The impact of age and sex on the inflammatory response during bone fracture healing

Abstract

Inflammation is thought to be dysregulated with age leading to impaired bone fracture healing. However, broad analyses of inflammatory processes during homeostatic bone aging and during repair are lacking. Here, we assessed changes in inflammatory cell and cytokine profiles in circulation and in bone tissue to identify age- and sex-dependent differences during homeostasis and repair. During homeostatic aging, male mice demonstrated accumulation of CD4+ helper T cells and CD8+ cytotoxic T cells within bone while both pro-inflammatory "M1" and anti-inflammatory "M2" macrophage numbers decreased. Female mice saw no age-associated changes in immune-cell population in homeostatic bone. Concentrations of IL-1β, IL-9, IFNγ, and CCL3/MIP-1α increased with age in both male and female mice, whereas concentrations of IL-2, TNFα, TNFR1, IL-4, and IL-10 increased only in female mice - thus we termed these "age-accumulated" cytokines. There were no notable changes in immune cell populations nor cytokines within circulation during aging. Sex-dependent analysis demonstrated slight changes in immune cell and cytokine levels within bone and circulation, which were lost upon fracture injury. Fracture in young male mice caused a sharp decrease in number of M1 macrophages; however, this was not seen in aged male mice nor in female mice of any age. Injury itself induced a decrease in the number of CD8+ T cells within the local tissue of aged male and of female mice but not of young mice. Cytokine analysis of fractured mice revealed that age-accumulated cytokines quickly dissipated after fracture injury, and did not re-accumulate in newly regenerated tissue. Conversely, CXCL1/KC-GRO, CXCL2/MIP-2, IL-6, and CCL2/MCP-1 acted as "fracture response" cytokines: increasing sharply after fracture, eventually returning to baseline. Collectively, we classify measured cytokines into three groups: (1) age-accumulated cytokines, (2) female-specific age-accumulated cytokines, and (3) fracture response cytokines. These inflammatory molecules represent potential points of intervention to improve fracture healing outcome.

Keywords: aging; cytokines; fracture healing; immune cells; inflammation; sex.

Graphical Abstract

Figure 1

Study design. (A) Young and old male and female mice underwent tibial fracture surgery and immunophenotyping was carried out for local fracture callus tissue and for systemic blood. Immunophenotyping involved analysis of inflammatory cells, inflammatory cytokines, and cytokine transcripts. (B) Cell surface markers were used to define inflammatory cell sub-types.

Figure 2

Inflammatory cells within bone are dysregulated with age. Tibial diaphyses from uninjured mice were homogenized and investigated for inflammatory cell populations using flow cytometry and related to total number of cells present within tissue. (A) Representative gating strategies shown. Total T cell number was measured using CD3 as a pan T cell marker; CD4+ helper and CD8+ cytotoxic T cell numbers were assessed. Macrophages were identified as CD11b+, Ly6G-. Pro-inflammatory macrophages (M1; Ly6c^high) and anti-inflammatory macrophages (M2; Ly6c^low) were assessed. Neutrophils were quantified as CD11b+, Ly6G+ cells and B cells were identified as CD19+ cells. (B) Percentage of CD3+, CD4+, and CD8+ T cells in the intact tibial diaphyses. (C) M1 and M2 macrophages were measured and ratio of M1:M2 was determined. (D) Neutrophils and (E) B cells were measured within intact tibial diaphyses. For all, n = 3; two-tailed t-tests were conducted. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001 (YM, young males; OM, old males; YF, young females; OF, old females).

Figure 3

Advanced age alters inflammatory response to fracture injury. Mice underwent tibial fracture surgery and fracture calluses were harvested 0dpf (prior to fracture), 3dpf, and 7dpf. Calluses were homogenized and investigated for inflammatory cell populations using flow cytometry and related to total number of cells present within the fracture callus. (A) Total T cell number was measured using CD3 as a pan T cell marker; CD4+ cytotoxic and CD8+ helper T cell numbers were assessed within the CD3+ T cell population. (B) Pro-inflammatory macrophage (CD11b+, Ly6G-, Ly6c^high) and anti-inflammatory macrophage (CD11b+, Ly6G-, Ly6c^low) number was measured and M1:M2 ratio was determined. (C) CD11b+, Ly6G+ neutrophils, and (D) CD19+ B cells amounts were measured within fracture calluses. N = 3; data analyzed by two-way ANOVA followed by Tukey’s test when significant; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001 (YM, young males; OM, old males; YF, young females; OF, old females).

Figure 4

The cytokine profile within bone changes with age. Lysates of tibial diaphyses from uninjured mice were investigated for cytokine profile using multiplex ELISA. Calculated cytokine amounts were normalized to total amount of protein within the lysate. (A) IL-1β; (B) IL-9; (C) IFNγ; (D) CCL3/MIP-1α; (E) IL-2; (F) TNFα; (G) TNFR1; (H) IL-4; (I) IL-10; (J) CXCL1/KC-GRO; (K) CXCL2/MIP-2; (L) IL-6; and (M) CCL2/MCP-1 were determined from lysates of young and old, male and female mice. N = 5–6; two-tailed t-tests were conducted. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 (YM, young males; OM, old males; YF, young females; OF, old females).

Figure 5

Transcript levels of age-accumulated cytokines fluctuate within bone tissue. Lysates of tibial diaphyses from uninjured mice were investigated for transcript levels of the cytokines which were found to be elevated with age using RT-PCR. (A) Infg, (B) Cccl3/Mipa, (C) Il9, and (D) Il1b were investigated from lysates of young and old, male and female mice. N = 5-6; two-tailed t-tests were conducted. ^**P < 0.01, ^***P < 0.001. (YM, young males; OM, old males; YF, young females; OF, old females; n.d., not detected).

Figure 6

Age-dependent changes are present in cytokine response to fracture injury. Mice underwent tibial fracture surgery and fracture calluses were harvested 0dpf (prior to fracture), 3dpf, and 7dpf. Calluses were homogenized and investigated for cytokine profile using multiplex ELISA. Calculated cytokine amounts were normalized to total amount of protein within the lysate. (A) IL-1β; (B) IL-9; (C) IFNγ; (D) CCL3/MIP-1α; (E) IL-2; (F) TNFα; (G) TNFR1; (H) IL-4; (I) IL-10; (J) CXCL1/KC-GRO; (K) CXCL2/MIP-2; (L) IL-6; and (M) CCL2/MCP were determined from lysates of young and old, male and female mice. N = 5–6; data analyzed by two-way ANOVA followed by Tukey’s test when significant; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001 (YM, young males; OM, old males; YF, young females; OF, old females).