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.

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.

Downregulation of protein phosphatase 2Aα in asthmatic airway smooth muscle.

Airway smooth muscle cell (ASM) is renowned for its involvement in airway hyperresponsiveness through impaired ASM relaxation and bronchoconstriction in asthma, which poses a significant challenge in the field. Recent studies have explored different targets in ASM to alleviate airway hyperresponsiveness, however, a sizeable portion of patients with asthma still experience poor control. In our study, we explored protein phosphatase 2 A (PP2A) in ASM as it has been reported to regulate cellular contractility by controlling intracellular calcium ([Ca2+]i), ion channels, and respective regulatory proteins. We obtained human ASM cells and lung tissues from healthy and patients with asthma and evaluated PP2A expression using RNA-Seq data, immunofluorescence, and immunoblotting. We further investigated the functional importance of PP2A by determining its role in bronchoconstriction using mouse bronchus and human ASM cell [Ca2+]i regulation. We found robust expression of PP2A isoforms in human ASM cells with PP2Aα being highly expressed. Interestingly, PP2Aα was significantly downregulated in asthmatic tissue and human ASM cells exposed to proinflammatory cytokines. Functionally, FTY720 (PP2A agonist) inhibited acetylcholine- or methacholine-induced bronchial contraction in mouse bronchus and further potentiated isoproterenol-induced bronchial relaxation. Mechanistically, FTY720 inhibited histamine-evoked [Ca2+]i response and myosin light chain (MLC) phosphorylation in the presence of interleukin-13 (IL-13) in human ASM cells. To conclude, we for the first time established PP2A signaling in ASM, which can be further explored to develop novel therapeutics to alleviate airway hyperresponsiveness in asthma.NEW & NOTEWORTHY This novel study deciphered the expression and function of protein phosphatase 2Aα (PP2Aα) in airway smooth muscle (ASM) during asthma and/or inflammation. We showed robust expression of PP2Aα in human ASM while its downregulation in asthmatic ASM. Similarly, we demonstrated reduced PP2Aα expression in ASM exposed to proinflammatory cytokines. PP2Aα activation inhibited bronchoconstriction of isolated mouse bronchi. In addition, we unveiled that PP2Aα activation inhibits the intracellular calcium release and myosin light chain phosphorylation in human ASM.

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.

DNMT3A-mediated high expression of circ_0057504 promotes benzo[a]pyrene-induced DNA damage via the NONO-SFPQ complex in human bronchial epithelial cells.

Benzo[a]pyrene (B[a]P) is a class I carcinogen and hazardous environmental pollutant with genetic toxicity. Understanding the molecular mechanisms underlying genetic deterioration and epigenetic alterations induced by environmental contaminants may contribute to the early detection and prevention of cancer. However, the role and regulatory mechanisms of circular RNAs (circRNAs) in the B[a]P-induced DNA damage response (DDR) have not been elucidated. In this study, human bronchial epithelial cell lines (16HBE and BEAS-2B) were exposed to various concentrations of B[a]P, and BALB/c mice were treated with B[a]P intranasally. B[a]P exposure was found to induce DNA damage and upregulate circular RNA hsa_circ_0057504 (circ_0057504) expression in vitro and in vivo. In addition, B[a]P upregulated TMEM194B mRNA and circ_0057504 expression through inhibition of DNA methyltransferase 3 alpha (DNMT3A) expression in vitro. Modulation (overexpression or knockdown) of circ_0057504 expression levels using a lentiviral system in human bronchial epithelial cells revealed that circ_0057504 promoted B[a]P-induced DNA damage. RNA pull-down and western blot assays showed that circ_0057504 interacted with non-POU domain-containing octamer-binding (NONO) and splicing factor proline and glutamine rich (SFPQ) proteins and regulated formation of the NONO-SFPQ protein complex. Thus, our findings indicate that circ_0057504 acts as a novel regulator of DNA damage in human bronchial epithelial cells exposed to B[a]P. The current study reveals novel insights into the role of circRNAs in the regulation of genetic damage, and describes the effect and regulatory mechanisms of circ_0057504 on B[a]P genotoxicity.

