic analysis was performed on exposed Vero E6 cells to assess differentially expressed genes and canonical pathways as compared to controls. ResultsSARS-CoV-2 Alpha, Beta and Delta variants are susceptible to UVA exposure prior to infection of Vero E6 cells. Importantly, the UVA-driven reduction in Delta variant load could be reproduced in human primary tracheal cells. Beta and Delta variants load also significantly decreased during Vero E6 cells intracellular experiments. UVA-driven reductions in viral loads ameliorate several host metabolic pathways, including canonical pathways related to viral infection and interferon signaling. ConclusionNarrow-band UVA exhibits both extracellular effects on SARS-CoV-2 viral particles and intracellular effects on infected cells with SARS-CoV-2. Efficacy appears to be variant independent.

SARS-CoV-2 particles promote airway epithelial differentiation and ciliation.

Introduction: The Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), which caused the coronavirus disease 2019 (COVID-19) pandemic, enters the human body via the epithelial cells of the airway tract. To trap and eject pathogens, the airway epithelium is composed of ciliated and secretory cells that produce mucus which is expelled through a process called mucociliary clearance. Methods: This study examines the early stages of contact between SARS-CoV-2 particles and the respiratory epithelium, utilizing 3D airway tri-culture models exposed to ultraviolet light-irradiated virus particles. These cultures are composed of human endothelial cells and human tracheal mesenchymal cells in a fibrin hydrogel matrix covered by mucociliated human tracheal epithelial cells. Results: We found that SARS-CoV-2 particles trigger a significant increase in ciliation on the epithelial surface instructed through a differentiation of club cells and basal stem cells. The contact with SARS-CoV-2 particles also provoked a loss of cell-cell tight junctions and impaired the barrier integrity. Further immunofluorescence analyses revealed an increase in FOXJ1 expression and PAK1/2 phosphorylation associated with particle-induced ciliation. Discussion: An understanding of epithelial responses to virus particles may be instrumental to prevent or treat respiratory infectious diseases such as COVID-19.

Mulberroside F improves airway hyperresponsiveness and inflammation in asthmatic mice.

Mulberroside F is isolated from the leaves and roots of Morus alba L. Here, we investigated whether mulberroside F could alleviate airway inflammation and eosinophil infiltration in the lungs of asthmatic mice. We also examined whether mulberroside F attenuated inflammatory responses in human tracheal epithelial BEAS-2B cells. Female BALB/c mice were sensitized and challenged with ovalbumin (OVA), and administered different doses of mulberroside F via intraperitoneal injection. Additionally, tumor necrosis factor (TNF)-α-stimulated BEAS-2B cells were treated with various doses of mulberroside F, followed by detection of the expressions of inflammatory cytokines and chemokines. The results demonstrated that mulberroside F mitigated the levels of proinflammatory cytokines and chemokines, and CCL11, in inflammatory BEAS-2B cells. Mulberroside F also suppressed reactive oxygen species (ROS) production and ICAM-1 expression in TNF-α-stimulated BEAS-2B cells, which effectively suppressed monocyte cell adherence. In an animal model of asthma, mulberroside F treatment attenuated airway hyperresponsiveness, eosinophil infiltration, and goblet cell hyperplasia. Mulberroside F treatment also decreased lung fibrosis and airway inflammation in OVA-sensitized mice. Moreover, mulberroside F significantly reduced expressions of Th2-associated cytokines (including interleukin(IL)-4, IL-5, and IL-13) in bronchoalveolar lavage fluid compared to OVA-sensitized mice. Our results confirmed that mulberroside F is a novel bioactive compound that can effectively reduce airway inflammation and eosinophil infiltration in asthmatic mice via inhibition of Th2-cell activation.

Validation of airway porcine epithelial cells as an alternative to human in vitro preclinical studies

Animal models are currently used in several fields of biomedical research as useful alternatives to human-based studies. However, the obtained results do not always effectively translate into clinical applications, due to interspecies anatomical and physiological differences. Detailed comparability studies are therefore required to verify whether the selected animal species could be a representative model for the disease or for cellular process under investigation. This has proven to be fundamental to obtaining reliable data from preclinical studies. Among the different species, swine is deemed an excellent animal model in many fields of biological research, and has been largely used in respiratory medicine, considering the high homology between human and swine airways. In the context of in vitro studies, the validation of porcine airway epithelial cells as an alternative to human epithelial cells is crucial. In this paper, porcine and human tracheal and bronchial epithelial cells are compared in terms of in vivo tissue architecture and in vitro cell behaviour under standard and airlifted conditions, analyzing the regenerative, proliferative and differentiative potentials of these cells. We report multiple analogies between the two species, validating the employment of porcine airway epithelial cells for most in vitro preclinical studies, although with some limitations due to species-related divergences.

