TRPV2 modulates mechanically Induced ATP Release from Human bronchial epithelial cells.

Repetitive bouts of coughing expose the large airways to significant cycles of shear stress. This leads to the release of alarmins and the tussive agent adenosine triphosphate (ATP) which may be modulated by the activity of ion channels present in the human airway. This study aimed to investigate the role of the transient receptor potential subfamily vanilloid member 2 (TRPV2) channel in mechanically induced ATP release from primary bronchial epithelial cells (PBECs).PBECs were obtained from individuals undergoing bronchoscopy. They were cultured in vitro and exposed to mechanical stress in the form of compressive and fluid shear stress (CFSS) or fluid shear stress (FSS) alone at various intensities. ATP release was measured using a luciferin-luciferase assay. Functional TRPV2 protein expression in human PBECs was investigated by confocal calcium imaging. The role of TRPV2 inhibition on FSS-induced ATP release was investigated using the TRPV2 inhibitor tranilast or siRNA knockdown of TRPV2. TRPV2 protein expression in human lung tissue was also determined by immunohistochemistry.ATP release was significantly increased in PBECs subjected to CFSS compared with control (unstimulated) PBECs (N = 3, ***P < 0.001). PBECs expressed functional TRPV2 channels. TRPV2 protein was also detected in fixed human lung tissue. ATP release from FFS stimulated PBECs was decreased by the TRPV2 inhibitor tranilast (N = 3, **P < 0.01) (vehicle: 159 ± 17.49 nM, tranilast: 25.08 ± 5.1 nM) or by TRPV2 siRNA knockdown (N = 3, *P < 0.05) (vehicle: 197 ± 24.52 nM, siRNA: 119 ± 26.85 nM).In conclusion, TRPV2 is expressed in the human airway and modulates ATP release from mechanically stimulated PBECs.

Airborne indoor allergen serine proteases and their contribution to sensitisation and activation of innate immunity in allergic airway disease.

Common airborne allergens (pollen, animal dander and those from fungi and insects) are the main triggers of type I allergic disorder in the respiratory system and are associated with allergic rhinitis, allergic asthma, as well as immunoglobulin E (IgE)-mediated allergic bronchopulmonary aspergillosis. These allergens promote IgE crosslinking, vasodilation, infiltration of inflammatory cells, mucosal barrier dysfunction, extracellular matrix deposition and smooth muscle spasm, which collectively cause remodelling of the airways. Fungus and insect (house dust mite and cockroaches) indoor allergens are particularly rich in proteases. Indeed, more than 40 different types of aeroallergen proteases, which have both IgE-neutralising and tissue-destructive activities, have been documented in the Allergen Nomenclature database. Of all the inhaled protease allergens, 85% are classed as serine protease activities and include trypsin-like, chymotrypsin-like and collagenolytic serine proteases. In this article, we review and compare the allergenicity and proteolytic effect of allergen serine proteases as listed in the Allergen Nomenclature and MEROPS databases and highlight their contribution to allergic sensitisation, disruption of the epithelial barrier and activation of innate immunity in allergic airways disease. The utility of small-molecule inhibitors of allergen serine proteases as a potential treatment strategy for allergic airways disease will also be discussed.

House dust mite allergens induce Ca2+ signalling and alarmin responses in asthma airway epithelial cells.

