Regulation of efferocytosis in the brain following traumatic injury - PROJECT SUMMARY Neuroinflammation and neuronal apoptosis are hallmarks of traumatic brain injury (TBI). Efferocytosis is a coordinated process where phagocytes remove apoptotic cell debris to resolve inflammation and begin tissue repair. However, a prolonged or excessive inflammatory response impairs efferocytosis, leading to delayed tissue recovery. The regulation of this process has not been evaluated following brain trauma, and its impact on injury outcomes is yet to be established. Our previous studies, using a controlled cortical impact (CCI) mouse model of TBI, showed evidence for apoptotic cell engulfment by both resident microglia and peripheral-derived macrophages (PDMs), the two ontogenetically distinct phagocytes present in the brain acutely after trauma. We also observed that an augmented peripheral inflammatory response impedes the clearance process. Initial findings revealed an upregulation of the efferocytosis receptor, MERTK, in both microglia and PDMs, suggesting its potential regulatory role in efferocytosis during brain injury. Additionally, we noted increased plasma levels of soluble MERTK (sol. MERTK), indicating the extracellular domain shedding of this receptor, which might restrict efferocytosis following CCI injury. The objective of this research application is to investigate the temporal course of efferocytosis by both microglia and PDMs in the acute, subacute, and chronic stages of brain injury and to determine the role of MERTK in regulating this process and controlling TBI outcome. We hypothesize that MERTK mediates efferocytosis in acute brain injury, and the cleavage of this receptor restricts the clearance process, delaying functional recovery. We will first employ a GFP bone marrow (BM) chimeric mouse model to distinguish the temporal efferocytosis response of PDMs from microglia (Aim 1). Then, we will determine the role of MERTK in regulating efferocytosis using Cx3cr1CreER-Eyfp/+Mertkf/f to induce conditional deletion of Mertk in Cx3cr1-expressing cells (including microglia and PDMs), and MertkY867F/Y867F mutant mice, to specifically inhibit MERTK phagocytosis signaling (Aim 2). Finally, we will determine if MERTK-cleavage restricts the ability of PDMs and microglia to clear apoptotic debris efficiently and delays the functional recovery following TBI. We will employ MERTK cleavage-resistant (MertkCR/CR) mice to generate BM chimeric mice (Mertk-WT+Mertk-CR/CR BM Cells and Mertk-CR/CR+Mertk-WT BM Cells) and parse out the peripheral and central role of MERTK-cleavage on TBI outcomes. Additionally, we will transfer MertkCR/CR macrophages, which retain MERTK function under cleavage- promoting conditions, into injured WT mice and assess their impact on apoptotic debris clearance and TBI outcomes after infiltrating damaged tissue (Aim 3). These studies will advance our understanding of efferocytosis regulation mechanisms, revealing novel therapeutic targets and identifying the optimal timeframe for treatment to mitigate secondary injury and behavior deficits after brain trauma.