A fixed 20:1 combination of cafedrine/theodrenaline increases cytosolic Ca2+ concentration in human tracheal epithelial cells via ryanodine receptor-mediated Ca2+ release

Mucociliary clearance is a pivotal physiological mechanism that protects the lung by cleaning the airways from pollution and colonization, thereby preventing infection. Ciliary function is influenced by various signal transduction cascades, and Ca2+ represents a key second messenger. A fixed 20:1 combination of cafedrine and theodrenaline has been widely used to treat perioperative hypotension and emergency hypotensive states since the 1960s; however, its effect on the intracellular Ca2+ concentration ([Ca2+]i) of respiratory epithelium remains unknown. Therefore, human tracheal epithelial cells were exposed to the clinically applied 20:1 mixture of cafedrine/theodrenaline and the individual substances separately. [Ca2+]i was assessed by FURA-2 340/380 fluorescence ratio. Pharmacological inhibitors were applied to elucidate relevant signal transduction cascades, and reverse transcription polymerase chain reaction (RT-PCR) was performed on murine tracheal epithelium to analyze ryanodine receptor (RyR) subtype expression. All three pharmacological preparations instantaneously induced a steep increase in [Ca2+]i that quickly returned to its baseline value despite the persistence of each substance. Peak [Ca2+]i following the administration of 20:1 cafedrine/theodrenaline, cafedrine alone, and theodrenaline alone increased in a dose-dependent manner, with median effective concentrations of 0.35 mM (7.32 mM cafedrine and 0.35 mM theodrenaline), 3.14 mM, and 3.45 mM, respectively. When extracellular Ca2+ influx was inhibited using a Ca2+-free buffer solution, the peak [Ca2+]i following the administration of cafedrine alone and theodrenaline alone were reduced but not abolished. No alteration in [Ca2+]i compared with baseline [Ca2+]i was observed during β-adrenergic receptor inhibition. Depletion of caffeine-sensitive stores and inhibition of RyR, but not IP3 receptors, completely abolished any increase in [Ca2+]i. However, [Ca2+]i still increased following the depletion of mitochondrial Ca2+ stores using 2,4-dinitrophenol. RT-PCR revealed RyR-2 and RyR-3 expression on murine tracheal epithelium. Although our experiments showed that cafedrine/theodrenaline, cafedrine alone, or theodrenaline alone release Ca2+ from intracellular stores through mechanisms that are exclusively triggered by β-adrenergic receptor stimulation, which most probably lead to RyR activation, clinical plasma concentrations are considerably lower than those used in our experiments to elicit an increase in [Ca2+]i; therefore, further studies are needed to evaluate the ability of cafedrine/theodrenaline to alter mucociliary clearance in clinical practice.

The dependence of particle size on cell toxicity for modern mining dust

Progressive massive pulmonary fibrosis among coal miners has unexpectedly increased. It would likely due to the greater generation of smaller rock and coal particles produced by powerful equipment used in modern mines. There is limited understanding of the relationship between micro- or nanoparticles with pulmonary toxicity. This study aims to determine whether the size and chemical characteristics of typical coal-mining dust contribute to cellular toxicity. Size range, surface features, morphology, and elemental composition of coal and rock dust from modern mines were characterized. Human macrophages and bronchial tracheal epithelial cells were exposed to mining dust of three sub- micrometer and micrometer size ranges at varying concentrations, then assessed for cell viability and inflammatory cytokine expression. Coal had smaller hydrodynamic size (180–3000 nm) compared to rock (495–2160 nm) in their separated size fractions, more hydrophobicity, less surface charge, and consisted of more known toxic trace elements (Si, Pt, Fe, Al, Co). Larger particle size had a negative association with in-vitro toxicity in macrophages (p < 0.05). Fine particle fraction, approximately 200 nm for coal and 500 nm for rock particles, explicitly induced stronger inflammatory reactions than their coarser counterparts. Future work will study additional toxicity endpoints to further elucidate the molecular mechanism causing pulmonary toxicity and determine a dose–response curve.