Potent necrosis effect of methanethiol mediated by METTL7B enzyme bioactivation mechanism in 16HBE cell.

Methanethiol is a widely existing malodorous pollutant with health effects on the human population. However, the cytotoxicity mechanism of methanethiol in vitro and its metabolic transformation (bioactivation or detoxification) have not been fully elucidated. Herein, the metabolites of methanethiol during cell culture and the cytotoxicity of methanethiol in human bronchial epithelial (16HBE) cells were investigated. Results indicate that methanethiol (10-50 µM) was partially converted into dimethyl sulfide, mainly catalyzed by thiol S-methyltransferase in the 16HBE cells, and then it induced potent cytotoxicity and cell membrane permeability. Moreover, methanethiol induced intracellular reactive oxygen species (ROS) up to 50 µM and further activated the tumor necrosis factor (TNF) signaling pathway, which eventually led to the decline in the mitochondrial membrane potential (MMP) and cell necrosis. However, all these effects were significantly alleviated with gene silencing of the methyltransferase-like protein 7B (METTL7B). These results indicate that methanethiol may induce cell necrosis in human respiratory tract cells mainly mediated by S-methyltransferase with interfering TNF and ROS induction. Non-target metabolomics results suggest that methanethiol potently affects expression of endogenous small molecule metabolites in 16HBE cells. To some extent, this work shows the possible conversion path and potential injury mechanism of human respiratory tract cells exposed to methanethiol.

Cellular and Molecular Signatures of Oxidative Stress in Bronchial Epithelial Cell Models Injured by Cigarette Smoke Extract.

Exposure of the airways epithelium to environmental insults, including cigarette smoke, results in increased oxidative stress due to unbalance between oxidants and antioxidants in favor of oxidants. Oxidative stress is a feature of inflammation and promotes the progression of chronic lung diseases, including Chronic Obstructive Pulmonary Disease (COPD). Increased oxidative stress leads to exhaustion of antioxidant defenses, alterations in autophagy/mitophagy and cell survival regulatory mechanisms, thus promoting cell senescence. All these events are amplified by the increase of inflammation driven by oxidative stress. Several models of bronchial epithelial cells are used to study the molecular mechanisms and the cellular functions altered by cigarette smoke extract (CSE) exposure, and to test the efficacy of molecules with antioxidant properties. This review offers a comprehensive synthesis of human in-vitro and ex-vivo studies published from 2011 to 2021 describing the molecular and cellular mechanisms evoked by CSE exposure in bronchial epithelial cells, the most used experimental models and the mechanisms of action of cellular antioxidants systems as well as natural and synthetic antioxidant compounds.

Arsenic activates STAT3 signaling during the transformation of the human bronchial epithelial cells.

Arsenic (As3+), a metalloid abundant in environment, is classified as a group I carcinogen associated with several common human cancers, including cancers in lung, skin, bladder, liver, and prostate (Wei et al., 2019). The mechanisms of As3+-induced carcinogenesis had been extensively studied, and different mechanisms might be involved in different types of cancer (Wei et al., 2019). Recent studies showed that exposure to a high dose of arsenic is able to induce lung cancer. Meanwhile, prolonged exposure to a low concentration of arsenic can increase the risk of lung cancer also (Liao et al., 2009; Fernández et al., 2012). Emerging evidence indicated that prolonged exposure to arsenic promotes malignant transformation and some of the transformed cells have cancer-stem-like properties (Ngalame et al., 2014). In the present report, we revealed that exposure to As3+ for short time period inhibited tyrosine-705 phosphorylation of signal transducer and activator of transcription 3 (pSTAT3Y705) and induced Src homology region 2 domain-containing phosphatase-1 (SHP-1) in bronchial epithelial cell line, BEAS-2B. In addition, we found that long term exposure of the cells to As3+ activates phosphorylation of STAT3 at serine 727 (pSTAT3S727) as well as pSTAT3Y705. Moreover, As3+ is able to induce the expression of miRNA-21 (miR-21) and decrease the expression of PDCD4. Taken together, our data suggest that activation of STAT3 and induction of miR-21 are important contributing factors to the reduced expression of PDCD4, which may play significant role in As3+-induced transformation of BEAS-2B cells.