Type 2 inflammation in asthma develops with exposure to stimuli to include inhaled allergens from house dust mites (HDM). Features include mucus hypersecretion and the formation of pro-secretory ion transport characterised by elevated basal Cl- current. Studies using human sinonasal epithelial cells treated with HDM extract report a higher protease activated receptor-2 (PAR-2) agonist-induced calcium mobilisation that may be related to airway sensitisation by allergen-associated proteases. Herein, this study aimed to investigate the effect of HDM on Ca2+ signalling and inflammatory responses in asthmatic airway epithelial cells. Primary bronchial epithelial cells (hPBECs) from asthma donors cultured at air-liquid interface were used to assess electrophysiological, Ca2+ signalling and inflammatory responses. Differences were observed regarding Ca2+ signalling in response to PAR-2 agonist 2-Furoyl-LIGRLO-amide (2-FLI), and equivalent short-circuit current (Ieq) in response to trypsin and 2-FLI, in ALI-asthma and healthy hPBECs. HDM treatment led to increased levels of intracellular cations (Ca2+, Na+) and significantly reduced the 2-FLI-induced change of Ieq in asthma cells. Apical HDM-induced Ca2+ mobilisation was found to mainly involve the activation of PAR-2 and PAR-4-associated store-operated Ca2+ influx and TRPV1. In contrast, PAR-2, PAR-4 antagonists and TRPV1 antagonist only showed slight impact on basolateral HDM-induced Ca2+ mobilisation. HDM trypsin-like serine proteases were the main components leading to non-amiloride sensitive Ieq and also increased interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP) from asthma hPBECs. These studies add further insight into the complex mechanisms associated with HDM-induced alterations in cell signalling and their relevance to pathological changes within asthma.

Furin as a therapeutic target in cystic fibrosis airways disease.

Clinical management of cystic fibrosis (CF) has been greatly improved by the development of small molecule modulators of the CF transmembrane conductance regulator (CFTR). These drugs help to address some of the basic genetic defects of CFTR; however, no suitable CFTR modulators exist for 10% of people with CF (PWCF). An alternative, mutation-agnostic therapeutic approach is therefore still required. In CF airways, elevated levels of the proprotein convertase furin contribute to the dysregulation of key processes that drive disease pathogenesis. Furin plays a critical role in the proteolytic activation of the epithelial sodium channel; hyperactivity of which causes airways dehydration and loss of effective mucociliary clearance. Furin is also responsible for the processing of transforming growth factor-ß, which is increased in bronchoalveolar lavage fluid from PWCF and is associated with neutrophilic inflammation and reduced pulmonary function. Pathogenic substrates of furin include Pseudomonas exotoxin A, a major toxic product associated with Pseudomonas aeruginosa infection and the spike glycoprotein of severe acute respiratory syndrome coronavirus 2, the causative pathogen for coronavirus disease 2019. In this review we discuss the importance of furin substrates in the progression of CF airways disease and highlight selective furin inhibition as a therapeutic strategy to provide clinical benefit to all PWCF.

The TMEM16A blockers benzbromarone and MONNA cause intracellular Ca2+-release in mouse bronchial smooth muscle cells.

We investigated effects of TMEM16A blockers benzbromarone, MONNA, CaCCinhA01 and Ani9 on isometric contractions in mouse bronchial rings and on intracellular calcium in isolated bronchial myocytes. Separate concentrations of carbachol (0.1-10 µM) were applied for 10 min periods to bronchial rings, producing concentration-dependent contractions that were well maintained throughout each application period. Benzbromarone (1 µM) markedly reduced the contractions with a more pronounced effect on their sustained component (at 10 min) compared to their initial component (at 2 min). Iberiotoxin (0.3 µM) enhanced the contractions, but they were still blocked by benzbromarone. MONNA (3 µM) and CaCCinhA01 (10 µM) had similar effects to benzbromarone, but were less potent. In contrast, Ani9 (10 µM) had no effect on carbachol-induced contractions. Confocal imaging revealed that benzbromarone (0.3 µM), MONNA (1 µM) and CaCCinhA01 (10 µM) increased intracellular calcium in isolated myocytes loaded with Fluo-4AM. In contrast, Ani9 (10 µM) had no effect on intracellular calcium. Benzbromarone and MONNA also increased calcium in calcium-free extracellular solution, but failed to do so when intracellular stores were discharged with caffeine (10 mM). Caffeine was unable to cause further discharge of the store when applied in the presence of benzbromarone. Ryanodine (100 µM) blocked the ability of benzbromarone (0.3 µM) to increase calcium, while tetracaine (100 µM) reversibly reduced the rise in calcium induced by benzbromarone. We conclude that benzbromarone and MONNA caused intracellular calcium release, probably by opening ryanodine receptors. Their ability to block carbachol contractions was likely due to this off-target effect.

Novel inhibitors and activity-based probes targeting serine proteases.

