Gurpreet K. Singhera

Intrinsic Phenotypic Differences of Asthmatic Epithelium and Its Inflammatory Responses to Respiratory Syncytial Virus and Air Pollution

A substantial proportion of healthcare cost associated with asthma is attributable to exacerbations of the disease. Within the airway, the epithelium forms the mucosal immune barrier, the first structural cell defense against common environmental insults such as respiratory syncytial virus (RSV) and particulate matter. We sought to characterize the phenotype of differentiated asthmatic-derived airway epithelial cultures and their intrinsic inflammatory responses to environmental challenges. Air-liquid interface (ALI) cultures were generated from asthmatic (n = 6) and nonasthmatic (n = 6) airway epithelial cells. Airway tissue and ALI cultures were analyzed by immunohistochemistry for cytokeratin-5, E-cadherin, Ki67, Muc5AC, NF-κB, the activation of p38, and apoptosis. ALI cultures were exposed to RSV (4 × 10(6) plaque forming unit/ml), particulate matter collected by Environmental Health Canada (EHC-93, 100 μg/ml), or mechanically wounded for 24, 48, and 96 hours and basolateral supernatants analyzed for inflammatory cytokines, using Luminex and ELISA. The airway epithelium in airway sections of patients with asthma as well as in vitro ALI cultures demonstrated a less differentiated epithelium, characterized by elevated numbers of basal cells marked by the expression of cytokeratin-5, increased phosphorylation of p38 mitogen-activated protein kinase, and less adherens junction protein E-cadherin. Transepithelial resistance was not different between asthmatic and nonasthmatic cultures. In response to infection with RSV, exposure to EHC-93, or mechanical wounding, asthmatic ALI cultures released greater concentrations of IL-6, IL-8, and granulocyte macrophage colony-stimulating factor, compared with nonasthmatic cultures (P < 0.05). This parallel ex vivo and in vitro study of the asthmatic epithelium demonstrates an intrinsically altered phenotype and aberrant inflammatory response to common environmental challenges, compared with nonasthmatic epithelium.

Toll-Like Receptor 4-Mediated Activation of p38 Mitogen-Activated Protein Kinase Is a Determinant of Respiratory Virus Entry and Tropism

ABSTRACT Respiratory viruses exert a heavy toll of morbidity and mortality worldwide. Despite this burden there are few specific treatments available for respiratory virus infections. Since many viruses utilize host cell enzymatic machinery such as protein kinases for replication, we determined whether pharmacological inhibition of kinases could, in principle, be used as a broad antiviral strategy for common human respiratory virus infections. A panel of green fluorescent protein (GFP)-expressing recombinant respiratory viruses, including an isolate of H1N1 influenza virus (H1N1/Weiss/43), was used to represent a broad range of virus families responsible for common respiratory infections (Adenoviridae, Paramyxoviridae, Picornaviridae, and Orthomyxoviridae). Kinase inhibitors were screened in a high-throughput assay that detected virus infection in human airway epithelial cells (1HAEo-) using a fluorescent plate reader. Inhibition of p38 mitogen-activated protein kinase (MAPK) signaling was able to significantly inhibit replication by all viruses tested. Therefore, the pathways involved in virus-mediated p38 and extracellular signal-regulated kinase (ERK) MAPK activation were investigated using bronchial epithelial cells and primary fibroblasts derived from MyD88 knockout mouse lungs. Influenza virus, which activated p38 MAPK to approximately 10-fold-greater levels than did respiratory syncytial virus (RSV) in 1HAEo- cells, was internalized about 8-fold faster and more completely than RSV. We show for the first time that p38 MAPK is a determinant of virus infection that is dependent upon MyD88 expression and Toll-like receptor 4 (TLR4) ligation. Imaging of virus-TLR4 interactions showed significant clustering of TLR4 at the site of virus-cell interaction, triggering phosphorylation of downstream targets of p38 MAPK, suggesting the need for a signaling receptor to activate virus internalization.