Involvement of m6A regulatory factor IGF2BP1 in malignant transformation of human bronchial epithelial Beas-2B cells induced by tobacco carcinogen NNK.

Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a Group 1 human carcinogen, as classified by the International Agency for Research of Cancer (IARC), and plays a significant role in lung carcinogenesis. However, its carcinogenic mechanism has not yet been fully elucidated. In this study, we performed colony formation assays, soft-agar assays, and tumor growth in nude mice to show that 100 mg/L NNK facilitates the malignant transformation of human bronchial epithelial Beas-2B cells. Transcriptome sequencing showed that insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), a post-transcriptional regulator, was differentially expressed in NNK-induced malignant transformed Beas-2B cells (2B-NNK cells). Small interfering RNA (SiRNA) was used to downregulate the expression of the IGF2BP1 gene. The reduction in protein expression, cell proliferation rate, and colony-forming ability and the increase in the apoptosis rate of Beas-2B cells transfected with the SiRNA indicated a role for IGF2BP1 in NNK-induced malignant transformation. IGF2BP1 is an N6-methyladenosine (m6A) regulatory factor, but it is not known whether its association with m6A mediates the malignant transformation of cells. Therefore, we measured the overall levels of m6A in Beas-2B cells. We found that the overall m6A level was lower in 2B-NNK cells, and knocking down IGF2BP1, the overall level of m6A was restored. Hence, we concluded that IGF2BP1 is involved in the NNK-induced malignant transformation of Beas-2B cells, possibly via m6A modification. This study therefore contributes novel insights into the environmental pathogenesis of lung cancer and the gene regulatory mechanisms of chemical carcinogenesis.

Repeated radon exposure induced lung damage via oxidative stress-mediated mitophagy in human bronchial epithelial cells and mice.

This study aimed to investigate the potential molecular mechanism underlying radon-induced lung damage. Our results showed that long-term radon exposure induced mitochondrial damage and redox imbalance in BEAS-2B cells and a time-dependent lung pathological injury in mice. The activation of Nrf-2 and its down-stream antioxidants, and the gene expression of the indicated markers at different stages of autophagy were found to be induced with the increasing of radon exposure time. Changes in the gene expression of PINK-1, Parkin, and p62 induced by radon showed differences in mechanisms of mitophagy activation and profiles of autophagic flux between BEAS-2B cells and mice. Our findings not only demonstrated that long-term radon exposure induced damages to bronchial epithelial cells and the mice lung through increasing oxidative stress, decreasing mitochondrial function and activating mitophagy with different profiles of autophagic flux, but also revealed Nrf-2 as a central regulator of mitochondrial homeostasis and lung damage.

Taxifolin improves inflammatory injury of human bronchial epithelial cells by inhibiting matrix metalloproteinase (MMP) 10 via Wnt/ß-catenin pathway.

Taxifolin (TXL), also known as dihydroquercetin, is one of the most important flavonoids prevalent across the plant kingdom. Increasing evidence has demonstrated its critical role in respiratory diseases. The present study aims to reveal the detailed mechanism in TNF-α-stimulated BEAS-2B cells by which TXL might exert effects on the development of asthma. Cell viability detection of BEAS-2B treated with TXL before and after TNF-α induction employed MMT. The expressions of inflammatory cytokines, MUC5AC and ICAM-1 were determined by quantitative reverse transcription PCR (RT-qPCR), enzyme-linked immunosorbent assay (ELISA) and Western blot after TXL was exposed to an in vitro asthma model. Then, light transmittance and apoptosis were then measured employing fluorescein transmittance, TUNEL and Western blot. After overexpressing MMP10, the abovementioned assays were performed again. Finally, the association between Wnt/ß-catenin pathway and MMP10 was confirmed by detecting the proteins in this pathway. TXL increases the cell viability of TNF-induced BEAS-2B cells. TXL suppressed the inflammation, mucus formation, and apoptosis in TNF-α-induced BEAS-2B cells. Furthermore, after the prediction of binding sites between TXL and MMP10, it was found that overexpression of MMP10 reversed the effects of TXL on suppressing the progression of TNF-α-induced BEAS-2B cells. Finally, TXL blocked Wnt/ß-catenin pathway by inhibiting MMP10 expression.TXL can be a promising drug for the treatment of asthma due to its inhibition of MMP10 expression by blocking Wnt/ß-catenin pathway. Future experimental in vivo studies of asthma on this commonly used bioactive flavonoid could open new avenues for the therapies of asthma.

