Abstract
BackgroundThe ability to distinguish resident microglia from infiltrating myeloid cells by flow cytometry-based surface phenotyping is an important technique for examining age-related neuroinflammation. The most commonly used surface markers for the identification of microglia include CD45 (low-intermediate expression), CD11b, Tmem119, and P2RY12.MethodsIn this study, we examined changes in expression levels of these putative microglia markers in in vivo animal models of stroke, cerebral amyloid angiopathy (CAA), and aging as well as in an ex vivo LPS-induced inflammation model.ResultsWe demonstrate that Tmem119 and P2RY12 expression is evident within both CD45int and CD45high myeloid populations in models of stroke, CAA, and aging. Interestingly, LPS stimulation of FACS-sorted adult microglia suggested that these brain-resident myeloid cells can upregulate CD45 and downregulate Tmem119 and P2RY12, making them indistinguishable from peripherally derived myeloid populations. Importantly, our findings show that these changes in the molecular signatures of microglia can occur without a contribution from the other brain-resident or peripherally sourced immune cells.ConclusionWe recommend future studies approach microglia identification by flow cytometry with caution, particularly in the absence of the use of a combination of markers validated for the specific neuroinflammation model of interest. The subpopulation of resident microglia residing within the “infiltrating myeloid” population, albeit small, may be functionally important in maintaining immune vigilance in the brain thus should not be overlooked in neuroimmunological studies.
Highlights
Resident microglia (MG) and infiltrating myeloid cells play a cooperative role in the initiation and resolution of inflammation after central nervous system (CNS) injuries [1]
The examination of brain myeloid populations reveals that a subpopulation of trans-membrane protein 119 (Tmem119)+Purinergic receptor P2Y12 (P2RY12)+ cells resides within the CD45highCD11b+ gate (Fig. 1)
Absolute numbers of Tmem119+ cells and P2RY12+ cells were almost identical in individual samples, and we verified that all Tmem119+ cells were P2RY12+ (Supplementary Fig 2); statistical analyses were carried out based on Tmem119+ counts as a proxy for Tmem119+P2RY12+ counts
Summary
Resident microglia (MG) and infiltrating myeloid cells play a cooperative role in the initiation and resolution of inflammation after central nervous system (CNS) injuries [1]. It has been suggested recently that MG possess a unique transcriptional signature that can be used in parallel with, or in lieu of, relative expression of CD45 for their reliable identification [9, 20,21,22,23,24] Two of these putative markers are the trans-membrane protein 119 (Tmem119) and the purinergic receptor P2Y12 (P2RY12). Tmem119 is a cellsurface protein with unknown function in the brain [25], and P2RY12 is a well-studied purinergic receptor [26, 27] These markers have not been thoroughly validated in various animal models of age-related neuroinflammation [28, 29]. The ability to distinguish resident microglia from infiltrating myeloid cells by flow cytometry-based surface phenotyping is an important technique for examining age-related neuroinflammation. The most commonly used surface markers for the identification of microglia include CD45 (low-intermediate expression), CD11b, Tmem119, and P2RY12
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
