Epigenetic Adaptation Drives Monocyte Differentiation into Microglia-Like Cells Upon Engraftment into the Central Nervous System

The identification of specific markers to distinguish resident microglia from infiltrating monocytes has been a long-standing challenge in neuroscience. Recently, proteins such as P2RY12, TMEM119, and FCRLS have been proposed as microglia-specific and are now widely used to define microglial populations in health and disease. The specificity of these markers was predicated on the assumption that circulating monocytes retain their distinct signatures after entering the central nervous system (CNS). Here, we challenge this paradigm. Using a combination of bone marrow chimeras, single-cell RNA sequencing, ATAC-seq, flow cytometry, and immunohistochemistry, we demonstrate that monocytes engrafting into the CNS acquire de novo expression of these established microglia markers. This phenotypic conversion is driven by profound epigenetic reprogramming, characterized by dynamic changes in chromatin accessibility at key gene loci, including P2ry12, Tmem119, and Aif1 (Iba1), and a shift in transcription factor binding motifs toward a microglial profile. We show this process occurs in the retina following injury and, remarkably, under physiological conditions in the brain and spinal cord, where blood-derived monocytes progressively contribute to the resident myeloid pool. Furthermore, engrafted monocytes downregulate canonical monocyte markers (Ly6C, CD45), eventually becoming indistinguishable from embryonic microglia based on conventional phenotyping. Our findings reveal that infiltrating monocytes undergo extensive epigenetic and transcriptional remodeling to adopt a microglia-like fate, challenging the specificity of current markers and necessitating a re-evaluation of the distinct roles of these two cell populations in CNS pathology.