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Respiratory syncytial virus (RSV) infection in elderly mice results in altered antiviral gene expression and enhanced pathology.

Wong TM, Boyapalle S, Sampayo V, Nguyen HD, Bedi R, Kamath SG, Moore ML, Mohapatra S, Mohapatra SS - PLoS ONE (2014)

Bottom Line: The expression of five antiviral genes, including pro-inflammatory cytokines IL-1β and osteopontin (OPN), was altered by both age and infection, while age was associated with the expression of 15 antiviral genes.Both kinetics and magnitude of antiviral gene expression were diminished as a result of older age.In conclusion, this study demonstrates inherent differences in response to RSV infection in young vs. aged mice, accompanied by delayed antiviral gene induction and cytokine signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, James A. Haley Veterans Affairs Hospital, Tampa, Florida, United States of America ; Division of Translational Medicine and Nanomedicine Research Center, Department of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida, United States of America.

ABSTRACT
Elderly persons are more susceptible to RSV-induced pneumonia than young people, but the molecular mechanism underlying this susceptibility is not well understood. In this study, we used an aged mouse model of RSV-induced pneumonia to examine how aging alters the lung pathology, modulates antiviral gene expressions, and the production of inflammatory cytokines in response to RSV infection. Young (2-3 months) and aged (19-21 months) mice were intranasally infected with mucogenic or non-mucogenic RSV strains, lung histology was examined, and gene expression was analyzed. Upon infection with mucogenic strains of RSV, leukocyte infiltration in the airways was elevated and prolonged in aged mice compared to young mice. Minitab factorial analysis identified several antiviral genes that are influenced by age, infection, and a combination of both factors. The expression of five antiviral genes, including pro-inflammatory cytokines IL-1β and osteopontin (OPN), was altered by both age and infection, while age was associated with the expression of 15 antiviral genes. Both kinetics and magnitude of antiviral gene expression were diminished as a result of older age. In addition to delays in cytokine signaling and pattern recognition receptor induction, we found TLR7/8 signaling to be impaired in alveolar macrophages in aged mice. In vivo, induction of IL-1β and OPN were delayed but prolonged in aged mice upon RSV infection compared to young. In conclusion, this study demonstrates inherent differences in response to RSV infection in young vs. aged mice, accompanied by delayed antiviral gene induction and cytokine signaling.

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Pathway network and heatmap assembly identified age-related changes to the activation of proinflammatory cytokine signaling.Expression of 84 antiviral gene targets in A2-infected young and aged mice was compared with RT2-PCR Profiler array analysis. (A) 27 genes from the PCR array analysis were found upregulated >2-fold on 1 dpi; a GeneMania network map was generated using the 27 gene symbols and the most relevant biological pathway recalled involved cytokine signaling (11 genes are involved in such pathway from the 27 genes initially entered as input). The pathway illustrates the networks linking genes through co-expression (purple), colocalization (light blue), predicted interaction (orange), shared protein domains (light yellow), or other known relationship (gray). Above or below each of the 11 gene symbols is a relative gene expression chart displaying relative gene expression from either aged (solid) or young (dotted) over the time course of RSV A2 infection (1 dpi, 3 dpi, or 5 dpi). Fold-regulation changes derived by ΔΔCt calculation were in reference to age-matched mock-infected control group and illustrate age-related changes in gene induction kinetics. (B) Heatmaps encompassing the complete 84-gene PCR array were generated using GENE-E software where rows were sorted in ascending gene expression order and processed for hierarchical clustering using one minus Pearson's correlation. Gradients from blue to red indicate minimum to maximum expression of the indicated gene, respectively, within each row. Columns indicate the aged and young groups at 1 dpi, 3 dpi, or 5 dpi. The heatmap was separated into two for size: the first column illustrates genes upregulated in young mice 1 dpi and the second column clustered genes that are commonly upregulated in young and aged groups. The lower half of the second column illustrates a cluster of genes that remain upregulated on 5 dpi in young mice. Individual mouse RNA array analysis was performed in a single experiment with three different time points (n = 3 mice/group).
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pone-0088764-g003: Pathway network and heatmap assembly identified age-related changes to the activation of proinflammatory cytokine signaling.Expression of 84 antiviral gene targets in A2-infected young and aged mice was compared with RT2-PCR Profiler array analysis. (A) 27 genes from the PCR array analysis were found upregulated >2-fold on 1 dpi; a GeneMania network map was generated using the 27 gene symbols and the most relevant biological pathway recalled involved cytokine signaling (11 genes are involved in such pathway from the 27 genes initially entered as input). The pathway illustrates the networks linking genes through co-expression (purple), colocalization (light blue), predicted interaction (orange), shared protein domains (light yellow), or other known relationship (gray). Above or below each of the 11 gene symbols is a relative gene expression chart displaying relative gene expression from either aged (solid) or young (dotted) over the time course of RSV A2 infection (1 dpi, 3 dpi, or 5 dpi). Fold-regulation changes derived by ΔΔCt calculation were in reference to age-matched mock-infected control group and illustrate age-related changes in gene induction kinetics. (B) Heatmaps encompassing the complete 84-gene PCR array were generated using GENE-E software where rows were sorted in ascending gene expression order and processed for hierarchical clustering using one minus Pearson's correlation. Gradients from blue to red indicate minimum to maximum expression of the indicated gene, respectively, within each row. Columns indicate the aged and young groups at 1 dpi, 3 dpi, or 5 dpi. The heatmap was separated into two for size: the first column illustrates genes upregulated in young mice 1 dpi and the second column clustered genes that are commonly upregulated in young and aged groups. The lower half of the second column illustrates a cluster of genes that remain upregulated on 5 dpi in young mice. Individual mouse RNA array analysis was performed in a single experiment with three different time points (n = 3 mice/group).

