Analysis of comorbidities and multimorbidity in adult patients in the International Severe Asthma Registry.

BACKGROUND: Investigation for the presence of asthma comorbidities is recommended by the Global Initiative for Asthma because their presence can complicate asthma management. OBJECTIVE: To understand the prevalence and pattern of comorbidities and multimorbidity in adults with severe asthma and their association with asthma-related outcomes. METHODS: This was a cross-sectional study using data from the International Severe Asthma Registry from 22 countries. A total of 30 comorbidities were identified and categorized a priori as any of the following: (1) potentially type 2-related comorbidities, (2) potentially oral corticosteroid (OCS)-related comorbidities, or (3) comorbidities mimicking or aggravating asthma. The association between comorbidities and asthma-related outcomes was investigated using multivariable models adjusted for country, age at enrollment, and sex (ie male or female). RESULTS: Of the 11,821 patients, 69%, 67%, and 55% had at least 1 potentially type 2-related, potentially OCS-related, or mimicking or aggravating comorbidities, respectively; 57% had 3 or more comorbidities, and 33% had comorbidities in all 3 categories. Patients with allergic rhinitis, nasal polyposis, and chronic rhinosinusitis experienced 1.12 (P = .003), 1.16 (P < .001), and 1.29 times (P < .001) more exacerbations per year, respectively, than those without. Patients with nasal polyposis and chronic rhinosinusitis were 40% and 46% more likely (P < .001), respectively, to have received long-term (LT) OCS. All assessed potential OCS-related comorbidities (except obesity) were associated with a greater likelihood of LTOCS use (odds ratios [ORs]: 1.23-2.77) and, except for dyslipidemia, with a greater likelihood of uncontrolled asthma (ORs: 1.29-1.68). All mimicking or aggravating comorbidities assessed were associated with more exacerbations (1.24-1.68 times more), all (except bronchiectasis) with increased likelihood of uncontrolled asthma (ORs: 1.57-1.81), and all (except chronic obstructive pulmonary disease) with increased likelihood of LTOCS use (ORs: 1.37-1.57). A greater number of comorbidities was associated with worse outcomes. CONCLUSION: In a global study, comorbidity or multimorbidity is reported in most adults with severe asthma and is associated with poorer asthma-related outcomes. CLINICAL TRIAL REGISTRATION: The International Severe Asthma Registry database has ethical approval from the Anonymous Data Ethics Protocols and Transparency (ADEPT) committee (ADEPT0218) and is registered with the European Union Electronic Register of Post-Authorization Studies (European Network Centres for Pharmacoepidemiology and Pharmacovigilance [ENCEPP]/DSPP/23720). The study was designed, implemented, and reported in compliance with the European Network Centres for Pharmacoepidemiology and Pharmacovigilance (ENCEPP) Code of Conduct (EMA 2014; EUPAS44024) and with all applicable local and international laws and regulations, and registered with ENCEPP (https://www.encepp.eu/encepp/viewResource.htm?id=48848). Governance was provided by ADEPT (registration number: ADEPT1121).

Association Between T2-related Comorbidities and Effectiveness of Biologics in Severe Asthma.

