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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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Aging results in diminished OPN production in response to 2-20 RSV infection.Young and aged BALB/c mice (n = 4/group) were intranasally infected with a dose of 106 pfu/mouse of A2 or 105 pfu/mouse of 2-20) and total lung RNA was collected on time points indicated. (A-B) RNA transcripts of OPN were analyzed with qRT-PCR and represented as a relative ratio of target gene expression to endogenous mouse HPRT and examined with rA2-L19F, 2-20, or A2. (B) 5 µm lung sections obtained at 4, 5 and 8 dpi with either rA2-L19F, 2-20, or A2 infected young and aged mice. Lung sections were immunostained for anti-mouse OPN and nickel-DAB reagent before counterstaining with hematoxylin and eosin. Representative images shown are at 100x magnification with inset [400x] displaying nickel-DAB (dark brown/black staining) positive cells and contrast the hematoxylin (light blue) nuclear stain. Representative images are shown with scale bar indicating 100 µm. (C) Enumeration of OPN-positive cells was performed with ImmunoRatio ImageJ analysis on 200X magnified lung sections from 8 dpi and values are shown as a percentage of total hematoxylin-stained cells in an individual box plot with mean interval bars. Within a single frame, at least 5 frames per mouse (n = 4/group) were collected and individual dot plots are shown of either aged or young mice with interval bars and significance, determined with ANOVA and Fisher's test (p<0.05). (D) qRT-PCR was performed on total lung RNA from young and aged 2–20 RSV infected mice for mRNA expression of OPN receptor CD44. Statistical significance was determine with ANOVA 2-way analysis with p<0.05. All experiments were performed in triplicate.
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pone-0088764-g007: Aging results in diminished OPN production in response to 2-20 RSV infection.Young and aged BALB/c mice (n = 4/group) were intranasally infected with a dose of 106 pfu/mouse of A2 or 105 pfu/mouse of 2-20) and total lung RNA was collected on time points indicated. (A-B) RNA transcripts of OPN were analyzed with qRT-PCR and represented as a relative ratio of target gene expression to endogenous mouse HPRT and examined with rA2-L19F, 2-20, or A2. (B) 5 µm lung sections obtained at 4, 5 and 8 dpi with either rA2-L19F, 2-20, or A2 infected young and aged mice. Lung sections were immunostained for anti-mouse OPN and nickel-DAB reagent before counterstaining with hematoxylin and eosin. Representative images shown are at 100x magnification with inset [400x] displaying nickel-DAB (dark brown/black staining) positive cells and contrast the hematoxylin (light blue) nuclear stain. Representative images are shown with scale bar indicating 100 µm. (C) Enumeration of OPN-positive cells was performed with ImmunoRatio ImageJ analysis on 200X magnified lung sections from 8 dpi and values are shown as a percentage of total hematoxylin-stained cells in an individual box plot with mean interval bars. Within a single frame, at least 5 frames per mouse (n = 4/group) were collected and individual dot plots are shown of either aged or young mice with interval bars and significance, determined with ANOVA and Fisher's test (p<0.05). (D) qRT-PCR was performed on total lung RNA from young and aged 2–20 RSV infected mice for mRNA expression of OPN receptor CD44. Statistical significance was determine with ANOVA 2-way analysis with p<0.05. All experiments were performed in triplicate.

Mentions: Gene expression of OPN was similarly examined with qRT-PCR and normalized relative to mouse HPRT. OPN expression was elevated in aged mice even in the absence of RSV, and by 8 dpi with any of the three RSV strains, the OPN mRNA transcript levels in aged mice significantly exceeded that of young mice (Fig.7A). Despite elevated OPN at baseline and at 8 dpi, the slope of OPN mRNA transcripts between 1 dpi and 5 dpi is diminished compared to young. We also found OPN to be slightly elevated in the BALF of mock-infected aged mice compared to mock-infected young mice and upon 2–20 infection, although BALF levels of OPN were often low or undetectable with ELISA (data not shown). OPN increased in young BALF but did not significantly increased in aged infected mice; no statistical difference was found between levels of OPN in the serum of young and aged mice. Immunohistochemical analysis also demonstrated constitutively higher expression of OPN in the airways of elderly mice prior to RSV infection (Fig. 7B). Infection with rA2-L19F results in increased expression of OPN in both young and aged mice on 5 dpi; however, as compared to a 7-fold increase in OPN-positive cells, aged mice had a 50% increase (Fig. 7C). By 8 dpi with rA2-L19F, OPN levels decrease in young but not aged mice, although number of OPN-positive cells remain higher than baseline expression. In contrast, 2-20 infection in both young yielded fewer OPN-positive cells than with rA2-L19F infections; moreover, the number of OPN-positive cells enumerated from the lungs of 2-20 infected aged mice remained unchanged from baseline. This suggests an age-dependent differential responses to the RSV stains examined here. Since neither severe cell infiltration nor mucus production [26] is associated with RSV A2, OPN-levels in the lung were not examined in this study.


