Extracellular DNA NET-Works With Dire Consequences for Health

Abstract

Neutrophils play a central role in innate immune defense. Advances in neutrophil biology have brought to light the capacity of neutrophils to release their decondensed chromatin and form large extracellular DNA networks called neutrophil extracellular traps (NETs). NETs are produced in response to many infectious and noninfectious stimuli and, together with fibrin, block the invasion of pathogens. However, their formation in inflamed blood vessels produces a scaffold that supports thrombosis, generates neo-antigens favoring autoimmunity, and aggravates damage in ischemia/reperfusion injury. NET formation can also be induced by cancer and promotes tumor progression. Formation of NETs within organs can be immediately detrimental, such as in lung alveoli, where they affect respiration, or they can be harmful over longer periods of time. For example, NETs initiate excessive deposition of collagen, resulting in fibrosis, thus likely contributing to heart failure. Here, we summarize the latest knowledge on NET generation and discuss how excessive NET formation mediates propagation of thrombosis and inflammation and, thereby, contributes to various diseases. There are many ways in which NET formation could be averted or NETs neutralized to prevent their detrimental consequences, and we will provide an overview of these possibilities.

Keywords: autoimmunity; cardiovascular disease; fibrosis; inflammation; neutrophils.

Figure 1:. Process of NET formation.

Different stimuli activate distinctive intracellular pathways that lead to NET formation. Stimuli may result in ROS production through activation of NADPH-oxidase via the Raf-MEK-ERK pathway. Increases in intracellular calcium leads to PAD4 activation. In the nucleus, PAD4 citrullinates histones promoting chromatin decondensation. Cytosolic or mitochondrial ROS production allows the release of neutrophil elastase (NE) and myeloperoxidase (MPO) from neutrophil granules. MPO and NE migrate to the nucleus, also contributing to chromatin decondensation. Several stimuli lead to the NLRP3 inflammasome pathway, potentially causing caspase-1 activation, while intracellular LPS and bacteria activate caspase-11. NE and caspases-1 and −11, cleave/activate gasdermin D (GSDMD) that forms pores in both nuclear and plasma membranes. Decondensation of chromatin and nuclear swelling, together with GSDMD pores, allow rupture of the nuclear membrane and release of the chromatin in the cytosol, resulting in decoration of chromatin with neutrophil proteins. Subsequently, plasma membrane ruptures at one or more sites and NETs, decorated with neutrophil proteins such as PAD4, MPO, NE, LL37 and Histones, are released.

Figure 2.. VWF and NETs released during ischemia/reperfusion injury contribute to occlusive thrombus formation in the microcirculation and to tissue fibrosis.

The relative roles of VWF and NETs to the complex process of fibrosis development are depicted in the following schematic: (A) Ischemia leads to the rapid release of storage granules containing P-selectin and UL-VWF by endothelial cells that, by self-aggregation, produce extremely long strings (red) which bind platelets (yellow). VWF is also released basolaterally into the underlying tissue (light pink). (B) P-selectin and VWF recruit neutrophils that, under the effect of hypoxia and/or platelet activation, readily release NETs. The large DNA strands (blue) covered with histones bind to VWF polymers through specific interactions, forming an extensive scaffold. VWF and P-selectin promote leukocyte activation and transmigration into the surrounding tissue. (C) Recruitment of additional platelets by NETs produces an occlusive clot that is rich in both VWF and DNA. This propagates ischemic response, while the transmigrated neutrophils produce NETs in the tissue. NETs stimulate other cells, such as fibroblasts (green), to release collagen and other extracellular matrix molecules in response to the perceived injury. Resolution of the clot proceeds by plasma and cellular release of DNases, cleaving the DNA scaffold, and ADAMTS13 fragmenting VWF. ADAMTS13 also detaches the scaffold from the vessel wall. (D) Clot resolution restores blood flow. The endothelium recovers its anti-thrombotic character; however, the fibrotic scar (dark green) persists with possible consequences on organ function.

Figure 3.. Emerging targets for NETs inhibition and direct consequences of NETs removal.

(A) Overview of potential druggable targets that interfere with NET formation. (B) Depiction of molecules associated with NETs injurious to tissues and summary of diseases discussed in this review with experimental and clinical evidence for direct involvement of NETs in their progression. (C) Cell types whose behavior NETs are known to influence. (D) Summary of beneficial and deleterious effects of NETs degradation by DNases.