Phillip Monk

Defective epithelial barrier function in asthma

BACKGROUND Asthma is a complex disease involving gene and environment interactions. Although atopy is a strong predisposing risk factor for asthma, local tissue susceptibilities are required for disease expression. The bronchial epithelium forms the interface with the external environment and is pivotally involved in controlling tissue homeostasis through provision of a physical barrier controlled by tight junction (TJ) complexes. OBJECTIVES To explain the link between environment exposures and airway vulnerability, we hypothesized that epithelial TJs are abnormal in asthma, leading to increased susceptibility to environmental agents. METHODS Localization of TJs in bronchial biopsies and differentiated epithelial cultures was assessed by electron microscopy or immunostaining. Baseline permeability and the effect of cigarette smoke and growth factor were assessed by measurement of transepithelial electrical resistance and passage of fluorescently labeled dextrans. RESULTS By using immunostaining, we found that bronchial biopsies from asthmatic subjects displayed patchy disruption of TJs. In differentiated bronchial epithelial cultures, TJ formation and transepithelial electrical resistance were significantly lower (P < .05) in cultures from asthmatic donors (n = 43) than from normal controls (n = 40) and inversely correlated with macromolecular permeability. Cultures from asthmatic donors were also more sensitive to disruption by cigarette smoke extract. Epidermal growth factor enhanced basal TJ formation in cultures from asthmatic subjects (P < .01) and protected against cigarette smoke-induced barrier disruption (P < .01). CONCLUSIONS Our results show that the bronchial epithelial barrier in asthma is compromised. This defect may facilitate the passage of allergens and other agents into the airway tissue, leading to immune activation and may thus contribute to the end organ expression of asthma.

Increased sputum and bronchial biopsy IL-13 expression in severe asthma.

BACKGROUND The importance of IL-13 in the asthma paradigm is supported by increased expression in human subjects, particularly in patients with mild-to-moderate asthma. However, the role of IL-13 in severe asthma needs to be further defined. OBJECTIVE We sought to assess IL-13 expression in sputum and bronchial biopsy specimens from subjects with mild-to-severe asthma. METHODS Sputum IL-13 concentrations were measured in 32 control subjects, 34 subjects with mild asthma, 21 subjects with moderate asthma, and 26 subjects with severe asthma. Enumeration of mast cells, eosinophils, and IL-13+ cells in the bronchial submucosa and airway smooth muscle (ASM) bundle was performed in 7 control subjects, 14 subjects with mild asthma, 7 subjects with moderate asthma, and 7 subjects with severe asthma. RESULTS The proportion of subjects with measurable IL-13 in the sputum was increased in the mild asthma group (15/34) and severe asthma group (10/26) compared with that seen in the control group (4/32; P = .004). IL-13+ cells were increased within the submucosa in all asthma severity groups compared with control subjects (P = .006). The number of IL-13+ cells were increased within the ASM bundle in the severe asthma group compared with that seen in the other groups (P < .05). Asthma control questionnaire scores positively correlated with sputum IL-13 concentrations (R(s) = 0.35, P = .04) and mast cells in the ASM bundle (R(s) = 0.7, P = .007). IL-13+ cells within the submucosa and ASM correlated with sputum eosinophilia (R(s) = 0.4, P < or = .05). CONCLUSIONS IL-13 overexpression in sputum and bronchial biopsy specimens is a feature of severe asthma.

The effect of inhaled IFN-β on worsening of asthma symptoms caused by viral infections. A randomized trial.

RATIONALE Ex vivo, bronchial epithelial cells from people with asthma are more susceptible to rhinovirus infection caused by deficient induction of the antiviral protein, IFN-β. Exogenous IFN-β restores antiviral activity. OBJECTIVES To compare the efficacy and safety of inhaled IFN-β with placebo administered to people with asthma after onset of cold symptoms to prevent or attenuate asthma symptoms caused by respiratory viruses. METHODS A total of 147 people with asthma on inhaled corticosteroids (British Thoracic Society Steps 2-5), with a history of virus-associated exacerbations, were randomized to 14-day treatment with inhaled IFN-β (n = 72) or placebo (n = 75) within 24 hours of developing cold symptoms and were assessed clinically, with relevant samples collected to assess virus infection and antiviral responses. MEASUREMENTS AND MAIN RESULTS A total of 91% of randomized patients developed a defined cold. In this modified intention-to-treat population, asthma symptoms did not get clinically significantly worse (mean change in six-item Asthma Control Questionnaire <0.5) and IFN-β treatment had no significant effect on this primary endpoint, although it enhanced morning peak expiratory flow recovery (P = 0.033), reduced the need for additional treatment, and boosted innate immunity as assessed by blood and sputum biomarkers. In an exploratory analysis of the subset of more difficult-to-treat, Step 4-5 people with asthma (n = 27 IFN-β; n = 31 placebo), Asthma Control Questionnaire-6 increased significantly on placebo; this was prevented by IFN-β (P = 0.004). CONCLUSIONS Although the trial did not meet its primary endpoint, it suggests that inhaled IFN-β is a potential treatment for virus-induced deteriorations of asthma in difficult-to-treat people with asthma and supports the need for further, adequately powered, trials in this population. Clinical trial registered with www.clinicaltrials.gov (NCT 01126177).