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)

Aging results in diminished OPN production in response to 2-20 RSV infection.Young and aged BALB/c mice (n = 4/group) were intranasally infected with a dose of 106 pfu/mouse of A2 or 105 pfu/mouse of 2-20) and total lung RNA was collected on time points indicated. (A-B) RNA transcripts of OPN were analyzed with qRT-PCR and represented as a relative ratio of target gene expression to endogenous mouse HPRT and examined with rA2-L19F, 2-20, or A2. (B) 5 µm lung sections obtained at 4, 5 and 8 dpi with either rA2-L19F, 2-20, or A2 infected young and aged mice. Lung sections were immunostained for anti-mouse OPN and nickel-DAB reagent before counterstaining with hematoxylin and eosin. Representative images shown are at 100x magnification with inset [400x] displaying nickel-DAB (dark brown/black staining) positive cells and contrast the hematoxylin (light blue) nuclear stain. Representative images are shown with scale bar indicating 100 µm. (C) Enumeration of OPN-positive cells was performed with ImmunoRatio ImageJ analysis on 200X magnified lung sections from 8 dpi and values are shown as a percentage of total hematoxylin-stained cells in an individual box plot with mean interval bars. Within a single frame, at least 5 frames per mouse (n = 4/group) were collected and individual dot plots are shown of either aged or young mice with interval bars and significance, determined with ANOVA and Fisher's test (p<0.05). (D) qRT-PCR was performed on total lung RNA from young and aged 2–20 RSV infected mice for mRNA expression of OPN receptor CD44. Statistical significance was determine with ANOVA 2-way analysis with p<0.05. All experiments were performed in triplicate.
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Related In: Results  -  Collection

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pone-0088764-g007: Aging results in diminished OPN production in response to 2-20 RSV infection.Young and aged BALB/c mice (n = 4/group) were intranasally infected with a dose of 106 pfu/mouse of A2 or 105 pfu/mouse of 2-20) and total lung RNA was collected on time points indicated. (A-B) RNA transcripts of OPN were analyzed with qRT-PCR and represented as a relative ratio of target gene expression to endogenous mouse HPRT and examined with rA2-L19F, 2-20, or A2. (B) 5 µm lung sections obtained at 4, 5 and 8 dpi with either rA2-L19F, 2-20, or A2 infected young and aged mice. Lung sections were immunostained for anti-mouse OPN and nickel-DAB reagent before counterstaining with hematoxylin and eosin. Representative images shown are at 100x magnification with inset [400x] displaying nickel-DAB (dark brown/black staining) positive cells and contrast the hematoxylin (light blue) nuclear stain. Representative images are shown with scale bar indicating 100 µm. (C) Enumeration of OPN-positive cells was performed with ImmunoRatio ImageJ analysis on 200X magnified lung sections from 8 dpi and values are shown as a percentage of total hematoxylin-stained cells in an individual box plot with mean interval bars. Within a single frame, at least 5 frames per mouse (n = 4/group) were collected and individual dot plots are shown of either aged or young mice with interval bars and significance, determined with ANOVA and Fisher's test (p<0.05). (D) qRT-PCR was performed on total lung RNA from young and aged 2–20 RSV infected mice for mRNA expression of OPN receptor CD44. Statistical significance was determine with ANOVA 2-way analysis with p<0.05. All experiments were performed in triplicate.
Mentions: Gene expression of OPN was similarly examined with qRT-PCR and normalized relative to mouse HPRT. OPN expression was elevated in aged mice even in the absence of RSV, and by 8 dpi with any of the three RSV strains, the OPN mRNA transcript levels in aged mice significantly exceeded that of young mice (Fig.7A). Despite elevated OPN at baseline and at 8 dpi, the slope of OPN mRNA transcripts between 1 dpi and 5 dpi is diminished compared to young. We also found OPN to be slightly elevated in the BALF of mock-infected aged mice compared to mock-infected young mice and upon 2–20 infection, although BALF levels of OPN were often low or undetectable with ELISA (data not shown). OPN increased in young BALF but did not significantly increased in aged infected mice; no statistical difference was found between levels of OPN in the serum of young and aged mice. Immunohistochemical analysis also demonstrated constitutively higher expression of OPN in the airways of elderly mice prior to RSV infection (Fig. 7B). Infection with rA2-L19F results in increased expression of OPN in both young and aged mice on 5 dpi; however, as compared to a 7-fold increase in OPN-positive cells, aged mice had a 50% increase (Fig. 7C). By 8 dpi with rA2-L19F, OPN levels decrease in young but not aged mice, although number of OPN-positive cells remain higher than baseline expression. In contrast, 2-20 infection in both young yielded fewer OPN-positive cells than with rA2-L19F infections; moreover, the number of OPN-positive cells enumerated from the lungs of 2-20 infected aged mice remained unchanged from baseline. This suggests an age-dependent differential responses to the RSV stains examined here. Since neither severe cell infiltration nor mucus production [26] is associated with RSV A2, OPN-levels in the lung were not examined in this study.

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