Forced turnover of aged microglia induces an intermediate phenotype but does not rebalance CNS environmental cues driving priming to immune challenge.

Microglia are the resident innate immune cells of the central nervous system. Limited turnover throughout the lifespan leaves microglia susceptible to age-associated dysfunction. Indeed, we and others have reported microglia develop a pro-inflammatory or "primed" profile with age, characterized by increased expression of inflammatory mediators (e.g., MHC-II, CD68, IL-1β). Moreover, immune challenge with lipopolysaccharide (LPS) causes an exaggerated and prolonged neuroinflammatory response mediated by primed microglia in the aged brain. Recent studies show colony-stimulating factor 1 receptor (CSF1R) antagonism results in rapid depletion of microglia without significant complications. Therefore, we hypothesized that CSF1R antagonist-mediated depletion of microglia in the aged brain would result in repopulation with new and unprimed microglia. Here we provide novel evidence that microglia in the brain of adult (6-8 weeks old) and aged (16-18 months old) BALB/c mice were depleted following 3-week oral PLX5622 administration. When CSF1R antagonism was stopped, microglia repopulated equally in the adult and aged brain. Microglial depletion and repopulation reversed age-associated increases in microglial CD68 lysosome enlargement and lipofuscin accumulation. Microglia-specific RNA sequencing revealed 511 differentially expressed genes with age. Of these, 117 genes were reversed by microglial repopulation (e.g., Apoe, Tgfb2, Socs3). Nevertheless, LPS challenge still induced an exaggerated microglial inflammatory response in the aged brain compared to adults. RNA sequencing of whole-brain tissue revealed an age-induced inflammatory signature, including reactive astrocytes, that was not restored by microglial depletion and repopulation. Furthermore, the microenvironment of the aged brain produced soluble factors that influenced developing microglia ex vivo and induced a profile primed to LPS challenge. Thus, the aged brain microenvironment promotes microglial priming despite repopulation of new microglia. Collectively, aged microglia proliferate and repopulate the brain, but these new cells still adopt a pro-inflammatory profile in the aged brain.