ABSTRACT
BACKGROUND: Delay in type II alveolar epithelial cell (AECII) regeneration has been linked to higher mortality in patients with acute respiratory distress syndrome (ARDS). However, the interaction between Doublecortin-like kinase 1 (DCLK1) and the Hippo signaling pathway in ARDS-associated AECII differentiation remains unclear. Therefore, the objective of this study was to understand the role of the DCLK1/Hippo pathway in mediating AECII differentiation in ARDS. MATERIALS AND METHODS: AECII MLE-12 cells were exposed to 0, 0.1, or 1 µg/mL of lipopolysaccharide (LPS) for 6 and 12 h. In the mouse model, C57BL/6JNarl mice were intratracheally (i.t.) injected with 0 (control) or 5 mg/kg LPS and were euthanized for lung collection on days 3 and 7. RESULTS: We found that LPS induced AECII markers of differentiation by reducing surfactant protein C (SPC) and p53 while increasing T1α (podoplanin) and E-cadherin at 12 h. Concurrently, nuclear YAP dynamic regulation and increased TAZ levels were observed in LPS-exposed AECII within 12 h. Inhibition of YAP consistently decreased cell levels of SPC, claudin 4 (CLDN-4), galectin 3 (LGALS-3), and p53 while increasing transepithelial electrical resistance (TEER) at 6 h. Furthermore, DCLK1 expression was reduced in isolated human AECII of ARDS, consistent with the results in LPS-exposed AECII at 6 h and mouse SPC-positive (SPC+) cells after 3-day LPS exposure. We observed that downregulated DCLK1 increased p-YAP/YAP, while DCLK1 overexpression slightly reduced p-YAP/YAP, indicating an association between DCLK1 and Hippo-YAP pathway. CONCLUSIONS: We conclude that DCLK1-mediated Hippo signaling components of YAP/TAZ regulated markers of AECII-to-AECI differentiation in an LPS-induced ARDS model.
Subject(s)
Hippo Signaling Pathway , Respiratory Distress Syndrome , Animals , Humans , Mice , Alveolar Epithelial Cells/metabolism , Cell Differentiation , Doublecortin-Like Kinases , Lipopolysaccharides/pharmacology , Mice, Inbred C57BL , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Tumor Suppressor Protein p53/metabolismABSTRACT
BACKGROUND: Orosomucoid 1-like protein 3 (ORMDL3), a transmembrane protein localized in the endoplasmic reticulum (ER), has been genetically associated with chronic obstructive pulmonary disease (COPD), in addition to childhood-onset asthma. However, the functional role of ORMDL3 in the pathogenesis of COPD is still unknown. OBJECTIVE: Because cigarette smoke is the major risk factor for COPD, we aimed to investigate the role of ORMDL3 in cigarette smoke-induced human airway smooth muscle cell (HASMC) injury. METHODS: The mRNA and protein expression of ORMDL3 was examined in HASMCs from nonsmokers and smokers without or with COPD. Knockdown of ORMDL3 in primary healthy HASMCs was performed using small interfering RNA before exposure to cigarette smoke medium (CSM) for 24 hours. Inflammatory, proliferative/apoptotic, ER stress, and mitochondrial markers were evaluated. RESULTS: Elevation of ORMDL3 mRNA and protein expression was observed in HASMCs of smokers without or with COPD. CSM caused significant upregulation of ORMDL3 expression in healthy nonsmokers. ORMDL3 knockdown regulated CSM-induced inflammation, cell proliferation, and apoptosis. Silencing ORMDL3 led to reduction of CSM-induced ER stress via inhibition of unfolded protein response pathways such as activating transcription factor 6 and protein kinase RNA-like ER kinase. ORMDL3 was also involved in CSM-induced mitochondrial dysfunction via the mitochondrial fission process. CONCLUSIONS: We report the induction of ORMDL3 in HASMCs after cigarette smoke exposure. ORMDL3 may mediate cigarette smoke-induced activation of unfolded protein response pathways during airway smooth muscle cell injury.
