Neutrophils induce proangiogenic T cells with a regulatory phenotype in pregnancy

Abstract

Although neutrophils are known to be fundamental in controlling innate immune responses, their role in regulating adaptive immunity is just starting to be appreciated. We report that human neutrophils exposed to pregnancy hormones progesterone and estriol promote the establishment of maternal tolerance through the induction of a population of CD4+ T cells displaying a GARP+CD127loFOXP3+ phenotype following antigen activation. Neutrophil-induced T (niT) cells produce IL-10, IL-17, and VEGF and promote vessel growth in vitro. Neutrophil depletion during murine pregnancy leads to abnormal development of the fetal-maternal unit and reduced empbryo development, with placental architecture displaying poor trophoblast invasion and spiral artery development in the maternal decidua, accompanied by significantly attenuated niT cell numbers in draining lymph nodes. Using CD45 congenic cells, we show that induction of niT cells and their regulatory function occurs via transfer of apoptotic neutrophil-derived proteins, including forkhead box protein 1 (FOXO1), to T cells. Unlike in women with healthy pregnancies, neutrophils from blood and placental samples of preeclamptic women fail to induce niT cells as a direct consequence of their inability to transfer FOXO1 to T cells. Finally, neutrophil-selective FOXO1 knockdown leads to defective placentation and compromised embryo development, similar to that resulting from neutrophil depletion. These data define a nonredundant function of neutrophil-T cell interactions in the regulation of vascularization at the maternal-fetal interface.

Keywords: T cells; neutrophils; placenta; pregnancy; regulatory.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Human neutrophils treated with estriol and progesterone display a distinct phenotype and induce Tregs from a naïve T cell population. (A) Neutrophils from healthy male donors were treated with progesterone (P; 100 ng/mL) or estriol (E3; 100 ng/mL) or in combination (E3P) for 30 min and then washed. Neutrophil phenotype was analyzed by flow cytometry for surface markers CD16 (FcγRIII), CD62L, CD11b, and the neutrophil anti-inflammatory protein AnxA1. *P < 0.05, **P < 0.01 compared with control-treated neutrophils. (B) Neutrophils were treated with hormones as described in A and then cocultured with autologous lymphocytes (labeled with 3 μM CFSE) at a 1:1 ratio for 5 d in the presence of 2 μg/mL soluble anti-CD3 and anti-CD28 antibodies. The scheme of cell treatment is reported in SI Appendix, Fig. S1B. *P < 0.05 compared with control. (C) Supernatants were collected from cocultures and from lymphocytes or neutrophils cultured on their own, in the presence or absence of P and E3 (100 ng/mL each). Cytokine levels were quantified using a multiplex assay. *P < 0.05 compared with control treatments. (D) After culture, lymphocytes or lymphocytes that had been cocultured with neutrophils (with or without P and E3) were stained for CD4, CD45RO, and FOXP3, as well as for GARP and CD127. *P < 0.05 compared with medium. (E) CD45RA-naïve T cells were isolated by negative selection from healthy male donors and cocultured at a 2:1 ratio with control- or E3P-treated neutrophils for 5 d (plus 2 μg/mL soluble anti-CD3 and anti-CD28 antibodies).

Fig. 2.

Human neutrophil-induced T cells secrete regulatory cytokines and are proangiogeneic. (A) Control (white bars) or E3P (green-lined bars) CD4+ lymphocytes cocultured with neutrophils were stained for intracellular FOXP3 and IL10 (Left; gated on CD4+FOXP3+; **P < 0.01 compared with medium) or IL17 (Right; *P < 0.01). **P < 0.01. (B) ELISA for VEGF carried out with the supernatants of cocultures described in Fig. 1A. In some cases, IL-17 was blocked during coculture at a concentration of 630 ng/mL, which was the optimal dose to inhibit the level of IL-17 released in these cocultures (950 pg/mL). As a positive control, recombinant human IL-17 was added to medium at the same concentration as in the cocultures. (C) Intracellular staining for VEGF in CD4+ T cells following the cocultures described above. (D) Vessel growth assay using growth factor-reduced Matrigel. *P < 0.05 compared with control; #P < 0.05 compared with E3P. In all cases, data are mean ± SEM of three to five experiments, with three to five donors per experiment.

Fig. 3.