Rationale: Previous studies investigating the impact of comorbidities on the effectiveness of biologic agents have been relatively small and of short duration and have not compared classes of biologic agents. Objectives: To determine the association between type 2-related comorbidities and biologic agent effectiveness in adults with severe asthma (SA). Methods: This cohort study used International Severe Asthma Registry data from 21 countries (2017-2022) to quantify changes in four outcomes before and after biologic therapy-annual asthma exacerbation rate, FEV1% predicted, asthma control, and long-term oral corticosteroid daily dose-in patients with or without allergic rhinitis, chronic rhinosinusitis (CRS) with or without nasal polyps (NPs), NPs, or eczema/atopic dermatitis. Measurements and Main Results: Of 1,765 patients, 1,257, 421, and 87 initiated anti-IL-5/5 receptor, anti-IgE, and anti-IL-4/13 therapies, respectively. In general, pre- versus post-biologic therapy improvements were noted in all four asthma outcomes assessed, irrespective of comorbidity status. However, patients with comorbid CRS with or without NPs experienced 23% fewer exacerbations per year (95% CI, 10-35%; P < 0.001) and had 59% higher odds of better post-biologic therapy asthma control (95% CI, 26-102%; P < 0.001) than those without CRS with or without NPs. Similar estimates were noted for those with comorbid NPs: 22% fewer exacerbations and 56% higher odds of better post-biologic therapy control. Patients with SA and CRS with or without NPs had an additional FEV1% predicted improvement of 3.2% (95% CI, 1.0-5.3; P = 0.004), a trend that was also noted in those with comorbid NPs. The presence of allergic rhinitis or atopic dermatitis was not associated with post-biologic therapy effect for any outcome assessed. Conclusions: These findings highlight the importance of systematic comorbidity evaluation. The presence of CRS with or without NPs or NPs alone may be considered a predictor of the effectiveness of biologic agents in patients with SA.

Hedgehog Signaling as a Therapeutic Target for Airway Remodeling and Inflammation in Allergic Asthma.

Genome-wide association studies (GWAS) have shown that variants of patched homolog 1 (PTCH1) are associated with lung function abnormalities in the general population. It has also been shown that sonic hedgehog (SHH), an important ligand for PTCH1, is upregulated in the airway epithelium of patients with asthma and is suggested to be involved in airway remodeling. The contribution of hedgehog signaling to airway remodeling and inflammation in asthma is poorly described. To determine the biological role of hedgehog signaling-associated genes in asthma, gene silencing, over-expression, and pharmacologic inhibition studies were conducted after stimulating human airway epithelial cells or not with transforming growth factor ß1 (TGFß1), an important fibrotic mediator in asthmatic airway remodeling that also interacts with SHH pathway. TGFß1 increased hedgehog-signaling-related gene expression including SHH, GLI1 and GLI2. Knockdown of PTCH1 or SMO with siRNA, or use of hedgehog signaling inhibitors, consistently attenuated COL1A1 expression induced by TGFß1 stimulation. In contrast, Ptch1 over-expression augmented TGFß1-induced an increase in COL1A1 and MMP2 gene expression. We also showed an increase in hedgehog-signaling-related gene expression in primary airway epithelial cells from controls and asthmatics at different stages of cellular differentiation. GANT61, an inhibitor of GLI1/2, attenuated TGFß1-induced increase in COL1A1 protein expression in primary airway epithelial cells differentiated in air-liquid interface. Finally, to model airway tissue remodeling in vivo, C57BL/6 wildtype (WT) and Ptch1+/- mice were intranasally challenged with house dust mite (HDM) or phosphate-buffered saline (PBS) control. Ptch1+/- mice showed reduced sub-epithelial collagen expression and serum inflammatory proteins compared to WT mice in response to HDM challenge. In conclusion, TGFß1-induced airway remodeling is partially mediated through the hedgehog signaling pathway via the PTCH1-SMO-GLI axis. The Hedgehog signaling pathway is a promising new potential therapeutic target to alleviate airway tissue remodeling in patients with allergic airways disease.

FAM13A as potential therapeutic target in modulating TGF-ß-induced airway tissue remodeling in COPD.