Subject(s)
Asthma , Cigarette Smoking , Pulmonary Disease, Chronic Obstructive , Asthma/metabolism , Child , Cigarette Smoking/adverse effects , Endoplasmic Reticulum Stress , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Myocytes, Smooth Muscle/metabolism , Pulmonary Disease, Chronic Obstructive/genetics , RNA, Messenger/metabolism , NicotianaABSTRACT
Mitochondrial dysfunction has been reported in chronic obstructive pulmonary disease (COPD). Transfer of mitochondria from mesenchymal stem cells to airway smooth muscle cells (ASMCs) can attenuate oxidative stress-induced mitochondrial damage. It is not known whether mitochondrial transfer can occur between structural cells in the lungs or what role this may have in modulating bioenergetics and cellular function in healthy and COPD airways. Here, we show that ASMCs from both healthy ex-smokers and subjects with COPD can exchange mitochondria, a process that happens, at least partly, via extracellular vesicles. Exposure to cigarette smoke induces mitochondrial dysfunction and leads to an increase in the donation of mitochondria by ASMCs, suggesting that the latter may be a stress response mechanism. Healthy ex-smoker ASMCs that receive mitochondria show increases in mitochondrial biogenesis and respiration and a reduction in cell proliferation, irrespective of whether the mitochondria are transferred from healthy ex-smoker or COPD ASMCs. Our data indicate that mitochondrial transfer between structural cells is a homeostatic mechanism for the regulation of bioenergetics and cellular function within the airways and may represent an endogenous mechanism for reversing the functional consequences of mitochondrial dysfunction in diseases such as COPD.
Subject(s)
Pulmonary Disease, Chronic Obstructive , Energy Metabolism , Humans , Lung/metabolism , Mitochondria/metabolism , Muscle, Smooth , Pulmonary Disease, Chronic Obstructive/metabolismABSTRACT
BACKGROUND: Oxidative stress-induced mitochondrial dysfunction can contribute to inflammation and remodeling in patients with chronic obstructive pulmonary disease (COPD). Mesenchymal stem cells protect against lung damage in animal models of COPD. It is unknown whether these effects occur through attenuating mitochondrial dysfunction in airway cells. OBJECTIVE: We sought to examine the effect of induced pluripotent stem cell-derived mesenchymal stem cells (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 cocultured with iPSC-MSCs in the presence of cigarette smoke medium (CSM), and mitochondrial reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔΨm), and apoptosis were measured. Conditioned medium from iPSC-MSCs and transwell cocultures were used to detect any paracrine effects. The effect of systemic injection of iPSC-MSCs on airway inflammation and hyperresponsiveness in ozone-exposed mice was also investigated. RESULTS: Coculture of iPSC-MSCs with ASMCs attenuated CSM-induced mitochondrial ROS, apoptosis, and ΔΨm loss in ASMCs. iPSC-MSC-conditioned medium or transwell cocultures 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 coculture and was enhanced by CSM. iPSC-MSCs attenuated ozone-induced mitochondrial dysfunction, airway hyperresponsiveness, and inflammation in mouse lungs. CONCLUSION: iPSC-MSCs offered protection against oxidative stress-induced mitochondrial dysfunction in human ASMCs and in mouse lungs while reducing airway inflammation and hyperresponsiveness. These effects are, at least in part, dependent on cell-cell contact, which allows for mitochondrial transfer, and paracrine regulation. Therefore iPSC-MSCs show promise as a therapy for oxidative stress-dependent lung diseases, such as COPD.
Subject(s)
Lung/pathology , Mesenchymal Stem Cells/pathology , Mitochondria/pathology , Mitochondrial Diseases/pathology , Oxidative Stress/physiology , Animals , Apoptosis/physiology , Coculture Techniques/methods , Culture Media, Conditioned/metabolism , Disease Models, Animal , Humans , Induced Pluripotent Stem Cells/metabolism , Inflammation/metabolism , Inflammation/pathology , Lung/metabolism , Male , Membrane Potential, Mitochondrial/physiology , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Mitochondria/metabolism , Mitochondrial Diseases/metabolism , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Disease, Chronic Obstructive/pathology , Reactive Oxygen Species/metabolism , Smoke/adverse effects , Nicotiana/adverse effectsABSTRACT
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 ß2-adrenergic receptor (AR) agonists (LABAs). We determined the effect of ß2-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 ß2-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 ß2-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 ß2-AR expression and were relatively insensitive to salmeterol but not to 8-Br-cAMP or rolipram. Dexamethasone increased ß2-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.
