The relation between age and airway epithelial barrier function.

BACKGROUND: The prevalence of age-associated diseases, such as chronic obstructive pulmonary disease (COPD), is increasing as the average life expectancy increases around the world. We previously identified a gene signature for ageing in the human lung which included genes involved in apical and tight junction assembly, suggesting a role for airway epithelial barrier dysfunction with ageing. AIM: To investigate the association between genes involved in epithelial barrier function and age both in silico and in vitro in the airway epithelium. METHODS: We curated a gene signature of 274 genes for epithelial barrier function and tested the association with age in two independent cohorts of bronchial brushings from healthy individuals with no respiratory disease, using linear regression analysis (FDR < 0.05). Protein-protein interactions were identified using STRING©. The barrier function of primary bronchial epithelial cells at air-liquid interface and CRISPR-Cas9-induced knock-down of target genes in human bronchial 16HBE14o-cells was assessed using Trans epithelial resistance (TER) measurement and Electric cell-surface impedance sensing (ECIS) respectively. RESULTS: In bronchial brushings, we found 55 genes involved in barrier function to be significantly associated with age (FDR < 0.05). EPCAM was most significantly associated with increasing age and TRPV4 with decreasing age. Protein interaction analysis identified CDH1, that was negatively associated with higher age, as potential key regulator of age-related epithelial barrier function changes. In vitro, barrier function was lower in bronchial epithelial cells from subjects > 45 years of age and significantly reduced in CDH1-deficient 16HBE14o-cells. CONCLUSION: The significant association between genes involved in epithelial barrier function and age, supported by functional studies in vitro, suggest a role for epithelial barrier dysfunction in age-related airway disease.

Airway epithelial barrier function regulates the pathogenesis of allergic asthma.

The integrity of the airway epithelium in patients with asthma is often disrupted, with loss of epithelial cell-cell contacts. Airway epithelial barrier dysfunction may have important implications for asthma, because structural epithelial barrier function is tightly interwoven with the ability of the epithelium to regulate the immune system. We propose that changes at the airway epithelial barrier play a central role in the sensitisation to allergens and pathogenesis of allergic asthma. Many of the recently identified susceptibility genes for asthma are expressed in airway epithelium. However, the exact mechanisms by which the expression of epithelial susceptibility genes translates into a functionally altered response to aeroallergens in asthma are still unknown. In this review, we will focus on the role of airway epithelial barrier function in the susceptibility to develop allergic asthma and discuss therapeutic strategies aimed at the epithelial barrier.

The composition of house dust mite is critical for mucosal barrier dysfunction and allergic sensitisation.

BACKGROUND: House dust mite (HDM) allergens have been reported to increase airway epithelial permeability, thereby facilitating access of allergens and allergic sensitisation. OBJECTIVES: The authors aimed to understand which biochemical properties of HDM are critical for epithelial immune and barrier responses as well as T helper 2-driven experimental asthma in vivo. METHODS: Three commercially available HDM extracts were analysed for endotoxin levels, protease and chitinase activities and effects on transepithelial resistance, junctional proteins and pro-inflammatory cytokine release in the bronchial epithelial cell line 16HBE and normal human bronchial cells. Furthermore, the effects on epithelial remodelling and airway inflammation were investigated in a mouse model. RESULTS: The different HDM extracts varied extensively in their biochemical properties and induced divergent responses in vitro and in vivo. Importantly, the Greer extract, with the lowest serine protease activity, induced the most pronounced effects on epithelial barrier function and CCL20 release in vitro. In vivo, this extract induced the most profound epithelial E-cadherin delocalisation and increase in CCL20, CCL17 and interleukin 5 levels, accompanied by the most pronounced induction of HDM-specific IgE, goblet cell hyperplasia, eosinophilic inflammation and airway hyper-reactivity. CONCLUSIONS: This study shows the ability of HDM extracts to alter epithelial immune and barrier responses is related to allergic sensitisation but independent of serine/cysteine protease activity.

Similar programmed death ligand 1 (PD-L1) expression profile in patients with mild COPD and lung cancer.

