Efferocytosis triggered proteomic reprogramming of macrophages reveals CD97 as a novel apoptotic receptor

During the daily process of healthy cellular turnover in the human body, billions of cells undergo apoptosis. 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) and alveolar macrophages following efferocytosis using stable isotope labelling by amino acids in cell culture (SILAC) combined with data-independent acquisition (DIA) mass spectrometry. Using this workflow, we dissected the mixed proteomes of BMDMs and apoptotic cells to map the reprogramming events occurring in macrophages in the later stages of efferocytosis. Specifically, we identified the adhesion G protein-coupled receptor Adgre5/CD97 as a novel efferocytosis-associated protein and showed that this evolutionary conserved protein plays a role in the uptake of apoptotic cells (ACs) by macrophages from mouse, human and fly. Additionally, we show that CD97 may act as a co-receptor for ACs with other efferocytic receptors such as MerTK. 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.