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.

Quercetin Attenuates Trauma-Induced Heterotopic Ossification by Tuning Immune Cell Infiltration and Related Inflammatory Insult

“…It is well documented in the literature that M1 and M2 synergistically participate in the healing response after musculoskeletal injury. Considering HO to be a result of aberrant healing, we also found that both M1 and M2 infiltrated the regenerated tissue at the early stage after tendon injury, similar to what was observed during the repair process of muscle injury and bone fracture (59). However, quercetin prominently inhibited macrophage polarization to either the M1 or M2 subtype both in vitro and in vivo.…”

Role of Macrophages and Plasminogen Activator Inhibitor-1 in Delayed Bone Repair Induced by Glucocorticoids in Mice

“…MacCauley et al recently reported that the percentage of M1 macrophages is elevated seven days after femoral fractures in mice [ 40 ], although the number of macrophages and expressions of macrophage-related genes other than TNF- α at the damaged site were not altered four days after femoral injury in our study. These differences might be due to the differences of time course for bone repair after bone injury between fracture model and pinhole bone defect method in mice, since bone repair after fracture slowly progresses in general, compared to that after pinhole bone defect.…”

A Meta-analysis of immune signaling pathways between human type 2 diabetic tissue and mouse bone repair

“…The Th1 response requires high levels of inflammation and causes a strong cell-mediated immune response [20]. The Th1 response is important to macrophage activation, which is essential to fracture repair [21]. We hypothesize that a reduction in Th1 signaling activity in T2DM could reduce available macrophages at the fracture site contributing to impaired fracture repair.…”