Microglial Heterogeneity in a Mouse Model of Alzheimer’s Disease: A Mass Cytometry Analysis

Abstract

Microglia, the resident immune cells of the central nervous system, have been subject to intense scrutiny recently because of their implicated role in the pathogenesis of Alzheimers disease, as well as many other neurological conditions. However, little is known about the diversity of microglial phenotypes, apart from the clear recognition that the M1-M2 distinction is an oversimplification. We used mass cytometry, a high dimensional analog of flow cytometry involving mass spectrometry-based analyses of single cells bound with metal-tagged antibodies, to characterize the diversity of microglia isolated from the brains of 3xTg-AD mice at 12, 18, and 24 months of age. Antibodies to microglial surface antigens CD11b, CD11c, CD14, CD18, CD24, CD35, CD38, CD45, CD51, CD64, CD68, CD86, CD124, CD172a, CD200R, CD206, CD274, CD301, CX3CR1, F4-80, MERTK, MHC class II, Siglec F, and TLR4 as well as intracellular antigens Arginase-1, IL10, IL12p40, iNOS, NADPH Oxidase, TGF-beta, and TNF-alpha were conjugated to unique metal isotopes, and used for mass cytometry. Phenograph-based clustering of the resulting high dimensional datasets identified 28 cell clusters of widely varying sizes, including clearly discrete phenotypes as well as pseudo-continuous/fractal phenotypic diversity. Monte Carlo simulations involving random reshuffling of the datasets revealed very different and much more uniform clustering patterns. Furthermore, control analyses of mouse peripheral blood mononuclear cells and brain microglia using the same markers revealed largely disjoint clusterings, indicating that these microglial clusters were not the result of peripheral blood contamination. Most of the microglial clusters were similar in samples from 3xTg-AD mice at all 3 ages, as well as from 12-month-old wild-type mice. The largest cluster expressed high levels of canonical microglial markers CD11b, CD11c, CD18, CD64, CD68, CD200R, CX3CR1, F4-80, NADPH oxidase, and TGF-beta but did not vary between experimental groups. However, 3 clusters did differ across experimental groups, one of which increased significantly with age in the 3xTg-AD mice as a fraction of the total microglial population. This cluster expressed high levels of CD18, CD38, CD45, CD86, and CD200R, but not other canonical markers. In summary, these mass cytometry-based analyses revealed previously uncharacterized microglial phenotypic heterogeneity in a mouse model of Alzheimers disease. The specific roles of the putative subclasses of microglia, their relevance to human disease, and many other questions remain to be addressed.