Human macrophages spontaneously differentiated in vitro are heterogeneous and show different proteomic profile

HUMAN MACROPHAGES SPONTANEOUSLY DIFFERENTIATED IN VITRO ARE HETEROGENEOUS AND SHOW DIFFERENT PROTEOMIC PROFILE S. Fiorelli1, M. Brioschi1, S. Eligini1, E. Tremoli1,2, S. Colli2, C. Banfi1 1Centro Cardiologico Monzino I.R.C.C.S, Milan, Italy. 2Dept. of Pharmacological and Biomolecular Science, Universit\ue0 degli Studi di Milano, Italy ABSTRACT Background Macrophages are critical components of host defense and their biological activities include phagocytosis, elimination of pathogens, inflammation, antigen presentation and tissue remodeling. Macrophages are hallmarked by morpho/phenotypic heterogeneity. For example, distinct populations were observed within atherosclerotic lesions where they contribute to plaque progression, stability and even regression. Tissue macrophages are not easily obtained and in vitro models commonly used might not adequately reflect the heterogeneity of tissue macrophages. We previously reported that human macrophages spontaneously differentiated from adherent monocytes (MDMs) show two dominant morphotypes (spindle and round) co-existing in the same culture. This study was conceived to define the proteome of these distinct morphotypes. Methods and Results Mononuclear cells were isolated from venous blood of healthy donors by Ficoll-Paque Plus and plated in DuplexDish50. Cells were cultured in medium 199 supplemented with 10% autologous serum. At the 7th day, MDMs were fixed and every single cells was dissected by means of a laser capture microdissection system (PALM MicroLaser, Zeiss) and catapulted into a cup of microcentrifuge tube. Proteins were extracted from a total of 6000 cells/morphotype and qualitatively and quantitatively compared using a label-free mass spectrometry-based method. One hundred-thirty two proteins were identified. Among them, 28 were more abundant in round and 28 in spindle MDMs. More in detail, 11 and 27 were unique in round and spindle MDMs, respectively. Of shared proteins, 17 were up-regulated in round MDMs whereas only 1 was up-regulated in spindle MDMs. Specifically, distinctive profiles were observed for proteins involved in cell motility, lipid metabolism, efferocytosis and protection against stress conditions. Conclusions The two MDM morphotypes show a different proteome. Proteome of round MDMs is reminiscent of a non-inflammatory and reparative phenotype. Results may be useful to focus on macrophage heterogeneity in health and disease and to address the effects of environmental challenges and drugs