Role of phosphoinositide 3-kinase–Akt signaling pathway in the age-related cytokine dysregulation in splenic macrophages stimulated via TLR-2 or TLR-4 receptors

Age-associated defects in both B-lymphocytes and macrophages in elderly result in a reduction in the efficacy of vaccines to many Gram positive bacteria like Streptococcus pneumoniae. Splenic macrophages from aged mice have been shown to have a defect in production of pro-inflammatory cytokines (IL-6, IL-12, IL-1β, TNF-α) but exhibit increased production of IL-10 upon TLR-4 ligation. Here we showed that aged macrophages demonstrate similar cytokine dysregulation phenotype upon stimulation with TLR-2 ligands, or killed S. pneumoniae. We hypothesized that an age-associated increase in activity of phosphatidyl inositol 3-kinase (PI3K)-Akt signaling pathway may be playing a causal role in the age-associated cytokine dysregulation. We found that gene expression of both the regulatory (p85β) and the catalytic (p110δ) subunits of Class IA PI3K is higher in aged than in young splenic macrophages. The age-associated increase in the activity of PI3K was also demonstrated by an upregulation of P-Akt and its downstream target, glycogen synthase kinase-3 (GSK-3). Inhibition of PI3K enhanced induction of pro-inflammatory cytokines, by TLR-2/TLR-1, TLR-2/TLR-6 and TLR-4 ligands as well as heat killed S. pneumoniae (HKSP). Therefore, targeting PI3-Kinase could rescue cytokine dysregulation in aged macrophages and enhance the relevant pro-inflammatory cytokines needed to support B-cell activation and differentiation.

► There is an age-associated heightened activity of the Class IA PI3K at the mRNA level in LPS stimulated splenic macrophages.
► The heightened activity of PI3K in the aged splenic macrophages can be validated indirectly by the increased phosphorylation of AKT and GSK-3 upon TLR-4 stimulation.
► Aged splenic macrophages exhibit cytokine dysregulation upon stimulation with ligands for TLR-2 and TLR-4 receptors.
► The age-related increase in activity of PI3K suppresses the pro-inflammatory cytokines but enhances IL-10 as shown by the ability of the PI3K pharmacological inhibitors, LY294002 and wortmannin to partially rescue this cytokine dysregulation in the presence of TLR-4 agonist.
► The aging phenomenon leading to increased PI3K activity can be recapitulated via western blots for P-Akt and P-GSK-3 in the context of stimulation with TLR-2/TLR-1 or TLR-2/TLR-6 heterodimers and HKSP.
► This finding is the first that directly links age-associated heightened activity of PI3K as a mechanism for cytokine dysregulation in macrophages in the context of TLR-2 and TLR-4 as well as HKSP.