Programmed Death Ligand 1 (PD-L1) is crucial in regulating the immunological tolerance in non-small cell lung cancer (NSCLC). Alveolar macrophage (AM)-derived PD-L1 binds to its receptor, PD-1, on surveilling lymphocytes, leading to lymphocyte exhaustion. Increased PD-L1 expression is associated with cigarette smoke (CS)-exposure. However, the PD-L1 role in CS-associated lung diseases associated with NSCLC, such as chronic obstructive pulmonary disease (COPD), is still unclear. In two different cohorts of ever smokers with COPD or NSCLC, and ever and never smoker controls, we evaluated PD-L1 expression: (1) via cutting-edge digital spatial proteomic and transcriptomic profiling (Geomx) of formalin-fixed paraffin-embedded (FFPE) lung tissue sections (n = 19); and (2) via triple immunofluorescence staining of bronchoalveolar lavage (BAL) AMs (n = 83). PD-L1 mRNA expression was also quantified in BAL AMs exposed to CS extract. PD-L1 expression was increased in the bronchiolar wall, parenchyma, and vascular wall from mild-moderate (GOLD 1-2) COPD patients compared to severe-very severe (GOLD 3-4) COPD patients and controls. Within all the COPD patients, PD-L1 protein expression was associated with upregulation of genes involved in tumor progression and downregulation of oncosuppressive genes, and strongly directly correlated with the FEV1% predicted, indicating higher PD-L1 expression in the milder vs. more severe COPD stages. In bronchioles, PD-L1 levels were strongly directly correlated with the number of functionally active AMs. In BAL, we confirmed that AMs from patients with both GOLD 1-2 COPD and NSCLC had the highest and similar, PD-L1 expression levels versus all the other groups, independently from active cigarette smoking. Intriguingly, AMs from patients with more severe COPD had reduced AM PD-L1 expression compared to patients with mild COPD. Acute CS extract stimulation increased PD-L1 mRNA expression only in never-and not in ever-smoker AMs. Lungs from patients with mild COPD and NSCLC are characterized by a similar strong PD-L1 expression signature in bronchioles and functionally active AMs compared to patients with severe COPD and controls. Active smoking does not affect PD-L1 levels. These observations represent a new resource in understanding the innate immune mechanisms underlying the link between COPD and lung cancer onset and progression and pave the way to future studies focused on the mechanisms by which CS promotes tumorigenesis and COPD.

Human airway smooth muscle is structurally and mechanically similar to that of other species.

Airway smooth muscle (ASM) plays a vital role in the exaggerated airway narrowing seen in asthma. However, whether asthmatic ASM is mechanically different from nonasthmatic ASM is unclear. Much of our current understanding about ASM mechanics comes from measurements made in other species. Limited data on human ASM mechanics prevents proper comparisons between healthy and asthmatic tissues, as well as human and animal tissues. In the current study, we sought to define the mechanical properties of healthy human ASM using tissue from intact lungs and compare these properties to measurements in other species. The mechanical properties measured included: maximal stress generation, force-length properties, the ability of the muscle to undergo length adaptation, the ability of the muscle to recover from an oscillatory strain, shortening velocity and maximal shortening. The ultrastructure of the cells was also examined. Healthy human ASM was found to be mechanically and ultrastructurally similar to that of other species. It is capable of undergoing length adaptation and responds to mechanical perturbation like ASM from other species. Force generation, shortening capacity and velocity were all similar to other mammalian ASM. These results suggest that human ASM shares similar contractile mechanisms with other animal species and provides an important dataset for comparisons with animal models of disease and asthmatic ASM.

Hedgehog signaling in the airway epithelium of patients with chronic obstructive pulmonary disease.

Genome-wide association studies have linked gene variants of the receptor patched homolog 1 (PTCH1) with chronic obstructive pulmonary disease (COPD). However, its biological role in the disease is unclear. Our objective was to determine the expression pattern and biological role of PTCH1 in the lungs of patients with COPD. Airway epithelial-specific PTCH1 protein expression and epithelial morphology were assessed in lung tissues of control and COPD patients. PTCH1 mRNA expression was measured in bronchial epithelial cells obtained from individuals with and without COPD. The effects of PTCH1 siRNA knockdown on epithelial repair and mucous expression were evaluated using human epithelial cell lines. Ptch1+/- mice were used to assess the effect of decreased PTCH1 on mucous expression and airway epithelial phenotypes. Airway epithelial-specific PTCH1 protein expression was significantly increased in subjects with COPD compared to controls, and its expression was associated with total airway epithelial cell count and thickness. PTCH1 knockdown attenuated wound closure and mucous expression in airway epithelial cell lines. Ptch1+/- mice had reduced mucous expression compared to wildtype mice following mucous induction. PTCH1 protein is up-regulated in COPD airway epithelium and may upregulate mucous expression. PTCH1 provides a novel target to reduce chronic bronchitis in COPD patients.

Characterization of a lung epithelium specific E-cadherin knock-out model: Implications for obstructive lung pathology.

The airway epithelium regulates responses to aeroallergens, acting as a physical and immunological barrier. In asthma, epithelial barrier function and the expression of adherens junction protein E-cadherin is compromised, but it is unknown whether this is cause or consequence of the disease. We hypothesized that airway epithelial loss of E-cadherin is a critical step in the development of manifestations of asthma. We generated a transgenic mouse model with conditional loss of E-cadherin in lung epithelial cells at birth and onwards. We observed normal lung development at the time of birth in mice lacking E-cadherin in the lung epithelium. However, E-cadherin deficiency led to progressive epithelial damage in mice growing into adulthood, as evidenced by airway epithelial denudation, decreased zonula occludens (ZO)-1 expression, loss of ciliated cells, and enlarged alveolar spaces. In addition, spontaneous goblet cell metaplasia with mucus production was observed. These epithelial changes were accompanied by elevated levels of the epithelial-derived chemokine CCL17, infiltration of eosinophils and dendritic cells, and mucus production. In conclusion, loss of E-cadherin induces features in the lung reminiscent of those observed in asthma, indicating that the disruption of E-cadherin-mediated cell-cell contacts may play a key role in the development of asthma manifestations.