Macrophage subtype and cytokine expression characterization during the acute inflammatory phase of mouse bone fracture repair

Abstract

Fracture repair is a complex process requiring heterotypic interactions between osteogenic cells and immune cells. Recent evidence indicates that macrophages are critically involved in fracture repair. Polarized macrophage populations differentially promote and regulate inflammation in other tissues, but little is known about the various macrophage subtypes and their signaling activities following a bone fracture. The authors hypothesized that classically activated (M1 subtype) and alternatively activated (M2 subtype) macrophages are active during the early repair process to initiate and regulate the inflammatory response. To test our hypothesis, bone marrow was collected from intact femurs (naïve group), contralateral and fractured femurs of mice on days 0, 1, 2, 4, and 7 postfracture. Macrophages were isolated from the bone marrow and macrophage subtypes were identified using flow cytometry with antibodies to F4/80, MHC II, CD86, CD11c, and CD40. Bone marrow cytokine levels were measured using xMAP. Flow cytometry revealed dynamic changes in M1 subtype (F4/80+/MHC II+/CD86+), M2 subtype (F4/80+/MHC II−/CD86−), and dendritic cell (DCs; MHCII+/CD11c+/CD40+) populations following fracture as compared to naïve controls. M1 subtype levels were correlated with IL-1α, IL-1ß, IL-2, IL-17, Eotaxin, and MCP-1, while DCs were correlated with IL-6, G-CSF, LIF, KC, and VEGF-A. The results indicate that M1 and M2 subtypes and DCs are recruited to the fracture site early during the repair process and consequently may work in tandem to regulate the inflammatory response required to recruit osteogenic cells needed for later stages of repair.