Serine proteases play varied and manifold roles in important biological, physiological, and pathological processes. These include viral, bacterial, and parasitic infection, allergic sensitization, tumor invasion, and metastasis. The use of activity-based profiling has been foundational in pinpointing the precise roles of serine proteases across this myriad of processes. A broad range of serine protease-targeted activity-based probe (ABP) chemotypes have been developed and we have recently introduced biotinylated and "clickable" peptides containing P1 N-alkyl glycine arginine N-hydroxy succinimidyl (NHS) carbamates as ABPs for detection/profiling of trypsin-like serine proteases. This present study provides synthetic details for the preparation of additional examples of this ABP chemotype, which function as potent irreversible inhibitors of their respective target serine protease. We describe their use for the activity-based profiling of a broad range of serine proteases including trypsin, the trypsin-like protease plasmin, chymotrypsin, cathepsin G, and neutrophil elastase (NE), including the profiling of the latter protease in clinical samples obtained from patients with cystic fibrosis.

Novel Inhibitors and Activity-Based Probes Targeting Trypsin-Like Serine Proteases.

The trypsin-like proteases (TLPs) play widespread and diverse roles, in a host of physiological and pathological processes including clot dissolution, extracellular matrix remodelling, infection, angiogenesis, wound healing and tumour invasion/metastasis. Moreover, these enzymes are involved in the disruption of normal lung function in a range of respiratory diseases including allergic asthma where several allergenic proteases have been identified. Here, we report the synthesis of a series of peptide derivatives containing an N-alkyl glycine analogue of arginine, bearing differing electrophilic leaving groups (carbamate and triazole urea), and demonstrate their function as potent, irreversible inhibitors of trypsin and TLPs, to include activities from cockroach extract. As such, these inhibitors are suitable for use as activity probes (APs) in activity-based profiling (ABP) applications.

Modulation of carbachol-induced Ca2+ oscillations in airway smooth muscle cells by PGE2.

PGE2 is a potent bronchodilator, but the mechanisms underlying this effect have not been fully elucidated. Acetylcholine-induced contractions of airway smooth muscle (ASM) are associated with the generation of repetitive Ca2+ oscillations in airway smooth muscle cells (ASMC) and the force of contraction is positively correlated with the frequency of the underlying Ca2+ oscillations. The purpose of the present study was to examine if carbachol-evoked Ca2+ oscillations in isolated ASMC were inhibited by PGE2. Isolated murine ASMC loaded with fluo4-AM were imaged with a Nipkow spinning disk confocal microscope. Cells responded to application of CCh (1 µM) by generating an initial Ca2+ transient followed by a series of Ca2+ oscillations. This activity was abolished by PGE2 (300 nM) and the EP2R agonist (R)-butaprost (3 µM) and the inhibitory effects of PGE2 were reversed by application of the EP2R antagonist PF-04418948 (100 nM). Activation of adenylate cyclase using forskolin (1 µM) mimicked the effects of PGE2. The PKA activator, 6-MB-cAMP (300 µM) reduced the frequency of CCh-induced Ca2+ oscillations by 33% and the PKA inhibitor Rp-8-CPT-cAMPs partially reversed the inhibitory effects of PGE2. The EPAC activator 007-AM (10 µM) reduced the frequency of the oscillations by 60% and joint application of 007-AM and 6-MB-cAMP reduced oscillation frequency by ∼85%. CCh-induced Ca2+ oscillations were inhibited by 2-APB and tetracaine, but caffeine-evoked Ca2+ transients were resistant to PGE2. These data suggest that PGE2 inhibits CCh-induced Ca2+ oscillations in murine ASMC via stimulation of EP2Rs and a mechanism involving activation of PKA and EPAC.

A highly selective, cell-permeable furin inhibitor BOS-318 rescues key features of cystic fibrosis airway disease.

