Mechanisms of airway epithelial injury and abnormal repair in asthma and COPD

The airway epithelium comprises of different cell types and acts as a physical barrier preventing pathogens, including inhaled particles and microbes, from entering the lungs. Goblet cells and submucosal glands produce mucus that traps pathogens, which are expelled from the respiratory tract by ciliated cells. Basal cells act as progenitor cells, differentiating into different epithelial cell types, to maintain homeostasis following injury. Adherens and tight junctions between cells maintain the epithelial barrier function and regulate the movement of molecules across it. In this review we discuss how abnormal epithelial structure and function, caused by chronic injury and abnormal repair, drives airway disease and specifically asthma and chronic obstructive pulmonary disease (COPD). In both diseases, inhaled allergens, pollutants and microbes disrupt junctional complexes and promote cell death, impairing the barrier function and leading to increased penetration of pathogens and a constant airway immune response. In asthma, the inflammatory response precipitates the epithelial injury and drives abnormal basal cell differentiation. This leads to reduced ciliated cells, goblet cell hyperplasia and increased epithelial mesenchymal transition, which contribute to impaired mucociliary clearance and airway remodelling. In COPD, chronic oxidative stress and inflammation trigger premature epithelial cell senescence, which contributes to loss of epithelial integrity and airway inflammation and remodelling. Increased numbers of basal cells showing deregulated differentiation, contributes to ciliary dysfunction and mucous hyperproduction in COPD airways. Defective antioxidant, antiviral and damage repair mechanisms, possibly due to genetic or epigenetic factors, may confer susceptibility to airway epithelial dysfunction in these diseases. The current evidence suggests that a constant cycle of injury and abnormal repair of the epithelium drives chronic airway inflammation and remodelling in asthma and COPD. Mechanistic understanding of injury susceptibility and damage response may lead to improved therapies for these diseases

Oxidative stress-induced mitochondria alteration in human airway smooth muscle cells and mesenchymal stem cells

Poster Discussion Session - D27. Mitochondria: Live and Let Die: no. A5544RATIONALE: Exposure to cigarette smoke (CS) is the primary cause of chronic obstructive pulmonary disease (COPD). Reactive oxygen species (ROS) produced by CS, as well as by infiltrating inflammatory cells, in conjunction with compromised antioxidant defenses in the lungs of COPD patients, results in oxidative stress. Oxidative stress leads to defective function of lung cells, such as airway smooth muscle cells (ASMCs), driving airway inflammation and remodelling. Mitochondrial dysfunction caused by oxidative stress leads to changes in cell survival and inflammatory responses. Mitochondrial transfer between mesenchymal stem cell (MSC) and airway cells has been shown to reverse mitochondrial dysfunction in lung disease models. We investigated the effect of oxidative stress on mitochondrial function and viability of …published_or_final_versio

DNA methylation modules in airway smooth muscle are associated with asthma severity

Abnormal DNA methylation patterns distinguish airway smooth muscle cell function in asthma and asthma severity

Mesenchymal stem cells alleviate oxidative stress-induced mitochondrial dysfunction in the airways

BACKGROUND: Oxidative stress-induced mitochondrial dysfunction may contribute to inflammation and remodeling in chronic obstructive pulmonary disease (COPD). Mesenchymal stem cells (MSCs) protect against lung damage in animal models of COPD. It is unknown whether these effects occur through attenuating mitochondrial dysfunction in airway cells. OBJECTIVE: To examine the effect of induced-pluripotent stem cell-derived MSCs (iPSC-MSCs) on oxidative stress-induce mitochondrial dysfunction in human airway smooth muscle cells (ASMCs) in vitro and in mouse lungs in vivo. METHODS: ASMCs were co-cultured with iPSC-MSCs in the presence of cigarette smoke medium (CSM), and mitochondrial reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm) and apoptosis were measured. Conditioned media from iPSC-MSCs and trans-well co-cultures were used to detect any paracrine effects. The effect of systemic injection of iPSC-MSCs on airway inflammation and hyper-responsiveness in ozone-exposed mice was also investigated. RESULTS: Co-culture of iPSC-MSCs with ASMCs attenuated CSM-induced mitochondrial ROS, apoptosis and ΔΨm loss in ASMCs. iPSC-MSC-conditioned media or trans-well co-cultures with iPSC-MSCs reduced CSM-induced mitochondrial ROS but not ΔΨm or apoptosis in ASMCs. Mitochondrial transfer from iPSC-MSCs to ASMCs was observed after direct co-culture and was enhanced by CSM. iPSC-MSCs attenuated ozone-induced mitochondrial dysfunction, airway hyper-responsiveness and inflammation in mouse lungs. CONCLUSION: iPSC-MSCs offered protection against oxidative stress-induced mitochondrial dysfunction in human ASMCs and in mouse lungs, whilst reducing airway inflammation and hyper-responsiveness. These effects are, at least partly, dependent on cell-cell contact that allows for mitochondrial transfer, and paracrine regulation. Therefore, iPSC-MSCs show promise as a therapy for oxidative stress-dependent lung diseases such as COPD

