Enhanced mitophagy in bronchial fibroblasts from severe asthmatic patients

BACKGROUND: Sub-epithelial fibrosis is a characteristic feature of airway remodeling in asthma which correlates with disease severity. Current asthma medications are ineffective in treating fibrosis. In this study, we aimed to investigate the mitochondrial phenotype in fibroblasts isolated from airway biopsies of non-asthmatic and severe asthmatic subjects by examining mitophagy as a mechanism contributing to fibroblast persistence and thereby, fibrosis in severe asthma. METHODS: Bioinformatics analysis of publicly available transcriptomic data was performed to identify the top enriched pathways in asthmatic fibroblasts. Endogenous expression of mitophagy markers in severe asthmatic and non-asthmatic fibroblasts was determined using qRT-PCR, western blot and immunofluorescence. Mitophagy flux was examined by using lysosomal protease inhibitors, E64d and pepstatin A. Mitochondrial membrane potential and metabolic activity were also evaluated using JC-1 assay and MTT assay, respectively. RESULTS: Bioinformatics analysis revealed the enrichment of Pink/Parkin-mediated mitophagy in asthmatic fibroblasts compared to healthy controls. In severe asthmatic fibroblasts, the differential expression of mitophagy genes, PINK1 and PRKN, was accompanied by the accumulation of PINK1, Parkin and other mitophagy proteins at baseline. The further accumulation of endogenous LC3BII, p62 and PINK1 in the presence of E64d and pepstatin A in severe asthmatic fibroblasts reinforced their enhanced mitophagy flux. Significantly reduced mitochondrial membrane potential and metabolic activity were also demonstrated at baseline confirming the impairment in mitochondrial function in severe asthmatic fibroblasts. Interestingly, these fibroblasts displayed neither an apoptotic nor senescent phenotype but a pro-fibrotic phenotype with an adaptive survival mechanism triggered by increased AMPKα phosphorylation and mitochondrial biogenesis. CONCLUSIONS: Our results demonstrated a role for mitophagy in the pathogenesis of severe asthma where the enhanced turnover of damaged mitochondria may contribute to fibrosis in severe asthma by promoting the persistence and pro-fibrotic phenotype of fibroblasts

Wnt Signaling Is Deranged in Asthmatic Bronchial Epithelium and Fibroblasts

Both canonical and non-canonical Wnt signaling pathway alterations have been documented in pulmonary disease pathogenesis and progression; therefore, they can be an attractive target for pharmaceutical management of severe asthma. Wnt/β-catenin signaling was shown to link early embryonic lung development impairment to later in life asthmatic airway remodeling. Here we explored the changes in Wnt signaling associated with asthma initiation and progression in epithelial and fibroblasts using a comprehensive approach based on in silico analysis and followed by in vitro validation. In summary, the in silico analysis showed that the bronchial epithelium of severe asthmatic patients showed a deranged balance between Wnt enhancer and Wnt inhibitors. A Th2-high phenotype is associated with upregulated Wnt-negative regulators, while inflammatory and neutrophilic severe asthmatics showed higher canonical Wnt signaling member enrichment. Most of these genes are regulators of healthy lung development early in life and, if disturbed, can make people susceptible to developing asthma early in life and prone to developing a severe phenotype. Most of the Wnt members are secreted, and their effect can be in an autocrine fashion on the bronchial epithelium, paracrine on nearby adjacent structural cells like fibroblasts and smooth muscles, or systemic in blood. Our results showed that canonical Wnt signaling is needed for the proper response of cells to proliferative stimuli, which puts cells under stress. Cells in response to this proliferative stress will activate the senescence mechanism, which is also dependent on Wnt signaling. Inhibition of Wnt signaling using FH535 inhibits both proliferation and senescence markers in bronchial fibroblasts compared to DMSO-treated cells. In fibroblasts from asthmatic patients, inhibition of Wnt signaling did not show that effect as the Wnt signaling is deranged besides other pathways that might be non-functional

IL-17 Induced Autophagy Regulates Mitochondrial Dysfunction and Fibrosis in Severe Asthmatic Bronchial Fibroblasts

The accumulation of fibroblasts, their synthesis of extracellular matrix (ECM) proteins and their innate resistance to apoptosis are characteristics of subepithelial fibrosis observed in severe asthma. Interleukin-17 (IL-17) is an important regulator of airway remodeling in asthma. However, the contribution of IL-17 to the pro-fibrotic phenotype of bronchial fibroblasts is not well-characterized. In this study, we investigated whether IL-17 induced autophagy regulates mitochondrial and pro-fibrotic function in bronchial fibroblasts. The primary cultured bronchial fibroblasts isolated from non-asthmatic (NHBF) and severe asthmatic (DHBF) subjects were treated with IL-17 in order to ascertain its effect on mitochondrial function, mitochondrial quality control, and apoptosis using immunoblotting and flow cytometric analyses. At baseline, DHBF exhibited higher levels of mitophagy and mitochondrial biogenesis compared to NHBF. Immunohistochemical evaluation of bronchial biopsies showed intense PINK1 immunoreactivity in severe asthma than in control. IL-17 intensified the mitochondrial dysfunction and impaired the mitochondrial quality control machinery in NHBF and DHBF. Moreover, IL-17 augmented a pro-fibrotic and anti-apoptotic response in both group of fibroblasts. Inhibition of autophagy using bafilomycin-A1 reduced PINK1 expression in NHBF and restored the IL-17 mediated changes in PINK1 to their basal levels in DHBF. Bafilomycin-A1 also reversed the IL-17 associated fibrotic response in these fibroblasts, suggesting a role for IL-17 induced autophagy in the induction of fibrosis in bronchial fibroblasts. Taken together, our findings suggest that IL-17 induced autophagy promotes mitochondrial dysfunction and fibrosis in bronchial fibroblasts from both non-asthmatic and severe asthmatic subjects. Our study provides insights into the therapeutic potential of targeting autophagy in ameliorating fibrosis, particularly in severe asthmatic individuals

