Graphical Abstract Highlights d Retina has 2 microglia pools differing by niche and IL-34 dependency d In homeostasis, IL-34-dependent microglia contribute to neuronal function d In degeneration, both pools relocate toward the retinal pigmented epithelium (RPE) d This transition reprograms microglia and is associated with RPE protection

Glaucoma is the most prevalent neurodegenerative disease and a leading cause of blindness worldwide. The mechanisms causing glaucomatous neurodegeneration are not fully understood. Here we show, using mice deficient in T and/or B cells and adoptive cell transfer, that transient elevation of intraocular pressure (IOP) is sufficient to induce T-cell infiltration into the retina. This T-cell infiltration leads to a prolonged phase of retinal ganglion cell degeneration that persists after IOP returns to a normal level. Heat shock proteins (HSP) are identified as target antigens of T-cell responses in glaucomatous mice and human glaucoma patients. Furthermore, retina-infiltrating T cells cross-react with human and bacterial HSPs; mice raised in the absence of commensal microflora do not develop glaucomatous T-cell responses or the associated neurodegeneration. These results provide compelling evidence that glaucomatous neurodegeneration is mediated in part by T cells that are pre-sensitized by exposure to commensal microflora.

The recent paradigm shift that microglia are yolk sac-derived, not hematopoietic-derived, is reshaping our knowledge about the isolated role of microglia in CNS diseases, including degenerative conditions of the retina. However, unraveling microglial-specific functions has been hindered by phenotypic overlap of microglia with monocyte-derived macrophages. The latter are differentiated from recruited monocytes in neuroinflammation, including retina. Here we demonstrate the use of fate mapping wherein microglia and monocyte-derived cells are endogenously labeled with different fluorescent reporters. Combining this method with 12-color flow cytometry, we show that these two populations are definitively distinguishable by phenotype in retina. We prove that retinal microglia have a unique CD45lo CD11clo F4/80lo I-A/I-E− signature, conserved in the steady state and during retinal injury. The latter was observed in the widely used light-induced retinal degeneration model and corroborated in other models, including whole-body irradiation/bone-marrow transplantation. The literature contains conflicting observations about whether microglia, including in the retina, increase expression of these markers in neuroinflammation. We show that monocyte-derived macrophages have elevated expression of these surface markers, not microglia. Our resolution of such phenotypic differences may serve as a robust way to help characterize isolated roles of these cells in retinal neuroinflammation and possibly elsewhere in CNS.