Genome-wide association studies have shown that a gene variant in the Family with sequence similarity 13, member A (FAM13A) is strongly associated with reduced lung function and the appearance of respiratory symptoms in patients with chronic obstructive pulmonary disease (COPD). A key player in smoking-induced tissue injury and airway remodeling is the transforming growth factor-ß1 (TGF-ß1). To determine the role of FAM13A in TGF-ß1 signaling, FAM13A-/- airway epithelial cells were generated using CRISPR-Cas9, whereas overexpression of FAM13A was achieved using lipid nanoparticles. Wild-type (WT) and FAM13A-/- cells were treated with TGF-ß1, followed by gene and/or protein expression analyses. FAM13A-/- cells augmented TGF-ß1-induced increase in collagen type 1 (COL1A1), matrix metalloproteinase 2 (MMP2), expression compared with WT cells. This effect was mediated by an increase in ß-catenin (CTNNB1) expression in FAM13A-/- cells compared with WT cells after TGF-ß1 treatment. FAM13A overexpression was partially protective from TGF-ß1-induced COL1A1 expression. Finally, we showed that airway epithelial-specific FAM13A protein expression is significantly increased in patients with severe COPD compared with control nonsmokers, and negatively correlated with lung function. In contrast, ß-catenin (CTNNB1), which has previously been linked to be regulated by FAM13A, is decreased in the airway epithelium of smokers with COPD compared with non-COPD subjects. Together, our data showed that FAM13A may be protective from TGF-ß1-induced fibrotic response in the airway epithelium via sequestering CTNNB1 from its regulation on downstream targets. Therapeutic increase in FAM13A expression in the airway epithelium of smokers at risk for COPD, and those with mild COPD, may reduce the extent of airway tissue remodeling.

Olodaterol exerts anti-inflammatory effects on COPD airway epithelial cells.

BACKGROUND: Airway inflammation is a key feature of chronic obstructive pulmonary disease (COPD) and inhaled corticosteroids (ICS) remain the main treatment for airway inflammation. Studies have noted the increased efficacy of ICS and long-acting beta 2 agonist (LABA) combination therapy in controlling exacerbations and improving airway inflammation than either monotherapy. Further studies have suggested that LABAs may have inherent anti-inflammatory potential, but this has not been well-studied. OBJECTIVE: We hypothesize that the LABA olodaterol can inhibit airway inflammation resulting from exposure to respiratory syncytial virus (RSV) via its binding receptor, the ß2-adrenergic receptor. METHODS: Human bronchial epithelial brushing from patients with and without COPD were cultured into air-liquid interface (ALI) cultures and treated with or without olodaterol and RSV infection to examine the effect on markers of inflammation including interleukin-8 (IL-8) and mucus secretion. The cell line NCI-H292 was utilized for gene silencing of the ß2-adrenergic receptor via siRNA as well as receptor blocking via ICI 118,551 and butaxamine. RESULTS: At baseline, COPD-ALIs produced greater amounts of IL-8 than control ALIs. Olodaterol reduced RSV-mediated IL-8 secretion in both COPD and control ALIs and also significantly reduced Muc5AC staining in COPD-ALIs infected with RSV. A non-significant reduction was seen in control ALIs. Gene silencing of the ß2-adrenergic receptor in NCI-H292 negated the ability of olodaterol to inhibit IL-8 secretion from both RSV infection and lipopolysaccharide stimulus, as did blocking of the receptor with ICI 118,551 and butaxamine. CONCLUSIONS: Olodaterol exhibits inherent anti-inflammatory properties on the airway epithelium, in addition to its bronchodilation properties, that is mediated through the ß2-adrenergic receptor and independent of ICS usage.

Structural and functional variations in human bronchial epithelial cells cultured in air-liquid interface using different growth media.

The human bronchial epithelium is an important barrier tissue that is damaged or pathologically altered in various acute and chronic respiratory conditions. To represent the epithelial component of respiratory disease, it is essential to use a physiologically relevant model of this tissue. The human bronchial epithelium is a highly organized tissue consisting of a number of specialized cell types. Primary human bronchial epithelial cells (HBEC) can be differentiated into a mucociliated tissue in air-liquid interface (ALI) cultures using appropriately supplemented media under optimized growth conditions. We compared the histology, ciliary length, and function, diffusion, and barrier properties of HBEC from donors with no respiratory disease grown in two different media, PneumaCult-ALI or Bronchial Epithelial Differentiation Medium (BEDM). In the former group, HBEC have a more physiological pseudostratified morphology and mucociliary differentiation, including increased epithelial thickness, intracellular expression of airway-specific mucin protein MUC5AC, and total expression of cilia basal-body protein compared with cells from the same donor grown in the other medium. Baseline expression levels of inflammatory mediators, thymic stromal lymphopoietin (TSLP), soluble ST2, and eotaxin-3 were lower in PneumaCult-ALI. Additionally, the physiological cilia beat frequency and electrical barrier properties with transepithelial electrical resistance were significantly different between the two groups. Our study has shown that these primary cell cultures from the same donor grown in the two media possess variable structural and functional characteristics. Therefore, it is important to objectively validate primary epithelial cell cultures before experimentation to ensure they are appropriate to answer a specific scientific question.

