title: Novel Molecular Insights into Classical and Alternative Activation States of Microglia as Revealed by Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Proteomics.
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.
authors.affiliation: From the ‡Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, 4202 E. Fowler Ave, Tampa, Florida 33620;
authors.forename: Harris
authors.initials: H
authors.lastname: Bell-Temin
authors.affiliation: From the ‡Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, 4202 E. Fowler Ave, Tampa, Florida 33620;
authors.forename: Ashley E
authors.initials: AE
authors.lastname: Culver-Cochran
authors.affiliation: From the ‡Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, 4202 E. Fowler Ave, Tampa, Florida 33620;
authors.forename: Dale
authors.initials: D
authors.lastname: Chaput
authors.affiliation: From the ‡Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, 4202 E. Fowler Ave, Tampa, Florida 33620;
authors.forename: Christina M
authors.initials: CM
authors.lastname: Carlson
authors.affiliation: From the ‡Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, 4202 E. Fowler Ave, Tampa, Florida 33620;
authors.forename: Melanie
authors.initials: M
authors.lastname: Kuehl
authors.affiliation: From the ‡Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, 4202 E. Fowler Ave, Tampa, Florida 33620;
authors.forename: Brant R
authors.initials: BR
authors.lastname: Burkhardt
authors.affiliation: §James A. Haley VA Hospital, Research Service and Department of Neurosurgery and Brain Repair, University of South Florida, 12901 Bruce B. Downs Blvd, Tampa, Florida 33612;
authors.forename: Paula C
authors.initials: PC
authors.lastname: Bickford
authors.affiliation: ¶Department of Pharmacodynamics, University of Florida, 1345 Center Drive, Gainesville, Florida 32610.
authors.forename: Bin
authors.initials: B
authors.lastname: Liu
authors.affiliation: From the ‡Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, 4202 E. Fowler Ave, Tampa, Florida 33620; smstevens@usf.edu.
authors.forename: Stanley M
authors.initials: SM
authors.lastname: Stevens
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism*; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D018929:Cell Culture Techniques; D002460:Cell Line; D005786:Gene Expression Regulation; D007553:Isotope Labeling; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D017628:Microglia / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D040901:Proteomics / Q000379:methods*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000596:Amino Acids; D051017:Apoptosis Regulatory Proteins; C478773:Dab2 protein, mouse; D008070:Lipopolysaccharides