Abstract
Microglia, the resident immune cells of the brain, have been shown to display a complex spectrum of roles that span from neurotrophic to neurotoxic depending on their activation status. Microglia can be classified into four stages of activation, M1, which most closely matches the classical (pro-inflammatory) activation stage, and the alternative activation stages M2a, M2b, and M2c. The alternative activation stages have not yet been comprehensively analyzed through unbiased, global-scale protein expression profiling. In this study, BV2 mouse immortalized microglial cells were stimulated with agonists specific for each of the four stages and total protein expression for 4644 protein groups was quantified using SILAC-based proteomic analysis. After validating induction of the various stages through a targeted cytokine assay and Western blotting of activation states, the data revealed novel insights into the similarities and differences between the various states. The data identify several protein groups whose expression in the anti-inflammatory, pro-healing activation states are altered presumably to curtail inflammatory activation through differential protein expression, in the M2a state including CD74, LYN, SQST1, TLR2, and CD14. The differential expression of these proteins promotes healing, limits phagocytosis, and limits activation of reactive nitrogen species through toll-like receptor cascades. The M2c state appears to center around the down-regulation of a key member in the formation of actin-rich phagosomes, SLP-76. In addition, the proteomic data identified a novel activation marker, DAB2, which is involved in clathrin-mediated endocytosis and is significantly different between M2a and either M1 or M2b states. Western blot analysis of mouse primary microglia stimulated with the various agonists of the classical and alternative activation states revealed a similar trend of DAB2 expression compared with BV2 cells.
Highlights
From the ‡Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, 4202 E
Western blot analysis of mouse primary microglia stimulated with the various agonists of the classical and alternative activation states revealed a similar trend of DAB2 expression compared with BV2 cells
M2a stimulation resulted in the quantification of 4224 protein groups across the three biological replicates: 2959 of these protein groups were quantified in all three biological replicates, and 3684 were found in at least two biological replicates
Summary
Chemicals and Reagents—All chemicals and reagents were obtained from Fisher Scientific unless otherwise noted. After a treatment to control ratio was calculated, protein group ratios were assigned using the median peptide expression ratio of unique peptides only. Raw files of these data are publicly available from Chorus Project, project #888, https://chorusproject.org. A volume of 50 l of Streptavidin conjugated to the fluorescent protein, R-Phycoerythrin, was added to each well and incubated at room temperature, protected from light, on a plate shaker set to 200 – 400 rpm for 30 min. 100 l of wash buffer was added to all wells and the beads will be resuspended on a plate shaker set at 200 – 400 rpm for 2 min. The plate was analyzed on the Luminex Magpix using a five-parameter logistic curve-fitting method for calculating cytokine concentrations in the media
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