Subject(s)
Adrenergic beta-2 Receptor Agonists/pharmacology , Asthma/physiopathology , Fibroblasts/drug effects , Leukocytes, Mononuclear/drug effects , Salmeterol Xinafoate/pharmacology , Severity of Illness Index , Adrenergic beta-2 Receptor Agonists/therapeutic use , Adult , Asthma/drug therapy , Asthma/immunology , Cells, Cultured , Dose-Response Relationship, Drug , Female , Fibroblasts/physiology , Humans , Leukocytes, Mononuclear/physiology , Male , Middle Aged , Salmeterol Xinafoate/therapeutic use , Treatment OutcomeABSTRACT
Oxidative stress, a pathogenetic factor in many conditions, including chronic obstructive pulmonary disease, arises due to accumulation of reactive oxygen species and defective antioxidant defenses in the lungs. The latter is due, at least in part, to impaired activation of NF-E2-related factor 2 (Nrf2), a transcription factor involved in the activation of antioxidant and cytoprotective genes. The bromodomain and extraterminal (BET) proteins, Brd2, Brd3, Brd4, and BrdT, bind to acetylated lysine residues on histone or nonhistone proteins recruiting transcriptional regulators and thus activating or repressing gene transcription. We investigated whether BET proteins modulate the regulation of Nrf2-dependent gene expression in primary human airway smooth muscle cells and the human monocytic cell line, THP-1. Inhibition of BET protein bromodomains using the inhibitor JQ1+ or attenuation of Brd2 and Brd4 expression using small interfering RNA led to activation of Nrf2-dependent transcription and expression of the antioxidant proteins heme oxygenase-1, NADPH quinone oxidoreductase 1, and glutamate-cysteine ligase catalytic subunit. Also, JQ1+ prevented H2O2-induced intracellular reactive oxygen species production. By coimmunoprecipitation, BET proteins were found to be complexed with Nrf2, whereas chromatin-immunoprecipitation studies indicated recruitment of Brd2 and Brd4 to Nrf2-binding sites on the promoters of heme oxygenase-1 and NADPH quinone oxidoreductase 1. BET proteins, particularly Brd2 and Brd4, may play a key role in the regulation of Nrf2-dependent antioxidant gene transcription and are hence an important target for augmenting antioxidant responses in oxidative stress-mediated diseases.
Subject(s)
Gene Expression Regulation, Enzymologic/immunology , Heme Oxygenase-1/immunology , NAD(P)H Dehydrogenase (Quinone)/immunology , NF-E2-Related Factor 2/immunology , Nuclear Proteins/immunology , Protein Serine-Threonine Kinases/immunology , Transcription Factors/immunology , Transcription, Genetic/immunology , Azepines/pharmacology , Cell Line, Tumor , Chromosomal Proteins, Non-Histone , Female , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/genetics , Heme Oxygenase-1/genetics , Humans , Hydrogen Peroxide/pharmacology , Male , NAD(P)H Dehydrogenase (Quinone)/genetics , NF-E2-Related Factor 2/genetics , Nuclear Proteins/genetics , Oxidants/pharmacology , Oxidative Stress/drug effects , Oxidative Stress/genetics , Oxidative Stress/immunology , Protein Serine-Threonine Kinases/genetics , Transcription Factors/genetics , Transcription, Genetic/genetics , Triazoles/pharmacologyABSTRACT
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.
Subject(s)
Adrenal Cortex Hormones/therapeutic use , Asthma/physiopathology , Myocytes, Smooth Muscle/drug effects , Receptors, Glucocorticoid/drug effects , Respiratory System/drug effects , Tumor Necrosis Factor-alpha/metabolism , Adrenal Cortex Hormones/immunology , Adrenal Cortex Hormones/pharmacology , Adult , Asthma/drug therapy , Asthma/immunology , Drug Resistance , Female , Humans , Male , Myocytes, Smooth Muscle/immunology , Myocytes, Smooth Muscle/metabolism , Receptors, Glucocorticoid/immunology , Receptors, Glucocorticoid/metabolism , Respiratory System/immunology , Respiratory System/physiopathology , Severity of Illness Index , Statistics, Nonparametric , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
BACKGROUND: Patients with severe asthma are less responsive to corticosteroid therapy and show increased airway remodeling. The mesenchymal progenitors, fibrocytes, may be involved in the remodeling of asthmatic airways. We propose that fibrocytes in severe asthma are different from those in nonsevere asthma. OBJECTIVES: To examine the survival, myofibroblastic differentiation, and C-C chemokine receptor 7 (CCR7) expression in blood fibrocytes from patients with severe and nonsevere asthma and study the effect of corticosteroids on fibrocyte function. METHODS: The nonadherent non-T-cell fraction of blood mononuclear cells was isolated from healthy subjects and patients with nonsevere and severe asthma. Total and differentiating fibrocytes were identified by their expression of CD45, collagen I, and α-smooth muscle actin using flow cytometry. The expression of CCR7 and of the glucocorticoid receptor was measured by using flow cytometry. RESULTS: Increased numbers of circulating fibrocytes, with greater myofibroblastic differentiation potential, were observed in patients with severe asthma. Dexamethasone induced apoptosis, leading to reduction in the number of cultured fibrocytes and total nonadherent non-T cells from healthy subjects and patients with nonsevere asthma but not from patients with severe asthma. Dexamethasone reduced CCR7 expression in fibrocytes from patients with nonsevere asthma but not from patients with severe asthma. Glucocorticoid receptor expression was attenuated in fibrocytes from patients with severe asthma. CONCLUSIONS: Patients with severe asthma have elevated numbers of circulating fibrocytes that show enhanced myofibroblastic differentiation and that are less responsive to the effects of corticosteroids.