Oxidative stress-induced mitochondrial dysfunction drives inflammation and airway smooth muscle remodeling in patients with chronic obstructive pulmonary disease

BackgroundInflammation 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 inflammation and cell hyperproliferation. Targeting mitochondrial ROS represents a promising therapeutic approach in patients with COPD

Reduced suppressive effect of beta(2)-adrenoceptor agonist on fibrocyte function in severe asthma

Background Patients with severe asthma have increased airway remodelling and elevated numbers of circulating fibrocytes with enhanced myofibroblastic differentiation capacity, despite being treated with high doses of corticosteroids, and long acting β2-adrenergic receptor (AR) agonists (LABAs). We determined the effect of β2-AR agonists, alone or in combination with corticosteroids, on fibrocyte function. Methods Non-adherent non-T cells from peripheral blood mononuclear cells isolated from healthy subjects and patients with non-severe or severe asthma were treated with the β2-AR agonist, salmeterol, in the presence or absence of the corticosteroid dexamethasone. The number of fibrocytes (collagen I+/CD45+ cells) and differentiating fibrocytes (α-smooth muscle actin+ cells), and the expression of CC chemokine receptor 7 and of β2-AR were determined using flow cytometry. The role of cyclic adenosine monophosphate (cAMP) was elucidated using the cAMP analogue 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) and the phosphodiesterase type IV (PDE4) inhibitor, rolipram. Results Salmeterol reduced the proliferation, myofibroblastic differentiation and CCR7 expression of fibrocytes from healthy subjects and non-severe asthma patients. Fibrocytes from severe asthma patients had a lower baseline surface β2-AR expression and were relatively insensitive to salmeterol but not to 8-Br-cAMP or rolipram. Dexamethasone increased β2-AR expression and enhanced the inhibitory effect of salmeterol on severe asthma fibrocyte differentiation. Conclusions Fibrocytes from patients with severe asthma are relatively insensitive to the inhibitory effects of salmeterol, an effect which is reversed by combination with corticosteroids

BET bromodomains regulate transforming growth factor-beta-induced proliferation and cytokine release in asthmatic airway smooth muscle

Airway smooth muscle (ASM) mass is increased in asthma, and ASM cells from patients with asthma are hyperproliferative and release more IL-6 and CXCL8. The BET (bromo- and extra-terminal) family of proteins (Brd2, Brd3, and Brd4) govern the assembly of histone acetylation-dependent chromatin complexes. We have examined whether they modulate proliferation and cytokine expression in asthmatic ASM cells by studying the effect of BET bromodomain mimics JQ1/SGCBD01 and I-BET762. ASM cells from healthy individuals and nonsevere and severe asthmatics were pretreated with JQ1/SGCBD01 and I-BET762 prior to stimulation with FCS and TGF-β. Proliferation was measured by BrdU incorporation. IL-6 and CXCL8 release was measured by ELISA, and mRNA expression was measured by quantitative RT-PCR. ChIP using a specific anti-Brd4 antibody and PCR primers directed against the transcriptional start site of IL-6 and CXCL8 gene promoters was performed. Neither JQ1/SGCBD01 nor I-BET762 had any effect on ASM cell viability. JQ1/SGCBD01 and I-BET762 inhibited FCS+TGF-β-induced ASM cell proliferation and IL-6 and CXCL8 release in healthy individuals (≥ 30 nm) and in nonsevere and severe asthma patients (≥100 nm), with the latter requiring higher concentrations of these mimics. JQ1/SGCBD01 reduced Brd4 binding to IL8 and IL6 promoters induced by FCS+TGF-β. Mimics of BET bromodomains inhibit aberrant ASM cell proliferation and inflammation with lesser efficiency in those from asthmatic patients. They may be effective in reducing airway remodeling in asthma

