The Role of Macrophage Efferocytosis in Bone

Abstract

Bone tissue can repair itself after injury and is constantly remodeled by bone-forming osteoblasts and bone-resorbing osteoclasts during homeostasis. As 50-70% of bone-lining osteoblasts ultimately undergo apoptosis, dead/dying cell clearance is crucial for normal tissue function. Macrophages are professional phagocytes that clear out apoptotic cells (AC) in an extremely efficient process called efferocytosis. In fact, billions of AC in the human body are cleared daily. Defective efferocytosis has been implicated in many chronic inflammatory diseases and in impaired bone repair. Though known to be crucial for bone remodeling during homeostasis and repair after injury, the mechanisms underlying post-efferocytosis macrophage reprogramming towards reparative activity in bone are still unclear. The inherent heterogeneity and plasticity of macrophages, which enables them to rapidly respond to environmental cues, poses an additional challenge in characterizing the effects of this important process. Efferocytic macrophages may aid in bone remodeling and repair in multiple ways: by producing various factors that promote an anti-inflammatory environment, facilitating continual efferocytosis, recruiting osteogenic precursor cells, and influencing the balance of bone anabolism vs. catabolism. The overall focus of this dissertation is to 1) characterize efferocytic macrophages with AC relevant to bone and 2) identify factors that were elevated in macrophages post-efferocytosis and investigate their impact on bone remodeling and repair.

This thesis firstly focuses on CCL2 and its role in mesenchymal stem/progenitor cell (MSPC) recruitment and early fracture repair. As a pleiotropic factor known to influence bone remodeling, AC-specific upregulation of CCL2 in efferocytic macrophages was notable. In vitro migration assays and Ccr2-/- (KO) murine models were utilized to investigate the relevance of CCL/R2 signaling to MSPC chemotaxis. Both collagen type 1 hydrogel migration assays and subcutaneous ossicle implantation in KO mice demonstrated that CCL/R2 signaling positively regulates MSPC migration. Next, the relevance of CCL/R2 signaling in early bone repair (5- & 7-days post injury) was probed using an ulnar stress fracture model in KO and WT mice and evaluated using standard micro-CT and immunohistochemistry techniques. While the callus formed in KO mice had increased bone, no significant changes in osteoprogenitors within the callus were seen at these early time points.

Subsequently, this thesis broadens its scope to further characterize efferocytic macrophages and elucidate the bone-specific impacts of efferocytosis-induced factors. Using transcriptomics data from bone marrow-derived macrophages engulfing apoptotic osteoblasts, single-cell RNA sequencing analysis revealed two subpopulations of macrophages unique to efferocytosis that exhibited profiles of enhanced glycolytic energy metabolism with anti-inflammatory activity. Various molecular biology methods confirmed increased expression of key enzymes, solute carriers, and metabolites involved in glycolysis, including lactate. As a metabolite with emerging roles in macrophage reprogramming, lactate was examined further for its effects on cells involved in bone homeostasis—osteoblasts, osteoclasts, and macrophages. Lactate exposure reduced osteoclast differentiation, while increasing anti-inflammatory and decreasing pro-inflammatory gene expression in macrophages.

Overall, the work presented in this dissertation utilizes molecular biology and bioinformatics approaches to study efferocytosis in bone remodeling and repair. The data reported demonstrate a clear transformation of macrophages post-efferocytosis, with ensuing modulation of various factors and processes that promote reparative activity in the osteogenic microenvironment. Furthermore, the approach of probing efferocytosis in a tissue-specific manner used in this thesis provides avenues for more in-depth investigation of macrophage contributions to bone, including several efferocytosis-modulated targets that require further exploration using multiomic tools and apoptosis & injury models.