Identification of the function and regulatory network of circ_009773 in DNA damage induced by nanoparticles of neodymium oxide.

The health hazards of nanoparticles of neodymium oxide (NPs-Nd2O3) have aroused public concern in recent years. Exposure to NPs-Nd2O3 can change the level of reactive oxygen species (ROS) that cause DNA damage and alter whole transcriptome expression profiles for micro (mi)RNA, circular (circ)RNA, long noncoding (lnc)RNA, and mRNA. However, there have been no reports to our knowledge about the role of circRNAs in DNA damage caused by NPs-Nd2O3. In our study, we analyzed the circRNA expression profile of human bronchial epithelial cells(16HBE)exposed to 40 µg/ml NPs-Nd2O3. Our results indicated that exposure produced 1025 up-regulated and 890 down-regulated circRNAs. Real-time quantitative polymerase chain reaction (qRT-PCR) was applied to verify some of the significantly changed circRNAs and demonstrated that circ_009773 was apparently down-regulated. Through exploration of its host gene function, we found that circ_009773 may be related to DNA damage. Functional experiments found that circ_009773 regulated NPs-Nd2O3-induced DNA damage in 16HBE cells. A circ_009773-associated competing endogenous (ce)RNA network was constructed based on one differentially expressed (DE) circRNA, 74 DE miRNAs and 208 DE mRNAs. Module analysis identified hub genes related to DNA damage and repair and a protein-protein interaction (PPI) network was created.

Vitamin D3 protects against nitrogen mustard-induced apoptosis of the bronchial epithelial cells via activating the VDR/Nrf2/Sirt3 pathway.

Respiratory system injury is the main cause of mortality for nitrogen mustard (NM)-induced damage. Previous studies indicate that reactive oxygen species (ROS) participates in NM-mediated respiratory injuries, but the detailed mechanism is not quite clear. Human bronchial epithelial cell lines 16HBE and BEAS-2B were treated with HN2, a type of NM. In detail, it was shown that HN2 treatment induced impaired cell viability, excessive mitochondrial ROS production and enhanced cellular apoptosis in bronchial epithelial cells. Moreover, impaired Sirt3/SOD2 axis was observed upon HN2 treatment, with decreased Sirt3 and increased acetylated SOD2 expression levels. Sirt3 overexpression partially ameliorated HN2-induced cell injury. Meanwhile, vitamin D3 treatment partially attenuated HN2-induced apoptosis and improved the mitochondrial functions upon HN2 intervention. In addition, HN2 exposure decreased VDR expression, thus inhibiting the Nrf2 phosphorylation and Sirt3 activation. Inhibition of Nrf2 or Sirt3 could decrease the protective effects of vitamin D3 and enhance mitochondrial ROS production via modulating mitochondrial redox balance. In conclusion, impaired VDR/Nrf2/Sirt3 axis contributed to NM-induced apoptosis, while vitamin D3 supplementation provides protective effects via the activation of VDR and the improvement of mitochondrial functions. This study provides novel mechanism and strategy for NM exposure-induced pulmonary injuries.

The protective effects of Omarigliptin against Lipopolysaccharide (LPS)- induced inflammatory response and expression of mucin 5AC (MUC5AC) in human bronchial epithelial cells.