Subject(s)
Adrenal Cortex Hormones/therapeutic use , Anti-Asthmatic Agents/therapeutic use , Asthma/diagnosis , Asthma/drug therapy , Drug Resistance , Phenotype , Adrenal Cortex Hormones/pharmacology , Adult , Anti-Asthmatic Agents/pharmacology , Apoptosis/drug effects , Case-Control Studies , Cell Count , Cell Differentiation/drug effects , Connective Tissue Cells/cytology , Connective Tissue Cells/drug effects , Connective Tissue Cells/metabolism , Female , Humans , Male , Middle Aged , Receptors, CCR7/metabolism , Receptors, Glucocorticoid/metabolism , Severity of Illness IndexABSTRACT
OBJECTIVES: Hydrogen sulfide (H2S) is a gas produced by respiratory cells including smooth muscle cells and may play a role as a cellular gasotransmitter. We evaluated whether H2S levels in serum or sputum could represent a new biomarker of COPD in a cross-sectional study. METHODS: H2S levels in sputum and serum samples were measured using a sulfide-sensitive electrode in 64 patients with stable COPD (S-COPD), 29 COPD subjects during acute exacerbation (AE-COPD), 14 healthy smokers and 21 healthy non-smokers. RESULTS: Sputum H2S levels in AE-COPD subjects were higher than those in S-COPD, healthy smoking and non-smoking subjects (p<0.001), but serum H2S levels in AE-COPD were lower than those in S-COPD (p<0.001). Thus, the sputum-to-serum ratio of H2S (H2S ratio) in AE-COPD subjects were higher than those in stable COPD, healthy smoking and non-smoking subjects (p<0.001). In 14 COPD subjects whose H2S ratios were measured during and after an exacerbation, the mean ratio was increased during exacerbation (p<0.05). H2S ratio was positively correlated with St. George's Respiratory Questionnaire score, sputum neutrophils and IL-6 and IL-8 levels in sputum and serum (p<0.01) but inversely correlated with sputum macrophages (%), FEV1%predicted and FEV1/FVC (p<0.01). The cut-off level of H2S ratio to indicate an exacerbation was ≥0.44 (sensitivity of 93.1% and specificity of 84.5%). CONCLUSIONS: The ratio of sputum-to-serum levels of H2S may provide a useful marker of COPD indicative of obstructive neutrophilic inflammation and of potential ongoing exacerbation.
Subject(s)
Hydrogen Sulfide/metabolism , Pulmonary Disease, Chronic Obstructive/metabolism , Sputum/chemistry , Aged , Biomarkers/metabolism , Cross-Sectional Studies , Female , Forced Expiratory Volume , Humans , Male , Middle Aged , Neutrophils/metabolism , Pulmonary Disease, Chronic Obstructive/physiopathology , Severity of Illness Index , Sputum/cytologyABSTRACT
A major goal of asthma management is maintaining optimal control. Current assessment is based on symptoms and lung function. We evaluated whether domiciliary daily home exhaled nitric oxide fraction (FeNO) monitoring could be useful as an index of asthma control. 50 asthmatic subjects and 15 healthy volunteers with a range of asthma severity underwent asthma control questionnaire (ACQ), spirometry before and after salbutamol and sputum induction. FeNO and peak expiratory flow (PEF) were measured twice daily for 2 weeks. A record of exacerbations was obtained 3 months later. Diurnal FeNO variation in uncontrolled asthmatics was significantly greater than in controlled asthmatics (p<0.01). PEF variation was not different. The daily variation of FeNO levels was also greater in uncontrolled asthmatics compared with controlled asthmatic and healthy subjects (p<0.01). 80% of uncontrolled asthmatics experienced at least one or more exacerbations over the 3 months after the enrolment. The combination of diurnal FeNO variation ≥ 16.6% and ACQ scores ≥ 1.8 was best at predicting uncontrolled asthma (area under curve 0.91, 95% CI 0.86-0.97; p<0.001). Diurnal variation in FeNO can be used as a biomarker of asthma control and as a predictor of the risk of future exacerbation. Prospective studies are warranted.
