NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin decondensation and nuclear envelope rupture

Abstract

Neutrophil extracellular traps (NETs) are web-like DNA structures decorated with histones and cytotoxic proteins that are released by activated neutrophils to trap and neutralize pathogens during the innate immune response, but also form in and exacerbate sterile inflammation. Peptidylarginine deiminase 4 (PAD4) citrullinates histones and is required for NET formation (NETosis) in mouse neutrophils. While the in vivo impact of NETs is accumulating, the cellular events driving NETosis and the role of PAD4 in these events are unclear. We performed high-resolution time-lapse microscopy of mouse and human neutrophils and differentiated HL-60 neutrophil-like cells (dHL-60) labeled with fluorescent markers of organelles and stimulated with bacterial toxins or Candida albicans to induce NETosis. Upon stimulation, cells exhibited rapid disassembly of the actin cytoskeleton, followed by shedding of plasma membrane microvesicles, disassembly and remodeling of the microtubule and vimentin cytoskeletons, ER vesiculation, chromatin decondensation and nuclear rounding, progressive plasma membrane and nuclear envelope (NE) permeabilization, nuclear lamin meshwork and then NE rupture to release DNA into the cytoplasm, and finally plasma membrane rupture and discharge of extracellular DNA. Inhibition of actin disassembly blocked NET release. Mouse and dHL-60 cells bearing genetic alteration of PAD4 showed that chromatin decondensation, lamin meshwork and NE rupture and extracellular DNA release required the enzymatic and nuclear localization activities of PAD4. Thus, NETosis proceeds by a stepwise sequence of cellular events culminating in the PAD4mediated expulsion of DNA. neutrophil | innate immunity | microscopy

Cross talk between intracellular pathogens and cell death

“…While ROS scavengers have minimal impact on PAD4‐dependent NETosis, neutrophil elastase‐deficient murine neutrophils display a slight defect in extruding NETs compared to WT cells upon ionomycin treatment 106,107 . PAD4‐driven NETosis and NE‐driven NETosis display similar characteristics including chromatin relaxation, nuclear delobulation, and disintegration of nuclear and plasma membrane rupture 108,109 . In agreement with a minor role of NE in driving PAD4‐dependent NETosis, the GSDMD inhibitor, LDC7559, provided minimal inhibition to ionophore‐driven NETosis 100 .…”

Lamin A/C and the Immune System: One Intermediate Filament, Many Faces

“…NETosis engages several cellular mechanisms, including disassembly of the actin, microtubule, and vimentin cytoskeletons; shedding of the plasma membrane; endoplasmic reticulum vesiculation; chromatin decondensation; plasma membrane and nuclear envelope permeabilization; nuclear lamin meshwork and nuclear envelope rupture to release DNA into the cytoplasm; and plasma membrane rupture and expulsion of extracellular DNA. The permeabilization and rupture of the nuclear lamin meshwork generates a punctate lamin A/C meshwork at the nuclear periphery that disassembles to allow DNA to expand throughout the cytoplasm [ 71 ]. The reduced lamin A/C content in activated neutrophils might facilitate nuclear envelope rupture, chromatin condensation, and NETosis [ 27 ] ( Figure 2 b).…”

The Trans-omics Landscape of COVID-19

“…So, the determinants of neutrophil transition from bene cial to detrimental effects deserves additional investigation. Furthermore, considering the major role excess NETs play in COVID-19 severity, targeting NETs formation by directly inhibiting critical molecules required for NET (neutrophil elastase (NE), PAD4, and gasdermin D [64][65][66] provide a promising therapeutic choice to reduce the clinical severity of COVID-19.…”