The epidemic of chronic inflammatory lung diseases such as asthma, bronchitis, and chronic obstructive pulmonary disease (COPD) has become a global public health problem. Oxidative stress, inflammation, and overproduction of airway mucus play critical roles in the progression of these diseases. Omarigliptin, an oral dipeptidyl peptidase 4 (DPP-4) inhibitor, has been demonstrated to have anti-inflammatory effects in patients with type II diabetes. However, its role in chronic inflammatory lung diseases remains enigmatic. This study is to investigate whether Omarigliptin possesses a beneficial effect against Lipopolysaccharide (LPS)-induced injuries in human BEAS-2B bronchial epithelial cells. Our results show that Omarigliptin suppressed LPS-induced oxidative stress by attenuating the generation of mitochondrial reactive oxygen species (ROS) and decrease in reduced glutathione (GSH) in BEAS-2B cells. Additionally, Omarigliptin mitigated inflammatory response by inhibiting the expression of pro-inflammatory mediators, including interleukin-1ß (IL-1ß), interleukin-12 (IL-12), and macrophage chemoattractant protein-1 (MCP-1) in LPS-challenged BEAS-2B cells. Moreover, Omarigliptin mitigated the LPS-induced overproduction of MUC5AC by rescuing the expression of the suppressor of cytokine signaling 1(SOCS1). Importantly, we found that this process is mediated by the Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway. Based on these findings, we conclude that Omarigliptin might be a promising agent for the treatment of chronic inflammatory lung diseases.

Exploring the molecular mechanisms of the inhibition of acrolein-induced BEAS-2B cytotoxicity by luteolin using network pharmacology and cell biology technology.

Acrolein is a highly reactive unsaturated hazardous air pollutant, which is extremely irritating to the respiratory tract. Luteolin, an active flavonoid compound, possesses multiple biological activities. The purpose of this study was to evaluate the mechanism of the inhibition of acrolein-induced human bronchial epithelial (BEAS-2B) cells cytotoxicity by luteolin using network pharmacology and cell biology technology. Firstly, network pharmacology results indicated that oxidative stress processes might play an important role in luteolin inhibiting lung injury. Next, it was verified at the cellular level. Reactive oxygen species (ROS) generation increased, glutathione (GSH) level decreased after exposure to acrolein. MAPK signaling pathways were activated, which activated downstream IκBα/NF-κB signaling pathways. Meanwhile, acrolein caused oxidative DNA damage and double-strand breaks, induced DNA damage response (DDR) and apoptosis. These adverse effects were significantly reversed by luteolin, which inhibited the activation of MAPK/IκBα/NF-κB and DDR pathways, and reduced the ratio of Bax/Bcl-2. Moreover, luteolin also had a similar effect to antioxidant N-acetyl cysteine (NAC) in the regulation of signaling transduction mechanisms, which indicated that the regulation of oxidative stress played an important role in the process. These results provide an experimental basis for elucidating the molecular mechanisms of the inhibition of acrolein-induced BEAS-2B cytotoxicity with luteolin.

Imiquimod Boosts Interferon Response, and Decreases ACE2 and Pro-Inflammatory Response of Human Bronchial Epithelium in Asthma.

