Molecular pharmacology of the capsaicin receptor (TRPV1) in the airways

The capsaicin receptor (vanilloid receptor I, transient receptor potential vanilloid 1 or TRPV I) is a member of the transient receptor potential (TRP) family of proteins. This cation channel is sensitive to a range of inflammatory mediators such as some lipoxygenase products, as well as the tussive agents capsaicin, resiniferatoxin and protons. It has been proposed that TRPV I is a cough receptor and may be important in airways inflammation.Rat TRPVI (rTRPVI) and human TRPVI (hTRPVl) permanently expressing cell lines were generated and successfully characterised by agonist triggered changes in intracellular calcium levels. Thapsigargin and/or removal of extracellular calcium revealed that, both rTRPVI and hTRPVI are not only expressed on the cell surface but on thapsigargin sensitive and insensitive intracellular stores respectively.Citric acid, an agent routinely used in the clinic for inhalation cough challenges, was investigated for its ability to activate TRPVl permanently expressed in a cell line. rTRPV I was activated by citric acid in a concentration and pH dependent manner. Citric acid activation of TRPVI was inhibited by iodoresiniferatoxin but not capsazepine. Mutation of the TRPVI putative proton binding site (E648 to A648) abolished citric acid activation of the channel without reducing the capsaicin evoked response. Thus, citric acid activates rTRPV I by a proton dependent mechanism.The role of N-linked glycosylation and sialylation on rTRPVI and hTRPVI was investigated. Treatment of rTRPVl with neuraminidase or tunicamycin dramatically reduced the channels' maximal responses to capsaicin. In addition mutation of the rTRPVI N-linked glycosylation site (N604 to Q604) or expression ofrTRPVI in the glycosylation mutant cell line, Lec2, also resulted in a striking reduction in the receptors' maximal calcium response to capsaicin. Flow cytometry data indicated that these differences in TRPVI function were unlikely to be linked to differences in receptor cell surface expression. Human TRPV I also displayed significant reductions in responsiveness to capsaicin following either neuraminidase or tunicamycin treatment. Thus, receptor sialylation regulates TRPVI activation by capsaicin.Finally, TRPVI expression on human primary bronchial fibroblasts (HPBF) was investigated. Negligible endogenous TRPVI expression was detected in HPBF. Interestingly, the inflammatory mediators tumour necrosis factor (TNF-a), lipopolysaccharide (LPS) and interleukin Ia (IL-Ia) all induced TRPVI expression in HPBF, as assessed by RT-PCR, flow cytometry and calcium signalling. TRPVI functional expression was observed as early as 6 hrs (for TNF-a) post challenge and remained elevated upto the final time point tested (96 hrs for IL-Ia). Thus, TRPVI may play an important role in the inflammatory process.In conclusion, TRPV I may play an important role in conditions where cough and inflammation have been implicated

Bleomycin increases neutrophil adhesion to human vascular endothelial cells independently of upregulation of ICAM-1 and E-selectin

© 2016 Taylor & Francis. Aim of the Study: Bleomycin-induced lung disease is a serious complication of therapy characterized by alveolar injury, cytokine release, inflammatory cell recruitment, and eventually pulmonary fibrosis. The mechanisms underlying bleomycin-induced pulmonary fibrosis may be relevant to other progressive scarring diseases of the lungs. Pulmonary vascular endothelial cells are critically involved in immune cell extravasation at sites of injury through adhesion molecule expression and cytokine release. We sought to determine the effects of bleomycin on adhesion molecule expression and cytokine release by pulmonary vascular endothelial cells, and their functional relevance to inflammatory cell recruitment. Materials and Methods: The effects of pharmacologically relevant concentrations of bleomycin on adhesion molecule expression and cytokine release by human vascular endothelial cells in vitro were studied by flow cytometry, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. A flow chamber model was used to assess the functional consequences on adhesion of flowing human neutrophils to endothelial cell monolayers. Results: Bleomycin increased intercellular adhesion molecule 1 (ICAM-1; CD54), vascular cell adhesion molecule (VCAM-1; CD106), and E-selectin (CD62E) expression, and increased monocyte chemoattractant protein (MCP-1) and interleukin (IL-8) release by endothelial cells. Increases in protein expression were accompanied by increased mRNA transcription. In contrast, there was no direct effect of bleomycin on the profibrotic cytokines transforming growth factor-beta (TGF-β), platelet-derived growth factor-BB (PDGF-BB), or endothelin-1. Under flow conditions, endothelial cells exposed to bleomycin supported increased neutrophil adhesion which was independent of ICAM-1 or E-selectin. Conclusion: Our findings demonstrate that bleomycin promotes endothelial-mediated inflammation and neutrophil adhesion. These mechanisms may contribute to the development of pulmonary fibrosis by supporting immune cell recruitment in the lungs

