Rhinovirus infections change DNA methylation and mRNA expression in children with asthma.

Martin Pech,Andre Franke,Brian Oliver,Matthias V Kopp,Gesine Hansen,The Alliance-Study Group ,Femke-Anouska Heinsen,Inke R König,Erika V Mutius,Isabell Ricklefs,Markus Weckmann,Klaus Rabe,Oliver Fuchs

doi:10.1371/journal.pone.0205275

Abstract

Human rhinovirus infection (HRVI) plays an important role in asthma exacerbations and is thought to be involved in asthma development during early childhood. We hypothesized that HRVI causes differential DNA methylation and subsequently differential mRNA expression in epithelial cells of children with asthma. Primary nasal epithelial cells from children with (n = 10) and without (n = 10) asthma were cultivated up to passage two and infected with Rhinovirus-16 (RV-16). HRVI-induced genome-wide differences of DNA methylation in asthmatics (vs. controls) and resulting mRNA expression were analyzed by the HumanMethylation450 BeadChip Kit (Illumina) and RNA sequencing. These results were further verified by pyrosequencing and quantitative PCR, respectively. 471 CpGs belonging to 268 genes were identified to have HRVI-induced asthma-specifically modified DNA methylation and mRNA expression. A minimum-change criteria was applied to restrict assessment of genes with changes in DNA methylation and mRNA expression of at least 3% and least 0.1 reads/kb per million mapped reads, respectively. Using this approach we identified 16 CpGs, including HLA-B-associated transcript 3 (BAT3) and Neuraminidase 1 (NEU1), involved in host immune response against HRVI. HRVI in nasal epithelial cells leads to specific modifications of DNA methylation with altered mRNA expression in children with asthma. The HRVI-induced alterations in DNA methylation occurred in genes involved in the host immune response against viral infections and asthma pathogenesis. The findings of our pilot study may partially explain how HRVI contribute to the persistence and progression of asthma, and aid to identify possible new therapeutic targets. The promising findings of this pilot study would benefit from replication in a larger cohort.

Highlights

Asthma is the most common chronic disease affecting children, with an increasing prevalence in westernized countries since the latter half of the 20th century [1,2,3]
The in vitro model of simulated Human Rhinovirus (HRV) infection (HRVI) in this study, with the infection of human nasal epithelial cells with RV-16 (RV-16 virus stock: 9x109 RV-16/mL in 10 mM EDTA, 1 mM Tris, HeLa origin, sucrose gradient purified) demonstrated increased IL-6 and IL-8 protein and mRNA expression in the HRVI-cell cultures compared with MOCK-infected cell cultures (Fig 2)
RIDM and RIRE are significantly different in asthmatic nasal epithelial cells The analysis of genome-wide methylation patterns after HRVI identified 27,517 CpGs, which showed significant RIDM (p-value

Summary

Introduction

Asthma is the most common chronic disease affecting children, with an increasing prevalence in westernized countries since the latter half of the 20th century [1,2,3]. Known risk factors for asthma development include a family history of atopic disease, passive smoke exposure, and allergic sensitization to food and/or inhaled allergens. The theory of early viral infections in the development of asthma has been well established. While Respiratory Syncytial Virus was initially considered to be the most important viral pathogen risk-factor in asthma development, Human Rhinovirus (HRV) has recently been recognized as a major etiologic factor in wheezing illnesses with a significant link to asthma development during childhood [5, 6]. HRV-A and -C have been linked to lower respiratory tract infections (LRTIs) with increased morbidity in affected children. Individuals with atopic asthma suffer from more frequent symptomatic respiratory tract infections (RTI) and have more severe and longer-lasting respiratory symptoms following a HRV infection (HRVI) [8]

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