Barrier responses of human bronchial epithelial cells to grass pollen exposure

The airway epithelium forms a physical, chemical and immunological barrier against inhaled environmental substances. In asthma, these barrier properties are thought to be abnormal. In this study, we analysed the effect of grass pollen on the physical and immunological barrier properties of differentiated human primary bronchial epithelial cells. Following exposure to Timothy grass (Phleum pratense) pollen extract, the integrity of the physical barrier was not impaired as monitored by measuring the transepithelial resistance and immunofluorescence staining of tight junction proteins. In contrast, pollen exposure affected the immunological barrier properties by modulating vectorial mediator release. CXC chemokine ligand (CXCL)8/interleukin (IL)-8 showed the greatest increase in response to pollen exposure with preferential release to the apical compartment. Inhibition of the extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase pathways selectively blocked apical CXCL8/IL-8 release via a post-transcriptional mechanism. Apical release of CC chemokine ligand (CCL)20/macrophage inflammatory protein-3&agr;, CCL22/monocyte-derived chemokine and tumour necrosis factor-&agr; was significantly increased only in severe asthma cultures, while CCL11/eotaxin-1 and CXCL10/interferon-&ggr;-induced protein-10 were reduced in nonasthmatic cultures. The bronchial epithelial barrier modulates polarised release of mediators in response to pollen without direct effects on its physical barrier properties. The differential response of cells from normal and asthmatic donors suggests the potential for the bronchial epithelium to promote immune dysfunction in asthma.

A novel IgE-neutralizing antibody for the treatment of severe uncontrolled asthma

The critical role played by IgE in allergic asthma is well-documented and clinically precedented, but some patients in whom IgE neutralization may still offer clinical benefit are excluded from treatment with the existing anti-IgE therapy, omalizumab, due to high total IgE levels or body mass. In this study, we sought to generate a novel high affinity anti-IgE antibody (MEDI4212) with potential to treat a broad severe asthma patient population. Analysis of body mass, total and allergen-specific IgE levels in a cohort of severe asthmatics was used to support the rationale for development of a high affinity IgE-targeted antibody therapeutic. Phage display technology was used to generate a human IgG1 lead antibody, MEDI4212, which was characterized in vitro using binding, signaling and functional assay systems. Protein crystallography was used to determine the details of the interaction between MEDI4212 and IgE. MEDI4212 bound human IgE with an affinity of 1.95 pM and was shown to target critical residues in the IgE Cε3 domain critical for interaction with FcεRI. MEDI4212 potently inhibited responses through FcεRI and also prevented the binding of IgE to CD23. When used ex vivo at identical concentration, MEDI4212 depleted free-IgE from human sera to levels ~1 log lower than omalizumab. Our results thus indicate that MEDI4212 is a novel, high affinity antibody that binds specifically to IgE and prevents IgE binding to its receptors. MEDI4212 effectively depleted free-IgE from human sera ex vivo to a level (1 IU/mL) anticipated to provide optimal IgE suppression in severe asthma patients.

Experimental rhinovirus 16 infection in moderate asthmatics on inhaled corticosteroids

To the Editor : The majority of asthma exacerbations are associated with respiratory virus infections, mostly rhinoviruses (RVs) [1], due to enhanced inflammation in the airways [2]. These occur despite symptom control with inhaled corticosteroids (ICS) [3]. Experimental RV infection is a valuable tool for studying virus-induced exacerbations [2, 4], but has, to date, involved only corticosteroid-naive asthmatics. We have, therefore, modified a validated infection protocol [4] to inoculate 11 subjects whose asthma was well controlled with ICS. As this was the first experimental infection in patients at risk of severe exacerbations, a cautious study design was implemented. All subjects were followed-up twice daily by SMS text messages during the study. We used RV16, a strain used safely in previous studies, which replicates in vitro to a similar extent but induces less inflammation and cell death than other strains [5]. We also chose a 10-fold lower inoculation dose of the same stock used in previous studies [6]. The design allowed for dose escalation if necessary (this proved to be unnecessary as all subjects developed cold symptoms at this dose). As a final precaution, the delivery device generated particles of 30–100 μm, restricting delivery to the nose (aerosols ≥16 μm are deposited in the upper respiratory tract (URT) [7]), thus closely mimicking natural infection, i.e. limiting direct lung exposure during inoculation. Symptoms of URT infection, asthma, and measurements of lower respiratory tract (LRT) function were recorded post-inoculation. Infection was confirmed by quantitative (q)PCR for RV16 in nasal lavage and sputum and by determining serum anti-RV16 titres. At least a ≥four-fold increase in titres in convalescent serum or shedding of RV16 in the airways was evidence of successful infection. We also studied innate immune responses …

Nanoscale dysregulation of collagen structure-function disrupts mechano-homeostasis and mediates pulmonary fibrosis

Matrix stiffening with downstream activation of mechanosensitive pathways is strongly implicated in progressive fibrosis; however, pathologic changes in extracellular matrix (ECM) that initiate mechano-homeostasis dysregulation are not defined in human disease. By integrated multiscale biomechanical and biological analyses of idiopathic pulmonary fibrosis lung tissue, we identify that increased tissue stiffness is a function of dysregulated post-translational collagen cross-linking rather than any collagen concentration increase whilst at the nanometre-scale collagen fibrils are structurally and functionally abnormal with increased stiffness, reduced swelling ratio, and reduced diameter. In ex vivo and animal models of lung fibrosis, dual inhibition of lysyl oxidase-like (LOXL) 2 and LOXL3 was sufficient to normalise collagen fibrillogenesis, reduce tissue stiffness, and improve lung function in vivo. Thus, in human fibrosis, altered collagen architecture is a key determinant of abnormal ECM structure-function, and inhibition of pyridinoline cross-linking can maintain mechano-homeostasis to limit the self-sustaining effects of ECM on progressive fibrosis.