PROJECT SUMMARY Macrophage fusion resulting in the formation of multinucleated giant cells (MGCs) accompanies a variety of disorders associated with chronic inflammation, including the foreign body response (FBR) elicited by implanted biomaterials. MGCs are a major factor contributing to long-term failures in human vascular prosthetic grafts, pacemaker leads, and other implanted medical devices. Despite the long history of research, the molecular and cellular mechanisms of macrophage fusion and, generally, cell-to-cell fusion remain poorly understood. During the current funding period, we have shown that fibrin polymer, but not its precursor, fibrinogen, deposited on the surface of implanted biomaterials, drives macrophage fusion. Our preliminary in vitro studies found that fibrinogen deposited on the surface at its physiological concentration does not support macrophage fusion, consistent with our previous findings that, upon contact with various surfaces, fibrinogen undergoes self-assembly forming nonadhesive soft matrices. Surprisingly, although the three-dimensional (3D) fibrin gel supports adhesion, it does not support fusion. However, removing the gel, leaving a "2D footprint" consisting of fibrils attached to the rigid surface, restores macrophage fusion. We hypothesize that adsorbed fibrinogen and deposited fibrin polymer form matrices with different mechanical properties and surface patterns, which macrophages sense, initiating different mechanotransduction responses. Specific Aim 1 is to determine why and how the fibrin polymer drives macrophage fusion during the foreign body reaction to implanted biomaterials. Using cell signaling assays with mechanosensitive molecules, ultrastructural studies, the hydrogels with different stiffness, and micropatterned surfaces, we will examine differential sensing by macrophages of the mechanical properties of 3D and 2D matrices prepared from wild-type and mutant fibrin(ogens) and characterize their architectural features. The knowledge obtained will be translated into generating biomaterials with a reduced ability to support macrophage fusion and testing their properties in vivo bioimplant models. Specific Aim 2 will determine the cellular and molecular organization of the macrophage fusion site. Following our finding that macrophage fusion is initiated by an actin-based protrusion and some preliminary data, we hypothesize that a strong actin-propelled protrusion is formed at the leading edge of a donor macrophage enriched in podosomes and situated within the interface restricted by zipper-forming proteins. Taking advantage of our methodological platform consisting of high-resolution microscopy, live cell imaging, mice with myeloid cell-specific knockouts, and macrophages with knockdowns of selected regulators of branched actin network, we will determine the cellular and molecular determinants of the fusion site and fusion pore formation. Overalls, these studies will define the novel biology of macro...