Mechanical ventilation is a life-saving therapy for critically ill patients, providing rest to the respiratory muscles and facilitating gas exchange in the lungs. Ventilator-induced lung injury (VILI) is an unfortunate side effect of mechanical ventilation that may lead to serious consequences for the patient and increase mortality. The four main injury mechanisms associated with VILI are: baro/volutrauma caused by overstretching the lung tissues; atelectrauma, caused by repeated opening and closing of the alveoli resulting in shear stress; oxygen toxicity due to use of high ratio of oxygen in inspired air, causing formation of free radicals; and biotrauma, the resulting biological response to tissue injury, that leads to a cascade of events due to excessive inflammatory reactions and may cause multi-organ failure. An often-overlooked part of the inflammatory reaction is oxidative stress. In this research, a mouse model of VILI was set up with three tidal volume settings (10, 20 and 30 mL/kg) at atmospheric oxygen level. Airway pressures and heart rate were monitored and bronchoalveolar lavage fluid (BALF) and lung tissue samples were taken.We show a correlation between increased inflammation and barrier failure, and higher tidal volumes, evidenced by increased IL-6 expression, high concentration of proteins in BALF along with changes in expression of adhesion molecules. Furthermore, swelling of mitochondria in alveolar type II cells was seen indicating their dysfunction and senescence-like state. RNA sequencing data present clear increases in inflammation, mitochondrial biogenesis and oxidative stress as tidal volume is increased, supported by degradation of Keap1, a redox-regulated substrate adaptor protein.Oxidative stress seems to be a more prominent mechanism of VILI than previously considered, indicating that possible treatment methods against VILI might be identified by impeding oxidative pathways.
Idiopathic interstitial pneumonias cause fibrosis in which lungs become scarred due to the increase number of myofibroblasts and excess extracellular matrix deposition. Structural alterations in the interstitium of lung parenchyma are associated with significant mortality and morbidity. Epithelial-to-mesenchymal transition (EMT) is a process whereby epithelial cells undergo transition to a mesenchymal phenotype. This process has been shown to contribute to idiopathic pulmonary fibrosis (IPF) but contribution to nonspecific interstitial pneumonitis (NSIP) is unknown We investigated whether EMT was present in patients with IPF (n = 10) or NSIP (n = 6), using immunohistochemistry and confocal microscopy. All patients had surgical lung biopsies. Normal lung tissue obtained from lobectomies was also evaluated.We performed immunostaining for E-cadherin, p63, CK14 and the signature EMT markers N-cadherin and Vimentin. In addition we performed immunofluorecent staining for N-cadherin, p63 and Vimentin. We show that p63 positive basal cells are present in the overlying epithelium adjacent to fibrotic foci in IPF. This basal epithelium has acquired increased mesenchymal markers (N-cadherin,Vimentin) and increased CK14 while retaining E-cadherin expression. The underlying fibrotic foci showed both E- and N-cadherin positive cells.Samples from patients with NSIP had much less responses to CK14, Vimentin, p63 and N-cadherin than patients with IPF. This was related to fewer numbers of fibroblastic foci. Cells positive for p63 were found in alveoli and terminal bronchioles in NSIP but not normal controls. Results suggest that EMT is differently involved in pathogenesis of IPF than NSIP.
Azithromycin (AZM) is a macrolide which is effective in the treatment of several airway diseases induced by bacterial or environmental insults. We have previously shown barrier enhancing effects of AZM on airway epithelium (AE). In order to analyze barrier enhancing effects of AZM in vivo we have challenged mice with inhalation of sulphur dioxide (SO2) to induce AE barrier failure. Mice were exposed to 50-400 ppm SO2 gas for 0.5-4 hours and monitored up to 7 days before bronchoalveolar lavage fluid (BALF) collection and analysis. AE barrier function was evaluated by measuring human serum albumin (HSA) leakage into BALF. HSA was injected into the tail vein of mice one hour prior to sacrifice, BALF was then harvested and HSA concentration measured by ELISA. AZM administered prior to exposure to SO2 resulted in a reduction of HSA, indicating protective effects of AZM on the AE. Increased intracellular vacuolization and lamellar body (LB) formations were seen in AZM treated animals. Glutathione-S-Transferases (GSTs), a class of enzymes that help maintain cellular integrity, were reduced in SO2 treated mice. Mass spectrometry analysis of BALF from SO2 exposed mice revealed that treating the mice with AZM lead to higher concentrations of GSTs in BALF, suggesting a link between AZM treatment and a reduction in GST concentration. In conclusion, we demonstrated a protective effect of AZM on the barrier integrity of the AE, possibly through stabilizing the intracellular microenvironment and by facilitating formation of LBs, providing further insight to its effectiveness in the treatment of airway diseases.
