Profiling of Differentially Expressed Genes Using Suppression Subtractive Hybridization in an Equine Model of Chronic Asthma

Jean-Pierre Lavoie,Jacques Lussier,Anouk Lavoie-Lamoureux,Josiane Lefebvre-Lavoie,Mathilde Leclere,Catherine Laprise,Annie Chamberland,Christian Schönbach

doi:10.1371/journal.pone.0029440

Jean-Pierre Lavoie, Jacques Lussier + Show 6 more

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https://doi.org/10.1371/journal.pone.0029440

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Abstract

BackgroundGene expression analyses are used to investigate signaling pathways involved in diseases. In asthma, they have been primarily derived from the analysis of bronchial biopsies harvested from mild to moderate asthmatic subjects and controls. Due to ethical considerations, there is currently limited information on the transcriptome profile of the peripheral lung tissues in asthma.ObjectiveTo identify genes contributing to chronic inflammation and remodeling in the peripheral lung tissue of horses with heaves, a naturally occurring asthma-like condition.MethodsEleven adult horses (6 heaves-affected and 5 controls) were studied while horses with heaves were in clinical remission (Pasture), and during disease exacerbation induced by a 30-day natural antigen challenge during stabling (Challenge). Large peripheral lung biopsies were obtained by thoracoscopy at both time points. Using suppression subtractive hybridization (SSH), lung cDNAs of controls (Pasture and Challenge) and asymptomatic heaves-affected horses (Pasture) were subtracted from cDNAs of horses with heaves in clinical exacerbation (Challenge). The differential expression of selected genes of interest was confirmed using quantitative PCR assay.ResultsHorses with heaves, but not controls, developed airway obstruction when challenged. Nine hundred and fifty cDNA clones isolated from the subtracted library were screened by dot blot array and 224 of those showing the most marked expression differences were sequenced. The gene expression pattern was confirmed by quantitative PCR in 15 of 22 selected genes. Novel genes and genes with an already defined function in asthma were identified in the subtracted cDNA library. Genes of particular interest associated with asthmatic airway inflammation and remodeling included those related to PPP3CB/NFAT, RhoA, and LTB4/GPR44 signaling pathways.ConclusionsPathways representing new possible targets for anti-inflammatory and anti-remodeling therapies for asthma were identified. The findings of genes previously associated with asthma validate this equine model for gene expression studies.

Highlights

Inflammation and remodeling of the airway wall are characteristic features of asthma
Nine hundred and fifty cDNA clones isolated from the subtracted library were screened by dot blot array and 224 of those showing the most marked expression differences were sequenced
The gene expression pattern was confirmed by quantitative PCR in 15 of 22 selected genes

Summary

Introduction

Inflammation and remodeling of the airway wall are characteristic features of asthma. Gene expression studies using human lung tissues have been primarily derived from the analysis of bronchial biopsies harvested from mild to moderate asthmatic subjects and controls [3]. These studies have identified candidate genes and pathways related to asthma pathogenesis. There is limited information on the transcriptome profile of the peripheral lung tissues where remodeling predominantly occurs in non-fatal asthma [4,5]. Gene expression analyses are used to investigate signaling pathways involved in diseases In asthma, they have been primarily derived from the analysis of bronchial biopsies harvested from mild to moderate asthmatic subjects and controls. There is currently limited information on the transcriptome profile of the peripheral lung tissues in asthma

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