Modulation of transient receptor potential (TRP) channels by plant derived substances used in over-the-counter cough and cold remedies

Background: Upper respiratory tract infections (URTIs) impact all age groups and have a significant economic and social burden on society, worldwide. Most URTIs are mild and self-limiting, but due to the wide range of possible causative agents, including Rhinovirus (hRV), Adenovirus, Respiratory Syncytial Virus (RSV), Coronavirus and Influenza, there is no single and effective treatment. Over-the-counter (OTC) remedies, including traditional medicines and those containing plant derived substances, help to alleviate symptoms including inflammation, pain, fever and cough. Purpose: This systematic review focuses on the role of the major plant derived substances in several OTC remedies used to treat cold symptoms, with a particular focus on the transient receptor potential (TRP) channels involved in pain and cough. Methods: Literature searches were done using Pubmed and Web of Science, with no date limitations, using the principles of the PRISMA statement. The search terms used were ‘TRP channel AND plant compound’, ‘cough AND plant compound’, ‘cough AND TRP channels AND plant compound’, ‘cough AND P2X3 AND plant compound’ and ‘P2X3 AND plant compound’ where plant compound represents menthol or camphor or eucalyptus or turpentine or thymol.Results: The literature reviewed showed that menthol activates TRPM8 and may inhibit respiratory reflexes reducing irritation and cough. Menthol has a bimodal action on TRPA1, but inhibition may have an analgesic effect. Eucalyptus also activates TRPM8 and inhibits TRPA1 whilst down regulating P2X3, aiding in the reduction of cough, pain and airway irritation. Camphor inhibits TRPA1 and the activation of TRPM8 may add to the effects of menthol. Activation of TRPV1 by camphor, may also have an analgesic effect. Conclusions: The literature suggests that these plant derived substances have multifaceted actions and can interact with the TRP ‘cough’ receptors. The plant derived substances used in cough and cold medicines have the potential to target multiple symptoms experienced during a cold

Rhinovirus-16 increases ATP release in A549 cells without concomitant increase in production

Human rhinovirus (RV) is the most common cause of upper respiratory tract infection (URTI) and chronic airway disease exacerbation. Cough is present in 50–80% of URTI cases, accompanied by heightened airway hypersensitivity, yet no effective treatment currently exists for this infectious cough. The mechanism by which RV causes cough and airway hypersensitivity in URTI is still unknown despite recent advances in potential therapies for chronic cough.The effect of RV-16 infection (MOI 1) on intracellular ATP stores and ATP release in A549 alveolar epithelial cells was measured.RV-16 infection was found to significantly increase (by 50% from basal at 24 h) followed by decrease (by 50% from basal at 48 and 72 h) intracellular ATP concentrations, while increasing ATP release (from 72 h) independently of secondary stimulation. This effect was mimicked by intercellular adhesion molecule 1 receptor binding alone through ultraviolet-inactivated sham control. In addition, RV-16-infected cells became more sensitive to secondary stimulation with both hypotonic and isotonic solutions, suggestive of a hypersensitive response. These responses were not mediated via increased TRPV4 or pannexin-1 whole-cell expression as determined by Western blotting. Interestingly, the increased ATP release seen was not a result of increased mitochondrial ATP production.Thus, this is the first report demonstrating that RV-16 infection of airway epithelial cells causes hypersensitivity by increasing ATP release. These finding provide a novel insight into the process by which viruses may cause cough and identify a potential target for treatment of viral and post-viral cough

ATP, an attractive target for the treatment of refractory chronic cough

Chronic cough is the most common complaint in respiratory clinics. Most of them have identifiable causes and some may respond to common disease-modifying therapies. However, there are many patients whose cough lacks effective aetiologically targeted treatments or remains unexplained after thorough assessments, which have been described as refractory chronic cough. Current treatments for refractory chronic cough are limited and often accompanied by intolerable side effects such as sedation. In recent years, various in-depth researches into the pathogenesis of chronic cough have led to an explosion in the development of drugs for the treatment of refractory chronic cough. There has been considerable progress in the underlying mechanisms of chronic cough targeting ATP, and ongoing or completed clinical studies have confirmed the promising antitussive efficacy of P2X3 antagonists for refractory cough. Herein, we review the foundation on which ATP target was developed as potential antitussive medications and provide an update on current clinical progresses

