Abstract WMP105: Human Monocyte Derived Macrophage Phagocytize Eryptotic Erythrocytes in a Phosphotidylserine Dependent Mechanism

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Abstract

Introduction: After intracerebral hemorrhage (ICH), erythrocytes contribute to secondary injury by releasing toxic hemoproteins. Our lab has previously shown that blood derived macrophages play an important role in ICH clearance but mechanisms of phagocytosis by human macrophages are unknown. This study aims to quantify eryptotic (phosphatidylserine (PtdSer)-expressing) red blood cells (RBCs) in an in vivo model of ICH, and to investigate the mechanisms that play a role in autologous eryptotic phagocytosis by human monocyte derived macrophages (huMDMs).

Methods: ICH was induced in mice by autologous blood injection. The mice were sacrificed at 1 day after ICH. The brains were separated into hemispheres and digested into a single cell suspension for analysis by flow cytometry. Cells were stained with antibodies to cell surface markers and annexin V to quantify externalized PtdSer expression. Human monocytes were cultured with M-CSF for 7 days to generate huMDMs. Autologous RBCs were heat shocked (HS) to induce eryptosis. The huMDMs were cocultured with HS RBCs, HS RBCs treated with annexin V, or control RBCs. After 1 hour of coculture, the huMDMs were washed, stained and erythrophagocytosis quantified by microscopy.

Results: The proportion of cells that externalized PtdSer increased by almost 20 fold at day 1 after ICH. Control brains mixed with fresh RBCs and subjected to tissue prep did not show PtdSer expression, ensuring that the PtdSer expression detected was induced in vivo (Fig A). HS RBCs increased PtdSer expression and were efficiently phagocytosed by huMDMs. Treatment of HS RBCs with annexin V to antagonize PtdSer-receptor interactions decreased RBC phagocytosis to levels comparable to control RBCs (Figs B and C).

Conclusions: In vivo after ICH, erythrocytes externalize PtdSer, a cue to be engulfed by macrophages. Human macrophages phagocytose RBCs in a PtdSer-dependent mechanism. These findings highlight potential targets to enhance ICH clearance.

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