Wilforol A inhibits human glioma cell proliferation and deactivates the PI3K/AKT signaling pathway.

To study the anti-proliferation activity of wilforol A against glioma cells and its possible molecular mechanisms. Human glioma cell lines U118 MG and A172, human tracheal epithelial cells (TECs) and astrocytes (HAs) were exposed to various concentrations of wilforol A and evaluated for viability, apoptosis, and levels of proteins using WST-8 assay, flow cytometry and Western blot analysis, respectively. Wilforol A inhibited the growth of U118 MG and A172 cells, but not TECs and HAs, in a concentration-dependent manner and the estimated IC50 were 6 to 11 μM after 4 h-exposure. Apoptosis was induced at an apoptotic rate of about 40% at 100 μM in U118 MG and A172 cells, but the rates were less than 3% in TECs and HAs. Co-exposure to caspase inhibitor Z-VAD-fmk significantly reduced wilforol A-induced apoptosis. Wilforol A treatment also reduced the colony formation ability of U118 MG cells and triggered a significant increase in ROS production. Elevated levelsof pro-apoptotic proteins p53, Bax and cleaved caspase 3 and reduced level of the anti-apoptotic protein Bcl-2 were observed in glioma cells exposed to wilforol A. The expression of PI3K and p-Akt genes in the PI3K/AKT pathways were significantly downregulated in glioma cellstreated with wilforol A. Wilforol A inhibits the growth of glioma cells, reduces the levels of proteins in the P13K/Akt signal transduction pathways and increases the levels of pro-apoptotic proteins.

IL-11 drives the phenotypic transformation of tracheal epithelial cells and fibroblasts to enhance abnormal repair after tracheal injury

Tracheal stenosis (TS) is a multifactorial and heterogeneous disease that can easily lead to respiratory failure and even death. Interleukin-11 (IL-11) has recently received increased attention as a fibrogenic factor, but its function in TS is uncertain. This study aimed to investigate the role of IL-11 in TS regulation based on clinical samples from patients with TS and a rat model of TS produced by nylon brush scraping. Using lentiviral vectors expressing shRNA (lentivirus-shRNA) targeting the IL-11 receptor (IL-11Rα), we lowered IL-11Rα levels in the rat trachea. Histological and immunostaining methods were used to evaluate the effects of IL-11Rα knockdown on tracheal injury, molecular phenotype, and fibrosis in TS rats. We show that IL-11 was significantly elevated in circulating serum and granulation tissue in patients with TS. In vitro, TGFβ1 dose-dependently stimulated IL-11 secretion from human tracheal epithelial cells (Beas-2b) and primary rat tracheal fibroblasts (PRTF). IL-11 transformed the epithelial cell phenotype to the mesenchymal cell phenotype by activating the β-catenin pathway. Furthermore, IL-11 activated the atypical ERK signaling pathway, stimulated fibroblasts proliferation, and transformed fibroblasts into alpha-smooth muscle actin (α-SMA) positive myofibroblasts. IL-11-neutralizing antibodies (IL-11NAb) or ERK inhibitors (U0126) inhibited IL-11 activity and downregulated fibrotic responses involving TGFβ/SMAD signaling. In vivo, IL-11Rα knockdown rats showed unobstructed tracheal lumen, relatively intact epithelial structure, and significantly reduced granulation tissue proliferation and collagen fiber deposition. Our findings confirm that IL-11 may be a target for future drug prevention and treatment of tracheal stenosis.

Recombinant Adeno-associated Viral Vectors Serotypes 6 and 9 are Able to Transduce Human Tracheal Epithelial Cells but Not Human Induced Pluripotent Stem Cells.

Recombinant adeno-associated viruses (rAAVs) may be useful for the development of gene therapy for hereditary diseases. Patient-specific human induced pluripotent stem cells (hiPSCs) can be differentiated into a variety of cells which are difficult or impossible to obtain by biopsy. To date, few research on the efficiency of rAAV transduction of hiPSCs has been published, but the obtained data are very contradictory and do not answer the actual question: how effective are rAAVs for the delivery of transgenes into hiPSCs. In this work, we used rAAV serotypes 5, 6, and 9 carrying the GFP transgene. The transduction efficiency of rAAV2/9-GFP and rAAV2/6-GFP for the immortalized tracheal epithelial cell line derived from a patient with cystic fibrosis (CFTE29o-) was relatively high. At the same time, the efficiency of transduction of iPSCs from a healthy donor and a cystic fibrosis (CF) donor was extremely low. Thus, our results show that the efficiency of hiPSC transduction by rAAV serotypes 5, 6, and 9 is not suitable for the delivery of transgenes.

