Cellular Mechanisms Of NETosis: From Microvesicle Shedding To Extracellular DNA Release
- Date: May 27, 2020
- Time: 15:00
- Speaker: Hawa Racine Thiam
- Visiting fellow at National Heart, Lung, and Blood Institute (NHLBI), Bethesda, Maryland, USA
- Room: Zoom Video Conference
- Host: Marcus Taylor
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Neutrophil extracellular traps (NETs) are web-like DNA structures decorated with histones and cytotoxic proteins released by activated neutrophils. Initially described as a way for neutrophils to trap and neutralize pathogens during innate immunity, NETs are now widely implicated in the detrimental effect of several autoimmune diseases. Peptidylarginine deiminase 4 (PAD4) citrullinates histones and is required for NETs formation (NETosis) in mouse neutrophils. While various molecular mechanisms mediating NETosis are being revealed, the cellular events driving NETs release are still unclear. Here, we determined the sequence of cellular events in NETosis, and examined the role of PAD4 in these events. We performed high resolution time-lapse microscopy of mouse and human neutrophils (PMN) and differentiated HL60 neutrophil-like cells (dHL-60) labelled with fluorescent markers of the cytoskeleton and organelles and stimulated with ionomycin, lipopolysaccharides or C. albicans to induce NETosis. We found that stimulated neutrophils eject extracellular DNA after decondensation of the nuclear DNA in the nucleus, rupture of the lamin meshwork and nuclear membrane allowing the release of decondensed chromatin to the cytosol, disassembly of the actin, microtubule and vimentin intermediate filaments networks, vesiculation of the endoplasmic reticulum and plasma membrane and finally rupture of the plasma membrane. More importantly, we found that these cellular pathways occur in a specific and well conserved temporal order suggesting a requirement for a precise sequence for progression through NETosis. Indeed, inhibition of actin disassembly, one of the first cellular events, blocked NET release. To examine the role of PAD4 in NETosis we isolated neutrophils from PAD4-deficient mice and generated a PAD4-knock down (KD) HL60 CRISPR line. We found that chromatin de-condensation, lamin meshwork and NE rupture and extracellular DNA release required the enzymatic and nuclear localization activities of PAD4. Thus, NETosis proceeds by a step-wise and well conserved sequence of cellular events culminating in the PAD4-mediated expulsion of DNA. Our data further suggest that targeting these cellular pathways might be a better approach for controlling NETosis progression than targeting the various divergent signaling pathways.