Blood and Salivary Amphiregulin Levels as Biomarkers for Asthma

BACKGROUND: mphiregulin (AREG) expression in asthmatic airways and sputum was shown to increase and correlate with asthma. However, no studies were carried out to evaluate the AREG level in blood and saliva of asthmatic patients. OBJECTIVE: To measure circulating AREG mRNA and protein concentrations in blood, saliva, and bronchial biopsies samples from asthmatic patients. METHODS: Plasma and Saliva AREG protein concentrations were measured using ELISA while PBMCs, and Saliva mRNA expression was measured by RT qPCR in non-severe, and severe asthmatic patients compared to healthy controls. Primary asthmatic bronchial epithelial cells and fibroblasts were assessed for AREG mRNA expression and released soluble AREG in their conditioned media. Tissue expression of AREG was evaluated using immunohistochemistry of bronchial biopsies from asthmatic patients and healthy controls. Publicly available transcriptomic databases were explored for the global transcriptomic profile of bronchial epithelium, and PBMCs were explored for AREG expression in asthmatic vs. healthy controls. RESULTS: Asthmatic patients had higher AREG protein levels in blood and saliva compared to control subjects. Higher mRNA expression in saliva and primary bronchial epithelial cells plus higher AREG immunoreactivity in bronchial biopsies were also observed. Both blood and saliva AREG levels showed positive correlations with allergic rhinitis status, atopy status, eczema status, plasma periostin, neutrophilia, Montelukast sodium use, ACT score, FEV1, and FEV1/FVC. In silico analysis showed that severe asthmatic bronchial epithelium with high AREG gene expression is associated with higher neutrophils infiltration. CONCLUSION: AREG levels measured in a minimally invasive blood sample and a non-invasive saliva sample are higher in non-allergic severe asthma

Derangement of cell cycle markers in peripheral blood mononuclear cells of asthmatic patients as a reliable biomarker for asthma control

In asthma, most of the identified biomarkers pertain to the Th2 phenotype and no known biomarkers have been verified for severe asthmatics. Therefore, identifying biomarkers using the integrative phenotype-genotype approach in severe asthma is needed. The study aims to identify novel biomarkers as genes or pathways representing the core drivers in asthma development, progression to the severe form, resistance to therapy, and tissue remodeling regardless of the sample cells or tissues examined. Comprehensive reanalysis of publicly available transcriptomic data that later was validated in vitro, and locally recruited patients were used to decipher the molecular basis of asthma. Our in-silicoanalysis revealed a total of 10 genes (GPRC5A, SFN, ABCA1, KRT8, TOP2A, SERPINE1, ANLN, MKI67, NEK2, and RRM2) related to cell cycle and proliferation to be deranged in the severe asthmatic bronchial epithelium and fibroblasts compared to their healthy counterparts. In vitro, RT qPCR results showed that (SERPINE1 and RRM2) were upregulated in severe asthmatic bronchial epithelium and fibroblasts, (SFN, ABCA1, TOP2A, SERPINE1, MKI67, and NEK2) were upregulated in asthmatic bronchial epithelium while (GPRC5A and KRT8) were upregulated only in asthmatic bronchial fibroblasts. Furthermore, MKI76, RRM2, and TOP2A were upregulated in Th2 high epithelium while GPRC5A, SFN, ABCA1 were upregulated in the blood of asthmatic patients. SFN, ABCA1 were higher, while MKI67 was lower in severe asthmatic with wheeze compared to nonasthmatics with wheezes. SERPINE1 and GPRC5A were downregulated in the blood of eosinophilic asthmatics, while RRM2 was upregulated in an acute attack of asthma. Validation of the gene expression in PBMC of locally recruited asthma patients showed that SERPINE1, GPRC5A, SFN, ABCA1, MKI67, and RRM2 were downregulated in severe uncontrolled asthma. We have identified a set of biologically crucial genes to the homeostasis of the lung and in asthma development and progression. This study can help us further understand the complex interplay between the transcriptomic data and the external factors which may deviate our understanding of asthma heterogeneity

Site of Allergic Airway Narrowing and the Influence of Exogenous Surfactant in the Brown Norway Rat

Background: The parameters RN (Newtonian resistance), G (tissue damping), and H (tissue elastance) of the constant phase model of respiratory mechanics provide information concerning the site of altered mechanical properties of the lung. The aims of this study were to compare the site of allergic airway narrowing implied from respiratory mechanics to a direct assessment by morphometry and to evaluate the effects of exogenous surfactant administration on the site and magnitude of airway narrowing. Methods: We induced airway narrowing by ovalbumin sensitization and challenge and we tested the effects of a natural surfactant lacking surfactant proteins A and D (InfasurfH) on airway responses. Sensitized, mechanically ventilated Brown Norway rats underwent an aerosol challenge with 5 % ovalbumin or vehicle. Other animals received nebulized surfactant prior to challenge. Three or 20 minutes after ovalbumin challenge, airway luminal areas were assessed on snap-frozen lungs by morphometry. Results: At 3 minutes, RN and G detected large airway narrowing whereas at 20 minutes G and H detected small airway narrowing. Surfactant inhibited RN at the peak of the early allergic response and ovalbumin-induced increase in bronchoalveolar lavage fluid cysteinyl leukotrienes and amphiregulin but not IgE-induced mast cell activation in vitro. Conclusion: Allergen challenge triggers the rapid onset of large airway narrowing, detected by RN and G, and subsequen