CDllb+ targeted depletion of macrophages negatively affects bone fracture healing

Inflammation is an important part of the fracture repair process which requires osteogenic cells to interact with innate immune cells such as macrophages. All murine macrophages express the F4/80 cell surface marker but they may be further subdivided into two main phenotypes: M1 (proinflammatory) or M2 (anti-inflammatory) based on surface marker expression and function. Macrophages polarize between these two main classes in response to inflammation while differentially regulating the healing process. Studies have shown that F4/80+ cell ablation impairs fracture healing, however, the distinct phenotypes that participate in the early healing process is unclear. We hypothesized that the M1 subtype is essential for the early steps of fracture healing and their depletion would impair fracture repair. To test this hypothesis, M1 (F4/80+/MHCII+/CD86+/CDllb+) macrophages were depleted using a saporin conjugated Mac-1 antibody (Mac1SAP) in vitro using primary macrophages and in vivo using a mouse femur fracture model. Primary macrophages isolated from mice femoral bone marrow were either left undifferentiated (+PBS), differentiated into M1 macrophages (+LPS), or differentiated to M2 macrophages (+IL-4), and then treated with either vehicle or 10 pM Mac1SAP. Samples were collected at day 2 and 5 post Mac1SAP treatment. Macrophage subtypes were identified by flow cytometry and cytokine secretion profiles were quantified using xMAP. For the in vivo model, mice were treated with Mac1SAP 24 h prior to fracture. Femur bone marrow samples were collected and analyzed by flow cytometry, xMAP, immunohistochemistry, MicroCT, and histology. The results demonstrated that Mac1SAP significantly depleted M1 macrophages both in vivo and in vitro. Mac1SAP treatment altered expression of 75% of cytokines in vitro and 30% of cytokines in vivo including IL-6, TNF-a, and IP-10. In both the in vitro and in vivo models, the M1 subtype correlated highly with cytokines G-CSF, IL-1α, IL-6, IL-10, LIX, KC, MCP-1, IP-10, MIP1α, MIP1β, RANTES, IL-9, IL-2 and TNFα. M1 depletion was also found to reduced callus properties at day 14 via microCT analysis. Overall, the data suggests that depletion of M1 macrophages by Mac1SAP treatment alters the cytokine expression profiles during early bone repair which ultimately impairs bone healing.