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Aging Impairs the Ability of Conventional Dendritic Cells to Cross-Prime CD8+ T Cells upon Stimulation with a TLR7 Ligand.

Zacca ER, Crespo MI, Acland RP, Roselli E, Núñez NG, Maccioni M, Maletto BA, Pistoresi-Palencia MC, Morón G - PLoS ONE (2015)

Bottom Line: Dendritic cells (DCs) are antigen-presenting cells that play a key role in both adaptive and innate immunity, but how aging affects DCs and their influence on immunity has not been thoroughly established.Using polyU, a synthetic ssRNA analog, as TLR7 ligand and OVA as an antigen (Ag) model, we found that cDCs from old mice have a poor ability to stimulate a CD8+ T cell-mediated cytotoxic response. cDCs from old mice exhibit alterations in Ag-processing machinery and TLR7 activation.Taken together, our results suggest that immunosenescence impacts cDC function, affecting the activation of naïve CD8+ T cells and the generation of effector cytotoxic T cells.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones en Bioquímica Clínica e Inmunología, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

ABSTRACT
The aging process is accompanied by altered immune system functioning and an increased risk of infection. Dendritic cells (DCs) are antigen-presenting cells that play a key role in both adaptive and innate immunity, but how aging affects DCs and their influence on immunity has not been thoroughly established. Here we examined the function of conventional DCs (cDCs) in old mice after TLR7 stimulation, focusing on their ability to cross-prime CD8+ T cells. Using polyU, a synthetic ssRNA analog, as TLR7 ligand and OVA as an antigen (Ag) model, we found that cDCs from old mice have a poor ability to stimulate a CD8+ T cell-mediated cytotoxic response. cDCs from old mice exhibit alterations in Ag-processing machinery and TLR7 activation. Remarkably, CD8α+ cDCs from old mice have an impaired ability to activate naïve CD8+ T cells and, moreover, a lower capacity to mature and to process exogenous Ag. Taken together, our results suggest that immunosenescence impacts cDC function, affecting the activation of naïve CD8+ T cells and the generation of effector cytotoxic T cells.

No MeSH data available.


Related in: MedlinePlus

Differential phosphorylation of IκB-α in cDCs from young and old mice after TLR7 stimulation.(A) Pooled splenic cDC isolated from 3 young or old mice were incubated with 20 μg/mL polyU/DO for the indicated time. Cells were then lysed, and the level of phosphorylation of IκB-α was evaluated by Western blot using an anti-pIκB-α antibody. β-actin levels were used for loading control. (B) IOD of phosphorylated IκB-α normalized to β-actin. Data represent mean ± SEM of 2 pooled independent experiments.
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pone.0140672.g006: Differential phosphorylation of IκB-α in cDCs from young and old mice after TLR7 stimulation.(A) Pooled splenic cDC isolated from 3 young or old mice were incubated with 20 μg/mL polyU/DO for the indicated time. Cells were then lysed, and the level of phosphorylation of IκB-α was evaluated by Western blot using an anti-pIκB-α antibody. β-actin levels were used for loading control. (B) IOD of phosphorylated IκB-α normalized to β-actin. Data represent mean ± SEM of 2 pooled independent experiments.

Mentions: NF-κB activation is crucial in TLR7-mediated DC maturation [34]. NF-κB activation via the canonical pathway is mediated by the upstream IκB kinase (IKK). Upon cellular stimulation, IKK is activated and phosphorylates IκB, which is then polyubiquitinated and degraded by proteasome. IκB degradation allows NF-κB to translocate to the nucleus, where it binds to its target sites [34,35]. As a read-out for NF-κB activation, we used Western blot to determine the phosphorylation of IκB-α in cDCs from young and old mice stimulated with polyU/DO. Unstimulated cDCs from young mice showed no phosphorylated IκB-α (pIκB-α), whereas 15 minutes after polyU/DO stimulation, cDCs from young mice showed a consistent pIκB-α band, which rapidly decreased to baseline values (Fig 6A and 6B). In contrast, unstimulated cDCs from old mice showed a strong band of pIκB-α. The presence of phosphorylated IκB-α in unstimulated cDCs from old mice was confirmed by the finding of nuclear immunoreactivity for the p65 subunit of NFκB (S4 Fig). Upon stimulation with polyU/DO, cDCs from old mice required 30 minutes of incubation to show an increase in pIκB-α level (Fig 6A and 6B), demonstrating an alteration in their downstream TLR7 signaling.