Abstract Background The airway epithelium (AE) forms the first line of defence against harmful particles and pathogens. Barrier failure of the airway epithelium contributes to exacerbations of a range of lung diseases that are commonly treated with Azithromycin (AZM). In addition to its anti-bacterial function, AZM has immunomodulatory effects which are proposed to contribute to its clinical effectiveness. In vitro studies have shown the AE barrier-enhancing effects of AZM. The aim of this study was to analyze whether AE damage caused by inhalation of sulfur dioxide (SO 2 ) in a murine model could be reduced by pre-treatment with AZM. Methods The leakiness of the AE barrier was evaluated after SO 2 exposure by measuring levels of human serum albumin (HSA) in bronchoalveolar lavage fluid (BALF). Protein composition in BALF was also assessed and lung tissues were evaluated across treatments using histology and gene expression analysis. Results AZM pre-treatment (2 mg/kg p.o. 5 times/week for 2 weeks) resulted in reduced glutathione-S-transferases in BALF of SO 2 injured mice compared to control (without AZM treatment). AZM treated mice had increased intracellular vacuolization including lamellar bodies and a reduction in epithelial shedding after injury in addition to a dampened SO 2 -induced inflammatory response. Conclusions Using a mouse model of AE barrier dysfunction we provide evidence for the protective effects of AZM in vivo, possibly through stabilizing the intracellular microenvironment and reducing inflammatory responses. Our data provide insight into the mechanisms contributing to the efficacy of AZM in the treatment of airway diseases.
Macrolides are frequently prescribed antibiotics, used to treat a spectrum of respiratory and skin infections. They are also known for their off-label use, some macrolides being effective against inflammation and oxidative stress, affecting several signaling pathways. Recently, their enhancing effects on the respiratory epithelial barrier have become more evident. In this study the non-antimicrobial effects of the traditional 14-membered macrolide antibiotics; erythromycin (ERY), clarithromycin (CLARI) and roxithromycin (ROXI), the 15-membered nitrogen containing macrolide, azithromycin (AZM) and the ketolide solithromycin (SOLI) were analyzed in vitro. A bronchial epithelial cell line, VA10, was treated with macrolides for 14 and 21 days in air-liquid interface (ALI) condition. Along with RNA sequencing, transepithelial electrical resistance (TEER), permeability and histological effects were observed to evaluate the epithelial barrier. Treatment with AZM differs from the other macrolides by a more pronounced increase in TEER, thickness of cell layer, decreased permeability and increased accumulation of phospholipids. ERY and CLARI showed both moderate increase in TEER and decreased permeability. Collectively, we have shown that macrolides have profound effects on gene expression and phenotype in human bronchial cells. AZM has distinctly greater epithelial barrier-inductive properties, although all macrolides tested affect gene expression, particularly gene sets involving inflammation, lipid metabolism and oxidative stress.
Azithromycin (Azm) is a macrolide recognized for its disease-modifying effects and reduction in exacerbation of chronic airway diseases. It is not clear whether the beneficial effects of Azm are due to its anti-microbial activity or other pharmacological actions. We have shown that Azm affects the integrity of the bronchial epithelial barrier measured by increased transepithelial electrical resistance. To better understand these effects of Azm on bronchial epithelia we have investigated global changes in gene expression. VA10 bronchial epithelial cells were treated with Azm and cultivated in air-liquid interface conditions for up to 22 days. RNA was isolated at days 4, 10 and 22 and analyzed using high-throughput RNA sequencing. qPCR and immunostaining were used to confirm key findings from bioinformatic analyses. Detailed assessment of cellular changes was done using microscopy, followed by characterization of the lipidomic profiles of the multivesicular bodies present. Bioinformatic analysis revealed that after 10 days of treatment genes encoding effectors of sterol and cholesterol metabolism were prominent. Interestingly, expression of genes associated with epidermal barrier differentiation, KRT1, CRNN, SPINK5 and DSG1, increased significantly at day 22. Together with immunostaining, these results suggest an epidermal differentiation pattern. We also found that Azm induced the formation of multivesicular and lamellar bodies in two different airway epithelial cell lines. Lipidomic analysis revealed that Azm was entrapped in multivesicular bodies linked to different types of lipids, most notably palmitate and stearate. Furthermore, targeted analysis of lipid species showed accumulation of phosphatidylcholines, as well as ceramide derivatives. Taken together, we demonstrate how Azm might confer its barrier enhancing effects, via activation of epidermal characteristics and changes to intracellular lipid dynamics. These effects of Azm could explain the unexpected clinical benefit observed during Azm-treatment of patients with various lung diseases affecting barrier function.
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