Human milk oligosaccharides promote immune tolerance via direct interactions with human dendritic cells

Abstract

Human milk oligosaccharides (HMOS) are a complex mixture of bioactive components supporting the immune development of breastfed-infants. Dendritic cells (DCs) play a central role in the regulation of immune responses, being specialized in antigen presentation and driving T-cell priming as well as differentiation. However, little is known about the direct effects of HMOS on human DC phenotypes and functions. Here, we report that HMOS mixture isolated from pooled human milk, induced semi-maturation of human monocytes-derived DCs (moDCs), and elevated levels of IL-10, IL-27 and IL-6 but not IL-12p70 and TNF-α. Consistently, HMOS-conditioned human moDCs promoted Treg generation from naïve CD4⁺ T cells. Interestingly, HMOS limited LPS-induced maturation of human moDCs, while maintained IL-10 and IL-27 secretion and reduced LPS-induced production of IL-12p70, IL-6 and TNF-α. Furthermore, HMOS+LPS-stimulated DCs induced a higher frequency of Tregs and increased IL-10 production, while a reduction in Tbet+Th1 frequency and IFN-γ production was detected as compared to LPS-DCs. The regulatory effects of HMOS seemed to be mediated by interactions of HMOS with receptors, including but not limited to TLR4 and DC-SIGN on human moDCs. In conclusion, HMOS contain tolerogenic factors influencing human moDCs and thereby modulating the development of the neonatal immune system.

Keywords: DC-SIGN; IL-10; TLR4; human milk oligosaccharides; regulatory T cells (Treg).

Figure 1

Effects of HMOS on the maturation status of human moDCs in the absence or presence of LPS. (A) Gating strategy of CD14‐APC negative DCs. (B) Representative histograms and (C) (Median Fluorescence Intensity (MFI)) of co‐stimulatory molecules CD80‐PE, CD86‐PE, CD40‐PE‐Cy7, and HLA‐DR‐PerCP‐Cy5.5, and inhibitory molecules PD‐L1‐PE‐Cy7 and OX40L‐PerCP‐Cy5.5. expression of DCs treated by medium, LPS, HMOS (0.8, 2, 5 mg/mL), and HMOS+LPS. Open histograms represent isotype mAb staining. The y‐axis of the column bar graphs shows the relative expression of surface markers obtained by setting medium control as 1‐fold within one experiment for each donor. (D) mRNA levels of co‐stimulatory molecules CD80, CD86, CD40, and HLA‐DRA, HLA‐DRB, and inhibitory molecule PD‐L1 expression of DCs treated by medium, LPS, HMOS (5mg/ml), and HMOS+LPS. The y‐axis of the column bar graphs shows the relative mRNA expression obtained by setting medium control as 1‐fold within one experiment for each donor. Immature DCs were generated from human PBMC‐derived monocytes, on day 6 immature DCs were stimulated with either medium (control), or HMOS (0.8, 2, 5 mg/mL) in the presence or absence of 100 ng/mL LPS. DCs were collected at 24 h for flow cytometry analysis or at 16 h for mRNA levels by qPCR analysis. Results are presented as mean ± SEM (7‐9 independent experiments (1‐2 donors/experiment) for flow cytometry analysis, and 2 independent experiments (2‐3 donors/experiment) for QPCR analysis). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001, paired Student's t‐test.

Figure 2

Effects of HMOS on cytokines release by human moDCs in the absence or presence of LPS. Production of (A) IL‐10, (B) IL‐27, (C) IL‐12p70, (D) IL‐4, (E) IL‐6, and (F) TNF‐α by DCs treated with medium, HMOS at 0.8, 2 and 5mg/mL in the presence or absence of 100 ng/mL LPS. mRNA expression of (G) IL‐10, (H) IL‐27, (I) IL‐12p40, (J) TGF‐β, (K) IL‐6, and (L) TNF‐α of DCs treated with medium, 5 mg/mL HMOS, LPS or 5 mg/mL HMOS+LPS. Immature DCs were stimulated with either medium (control), 100 ng/mL LPS (positive control), (0.8, 2 or 5 mg/mL) HMOS or HMOS in the presence of 100 ng/mL LPS. After 24 h, supernatants were collected and analyzed for IL‐10 (A), IL‐12p70 (B), IL‐6 (C), TNF‐a, and IL‐27 release by ELISA assays and cells were collected for QPCR analysis. Results are presented as mean ± SEM (3 independent experiments (2‐3 donors/experiment) for Elisa assays and 2 independent experiments (2‐3 donors/experiment) for QPCR analysis). For panel G‐L, the y‐axis of the column bar graphs shows the relative mRNA expression obtained by setting medium control as 1‐fold within one experiment and for each donor. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Paired Student's t‐test.