Mentions: A GeneMania network map was then constructed to summarize the findings from the GeneGo pathway analysis (Fig. 3A). Despite fewer annotated pathways within the GeneMania database, the GeneMania pathway maps shared similar identity with the GeneGo analysis (data not shown). The 27 genes found to be upregulated >2-fold upon RSV infection in young mice at 1 dpi were used as inputs for GeneMania interaction pathway interaction analysis, where it was predicted that 11 of the 27 genes are associated with inflammatory cytokine signaling (Fig. 3A) with a low false discovery rate (FDR) of 8.69e−24 (coverage spanned 16 of 88 genes associated with cytokine signaling) [34]. Relative age-specific induction of antiviral genes between aged (solid) and young (dotted) was compared to age-matched mock-infected controls and displayed as miniaturized line graphs alongside each of the 11 genes. In all but one of the targets, young mice had greater induction at 1 dpi or 3 dpi. When comparing fold-changes in age-matched normalized gene expression, magnitude and kinetics of antiviral gene induction were affected by substantially by age. Heatmaps were assembled using GENE-E Heatmap software using the fold-change values obtained from the PCR array analysis; genes were then sorted for hierarchical clustering using one-minus Pearson’s correlation (Fig. 3B). RSV infection in young mice induced more innate antiviral gene expression earlier in infection, between 1 and 3 dpi, as seen in the first cluster on the column on the left. Innate antiviral gene expression levels generally tapered down by 5 dpi in young mice, with exception of a minor cluster of NLR- and TLR-associated genes, shown on the bottom-half of the second column; these genes remained elevated at 5 dpi. Alternatively, RSV infections in aged mice are diminished for the majority of the TLR- associated genes, and induction often peaks much later during infection at 5 dpi. It should be noted, however, that the heatmap illustrates relative induction of an individual gene over time after infection in each age group, even if the change of expression not found to be statistically significant.


Respiratory syncytial virus (RSV) infection in elderly mice results in altered antiviral gene expression and enhanced pathology.

Wong TM, Boyapalle S, Sampayo V, Nguyen HD, Bedi R, Kamath SG, Moore ML, Mohapatra S, Mohapatra SS - PLoS ONE (2014)

Pathway network and heatmap assembly identified age-related changes to the activation of proinflammatory cytokine signaling.Expression of 84 antiviral gene targets in A2-infected young and aged mice was compared with RT2-PCR Profiler array analysis. (A) 27 genes from the PCR array analysis were found upregulated >2-fold on 1 dpi; a GeneMania network map was generated using the 27 gene symbols and the most relevant biological pathway recalled involved cytokine signaling (11 genes are involved in such pathway from the 27 genes initially entered as input). The pathway illustrates the networks linking genes through co-expression (purple), colocalization (light blue), predicted interaction (orange), shared protein domains (light yellow), or other known relationship (gray). Above or below each of the 11 gene symbols is a relative gene expression chart displaying relative gene expression from either aged (solid) or young (dotted) over the time course of RSV A2 infection (1 dpi, 3 dpi, or 5 dpi). Fold-regulation changes derived by ΔΔCt calculation were in reference to age-matched mock-infected control group and illustrate age-related changes in gene induction kinetics. (B) Heatmaps encompassing the complete 84-gene PCR array were generated using GENE-E software where rows were sorted in ascending gene expression order and processed for hierarchical clustering using one minus Pearson's correlation. Gradients from blue to red indicate minimum to maximum expression of the indicated gene, respectively, within each row. Columns indicate the aged and young groups at 1 dpi, 3 dpi, or 5 dpi. The heatmap was separated into two for size: the first column illustrates genes upregulated in young mice 1 dpi and the second column clustered genes that are commonly upregulated in young and aged groups. The lower half of the second column illustrates a cluster of genes that remain upregulated on 5 dpi in young mice. Individual mouse RNA array analysis was performed in a single experiment with three different time points (n = 3 mice/group).
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Related In: Results  -  Collection

