Potentiation of long-acting ß2-agonist and glucocorticoid responses in human airway epithelial cells by modulation of intracellular cAMP.

INTRODUCTION: Over 300 million people in the world live with asthma, resulting in 500,000 annual global deaths with future increases expected. It is estimated that around 50-80% of asthma exacerbations are due to viral infections. Currently, a combination of long-acting beta agonists (LABA) for bronchodilation and glucocorticoids (GCS) to control lung inflammation represent the dominant strategy for the management of asthma, however, it is still sub-optimal in 35-50% of moderate-severe asthmatics resulting in persistent lung inflammation, impairment of lung function, and risk of mortality. Mechanistically, LABA/GCS combination therapy results in synergistic efficacy mediated by intracellular cyclic adenosine monophosphate (cAMP). HYPOTHESIS: Increasing intracellular cAMP during LABA/GCS combination therapy via inhibiting phosphodiesterase 4 (PDE4) and/or blocking the export of cAMP by ATP Binding Cassette Transporter C4 (ABCC4), will potentiate anti-inflammatory responses of mainstay LABA/GCS therapy. METHODS: Expression and localization experiments were performed using in situ hybridization and immunohistochemistry in human lung tissue from healthy subjects, while confirmatory transcript and protein expression analyses were performed in primary human airway epithelial cells and cell lines. Intervention experiments were performed on the human airway epithelial cell line, HBEC-6KT, by pre-treatment with combinations of LABA/GCS with PDE4 and/or ABCC4 inhibitors followed by Poly I:C or imiquimod challenge as a model for viral stimuli. Cytokine readouts for IL-6, IL-8, CXCL10/IP-10, and CCL5/RANTES were quantified by ELISA. RESULTS: Using archived human lung and human airway epithelial cells, ABCC4 gene and protein expression were confirmed in vitro and in situ. LABA/GCS attenuation of Poly I:C or imiquimod-induced IL-6 and IL-8 were potentiated with ABCC4 and PDE4 inhibition, which was greater when ABCC4 and PDE4 inhibition was combined. Modulation of cAMP levels had no impact on LABA/GCS modulation of Poly I:C-induced CXCL10/IP-10 or CCL5/RANTES. CONCLUSION: Modulation of intracellular cAMP levels by PDE4 or ABCC4 inhibition potentiates LABA/GCS efficacy in human airway epithelial cells challenged with viral stimuli. The data suggest further exploration of the value of adding cAMP modulators to mainstay LABA/GCS therapy in asthma for potentiated anti-inflammatory efficacy.

Effects of environmental air pollutants on CFTR expression and function in human airway epithelial cells.

The airway epithelium is exposed to a variety of air pollutants, which have been associated with the onset and worsening of respiratory diseases. These air pollutants can vary depending on their composition and associated chemicals, leading to different molecular interactions and biological effects. Mucociliary clearance is an important host defense mechanism against environmental air pollutants and this process is regulated by various ion transporters including the cystic fibrosis transmembrane conductance regulator (CFTR). With evidence suggesting that environmental air pollutants can lead to acquired CFTR dysfunction, it may be possible to leverage therapeutic approaches used in cystic fibrosis (CF) management. The aim of our study was to test whether environmental air pollutants tobacco smoke extract, urban particulate matter, and diesel exhaust particles lead to acquired CFTR dysfunction and whether it could be rescued with pharmacological interventions. Human airway epithelial cells (Calu-3) were exposed to air pollutant extracts for 24 h, with and without pharmacological interventions, with readouts of CFTR expression and function. We demonstrate that both tobacco smoke extract and diesel exhaust particles led to acquired CFTR dysfunction and that rescue of acquired CFTR dysfunction is possible with pharmacological interventions in diesel exhaust particle models. Our study emphasizes that CFTR function is not only important in the context of CF but may also play a role in other respiratory diseases impacted by environmental air pollutants. In addition, the pharmacological interventions approved for CF management may be more broadly leveraged for chronic respiratory disease management.

