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.

Neutrophils are generally the first leukocytes to arrive at sites of inflammation or injury, where they release a variety of inflammatory mediators, which contribute to shaping the ensuing immune response. Here, we show that in neutrophils exposed to physiological stimuli (i.e. LPS and TNF‐α), inhibition of the PI3K signaling pathway impairs the synthesis and secretion of IL‐8, Mip‐1α, and Mip‐1β. Further investigation showed that Mip‐1α and Mip‐1β gene transcription was similarly decreased, whereas IL‐8 transcription and steady‐state mRNA levels were unaffected. Accordingly, PI3K inhibition had no impact on NF‐κB or C/EBP activation, which are essential for IL‐8 transcription, but the basis for this selective inhibition of chemokine transcription remains elusive. We nevertheless identified translational targets of the PI3K pathway (S6, S6 kinase, 4E‐BP1). Inhibitor studies and overexpression experiments further established that the various effects of PI3K on chemokine production can be ascribed to p85α and p110δ subunits. Finally, we show that in LPS‐ and TNF‐activated neutrophils, PI3K acts downstream of the kinases p38 MAPK and TAK1. Given the importance of neutrophils and their products in numerous chronic inflammatory disorders, the PI3K pathway could represent an attractive therapeutic target.

Hypoxia in the tumor microenvironment is a negative prognostic factor associated with tumor progression and metastasis, and therefore represents an attractive therapeutic target for anti-tumor therapy. To test the effectiveness of novel hypoxia-targeting drugs, appropriate preclinical models that recreate tumor hypoxia are essential. The chicken ChorioAllantoic Membrane (CAM) assay is increasingly used as a rapid cost-effective in vivo drug-testing platform that recapitulates many aspects of human cancers. However, it remains to be determined whether this model recreates the hypoxic microenvironment of solid tumors. To detect hypoxia in the CAM model, the hypoxic marker pimonidazole was injected into the vasculature of tumor-bearing CAM, and hypoxia-dependent gene expression was analyzed. We observed that the CAM model effectively supports the development of hypoxic zones in a variety of human tumor cell line-derived and patient’s tumor fragment-derived xenografts. The treatment of both patient and cell line-derived CAM xenografts with modulators of angiogenesis significantly altered the formation of hypoxic zones within the xenografts. Furthermore, the changes in hypoxia translated into modulated levels of chick liver metastasis as measured by Alu-based assay. These findings demonstrate that the CAM xenograft model is a valuable in vivo platform for studying hypoxia that could facilitate the identification and testing of drugs targeting this tumor microenvironment.

Human neutrophils are key players of innate immunity, and influence inflammatory and immune reactions through the production of numerous cytokines and chemokines. Despite major advances in our understanding of this important functional response of neutrophils, the short lifespan of these cells and their resistance to transfection have always been an obstacle to the detailed dissection of signaling pathways and effector responses that is often possible in other cell types.

Gout is a prevalent and incapacitating disease triggered by the deposition of monosodium urate (MSU) crystals in joints, which are also massively infiltrated by neutrophils. The interaction of the latter with MSU crystals triggers several responses, including the generation of inflammatory mediators and of neutrophil extracellular traps (NETs). Though some of the signaling events mobilized by MSU in neutrophils have been described (e.g., Src family kinases, Syk, PKC, PI3K), the picture remains fragmentary. Likewise, the impact of these signaling events on cellular responses is incompletely understood. In this study, we examined transcriptomic changes triggered by MSU in neutrophils and their impact on the corresponding proteins, as well as the role of various signaling pathways in prominent functional responses. We report for the first time that neutrophils can secrete the monocyte chemoattractant, CCL4, in response to MSU. Accordingly, we found that transcription factors NF-κB, CREB, and C/EBP are belatedly activated by MSU crystals, and at least the former is involved in chemokine generation. Moreover, we show that MAPKs and Akt are activated by MSU in neutrophils, that they are under the control of TAK1 and Syk, and that they participate in cytokine generation and NETosis. In the latter instance, we found the phenomenon to be independent of endogenous ROS, but under the control of PAD4. We finally provide evidence that endogenous factors contribute to the belated phosphorylation of kinases and transcription factors in response to MSU. Collectively, our findings unveil potentially important therapeutic targets for gouty arthritis.

Neutrophils influence innate and adaptative immunity, notably through the generation of numerous cytokines and chemokines and through the modulation of their constitutive apoptosis. Several signaling cascades are known to control neutrophil responses, including the MEK pathway, which is normally coupled to ERK. However, we show here that in human neutrophils stimulated with cytokines or TLR ligands, MEK and ERK are activated independently of each other. Pharmacological blockade of MEK had no effect on the induction of ERK kinase activity and vice versa. In autologous PBMC exposed to the same stimuli or in neutrophils exposed to chemoattractants, this uncoupling of MEK and ERK was not observed. Whereas we had shown before that MEK inhibition impairs cytokine generation translationally in LPS- or TNF-stimulated neutrophils, ERK inhibition affected this response transcriptionally and translationally. Transcriptional targets or ERK include the mitogen- and stress-activated protein kinase 1 (MSK-1) and its substrates, C/EBPβ and CREB, whereas translational targets include the S6 kinase and its substrate, the S6 ribosomal protein. In addition to affecting cytokine production, ERK inhibition interfered with how LPS or TNF promotes neutrophil survival and levels of the myeloid cell leukemia 1 (Mcl-1) antiapoptotic protein. Whereas the ERK-activating kinase was not identified, we found that the MAP3K, TGF-β-activated kinase 1 (TAK1), acts upstream of ERK and MEK in neutrophils. Our results document a functional uncoupling of the MEK/ERK module under certain stimulatory conditions and suggest that therapeutic strategies based on MEK inhibition might benefit from being complemented by ERK inhibition, particularly in chronic inflammatory conditions featuring a strong neutrophilic component.