Phagocytosis of Aspergillus fumigatus by Human Bronchial Epithelial Cells Is Mediated by the Arp2/3 Complex and WIPF2.

Aspergillus fumigatus is an opportunistic fungal pathogen capable of causing severe infection in humans. One of the limitations in our understanding of how A. fumigatus causes infection concerns the initial stages of infection, notably the initial interaction between inhaled spores or conidia and the human airway. Using publicly-available datasets, we identified the Arp2/3 complex and the WAS-Interacting Protein Family Member 2 WIPF2 as being potentially responsible for internalization of conidia by airway epithelial cells. Using a cell culture model, we demonstrate that RNAi-mediated knockdown of WIPF2 significantly reduces internalization of conidia into airway epithelial cells. Furthermore, we demonstrate that inhibition of Arp2/3 by a small molecule inhibitor causes similar effects. Using super-resolution fluorescence microscopy, we demonstrate that WIPF2 is transiently localized to the site of bound conidia. Overall, we demonstrate the active role of the Arp2/3 complex and WIPF2 in mediating the internalization of A. fumigatus conidia into human airway epithelial cells.

IL-13 signaling through IL-13 receptor α2 mediates airway epithelial wound repair.

Asthma is an airway inflammatory disease characterized by epithelial barrier dysfunction and airway remodeling. Interleukin-13 (IL-13) is a pleiotropic cytokine shown to contribute to features of airway remodeling. We have previously demonstrated that IL-13 is an important mediator of normal airway epithelial repair and health. The role of IL-13 signaling via its receptor subunits (IL-13Rα1/IL-4Rα and IL-13Rα2) in airway epithelial repair and restoration of intact barrier function is not well understood and was investigated in this study using in vitro models. The blocking of IL-13 signaling via IL-13Rα2 significantly reduced airway epithelial repair by 24 h post-mechanical wounding in 1HAEo- cells. Expression and release of repair-mediating growth factor, heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), and subsequent activation of EGF receptor (EGFR) were also significantly reduced in response to wounding when IL-13Rα2 was blocked. Our data support that IL-13 signals via IL-13Rα2 to mediate normal airway epithelial repair via HB-EGF-dependent activation of EGFR. In human donor lung tissues, we observed that airway epithelium of asthmatics expressed significantly decreased levels of IL-13Rα2 and increased levels of IL-13Rα1 compared with nonasthmatics. Dysregulated expression of IL-13 receptor subunits in the airways of asthmatics may thus contribute to the epithelial barrier dysfunction observed in asthma.-Yang, S. J., Allahverdian, S., Saunders, A. D. R., Liu, E., Dorscheid, D. R. IL-13 signaling through IL-13 receptor α2 mediates airway epithelial wound repair.

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

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-1ß 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-1ß 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-1ß responses to RSV exposure. Finally we demonstrate that PM10-priming of human airway epithelial cell IL-1ß 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.

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

BACKGROUND: Surfactant 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. METHODS: Expression 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. RESULTS: Airway 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. CONCLUSIONS: SP-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.

Estradiol increases mucus synthesis in bronchial epithelial cells.

