Tumor-associated neutrophils (TANs) regulate many processes associated with tumor progression, and depending on the microenvironment, they can exhibit pro- or antitumor functions. However, the molecular mechanisms regulating their tumorigenicity are not clear. Using transplantable tumor models, we showed here that nicotinamide phosphoribosyltransferase (NAMPT), a molecule involved in CSF3R downstream signaling, is essential for tumorigenic conversion of TANs and their pro-angiogenic switch. As a result tumor vascularization and growth are strongly supported by these cells. Inhibition of NAMPT in TANs leads to their antitumor conversion. Adoptive transfer of such TANs into B16F10-tumor bearing mice attenuates tumor angiogenesis and growth. Of note, we observe that the regulation of NAMPT signaling in TANs, and its effect on the neutrophil tumorigenicity, are analogous in mice and human. NAMPT is up-regulated in TANs from melanoma and head-and-neck tumor patients, and its expression positively correlates with tumor stage. Mechanistically, we found that targeting of NAMPT suppresses neutrophil tumorigenicity by inhibiting SIRT1 signaling, thereby blocking transcription of pro-angiogenic genes. Based on these results, we propose that NAMPT regulatory axis is important for neutrophils to activate angiogenic switch during early stages of tumorigenesis. Thus, identification of NAMPT as the critical molecule priming protumor functions of neutrophils provides not only mechanistic insight into the regulation of neutrophil tumorigenicity, but also identifies a potential pathway that may be targeted therapeutically in neutrophils. This, in turn, may be utilized as a novel mode of cancer immunotherapy.

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Pseudomonas aeruginosa is an opportunistic multidrug-resistant pathogen, able to grow in biofilms. It causes life-threatening complications in diseases characterized by the up-regulation of type I interferon (IFN) signaling, such as cancer or viral infections. Since type I IFNs regulate multiple functions of neutrophils, which constitute the first line of anti-bacterial host defense, in this work we aimed to study how interferon-activated neutrophils influence the course of P. aeruginosa infection of the lung. In lungs of infected IFN-sufficient WT mice, significantly elevated bacteria load was observed, accompanied by the prominent lung tissue damage. At the same time IFN-deficient animals seem to be partly resistant to the infection. Lung neutrophils from such IFN-deficient animals release significantly lower amounts of neutrophil extracellular traps (NETs) and reactive oxygen species (ROS), as compared to WT neutrophils. Of note, such IFN-deficient neutrophils show significantly decreased capacity to stimulate biofilm formation by P. aeruginosa. Reduced biofilm production impairs in turn the survival of bacteria in a lung tissue. In line with that, treatment of neutrophils with recombinant IFN-β enhances their NETosis and stimulates biofilm formation by Pseudomonas after co-incubation with such neutrophils. Possibly, bacteria utilizes neutrophil-derived NETs as a scaffold for released biofilms. In agreement with this, in vivo treatment with ROS-scavengers, NETs disruption or usage of the bacterial strains unable to bind DNA, suppress neutrophil-mediated biofilm formation in the lungs. Together, our findings indicate that the excessive activation of neutrophils by type I IFNs leads to their boosted NETosis that in turn triggers biofilm formation by P. aeruginosa and supports its persistence in the infected lung. Targeting these mechanisms could offer a new therapeutic approach to prevent persistent bacterial infections in patients with diseases associated with the up-regulation of type I IFNs.

Angiogenesis plays an important role during tumor growth and metastasis. We could previously show that Type I interferon (IFN)‐deficient tumor‐associated neutrophils (TANs) show strong pro‐angiogenic activity, and stimulate tumor angiogenesis and growth. However, the exact mechanism responsible for their pro‐angiogenic shift is not clear. Here, we set out to delineate the molecular mechanism and factors regulating pro‐angiogenic properties of neutrophils in the context of Type I IFN availability. We demonstrate that neutrophils from IFN‐deficient (Ifnar1−/−) mice efficiently release pro‐angiogenic factors, such as VEGF, MMP9 or BV8, and thus significantly support the vascular normalization of tumors by increasing the maturation of perivascular cells. Mechanistically, we could show here that the expression of pro‐angiogenic factors in neutrophils is controlled by the transcription factor forkhead box protein O3a (FOXO3a), which activity depends on its post‐translational modifications, such as deacetylation or phosphorylation. In TANs isolated from Ifnar1−/− mice, we observe significantly elevated SIRT1, resulting in SIRT1‐mediated deacetylation of FOXO3a, its nuclear retention and activation. Activated FOXO3a supports in turn the transcription of pro‐angiogenic genes in TANs. In the absence of SIRT1, or after its inhibition in neutrophils, elevated kinase MEK/ERK and PI3K/AKT activity is observed, leading to FOXO3a phosphorylation, cytoplasmic transfer and inactivation. In summary, we have found that FOXO3a is a key transcription factor controlling the angiogenic switch of neutrophils. Post‐translational FOXO3a modifications regulate its transcriptional activity and, as a result, the expression of pro‐angiogenic factors supporting development of vascular network in growing tumors. Therefore, targeting FOXO3a activity could provide a novel strategy of antiangiogenic targeted therapy for cancer.