Cellular and Molecular Mechanisms of Macrophage Fusion

Macrophage fusion resulting multinucleated giant cells (MGCs) formation is associated with numerous chronic inflammatory diseases including the foreign body reaction to implanted biomaterials. Despite long-standing predictions, there have been attempts to use live-cell imaging to investigate the morphological features initiating macrophage fusion because macrophages do not fuse on clean glass required for most imaging techniques. Consequently, the mechanisms of macrophage fusion remain poorly understood. The goal of this research project was to characterize the early and late stages of macrophage multinucleation using fusogenic optical quality substrate. Live-cell imaging with phase-contrast and lattice-light sheet microscopy revealed that an actin-based protrusion initiates macrophage fusion. WASpdeficient macrophages and macrophages isolated from myeloid cell-specific Cdc42-/- mice fused at very low rates. In addition, inhibiting the Arp2/3 complex impaired both the formation of podosomes and macrophage fusion. Analyses of the late stages of macrophage multinucleation on biomaterials implanted into mice revealed novel actin-based zipper-like structures (ZLSs) formed at contact sites between MGCs. The model system that was developed for the induction of ZLSs in vitro allowed for the characterization of protein composition using confocal and super-resolution microscopy. Live-cell imaging demonstrated that ZLSs are dynamic formations undergoing continuous assembly and disassembly and that podosomes are precursors of these structures. It was further found that E-cadherin and nectin-2 are involved in ZLS formation by bridging the plasma membranes together. ii Macrophage fusion on implanted biomaterials inherently involves their adhesion to the implant surface. While biomaterials rapidly acquire a layer of host proteins, a biological substrate that is required for macrophage fusion is unknown. It was shown that mice with fibrinogen deficiency as well as mice expressing fibrinogen incapable of fibrin polymerization displayed a dramatic reduction of macrophage fusion on biomaterials. Furthermore, these mice were protected from the formation of the dense collagenous capsule enveloping the implant. It was also found that the main cell type responsible for the deposition of collagen in the capsule were mononuclear macrophages but not myofibroblasts. Together, these findings reveal a critical role of the actin cytoskeleton in macrophage fusion and identify potential targets to reduce the drawbacks of macrophage fusion on implanted biomaterials.