Neutrophil extracellular traps: from physiology to pathology

Abstract

At the frontline of the host defence response, neutrophil antimicrobial functions have adapted to combat infections and injuries of different origins and magnitude. The release of web-like DNA structures named neutrophil extracellular traps (NETs) constitutes an important mechanism by which neutrophils prevent pathogen dissemination or deal with microorganisms of a bigger size. At the same time, nuclear and granule proteins with microbicidal activity bind to these DNA structures promoting the elimination of entrapped pathogens. However, these toxic properties may produce unwanted effects in the host, when neutrophils uncontrollably release NETs upon persistent inflammation. As a consequence, NET accumulation can produce vessel occlusion, tissue damage, and prolonged inflammation associated with the progression and exacerbation of multiple pathologic conditions. This review outlines recent advances in understanding the mechanisms of NET release and functions in sterile disease. We also discuss mechanisms of physiological regulation and the importance of neutrophil heterogeneity in NET formation and composition.

Keywords: NETosis; Neutrophil; Sterile inflammation.

Figure 1

Pathways and mechanisms regulating lytic NETosis. NETosis is triggered by microbial and endogenous stimuli via several activating molecules such as receptor for advanced glycation end products (RAGE), P-selectin–P-selectin glycoprotein ligand 1 (PSGL1), toll-like receptors (TLR), low-affinity immunoglobulin gamma receptor (FcγR), or sialic acid-binding immunoglobulin-type lectins (Siglec), among others. Activation of MAP kinase signalling induces reactive oxygen species (ROS) generation by the NADPH oxidase 2 (Nox2). Alternative ROS can be generated by mitochondria. ROS plays a central role in NETosis triggering NE release from the azurosome complex, a process aided by gasdermin D (GSDMD) which is activated by caspase-11 upon exposure to intracellular cytosolic bacteria. NE degrades F-actin and translocates to the nucleus where it will partially cleave histones promoting chromatin decondensation. Chromatin decondensation is also enhanced by the binding of cationic proteins like MPO or DEK and by protein-arginine deiminase type 4 (PAD4)-mediated histone citrullination. Phosphorylation of the lamin network drives its disassembly and the breakdowns of the nuclear envelope. High levels of ROS promote DNA damage triggering DNA repair via ataxia-telangiectasia mutated (ATM) and BReast CAncer gene (BRCA)-1. NETosis also depends on cell cycle cyclin-dependent kinase 4/6 (CDK4/6) and the duplication of centrosomes and autophagy. Inhibitory receptors such as sialic acid-binding immunoglobulin-type lectin-5 and 9 (Siglec-5,9) or signal inhibitory receptor on leukocytes 1 (SIRL1) block NEtosis. Phagocytic receptors like Dectin-1 inhibit NETosis in response to small microorganisms by sequestering NE to phagosomes. ATG7, autophagy-related protein 7; AZU, azurophilic granule; CG, cathepsin G; CR3, complement receptor 3; IRAK, IL-1 receptor-associated kinase; MEK, MAPK/ERK kinase; mTOR, mechanistic target of rapamycin; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; RIPK1/3, receptor-interacting serine/threonine-protein kinase 1/3.

Figure 2

Pathomechanisms of NETosis: excessive release of NETs drives disease through multiple mechanisms involving vessel occlusion, tissue injury, modulation of immune cell function, and pro-tumorigenic and pro-metastatic functions. NETs promote thrombosis through a coagulant activity by the induction of tissue factor release by activated platelets and monocytes and by providing a physical scaffold for platelets and thrombotic molecules (fibrin) to deposit and aggregate to form the thrombus. NET aggregation can also occlude other tube-shaped structures such as the bile and pancreatic ducts provoking alterations of organ function and inflammation. NET components cause different effects depending on their nature, amount, and targeted cell. The toxic cargo (histones or granule proteins such as LL37) of NETs induces cell apoptosis and lysis causing death and promoting tissue injury and inflammation. However, NETs also contain multiple proteases that degrade (‘sink effect’) or activate (‘solid-state reactor’) entrapped cytokines and chemokines through a proteolytic activity, hence modulating the inflammatory response. The immunomodulatory function of NETs occurs upon interaction with phagocytes such as macrophages and dendritic cells resulting in activation cell activation, and the release of inflammasome-dependent IL-1β or TLR9-mediated signalling release of IFNα. NETs can also activate T-cell to release IFNα and IFNγ and serve as autoantigens to synovial fibroblasts to initiate autoimmune responses. Among the NET-driven tumorigenic activities, NETs promote tumour growth through direct induction of cell proliferation or the awakening of dormant tumour cells after the remodelling of the surrounding extracellular matrix. The metastatic function of NETs involves their ability to attract and trap circulating tumour cells, providing a physical niche for the development of the metastasis. Finally, NETs can be released by recruited neutrophils around the primary tumour, thus preventing the entrance and function of anti-tumoural cytotoxic T cells and natural killer cells. LPS, lipopolysaccharide; NET, neutrophil extracellular trap.