Neutrophil depletion during allogeneic pregnancy leads to abnormal placentation. Balb/C males were mated with C57 BL/6 females. After identification of vaginal plugs, circulating maternal neutrophils were depleted using a monoclonal neutralizing antibody, or some pregnant females were treated with isotype control antibody (both antibodies at 50 μg i.v.), at days 5 and 8, and pregnant animals were killed at day 12. (A) Absolute numbers of Ly6G+ neutrophils, CD4+ T cells, and CD4+FOXP3+ Tregs in the draining para-aortic lymph nodes. *P < 0.05; **P < 0.01 compared with isotype control-treated animals. (B) Crown-to-rump measurements obtained to determine embryo lengths. (C) Differences in placenta diameter from isotype control and neutrophil-depleted mice. *P < 0.05; **P < 0.01 compared with isotype control-treated animals. (D) Representative images of embryos and placentas from isotype control and neutrophil-depleted animals. (Scale bar: 2.5 mm.) (E) H&E staining of placentas from two groups of mice to examine the organization of placental layers (dotted lines). Gi, giant cell trophoblasts; Db, decidua basalis; Jz, junctional zone; Lz, labyrinthine zone. (Scale bar: 1,000 μm.) (F) Immunofluorescent staining of placentas for CD31 (red), trophoblasts (cytokeratin-7; green), and cell nuclei with DAPI (blue). Dotted lines indicate placental layers as described in E. Yellow dotted square indicates zoomed-in section of the Db layer to more closely examine trophoblast invasion. Invasion of trophoblasts into the maternal layer was quantified using ImageJ software. **P < 0.01 compared with isotype control. In all cases, data are mean ± SEM of two to three experiments with three to four animals per group.

Fig. 4.

Human neutrophils induce Tregs via apoptotic bodies. (A) Control- and E3P-treated (Fig. 1) neutrophils were incubated with or without GMCSF (50 ng/mL) in coculture with autologous T cells. After a 24-h coculture, cells were stained with annexin-V and PI. Based on forward/side scatter, neutrophils were gated, and the degree of apoptosis (**P < 0.01) was compared with control. (B) T cells were stained for CD4 (yellow) MRP8 (red), and DAPI (blue) and analyzed by confocal microscopy. ***P < 0.001 compared with without GMCSF. (Scale bar: 7 μm.) (C) Flow cytometry analyses of niT cell induction in the presence or absence of GMCSF with respect to CD45RO, FOXP3, CD127, and GARP. ***P < 0.001. In all cases, data are mean ± SEM of four to five experiments with more than three distinct donors per experiment.

Fig. 5.

Neutrophils induce FOXP3 to T cells via the transfer of FOXO1. (A) The effect of recombinant human AnxA1 on niT cell induction was assessed by treating either T cells alone (white bars) or in coculture following direct treatment of neutrophils (green bar). **P < 0.01 compared with neutrophil coculture in the absence of AnxA1 treatment. (B) Neutrophils and splenic T cells were isolated from male WT and AnxA1 KO mice. Neutrophils were treated with E3P (green lined bars) or left untreated (white bars) and then cocultured (at a 1:1 ratio) for 3 d with autologous or counterpart (WT or KO) T cells, stimulated with 2 μg/mL anti-CD3 and anti-CD28 antibodies. T cells were stained for CD4 and FOXP3. ***P < 0.001 compared with T cells in the absence of neutrophils. (C) Neutrophils from male donors were treated with progesterone and estriol (E3P; 100 ng/mL each) for 30 min, washed, and stained with DAPI and mouse anti-human FOXO1 (10 μg/mL) in 0.1% Triton X-100 to gently permeablize the cells, followed by the addition of secondary Alexa Fluor 555 antibody. FOXO1 expression by neutrophils was quantified by confocal microscopy. (Scale bar: 7 μM.) **P < 0.001 compared with control. (D) FOXO1 protein contained by E3P neutrophils was labeled with a fluorochrome-conjugated antibody as in B. After an 18-h incubation, FOXO1-labeled apoptotic bodies were collected and cocultured with autologous T cells for 3 d in the presence of 2 μg/mL soluble anti-CD3 and anti-CD28 antibodies. FOXO1 transfer to T cells was quantified by confocal microscopy and measured as corrected total cell fluorescence (CTCF). (Scale bar: 3.5 μM.) ***P < 0.001 compared with control. (E) T cells, cultured as described in D, were stained for CD4 (pink), FOXP3 (green), and FOXO1 (red) expression and then counterstained with DAPI (purple). The intensity of FOXP3 and FOXO1 expression in the same cell was determined using ImageStreamX analysis by comparing bright detail similarity (median fluorescence) of the two. **P < 0.01 compared with control. (Scale bar: 7 μm.) (F) The ability of E3P-treated neutrophils to induce Tregs was determined following neutrophil-specific inhibition of FOXO1. Log scale is shown. (G) T cells were cocultured with neutrophil apoptotic bodies as described in D. In addition, AnxA1 was neutralized on neutrophils before apoptotic body formation, and the ability of neutrophil-derived FOXO1 transfer to T cells was measured by confocal microscopy. Data are mean ± SEM of two experiments conducted with three donors per experiment.

