The Formin FMNL1 Contributes to the Macrophage Inflammatory Response by Regulating Podosome-dependent Adhesion and Migration.

Abstract

Macrophages are indispensible white blood cells (leukocytes) that contribute to both the innate and adaptive immune response. They are crucial for resolving inflammatory events by clearing pathogens and cellular debris, in addition to promoting wound repair. Macrophages are derived from peripherally circulating monocytes, which after stimulation undergo diapedesis from the vasculature into the underlying complex extracellular matrix, where they can become fully differentiated macrophages and migrate to inflammatory loci. Tissues also contain residential populations of macrophages that aid in immediate immune responses and maintain tissue homeostasis. Conversely, unwarranted macrophage activation largely contributes to the onset and progression of inflammatory diseases, such as atherosclerosis and rheumatoid arthritis, in addition to aiding cancer metastasis and facilitating organ transplant rejection. In order for macrophages to effectively resolve inflammatory events or contribute to disease pathology, they must be able to undergo directional migration, which is mediated by integrin-dependent adhesion complexes termed podosomes. Macrophage podosomes are the most prominent structure of the macrophage actin cytoskeleton, containing a pillar-like core of dense filamentous actin that is tethered to the cortical actincytoskeleton via radial actin filaments. Podosomes also contain a variety of proteins that are circumferentially arranged orassociated with the core, and thatare involved in signaling, linking, and scaffolding,as well as modulating the actin cytoskeleton.Historically, our lab has been interested in leukocyte integrin biology and understanding how these receptors mediate adhesion and migration through complex extracellular matrices. Previous studies in our lab demonstrated the novel podosomal association of an actin modulating protein with the ability to processively elongate unbranched linear actin filaments. Subsequent studies determined this protein to be the formin FMNL1, which is predominantly expressed in hematopoietic cells. Consequently, we further revealed that FMNL1 localizes to the apex of the dense actin core, and is required for podosome stability and macrophage adhesion.The work described in this dissertation has greatly expanded on these findings, demonstrating for the first time that primary macrophage migration is dependent on the formin FMNL1. Utilizing in vitro and in vivotechniques with aid of a novel conditional murine FMNL1 KO, we have observed that macrophage podosome formation, migration, and tissue distribution are dependent on FMNL1. Additionally, we have indicated that FMNL1 is required for embryonic development. Remarkably, our findings also suggest that FMNL1-dependent macrophage migration and podosome localization rely on the specific isoform FMNL1γ. Foremost, we have demonstrated that barbed end binding by the FMNL1γ FH2 domain is dispensable for its cellularfunction in macrophages, which has not been previously shown for any other cellular formin function. Thus, these findings, in addition to current formin knowledge, have allowed us develop a working model for FMNL1 function at macrophage podosomes. This work has distinguished FMNL1 as a unique therapeutic target to restrict macrophage migration that contributes to macrophage-mediated diseases. Furthermore, this could translate to treatment of certain cancers, since FMNL1 has been suggested to promote leukemic cell migration.