Erroneous efferocytosis of live neutrophils during infection prevents the resolution of inflammation [RNAseq_single_species]

Here, we show that neutrophils infected with the bacterial pathogenPorphyromonas gingivalis (Pg) are internalized alive by macrophages in a manner that bypasses all known efferocytic receptor-ligand interactions. Live cell uptake was mediated by the proteolysis of neutrophil granule proteins by the Pg protease RgpB. Subsequent translocation and recognition of these RgpB-generated efferocytic ligands by macrophage complement receptor 3 (CR3) receptors led to the internalization of live neutrophils by macrophages. Contrary to the immunosuppression induced by apoptotic cells, efferocytosis of live neutrophils was highly inflammatory. Thus, our data show a novel immune subversion strategy employed by a bacterial pathogen that utilizes a previously unknown receptor-ligand interaction for the efferocytosis of live cells and consequently dysregulates the resolution of inflammation. Overall design: To examine the consequences of atypical (live neutrophil) efferocytosis in vivo. We fluorescently labeled CD45.1 bone marrow neutrophils, then treated with bacterial protease (RgpB) or induced apoptosis. We injected RgpB treated live neutrophils (gLN) or apoptotic neutrophils (AN) into the inflammed peritoneum of CD45.2 mice. 4 h following injection, we collected peritoneal cells by lavage and sorted for macrophage populations that were positive for fluorescently labeled BMN or macrophage populations that were negative for BMN (control macrophages). We collected peritoneal exudate macrophages (PEMs) from the inflammed peritoneum of CD45.2 mice (no BMN injection) and resident peritoneal macrophages (RPM) from peritoneal cavities of naive CD45.2 mice. We profiled gene expression in efferocytosing macrophages vs control macrophages in gLN vs AN injected mice in order to characterize macrophage transcription and polarization during live cell efferocytosis.