Increasing production of reactive oxygen species by Nox2 enables bone marrow-derived macrophages to target Listeria monocytogenes by LC3-associated phagocytosis Alina Farid1, Alexander Gluschko1, Marc Herb1, Daniela Grumme1, Martin Krönke1,2,3,4, Michael Schramm1 1Institute for Medical Microbiology, Immunology and Hygiene, Faculty of Medicine and University Hospital Cologne, University of Cologne, 50935 Cologne, Germany. 2Center of Molecular Medicine Cologne, 50931 Cologne, Germany. 3Cologne Cluster of Excellence on Cellular Stress Responses in Aging-associated Diseases (CECAD), 50931 Cologne, Germany. 4German Center for Infection Research (DZIF), 50931 Cologne, Germany Non-canonical autophagy pathways decorate single-membrane vesicles with Atg8 family proteins such as ‘microtubule-associated proteins 1A/1B light chain 3B’ (LC3). LC3-associated phagocytosis (LAP) is a non-canonical autophagy pathway that is essential for elimination of intracellular bacterial pathogens such as Listeria monocytogenes (L.m.) by tissue macrophages and thereby substantially contributes to anti-listerial immunity of mice. LAP induction essentially requires the production of reactive oxygen species (ROS) by the phagocyte NADPH oxidase 2 (Nox2). Here, we show that bone marrow-derived macrophages (BMDM) fail to target L.m. by LAP. Instead, BMDM exclusively target L.m. by a damage-induced non-canonical autophagy pathway (DINCA) that is induced by phagosomal membrane damage caused by the pore-forming toxin of L.m., listeriolysin O (LLO). However, we found that DINCA does not have anti-listerial activity at all. The inability of BMDM to target L.m. by LAP originated from insufficient ROS production by Nox2. By contrast, peritoneal macrophages (PM) produced high amounts of ROS and could therefore effectively target L.m. by LAP. These data indicate that not all commonly used macrophage models can use LAP against intracellular pathogens. Next, we investigated if BMDM can be endowed with the ability to use LAP against L.m. by increasing ROS production by Nox2. Priming of BMDM with the proinflammatory cytokines TNF or IFNγ increased Nox2 expression leading to increased production of Nox2-derived ROS. This increase in ROS production enabled BMDM to target L.m. by LAP. In addition, priming diminished phagosomal membrane damage and escape of L.m. from LC3-positive phagosomes. However, targeting of L.m. by LAP remained relatively rare and therefore made only a minor contribution to overall increased anti-listerial activity. Therefore, we conclude that in contrast to tissue macrophages, BMDM are not a good model to study LAP because even primed BMDM do not produce sufficient amounts of ROS by Nox2 for effective targeting of L.m. by LAP.
Institute for Medical Microbiology, Immunology and Hygiene, Faculty of Medicine and University Hospital Cologne, University of Cologne
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