RSV vs. rhinovirus bronchiolitis: difference in nasal airway microRNA profiles and NFκB signaling

Abstract

BackgroundAlthough rhinovirus infection is associated with increased risks of acute and chronic respiratory outcomes during childhood compared with respiratory syncytial virus (RSV), the underlying mechanisms remain unclear. We aimed to determine the differences in nasal airway microRNA profiles and their downstream effects between infants with rhinovirus and RSV bronchiolitis.MethodsAs part of a multicenter cohort study of infants hospitalized for bronchiolitis, we examined nasal samples obtained from 16 infants with rhinovirus and 16 infants with RSV. We tested nasal airway samples using microarrays to profile global microRNA expression and determine the predicted regulation of targeted transcripts. We also measured gene expression and cytokines for NFκB pathway components.ResultsBetween the virus groups, 386 microRNAs were differentially expressed (false discovery rate (FDR)<0.05). In infants with rhinovirus, the NFκB pathway was highly ranked as a predicted target for these differentially expressed microRNAs compared with RSV. Pathway analysis using measured mRNA expression data validated that rhinovirus infection had upregulation of NFκB family (RelA and NFκB2) and downregulation of inhibitor κB family. Infants with rhinovirus had higher levels of NFκB-induced type-2 cytokines (IL-10 and IL-13; FDR<0.01).ConclusionIn infants with bronchiolitis, rhinovirus and RSV infections had different nasal airway microRNA profiles associated with NFκB signaling.

Figure 1.. Principal coordinates analysis plot comparing nasal airway microRNA profiles in infants with rhinovirus bronchiolitis and those with RSV bronchiolitis

To show the differences in nasal airway microRNA profiles among infants with bronchiolitis, principal coordinates analysis (PCoA) plot based on the Bray-Curtis distance was generated. Each dot represents the overall microRNA expression in each infant. The distance between infants indicates their dissimilarity. The PCoA revealed that infants cluster together according to their viral etiology. In addition to 16 samples with rhinovirus and 16 with RSV, six technical replicates were also included in the analysis. Abbreviations: RV, rhinovirus; RSV, respiratory syncytial virus.

Figure 2.. Unsupervised hierarchical clustering of the expression of identified microRNAs in nasal airway of infants with bronchiolitis

The heatmap of 2,758 microRNAs that are identified in the nasal airway was generated using the Spearman’s rank correlation similarity and the Ward’s clustering algorithm. The microRNA expression profiles almost completely separated infants with rhinovirus bronchiolitis from those with RSV bronchiolitis. The color bar indicates the standardized expression of each microRNA to a mean of 0. Upregulated microRNAs have positive values and are displayed as red. Downregulated microRNAs have negative values and are displayed as blue. The differences in microRNA expression between rhinovirus and RSV are summarized in Supplemental Table S2. Abbreviations: RV, rhinovirus; RSV, respiratory syncytial virus.

Figure 3.. NFκB signaling pathway in the nasal airway comparing infants with rhinovirus bronchiolitis to those with RSV bronchiolitis.

a) Predicted up- and down-regulation of target transcripts in the NFκB signaling pathway. The canonical pathway for NFκB signaling was highly ranked as a target for the microRNAs in infants with rhinovirus bronchiolitis compared to those with RSV bronchiolitis. The green color indicates predicted down-regulation of transcripts targeted by differentially expressed microRNAs in the nasal airway of infants with rhinovirus bronchiolitis compared to those in infants with RSV; the red color indicates predicted up-regulation. Genes are targeted by multiple microRNAs. b) Measured up- and down-regulation of target transcripts in the NFκB signaling pathway via RT-PCR.

Figure 4.. Integrated analysis of microRNA and mRNA expressions of NFκB signaling pathway in nasal airway of infants with bronchiolitis

Treemap of predicted microRNA-target (mRNA) interaction was depicted by the use of miRTarVis, which identifies microRNA-mRNA pairs with an expression value. Normalized, background-subtracted microRNA-mRNA expression profile data were imported into miRTarVis (rhinovirus infection compared to RSV infection). The color gradient indicates the magnitude of the fold change in microRNA and mRNA expression (red, up-regulation; blue, down-regulation). The size (area) of each box represents the frequency of that finding. The shape is designed to automatically fit into the overall structure of the figure. a) Pairs of up-regulated microRNAs (in red) and down-regulated mRNA (in blue). Notably, infants with rhinovirus bronchiolitis had up-regulation of multiple microRNAs (e.g., hsa-miR-149–3p, hsa-miR-197–3p, hsa-miR-197–5p, hsa-miR-296–3p) targeting NFKBIB, thereby predicting the down-regulation of NFKBIB, a gene encoding inhibitor κB family. b) Pairs of down-regulated microRNAs (in blue) and up-regulated mRNA (in red). Notably, infants with rhinovirus bronchiolitis had down-regulation of multiple microRNAs targeting RELA, thereby predicting the up-regulation of RELA, the gene encoding RelA (one of the proteins in the NFκB family).