Human monocytes undergo functional re-programming during differentiation to dendritic cell mediated by human extravillous trophoblasts

Abstract

Maternal immune adaptation is required for a successful pregnancy to avoid rejection of the fetal-placental unit. Dendritic cells within the decidual microenvironment lock in a tolerogenic profile. However, how these tolerogenic DCs are induced and the underlying mechanisms are largely unknown. In this study, we show that human extravillous trophoblasts redirect the monocyte-to-DC transition and induce regulatory dendritic cells. DCs differentiated from blood monocytes in the presence of human extravillous trophoblast cell line HTR-8/SVneo displayed a DC-SIGN(+)CD14(+)CD1a(-) phenotype, similar with decidual DCs. HTR8-conditioned DCs were unable to develop a fully mature phenotype in response to LPS, and altered the cytokine secretory profile significantly. Functionally, conditioned DCs poorly induced the proliferation and activation of allogeneic T cells, whereas promoted CD4(+)CD25(+)Foxp3(+) Treg cells generation. Furthermore, the supernatant from DC and HTR-8/SVneo coculture system contained significant high amount of M-CSF and MCP-1. Using neutralizing antibodies, we discussed the role of M-CSF and MCP-1 during monocyte-to-DCs differentiation mediated by extravillous trophoblasts. Our data indicate that human extravillous trophoblasts play an important role in modulating the monocyte-to-DC differentiation through M-CSF and MCP-1, which facilitate the establishment of a tolerogenic microenvironment at the maternal-fetal interface.

Figure 1. EVTs redirect the differentiation of DC to a CD1a⁻CD14⁺ phenotype.

(A) CD14⁺ monocytes were cultured for 5 d with GM-CSF and IL-4 in the absence (imDC) or presence of seminal plasma (H-imDC). The HTR8:CD14⁺ cells ratio of 1:10 was used in coculture system. Cells obtained from day3 and day5 of culture were analyzed by flow cytometry. The expression of CD1a and CD14 was displayed gated on CD11c⁺ cells. Dot plots from a representative experiment (n = 8) are shown. (B)The percentages of CD1a⁺ and CD14⁺ cells in DCs in HTR-8/ monocytes cocultures at ratios ranging from 1:2 to 1:50 were assessed by flow cytometry at day 5. Experiments were performed using monocyte from 8 distinct donors. (C) The phenotype of imDC and H-imDC (the ratio of HTR-8:monocyte is 1:10, day5) were analyzed by flow cytometry. Representative histograms from at least 5 individual experiments are shown. (isotype control Ab: gray filled histogram; indicated mAbs: open histogram) (D,E) The summarized results of surface markers expression are shown as the percentage (D) and the mean fluorescence intensity (E). The bar graphs represent mean ± EM of five individual experiments (*P < 0.05; **P < 0.01)

Figure 2. DCs differentiated in the presence of EVTs show reduced CD83 and CCR7 expression and altered cytokine profile upon stimulation by inflammatory stimuli.

At day 5, cells were collected, washed and induced to mature by exposure to 100 ng/ml LPS for 48 h in the absence of HTR8, and analyzed at day 7 for CD83 and CCR7 expression. (A) Histograms from a representative experiment are showed (n = 5). isotype control Ab: gray filled histogram; indicated mAbs: open histogram (B) Graph bars indicating the percentages of CD83⁺ and CCR7⁺ cells (mean ± SEM, n = 5) are shown. (*P < 0.05) (C) Supernatants were collected from the indicated culture and cytokine production was evaluated by ELISA. Results represent mean ± SEM of 5 experiments (*P < 0.05).

Figure 3. EVT-conditioned DCs show reduced capacity to prime naive T cell proliferation and activation, but enhanced capacity to induce Tregs generation.

(A) DCs and H-DCs were harvested and washed after treated with LPS (100 ng/ml, 48 h), then seeded with CFSE-labeled allogeneic naive CD4⁺T lymphocytes at a DC/lymphocyte ratio of 1:5 in the present of IL-2(50 U/ml). The dilution of CFSE staining was analyzed at day 5 by flow cytometry. T cells cultured alone with or without PMA (10 mg/ml) were used as positive and negative controls, respectively. Histograms from a representative experiment (n = 5) are shown. (B) Graphical representation of data in (A) is shown as mean ± SEM from 3 to 5 independent experiments with consistent results. (C) Mature DCs and H-DCs were incubated with allogeneic naive CD4⁺T cells using a DC/lymphocyte ratio of 1:2. At day 5, cells were harvested for surface staining of CD4 and CD69. For intracellular staining of IFN-γ, cells were resuspended in fresh medium, stimulated with PMA/ionomycin for 6 h and treated with brefeldin A for the last 4 h of cultures. The representative flow cytometry pictures are depicted from a representative experiment (n = 5). Gray filled histograms, isotype control Ab staining; Solid lines, anti-CD69 Ab staining. (D) Summary of the proportion of CD69 (upper) and IFN-γ (lower) on CD4⁺T cells. The results are shown as mean ± SEM from 5 independent experiments. (E) Mature DCs and H-DCs were recovered and then cultured for 5 days with isolated naive CD4⁺T cells in the present of IL-2(50U/ml). Cells were stained with anti-CD4, anti-CD25 and anti-Foxp3. Flow cytometry dot plots showing that CD25⁺Foxp3⁺ Tregs increased in the CD4⁺T cell population were shown. (F) The percentage of CD25⁺Foxp3⁺ cells in the CD4⁺T cell population was summarized in the bar graph(mean ± SEM, n = 5). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4. EVTs modulate the phenotype of DCs though soluble factors.

CD14⁺ monocytes were cultured with GM-CSF and IL-4 in the absence (DC)or presence of HTR8(H-DC), or separated with HTR8 in transwell chamber(trans-DC). (A) Cells were harvested at day 5 and examined for the expression of CD1a and CD14. Representative dot plots are shown. (B) The phenotypes of the indicated DCs were analyzed by flow cytometry. Representative histographs from 5 individual experiments were depicted.

Figure 5. High concentration of MCP-1 and M-CSF in EVT-DC coculture system.

CD14⁺ monocytes were cultured with GM-CSF and IL-4 in the absence (DC)or presence of HTR8(H-DC), or separated with HTR8 in a transwell system(trans-DC). The cell-free supernatants of different cultures were collected at day 5 of differentiation process. The concentration of MCP-1, IL-6, IL-10, G-CSF and IFN-γ were analyzed by the Bio-Plex Protein Array system. The production of M-CSF was examined by ELISA. Data are the mea ± SEM from 3 independent experiments. *p < 0.05, ***p < 0.001.

Figure 6. The modulating effect of EVTs on DCs is partially reversed by anti–MCP-1 and anti-M-CSF neutralizing Ab.

Neutralizing Abs against MCP-1 or M-CSF or both were added in HTR8-DC coculture system during the differentation of DCs. The isotype Abs were added as control at the same time. Cells from different culture were harvested at day 5, and the expression of CD14 (A), CD1a (B) and DC-SIGN (C) by the indicated DCs were summarized. The production of IL-10 (D) and IL-12 (E) by the indicated DCs upon LPS stimulation were measured and shown in the bar graph. Graphs show mean ± SEM of 5 individual experiments. *p < 0.05, **p < 0.01.