Airway epithelial mucus hypersecretion and mucus plugging are prominent pathologic features of chronic inflammatory conditions of the airway (e.g. asthma and cystic fibrosis) and in most of these conditions, women have worse prognosis compared with male patients. We thus investigated the effects of estradiol on mucus expression in primary normal human bronchial epithelial cells from female donors grown at an air liquid interface (ALI). Treatment with estradiol in physiological ranges for 2 weeks caused a concentration-dependent increase in the number of PAS-positive cells (confirmed to be goblet cells by MUC5AC immunostaining) in ALI cultures, and this action was attenuated by estrogen receptor beta (ER-ß) antagonist. Protein microarray data showed that nuclear factor of activated T-cell (NFAT) in the nuclear fraction of NHBE cells was increased with estradiol treatment. Estradiol increased NFATc1 mRNA and protein in ALI cultures. In a human airway epithelial (1HAE0) cell line, NFATc1 was required for the regulation of MUC5AC mRNA and protein. Estradiol also induced post-translational modification of mucins by increasing total fucose residues and fucosyltransferase (FUT-4, -5, -6) mRNA expression. Together, these data indicate a novel mechanism by which estradiol increases mucus synthesis in the human bronchial epithelium.

Formation of a stable mimic of ambient particulate matter containing viable infectious respiratory syncytial virus and its dry-deposition directly onto cell cultures.

Epidemiological associations of worse respiratory outcomes from combined exposure to ambient particulate matter (PM) and respiratory viral infection suggest possible interactions between PM and viruses. To characterize outcomes of such exposures, we developed an in vitro mimic of the in vivo event of exposure to PM contaminated with respiratory syncytial virus (RSV). Concentration of infectious RSV stocks and a particle levitation apparatus were the foundations of the methodology developed to generate specific numbers of PM mimics (PM(Mimics)) of known composition for dry, direct deposition onto airway epithelial cell cultures. Three types of PM(Mimics) were generated for this study: (i) carbon alone (P(C)), (ii) carbon and infectious RSV (P(C+RSV)), and (iii) aerosols consisting of RSV (A(RSV)). P(C+RSV) were stable in solution and harbored infectious RSV for up to 6 months. Unlike A(RSV) infection, P(C+RSV) infection was found to be dynamin dependent and to cause lysosomal rupture. Cells dosed with PM(Mimics) comprised of RSV (A(RSV)), carbon (P(C)), or RSV and carbon (P(C+RSV)) responded differentially as exemplified by the secretion patterns of IL-6 and IL-8. Upon infection, and prior to lung cell death due to viral infection, regression analysis of these two mediators in response to incubation with A(RSV), P(C), or P(C+RSV) yielded higher concentrations upon infection with the latter and at earlier time points than the other PM(Mimics). In conclusion, this experimental platform provides an approach to study the combined effects of PM-viral interactions and airway epithelial exposures in the pathogenesis of respiratory diseases involving inhalation of environmental agents.

Wound-induced TGF-ß1 and TGF-ß2 enhance airway epithelial repair via HB-EGF and TGF-α.

The abundance of transforming growth factor-beta (TGF-ß) in normal airway epithelium suggests its participation in physiological processes to maintain airway homeostasis. The current study was designed to address the hypothesis that TGF-ß1 and TGF-ß2 might contribute to normal reparative response of airway epithelial cells (AECs). Treatments with exogenous TGF-ß1 or TGF-ß2 significantly enhanced wound repair of confluent AEC monolayers. Mechanical injury of AEC monolayers induced production of both TGF-ß1 and TGF-ß2. Wound repair of AECs was significantly reduced by a specific inhibitor of TGF-ß type I receptor kinase activity. We investigated whether the TGF-ß-enhanced repair required epidermal growth factor receptor (EGFR) transactivation and secretion of EGFR ligands. Both TGF-ß1 and TGF-ß2 enhanced EGFR phosphorylation and induced production of heparin-binding EGF-like growth factor (HB-EGF) and transforming growth factor-alpha (TGF-α) in AECs. Moreover, treatment with a broad-spectrum metalloproteinase inhibitor or anti-HB-EGF and anti-TGF-α antibodies inhibited the wound repair and the EGFR phosphorylation by TGF-ß1 and TGF-ß2, indicating that the TGF-ß1 and TGF-ß2 effects on wound repair required the release of HB-EGF and TGF-α. Our data, for the first time, have shown that both TGF-ß1 and TGF-ß2 play a stimulatory role in airway epithelial repair through EGFR phosphorylation following autocrine production of HB-EGF and TGF-α. These findings highlight an important collaborative mechanism between TGF-ß and EGFR in maintaining airway epithelial homeostasis.