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pone-0088764-g003: Pathway network and heatmap assembly identified age-related changes to the activation of proinflammatory cytokine signaling.Expression of 84 antiviral gene targets in A2-infected young and aged mice was compared with RT2-PCR Profiler array analysis. (A) 27 genes from the PCR array analysis were found upregulated >2-fold on 1 dpi; a GeneMania network map was generated using the 27 gene symbols and the most relevant biological pathway recalled involved cytokine signaling (11 genes are involved in such pathway from the 27 genes initially entered as input). The pathway illustrates the networks linking genes through co-expression (purple), colocalization (light blue), predicted interaction (orange), shared protein domains (light yellow), or other known relationship (gray). Above or below each of the 11 gene symbols is a relative gene expression chart displaying relative gene expression from either aged (solid) or young (dotted) over the time course of RSV A2 infection (1 dpi, 3 dpi, or 5 dpi). Fold-regulation changes derived by ΔΔCt calculation were in reference to age-matched mock-infected control group and illustrate age-related changes in gene induction kinetics. (B) Heatmaps encompassing the complete 84-gene PCR array were generated using GENE-E software where rows were sorted in ascending gene expression order and processed for hierarchical clustering using one minus Pearson's correlation. Gradients from blue to red indicate minimum to maximum expression of the indicated gene, respectively, within each row. Columns indicate the aged and young groups at 1 dpi, 3 dpi, or 5 dpi. The heatmap was separated into two for size: the first column illustrates genes upregulated in young mice 1 dpi and the second column clustered genes that are commonly upregulated in young and aged groups. The lower half of the second column illustrates a cluster of genes that remain upregulated on 5 dpi in young mice. Individual mouse RNA array analysis was performed in a single experiment with three different time points (n = 3 mice/group).
Mentions: A GeneMania network map was then constructed to summarize the findings from the GeneGo pathway analysis (Fig. 3A). Despite fewer annotated pathways within the GeneMania database, the GeneMania pathway maps shared similar identity with the GeneGo analysis (data not shown). The 27 genes found to be upregulated >2-fold upon RSV infection in young mice at 1 dpi were used as inputs for GeneMania interaction pathway interaction analysis, where it was predicted that 11 of the 27 genes are associated with inflammatory cytokine signaling (Fig. 3A) with a low false discovery rate (FDR) of 8.69e−24 (coverage spanned 16 of 88 genes associated with cytokine signaling) [34]. Relative age-specific induction of antiviral genes between aged (solid) and young (dotted) was compared to age-matched mock-infected controls and displayed as miniaturized line graphs alongside each of the 11 genes. In all but one of the targets, young mice had greater induction at 1 dpi or 3 dpi. When comparing fold-changes in age-matched normalized gene expression, magnitude and kinetics of antiviral gene induction were affected by substantially by age. Heatmaps were assembled using GENE-E Heatmap software using the fold-change values obtained from the PCR array analysis; genes were then sorted for hierarchical clustering using one-minus Pearson’s correlation (Fig. 3B). RSV infection in young mice induced more innate antiviral gene expression earlier in infection, between 1 and 3 dpi, as seen in the first cluster on the column on the left. Innate antiviral gene expression levels generally tapered down by 5 dpi in young mice, with exception of a minor cluster of NLR- and TLR-associated genes, shown on the bottom-half of the second column; these genes remained elevated at 5 dpi. Alternatively, RSV infections in aged mice are diminished for the majority of the TLR- associated genes, and induction often peaks much later during infection at 5 dpi. It should be noted, however, that the heatmap illustrates relative induction of an individual gene over time after infection in each age group, even if the change of expression not found to be statistically significant.

Bottom Line: The expression of five antiviral genes, including pro-inflammatory cytokines IL-1β and osteopontin (OPN), was altered by both age and infection, while age was associated with the expression of 15 antiviral genes.Both kinetics and magnitude of antiviral gene expression were diminished as a result of older age.In conclusion, this study demonstrates inherent differences in response to RSV infection in young vs. aged mice, accompanied by delayed antiviral gene induction and cytokine signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, James A. Haley Veterans Affairs Hospital, Tampa, Florida, United States of America ; Division of Translational Medicine and Nanomedicine Research Center, Department of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida, United States of America.

ABSTRACT
Elderly persons are more susceptible to RSV-induced pneumonia than young people, but the molecular mechanism underlying this susceptibility is not well understood. In this study, we used an aged mouse model of RSV-induced pneumonia to examine how aging alters the lung pathology, modulates antiviral gene expressions, and the production of inflammatory cytokines in response to RSV infection. Young (2-3 months) and aged (19-21 months) mice were intranasally infected with mucogenic or non-mucogenic RSV strains, lung histology was examined, and gene expression was analyzed. Upon infection with mucogenic strains of RSV, leukocyte infiltration in the airways was elevated and prolonged in aged mice compared to young mice. Minitab factorial analysis identified several antiviral genes that are influenced by age, infection, and a combination of both factors. The expression of five antiviral genes, including pro-inflammatory cytokines IL-1β and osteopontin (OPN), was altered by both age and infection, while age was associated with the expression of 15 antiviral genes. Both kinetics and magnitude of antiviral gene expression were diminished as a result of older age. In addition to delays in cytokine signaling and pattern recognition receptor induction, we found TLR7/8 signaling to be impaired in alveolar macrophages in aged mice. In vivo, induction of IL-1β and OPN were delayed but prolonged in aged mice upon RSV infection compared to young. In conclusion, this study demonstrates inherent differences in response to RSV infection in young vs. aged mice, accompanied by delayed antiviral gene induction and cytokine signaling.

Show MeSH
Related in: MedlinePlus