Peptidoglycan from Bacillus anthracis Inhibits Human Macrophage Efferocytosis in Part by Reducing Cell Surface Expression of MERTK and TIM-3

Bacillus anthracis peptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen-associated molecular pattern (PAMP) contributing to anthrax pathology, including organ dysfunction and coagulopathy. Increases in apoptotic lymphocytes are a late-stage feature of anthrax and sepsis, suggesting there is a defect in apoptotic clearance. Here, we tested the hypothesis that B. anthracis PGN inhibits the capacity of human monocyte-derived macrophages (M{Phi}) to efferocytose apoptotic cells. Exposure of CD163+CD206+ M{Phi} to PGN for 24h impaired efferocytosis in a manner dependent on human serum opsonins but independent of complement component C3. PGN treatment reduced cell surface expression of the pro-efferocytic signaling receptors MERTK, TYRO3, AXL, integrin V{beta}5, CD36 and TIM-3, whereas TIM-1, V{beta}3, CD300b, CD300f, STABILIN-1 and STABILIN-2 were unaffected. ADAM17 is a major membrane-bound protease implicated in mediating efferocytotic receptor cleavage. We found multiple ADAM17-mediated substrates increased in PGN-treated supernatant suggesting involvement of membrane-bound proteases. ADAM17 inhibitors TAPI-0 and Marimastat prevented TNF release, indicating effective protease inhibition, and modestly increased cell-surface levels of MerTK and TIM-3 but only partially restored efferocytic capacity by PGN-treated M{Phi}. We conclude that human serum factors are required for optimal recognition of PGN by human M{Phi} and that B. anthracis PGN inhibits efferocytosis in part by reducing cell surface expression of MERTK and TIM-3.