The Mechanism of Type I Interferon-Mediated Polarization of Tumor-Associated Neutrophils in Mice and Human

Neutrophils are the most abundant cells of all white blood cells and play a key role in host inflammatory responses. Importantly, inflammation has been associated with increased susceptibility for cancer and neutrophils, as a crucial component of this process, play essential role in inflammation-driven tumorigenesis. Neutrophils also represent an independent prognostic marker in a broad variety of neoplasias. The tumor microenvironment represents a special niche that is extremely influencing infiltrating immune cells. The concept of immune cell polarization was first described for macrophages (anti-tumor M1/pro-tumor M2). Recently neutrophil polarization has been postulated. Neutrophils appear to have diverse phenotypes in the tumor microenvironment i.e. tumor promoting (N2) or inhibiting (N1).

Previously, we could show that significantly elevated numbers of neutrophils accumulate in tumors of mice that lack endogenous type I IFNs (Ifnb1^-/-). Such tumor associated neutrophils (TANs) do not only efficiently support tumor angiogenesis and growth by up-regulating pro-angiogenic molecules (VEGF and MMP9), but also secrete higher amounts of neutrophil-attracting chemokines and display prolonged survival, compared to their WT counterparts thus representing pro-tumor N2 phenotype. Moreover, we could show that these N2 neutrophils efficiently support metastatic processes, due to up-regulation of pro-metastatic proteins, like Bv8, MMP9, S100A8 and S100A9, and due to impaired tumor cell killing. Treatment of such cells with rmIFNb reversed such an effect leading to anti-tumor N1 polarization.

Here, we add further evidence emphasizing the importance of type I IFNs for neutrophil polarization in tumor microenvironment and reveal possible mechanism responsible for this phenomenon. In Ifnb1^-/- mice, we observe a significant down-regulation of anti-tumor neutrophil markers, like ICAM1 and TNF-α. Moreover, neutrophils show reduced formation of NETs, accompanied by lower tumor killing capacity. Under these conditions, massively enhanced neutrophil turnover in combination with accumulation of immature neutrophils is observed. Importantly, therapeutic intervention in both Ifnb1^-/-and WT mice using low dose IFN-β, induced anti-tumor activation of neutrophils. Correspondingly, in human melanoma patients undergoing type I IFN therapy, neutrophil anti-tumor characteristics were augmented, compared to untreated patients, suggesting effective outcome of this therapy.

Further, we evaluated the mechanism of prolonged neutrophil survival in the absence of endogenous IFN-β. Importantly, we found that G-CSF mRNA expression levels in Ifnb1^-/- neutrophils from different anatomical compartments as well as G-CSF blood serum levels were markedly up-regulated in such mice. G-CSF-expression levels were strongly reduced when the Ifnb1^-/- neutrophils were incubated with rmIFN-β suggesting involvement of type I interferons in G-CSF down-regulation.  Notably, we could recently show that G-CSF induces synthesis of enzyme Nicotinamide phosphoribosyltransferase (NAMPT), which is a rate-limiting enzyme converting nicotinamide (NA) into NAD⁺ that in turn activates NAD⁺-dependent protein deacetylases sirtuins (SIRTs). NAMPT serves as an inhibitor of neutrophil apoptosis and as neutrophil chemoattractant by upregulation of CXCL8. It is a potent pro-inflammatory factor (upregulation of ROS release) and pro-angiogenic factor (smooth muscle maturation). At the same time, NAMPT was found to be strongly overexpressed in tumors and serum of leukemia patients. Analysis of NAMPT and SIRTs levels in tumor bearing Ifnb1^-/-mice revealed highly upregulated levels of NAMPT and SIRT1 in blood neutrophils of these animals, in comparison to WT mice, which was in line with elevated levels of G-CSF. It also correlates with enhanced tumor angiogenesis, growth and metastasis. Based on these observations, we identified a new mechanism of interferon-mediated activation of pro-tumor neutrophils. Since tumor associated neutrophils represent a highly potent therapeutic target, these data highlight the therapeutic potential of interferons and NAMPT inhibitors, suggesting optimization of their clinical use as potent anti-tumor agent.