In cystic fibrosis (CF), excessive furin activity plays a critical role in the activation of the epithelial sodium channel (ENaC), dysregulation of which contributes to airway dehydration, ineffective mucociliary clearance (MCC), and mucus obstruction. Here, we report a highly selective, cell-permeable furin inhibitor, BOS-318, that derives selectivity by eliciting the formation of a new, unexpected binding pocket independent of the active site catalytic triad. Using human ex vivo models, BOS-318 showed significant suppression of ENaC, which led to enhanced airway hydration and an ∼30-fold increase in MCC rate. Furin inhibition also protected ENaC from subsequent activation by neutrophil elastase, a soluble protease dominant in CF airways. Additional therapeutic benefits include protection against epithelial cell death induced by Pseudomonas aeruginosa exotoxin A. Our findings demonstrate the utility of selective furin inhibition as a mutation-agnostic approach that can correct features of CF airway pathophysiology in a manner expected to deliver therapeutic value.

Investigating the Transcriptome of Candida albicans in a Dual-Species Staphylococcus aureus Biofilm Model.

Candida albicans is an opportunistic pathogen found throughout multiple body sites and is frequently co-isolated from infections of the respiratory tract and oral cavity with Staphylococcus aureus. Herein we present the first report of the effects that S. aureus elicits on the C. albicans transcriptome. Dual-species biofilms containing S. aureus and C. albicans mutants defective in ALS3 or ECE1 were optimised and characterised, followed by transcriptional profiling of C. albicans by RNA-sequencing (RNA-seq). Altered phenotypes in C. albicans mutants revealed specific interaction profiles between fungus and bacteria. The major adhesion and virulence proteins Als3 and Ece1, respectively, were found to have substantial effects on the Candida transcriptome in early and mature biofilms. Despite this, deletion of ECE1 did not adversely affect biofilm formation or the ability of S. aureus to interact with C. albicans hyphae. Upregulated genes in dual-species biofilms corresponded to multiple gene ontology terms, including those attributed to virulence, biofilm formation and protein binding such as ACE2 and multiple heat-shock protein genes. This shows that S. aureus pushes C. albicans towards a more virulent genotype, helping us to understand the driving forces behind the increased severity of C. albicans-S. aureus infections.

The Serpin Superfamily and Their Role in the Regulation and Dysfunction of Serine Protease Activity in COPD and Other Chronic Lung Diseases.

Chronic obstructive pulmonary disease (COPD) is a debilitating heterogeneous disease characterised by unregulated proteolytic destruction of lung tissue mediated via a protease-antiprotease imbalance. In COPD, the relationship between the neutrophil serine protease, neutrophil elastase, and its endogenous inhibitor, alpha-1-antitrypsin (AAT) is the best characterised. AAT belongs to a superfamily of serine protease inhibitors known as serpins. Advances in screening technologies have, however, resulted in many members of the serpin superfamily being identified as having differential expression across a multitude of chronic lung diseases compared to healthy individuals. Serpins exhibit a unique suicide-substrate mechanism of inhibition during which they undergo a dramatic conformational change to a more stable form. A limitation is that this also renders them susceptible to disease-causing mutations. Identification of the extent of their physiological/pathological role in the airways would allow further expansion of knowledge regarding the complexity of protease regulation in the lung and may provide wider opportunity for their use as therapeutics to aid the management of COPD and other chronic airways diseases.

Trypsin-Like Proteases and Their Role in Muco-Obstructive Lung Diseases.

Trypsin-like proteases (TLPs) belong to a family of serine enzymes with primary substrate specificities for the basic residues, lysine and arginine, in the P1 position. Whilst initially perceived as soluble enzymes that are extracellularly secreted, a number of novel TLPs that are anchored in the cell membrane have since been discovered. Muco-obstructive lung diseases (MucOLDs) are characterised by the accumulation of hyper-concentrated mucus in the small airways, leading to persistent inflammation, infection and dysregulated protease activity. Although neutrophilic serine proteases, particularly neutrophil elastase, have been implicated in the propagation of inflammation and local tissue destruction, it is likely that the serine TLPs also contribute to various disease-relevant processes given the roles that a number of these enzymes play in the activation of both the epithelial sodium channel (ENaC) and protease-activated receptor 2 (PAR2). More recently, significant attention has focused on the activation of viruses such as SARS-CoV-2 by host TLPs. The purpose of this review was to highlight key TLPs linked to the activation of ENaC and PAR2 and their association with airway dehydration and inflammatory signalling pathways, respectively. The role of TLPs in viral infectivity will also be discussed in the context of the inhibition of TLP activities and the potential of these proteases as therapeutic targets.

