Project Summary For almost 50 years it has been known that neutrophils regularly appear within megakaryocytes (MKs), a histological phenomenon termed emperipolesis. Using new tools we developed to study this process in vitro and in vivo, we found that emperipolesis is common and increases with experimental inflammatory stress in a murine system. Neutrophils penetrate into the MK cytoplasm, donating a fraction of their surface membrane to platelets before emerging viable and intact. Our preliminary data show that neutrophils take up MK-derived exosomes during emperipolesis and emerge with enhanced migratory capacity. Building on these results, we explore the hypothesis that emperipolesis is a novel cell-in-cell interaction that modulates the function of neutrophils. We test this hypothesis in two independent but complementary aims. In Aim I, we examine the effect of emperipolesis, and in particular of MK exosomes, on neutrophil effector functions, metabolism, and in vivo migration in neutrophil-dependent K/BxN serum transfer arthritis. In Aim II, we employ whole-mount 3D marrow imaging, 2-photon in vivo microscopy, and specific MK deletion and potentially emperipolesis blockade to test the possibility that emperipolesis represents a quantitatively important pathway of neutrophil egress from bone marrow in vivo, including in experimental arthritis. Together, these studies will define a new form of cooperation between MKs and neutrophils that modulates the ability of neutrophils to participate in health and disease. More generally, building upon our recent identification of MKs as a source of IL-1 in arthritis, these studies continue to develop the understanding of MKs and neutrophils as participants in systemic inflammatory disease. These “high risk, high reward” studies of a previously overlooked biological process, widely conserved across mammalian species, will set the stage for an extended investigation of emperipolesis as a novel cell-in-cell phenomenon.