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Journal articleTelcian AG, Zdrenghea MT, Edwards MR, et al., 2016,
Vitamin D increases the antiviral activity of bronchial epithelial cells in vitro
, Antiviral Research, Vol: 137, Pages: 93-101, ISSN: 1872-9096BACKGROUND: By modulating the antiviral immune response via vitamin D receptor, the active form of vitamin D (1,25-dihydroxyvitamin D, calcitriol) could play a central role in protection against respiratory virus infections. This in vitro study tested the hypothesis that respiratory viruses modulate vitamin D receptor expression in human bronchial epithelial cells and this modulation affects the antiviral response to exogenous vitamin D. METHODS: Human primary bronchial epithelial cells were infected with rhinoviruses and respiratory syncytial virus in the presence or absence of vitamin D. Expression of vitamin D receptor, 1α-hydroxylase (1α(OH)ase), 24-hydroxylase (24(OH)ase), innate interferons, interferon stimulated genes and cathelicidin were measured by quantitative polymerase chain reaction. The antiviral effect of vitamin D on rhinovirus replication was determined by measurement of virus load. A direct inactivation assay was used to determine the antiviral activity of cathelicidin. RESULTS: Both RV and RSV decreased vitamin D receptor and 24(OH)ase and, in addition, RSV increased 1α(OH)ase expression in epithelial cells. Vitamin D decreased rhinovirus replication and release, and increased rhinovirus-induced interferon stimulated genes and cathelicidin. Furthermore, cathelicidin had direct anti-rhinovirus activity. CONCLUSIONS: Despite lower vitamin D receptor levels in rhinovirus-infected epithelial cells, exogenous vitamin D increased antiviral defences most likely via cathelicidin and innate interferon pathways.
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Journal articleCarugo D, Aron M, Sezgin E, et al., 2016,
Modulation of the molecular arrangement in artificial and biological membranes by phospholipid-shelled microbubbles
, Biomaterials, Vol: 113, Pages: 105-117, ISSN: 1878-5905The transfer of material from phospholipid-coated microbubbles to cell membranes has been hypothesized to play a role in ultrasound-mediated drug delivery. In this study, we employed quantitative fluorescence microscopy techniques to investigate this phenomenon in both artificial and biological membrane bilayers in an acoustofluidic system. The results of the present study provide strong evidence for the transfer of material from microbubble coatings into cell membranes. Our results indicate that transfer of phospholipids alters the organization of molecules in cell membranes, specifically the lipid ordering or packing, which is known to be a key determinant of membrane mechanical properties, protein dynamics, and permeability. We further show that polyethylene-glycol, used in many clinical microbubble formulations, also has a major impact on both membrane lipid ordering and the extent of lipid transfer, and that this occurs even in the absence of ultrasound exposure.
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Journal articleBaker JR, Vuppusetty C, Colley T, et al., 2016,
Oxidative stress dependent microRNA-34a activation via PI3Kα reduces the expression of sirtuin-1 and sirtuin-6 in epithelial cells
, Scientific Reports, Vol: 6, ISSN: 2045-2322Sirtuin-1 (SIRT1) and SIRT6, NAD(+)-dependent Class III protein deacetylases, are putative anti-aging enzymes, down-regulated in patients with chronic obstructive pulmonary disease (COPD), which is characterized by the accelerated ageing of the lung and associated with increased oxidative stress. Here, we show that oxidative stress (hydrogen peroxide) selectively elevates microRNA-34a (miR-34a) but not the related miR-34b/c, with concomitant reduction of SIRT1/-6 in bronchial epithelial cells (BEAS2B), which was also observed in peripheral lung samples from patients with COPD. Over-expression of a miR-34a mimic caused a significant reduction in both mRNA and protein of SIRT1/-6, whereas inhibition of miR-34a (antagomir) increased these sirtuins. Induction of miR-34a expression with H2O2 was phosphoinositide-3-kinase (PI3K) dependent as it was associated with PI3Kα activation as well as phosphatase and tensin homolog (PTEN) reduction. Importantly, miR-34a antagomirs increased SIRT1/-6 mRNA levels, whilst decreasing markers of cellular senescence in airway epithelial cells from COPD patients, suggesting that this process is reversible. Other sirtuin isoforms were not affected by miR-34a. Our data indicate that miR-34a is induced by oxidative stress via PI3K signaling, and orchestrates ageing responses under oxidative stress, therefore highlighting miR-34a as a new therapeutic target and biomarker in COPD and other oxidative stress-driven aging diseases.