IL-13 and TH2 cytokine exposure triggers matrix metalloproteinase 7–mediated Fas ligand cleavage from bronchial epithelial cells

BACKGROUND Bronchial epithelial damage and activation likely contribute to the inflammatory and airway-remodeling events characteristic of severe asthma. Interaction of Fas receptor (CD95) with its ligand (FasL; CD95L) is an important mechanism of cell-mediated apoptosis. Bronchial epithelial FasL expression provides immune barrier protection from immune cell-mediated damage. OBJECTIVES Membrane FasL (mFasL) is a cleavage target of matrix metalloproteinases (MMPs). We investigated whether the asthmatic T(H)2 environment might influence disease processes by increasing airway epithelial MMP-mediated cleavage of mFasL into proinflammatory soluble FasL. METHODS We used human airway epithelial cell lines and primary cells to model the human airway epithelium in vitro. Airway tissue from healthy subjects and patients with severe asthma was used to investigate MMP expression patterns in diseased airways. RESULTS We demonstrate that active MMP-7 is present in the ciliated epithelial cells of normal human airways. In patients with severe asthma, MMP-7 levels are increased in basal epithelial cells. Airway epithelial cell lines (1HAEo(-) and 16HBE14o(-)) in vitro express constitutively high levels of MMP-2 and MMP-9 but relatively low levels of MMP-7. T(H)2 cytokine (IL-4, IL-9, and IL-13) treatment of 1HAEo(-) cells increased MMP-7 mRNA and activity, triggered colocalization of intracellular MMP-7 with FasL, and caused mFasL cleavage with soluble FasL release. Small interfering RNA knockdown shows that cytokine-induced mFasL cleavage is dependent on MMP-7 activity. CONCLUSIONS MMPs serve multiple beneficial roles in the lung. However, chronic disordered epithelial expression of MMP-7 in patients with asthma might increase mFasL cleavage and contribute to airway epithelial damage and inflammation.

Erythropoietin inhibits respiratory epithelial cell apoptosis in a model of acute lung injury

Fas-mediated apoptosis of the alveolar epithelium is important in the pathogenesis of acute respiratory distress syndrome. Erythropoietin (EPO) has cytoprotective properties in other organ systems, and is relatively deficient in critical illness. This study investigates a potential role for EPO in reducing apoptosis in a model of acute lung injury. Apoptosis was induced in human alveolar epithelial (A549) cells or normal human bronchial epithelial (NHBE) cells by Fas activation with CH-11 Fas-crosslinking antibody or by co-culture with polymorphonuclear neutrophils in a transwell system. The effect of recombinant human (rh)EPO on apoptosis was measured by poly(ADP-ribose) polymerase cleavage and cell death detection assay. The specific EPO–EPO receptor (EPOR)-mediated effect was determined using an EPO-blocking antibody or EPOR small interfering RNA. Expression of EPOR was demonstrated in A549, NHBE and normal human alveolar epithelium. Fas- and neutrophil-mediated apoptosis of A549 and NHBE cells was inhibited by rhEPO by a specific EPO–EPOR-mediated mechanism. This anti-apoptotic effect was associated with induction of a pro-apoptotic Bcl-xL/Bax ratio. EPO has cytoprotective properties in respiratory epithelium in an in vitro model, which may indicate a potential therapeutic role in acute lung injury.

Conjugated linoleic acid improves airway hyper-reactivity in overweight mild asthmatics.

Background Conjugated linoleic acids (CLA) are naturally occurring fatty acids that have multiple biological properties including the regulation of metabolic, proliferative and immune processes.