Subject(s)
Asthma/therapy , Circadian Rhythm , Exhalation , Nitric Oxide/metabolism , Adult , Asthma/physiopathology , Bronchodilator Agents/administration & dosage , Case-Control Studies , Female , Humans , Male , Middle Aged , Monitoring, Ambulatory , Peak Expiratory Flow Rate , ROC Curve , Respiratory Function Tests , Sensitivity and Specificity , Spirometry , Surveys and QuestionnairesABSTRACT
BACKGROUND: Bacteria are frequently cultured from sputum samples of severe asthma patients suggesting a defect in bacterial clearance from the airway. We measured the capacity of macrophages from patients with asthma to phagocytose bacteria. METHODS: Phagocytosis of fluorescently-labelled polystyrene beads, Haemophilus influenzae or Staphylococcus aureus by broncholaveolar lavage alveolar macrophages (AM) and by monocyte-derived macrophages (MDM) from non-asthmatics, mild-moderate and severe asthmatic patients was assessed using fluorimetry. RESULTS: There were no differences in phagocytosis of polystyrene beads by AMs or MDMs from any of the subject groups. There was reduced phagocytosis of Haemophilus influenzae and Staphylococcus aureus in MDMs from patients with severe asthma compared to non-severe asthma (p < 0.05 and p < 0.01, respectively) and healthy subjects (p < 0.01and p < 0.001, respectively). Phagocytosis of Haemophilus influenzae and Staphylococcus aureus by AM was also reduced in severe asthma compared to normal subjects (p < 0.05). Dexamethasone and formoterol did not suppress phagocytosis of bacteria by MDMs from any of the groups. CONCLUSIONS: Persistence of bacteria in the lower airways may result partly from a reduced phagocytic capacity of macrophages for bacteria. This may contribute to increased exacerbations, airway colonization and persistence of inflammation.
Subject(s)
Asthma/metabolism , Haemophilus influenzae/metabolism , Macrophages, Alveolar/metabolism , Phagocytosis/physiology , Severity of Illness Index , Staphylococcus aureus/metabolism , Adult , Asthma/microbiology , Bronchoalveolar Lavage Fluid/microbiology , Female , Humans , Macrophages, Alveolar/microbiology , Male , Middle AgedABSTRACT
BACKGROUND: IL-22 controls tissue homeostasis by both proinflammatory and anti-inflammatory effects. However, the anti-inflammatory mechanisms of IL-22 remain poorly investigated. OBJECTIVE: We sought to investigate the anti-inflammatory role for IL-22 in human asthma. METHODS: T-cell lines derived from lung biopsy specimens of asthmatic patients were characterized by means of flow cytometry. Human bronchial epithelial cells from healthy and asthmatic subjects were stimulated with IL-22, IFN-γ, or the combination of both cytokines. Effects of cytokine stimulation were investigated by using whole-genome analysis, ELISA, and flow cytometry. The functional consequence of cytokine stimulation was evaluated in an in vitro wound repair model and T cell-mediated cytotoxicity experiments. In vivo cytokine expression was measured by using immunohistochemistry and Luminex assays in bronchoalveolar lavage fluid of healthy and asthmatic patients. RESULTS: The current study identifies a tissue-restricted antagonistic interplay of IL-22 and the proinflammatory cytokine IFN-γ. On the one hand, IFN-γ antagonized IL-22-mediated induction of the antimicrobial peptide S100A7 and epithelial cell migration in bronchial epithelial cells. On the other hand, IL-22 decreased epithelial susceptibility to T cell-mediated cytotoxicity by inhibiting the IFN-γ-induced expression of MHC-I, MHC-II, and CD54/intercellular adhesion molecule 1 molecules. Likewise, IL-22 inhibited IFN-γ-induced secretion of the proinflammatory chemokines CCL5/RANTES and CXCL10/interferon-inducible protein 10 in vitro. Consistently, the IL-22 expression in bronchoalveolar lavage fluid of asthmatic patients inversely correlated with the expression of CCL5/RANTES and CXCL10/interferon-inducible protein 10 in vivo. CONCLUSIONS: IL-22 might control the extent of IFN-γ-mediated lung inflammation and therefore play a tissue-restricted regulatory role.