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Conference paperJarrett R, Ogg G, Salio M, et al., 2016,
Filaggrin inhibits house dust mite phospholipase generation of CD1a lipid antigens for recognition by T cells
, 20th Anniversary Conference of the British-Skin-Foundation on Skin Deep - 20 Years of Research, Publisher: WILEY-BLACKWELL, Pages: 50-50, ISSN: 0007-0963 -
Journal articleBernardino de la Serna J, Schuetz GJ, Eggeling C, et al., 2016,
There is no simple model of the plasma membrane organizationyy
, Frontiers in Cell and Developmental Biology, Vol: 4, ISSN: 2296-634XEver since technologies enabled the characterization of eukaryotic plasma membranes,heterogeneities in the distributions of its constituents were observed. Over the years thisled to the proposal of various models describing the plasma membrane organizationsuch as lipid shells, picket-and-fences, lipid rafts, or protein islands, as addressed innumerous publications and reviews. Instead of emphasizing on one model we in thisreview give a brief overview over current models and highlight how current experimentalwork in one or the other way do not support the existence of a single overarching model.Instead, we highlight the vast variety of membrane properties and components, theirinfluences and impacts. We believe that highlighting such controversial discoveries willstimulate unbiased research on plasma membrane organization and functionality, leadingto a better understanding of this essential cellular structure.
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Journal articleStanly TA, Fritzsche M, Banerji S, et al., 2016,
Critical importance of appropriate fixation conditions for faithful imaging of receptor microclusters
, Biology Open, Vol: 5, Pages: 1343-1350, ISSN: 2046-6390Receptor clustering is known to trigger signalling events that contributeto critical changes in cellular functions. Faithful imaging of suchclusters by means of fluorescence microscopy relies on the applicationof adequate cell fixation methods prior to immunolabelling in order toavoid artefactual redistribution by the antibodies themselves. Previouswork has highlighted the inadequacy of fixation with paraformaldehyde(PFA) alone for efficient immobilisation of membrane-associatedmolecules, and the advantages of fixation with PFA in combinationwith glutaraldehyde (GA). Using fluorescence microscopy, we herehighlight how inadequate fixation can lead to the formation ofartefactual clustering of receptors in lymphatic endothelial cells,focussing on the transmembrane hyaluronan receptors LYVE-1 andCD44, and the homotypic adhesion molecule CD31, each of whichdisplays their native diffuse surface distribution pattern only whenvisualised with the right fixation techniques, i.e. PFA/GA incombination. Fluorescence recovery after photobleaching (FRAP)confirms that the artefactual receptor clusters are indeed introduced byresidual mobility. In contrast, we observed full immobilisation ofmembrane proteins in cells that were fixed and then subsequentlypermeabilised, irrespective of whether the fixative was PFA or PFA/GAin combination. Our study underlines the importance of choosingappropriate sample preparation protocols for preserving authenticreceptor organisation in advanced fluorescence microscopy
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Journal articleBarnes PJ, 2016,
Kinases as Novel Therapeutic Targets in Asthma and Chronic Obstructive Pulmonary Disease
, Pharmacological Reviews, Vol: 68, Pages: 788-815, ISSN: 1521-0081Multiple kinases play a critical role in orchestrating the chronic inflammation and structural changes in the respiratory tract of patients with asthma and chronic obstructive pulmonary disease (COPD). Kinases activate signaling pathways that lead to contraction of airway smooth muscle and release of inflammatory mediators (such as cytokines, chemokines, growth factors) as well as cell migration, activation, and proliferation. For this reason there has been great interest in the development of kinase inhibitors as anti-inflammatory therapies, particular where corticosteroids are less effective, as in severe asthma and COPD. However, it has proven difficult to develop selective kinase inhibitors that are both effective and safe after oral administration and this has led to a search for inhaled kinase inhibitors, which would reduce systemic exposure. Although many kinases have been implicated in inflammation and remodeling of airway disease, very few classes of drug have reached the stage of clinical studies in these diseases. The most promising drugs are p38 MAP kinases, isoenzyme-selective PI3-kinases, Janus-activated kinases, and Syk-kinases, and inhaled formulations of these drugs are now in development. There has also been interest in developing inhibitors that block more than one kinase, because these drugs may be more effective and with less risk of losing efficacy with time. No kinase inhibitors are yet on the market for the treatment of airway diseases, but as kinase inhibitors are improved from other therapeutic areas there is hope that these drugs may eventually prove useful in treating refractory asthma and COPD.