Modulation of Ion Transport to Restore Airway Hydration in Cystic Fibrosis.

Cystic fibrosis (CF) is a life-limiting genetic disorder caused by loss-of-function mutations in the gene which codes for the CF transmembrane conductance regulator (CFTR) Cl- channel. Loss of Cl- secretion across the apical membrane of airway lining epithelial cells results in dehydration of the airway surface liquid (ASL) layer which impairs mucociliary clearance (MCC), and as a consequence promotes bacterial infection and inflammation of the airways. Interventions that restore airway hydration are known to improve MCC. Here we review the ion channels present at the luminal surface of airway epithelial cells that may be targeted to improve airway hydration and MCC in CF airways.

Non-typeable Haemophilus influenzae chronic colonization in chronic obstructive pulmonary disease (COPD).

Haemophilus influenzae is the most common cause of bacterial infection in the lungs of chronic obstructive pulmonary disease (COPD) patients and contributes to episodes of acute exacerbation which are associated with increased hospitalization and mortality. Due to the ability of H. influenzae to adhere to host epithelial cells, initial colonization of the lower airways can progress to a persistent infection and biofilm formation. This is characterized by changes in bacterial behaviour such as reduced cellular metabolism and the production of an obstructive extracellular matrix (ECM). Herein we discuss the multiple mechanisms by which H. influenzae contributes to the pathogenesis of COPD. In particular, mechanisms that facilitate bacterial adherence to host airway epithelial cells, biofilm formation, and microbial persistence through immune system evasion and antibiotic tolerance will be discussed.

Perspectives on neuroinflammation contributing to chronic cough.

Chronic cough can be a troublesome clinical problem. Current thinking is that increased activity and/or enhanced sensitivity of the peripheral and central neural pathways mediates chronic cough via processes similar to those associated with the development of chronic pain. While inflammation is widely thought to be involved in the development of chronic cough, the true mechanisms causing altered neural activity and sensitisation remain largely unknown. In this back-to-basics perspective article we explore evidence that inflammation in chronic cough may, at least in part, involve neuroinflammation orchestrated by glial cells of the nervous system. We summarise the extensive evidence for the role of both peripheral and central glial cells in chronic pain, and hypothesise that the commonalities between pain and cough pathogenesis and clinical presentation warrant investigations into the neuroinflammatory mechanisms that contribute to chronic cough. We open the debate that glial cells may represent an underappreciated therapeutic target for controlling troublesome cough in disease.

A Novel Serine Protease Inhibitor PE-BBI Ameliorates Cockroach Extract-Mediated Airway Epithelial Barrier Dysfunction.

Epithelial barrier dysfunction, characteristic of allergic airway disease may be, at least in part, due to the action of allergen-associated protease activities. Cockroach allergy is a major global health issue, with cockroaches containing considerable serine trypsin-like protease (TLP) activity. The present study sought to evaluate two novel protease inhibitors (PE-BBI and pLR-HL), recently isolated from amphibian skin secretions, for their potential to neutralise cockroach TLP activity and to determine any protective effect on cockroach-induced airway epithelial barrier disruption. Inhibitor potencies against the cockroach-associated activities were determined using a fluorogenic peptide substrate-based activity assay. 16HBE14o- cells (16HBE; a bronchial epithelial cell line) were treated with cockroach extract (CRE) in the presence or absence of the compounds in order to assess cell viability (RealTime Glo luminescent assay) and epithelial barrier disruption (transepithelial resistance and paracellular dextran flux). PE-BBI potently and selectively inhibited CRE TLP activity (pIC50 -8), but not host (16HBE) cell surface activity, which conferred protection of 16HBE cells from CRE-induced cell damage and barrier disruption. Novel protease inhibitor strategies such as PE-BBI may be useful for the treatment of allergic airway disease caused by cockroach proteases.

Ion channels as targets to treat cystic fibrosis lung disease.

