Retinal perivascular macrophages: ontology and function during neuroinflammation - PROJECT SUMMARY: The blood-retina barrier is a key anatomical characteristic of the retina, which is disrupted in diabetic retinopathy (DR), retinal vein occlusion, and posterior uveitis. Retinal perivascular macrophages are components of the blood-retina barrier, yet their function is unknown. Brain perivascular macrophages are long-lived tissue resident macrophages that reside on arterioles and venules, which regulate blood-brain barrier hyperpermeability. Our preliminary data demonstrate that retinal perivascular macrophages are the major antigen presenting cells in the retina and reside on post-capillary venules. Furthermore, retinal perivascular macrophages express a pro- chemotactic transcriptome for monocytes, neutrophils, and lymphocytes. Additionally, we developed an effective tool to target and deplete (Pf4Cre) perivascular macrophages without affecting microglia. Finally, after intravitreal CCL2 injection, perivascular macrophage depletion severely hampers Ly6C+ monocyte infiltration. Based upon these data, we hypothesize that retinal perivascular macrophages are long-lived tissue resident macrophages that orchestrate transendothelial migration of immune cells during DR and uveitis. To test this hypothesis, we formulated the following specific aims: 1) Determine the ontological origin of retinal perivascular macrophages. In this aim, we will use Cx3cr1CreER x Rosa26GFP mice to fate map perivascular macrophages. We will label macrophages at the yolk sac and fetal liver stages to determine perivascular macrophage origin. Additionally, we will perform pulse-chase labeling studies to determine the longevity and bone marrow contribution over 1 year. 2) Determine the role of perivascular macrophages during experimental autoimmune uveitis (EAU). We will use Pf4Cre x Rosa26LSL-DTR mice to deplete perivascular macrophages during EAU. Additionally, we will use Pf4Cre x MHCIIflox mice to conditionally knockout MHCII in perivascular macrophages during EAU. 3) Determine the role of perivacular macrophages during diabetic retinopathy (DR). In this aim, we will use diabetic Pf4Cre :: Csf1rflox/flox mice to chronically deplete perivascular macrophages and perform confocal immunofluorescence of retinal flatmounts to quantitate immune cell infiltration at Month 1 and both pericyte numbers and avascular capillaries at Month 9. Completion of these aims will determine that perivascular macrophages are derived from fetal progenitors without bone marrow contribution, are necessary for EAU initiation, and regulate immune cell infiltration during DR. The blood-retina barrier is a key component in health and blinding diseases like uveitis and DR. These studies will significantly advance our knowledge of perivascular macrophages and their blood-retina barrier functions. Furthermore, they will potentially open a new therapeutic space, targeting perivascular macrophages during inflammation and inflammatory retinal vascular diseases.
