An Attempt to Polarize Human Neutrophils Toward N1 and N2 Phenotypes in vitro

Abstract

Neutrophils act as the first line of defense against invading pathogens. Although traditionally considered in context of their antimicrobial effector functions, the importance of tumor-associated neutrophils (TANs) in the development of cancer has become increasingly clear during the last decade. With regard to their high plasticity, neutrophils were shown to acquire an anti-tumorigenic N1 or a pro-tumorigenic N2 phenotype. Despite the urgent need to get a comprehensive understanding of the interaction of TANs with their tumor microenvironment, most studies still rely on murine tumor models. Here we present for the first time a polarization attempt to generate N1 and N2 neutrophils from primary human neutrophils in vitro. Our results underscore that N1-polarized neutrophils have a pro-inflammatory phenotype characterized among others by a higher level of intercellular adhesion molecule (ICAM)-1 and high secretion of interferon (IFN)γ-induced protein 10 (IP-10)/C-X-C motif chemokine 10 (CXCL10) and tumor necrosis factor (TNF). Further, we demonstrate that neutrophils incubated under a tumor-mimicking in vitro environment show a high cell surface expression of C-X-C motif chemokine receptor 2 (CXCR2) and secrete high levels of interleukin (IL)-8. These findings suggest that it is feasible to polarize blood-derived primary human neutrophils toward N1- and N2-like phenotypes in vitro. Further, we hypothesized that the presence of anti-inflammatory neutrophil phenotype is not a phenomenon limited to cancer but also occurs when neutrophils are infected with intracellular pathogens. Indeed, our findings indicate that N2-polarized neutrophils exert a markedly decreased capacity to kill the protozoan parasite Leishmania donovani and therefore permit parasite persistence.

Keywords: Leishmania donovani; N1; N2; neutrophil heterogeneity; neutrophils; polarization; tumor-associated neutrophils; visceral leishmaniasis.

FIGURE 1

Cell surface expression of typical N1 and N2 markers on in vitro polarized neutrophils. Primary human neutrophils were incubated for 24 or 48 h in the presence of an N1 or N2 polarization cocktail containing the Pan-caspase inhibitor QVD-OPh. Neutrophils which were only treated with QVD-OPh (N0) served as control. The cell surface expression of the typical N1 markers FasR (CD95) (A) and intercellular adhesion molecule (ICAM)-1 (CD54) (B), and the typical N2 marker C-X-C motif chemokine receptor 2 (CXCR2) (CD182) (C) was assessed by using flow cytometry. Bar diagrams show mean fluorescence intensity (MFI) ± SD (n = 3). *p < 0.05, **p < 0.01, ****p < 0.0001, ns = not significant.

FIGURE 2

Cell surface expression of the activation marker CD62L and of the degranulation markers CEACAM8 (CD66b), integrin alpha M (CD11b), as well as secretion of myeloperoxidase (MPO) by in vitro polarized neutrophils. Primary human neutrophils were incubated for 24 and 48 h in the presence of an N1 or N2 polarization cocktail including the Pan-caspase inhibitor QVD-OPh. The cell surface expression of CD62L (A), CD66b (C), and CD11b (D) was assessed by using flow cytometry. For the detection of MPO (B), cell-free supernatants were collected after 24 and 48 h. The concentration of MPO was measured by ELISA. Bar diagrams show mean fluorescence intensity (MFI) ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.

FIGURE 3

Secretion of tumor necrosis factor (TNF), interferon (IFN)γ-induced protein 10 (IP-10), and interleukin (IL)-8 by N1- and N2-polarized neutrophils. Primary human neutrophils were incubated in the presence of an N1 or N2 polarization cocktail including the Pan-caspase inhibitor QVD-OPh. Neutrophils which were only treated with QVD-OPh (N0) served as control. Cell-free culture supernatants were collected after 24 and 48 h. The concentrations of TNF (A), IP-10 (B), and IL-8 (C) were measured by ELISA. Bar diagrams show mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns = not significant.

FIGURE 4

Myeloperoxidase (MPO)-derived reactive oxygen species (ROS) release by in vitro polarized neutrophils. Primary human neutrophils were incubated in the presence of an N1 or N2 polarization cocktail including the Pan-caspase inhibitor QVD-OPh. Neutrophils which were only treated with QVD-OPh (N0) served as control. After 24 and 48 h, the MPO-derived ROS release was measured for 1 h at 37°C in medium alone or in the presence or absence of 20 nM phorbol 12-myristate 13-acetate (PMA) by using the luminol-based chemiluminescence assay. Representative curves of luminol chemiluminescence are shown in panels (A–D). Bar diagrams of the area under the curve (AUC) values normalized to N0 (mean ± SD; n = 3) are shown in panels (E) and (F). *p < 0.05, **p < 0.01, ns = not significant.

FIGURE 5

Superoxide release by in vitro polarized neutrophils. Primary human neutrophils were incubated in the presence of an N1 or N2 polarization cocktail including the Pan-caspase inhibitor QVD-OPh. Neutrophils which were only treated with QVD-OPh (N0) served as control. After 24 and 48 h, the reactive oxygen species (ROS) release was measured for 1 h at 37°C in medium alone or in the presence of 20 nM phorbol 12-myristate 13-acetate (PMA) by using the lucigenin-based chemiluminescence assay. Representative curves of the time kinetics of lucigenin chemiluminescence are shown in panels (A–D). Bar diagrams of the area under the curve (AUC) values normalized to N0 (mean ± SD; n = 3) are shown in panels (E) and (F). *p < 0.05, **p < 0.01, ns = not significant.

FIGURE 6

Enhanced survival of Leishmania donovani promastigotes in N2-polarized neutrophils in vitro. Primary human neutrophils were infected with L. donovani promastigotes (ratio 1:10) for 3 h at 37°C, 5% carbon dioxide (CO₂). Successful infection was determined by Giemsa staining of cytocentrifuged samples, representative microscopy shown in (A). Arrows show representative ingested (intracellular) Leishmania. After removing the free, non-ingested parasites, the infected cells were incubated in the presence of an N1 or N2 polarization cocktail at 37°C, 5% CO₂. Survival of parasites was assessed after 24 and 48 h by the limiting dilution assay (B). Bar diagrams show mean ± SD (n = 4). *p < 0.05.