The Fatal Circle of NETs and NET-Associated DAMPs Contributing to Organ Dysfunction

Abstract

The innate immune system is the first line of defense against invading pathogens or sterile injuries. Pattern recognition receptors (PRR) sense molecules released from inflamed or damaged cells, or foreign molecules resulting from invading pathogens. PRRs can in turn induce inflammatory responses, comprising the generation of cytokines or chemokines, which further induce immune cell recruitment. Neutrophils represent an essential factor in the early immune response and fulfill numerous tasks to fight infection or heal injuries. The release of neutrophil extracellular traps (NETs) is part of it and was originally attributed to the capture and elimination of pathogens. In the last decade studies revealed a detrimental role of NETs during several diseases, often correlated with an exaggerated immune response. Overwhelming inflammation in single organs can induce remote organ damage, thereby further perpetuating release of inflammatory molecules. Here, we review recent findings regarding damage-associated molecular patterns (DAMPs) which are able to induce NET formation, as well as NET components known to act as DAMPs, generating a putative fatal circle of inflammation contributing to organ damage and sequentially occurring remote organ injury.

Keywords: CIRP; HMGB1; LL37; cfDNA; damage associated molecular pattern; histone; inflammation; innate immune response; neutrophil extracellular traps; remote organ damage.

Figure 3

Schematic illustration of NET-associated DAMPs contributing to inflammation. During inflammation, resident cells such as macrophages or endothelial cells lining the vessel release pro-inflammatory cytokines and chemokines, inducing neutrophil recruitment. Activated neutrophils may release NETs decorated with diverse proteins such as neutrophil elastase, myeloperoxidase, LL37 or mCramp in murine cells, HMGB1, and histones. Some of them have been reported to act as DAMPs. Arrows indicate the contribution of the respective proteins to the pro-inflammatory features. HMGB1 induces NET formation, as well as cytokine/chemokine release. LL37 protects NET fibers from degradation, and also facilitates the internalization of cfDNA, which in turn also promotes cytokine release. Histones have cytotoxic effects on endothelial cells, resulting in additional cfDNA, and are also capable of directly inducing NET formation. The molecule eCIRP has recently been described as promoting NET formation.

Figure 1

Inducers and mechanism of the lytic NET formation. Gram-negative bacteria and bacteria-deriving molecules, antibodies, phorbol-12-myristat-13-acetat (PMA), monosodium urate crystals (MSU) or damage-associated molecular patterns (DAMPs) are capable of activating neutrophils via different receptors and initiating NET formation. Histones induce NET formation via the toll-like receptors (TLR) 4 and 9, eCIRP via parallel binding of myeloid differentiation factor 2 (MD2) and TLR4, and high-mobility group box 1 (HMGB1) via RAGE and TLR4. Calcium is released into the cytosol, followed by activation of the NADPH-oxidase complex (NOX), which generates reactive oxygen species (ROS). In a ROS-dependent step, neutrophil elastase (NE) gets released from the membranes of azurophilic granules and translocates into the nucleus and in parallel degrading actin fibers. NE activity induces the decondensation of chromatin, further supported by the PAD4-dependent citrullination of histones. The activation of gasdermin D (GSDMD) leads to the formation of pores in the cell membrane, thereby enabling the release of chromatin, which has been decorated with cytosolic or granule-associated molecules such as histones, LL37, HMGB1, MPO and NE into the environment.

Figure 2

Inducers and mechanism of non-lytic NET formation. To date, only S. aureus, C. albicans, and activated platelets have been proven to induce early non-lytic NET formation in neutrophils. C. albicans induces NET formation via MAC1-signaling in presence of fibronectin, S. aureus via TLR9 and MAC1, and activated platelets require LFA-1 and LPS. During early NET formation, NE and MPO are released in an NADPH-independent manner into the cytosol and the nucleus, resulting the decondensation of chromatin, supported by PAD4-activity. Nuclear DNA fibers are finally released into the cytosol via vesicles, leaving an anucleated but functional cytoplast.