Impact of Changes in Human Airway Epithelial Cellular Composition and Differentiation on SARS-CoV-2 Infection Biology

The consequences of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can range from asymptomatic to fatal disease. Variations in epithelial susceptibility to SARS-CoV-2 infection depend on the anatomical location from the proximal to distal respiratory tract. However, the cellular biology underlying these variations is not completely understood. Thus, air-liquid interface cultures of well-differentiated primary human tracheal and bronchial epithelial cells were employed to study the impact of epithelial cellular composition and differentiation on SARS-CoV-2 infection by transcriptional (RNA sequencing) and immunofluorescent analyses. Changes of cellular composition were investigated by varying time of differentiation or by using specific compounds. We found that SARS-CoV-2 primarily infected not only ciliated cells but also goblet cells and transient secretory cells. Viral replication was impacted by differences in cellular composition, which depended on culturing time and anatomical origin. A higher percentage of ciliated cells correlated with a higher viral load. However, DAPT treatment, which increased the number of ciliated cells and reduced goblet cells, decreased viral load, indicating the contribution of goblet cells to infection. Cell entry factors, especially cathepsin L and transmembrane protease serine 2, were also affected by differentiation time. In conclusion, our study demonstrates that viral replication is affected by changes in cellular composition, especially in cells related to the mucociliary system. This could explain in part the variable susceptibility to SARS-CoV-2 infection between individuals and between anatomical locations in the respiratory tract.

Nearly Complete Genome Sequences of 12 Types of Human Rhinoviruses Isolated from Pediatric Inpatients in Fukushima, Japan.

ABSTRACTWe reported nearly complete genomic sequences of 12 serotypes of human rhinoviruses (HRVs) isolated from pediatric inpatients in Fukushima, Japan using an air-liquid interface culture of human bronchial tracheal epithelial cells. We found that various serotypes of HRV circulated locally and simultaneously from 2018 to 2021.

Ten Nearly Complete Genome Sequences of Human Orthorubulavirus 4 Isolated from Pediatric Inpatients in Fukushima, Japan.

ABSTRACTWe report 10 nearly complete genomic sequences of human orthorubulavirus 4, also called human parainfluenza virus 4 (HPIV4), isolated from pediatric inpatients with respiratory infections in Fukushima, Japan, by using an air-liquid interface culture of human bronchial and tracheal epithelial cells.

Sophoraflavanone G from Sophora flavescens Ameliorates Allergic Airway Inflammation by Suppressing Th2 Response and Oxidative Stress in a Murine Asthma Model.

Sophoraflavanone G (SG), isolated from Sophora flavescens, has anti-inflammatory and anti-tumor bioactive properties. We previously showed that SG promotes apoptosis in human breast cancer cells and leukemia cells and reduces the inflammatory response in lipopolysaccharide-stimulated macrophages. We investigated whether SG attenuates airway hyper-responsiveness (AHR) and airway inflammation in asthmatic mice. We also assessed its effects on the anti-inflammatory response in human tracheal epithelial cells. Female BALB/c mice were sensitized with ovalbumin, and asthmatic mice were treated with SG by intraperitoneal injection. We also exposed human bronchial epithelial BEAS-2B cells to different concentrations of SG to evaluate its effects on inflammatory cytokine levels. SG treatment significantly reduced AHR, eosinophil infiltration, goblet cell hyperplasia, and airway inflammation in the lungs of asthmatic mice. In the lungs of ovalbumin-sensitized mice, SG significantly promoted superoxide dismutase and glutathione expression and attenuated malondialdehyde levels. SG also suppressed levels of Th2 cytokines and chemokines in lung and bronchoalveolar lavage samples. In addition, we confirmed that SG decreased pro-inflammatory cytokine, chemokine, and eotaxin expression in inflammatory BEAS-2B cells. Taken together, our data demonstrate that SG shows potential as an immunomodulator that can improve asthma symptoms by decreasing airway-inflammation-related oxidative stress.

