Identification of novel protein biomarkers of macrophage polarization using comparative proteomic analyses of murine primary macrophages

Macrophages comprise the first line of host responses against injury and pathogens and therefore are critically engaged in tissue repair, host defense, and homeostasis maintenance. Depending on the surrounding microenvironment, macrophages polarize into a wide spectrum of immunophenotypes with 2 extreme opposite ends-proinflammatory M1 and anti-inflammatory M2. Elucidating the biochemical bases of distinct macrophage immunophenotypes, as well as discriminating between these phenotypes, are paramount to understanding the contributions of macrophage subpopulations to health and diseases. In this study, murine bone marrow-derived macrophages were treated with LPS or IL-4 to induce the M1/M(LPS) or M2/M(IL-4) state, respectively. Comparative proteomic analyses demonstrate that M1 and M2 macrophages have their own unique protein landscapes. The signature proteins of M1 and M2 macrophages are engaged in distinct signaling pathways, which offer the biochemical bases for their specialized functions. The plasma membrane proteins Clec4e and Cd72 are identified as new biomarkers to discriminate murine M1 and M2 macrophages, respectively. Comparison of the proteomes of murine and human macrophages leads to identification of 2 new shared M1 biomarkers, Gbp2/GBP2 and Acod1/ACOD1. In addition, CLEC4E is validated as a new M1 biomarker for human primary macrophages. This study provides an unbiased protein dataset of murine primary M1/M(LPS) and M2/M(IL-4) macrophages for future research in macrophage biology. The plasma membrane localization of the new biomarkers Clec4e and Cd72 facilitates their labeling and detection. The new M1 biomarkers shared by human and mouse primary macrophages have potential broad applications in both basic research and clinical practice.