The Role of Turnover and Inflammation in Bone

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URL: https://hdl.handle.net/2027.42/177887

Abstract

Bone is in a constant state of remodeling in response to metabolic needs and to maintain structure. While osteoblasts and osteoclasts are under tight temporal and spatial regulation to maintain bone mass, macrophages traditionally associated with immune responsibilities also contribute to homeostasis. Bone formation is supported by osteal macrophage efferocytosis (phagocytosis of apoptotic cells). There are numerous clinical conditions associated with both defective macrophage function and bone, including osteoporosis, in which bone resorption outpaces bone formation. It is estimated that more than 10 million patients are living with osteoporosis in the US, resulting in decreased bone mineral density and increased stress fracture risk. Although parathyroid hormone (PTH) has been FDA approved since 2002 as an anabolic agent for the treatment of osteoporosis and has been investigated for decades, its mechanism is still not fully defined. This thesis sought to contribute to the understanding of macrophage function in bone biology, in conjunction with anabolic PTH and stress fracture healing. We evaluated more than 90 murine models relative to bone formative actions with a primary focus on the trabecular compartment, revealing trends in critical genes and gene families relevant to PTH anabolic actions. Gene deletions with the greatest increase in trabecular bone volume in response to PTH include PTH and 1-α-hydroxylase, amphiregulin, and PTH-related protein. Effects of PTH treatment in macrophage depletion models was dependent on the target. While early lineage depletion models (macrophage Fas-induced apoptosis mouse) had a decreased anabolic response, late lineage depletion models (clodronate liposome induced apoptosis) had an enhanced anabolic response attributed to macrophage recruitment, indicating osteal macrophages play an integral role in the osteoinductive effect of PTH. Next, we evaluated stress fracture healing with PTH treatment. By one week of healing, PTH enhanced callus bone volume. Earlier histological evaluation showed significantly more macrophages in the callus with PTH. Treatment with FDA-approved chemotherapeutic trabectedin showed a decrease in callus macrophages and smaller callus bone volume. The callus macrophages and bone volume were synergistic, supporting a role for macrophages in enhanced stress fracture healing. To have a better understanding of macrophages in bone, we considered one of their main functions, efferocytosis. When macrophages efferocytosed apoptotic bone marrow stromal cells (apBMSCs), 1219 genes significantly up- or down-regulated genes compared to macrophages alone, with SerpinB2 being the most upregulated (9.69x higher during efferocytosis). SerpinB2 is a serine protease inhibitor, but its biological role in bone has not been fully elucidated. We hypothesized that stress fracture healing, under global inflammatory conditions with LPS treatment, would be delayed in SerpinB2 KO mice. With loss of SerpinB2, there was a greater reduction in stress fracture healing with LPS treatment. WT and KO macrophages in vitro had similar phagocytic function. Macrophages exist on a continuum from M1-like (pro-inflammatory) to M2-like (pro-resolving). LPS-treated KO macrophages had lower expression of M1 marker Cd86 but no change in M2 marker Cd206, suggesting that M1-like macrophages are more impacted by the loss of SerpinB2. This is supported by our data showing no differences in outcomes where the M2-like macrophage dominates: one week of stress fracture healing and phagocytosis, suggesting a differential role of SerpinB2 in M1-like and M2-like macrophages. Overall, this dissertation contributes to the understanding of bone biology with clinical motivation, including the anabolic actions of PTH, macrophages in stress fracture healing, and SerpinB2 in bone and macrophage physiology.

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