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Journal articleBarnes PJ, 2016,
Inflammatory mechanisms in patients with chronic obstructive pulmonary disease
, Journal of Allergy and Clinical Immunology, Vol: 138, Pages: 16-27, ISSN: 1097-6825Chronic obstructive pulmonary disease (COPD) is associated with chronic inflammation affecting predominantly the lung parenchyma and peripheral airways that results in largely irreversible and progressive airflow limitation. This inflammation is characterized by increased numbers of alveolar macrophages, neutrophils, T lymphocytes (predominantly TC1, TH1, and TH17 cells), and innate lymphoid cells recruited from the circulation. These cells and structural cells, including epithelial and endothelial cells and fibroblasts, secrete a variety of proinflammatory mediators, including cytokines, chemokines, growth factors, and lipid mediators. Although most patients with COPD have a predominantly neutrophilic inflammation, some have an increase in eosinophil counts, which might be orchestrated by TH2 cells and type 2 innate lymphoid cells though release of IL-33 from epithelial cells. These patients might be more responsive to corticosteroids and bronchodilators. Oxidative stress plays a key role in driving COPD-related inflammation, even in ex-smokers, and might result in activation of the proinflammatory transcription factor nuclear factor κB (NF-κB), impaired antiprotease defenses, DNA damage, cellular senescence, autoantibody generation, and corticosteroid resistance though inactivation of histone deacetylase 2. Systemic inflammation is also found in patients with COPD and can worsen comorbidities, such as cardiovascular diseases, diabetes, and osteoporosis. Accelerated aging in the lungs of patients with COPD can also generate inflammatory protein release from senescent cells in the lung. In the future, it will be important to recognize phenotypes of patients with optimal responses to more specific therapies, and development of biomarkers that identify the therapeutic phenotypes will be important.
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Conference paperJarrett R, Salio M, Lloyd-Lavery A, et al., 2016,
Filaggrin inhibits generation of CD1a neolipid antigens by house dust mite-derived phospholipase
, Annual Meeting of the British-Society-for-Investigative-Dermatology (BSID), Publisher: WILEY-BLACKWELL, Pages: E49-E50, ISSN: 0007-0963 -
Journal articleGarcia E, Ragazzini C, Yu X, et al., 2016,
WIP and WICH/WIRE co-ordinately control invadopodium formation and maturation in human breast cancer cell invasion
, Scientific Reports, Vol: 6, ISSN: 2045-2322Cancer cells form actin-rich degradative protrusions (invasive pseudopods and invadopodia), whichallows their efficient dispersal during metastasis. Using biochemical and advanced imaging approaches,we demonstrate that the N-WASP-interactors WIP and WICH/WIRE play non-redundant roles incancer cell invasion. WIP interacts with N-WASP and cortactin and is essential for invadopodiumassembly, whereas WICH/WIRE regulates N-WASP activation to control invadopodium maturationand degradative activity. Our data also show that Nck interaction with WIP and WICH/WIRE modulatesinvadopodium maturation; changes in WIP and WICH/WIRE levels induce differential distribution ofNck. We show that WIP can replace WICH/WIRE functions and that elevated WIP levels correlate withhigh invasiveness. These findings identify a role for WICH/WIRE in invasiveness and highlight WIP asa hub for signaling molecule recruitment during invadopodium generation and cancer progression, aswell as a potential diagnostic biomarker and an optimal target for therapeutic approaches.
