Selectin’ for NETs

In this issue of Blood, Etulain et al have added to the knowledge of neutrophil extracellular traps (NETs) by defining the mechanisms involved in platelet-triggered NET formation.¹ 

NETs have been the subject of intense investigation since their description over a decade ago.²  Platelets have been a late arrival to the immunologic roundtable, their small size and lack of a nucleus having contributed to the underestimation of their versatility. Platelets have the capacity to intimately interact with immune cells, including neutrophils, and the consequences of these interactions are still being defined but range from enhancement of neutrophil activation to the transcellular production of bioactive lipid mediators.³  An important ligand/receptor pairing is the interaction between platelet P-selectin and neutrophil P-selectin glycoprotein ligand-1 (PSGL-1).⁴ 

Perhaps it should not be surprising that NETs have been implicated in an increasingly large number of human diseases. The release of unraveled chromatin decorated with potentially toxic neutrophil granular enzymes is a dramatic event, and the list of NET-associated diseases includes sepsis, acute lung injury, thrombosis, autoimmunity, and malignancy.^5,6  The diversity of these conditions suggests a fundamental role of NETs in immunity. The triggers for NET production are still being defined and likely vary depending on the disease process. Activated platelets were described in 2007 to be a potent stimulator of NETs.⁷  The potential mechanisms by which platelets induce NETs include the release of thromboxane A₂⁸  and β-defensin-1.⁹  Etulain et al have extended these mechanisms to include P-selectin/PSGL-1. In a series of experiments using isolated cells from mice, the authors convincingly demonstrate that thrombin-activated platelets can trigger NET formation, and this process can be inhibited by blocking either P-selectin or PSGL-1. Activated platelets from P-selectin null mice were unable to trigger NETs, which implies that the platelet releasate alone is insufficient. Cellular contact was demonstrated between platelets and NET-releasing cells, but it was also apparent that soluble P-selectin promoted NET formation. Using neutrophils obtained from mice that overproduce soluble P-selectin, these neutrophils (with bound P-selectin) had exaggerated agonist-induced NET formation, which suggests a priming effect by P-selectin. The authors conclude that interrupting P-selectin/PSGL-1 interactions is a therapeutic target in NET-forming diseases.

Several questions emerge from these important findings. First, what are the intracellular events downstream of PSGL-1 that drive histone citrullination,¹⁰  chromatin decondensation, mixing of the nuclear and cytoplasmic compartments, and the ultimate release of NETs? These intracellular events are still poorly understood. Second, what is the nature of the priming effect by P-selectin? NET formation has been classically associated with a reactive oxygen species (ROS)-dependent process, although ROS-independent NET formation is also recognized.⁶  Indeed, P-selectin–dependent NET formation was shown by the authors to be ROS dependent.¹  Therefore, soluble P-selectin may prime the neutrophil NADPH oxidase for NET formation. Finally, how do we explain the significant (although minor) fraction of circulating neutrophils in healthy humans that exist as heterotypic aggregates with platelets? What is the function of these aggregates relative to NET formation? Perhaps neutrophils circulating as heterotypic aggregates represent a “primed” population that, in the setting of “activating” signals, can rapidly release NETs. A “2-event” model may emerge, with P-selectin being critical to the priming effect.

Ultimately, the big question that remains in the field of NET biology is the relative importance of NETs during in vivo host defense vs their potential for tissue injury. The answers will come from continuing to define the basic biology of NETs, including the relevant triggers, the intracellular events that culminate in the expulsion of the chromatin lattice, and the mechanisms by which NETs provoke cellular injury. The reported findings are a conceptual advancement, and position events on the neutrophil surface as a target of intervention to prevent or treat NET-associated diseases.