Adhesive-Based Fabrication Technique for Culture of Lung Airway Epithelial Cells with Applications in Cell Patterning and Microfluidics.

This work describes a versatile and cost-effective cell culture method for micropatterning and growing adherent cells on porous membranes using pressure-sensitive double-sided adhesives. This technique also allows cell culture using conventional methods and their easy integration into microfluidic chip devices. Adhesives can be used to form different patterns of cultured cells, which can be used for cell proliferation and wound-healing models. To demonstrate the viability of our system, we evaluate the toxicity effect of five different adhesives on two distinct airway epithelial cell lines and show functional applications for cell patterning and microfluidic cell culture chip fabrication. We developed a sandwiched microfluidic device that enabled us to culture cells in a submerged condition and transformed it into a dynamic platform when required. The viability of cells and their inflammatory responses to IL-1ß stimulation were investigated. Our technique is applicable for conventional culturing of cells, widely available in biomedical research labs, while enabling the introduction of perfusion for an advanced dynamic cell culture model when needed.

Development and validation of an open-source, disposable, 3D-printed in vitro environmental exposure system for Transwell culture inserts.

Accessible in vitro models recapitulating the human airway that are amenable to study whole cannabis smoke exposure are needed for immunological and toxicological studies that inform public health policy and recreational cannabis use. In the present study, we developed and validated a novel three-dimensional (3D)-printed in vitro exposure system (IVES) that can be directly applied to study the effect of cannabis smoke exposure on primary human bronchial epithelial cells. Using commercially available design software and a 3D printer, we designed a four-chamber Transwell insert holder for exposures to whole smoke. COMSOL Multiphysics software was used to model gas distribution, concentration gradients, velocity profile and shear stress within IVES. Following simulations, primary human bronchial epithelial cells cultured at the air-liquid interface on Transwell inserts were exposed to whole cannabis smoke using a modified version of the Foltin puff procedure. Following 24 h, outcome measurements included cell morphology, epithelial barrier function, lactate dehydrogenase (LDH) levels, cytokine expression and gene expression. Whole smoke delivered through IVES possesses velocity profiles consistent with uniform gas distribution across the four chambers and complete mixing. Airflow velocity ranged between 1.0 and 1.5 µm·s-1 and generated low shear stresses (<<1 Pa). Human airway epithelial cells exposed to cannabis smoke using IVES showed changes in cell morphology and disruption of barrier function without significant cytotoxicity. Cannabis smoke elevated interleukin-1 family cytokines and elevated CYP1A1 and CYP1B1 expression relative to control, validating IVES smoke exposure impacts in human airway epithelial cells at a molecular level. The growing legalisation of cannabis on a global scale must be paired with research related to potential health impacts of lung exposures. IVES represents an accessible, open-source, exposure system that can be used to model varying types of cannabis smoke exposures with human airway epithelial cells grown under air-liquid interface culture conditions.

Modulation of cAMP metabolism for CFTR potentiation in human airway epithelial cells.

Cystic fibrosis (CF) is a genetic disease characterized by CF transmembrane regulator (CFTR) dysfunction. With over 2000 CFTR variants identified, in addition to known patient to patient variability, there is a need for personalized treatment. The discovery of CFTR modulators has shown efficacy in certain CF populations, however there are still CF populations without valid therapeutic options. With evidence suggesting that single drug therapeutics are insufficient for optimal management of CF disease, there has been an increased pursuit of combinatorial therapies. Our aim was to test cyclic AMP (cAMP) modulation, through ATP Binding Cassette Transporter C4 (ABCC4) and phosphodiesterase-4 (PDE-4) inhibition, as a potential add-on therapeutic to a clinically approved CFTR modulator, VX-770, as a method for increasing CFTR activity. Human airway epithelial cells (Calu-3) were used to test the efficacy of cAMP modulation by ABCC4 and PDE-4 inhibition through a series of concentration-response studies. Our results showed that cAMP modulation, in combination with VX-770, led to an increase in CFTR activity via an increase in sensitivity when compared to treatment of VX-770 alone. Our study suggests that cAMP modulation has potential to be pursued as an add-on therapy for the optimal management of CF disease.

