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An Innovative Lipidomic Workflow to Investigate the Lipid Profile in a Cystic Fibrosis Cell Line.

ABSTRACT: Altered lipid metabolism has been associated to cystic fibrosis disease, which is characterized by chronic lung inflammation and various organs dysfunction. Here, we present the validation of an untargeted lipidomics approach based on high-resolution mass spectrometry aimed at identifying those lipid species that unequivocally sign CF pathophysiology. Of n.13375 mass spectra recorded on cystic fibrosis bronchial epithelial airways epithelial cells IB3, n.7787 presented the MS/MS data, and, after software and manual validation, the final number of annotated lipids was restricted to n.1159. On these lipids, univariate and multivariate statistical approaches were employed in order to select relevant lipids for cellular phenotype discrimination between cystic fibrosis and HBE healthy cells. In cystic fibrosis IB3 cells, a pervasive alteration in the lipid metabolism revealed changes in the classes of ether-linked phospholipids, cholesterol esters, and glycosylated sphingolipids. Through functions association, it was evidenced that lipids variation involves the moiety implicated in membrane composition, endoplasmic reticulum, mitochondria compartments, and chemical and biophysical lipids properties. This study provides a new perspective in understanding the pathogenesis of cystic fibrosis and strengthens the need to use a validated mass spectrometry-based lipidomics approach for the discovery of potential biomarkers and perturbed metabolism.

SUBMITTER: Dei Cas M

PROVIDER: S-EPMC7291020 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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An Innovative Lipidomic Workflow to Investigate the Lipid Profile in a Cystic Fibrosis Cell Line.

Dei Cas Michele M Zulueta Aida A Mingione Alessandra A Caretti Anna A Ghidoni Riccardo R Signorelli Paola P Paroni Rita R

Cells 20200512 5

Altered lipid metabolism has been associated to cystic fibrosis disease, which is characterized by chronic lung inflammation and various organs dysfunction. Here, we present the validation of an untargeted lipidomics approach based on high-resolution mass spectrometry aimed at identifying those lipid species that unequivocally sign CF pathophysiology. Of n.13375 mass spectra recorded on cystic fibrosis bronchial epithelial airways epithelial cells IB3, n.7787 presented the MS/MS data, and, after ...[more]

PMID: 32408521

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Project description:Antimicrobial-resistant microbes are an increasing threat to human health. In cystic fibrosis (CF), airway infections with Pseudomonas aeruginosa remain a key driver of lung damage. With few new antibiotics on the development horizon, alternative therapeutic approaches are needed against antimicrobial-resistant pathogens. Phage therapy, or the use of viruses that infect bacteria, is one proposed novel therapy to treat bacterial infections. However, the airways are complex microenvironments with unique characteristics that may affect the success of novel therapies. Here, three phages of P. aeruginosa (E79, F116, and one novel clinically derived isolate, designated P5) were screened for activity against 21 P. aeruginosa strains isolated from children with CF. Of these, phage E79 showed broad antibacterial activity (91% of tested strains sensitive) and was selected for further assessment. E79 genomic DNA was extracted, sequenced, and confirmed to contain no bacterial pathogenicity genes. High titre phage preparations were then purified using ion-exchange column chromatography and depleted of bacterial endotoxin. Primary airway epithelial cells derived from children with CF (n = 8, age range 0.2-5.5 years, 5 males) or healthy non-CF controls (n = 8, age range 2.5-4.0 years, 4 males) were then exposed to purified phage for 48 h. Levels of inflammatory IL-1?, IL-6, and IL-8 cytokine production were measured in culture supernatant by immunoassays and the extent of cellular apoptosis was measured using a ssDNA kit. Cytokine and apoptosis levels were compared between E79-stimulated and unstimulated controls, and, encouragingly, purified preparations of E79 did not stimulate any significant inflammatory cytokine responses or induce apoptosis in primary epithelial cells derived from children with or without CF. Collectively, this study demonstrates the feasibility of utilizing pre-clinical in vitro culture models to screen therapeutic candidates, and the potential of E79 as a therapeutic phage candidate in CF.

Project description:Hallmarks of cystic fibrosis (CF) are increased viscosity of mucus and impaired mucociliary clearance within the airways due to mutations of the cystic fibrosis conductance regulator gene. This facilitates the colonization of the lung by microbial pathogens and the concomitant establishment of chronic infections leading to tissue damage, reduced lung function, and decreased life expectancy. Although the interplay between key CF pathogens plays a major role during disease progression, the pathophysiology of the microbial community in CF lungs remains poorly understood. Particular challenges in the analysis of the microbial population present in CF sputum is (I) the inhomogeneous, viscous, and slimy consistence of CF sputum, and (II) the high number of human proteins masking comparably low abundant microbial proteins. To address these challenges, we used 21 CF sputum samples to develop a reliable, reproducible and widely applicable protocol for sputum processing, microbial enrichment, cell disruption, protein extraction and subsequent metaproteomic analyses. As a proof of concept, we selected three sputum samples for detailed metaproteome analyses and complemented and validated metaproteome data by 16S sequencing, metabolomic as well as microscopic analyses. Applying our protocol, the number of bacterial proteins/protein groups increased from 199-425 to 392-868 in enriched samples compared to nonenriched controls. These early microbial metaproteome data suggest that the arginine deiminase pathway and multiple proteases and peptidases identified from various bacterial genera could so far be underappreciated in their contribution to the CF pathophysiology. By providing a standardized and effective protocol for sputum processing and microbial enrichment, our study represents an important basis for future studies investigating the physiology of microbial pathogens in CF in vivo - an important prerequisite for the development of novel antimicrobial therapies to combat chronic recurrent airway infection in CF.

Dataset Information

An Innovative Lipidomic Workflow to Investigate the Lipid Profile in a Cystic Fibrosis Cell Line.

Publications

An Innovative Lipidomic Workflow to Investigate the Lipid Profile in a Cystic Fibrosis Cell Line.

Similar Datasets

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