The role of female hormones on lung function in chronic lung diseases.

BACKGROUND: The prevalence, morbidity, and mortality of inflammatory lung diseases such as asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) are increasing in women. There is a dearth of data on the biological mechanisms to explain such observations. However, some large epidemiologic studies suggest that lung function fluctuates during the menstrual cycle in female patients with airways disease but not in women without disease, suggesting that circulating estradiol and progesterone may be involved in this process. DISCUSSION: In asthma, estradiol shuttles adaptive immunity towards the TH2 phenotype while in smokers estrogens may be involved in the generation of toxic intermediate metabolites in the airways of female smokers, which may be relevant in COPD pathogenesis. In CF, estradiol has been demonstrated to up-regulate MUC5B gene in human airway epithelial cells and inhibit chloride secretion in the airways. Progesterone may augment airway inflammation. SUMMARY: Taken together, clinical and in-vivo data have demonstrated a sex-related difference in that females may be more susceptible to the pathogenesis of lung diseases. In this paper, we review the effect of female sex hormones in the context of these inflammatory airway diseases.

The airway epithelium: more than just a structural barrier.

The mammalian airway is lined by a variety of specialized epithelial cells that not only serve as a physical barrier but also respond to environment-induced damage through the release of biologically active factors and constant cellular renewal. The lung epithelium responds to environmental insults such as pathogens, cigarette smoke and pollution by secreting inflammatory mediators and antimicrobial peptides, and by recruiting immune cells to the site of infection or damage. When the epithelium is severely damaged, basal cells and Clara cells that have stem-cell-like properties are capable of self-renewal and proliferation in the affected area, to repair the damage. In order to effectively fight off infections, the epithelium requires the assistance of neutrophils recruited from the peripheral circulation through transendothelial followed by transepithelial migration events. Activated neutrophils migrate across the epithelium through a series of ligand-receptor interactions to the site of injury, where they secrete proteolytic enzymes and oxidative radicals for pathogen destruction. However, chronic activation and recruitment of neutrophils in airway diseases such as chronic obstructive pulmonary disease and asthma has been associated with tissue damage and disease severity. In this paper, we review the current understanding of the airway epithelial response to injury and its interaction with inflammatory cells, in particular the neutrophil.

Toll-like receptor 4-mediated activation of p38 mitogen-activated protein kinase is a determinant of respiratory virus entry and tropism.

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.

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.

Secretion of IL-13 by airway epithelial cells enhances epithelial repair via HB-EGF.

Inappropriate repair after injury to the epithelium generates persistent activation, which may contribute to airway remodeling. In the present study we hypothesized that IL-13 is a normal mediator of airway epithelial repair. Mechanical injury of confluent airway epithelial cell (AEC) monolayers induced expression and release of IL-13 in a time-dependent manner coordinate with repair. Neutralizing of IL-13 secreted from injured epithelial cells by shIL-13Ralpha2.FC significantly reduced epithelial repair. Moreover, exogenous IL-13 enhanced epithelial repair and induced epidermal growth factor receptor (EGFR) phosphorylation. We examined secretion of two EGFR ligands, epidermal growth factor (EGF) and heparin-binding EGF (HB-EGF), after mechanical injury. Our data showed a sequential release of the EGF and HB-EGF by AEC after injury. Interestingly, we found that IL-13 induces HB-EGF, but not EGF, synthesis and release from AEC. IL-13-induced EGFR phosphorylation and the IL-13-reparative effect on AEC are mediated via HB-EGF. Finally, we demonstrated that inhibition of EGFR tyrosine kinase activity by tyrphostin AG1478 increases IL-13 release after injury, suggesting negative feedback between EGFR and IL-13 during repair. Our data, for the first time, showed that IL-13 plays an important role in epithelial repair, and that its effect is mediated through the autocrine release of HB-EGF and activation of EGFR. Dysregulation of EGFR phosphorylation may contribute to a persistent repair phenotype and chronically increased IL-13 release, and in turn result in airway remodeling.