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Conference paperBernardino de la Serna J, Chang VT, Waithe D, et al., 2016,
T-Cells in Suspension Do Not Show Pre-Clustered LCK
, 60th Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 570A-570A, ISSN: 0006-3495 -
Journal articleJarrett R, Salio M, Lloyd-Lavery A, et al., 2016,
Filaggrin inhibits generation of CD1a neolipid antigens by house dust mite-derived phospholipase
, Science Translational Medicine, Vol: 8, ISSN: 1946-6234Atopic dermatitis is a common pruritic skin disease in which barrier dysfunction and cutaneous inflammation contribute to pathogenesis. Mechanisms underlying the associated inflammation are not fully understood, and although Langerhans cells expressing the nonclassical major histocompatibility complex (MHC) family member CD1a are known to be enriched within lesions, their role in clinical disease pathogenesis has not been studied. We observed that house dust mite (HDM) allergen generates neolipid antigens presented by CD1a to T cells in the blood and skin lesions of affected individuals. HDM-responsive CD1a-reactive T cells increased in frequency after birth in individuals with atopic dermatitis and showed rapid effector function, consistent with antigen-driven maturation. In HDM-challenged human skin, we observed phospholipase A2 (PLA2) activity in vivo. CD1a-reactive T cell activation was dependent on HDM-derived PLA2, and such cells infiltrated the skin after allergen challenge. Moreover, we observed that the skin barrier protein filaggrin, insufficiency of which is associated with atopic skin disease, inhibited PLA2 activity and decreased CD1a-reactive PLA2-generated neolipid-specific T cell activity from skin and blood. The most widely used classification schemes of hypersensitivity suggest that nonpeptide stimulants of T cells act as haptens that modify peptides or proteins; however, our results show that HDM proteins may also generate neolipid antigens that directly activate T cells. These data define PLA2 inhibition as a function of filaggrin, supporting PLA2 inhibition as a therapeutic approach.
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Journal articleMitani A, Ito K, Vuppusetty C, et al., 2016,
Restoration of corticosteroid sensitivity in chronic obstructive pulmonary disease by inhibition of mammalian target of rapamycin
, American Journal of Respiratory and Critical Care Medicine, Vol: 193, Pages: 143-153, ISSN: 1073-449XRationale: Corticosteroid resistance is a major barrier to the effective treatment of chronic obstructive pulmonary disease (COPD). Several molecular mechanisms have been proposed, such as activations of the phosphoinositide-3-kinase/Akt pathway and p38 mitogen-activated protein kinase. However, the mechanism for corticosteroid resistance is still not fully elucidated.Objectives: To investigate the role of mammalian target of rapamycin (mTOR) in corticosteroid sensitivity in COPD.Methods: The corticosteroid sensitivity of peripheral blood mononuclear cells collected from patients with COPD, smokers, and nonsmoking control subjects, or of human monocytic U937 cells exposed to cigarette smoke extract (CSE), was quantified as the dexamethasone concentration required to achieve 30% inhibition of tumor necrosis factor-α–induced CXCL8 production in the presence or absence of the mTOR inhibitor rapamycin. mTOR activity was determined as the phosphorylation of p70 S6 kinase, using Western blotting.Measurements and Main Results: mTOR activity was increased in peripheral blood mononuclear cells from patients with COPD, and treatment with rapamycin inhibited this as well as restoring corticosteroid sensitivity. In U937 cells, CSE stimulated mTOR activity and c-Jun expression, but pretreatment with rapamycin inhibited both and also reversed CSE-induced corticosteroid insensitivity.Conclusions: mTOR inhibition by rapamycin restores corticosteroid sensitivity via inhibition of c-Jun expression, and thus mTOR is a potential novel therapeutic target for COPD.
