Advanced age impairs macrophage polarization

Abstract

Aging affects many aspects of the cellular function of macrophages. Macrophages play a critical role in innate immunity, acting as sentinels to fight pathogens, promoting wound healing, and orchestrating the development of the specific acquired immune response. However, little is known about how age influences the ability of macrophage to change phenotypes in response to environmental factors. This study examined the age-associated defects on macrophage polarization toward a pro-inflammatory (M1) or an anti-inflammatory (M2) phenotype. Adherent splenocytes enriched for macrophages were cultured with or without lipopolysaccharide (LPS), a combination of interferon (IFN)-γ and tumor necrosis factor (TNF)-α or interleukin (IL)-4. A panel of M1 markers, inducible nitric oxide synthase (iNOS), IL-6, IL-1β, and TNF-α, and M2 markers, including arginase-1 (Arg1), Ym1, and Found In Inflammatory Zone 1 (FIZZ1), were analyzed. IL-6 mRNA in cells from aged mice was decreased by 78% and 58% compared with young after stimulation with LPS or IFN-γ and TNF-α (P<0.05), respectively. Also, there was a marked reduction in the induced levels of iNOS, IL-1β, and TNF-α in cells from aged mice relative to young controls. Similarly, IL-4 exposure resulted in a reduction of M2 markers in adherent splenocytes from aged mice compared with younger animals. This was consistent with a 28% decrease in splenic F4/80(+)IL-4R(+) cells in aged mice relative to controls, although IL-4R expression on these cells did not vary between age groups. In contrast, levels of M1 and most M2 markers, save for FIZZ1, in bone marrow-derived macrophages were similar between the age groups, irrespective of stimuli. These data imply that impaired macrophage polarization in the elderly may dysregulate the development of the host response, making them more susceptible to infectious diseases and that the aging microenvironment may be a key modulator of these macrophage-elicited responses.

FIG. 1.

M1 responses in adherent splenocytes from young and aged mice. (A) Relative iNOS and (B) IL-6 expression was measured via real-time PCR using cDNA from adherent cells obtained from the spleens of young and aged mice. The cells were stimulated for 20 h in the presence of IL-4, LPS, or a combination of IFNγ and TNFα. Protein levels of (C) IL-1β and (D) TNF-α in cell supernatants harvested 20 h after stimulation were quantified by multiplex bead array. N=4–8 mice, *P<0.05 compared with young of the same treatment group. All data are normalized to values from media-treated samples from young mice to highlight the baseline differences between the age groups, if any. iNOS, inducible nitric oxide synthase; interferon-γ, IFN-γ; TNF-α, tumor necrosis factor-α; IL, interleukin; LPS, lipopolysaccharide; PCR, polymerase chain reaction; ND, not detectable.

FIG. 2.

M2 responses in adherent splenocytes from young and aged mice. Relative mRNA expression of (A) Arg1, (B) FIZZ1, and (C) Ym1 was quantified by real-time PCR using cDNA from adherent cells obtained from the spleens of young and aged mice. Cells were stimulated in vitro for 20 h in the presence of IL-4, LPS, or a combination of IFNγ and TNF-α. N=3–7 mice *P<0.05 compared with young of the same treatment group. All data are normalized to values from media-treated samples from young mice to highlight the baseline differences between the age groups, if any. Arg1, arginase-1; FIZZ1, Found In Inflammatory Zone 1.

FIG. 3.

Percentage and expression of IL-4 receptors on F4/80+ cells in the spleen. Spleen cells from young and aged mice were stained with F4/80 (pan macrophage marker) and IL-4R to assess co-expression as well as MFI of IL-4R in F4/80⁺ cells. (A) Analysis showing total percentage of F4/80⁺ cells from the spleen of young and aged mice. (B) Representative dot plots showing total F4/80⁺ macrophage population in the spleen expressing IL-4R (inset). (C) Graphical representation of the total percentage of cells that co-expresses F4/80 and IL-4R in the spleen from young and aged mice. (D) Plot showing IL-4R expression as MFI on F4/80⁺IL-4R⁺ double-positive cells in the spleen from young and aged mice lower right. N=5 mice per group, P<0.05, when compared with young mice. Gating strategies were based on unstained and single color controls and the gates were kept constant among all age and treatment groups. MFI, mean fluorescence intensity.