Subject(s)
Asthma/immunology , Asthma/pathology , Interferon-gamma/immunology , Interleukins/immunology , Pneumonia/immunology , Pneumonia/pathology , Adult , Asthma/metabolism , Bronchi/immunology , Bronchi/metabolism , Bronchi/pathology , Bronchoalveolar Lavage Fluid/chemistry , Case-Control Studies , Cell Movement/immunology , Cells, Cultured , Chemokine CCL5/immunology , Chemokine CCL5/metabolism , Chemokine CXCL10/immunology , Chemokine CXCL10/metabolism , Epithelial Cells/immunology , Epithelial Cells/metabolism , Epithelial Cells/pathology , Female , Genes, MHC Class I , Genes, MHC Class II , Humans , Intercellular Adhesion Molecule-1/immunology , Intercellular Adhesion Molecule-1/metabolism , Interferon-gamma/metabolism , Interleukins/metabolism , Male , Pneumonia/metabolism , Respiratory Function Tests , T-Lymphocytes/metabolism , Wound Healing/immunology , Interleukin-22ABSTRACT
BACKGROUND: Eosinophilic and neutrophilic asthma defined by high levels of blood and sputum eosinophils and neutrophils exemplifies the inflammatory heterogeneity of asthma, particularly severe asthma. We analysed the serum and sputum proteome to identify biomarkers jointly associated with these different phenotypes. METHODS: Proteomic profiles (N = 1129 proteins) were assayed in sputum (n = 182) and serum (n = 574) from two cohorts (U-BIOPRED and ADEPT) of mild-moderate and severe asthma by SOMAscan. Using least absolute shrinkage and selection operator (LASSO)-penalised logistic regression in a stability selection framework, we sought sparse sets of proteins associated with either eosinophilic or neutrophilic asthma with and without adjustment for established clinical factors including oral corticosteroid use and forced expiratory volume. FINDINGS: We identified 13 serum proteins associated with eosinophilic asthma, including 7 (PAPP-A, TARC/CCL17, ALT/GPT, IgE, CCL28, CO8A1, and IL5-Rα) that were stably selected while adjusting for clinical factors yielding an AUC of 0.84 (95% CI: 0.83-0.84) compared to 0.62 (95% CI: 0.61-0.63) for clinical factors only. Sputum protein analysis selected only PAPP-A (AUC = 0.81 [95% CI: 0.80-0.81]). 12 serum proteins were associated with neutrophilic asthma, of which 5 (MMP-9, EDAR, GIIE/PLA2G2E, IL-1-R4/IL1RL1, and Elafin) complemented clinical factors increasing the AUC from 0.63 (95% CI: 0.58-0.67) for the model with clinical factors only to 0.89 (95% CI: 0.89-0.90). Our model did not select any sputum proteins associated with neutrophilic status. INTERPRETATION: Targeted serum proteomic profiles are a non-invasive and scalable approach for subtyping of neutrophilic and eosinophilic asthma and for future functional understanding of these phenotypes. FUNDING: U-BIOPRED has received funding from the Innovative Medicines Initiative (IMI) Joint Undertaking under grant agreement no. 115010, resources of which are composed of financial contributions from the European Union's Seventh Framework Programme (FP7/2007-2013), and European Federation of Pharmaceutical Industries and Associations (EFPIA) companies' in-kind contributions (www.imi.europa.eu). ADEPT was funded by Johnson & Johnson/Janssen pharmaceutical Company.
Subject(s)
Asthma , Sputum , Humans , Proteomics , Pregnancy-Associated Plasma Protein-A/metabolism , Asthma/metabolism , Neutrophils/metabolism , Blood Proteins/metabolismABSTRACT
A previous genome-wide association study in asthma revealed putative associations that merit further investigation. In this study, the genome-wide significant associations of SNPs at the 5% false discovery rate were examined in independent groups of severe asthmatics. The panel consisted of 397 severe asthmatic adults, 116 severe asthmatic children, and a collection of 207 family-trios with an asthmatic proband. Three SNPs in the PDCD4 gene (rs6585018:G>A, rs1322997:C>A, and rs34104444:G>A) were significantly associated with severe childhood asthma (P values: 0.003, 0.002, 0.004) and total immunoglobulin E (IgE) levels (P values: 0.034, 0.041, 0.052). In an independent group of 234 asthmatic children and 652 controls, PDCD4 SNPs rs1407696:T>G and rs11195360:T>C were associated with total IgE levels (P values: 0.006, 0.014). In silico analysis of PDCD4 locus showed that rs6585018:G>A had the potential to affect MYB transcription factor binding, shown to act as a PDCD4-transcription inducer. Electromobility shift assays and reporter assays revealed that rs6585018:G>A alters MYB binding thereby influencing the expression of PDCD4. SNPs within MYB itself confer susceptibility to eosinophilia and asthma. Our association between a variant MYB binding site in PDCD4 and the severest form of childhood asthma therefore suggests that PDCD4 is a novel molecule of importance to asthmatic inflammatory responses.
