Proteomic mapping of macrophages in response to the clearance of apoptotic cells reveals a unique reprogramming profile

During the daily process of healthy cellular turnover, billions of cells undergo apoptosis in the human body. These cells are removed by phagocytic cells, namely macrophages through a process known as efferocytosis, which triggers a cascade of reprogramming events in the cell, with a shift towards a pro-resolving or ‘wound healing’ phenotype. To date, no study has attempted to investigate these phenotypic changes from a proteomic perspective. Here, we present a novel and robust workflow for the investigation of proteome and secretome changes in bone marrow-derived macrophages (BMDMs) following efferocytosis using stable isotope labelling by amino acids in cell culture (SILAC) combined with data-independent acquisition (DIA) mass spectrometry. We show that using this workflow we can dissect the mixed proteomes of BMDMs and apoptotic cells to specifically map the reprogramming events occurring in macrophages in the later stages of efferocytosis. Specifically, we find that efferocytic macrophages adopt an alternatively activated phenotype underpinned by an increase in efferocytic and anti-inflammatory markers. We also show that the secretome contains factors that can reprogram naïve BMDMs towards an efferocytosis-like, pro-resolving, phenotype. Our results provide an unprecedented view of the efferocytic landscape of macrophages and will aid in further understanding this important immunological process in the larger context of immune homeostasis and inflammatory disorders.