Physiological Stimuli Induce PAD4-Dependent, ROS-Independent NETosis, With Early and Late Events Controlled by Discrete Signaling Pathways

Abstract

Neutrophils are known to extrude decondensed chromatin, thus forming NETs (neutrophil extracellular traps). These structures immobilize pathogens, thereby preventing their spreading, and are also adorned with antimicrobial molecules. NETs can also influence pathogenesis in chronic inflammation, autoimmunity, and cancer. Despite the importance of NETs, the molecular mechanisms underlying their formation, as well as the upstream signaling pathways involved, are only partially understood. Likewise, current methodological approaches to quantify NETs suffer from significant drawbacks, not the least being the inclusion of a significant non-specific signal. In this study, we used novel, fluorescent polymers that only bind extruded chromatin, allowing a specific and standardized quantification of NETosis. This allowed us to reliably rank the relative potency of various physiologic NET inducers. In neutrophils activated with such stimuli, inhibition of the Syk or PI3K pathways blocked NETosis by acting upon late events in NET formation. Inhibition of the TAK1, p38 MAPK, or MEK pathways also hindered NETosis, but by acting on early events. By contrast, inhibiting PKC, Src family kinases, or JNK failed to prevent NETosis; cycloheximide or actinomycin D were also ineffective. Expectedly, NET formation was deeply compromised following inhibition of the NADPH oxidase in PMA-activated neutrophils, but was found to be ROS-independent in response to physiological agonists. Conversely, we show for the first time in human neutrophils that selective inhibition of PAD4 potently prevents NETosis by all stimuli tested. Our data substantially extends current knowledge of the signaling pathways controlling NETosis, and reveals how they affect early or late stages of the phenomenon. In view of the involvement of NETs in several pathologies, our findings also identify molecular targets that could be exploited for therapeutic intervention.

Primary tumors induce neutrophil extracellular traps with targetable metastasis-promoting effects

“…PAD4 is an important mediator of innate immunity since PAD4 -/mice were shown to be more vulnerable to bacterial infection (34). Moreover, it was recently shown that many known physiological NET inducers (N-Formylmethionyl-leucyl-phenylalanine [fMLP], GM-CSF, TNFα, or PMA) are PAD4 dependent (35). Here again, we found that PAD4 -/-TBM had significantly lower NET levels compared with untreated TBM ( Figure 2D).…”

Cytokine Production and NET Formation by Monosodium Urate-Activated Human Neutrophils Involves Early and Late Events, and Requires Upstream TAK1 and Syk

“…In view of the prevalence of gouty arthritis and of the neutrophil involvement in its pathogenesis, a better understanding of both MSU-elicited responses and of their molecular bases is clearly needed. In this regard, our previous work has provided several potential clues, insofar as we have shown the crucial involvement of TAK1, MAPKs, PI3K, and Syk in cytokine generation, delayed apoptosis, and NETosis in response to several physiological neutrophil stimuli (23)(24)(25)(26)(27). Under the same stimulatory conditions, we have also established that several transcription factors (e.g., NF-κB, C/EBP, CREB) drive cytokine production in neutrophils (23,26,28,29).…”

“…Conversely, our finding that MSU-induced NET formation depends on PAD4, is to our knowledge a first. Thus, MSU appears to function like most other physiological neutrophil agonists (e.g., TNFα, GM-CSF, fMLP, PAF, C5a, CXCL8) with respect to the involvement of endogenous ROS and PAD4 (27). Overall, our findings substantially extend our understanding of the mechanisms underlying NET generation, by showing that MSU crystals represent yet another class of physiological stimuli (in addition to growth factors, chemoattractants, and cytokines) (27) that employ common signaling components, as well as PAD4.…”

“…As shown in Figure 3A, TAK1 inhibition mostly blocked the phosphorylation of all three kinases in response to MSU. We also reported that Syk and Src family tyrosine kinases can affect at least some neutrophil responses (26,27) and our observation that MSU rapidly activates these kinases (Figure 2A) prompted us to examine whether they may also act upstream of MAPKs and Akt. As shown in Figure 3B, Syk inhibition profoundly hindered the phosphorylation of all three kinases, while Src inhibition only significantly affected that of p38 MAPK.…”

Root extracellular traps versus neutrophil extracellular traps in host defence, a case of functional convergence?

“…This may in part be explained by the fact that neutrophils were shown to produce NETs in response to high quantities of invading pathogens and their virulence factors (DAMPs or PAMPs) (Papayannopoulos, 2018) or pathogens too large to be cleared by phagocytosis alone (Branzk et al, 2014). However, recent evidence also suggests that NET formation can occur independently of MPO and NOx (Kenny et al, 2017) and ROS (Pilsczek et al, 2010;Tatsiy & McDonald, 2018). The relative importance of these different NET production pathways remains debated.…”