Lung health relies on effective mucociliary clearance and innate immune defence mechanisms. In cystic fibrosis (CF), an imbalance in ion transport due to an absence of chloride ion secretion, caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) and a concomitant sodium hyperabsorption, caused by dyregulation of the epithelial sodium channel (ENaC), results in mucus stasis which predisposes the lungs to cycles of chronic infection and inflammation leading to lung function decline. An increased understanding of CFTR structure and function has provided opportunity for the development of a number of novel modulators targeting mutant CFTR however, it is important to also consider other ion channels and transporters present in the airways as putative targets for drug development. In this review, we discuss recent advances in CFTR biology which will contribute to further drug discovery in the field. We also examine developments to inhibit the epithelial sodium channel (ENaC) and potentially activate alternative chloride channels and transporters as a multi-tracked strategy to hydrate CF airways and restore normal mucociliary clearance mechanisms in a manner independent of CFTR mutation.

Neutrophil Elastase Activity Is Associated with Exacerbations and Lung Function Decline in Bronchiectasis.

RATIONALE: Sputum neutrophil elastase and serum desmosine, which is a linked marker of endogenous elastin degradation, are possible biomarkers of disease severity and progression in bronchiectasis. This study aimed to determine the association of elastase activity and desmosine with exacerbations and lung function decline in bronchiectasis. METHODS: This was a single-center prospective cohort study using the TAYBRIDGE (Tayside Bronchiectasis Registry Integrating Datasets, Genomics, and Enrolment into Clinical Trials) registry in Dundee, UK. A total of 433 patients with high-resolution computed tomography-confirmed bronchiectasis provided blood samples for desmosine measurement, and 381 provided sputum for baseline elastase activity measurements using an activity-based immunosassay and fluorometric substrate assay. Candidate biomarkers were tested for their relationship with cross-sectional markers of disease severity, and with future exacerbations, mortality and lung function decline over 3 years. MEASUREMENT AND MAIN RESULTS: Elastase activity in sputum was associated with the bronchiectasis severity index (r = 0.49; P < 0.0001) and was also correlated with the Medical Research Council dyspnea score (r = 0.34; P < 0.0001), FEV1% predicted (r = -0.33; P < 0.0001), and the radiological extent of bronchiectasis (r = 0.29; P < 0.0001). During a 3-year follow-up, elevated sputum elastase activity was associated with a higher frequency of exacerbations (P < 0.0001) but was not independently associated with mortality. Sputum elastase activity was independently associated with FEV1 decline (ß coefficient, -0.139; P = 0.001). Elastase showed good discrimination for severe exacerbations with an area under the curve of 0.75 (95% confidence interval [CI], 0.72-0.79) and all-cause mortality (area under the curve, 0.70; 95% CI, 0.67-0.73). Sputum elastase activity increased at exacerbations (P = 0.001) and was responsive to treatment with antibiotics. Desmosine was correlated with sputum elastase (r = 0.42; P < 0.0001) and was associated with risk of severe exacerbations (hazard ratio 2.7; 95% CI, 1.42-5.29; P = 0.003) but not lung function decline. CONCLUSIONS: Sputum neutrophil elastase activity is a biomarker of disease severity and future risk in adults with bronchiectasis.

Bacterial proteases and haemostasis dysregulation in the CF lung.

BACKGROUND: Pathogenic bacteria which chronically colonise the cystic fibrosis (CF) lung produce a number of virulence determinants, including distinct proteolytic activities. The potential role bacterial proteases play on haemostatic dysregulation within the CF lung is, however, poorly defined, despite haemoptysis being a common complication in CF. METHODS: The potential impact of known CF pathogens (Pseudomonas aeruginosa and Burkholderia cepacia complex spp.) on haemostasis was examined for their ability to degrade fibrinogen and dysregulate fibrin clot formation and platelet aggregation. RESULTS: Results demonstrate that key CF pathogens growing as a biofilm on mucin exhibit considerable fibrinogenolytic activity, resulting in fibrinogen breakdown, impaired clot formation, and modulation of platelet aggregation. Human neutrophil elastase may also contribute to fibrinogen breakdown and dysregulated clot formation at high concentration. CONCLUSION: Bacterial-derived proteases may play an important role in the dysregulation of airway haemostasis, and potentially contribute to episodes of haemoptysis within the CF lung.