Background: Both anti-viral and anti-inflammatory bronchial effects are warranted to treat viral infections in asthma. We sought to investigate if imiquimod, a TLR7 agonist, exhibits such dual actions in ex vivo cultured human bronchial epithelial cells (HBECs), targets for SARS-CoV-2 infectivity. Objective: To investigate bronchial epithelial effects of imiquimod of potential importance for anti-viral treatment in asthmatic patients. Methods: Effects of imiquimod alone were examined in HBECs from healthy (N=4) and asthmatic (N=18) donors. Mimicking SARS-CoV-2 infection, HBECs were stimulated with poly(I:C), a dsRNA analogue, or SARS-CoV-2 spike-protein 1 (SP1; receptor binding) with and without imiquimod treatment. Expression of SARS-CoV-2 receptor (ACE2), pro-inflammatory and anti-viral cytokines were analyzed by RT-qPCR, multiplex ELISA, western blot, and Nanostring and proteomic analyses. Results: Imiquimod reduced ACE2 expression at baseline and after poly(I:C) stimulation. Imiquimod also reduced poly(I:C)-induced pro-inflammatory cytokines including IL-1ß, IL-6, IL-8, and IL-33. Furthermore, imiquimod increased IFN-ß expression, an effect potentiated in presence of poly(I:C) or SP1. Multiplex mRNA analysis verified enrichment in type-I IFN signaling concomitant with suppression of cytokine signaling pathways induced by imiquimod in presence of poly(I:C). Exploratory proteomic analyses revealed potentially protective effects of imiquimod on infections. Conclusion: Imiquimod triggers viral resistance mechanisms in HBECs by decreasing ACE2 and increasing IFN-ß expression. Additionally, imiquimod improves viral infection tolerance by reducing viral stimulus-induced epithelial cytokines involved in severe COVID-19 infection. Our imiquimod data highlight feasibility of producing pluripotent drugs potentially suited for anti-viral treatment in asthmatic subjects.

Budesonide promotes airway epithelial barrier integrity following double-stranded RNA challenge.

Airway epithelial barrier dysfunction is increasingly recognized as a key feature of asthma and other lung diseases. Respiratory viruses are responsible for a large fraction of asthma exacerbations, and are particularly potent at disrupting epithelial barrier function through pattern recognition receptor engagement leading to tight junction dysfunction. Although different mechanisms of barrier dysfunction have been described, relatively little is known about whether barrier integrity can be promoted to limit disease. Here, we tested three classes of drugs commonly prescribed to treat asthma for their ability to promote barrier function using a cell culture model of virus-induced airway epithelial barrier disruption. Specifically, we studied the corticosteroid budesonide, the long acting beta-agonist formoterol, and the leukotriene receptor antagonist montelukast for their ability to promote barrier integrity of a monolayer of human bronchial epithelial cells (16HBE) before exposure to the viral mimetic double-stranded RNA. Of the three, only budesonide treatment limited transepithelial electrical resistance and small molecule permeability (4 kDa FITC-dextran flux). Next, we used a mouse model of acute dsRNA challenge that induces transient epithelial barrier disruption in vivo, and studied the effects budesonide when administered prophylactically or therapeutically. We found that budesonide similarly protected against dsRNA-induced airway barrier disruption in the lung, independently of its effects on airway inflammation. Taken together, these data suggest that an under-appreciated effect of inhaled budesonide is to maintain or promote airway epithelial barrier integrity during respiratory viral infections.

SB203580-A Potent p38 MAPK Inhibitor Reduces the Profibrotic Bronchial Fibroblasts Transition Associated with Asthma.

Subepithelial fibrosis is a component of the remodeling observed in the bronchial wall of patients diagnosed with asthma. In this process, human bronchial fibroblasts (HBFs) drive the fibroblast-to-myofibroblast transition (FMT) in response to transforming growth factor-ß1 (TGF-ß1), which activates the canonical Smad-dependent signaling. However, the pleiotropic properties of TGF-ß1 also promote the activation of non-canonical signaling pathways which can affect the FMT. In this study we investigated the effect of p38 mitogen-activated protein kinase (MAPK) inhibition by SB203580 on the FMT potential of HBFs derived from asthmatic patients using immunocytofluorescence, real-time PCR and Western blotting methods. Our results demonstrate for the first time the strong effect of p38 MAPK inhibition on the TGF-ß1-induced FMT potential throughout the strong attenuation of myofibroblast-related markers: α-smooth muscle actin (α-SMA), collagen I, fibronectin and connexin 43 in HBFs. We suggest the pleiotropic mechanism of SB203580 on FMT impairment in HBF populations by the diminishing of TGF-ß/Smad signaling activation and disturbances in the actin cytoskeleton architecture along with the maturation of focal adhesion sites. These observations justify future research on the role of p38 kinase in FMT efficiency and bronchial wall remodeling in asthma.