Chronic cough—the limitation and advances in assessment techniques

Accurate and consistent assessments of cough are essential to advance the understanding of the mechanisms of cough and individualised the management of patients. Considerable progress has been made in this work. Here we reviewed the currently available tools for subjectively and objectively measuring both cough sensitivity and severity. We also provided some opinions on the new techniques and future directions. The simple and practical Visual Analogue Scale (VAS), the Leicester Cough Questionnaire (LCQ), and the Cough Specific Quality of Life Questionnaire (CQLQ) are the most widely used self-reported questionnaires for evaluating and quantifying cough severity. The Hull Airway Reflux Questionnaire (HARQ) is a tool to elucidate the constellation of symptoms underlying the diagnosis of chronic cough. Chemical excitation tests are widely used to explore the pathophysiological mechanisms of the cough reflex, such as capsaicin, citric acid and adenosine triphosphate (ATP) challenge test. Cough frequency is an ideal primary endpoint for clinical research, but the application of cough counters has been limited in clinical practice by the high cost and reliance on aural validation. The ongoing development of cough detection technology for smartphone apps and wearable devices will hopefully simplify cough counting, thus transitioning it from niche research to a widely available clinical application

Lipid laden macrophages in respiratory disease

Letter to the edito

Outdoor Microplastic Analysis Using Inlet Filters from an NOx Regulatory Air Quality Monitoring Device

Atmospheric microplastics (MPs) are a ubiquitous environmental contaminant of emerging concern. Sampling methods provide information relating to surface area concentration and MP characteristics, without direct comparison with routinely measured standard air quality parameters. This study analysed 6 active air samples generated by a local authority as part of their routine air quality monitoring activities. Continuous sampling totalled 10 months, within the city centre of Kingston-upon-Hull. By using μFTIR analysis, levels of total particles detected using the NOx inlet filters ranged from 5139 ± 2843 particles m−2 day−1, comprising 1029 ± 594 MPs m−2 day−1. The controls displayed a mean level of 2.00 ± 3.49 MPs. The polymers nylon (32%) and polypropylene, PP (22%) were the most abundant. Small fragments of 47.42 ± 48.57 μm (length) and 21.75 ± 13.62 μm (width) were most common. An increase in MP levels during April 2020 coincided with an increase in PM10 levels. This study used robust procedures to measure MPs in the air by exploiting existing air quality monitoring equipment. Knowing the levels, types, and characteristics of MPs can inform toxicity studies to provide more environmentally relevant exposures, which is urgent now that MPs have been reported in human lung tissu

TRPA1 mediates aromatase inhibitor-evoked pain by the aromatase substrate androstenedione

Aromatase inhibitors (AI) induce painful musculoskeletal symptoms (AIMSS), which are dependent upon the pain transducing receptor TRPA1. However, as the AI concentrations required to engage TRPA1 in mice are higher than those found in the plasma of patients, we hypothesized that additional factors may cooperate to induce AIMSS. Here we report that the aromatase substrate androstenedione, unique among several steroid hormones, targeted TRPA1 in peptidergic primary sensory neurons in rodent and human cells expressing the native or recombinant channel. Androstenedione dramatically lowered the concentration of letrozole required to engage TRPA1. Notably, addition of a minimal dose of androstenedione to physiologically ineffective doses of letrozole and oxidative stress byproducts produces AIMSS-like behaviors and neurogenic inflammatory responses in mice. Elevated androstenedione levels cooperated with low letrozole concentrations and inflammatory mediators were sufficient to provoke AIMSS-like behaviors. The generation of such painful conditions by small quantities of simultaneously administered TRPA1 agonists justifies previous failure to identify a precise link between AIs and AIMSS, underscoring the potential of channel antagonists to treat AIMSS

Characterisation of a New Human Alveolar Macrophage-Like Cell Line (Daisy)

Purpose: There is currently no true macrophage cell line and in vitro experiments requiring these cells currently require mitogenic stimulation of a macrophage precursor cell line (THP-1) or ex vivo maturation of circulating primary monocytes. In this study, we characterise a human macrophage cell line, derived from THP-1 cells, and compare its phenotype to the THP-1 cells. Methods: THP-1 cells with and without mitogenic stimulation were compared to the newly derived macrophage-like cell line (Daisy) using microscopy, flow cytometry, phagocytosis assays, antigen binding assays and gene microarrays. Results: We show that the cell line grows predominantly in an adherent monolayer. A panel of antibodies were chosen to investigate the cell surface phenotype of these cells using flow cytometry. Daisy cells expressed more CD11c, CD80, CD163, CD169 and CD206, but less CD14 and CD11b compared with mitogen-stimulated THP-1 cells. Unlike stimulated THP-1 cells which were barely able to bind immune complexes, Daisy cells showed large amounts of immune complex binding. Finally, although not statistically significant, the phagocytic ability of Daisy cells was greater than mitogen-stimulated THP-1 cells, suggesting that the cell line is more similar to mature macrophages. Conclusions: The observed phenotype suggests that Daisy cells are a good model of human macrophages with a phenotype similar to human alveolar macrophages