Pre-existing atopy prevents mortality from respiratory viral infection via induction of neuregulin-1

Abstract Respiratory viral infections are associated with significant morbidity and mortality. RNA viruses, such as Respiratory Syncytial Virus (RSV) have been implicated in the development of asthma. We have a high-fidelity model of respiratory viral infection. Our mouse model utilizes a natural rodent pathogen, Sendai virus (SeV) similar to RSV, which faithfully replicates in mouse airway epithelial cells. We have shown that making these mice atopic (i.e, sensitized and challenged with house dust mite extracts) before viral infection prevents development of disease and more importantly prevents mice from dying of a normally lethal dose of SeV. Further, lungs and bronchoalveolar lavage fluid of atopic mice have significant increased levels of Neuregulin-1 (NRG1), potentially due to high expression of Nrg1 in dendritic cells as determined by RNAseq of CD11c+ cells. To determine a temporal relationship between NRG1 expression and respiratory viral infection, we utilized an in vitro system of well-differentiated human bronchial epithelial cells and mouse tracheal epithelial cells. Cells were treated on the basolateral side with NRG1 (10 to 100ng) for six-days before 4000 pfu of RSV-GFP or SeV-GFP inoculation, respectively. There was marked reduction in viral spreading (quantified by reduced GFP expression) with NRG1 treatment. Interleukin-6 (IL6) expression was reduced in NRG1 treated cells at 48h PI, as well. IL6 has been implicated with cellular stress and is involved in disease pathology. Therefore, reducing IL6 might affect the clinical presentation of serious infections. In conclusion our studies show that atopy induced NRG1 plays a novel role with a therapeutic potential in severe respiratory viral infections. Supported by Abigail Wexner Research Institute at Nationwide Children’s Hospital, Start-up fund to MHG

The Effects of Rapamycin on the Proliferation, Migration, and Apoptosis of Human Tracheal Fibroblasts (HTrF) and Human Tracheal Epithelial Cells (HTEpiC).

Background: Restenosis after airway stenting needs to be addressed urgently. Rapamycin has been proven to inhibit restenosis elsewhere. This study aimed at observing its effects on the respiratory tract. Methods: CCK-8, wound healing, Transwell and apoptosis assays were performed to detect the effects of rapamycin on the survival, migration, and apoptosis, respectively, of human tracheal fibroblasts (HTrF) and human tracheal epithelial cells (HTEpiC). Results: The effective concentrations of paclitaxel, mitomycin C and rapamycin on HTrF were 10−7–10−4 mol/L, 10−6–10−4 mol/L, and 10−5–10−4 mol/L, respectively. At the effective concentrations, the inhibition rates of paclitaxel on HTEpiC were (43.03 ± 1.12)%, (49.49 ± 0.86)%, (55.22 ± 1.43)%, and (93.19 ± 0.45)%; the inhibition rates of mitomycin C on HTEpiC were (88.11 ± 0.69)%, (93.82 ± 0.96)%, and (94.94 ± 0.54)%; the inhibition rates of rapamycin on HTEpiC were (10.19 ± 0.35)% and (94.55 ± 0.71)%. At the concentration of (1–4) × 10−5 mol/L, the inhibition rate of rapamycin on HTrF was more than 50%, and that on HTEpiC was less than 20% (p < 0.05). Conclusions: Compared to paclitaxel and mitomycin C, rapamycin had the least effect on HTEpiC while effectively inhibiting HTrF. The optimum concentration range was (1–4) × 10−5 mol/L.

Thirteen Nearly Complete Genome Sequences of Human Bocavirus 1 Isolated from Pediatric Inpatients in Fukushima, Japan.

ABSTRACTWe report 13 genomic sequences of human bocavirus 1 isolated from pediatric inpatients in Fukushima, Japan, using an air-liquid interface culture of human bronchial tracheal epithelial cells. This work suggests the endemic circulation of a human bocavirus variant with a unique amino acid signature in Fukushima.

Planar organization of airway epithelial cell morphology using hydrogel grooves during ciliogenesis fails to induce ciliary alignment.

