Placental Protein 13 (Galectin-13) Polarizes Neutrophils Toward an Immune Regulatory Phenotype

Abstract

Termed as galectin-13, placental protein 13 (PP13) is exclusively expressed in the placenta of anthropoid primates. Research on PP13 in normal and pathologic pregnancies show alteration of PP13 concentrations in pregnancy affected by preeclampsia or gestational diabetes. Galectins are also described as potent immunomodulators, and PP13 regulates T cell function in the placenta. Therefore, this study aims to investigate the effects of PP13 on neutrophils; a cell type often ignored in pregnancy, but present in the uterus and placenta from the early stages of pregnancy. Since neutrophil function is dysregulated during pathologic pregnancies, a link between PP13 and neutrophil activity is possible. We determined that PP13 reduces the apoptosis rate in neutrophils. Also, PP13 increases the expression of PD-L1 and production of HGF, TNF-α, reactive oxygen species (ROS), and MMP-9 in these cells. This phenotype resembles one observed in permissive tumor neutrophils; able to sustain tissue and vessel growth, and inhibit T cell activation. At the same time, PP13 does not alter all neutrophil functions, i.e., extrusion of neutrophil extracellular traps, degranulation, phagocytosis, and ROS production following bacterial exposure. PP13 seems to play an essential role in regulating the activity of neutrophils in the placenta by polarizing them toward a placental-growth-permissive phenotype.

Keywords: galectin; immunoregulation; neutrophils; placental protein 13; preeclampsia; pregnancy; tolerance; tumor permissive phenotype.

Figure 1

PP13 binds to neutrophils and increases their survival. (A) PP13 binding to freshly isolated neutrophils at 4°C and 37°C for 60 min. Flow cytometry plot analysis (red: control, blue: treated with PP13) is represented as the relative mean of fluorescence (RMF) and percentage of positive cells (n = 3). (B) Apoptosis of neutrophils represented as AnnexinV⁺ and 7-AAD^+/− cells after 24 h incubation with PP13 (3 μg/ml) or control Gal (3 μg/ml), in the presence (empty squares, n = 6) or absence (full circles, n = 8) of pregnancy hormones (progesterone, estradiol, estriol). One way-ANOVA, unpaired samples, ^*p < 0.05. (C) Taqman assay for the expression of SERPINB1 mRNA in neutrophils after overnight culture with or without PP13 (3 μg/ml) (n = 5). Data are presented as ΔCt values (normalization to GAPDH). Student's t-test, ^**p < 0.01.

Figure 2

PP13 does not influence the functionality of neutrophils. (A) Spontaneous or induced NET formation after 2 h stimulation of neutrophils with or without PP13 (3 μg/ml) and 1 h ± PMA or A23187 (A23) was quantified by immunostaining of neutrophils (n = 4). The quantification analysis was performed by NETQUANT. (B) NE activity assay performed on supernatants from neutrophils cultured in the presence (n = 4) or absence (n = 5) of PP13 (3 μg/ml) or control Gal (n = 5) for 24 h. (C) Phagocytosis of FITC-dextran measured with flow cytometry in neutrophils cultured in the presence or absence of PP13 (3 μg/ml) for 3 days and exposed to FITC-dextran for 60 min (n = 5). (D) Production of ROS by neutrophils cultured with PP13 (3 μg/ml) or a control Galectin for 24 h (n = 6). (E) ROS production by neutrophils stimulated with PP13 (3 μg/ml) (triangles) for 30 min with (triangles) or without (circles) heat-killed E. coli or S. aureus (n = 5). All analysis was performed with One-way ANOVA unpaired test, ^***p < 0.001.

Figure 3

PP13 polarizes neutrophils toward a regulatory phenotype. (A) Expression of CD66 or CD11b on the surface of neutrophils co-cultured with BeWo cells and exposed to PP13 (3 μg/ml) (n = 3) or left untreated (n = 3) for 24 h (red: control, blue: treated with PP13). This is a representative flow cytometry experiment. (B) Migration assay of neutrophils in a transwell plate toward PP13, control Gal, or fMLP. (C) Expression of PD-L1 on the surface of neutrophils co-cultured with BeWo cells and exposed to PP13 (3 μg/ml) or left untreated for 24 h (n = 3) (red: control, blue: treated with PP13). This is a representative flow cytometry experiment. (D) De novo synthesis of TNFA mRNA in neutrophils cultured with or without PP13 for 6 h (n = 6). Data are presented as ΔCt values (normalization to GAPDH). (E) ELISA assay for the secretion of HGF or VEGF-α by neutrophils treated with PP13 (3 μg/ml) or Control Gal for 6 h (n = 9). (F) ELISA assay for the secretion of MMP-9 or IL-4 by neutrophils treated with PP13 (3 μg/ml) for 6 h (n = 5) (^**p < 0.01). All analysis was performed with Student's t-test (number of groups are 2) or One-way ANOVA unpaired test (number of groups are more than 2), ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.