Investigation of Osteoclasts as Immune Regulators in the Bone Marrow Microenvironment

Abstract

Osteoclasts are myeloid-derived, multinucleated, bone cells that are primarily known for their bone resorption function. Osteoclast-mediated bone resorption followed by osteoblast-mediated bone formation is essential for proper bone health during adult bone remodeling. While most osteoclast studies have focused on differentiation, bone resorption, and the “coupling” of resorption to bone formation, there has recently been increased interest in potential osteoclast immune functions. The focus of this thesis work was to better understand osteoclast-immune cell relationships in the context of osteoclast resorption and bone remodeling. In Chapter 1, I introduce osteoclast-osteoblast coupling and bone remodeling, as well as what is known regarding osteoclast-immune interactions. I outline the currently established bone-immune connections and walk through data from my experiments assessing the antigen presenting capabilities of osteoclasts in-vitro. These in-vitro assays played a major role in shaping the development of my thesis, to dive deeper into the impact of osteoclast resorption on T cell populations. We found that the lineage of osteoclasts altered their ability to uptake antigens, as the initial step in the innate immune response. In Chapter 2, I describe the development and validation of the CtskDTA inducible osteoclast ablation mouse model that we generated to evaluate how T cells are influenced by osteoclasts. This model is used to temporally reduce osteoclasts, leading to decreased bone resorption and formation. We observed increased Bone Volume per Tissue Volume (BV/TV%) and decreased number of osteoclasts lining the trabecular bone surface in our model. I also discuss various genetic models that can affect osteoclasts or their function, and outline positives and negatives from these different models, highlighting the strengths and weakness of our CtskDTA model. In Chapter 3, we evaluated T cell subsets in the bone marrow of these CtskDTA osteoclast ablated mice. These experiments uncovered a link between osteoclasts and cytotoxic T cell populations, with osteoclast-ablated mice exhibiting decreased activated CD8+ T cells and regulatory CD8 T cells in the bone marrow. To assess the mechanism behind this connection, in Chapter 4, we used pharmacological approaches to specifically inhibit resorption (cathepsin K, Ctsk, inhibition), decrease osteoclasts and resorption (alendronate), or increase resorption (intermittent parathyroid hormone, PTH) to determine if the cytotoxic T cell population changes were influenced by osteoclasts or resorption specifically. We found that cytotoxic T cell populations, CD8+CD25+ and Tcreg populations decreased upon decreased osteoclast resorption, indicating an important link between osteoclast activity and T cell activation. Chapter 5 discusses a model of Rheumatoid arthritis using the KbXN serum transfer mouse model and its preliminary use in our studies to evaluate the role of osteoclasts in a regulated inflammatory model of disease. These data suggested that mice that underwent osteoclast ablation did not need an additional inflammatory stimulus in the context of Rheumatoid arthritis to show altered T cell populations in the bone marrow. In Chapter 6, I show methodology established for identifying and quantifying osteoclast resorption in-vitro using NHS ester mediated bone labeling. The identification and quantification of resorption area is a rigorous way to identify the resorptive state of osteoclast cultures. These studies show some advances towards visualizing, characterizing, and quantifying osteoclast resorption, which increases the knowledge in the field bone biology and resorption-associated diseases regarding controlled osteoclast resorption. Overall, the data presented here advance our knowledge of the biology underlying connections between osteoclast resorption and T cell activation.