Defining the PS neighborhood proteins on apoptotic cells and their impact on efferocytosis

Phosphatidylserine (PS), which constitutes only 2-10% of plasma membrane lipids, is a key phospholipid involved in intercellular communication/signaling. PS is actively sequestered to the inner leaflet of the plasma membrane and cells spend significant energy to maintain this asymmetry. PS externalization to the outer leaflet under specific contexts, such as after induction of apoptosis, allows phagocytes to recognize and engulf the dying cells via efferocytosis. Further, the engagement of PS on apoptotic cells by PS receptors on phagocytes (such as macrophages) induces anti-inflammatory signaling, which is a hallmark of efferocytosis during homeostasis. Despite our recognition of PS as a key apoptotic marker, nearly all studies consider PS as an isolated entity, and the PS-proximal proteins influencing PS recognition and signaling remain largely unexplored. Our exciting preliminary studies reveal that PS exposure on apoptotic cells occurs in patches and occurs adjacent to specific proteins, which can, in turn, influence efferocytosis. Our overarching hypothesis tested in this proposal is that proteins in the PS neighborhood on apoptotic cells critically influence/modulate the efferocytosis of dying cells, and the downstream signaling/responses elicited within phagocytes. In Aim 1, based on preliminary data identifying specific PS-proximal proteins, we test how they differ between apoptotic versus 'live’ cells engineered to expose PS (without caspase activation), the proximity of PS to specific scramblases that mediate PS exposure, and how different cell death modalities may alter PS- proximal proteins. In Aim 2, we test specific PS-proximal tetraspanin proteins in regulating efferocytosis ex vivo and in vivo, via models of tissue inflammation. The novel approaches taken here to define the PS neighborhood on apoptotic cells and its influence on efferocytosis address a long-standing key gap in our knowledge, with relevance to auto-inflammatory diseases. Project Number: 1R01GM163049-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Kodi Ravichandran | Institution: WASHINGTON UNIVERSITY, SAINT LOUIS, MO | Award Amount: $388,750 | Activity Code: R01 | Study Section: Special Emphasis Panel[ZRG1 IIDA-A (81)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11289947