Figure 6. Reduction of FoxO3 expression with aging contributes to decrease in anti-inflammatory phenotype.

Western blot revealed a modest but statistically significant reduction of FoxO3 expression in protein extract of LMMP from old compared to young mice based on pixel intensity normalized to GAPDH (A). A decline in FoxO3 expression was also found in sorted MMs (B) and IL-4/IL-13-treated BMDMs (C) from old mice. To evaluate whether decreased FoxO3 expression might be responsible for alterations in macrophage phenotype, BMDMs were derived from FoxO3^−/− and WT mice and treated with either IFNγ (M₁) or IL-4 and IL-13 (M₂). M₂ BMDMs from FoxO3^−/− mice demonstrated reduced expression of M2 markers particularly CD206 compared to WT (D). Decreased expression of pro-inflammatory markers iNOS and TNFα was observed in M₁ BMDMs from FoxO3^−/− mice (E) although higher expression of M1 markers was seen following LPS stimulation (see supplemental figure 5D). For (C–E) RNA expression was normalized to untreated (M₀) macrophages from WT mice. *p<.05, **p<.01 by t-test.