Neutrophil Extracellular Traps Orchestrate Necroinflammation

In 1869, a young Swiss student who was interested in the study of the nuclei of cells succeeded in separating the nuclei from the cytoplasm of white blood cell from pus, which he obtained from discarded surgical bandages from the local surgical clinic.¹ He found that the nuclei were made of an acidic substance that was rich in phosphorus, which he called nuclein and was, in fact, chromatin (i.e., DNA embedded with proteins, such as histones). This sticky and viscous substance was long considered to be stupid. The outcome is well known; the discovery of the double helix led to the immediate realization that the structure of DNA determines its functions. Consequently, DNA is both autocatalytic, which implies that it dictates the construction of more DNA that is identical to itself, and heterocatalytic, in that it dictates the construction of proteins that are very different from itself. Another critical aspect of the physicochemical properties of chromatin is its high immunogenicity, and if similar to many intracellular components, it is located in the wrong place at the wrong time; for example, in the extracellular milieu, it will engage specific pattern recognition receptors and activate the immune system. For example, histones engage Toll Like Receptor 2 (TLR2) and TLR4 at the surface of cells that will activate NF-κB– and IFN Regulatory Transcription Factor–mediated signaling pathways, leading to inflammation.² Moreover, under some circumstances, some TLRs promote a form of programmed cell death called necroptosis: on fixation to ligand, TLR4 will recruit the adaptor Toll/interleukin-1 receptor domain–containing adapter-inducing IFN-β that participates in the formation of a signaling platform called the necrosome.³ The necrosome activates the pronecroptotic protein Mixed Lineage Kinase Domain Like via phosphorylation that ultimately dismantles plasma membranes.^4,5

In 2004, an extension of the immune functions of chromatin was described. Slowly released from dying neutrophils or budding from live cells, decondensed chromatin fibers containing histones serve as scaffolds for enzymes and antimicrobial peptides and are able to entrap microorganisms, cleave their virulence factors, and kill them.⁶ These web-like structures that trap and kill microbes are called neutrophil extracellular traps (NETs). The neologism NETosis refers to the process that leads to the production of NETs.

Since their original description, it soon became apparent that dysregulated NETs formation has important consequences in human diseases, which suggest that the tight regulation of NETosis is critical to minimizing damage to the host. NET constituents have been identified in injured kidneys in lupus and ANCA-associated vasculitides.^7–9 In addition to making potential autoantigens accessible to the immune system and creating the milieu in which an autoimmune response may be triggered, NETs may be involved in the induction of manifestations of systemic inflammatory disorders, and they may contribute to the pathogeneses of autoimmune diseases, such as rheumatoid arthritis. In the latter case, citrulline, which is a byproduct of the arginine on histones after the enzymatic activity of NO synthase, favors immunogenicity and may prompt the formation of antibodies against citrullinated histones.¹⁰

Although NETs have been observed in various models of kidney injury and are suspected to play a role in sterile inflammation, their exact function in AKI remains unknown. AKI is an inflammatory disorder; stressed and necrotic renal cells secrete and release molecules termed danger-associated molecular patterns that can prime innate immune cells and thereby, activate innate immunity, inflammation, and occasionally, adaptive immunity as predicted by the danger theory in kidney allografts. An inflammatory response can be considered to be at the extreme end of a spectrum that ranges from a homeostatic state to the stress response (a cell-autonomous adaptive response) and finally, inflammation and is engaged to eliminate the stressor, promote adaptations to the stressor, and ultimately, return the system to the homeostatic state.¹¹ In turn, disease reflects the inability of these responses to restore tissue homeostasis. In this context, critical points for which there is still room for explanation are when and why inflammation fueled by AKI becomes deleterious both locally (in the tissue) and at the systemic level (remote injury). This issue is of clinical relevance, because the mediators of these autoamplification loops are potential (at least theoretically) therapeutic targets.

It is from this perspective that the study by Nakazawa et al.¹² proves its true worth. The authors provide clear evidence that tubular epithelial cell death promotes NET formation, which in turn, exacerbates kidney injury and induces remote organ injury during ischemia-reperfusion injury. These authors showed that the DNA and histones contained in NETs are direct mediators of necroinflammation, which is an autoamplification loop of tubular cell death,¹³ and the release of danger-associated molecular patterns, including histones, embedded in NETs induces additional injuries of the tubular epithelial cells. In this respect, NETs seem to be critical players in necroinflammation loops. Importantly, after they are released in the circulation, NETs can promote lung injury. Finally, the inhibition of NETosis seems to be a valuable method for dampening necroinflammation. Notably, this model has been raised in mice subjected to ischemia-reperfusion injury, and it would be interesting to evaluate whether NETosis is a ubiquitous phenomenon that occurs from the moment at which cell death occurs in the kidney or whether it is more specifically restricted to certain types of kidney injury.

These important results pave the way for further stimulating issues related to necroinflammation. For example, it will be important to determine whether NETs that are released into the circulation on AKI predispose individuals to systemic inflammatory disorders and determine the manners in which NET clearance can be manipulated to break the necroinflammation autoamplification loop. Although NETs play a critical role in the host defense, the excessive formation or persistence of NETs may lead to adverse effects. Thus, the clearance of NETs is an important physiologic process that helps to minimize the excessive presentation of both toxic products and potential self-antigens. The degradation of NETs by DNase is one mechanism by which NETs are cleared, and the impairment of this process leads to lupus-related lesions in mice. Consistently, inadequate DNase activity has also been detected in the blood of patients with both lupus and autoimmune vasculitis.¹⁴ Beyond the enzymatic activity of DNase, macrophages also play a role in the clearance of NETs. The DNase-mediated processing of NETs prepares them for engulfment by macrophages, and this process is further facilitated by the opsonization of NETs by C1q.¹⁵ These observations raise another line of questions with clinically relevant influences that are related to the individual susceptibility to produce and clear NETs and the net effect of this equilibrium. The identification of individuals with propensities to accumulate NETs (for example, on a genetic basis) on kidney injury could explain interindividual differences in the local and systemic prognoses of AKIs of similar severity. Finally, on the basis of the observation made that circulating NETs are directly responsible for remote injury after AKI, their detection as minimally invasive biomarkers could help the early identification of individuals who are at risk for severe local and systemic consequences of AKI and thus, lead to adapted management.

Clearly, the field of cell death is very much alive, and many concepts and approaches, such as those presented by Nakazawa et al.,¹² have flourished in this burgeoning area of research and debate.

Disclosures

None.