Figure 3

Effects of HMOS on the migratory marker's expression of human moDCs in the absence or presence of LPS. (A) Representative histograms of CCR7‐PerCP‐Cy5.5 and CXCR3‐FITC expression on the DCs treated by medium, LPS, HMOS (5 mg/ml), and HMOS (5 mg/ml) + LPS. Open histograms represent isotype mAb staining. MFI of (B) CCR7 and (D) CXCR3 expression on human DCs. mRNA expression of (C) CCR7 and (E) CXCR3 on human DCs. iDCs were generated and treated as described in the figure legend 1, after 24 h, DCs were analyzed by flow cytometry and qPCR for the expression of the migration markers CCR7 and CXCR3 at protein and mRNA levels, respectively. Results are presented as mean ± SEM (3 independent experiments (1–2 donors/experiment)). *p < 0.05, **p < 0.01, paired Student's t‐test.

Figure 4

Effects of HMOS‐conditioned human moDCs on the induction of Tregs and IL‐10 production by T‐cell. (A) Representative plots of CD4⁺CD25+Foxp3+ Tregs induced by control DCs treated with medium, LPS, HMOS (5 mg/mL), and HMOS (5 mg/mL) +LPS matured DCs. Gating strategy is shown in Supporting Information Fig. 4. (B) Percentage of CD25+Foxp3+ Tregs from CD4⁺ T‐cell 6–7 days’ post MLR assay. Stimulated DCs were collected at 24 h and co‐cultured with fresh isolated naïve CD4⁺ T‐cell in a 1:10 ratio. After 6–7 days of co‐culture, T‐cell was harvested and analyzed for the expression of CD4‐PerCP‐Cy5.5, CD25‐FITC and Foxp3‐PE‐Cy7 by flow cytometry. (C) IL‐10 secretion in the supernatant of MLR. Supernatant was collected for IL‐10 measurement by ELISA assay. Results are presented as mean ± SEM (4 independent experiments (2‐3 donors/experiment) for panel B, (3 independent experiments (1‐2 donors/experiment) for panel C), *p < 0.05, **p < 0.01, ****p < 0.0001, paired Student's t‐test.

Figure 5

Effects of HMOS‐conditioned human moDCs on the prime of Th1 and Th2 responses. (A) Representative plots of CD69+Tbet+ (left panels) and CD69+GATA3+ (right panels) CD4 T‐cell. Gating strategy is shown in Supporting Information Fig. 4. Percentage of (B) CD69+Tbet+ (Th1) and (C) CD69+GATA3+ (Th2) cells of the CD4⁺ T‐cell population. (D) Ratio of Th1 and Th2 cells. Production of (E) IFN‐γ and (F) IL‐4 by co‐cultured T‐cell. The expression of CD4‐PerCp‐Cy5.5, CD69‐FITC, Tbet‐PE‐Cy7, and GATA3‐PE was analyzed by flow cytometry. Results are presented as mean ± SEM, (4 independent experiments (2‐3 donors/experiment) for panel B‐D, (3 independent experiments (1‐2 donors/experiment) for panel E,F). *p < 0.05, **p < 0.01, paired Student's t‐test.

Figure 6

Suppressive capacity of HMOS‐conditioned human moDCs on activated responder CD4⁺ T‐cell proliferation. (A) Representative plots of T‐responder cell (Tresp) co‐cultured with CD4⁺T‐cell primed by control DCs, LPS treated DCs, HMOS (5 mg/ml) +LPS treated DCs, or HMOS (5 mg/ml) treated DCs at the ratio of 1:1. (B) The degree of Tresp proliferation after co‐culturing with CD4⁺T‐cell primed by different DCs. CD3/CD28 activated CFSE‐labelled responder CD4⁺T‐cell was co‐cultured with CD4⁺T‐cell primed by different DCs at ratio 4:1, 2:1, and 1:1 for 5 days. Proliferation of FITC‐positive cells was analyzed by flow cytometry and suppressive functionality was determined by comparing the proliferated CD4‐PerCP‐Cy5.5 positive T cell. Results are presented as mean ± SEM, (3 independent experiments (1‐2 donors/experiment). *p < 0.05, **p < 0.01, ***p < 0.01, ****p < 0.001, paired Student's t‐test.

Figure 7

Effects of HMOS on TLR4 and DC‐SIGN expression and activity. (A) DC‐SIGN‐PE and (B) TLR4‐PE‐Cy7 expression on human monocyte‐derived immature DCs at protein and mRNA levels assessed by flow cytometry and QPCR assays, respectively. The effects of blockage of TLR4 or/and DC‐SIGN receptors on the production of (C,E) IL‐10, (D,E) IL‐12p70, and (F) Tregs generation. iDCs were pre‐incubated with anti‐TLR4 or/and DC‐SIGN antibodies for 2h before adding HMOS or/and LPS treatment, supernatant was collected at 24 h for the measurement of IL‐10 and IL‐12p70 by Elisa assays, DCs were collected and co‐cultured with naïve CD4⁺ T‐cell as described above. Results are presented as mean ± SEM, (4 independent experiments (1‐3 donors/experiment). *p < 0.05, **p < 0.01, paired Student's t‐test.