Controversy surrounds neutrophil function in cancer because neutrophils were shown to provide both pro- and antitumor functions. We identified a heterogeneous subset of low-density neutrophils (LDNs) that appear transiently in self-resolving inflammation but accumulate continuously with cancer progression. LDNs display impaired neutrophil function and immunosuppressive properties, characteristics that are in stark contrast to those of mature, high-density neutrophils (HDNs). LDNs consist of both immature myeloid-derived suppressor cells (MDSCs) and mature cells that are derived from HDNs in a TGF-β-dependent mechanism. Our findings identify three distinct populations of circulating neutrophils and challenge the concept that mature neutrophils have limited plasticity. Furthermore, our findings provide a mechanistic explanation to mitigate the controversy surrounding neutrophil function in cancer.

Summary During the resolution phase of inflammation apoptotic leukocytes are efferocytosed by macrophages in a nonphlogistic fashion that results in diminished responses to bacterial moieties and production of anti-inflammatory cytokines. Complement receptor 3 (CR3) and pro-resolving lipid mediators promote the engulfment of apoptotic leukocytes by macrophages. Here, we present evidence for the emergence of pro-resolving, CD11blow macrophages in vivo during the resolution of murine peritonitis. These macrophages are distinct from the majority of peritoneal macrophages in terms of their functional protein expression profile, as well as pro-resolving properties, such as apoptotic leukocyte engulfment, indifference to TLR ligands, and emigration to lymphoid organs. Notably, we also found macrophages convert from the CD11bhigh to the CD11blow phenotype upon interaction with apoptotic cells ex vivo. In addition, we found that the pro-resolving lipid mediators resolvin (Rv) E1 and RvD1, and the glucocorticoid dexamethasone (Dex) regulated pro-resolving macrophage functions in vivo. This regulation culminated in a novel pro-resolving function, namely reducing the apoptotic leukocyte ingestion requirement for CD11blow macrophage generation. These new phenotype and molecular pathway markers define the new satiated-macrophage. Thus, we suggest that satisfying-efferocytosis generates CD11blow macrophages that are essential for complete non-phlogistic containment of inflammatory agents and the termination of acute inflammation.

The uptake of apoptotic polymorphonuclear cells (PMN) by macrophages is critical for timely resolution of inflammation. High-burden uptake of apoptotic cells is associated with loss of phagocytosis in resolution phase macrophages. Here, using a transcriptomic analysis of macrophage subsets, we show that non-phagocytic resolution phase macrophages express a distinct IFN-β-related gene signature in mice. We also report elevated levels of IFN-β in peritoneal and broncho-alveolar exudates in mice during the resolution of peritonitis and pneumonia, respectively. Elimination of endogenous IFN-β impairs, whereas treatment with exogenous IFN-β enhances, bacterial clearance, PMN apoptosis, efferocytosis and macrophage reprogramming. STAT3 signalling in response to IFN-β promotes apoptosis of human PMNs. Finally, uptake of apoptotic cells promotes loss of phagocytic capacity in macrophages alongside decreased surface expression of efferocytic receptors in vivo. Collectively, these results identify IFN-β produced by resolution phase macrophages as an effector cytokine in resolving bacterial inflammation.

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or naïve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a > 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

The engulfment of apoptotic polymorphonuclear cells (PMN) during the resolution of inflammation leads to macrophage reprogramming culminating in reduced proinflammatory and increased anti-inflammatory mediator secretion. The atypical chemokine receptor D6/ACKR2 is expressed on apoptotic PMN and plays an important role in regulating macrophage properties during and after engulfment. In this study, we found that the inflammatory chemokine CCL5 is mostly retained (75%) during the resolution of zymosan A peritonitis in mice. Moreover, this chemokine is secreted by resolution-phase macrophages (2.5 ng/ml) and promotes their reprogramming in vivo in D6 mice (2-fold increase in IL-10/IL-12 ratio) but not their D6 counterparts. In addition, CCL5 enhanced macrophage reprogramming ex vivo exclusively when bound to D6 apoptotic PMN. Signaling through p38MAPK and JNK in reprogrammed macrophages was enhanced by CCL5-bound apoptotic PMN (3.6-4 fold) in a D6-dependent manner, and was essential for reprogramming. Thus, CCL5 exerts a novel proresolving role on macrophages when acting in concert with apoptotic PMN-expressed D6.

Monocyte-derived macrophages are readily differentiating cells that adapt their gene expression profile to environmental cues and functional needs. During the resolution of inflammation, monocytes initially differentiate to reparative phagocytic macrophages and later to pro-resolving non-phagocytic macrophages that produce high levels of IFNβ to boost resolutive events. Here, we performed in-depth analysis of phagocytic and non-phagocytic myeloid cells to reveal their distinct features. Unexpectedly, our analysis revealed that the non-phagocytic compartment of resolution phase myeloid cells is composed of Ly6C med F4/80 − and Ly6C hi F4/80 lo monocytic cells in addition to the previously described Ly6C − F4/80 + satiated macrophages. In addition, we found that both Ly6C + monocytic cells differentiate to Ly6C − F4/80 + macrophages, and their migration to the peritoneum is CCR2 dependent. Notably, satiated macrophages expressed high levels of IFNβ, whereas non-phagocytic monocytes of either phenotype did not. A transcriptomic comparison of phagocytic and non-phagocytic resolution phase F4/80 + macrophages showed that both subtypes express similar gene signatures that make them distinct from other myeloid cells. Moreover, we confirmed that these macrophages express closer transcriptomes to monocytes than to resident peritoneal macrophages (RPM) and resemble resolutive Ly6C lo macrophages and monocyte-derived macrophages more than their precursors, inflammatory Ly6C hi monocytes, recovered following liver injury and healing, and thioglycolate-induced peritonitis, respectively. A direct comparison of these subsets indicated that the non-phagocytic transcriptome is dominated by satiated macrophages and downregulate gene clusters associated with excessive tissue repair and fibrosis, ROS and NO synthesis, glycolysis, and blood vessel morphogenesis. On the other hand, non-phagocytic macrophages enhance the expression of genes associated with migration, oxidative phosphorylation, and mitochondrial fission as well as anti-viral responses when compared to phagocytic macrophages. Notably, conversion from phagocytic to satiated macrophages is associated with a reduction in the expression of extracellular matrix constituents that Butenko et al. Non-Phagocytic Macrophages Express an Anti-Fibrotic Transcriptomewere demonstrated to be associated with idiopathic pulmonary fibrosis (IPF). Thus, macrophage satiation during the resolution of inflammation seems to bring about a transcriptomic transition that resists tissue fibrosis and oxidative damage while promoting the restoration of tissue homeostasis to complete the resolution of inflammation.

No abstract

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibodydependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or naïve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a > 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.