Aging Affects Bone Marrow Macrophage Polarization: Relevance to Bone Healing

Abstract

Macrophages are an important component of the inflammatory cascade by initiating and modulating the processes leading to tissue regeneration and bone healing. Depending on the local environment, macrophages can be polarized into M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotypes. In order to assess the effects of aging on macrophage function, bone marrow macrophage polarization using primary bone marrow macrophages (BMMs) from young (8 weeks old) and aged (72 weeks old) wild-type male C57BL/6J mice was analyzed. Fluorescence-activated cell sorting (FACS) analysis (CD11b, iNOS, CD206), qRT-PCR (iNOS, TNF-α, CD206, Arginase 1), and ELISA (TNF-α, IL-1ra) were performed to compare the M1 and M2 phenotypic markers in young and aged mouse macrophages. Once M1 and M2 macrophage phenotypes were confirmed, the results showed that TNF-α mRNA was significantly upregulated in aged M1s after interferon gamma (INF-γ) exposure. Arginase 1 and CD206 mRNA expression were still upregulated with IL4 stimulation in aged macrophages, but to a lesser extend than those from younger animals. TNF-α secretion was also significantly increased in aged M1s compared to young M1s, following lipopolysaccharide (LPS) exposure. However, the IL-1ra secretion did not increase accordingly in aged mice. The results demonstrate that, compared to younger animals, aging of bone marrow derived macrophages increases the resting levels of oxidative stress, and the ratios of pro- to anti-inflammatory markers. These age-related changes in macrophage polarization may explain in part the attenuated response to adverse stimuli and delay in processes such as fracture healing seen in the elderly.

Lay summary: Bone healing is a complex process that involves both biological and mechanical factors. Macrophages are key cells that regulate the events involved in bone healing, especially the initial inflammatory phase. In this biological cascade of events, macrophages present as different functional phenotypes including uncommitted (M0), pro-inflammatory (M1), and anti-inflammatory (M2), a process called macrophage polarization. A clear understanding of the effects of aging on macrophage polarization is critical to modulating adverse events such as fractures, atraumatic bone loss, and tissue regeneration in an aging population.

Keywords: Aging; Bone healing; Bone marrowmacrophage; Polarization.

Fig. 1

Characterization of mouse bone marrow-derived macrophages (a young; b aged) after polarization. Most of the aged M0s are both iNOS+ and CD206+ where as most of the young M0s are iNOS− and CD206−. There was no difference between young and aged M2 polarized cells and no difference between LPS and IFN-γ induced M1 polarization

Fig. 2

qRT-PCR for iNOS, TNF-α, and arginase 1, and CD206 after polarization. Aged M1s significantly overreacted to polarization stimuli with IFN-γ with increased TNF-α mRNA expression underlying a higher sensitivity to the microenvironment. CD206 and arginase 1 mRNA are downregulated in aged M2s after IL-4 polarization compared to young M2s. Data reported as mean ± standard error of the mean. Fold refers to change over M0

Fig. 3

TNF-α secretion is significantly increased in aged M1s polarized with LPS whereas the IL-1ra increase was significantly lower in aged M1s polarized with LPS. Aged M1s have a dysregulated negative feedback control over inflammation. Data reported as mean ± standard error of the mean