INTERLEUKIN-9 AND -13 INHIBIT SPONTANEOUS AND CORTICOSTEROID INDUCED APOPTOSIS OF NORMAL AIRWAY EPITHELIAL CELLS

The airway epithelium is the target of physical and allergic insults. The resulting inflammatory signals from Th2 cytokines including interleukin (IL)-9 and IL-13 have pleiotropic activities and have been implicated in airway remodeling in asthmatics. The objective of this study was to determine the role of IL-9 and IL-13 in the regulation of normal airway epithelial cell death and epithelial repair. In a cell culture model, a normal human airway epithelial cell line and primary airway epithelial cells were treated with IL-9 or IL-13 alone and in combination. Apoptosis was determined by multiple techniques, including enrichment of nucleosomes released into the cytoplasm, mitochondrial membrane polarity perturbation, cytosolic cytochrome c released and the detection of cleaved p85-poly(ADP-ribose)polymerase (PARP). Proliferation was quantified by BrdU incorporation. IL-9 and IL-13 treatment, alone and in combination, resulted in a significant reduction in spontaneous airway epithelial cell apoptosis when compared to controls. The cytoprotective effect of IL-9 was associated with up-regulation of the antiapoptotic molecule Bcl-2. IL-13 also demonstrated coordinate pro-proliferative activity .Dexamethasone induces apoptosis in airway epithelial cells. Coincubation with IL-9 or IL-13 was protective against this corticosteroid-induced apoptosis by up-regulation of Bcl-2. These data demonstrate that IL-9 and IL-13 may be critical to normal cellular homeostasis in the setting of airway epithelial injury. A dysregulated response to these cytokines may contribute to airway remodeling in asthma.

Apoptosis of viral-infected airway epithelial cells limit viral production and is altered by corticosteroid exposure

BackgroundEffects of respiratory viral infection on airway epithelium include airway hyper-responsiveness and inflammation. Both features may contribute to the development of asthma. Excessive damage and loss of epithelial cells are characteristic in asthma and may result from viral infection.ObjectiveTo investigate apoptosis in Adenoviral-infected Guinea pigs and determine the role of death receptor and ligand expression in the airway epithelial response to limit viral infection.MethodsAnimal models included both an Acute and a Chronic Adeno-infection with ovalbumin-induced airway inflammation with/without corticosteroid treatment. Isolated airway epithelial cells were cultured to study viral production after infection under similar conditions. Immunohistochemistry, western blots and viral DNA detection were used to assess apoptosis, death receptor and TRAIL expression and viral release.ResultsIn vivo and in vitro Adeno-infection demonstrated different apoptotic and death receptors (DR) 4 and 5 expression in response to corticosteroid exposure. In the Acute Adeno-infection model, apoptosis and DR4/5 expression was coordinated and were time-dependent. However, in vitro Acute viral infection in the presence of corticosteroids demonstrated delayed apoptosis and prolonged viral particle production. This reduction in apoptosis in Adeno-infected epithelial cells by corticosteroids exposure induced a prolonged virus production via both DR4 and TRAIL protein suppression. In the Chronic model where animals were ovalbumin-sensitized/challenged and were treated with corticosteroids, apoptosis was reduced relative to adenovirus-infected or corticosteroid alone.ConclusionOur data suggests that apoptosis of infected cells limits viral production and may be mediated by DR4/5 and TRAIL expression. In the Acute model of Adeno-infection, corticosteroid exposure may prolong viral particle production by altering this apoptotic response of the infected cells. This results from decreased DR4 and TRAIL expression. In the Chronic model treated with corticosteroids, a similar decreased apoptosis was observed. This data suggests that DR and TRAIL modulation by corticosteroids may be important in viral infection of airway epithelium. The prolonged virus release in the setting of corticosteroids may result from reduced apoptosis and suppressed DR4/TRAIL expression by the infected cells.

Sialyl Lewis X modification of the epidermal growth factor receptor regulates receptor function during airway epithelial wound repair

Background Epidermal growth factor receptor (EGFR) is a major regulator of airway epithelial cell (AEC) functions such as migration, proliferation and differentiation, which play an essential role in epithelial repair. EGFR is a glycoprotein with 12 potential N‐glycosylation sites in its extracellular domain. Glycosylation of EGFR has been shown to modulate its function. Previously, our laboratory demonstrated an important role of the carbohydrate structure sialyl Lewis x (sLex) in airway epithelial repair.