Subject(s)
Apoptosis Regulatory Proteins/genetics , Asthma/genetics , Polymorphism, Single Nucleotide , Promoter Regions, Genetic , Proto-Oncogene Proteins c-myb/genetics , RNA-Binding Proteins/genetics , Adult , Apoptosis Regulatory Proteins/metabolism , Binding Sites , Case-Control Studies , Cell Line , Child , Female , Genome-Wide Association Study , Haplotypes , Humans , Immunoglobulin E/blood , Male , Proto-Oncogene Proteins c-myb/metabolism , RNA-Binding Proteins/metabolismABSTRACT
BACKGROUND: Patients with severe asthma are less responsive to the beneficial effects of corticosteroid therapy. OBJECTIVE: We investigated whether corticosteroid insensitivity was present in airway smooth muscle cells (ASMCs) of patients with severe asthma. METHODS: ASMCs cultured from bronchial biopsy specimens of nonasthmatic control subjects (n = 12) and patients with nonsevere (n = 10) or severe (n = 10) asthma were compared for the effect of dexamethasone on suppression of TNF-α- and IFN-γ-induced CCL11 (eotaxin), CXCL8 (IL-8), and CX3CL1 (fractalkine) expression. The mechanisms of corticosteroid insensitivity are also determined. RESULTS: CCL11 release was higher in ASMCs of patients with nonsevere but not severe asthma and nonasthmatic control subjects; CXCL8 and CX3CL1 release were similar in all groups. In patients with severe asthma, dexamethasone caused less suppression of CCL11 and CXCL8 release induced by TNF-α. Dexamethasone potentiated TNF-α- and IFN-γ-induced CX3CL1 release equally in all 3 groups. TNF-α-induced phosphorylated p38 mitogen-activated protein kinase levels were increased in ASMCs from patients with severe asthma compared with those from patients with nonsevere asthma and nonasthmatic subjects, whereas TNF-α-induced phosphorylated c-Jun N-terminal kinase and phosphorylated extracellular signal-related kinase levels were increased in all asthmatic groups. A p38 inhibitor increased the inhibitory effect of dexamethasone. CONCLUSIONS: ASMCs of patients with severe asthma are corticosteroid insensitive; this might be secondary to heightened p38 mitogen-activated protein kinase levels.
Subject(s)
Asthma/drug therapy , Bronchi/drug effects , Chemokines/genetics , Dexamethasone/pharmacology , Myocytes, Smooth Muscle/drug effects , Adult , Asthma/immunology , Bronchi/immunology , Cells, Cultured , Chemokines/metabolism , Female , Humans , Male , Middle Aged , Mitogen-Activated Protein Kinases/metabolism , Myocytes, Smooth Muscle/immunology , Myocytes, Smooth Muscle/metabolism , Promoter Regions, Genetic , Transcription Factor RelA/physiologyABSTRACT
Acute respiratory distress syndrome (ARDS) contributes to higher mortality worldwide. Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have immunomodulatory and regenerative potential. However, the effects of hUC-MSCs as an ARDS treatment remain unclear. We investigated the role of hUC-MSCs in the differentiation of type II alveolar epithelial cells (AECII) by regulating Yes-associated protein (YAP) in ARDS. Male C57BL/6JNarl mice were intratracheally (i.t.) administered lipopolysaccharide (LPS) to induce an ARDS model, followed by a single intravenous (i.v.) dose of hUC-MSCs. hUC-MSCs improved pulmonary function, decreased inflammation on day 3, and mitigated lung injury by reducing the lung injury score and increasing lung aeration (%) in mice on day 7 (p < 0.05). hUC-MSCs inactivated YAP on AECII and facilitated cell differentiation by decreasing Pro-surfactant protein C (Pro-SPC) and galectin 3 (LGALS3) while increasing podoplanin (T1α) in lungs of mice (p < 0.05). In AECII MLE-12 cells, both coculture with hUC-MSCs after LPS exposure and the YAP inhibitor, verteporfin, reduced Pro-SPC and LGALS3, whereas the YAP inhibitor increased T1α expression (p < 0.05). In conclusion, hUC-MSCs ameliorated lung injury of ARDS and regulated YAP to facilitate AECII differentiation.
