Retinal neurovascular protection by boosting efferocytosis - Project summary/abstract: Our research focuses on identifying cellular mechanisms and therapeutic targets for retinopathies caused by ischemia or trauma including diabetic retinopathy, retinal artery or vein occlusion and traumatic optic neuropathy. A shared pathogenic event of these diseases is the myeloid cell response to retinal injury. In this project, we will study ‘efferocytosis’, which refers to the process by which myeloid cells (macrophages/microglia) clear dead cells by ‘eating’ them. In pathological conditions including stroke, atherosclerosis, and cancer, efferocytosis is thought to promote resolution of inflammation by clearance of dying cells; however, its role in retinopathy is poorly understood. We hypothesize that impaired timely clearance of dead cells in the injured retina contributes to progressive neurovascular damage. Our preliminary data show strong upregulation of the enzyme histone deacetylase 3 (HDAC3) in retinal myeloid cells after injury. Deleting HDAC3 from macrophages boosts their efferocytic capacity. Unbiased RNA-seq screening showed upregulation of the pro-efferocytic secreted protein, CD5 Molecule Like (CD5L) in stimulated macrophages that lack HDAC3. We also observed that the ‘don’t eat me’ signal cluster of differentiation 47 (CD47), which impairs efferocytosis of dead cells, is upregulated in the injured retina and this was ameliorated with myeloid HDAC3 deletion. CD47 impairs efferocytosis by binding to signal regulatory protein α (SIRPα) on myeloid cells. We plan to induce retinal injury by ischemia or trauma, and then evaluate the impact on retinal outcome of two potential therapeutic strategies that enhance efferocytosis: deleting HDAC3 in myeloid cells, and neutralizing CD47 or its ligand SIRPα with antibodies. Our central hypothesis is that: Upregulation of HDAC3 in myeloid cells after retinal injury increases expression of the “don’t eat me” signal CD47 on apoptotic cells and concurrently downregulates the pro- efferocytotic molecule CD5L, contributing to defective efferocytosis and worsened retinal outcomes. This hypothesis will be tested by three related yet independent specific aims: Aim 1 will determine whether myeloid HDAC3 signals through tumor necrosis factor α (TNF-α) to upregulate CD47 on apoptotic cells and inhibit efferocytosis in the injured retina. Aim 2 will determine whether myeloid HDAC3 deletion enhances efferocytosis and retinal injury resolution by upregulating CD5L. Aim 3 will assess whether enhancing efferocytosis by disrupting the ‘don’t eat me’ CD47-SIRPα axis promotes resolution of retinal injury. We will employ novel methods to test our hypothesis including high-resolution microscopy, flow cytometry, monitoring of diverse in vivo retinal neurovascular endpoints, imaging using optical coherence tomography, and assessment of retina function by electroretinography. Our proposal is designed to provide new insights leading to the development of new treatments for ischemic and traumatic retinal injury and it aligns with the NEI mission to support research studying visual disorders and their mechanisms.