Topographical cues are known to influence cell organization both in native tissues and in vitro. In the trachea, the matrix beneath the epithelial lining is composed of collagen fibres that run along the long axis of the airway. Previous studies have shown that grooved topography can induce morphological and cytoskeletal alignment in epithelial cell lines. In the present work we assessed the impact of substrate topography on the organization of primary human tracheal epithelial cells (HTECs) and human induced pluripotent stem cell (hiPSC)-derived airway progenitors and the resulting alignment of cilia after maturation of the airway cells under Air-Liquid-Interface (ALI) culture. Grooves with optimized dimensions were imprinted into collagen vitrigel membranes (CVM) to produce gel inserts for ALI culture. Grooved CVM substrates induced cell alignment in HTECs and hiPSC airway progenitors in submerged culture. Further, both cell types were able to terminally differentiate into a multi-ciliated epithelium on both flat and groove CVM substrates. When exposed to ALI conditions, HTECs lost alignment after 14 days. Meanwhile, hiPSC-derived airway progenitors maintained their alignment throughout 31 days of ALI culture. Interestingly, neither initial alignment on the grooves, nor maintained alignment on the grooves induced alignment of cilia basal bodies, an indication of the direction of ciliary beating direction in the airway cells. Planar organization of airway cells during or prior to ciliogenesis therefore does not appear to be a feasible strategy to control cilia organization and subsequent airway epithelial function and additional cues are likely necessary to produce cilia alignment.

Ultraviolet-A light increases mitochondrial anti-viral signaling protein in confluent human tracheal cells via cell-cell signaling.

Mitochondrial antiviral signaling (MAVS) protein mediates innate antiviral responses, including responses to certain coronaviruses such as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We have previously shown that ultraviolet-A (UVA) therapy can prevent virus-induced cell death in human ciliated tracheal epithelial cells (HTEpC) infected with coronavirus-229E (CoV-229E), and results in increased intracellular levels of MAVS. In this study, we explored the mechanisms by which UVA light can activate MAVS, and whether local UVA light application can activate MAVS at locations distant from the light source (e.g. via cell-to-cell communication). MAVS levels were compared in HTEpC exposed to 2 mW/cm2 narrow band (NB)-UVA for 20 min and in unexposed controls at 30–40% and at 100% confluency, and in unexposed HTEpC treated with supernatants or lysates from UVA-exposed cells or from unexposed controls. MAVS was also assessed in different sections of confluent monolayer plates where only one section was exposed to NB-UVA. Our results showed that UVA increases the expression of MAVS protein. Further, cells in a confluent monolayer exposed to UVA conferred an elevation in MAVS in cells adjacent to the exposed section, and also in cells in the most distant sections which were not exposed to UVA. In this study, human ciliated tracheal epithelial cells exposed to UVA demonstrate increased MAVS protein, and also appear to transmit this influence to confluent cells not exposed to UVA, likely via cell-cell signaling.

Effective-component compatibility of Bufei Yishen formula protects COPD rats against PM2.5-induced oxidative stress via miR-155/FOXO3a pathway

Ambient particulate matter <2.5 µm (PM2.5) has been identified as a critical risk factor in chronic obstructive pulmonary disease (COPD) exacerbation, but therapies for this condition are limited. Effective-component compatibility of Bufei Yishen formula (ECC-BYF) exhibits beneficial efficacy on COPD rats. However, its effect on PM2.5-aggravated COPD rats are considered to be uncertain. In this study, we used an established PM2.5-aggravated COPD rat model in vivo to evaluate the protective effect of ECC-BYF, and focused on its antioxidative role in PM2.5-stimulated bronchial epithelial cells via regulating microRNA (miR)−155/ forkhead box class O3a (FOXO3a) pathway. As expected, PM2.5-aggravated COPD rats showed a reduction of lung function, persistent lung inflammation, and remodeling of lung tissue. In comparison, ECC-BYF administration significantly enhanced lung function, alleviated alveolar destruction, inflammatory cell infiltration, mucus hypersecretion, and collagen deposition, along with diminishing inflammatory cytokine production and oxidative stress. Furthermore, ECC-BYF pretreatment markedly decreased the fluorescence intensity of reactive oxygen species (ROS) in PM2.5-induced human bronchial epithelial (Beas-2B) cells and primary mouse tracheal epithelial cells (MTECs), as well as reversing the imbalance between oxidants and antioxidants in Beas-2B. Meanwhile, ECC-BYF elevated FOXO3a while inhibiting miR-155 expression dose -dependently. In vitro transfection of miR-155 mimic into Beas-2B significantly decreased FOXO3a protein expression, accompanied by the reduced superoxide dismutase 2 (SOD2) and catalase (CAT) expressions, thus eliminating the protective effect of ECC-BYF on PM2.5-evoked oxidative stress. Nonethless, FOXO3a overexpression could partially restore the antioxidative effect of ECC-BYF. In conclusion, ECC-BYF can protect pre-existing COPD against PM2.5 contamination by exerting a profound antioxidative influence via regulating miR-155/FOXO3a signaling.