Subject(s)
Lung Injury , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Respiratory Distress Syndrome , Animals , Humans , Male , Mice , Alveolar Epithelial Cells/metabolism , Cell Differentiation , Galectin 3/metabolism , Lipopolysaccharides/pharmacology , Lipopolysaccharides/metabolism , Lung Injury/metabolism , Mesenchymal Stem Cells/metabolism , Mice, Inbred C57BL , Respiratory Distress Syndrome/therapy , Respiratory Distress Syndrome/metabolism , Umbilical CordABSTRACT
BACKGROUND: Spread of SARS-CoV2 by aerosol is considered an important mode of transmission over distances >2 m, particularly indoors. OBJECTIVES: We determined whether SARS-CoV2 could be detected in the air of enclosed/semi-enclosed public spaces. METHODS AND ANALYSIS: Between March 2021 and December 2021 during the easing of COVID-19 pandemic restrictions after a period of lockdown, we used total suspended and size-segregated particulate matter (PM) samplers for the detection of SARS-CoV2 in hospitals wards and waiting areas, on public transport, in a university campus and in a primary school in West London. RESULTS: We collected 207 samples, of which 20 (9.7%) were positive for SARS-CoV2 using quantitative PCR. Positive samples were collected from hospital patient waiting areas, from hospital wards treating patients with COVID-19 using stationary samplers and from train carriages in London underground using personal samplers. Mean virus concentrations varied between 429 500 copies/m3 in the hospital emergency waiting area and the more frequent 164 000 copies/m3 found in other areas. There were more frequent positive samples from PM samplers in the PM2.5 fractions compared with PM10 and PM1. Culture on Vero cells of all collected samples gave negative results. CONCLUSION: During a period of partial opening during the COVID-19 pandemic in London, we detected SARS-CoV2 RNA in the air of hospital waiting areas and wards and of London Underground train carriage. More research is needed to determine the transmission potential of SARS-CoV2 detected in the air.
Subject(s)
COVID-19 , Chlorocebus aethiops , Animals , Humans , COVID-19/epidemiology , RNA, Viral , SARS-CoV-2 , London/epidemiology , Pandemics , Vero Cells , Communicable Disease Control , Respiratory Aerosols and Droplets , Particulate Matter/analysisABSTRACT
BACKGROUND: COVID-19 has overwhelmed health services globally. Oral antiviral therapies are licensed worldwide, but indications and efficacy rates vary. We aimed to evaluate the safety and efficacy of oral favipiravir in patients hospitalised with COVID-19. METHODS: We conducted a multicentre, open-label, randomised controlled trial of oral favipiravir in adult patients who were newly admitted to hospital with proven or suspected COVID-19 across five sites in the UK (n=2), Brazil (n=2) and Mexico (n=1). Using a permuted block design, eligible and consenting participants were randomly assigned (1:1) to receive oral favipiravir (1800 mg twice daily for 1 day; 800 mg twice daily for 9 days) plus standard care, or standard care alone. All caregivers and patients were aware of allocation and those analysing data were aware of the treatment groups. The prespecified primary outcome was the time from randomisation to recovery, censored at 28 days, which was assessed using an intention-to-treat approach. Post-hoc analyses were used to assess the efficacy of favipiravir in patients aged younger than 60 years, and in patients aged 60 years and older. The trial was registered with clinicaltrials.gov, NCT04373733. FINDINGS: Between May 5, 2020 and May 26, 2021, we assessed 503 patients for eligibility, of whom 499 were randomly assigned to favipiravir and standard care (n=251) or standard care alone (n=248). There was no significant difference between those who received favipiravir and standard care, relative to those who received standard care alone in time to recovery in the overall study population (hazard ratio [HR] 1·06 [95% CI 0·89-1·27]; n=499; p=0·52). Post-hoc analyses showed a faster rate of recovery in patients younger than 60 years who received favipiravir and standard care versus those who had standard care alone (HR 1·35 [1·06-1·72]; n=247; p=0·01). 36 serious adverse events were observed in 27 (11%) of 251 patients administered favipiravir and standard care, and 33 events were observed in 27 (11%) of 248 patients receiving standard care alone, with infectious, respiratory, and cardiovascular events being the most numerous. There was no significant between-group difference in serious adverse events per patient (p=0·87). INTERPRETATION: Favipiravir does not improve clinical outcomes in all patients admitted to hospital with COVID-19, however, patients younger than 60 years might have a beneficial clinical response. The indiscriminate use of favipiravir globally should be cautioned, and further high-quality studies of antiviral agents, and their potential treatment combinations, are warranted in COVID-19. FUNDING: LifeArc and CW+.