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Journal articleClausen MP, Sezgin E, Bernardino de la Serna J, et al., 2015,
A straightforward approach for gated STED-FCS to investigate lipid membrane dynamics
, Methods, Vol: 88, Pages: 67-75, ISSN: 1046-2023Recent years have seen the development of multiple technologies to investigate, with great spatial and temporal resolution, the dynamics of lipids in cellular and model membranes. One of these approaches is the combination of far-field super-resolution stimulated-emission-depletion (STED) microscopy with fluorescence correlation spectroscopy (FCS). STED-FCS combines the diffraction-unlimited spatial resolution of STED microscopy with the statistical accuracy of FCS to determine sub-millisecond-fast molecular dynamics with single-molecule sensitivity. A unique advantage of STED-FCS is that the observation spot for the FCS data recordings can be tuned to sub-diffraction scales, i.e. <200 nm in diameter, in a gradual manner to investigate fast diffusion of membrane-incorporated labelled entities. Unfortunately, so far the STED-FCS technology has mostly been applied on a few custom-built setups optimised for far-red fluorescent emitters. Here, we summarise the basics of the STED-FCS technology and highlight how it can give novel details into molecular diffusion modes. Most importantly, we present a straightforward way for performing STED-FCS measurements on an unmodified turnkey commercial system using a time-gated detection scheme. Further, we have evaluated the STED-FCS performance of different commonly used green emitting fluorescent dyes applying freely available, custom-written analysis software.
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Journal articleWiegman CH, Michaeloudes C, Haji G, et al., 2015,
Oxidative stress-induced mitochondrial dysfunction drives inflammation and airway smooth muscle remodeling in patients with chronic obstructive pulmonary disease
, Journal of Allergy and Clinical Immunology, Vol: 136, Pages: 769-780, ISSN: 1097-6825BackgroundInflammation and oxidative stress play critical roles in patients with chronic obstructive pulmonary disease (COPD). Mitochondrial oxidative stress might be involved in driving the oxidative stress–induced pathology.ObjectiveWe sought to determine the effects of oxidative stress on mitochondrial function in the pathophysiology of airway inflammation in ozone-exposed mice and human airway smooth muscle (ASM) cells.MethodsMice were exposed to ozone, and lung inflammation, airway hyperresponsiveness (AHR), and mitochondrial function were determined. Human ASM cells were isolated from bronchial biopsy specimens from healthy subjects, smokers, and patients with COPD. Inflammation and mitochondrial function in mice and human ASM cells were measured with and without the presence of the mitochondria-targeted antioxidant MitoQ.ResultsMice exposed to ozone, a source of oxidative stress, had lung inflammation and AHR associated with mitochondrial dysfunction and reflected by decreased mitochondrial membrane potential (ΔΨm), increased mitochondrial oxidative stress, and reduced mitochondrial complex I, III, and V expression. Reversal of mitochondrial dysfunction by the mitochondria-targeted antioxidant MitoQ reduced inflammation and AHR. ASM cells from patients with COPD have reduced ΔΨm, adenosine triphosphate content, complex expression, basal and maximum respiration levels, and respiratory reserve capacity compared with those from healthy control subjects, whereas mitochondrial reactive oxygen species (ROS) levels were increased. Healthy smokers were intermediate between healthy nonsmokers and patients with COPD. Hydrogen peroxide induced mitochondrial dysfunction in ASM cells from healthy subjects. MitoQ and Tiron inhibited TGF-β–induced ASM cell proliferation and CXCL8 release.ConclusionsMitochondrial dysfunction in patients with COPD is associated with excessive mitochondrial ROS levels, which contribute to enhanced inflammat
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Journal articleBarnes PJ, 2015,
Therapeutic approaches to asthma-chronic obstructive pulmonary disease overlap syndromes
, JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY, Vol: 136, Pages: 531-545, ISSN: 0091-6749- Author Web Link
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- Citations: 120
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Journal articleBarnes PJ, 2015,
Identifying Molecular Targets for New Drug Development for Chronic Obstructive Pulmonary Disease: What Does the Future Hold?
