Neutrophil extracellular trap (NET) formation has emerged as an important response against various pathogens; it also plays a role in chronic inflammation, autoimmunity, and cancer. Despite a growing understanding of the mechanisms underlying NET formation, much remains to be elucidated. We previously showed that in human neutrophils activated with different classes of physiological stimuli, NET formation features both early and late events that are controlled by discrete signaling pathways. However, the nature of these events has remained elusive. We now report that PAD4 inhibition only affects the early phase of NET generation, as do distinct signaling intermediates (TAK1, MEK, p38 MAPK). Accordingly, the inducible citrullination of residue R2 on histone H3 is an early neutrophil response that is regulated by these kinases; other arginine residues on histones H3 and H4 do not seem to be citrullinated. Conversely, elastase blockade did not affect NET formation by several physiological stimuli, though it did so in PMA-activated cells. Among belated events in NET formation, we found that chromatin decondensation is impaired by the inhibition of signaling pathways controlling both early and late stages of the phenomenon. In addition to chromatin decondensation, other late processes were uncovered. For instance, unstimulated neutrophils can condition themselves to be poised for rapid NET induction. Similarly, activated neutrophils release endogenous proteic factors that promote and largely mediate NET generation. Several such factors are known RAGE ligands and accordingly, RAGE inbibition largely prevents both NET formation and the conditioning of neutrophils to rapidly generate NETs upon stimulation. Our data shed new light on the cellular processes underlying NET formation, and unveil unsuspected facets of the phenomenon that could serve as therapeutic targets. In view of the involvement of NETs in both homeostasis and several pathologies, our findings are of broad relevance.

中性粒细胞胞外陷阱 (NET) 的形成已成为对抗各种病原体的重要反应。它还在慢性炎症、自身免疫和癌症中发挥作用。尽管人们对 NET 形成的潜在机制有了越来越多的了解，但仍有许多需要阐明的地方。我们之前表明，在用不同类别的生理刺激激活的人类中性粒细胞中，NET 形成具有由离散信号通路控制的早期和晚期事件。然而，这些事件的性质仍然难以捉摸。我们现在报告说 PAD4 抑制只影响 NET 生成的早期阶段，不同的信号中间体（TAK1、MEK、p38 MAPK）也是如此。因此，组蛋白 H3 上残基 R2 的可诱导瓜氨酸化是受这些激酶调节的早期中性粒细胞反应；组蛋白 H3 和 H4 上的其他精氨酸残基似乎没有被瓜氨酸化。相反，弹性蛋白酶阻断不会通过几种生理刺激影响 NET 形成，尽管它在 PMA 激活的细胞中会影响。在 NET 形成的迟来事件中，我们发现染色质解聚受到控制该现象早期和晚期的信号通路的抑制。除了染色质去凝聚之外，还发现了其他晚期过程。例如，未受刺激的中性粒细胞可以使自己准备好快速诱导 NET。同样，激活的中性粒细胞会释放内源性蛋白质因子，促进并在很大程度上介导 NET 的产生。几个这样的因素是已知的 RAGE 配体，因此，RAGE 抑制在很大程度上阻止了 NET 的形成和调节中性粒细胞在刺激时快速生成 NET。我们的数据揭示了 NET 形成背后的细胞过程，并揭示了可以作为治疗靶点的现象的意想不到的方面。鉴于 NETs 参与稳态和几种病理，我们的发现具有广泛的相关性。