Impaired nuclear translocation of the glucocorticoid receptor in corticosteroid-insensitive airway smooth muscle in severe asthma

Rationale: Patients with severe asthma (SA) are less responsive to the beneficial effects of corticosteroid (CS) therapy, and relative CS insensitivity has been shown in airway smooth muscle cells (ASMC) from patients with SA. Objectives: We investigated whether there was a defect in the actions of the glucocorticoid receptor (GR) underlying the ability of CS to suppress the inflammatory response in ASMC of patients with SA. ASMC from healthy subjects (n = 10) and subjects with severe (n = 8) and nonsevere asthma (N-SA; n = 8) were cultured from endobronchial biopsies. Measurements and Main Results: GR expression in ASMC from SA and N-SA was reduced compared with that from healthy subjects by 49% (P < 0.01). Although baseline levels of nuclear GR were similar, GR nuclear translocation induced by dexamethasone (10−7 M) in SA was 60% of that measured in either healthy subjects or subjects with N-SA. Tumor necrosis factor (TNF)-α induced greater nuclear factor (NF)-κB (p65) mRNA expression in ASMC from subjects with SA (5.6- vs. 2.0-fold; P < 0.01), whereas baseline and TNF-α–induced nuclear translocation and dexamethasone-mediated suppression of p65 expression were similar between groups. Dexamethasone, although not modulating TNF-α–induced p65 nuclear translocation, attenuated p65 recruitment to the CCL11 promoter in the healthy and N-SA groups, but this suppressive effect was impaired in subjects with SA. Conclusions: Decreased GR expression with impaired nuclear translocation in ASMC, associated with reduced dexamethasone-mediated attenuation of p65 recruitment to NF-κB–dependent gene promoters, may underlie CS insensitivity of severe asthma

Oxidants induce a corticosteroid-insensitive phosphorylation of histone 3 at serine 10 in monocytes

Oxidative stress enhances inflammation and reduces the effectiveness of corticosteroids, but the inflammatory signalling pathways induced by oxidants remain ill-defined. Phosphorylation of histone 3 at serine 10 (H3-Pser10) marks out a subset of inflammatory genes for transcription, several of which are induced in oxidant-associated inflammation. However, the influence of oxidants or of corticosteroids on this modification remains unknown. We assessed the regulation of H3-Pser10 by oxidants and lipopolysaccharide (LPS) in human blood monocytes and lung macrophages and the effectiveness of its abolition in controlling inflammatory gene expression in cells from asthmatic subjects compared to corticosteroids alone. Both oxidants and LPS promoted the induction of H3-Pser10 which was unaffected by corticosteroids. The induction of H3-Pser10 was mediated through p38α mitogen-activated protein kinase (MAPK) and IκB kinase 2 (IKK-2) signalling. Consequently, inhibitors of p38α MAPK or IKK-2 used in combination with dexamethasone were more effective at controlling inflammatory gene expression from monocytes and lung macrophages from asthmatic patients than the corticosteroid alone. Therefore, reduction of H3-Pser10 by inhibition of p38α MAPK or of IKK-2 may provide greater anti-inflammatory control than corticosteroids alone in oxidant-associated inflammation such as severe asthma

Chemical genetics approaches for selective intervention in epigenetics

Chemical genetics is the use of biologically active small molecules (chemical probes) to investigate the functions of gene products, through the modulation of protein activity. Recent years have seen significant progress in the application of chemical genetics to study epigenetics, following the development of new chemical probes, a growing appreciation of the role of epigenetics in disease and a recognition of the need and utility of high-quality, cell-active chemical probes. In this review, we single out the bromodomain reader domains as a prime example of both the success, and challenges facing chemical genetics. The difficulty in generating single-target selectivity has long been a thorn in the side of chemical genetics, however, recent developments in advanced forms of chemical genetics promise to bypass this, and other, limitations. The ‘bump-and-hole’ approach has now been used to probe — for the first time — the BET bromodomain subfamily with single-target selectivity and may be applicable to other epigenetic domains. Meanwhile, PROTAC compounds have been shown to be significantly more efficacious than standard domain inhibitors, and have the potential to enhance target selectivity