, SEMINARS IN RESPIRATORY AND CRITICAL CARE MEDICINE, Vol: 36, Pages: 508-522, ISSN: 1069-3424- Author Web Link
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- Citations: 23
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Journal articleTetley TD, Ruenraroengsak P, 2015,
Differential bioreactivity of neutral, cationic and anionic polystyrene nanoparticles with cells from the human alveolar compartment: robust response of alveolar type 1 epithelial cells
, Particle and Fibre Toxicology, Vol: 12, ISSN: 1743-8977BackgroundEngineered nanoparticles (NP) are being developed for inhaled drug delivery. This route is non-invasive and the major target; alveolar epithelium provides a large surface area for drug administration and absorption, without first pass metabolism. Understanding the interaction between NPs and target cells is crucial for safe and effective NP-based drug delivery. We explored the differential effect of neutral, cationic and anionic polystyrene latex NPs on the target cells of the human alveolus, using primary human alveolar macrophages (MAC) and primary human alveolar type 2 (AT2) epithelial cells and a unique human alveolar epithelial type I-like cell (TT1). We hypothesized that the bioreactivity of the NPs would relate to their surface chemistry, charge and size as well as the functional role of their interacting cells in vivo.MethodsAmine- (ANP) and carboxyl- surface modified (CNP) and unmodified (UNP) polystyrene NPs, 50 and 100 nm in diameter, were studied. Cells were exposed to 1–100 μg/ml (1.25-125 μg/cm 2 ; 0 μg/ml control) NP for 4 and 24 h at 37 °C with or without the antioxidant, N-acetyl cysteine (NAC). Cells were assessed for cell viability, reactive oxygen species (ROS), oxidised glutathione (GSSG/GSH ratio), mitochondrial integrity, cell morphology and particle uptake (using electron microscopy and laser scanning confocal microscopy).ResultsANP-induced cell death occurred in all cell types, inducing increased oxidative stress, mitochondrial disruption and release of cytochrome C, indicating apoptotic cell death. UNP and CNP exhibited little cytotoxicity or mitochondrial damage, although they induced ROS in AT2 and MACs. Addition of NAC reduced epithelial cell ROS, but not MAC ROS, for up to 4 h. TT1 and MAC cells internalised all NP formats, whereas only a small fraction of AT2 cells internalized ANP (not UNP or CNP). TT1 cells were the most resistant to the effects of UNP and CNP.ConclusionANP induced marked oxidative damage
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Journal articleSezgin E, Waithe D, Bernardino de la Serna J, et al., 2015,
Spectral imaging to measure heterogeneity in membrane lipid packing
, ChemPhysChem, Vol: 16, Pages: 1387-1394, ISSN: 1439-7641Physicochemical properties of the plasma membrane have been shown to play an important role in cellular functionality. Among those properties, the molecular order of the lipids, or the lipid packing, is of high importance. Changes in lipid packing are believed to compartmentalize cellular signaling by initiating coalescence and conformational changes of proteins. A common way to infer membrane lipid packing is by using membrane‐embedded polarity‐sensitive dyes, whose emission spectrum is dependent on the molecular order of the immediate membrane environment. Here, we report on an improved determination of such spectral shifts in the emission spectrum of the polarity‐sensitive dyes. This improvement is based on the use of spectral imaging on a scanning confocal fluorescence microscope in combination with an improved analysis, which considers the whole emission spectrum instead of just single wavelength ranges. Using this approach and the polarity‐sensitive dyes C‐Laurdan or Di‐4‐ANEPPDHQ, we were able to image—with high accuracy—minute differences in the lipid packing of model and cellular membranes.
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Journal articleBarnes PJ, 2015,
Mechanisms of development of multimorbidity in the elderly
, EUROPEAN RESPIRATORY JOURNAL, Vol: 45, Pages: 790-806, ISSN: 0903-1936- Author Web Link
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- Citations: 120
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