Erroneous efferocytosis of live neutrophils during infection prevents the resolution of inflammation [RNAseq_dual_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. To examine the consequences of live neutrophil efferocytosis in vivo using a dual species model. We depleted C57BL6/J mice of their endogenous neutrophil populations (anti-Ly6G), then induced peritonitis. Human peripheral neutrophils were treated with bacterial protease (RgpB) or apoptosis was induced. RgpB-treated neutrophils (gLN) or apoptotic neutrophils (AN) were injected into the inflammed peritoneum. 4 h or 24 h after injection, peritoneal cell populations were collected by lavage and sorted to obtain a pure macrophage population. Peritoneal exudate macrophages (PEM) were obtained from the inflammed peritoneum of mice that did not receive an adoptive transfer of neutrophils. Isotype control (iso ctrl) indicates PEMs obtained from the inflammed peritoneum that were not neutrophil depleted. We profiled differential gene expression in macrophages from gLN vs AN injected mice to determine how live cell efferocytosis effects macrophage polarization.