Expression of surfactant protein D in airways of asthmatics and interleukin-13 modulation of surfactant protein D in human models of airway epithelium

BackgroundSurfactant protein D (SP-D), a pattern recognition molecule, has been shown to play roles in host defense such as opsonisation, aggregation of pathogens, and modulation of the inflammatory response. In light of infection-induced exacerbations and damage to the airway epithelium from inflammation, these functions of SP-D make it relevant in the development and pathogenesis of asthma.MethodsExpression of SP-D was examined in human airway sections and primary airway epithelial cells (AEC) grown in air-liquid interface (ALI) cultures and comparisons were made between those from asthmatic and non-asthmatic donors. ALI cultures of AEC from non-asthmatic donors were examined for SP-D, Mucin 5AC, and cytokeratin-5 expression at different stages of differentiation. Interleukin-13 (IL-13) treatment of airway epithelium and its effect on SP-D expression was studied using ALI and monolayer cultures of primary AEC from non-asthmatic and asthmatic donors.ResultsAirway epithelium of asthmatics, compared to that of non-asthmatics, expressed increased levels of SP-D as demonstrated in airway tissue sections (fraction of epithelium 0.66 ± 0.026 vs. 0.50 ± 0.043, p = 0.004) and ALI cultures (fraction of epithelium 0.50 ± 0.08 vs. 0.25 ± 0.07). SP-D expression decreased as ALI cultures differentiated from 7 days to 21 days (fraction of epithelium 0.62 ± 0.04 to 0.23 ± 0.03, p = 0.004). Treatment with IL-13 decreased SP-D expression in both ALI cultures (fraction of epithelium 0.21 ± 0.06 vs. 0.62 ± 0.04, p = 0.0005) and monolayer cultures (protein expression fold change 0.62 ± 0.05) of non-asthmatic AEC; however, IL-13 had no significant effect on SP-D expression in monolayer cultures of asthmatic AEC. Experiments with non-asthmatic monolayer cultures indicate IL-13 exert its effect on SP-D through the IL-13 receptor alpha1 and transcription factor STAT6.ConclusionsSP-D is expressed differently in airways of asthmatics relative to that of non-asthmatics. This can have implications on the increased susceptibility to infections and altered inflammatory response in asthmatic patients. Future functional studies on the role of SP-D in asthma can provide better insight into defects in the structure and regulation of SP-D.

Urban particulate matter increases human airway epithelial cell IL-1β secretion following scratch wounding and H1N1 influenza A exposure in vitro.

ABSTRACT Purpose: The airway epithelium represents the first line of defense against inhaled environmental insults including air pollution, allergens, and viruses. Epidemiological and experimental evidence has suggested a link between air pollution exposure and the symptoms associated with respiratory viral infections. We hypothesized that multiple insults integrated by the airway epithelium NLRP3 inflammasome would result in augmented IL-1β release and downstream cytokine production following respiratory virus exposure. Materials and Methods: We performed in vitro experiments with a human airway epithelial cell line (HBEC-6KT) that involved isolated or combination exposure to mechanical wounding, PM10, house dust mite, influenza A virus, and respiratory syncytial virus. We performed confocal microscopy to image the localization of PM10 within HBEC-6KT and ELISAs to measure soluble mediator production. Results: Airway epithelial cells secrete IL-1β in a time-dependent fashion that is associated with internalization of PM10 particles. PM10 exposure primes human airway epithelial cells to subsequent models of cell damage and influenza A virus exposure. Prior PM10 exposure had no effect on IL-1β responses to RSV exposure. Finally we demonstrate that PM10-priming of human airway epithelial cell IL-1β and GM-CSF responses to influenza A exposure are sensitive to NLRP3 inflammasome inhibition. Conclusions: Our results suggest the NLRP3 inflammasome may contribute to exaggerated immune responses to influenza A virus following periods of poor air quality. Intervention strategies targeting the NLRP3 inflammasome in at risk individuals may restrict poor air quality priming of mucosal immune responses that result from subsequent viral exposures.