Aging Impairs the Ability of Conventional Dendritic Cells to Cross-Prime CD8+ T Cells upon Stimulation with a TLR7 Ligand.

Zacca ER, Crespo MI, Acland RP, Roselli E, Núñez NG, Maccioni M, Maletto BA, Pistoresi-Palencia MC, Morón G - PLoS ONE (2015)

Differential phosphorylation of IκB-α in cDCs from young and old mice after TLR7 stimulation.(A) Pooled splenic cDC isolated from 3 young or old mice were incubated with 20 μg/mL polyU/DO for the indicated time. Cells were then lysed, and the level of phosphorylation of IκB-α was evaluated by Western blot using an anti-pIκB-α antibody. β-actin levels were used for loading control. (B) IOD of phosphorylated IκB-α normalized to β-actin. Data represent mean ± SEM of 2 pooled independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608578&req=5

pone.0140672.g006: Differential phosphorylation of IκB-α in cDCs from young and old mice after TLR7 stimulation.(A) Pooled splenic cDC isolated from 3 young or old mice were incubated with 20 μg/mL polyU/DO for the indicated time. Cells were then lysed, and the level of phosphorylation of IκB-α was evaluated by Western blot using an anti-pIκB-α antibody. β-actin levels were used for loading control. (B) IOD of phosphorylated IκB-α normalized to β-actin. Data represent mean ± SEM of 2 pooled independent experiments.
Mentions: NF-κB activation is crucial in TLR7-mediated DC maturation [34]. NF-κB activation via the canonical pathway is mediated by the upstream IκB kinase (IKK). Upon cellular stimulation, IKK is activated and phosphorylates IκB, which is then polyubiquitinated and degraded by proteasome. IκB degradation allows NF-κB to translocate to the nucleus, where it binds to its target sites [34,35]. As a read-out for NF-κB activation, we used Western blot to determine the phosphorylation of IκB-α in cDCs from young and old mice stimulated with polyU/DO. Unstimulated cDCs from young mice showed no phosphorylated IκB-α (pIκB-α), whereas 15 minutes after polyU/DO stimulation, cDCs from young mice showed a consistent pIκB-α band, which rapidly decreased to baseline values (Fig 6A and 6B). In contrast, unstimulated cDCs from old mice showed a strong band of pIκB-α. The presence of phosphorylated IκB-α in unstimulated cDCs from old mice was confirmed by the finding of nuclear immunoreactivity for the p65 subunit of NFκB (S4 Fig). Upon stimulation with polyU/DO, cDCs from old mice required 30 minutes of incubation to show an increase in pIκB-α level (Fig 6A and 6B), demonstrating an alteration in their downstream TLR7 signaling.

Bottom Line: Dendritic cells (DCs) are antigen-presenting cells that play a key role in both adaptive and innate immunity, but how aging affects DCs and their influence on immunity has not been thoroughly established.Using polyU, a synthetic ssRNA analog, as TLR7 ligand and OVA as an antigen (Ag) model, we found that cDCs from old mice have a poor ability to stimulate a CD8+ T cell-mediated cytotoxic response. cDCs from old mice exhibit alterations in Ag-processing machinery and TLR7 activation.Taken together, our results suggest that immunosenescence impacts cDC function, affecting the activation of naïve CD8+ T cells and the generation of effector cytotoxic T cells.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones en Bioquímica Clínica e Inmunología, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

ABSTRACT
The aging process is accompanied by altered immune system functioning and an increased risk of infection. Dendritic cells (DCs) are antigen-presenting cells that play a key role in both adaptive and innate immunity, but how aging affects DCs and their influence on immunity has not been thoroughly established. Here we examined the function of conventional DCs (cDCs) in old mice after TLR7 stimulation, focusing on their ability to cross-prime CD8+ T cells. Using polyU, a synthetic ssRNA analog, as TLR7 ligand and OVA as an antigen (Ag) model, we found that cDCs from old mice have a poor ability to stimulate a CD8+ T cell-mediated cytotoxic response. cDCs from old mice exhibit alterations in Ag-processing machinery and TLR7 activation. Remarkably, CD8α+ cDCs from old mice have an impaired ability to activate naïve CD8+ T cells and, moreover, a lower capacity to mature and to process exogenous Ag. Taken together, our results suggest that immunosenescence impacts cDC function, affecting the activation of naïve CD8+ T cells and the generation of effector cytotoxic T cells.

No MeSH data available.


Related in: MedlinePlus