ABCF1 Regulates dsDNA-induced Immune Responses in Human Airway Epithelial Cells.

Background: The airway epithelium represents a critical component of the human lung that helps orchestrate defenses against respiratory tract viral infections, which are responsible for more than 2.5 million deaths/year globally. Innate immune activities of the airway epithelium rely on Toll-like receptors (TLRs), nucleotide binding and leucine-rich-repeat pyrin domain containing (NLRP) receptors, and cytosolic nucleic acid sensors. ATP Binding Cassette (ABC) transporters are ubiquitous across all three domains of life-Archaea, Bacteria, and Eukarya-and expressed in the human airway epithelium. ABCF1, a unique ABC family member that lacks a transmembrane domain, has been defined as a cytosolic nucleic acid sensor that regulates CXCL10, interferon-ß expression, and downstream type I interferon responses. We tested the hypothesis that ABCF1 functions as a dsDNA nucleic acid sensor in human airway epithelial cells important in regulating antiviral responses. Methods: Expression and localization experiments were performed using in situ hybridization and immunohistochemistry in human lung tissue, while confirmatory transcript and protein expression was performed in human airway epithelial cells. Functional experiments were performed with siRNA methods in a human airway epithelial cell line. Complementary transcriptomic analyses were performed to explore the contributions of ABCF1 to gene expression patterns. Results: Using archived human lung and human airway epithelial cells, we confirm expression of ABCF1 gene and protein expression in these tissue samples, with a role for mediating CXCL10 production in response to dsDNA viral mimic challenge. Although, ABCF1 knockdown was associated with an attenuation of select genes involved in the antiviral responses, Gene Ontology analyses revealed a greater interaction of ABCF1 with TLR signaling suggesting a multifactorial role for ABCF1 in innate immunity in human airway epithelial cells. Conclusion: ABCF1 is a candidate cytosolic nucleic acid sensor and modulator of TLR signaling that is expressed at gene and protein levels in human airway epithelial cells. The precise level where ABCF1 protein functions to modulate immune responses to pathogens remains to be determined but is anticipated to involve IRF-3 and CXCL10 production.

Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue.

In December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, causing the coronavirus disease 2019 (COVID-19) pandemic. SARS-CoV, the agent responsible for the 2003 SARS outbreak, utilises angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) host molecules for viral entry. ACE2 and TMPRSS2 have recently been implicated in SARS-CoV-2 viral infection. Additional host molecules including ADAM17, cathepsin L, CD147 and GRP78 may also function as receptors for SARS-CoV-2.To determine the expression and in situ localisation of candidate SARS-CoV-2 receptors in the respiratory mucosa, we analysed gene expression datasets from airway epithelial cells of 515 healthy subjects, gene promoter activity analysis using the FANTOM5 dataset containing 120 distinct sample types, single cell RNA sequencing (scRNAseq) of 10 healthy subjects, proteomic datasets, immunoblots on multiple airway epithelial cell types, and immunohistochemistry on 98 human lung samples.We demonstrate absent to low ACE2 promoter activity in a variety of lung epithelial cell samples and low ACE2 gene expression in both microarray and scRNAseq datasets of epithelial cell populations. Consistent with gene expression, rare ACE2 protein expression was observed in the airway epithelium and alveoli of human lung, confirmed with proteomics. We present confirmatory evidence for the presence of TMPRSS2, CD147 and GRP78 protein in vitro in airway epithelial cells and confirm broad in situ protein expression of CD147 and GRP78 in the respiratory mucosa.Collectively, our data suggest the presence of a mechanism dynamically regulating ACE2 expression in human lung, perhaps in periods of SARS-CoV-2 infection, and also suggest that alternative receptors for SARS-CoV-2 exist to facilitate initial host cell infection.