Fig. 6.

Neutrophils from preeclamptic pregnancies fail to induce niT cells. (A) Peripheral blood neutrophils from patients with preeclampsia were stained for CD15, CD16, CD62L, CD11b, and AnxA1 to establish their phenotype compared with cells from age- and gestation-matched healthy pregnant women (SI Appendix, Table S1). Data are mean ± SEM of 15 healthy and 10 preeclamptic samples. ***P < 0.001; **P < 0.01 using Student’s t test for each marker. (B) Comparison of gestation-adjusted birth weight percentiles for babies born from normotensive mothers (n = 15; mean, 52nd percentile; range, 44th–68th percentile) and preeclamptic mothers (n = 10; mean, 22nd percentile; range, 11th–35th percentile). (C) Extent of apoptosis of peripheral blood neutrophils from healthy and preeclamptic samples measured by annexin-V and PI labeling. Data are mean ± SEM of 15 healthy and 10 preeclamptic samples. **P < 0.01 compared with healthy pregnancy. (D) T cells from healthy and preeclamptic pregnancies were cocultured for 5 d with autologous neutrophils (no additional hormones added) and subsequently stained for DAPI (blue), AnxA1 (red), and MRP8 (green). The amount of neutrophil protein transferred to T cells was analyzed by confocal microscopy, and colocalization of AnxA1 and MRP8 was analyzed using Mander’s overlap coefficient indices. Data are mean ± SEM of 15 healthy and 10 preeclamptic samples.***P < 0.001. (Scale bar: 7 μm.) (E) Neutrophils from healthy and preeclamptic donors were cocultured with autologous T cells as described in B. T cells were then stained for DAPI (blue) and FOXO1 (red) and analyzed by confocal microscopy to establish the efficiency of FOXO1 transfer to the T cells, as measured by CTCF. Data are mean ± SEM of 15 healthy and 10 preeclamptic samples. ***P < 0.001. (Scale bar: 7 μm.) (F) Neutrophils from healthy and preeclamptic patients were cocultured with autologous T cells at a 1:1 ratio for 5 d in the presence of 2 μg/mL soluble anti-CD3 and anti-CD28 antibodies. T cells were stained for niTreg markers as in Fig. 2. In addition, neutrophils from preeclamptic patients were treated with exogenous E3P and then cocultured with T cells (blue lined bar). Data are mean ± SEM of three experiments with 15 healthy and 10 preeclamptic samples; **P < 0.01.

Fig. 7.

Knockdown of FOXO1 in neutrophils leads to abnormal pregnancy. (A) Balb/C males were mated with C57 BL/6 females and neutrophil-depleted as described in Fig. 3. Bone marrow progenitors were transduced to knock down FOXO1, followed by culture with 100 ng/mL GCSF for neutrophil differentiation. Then 3 × 106 transduced neutrophils were injected i.v into the tail vein at days 6 and 9 of pregnancy, and mothers were killed at day 12. Absolute numbers of Ly6G+ neutrophils, CD4+ T cells, and CD4+FOXP3+ Tregs in the uterine-draining lymph nodes were counted. n = 3 mice per group from three distinct timed matings. P < 0.01. (B) Bone marrow from CD45.1+ donor mice was transduced with nonspecific neutrophils reconstituted as described above, and the presence of donor CD45.1 Ly6G+ (clone Gr-1) F4/80− neutrophils was analyzed by flow cytometry after 16 h (contour plots) and after 72 h (day 12) postinjection. (C) The presence of transferred CD45.1 donor neutrophil material to the CD4+FOXP3+ population was also analyzed by flow cytometry, and its origin was confirmed by Ly6G staining, compared with CD45.2+ cells. n = 3–4 mice per group from three distinct timed matings. (D and E) Embryo size (D) and placenta diameter (E) were measured as described in Fig. 3. n = 22 embryos and placentas from nonspecific, n = 16 from FOXO1 30, and n = 12 from FOXO1 42 from three distinct pregnancies per group. **P < 0.01 compared with nonspecific. (F) Representative images of embryo and placenta of all three groups. (Scale bar: 2.5 mm.) (G) Representative immunofluorescence images of placentas for CD31 (red), trophoblasts (cytokeratin-7; green), and cell nuclei with DAPI (blue). Dotted lines indicate placental layers as described in E. The yellow dotted square indicates the zoomed-in section of the Db layer to more closely examine trophoblast invasion. Invasion of trophoblasts into the maternal layer was quantified using ImageJ software. n = 3 placentas from each group. *P < 0.05 compared with nonspecific. In A and F, data are mean ± SEM; in B and C, the line indicates the mean.