Fig 1.
Fatty acid uptake by dendritic cells (DCs).
DC cultures were supplemented with fatty acids (50 μM); arachidonic acid (AA), docosahexaenoic acid (DHA) or oleic acid (OA) for 3 days and thereafter the cells were analyzed by gas chromatography. The proportion of (A) oleic acid, (B) arachidonic acid and (C) DHA of all lipid content in the cells. Black dots denote samples supplemented with fatty acid while white dots with black borders denote control (ethanol only). Horizontal solid black lines show median value. The median from the control group has been extended with a dotted line for easy comparison to the other groups. Statistical mean difference was compared to the control group. p-values: ** <0.01, *** <0.001, **** <0.0001.
Fig 2.
Distribution of dendritic cells (DCs) in fatty-acid supplemented cell cultures.
DC cultures were supplemented with fatty acids (50 μM); α-linolenic acid (ALA), arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA) or oleic acid (OA) for 3 days and thereafter DCs were analyzed by flow cytometry. (A) Dot plots showing gating strategy for CD11c+ CD11bneg (lower gate) and CD11c+ CD11b+ (upper gate) DCs based on FMO (fluorescence minus one) samples. To the right a representative sample, in the middle FMO for CD11b and to the right FMO for CD11c. (B) Proportion of CD11c+ DCs, both CD11bneg and CD11b+, in fatty-acid supplemented cell cultures. (C) Proportion of CD11b+ cells within the CD11c+ population shown in Fig 2B. Black bars denote samples supplemented with fatty acid while white bar with black border denotes control (ethanol only). For each group n = 8. Error bars show standard deviation.
Fig 3.
Expression of MHC class II and costimulatory molecules on fatty-acid supplemented dendritic cells (DCs).
DC cultures were supplemented with fatty acids (50 μM); α-linolenic acid (ALA), arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA), oleic acid (OA) or ethanol only (Ctrl) for 3 days and thereafter analyzed by flow cytometry. (A) Mean fluorescence intensity (MFI) of IAd, CD80, CD86 and CD40. Left panel show MFI of CD11c+CD11bneg and right panel on CD11c+CD11b+ DCs. Each dot represents one individual. Black dots denote samples supplemented with fatty acid while white dots with black borders denote control (ethanol only). Horizontal solid black lines show median value. The median from the control group has been extended with a dotted line for easy comparison to the other groups. Statistical mean difference was compared to the control group. Data are representative of two independent experiments. (B) Representative histograms for CD80 and CD86. (C) Representative histograms for CD40 and IAd. In the histograms FMO samples are shown with grey color, DHA samples are transparent with solid black border and control samples (ethanol only) are transparent with dotted black border. p-values: ** <0.01, *** <0.001, **** <0.0001.
Fig 4.
Expression of programmed death ligand-1 (PDL-1) and CD83 on fatty-acid supplemented dendritic cells (DCs).
DC cultures were supplemented with fatty acids (50 μM); α-linolenic acid (ALA), arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA), oleic acid (OA) or ethanol only (Ctrl) for 3 days and thereafter analyzed by flow cytometry. (A) Mean fluorescence intensity (MFI) of PDL-1 and CD83. Left panel show MFI of CD11c+CD11bneg and right panel of CD11c+CD11b+ DCs. Each dot represents one individual. Black dots denote samples supplemented with fatty acid while white dots with black borders denote control (ethanol only). Horizontal solid black lines show median value. The median from the control group has been extended with a dotted line for easy comparison to the other groups. Statistical mean difference was compared to the control group. Data are representative of two independent experiments. (B) Representative histogram of each marker. In the histograms FMO samples are shown with grey color, DHA samples are transparent with solid black border and control samples (ethanol only) are transparent with dotted black border. p-values: ** <0.01, *** <0.001, **** <0.0001.
Fig 5.
Soluble CD83 (sCD83) and prostaglandin E2 (PGE2) in dendritic cell (DC) culture supernatant.
(A) Levels of sCD83 (pg/ml). (B) Levels of PGE2 (pg/ml). DC cultures were supplemented with fatty acids (50 μM); arachidonic acid (AA), docosahexaenoic acid (DHA), oleic acid (OA) or ethanol (Ctrl) for 3 days and supernatants analyzed by ELISA. Black dots denote samples supplemented with fatty acid while white dots with black borders denote control (ethanol only). Horizontal solid black lines show median value. The median from the control group has been extended with a dotted line for easy comparison to the other groups. Statistical mean difference was compared to the control group. Data are representative of one experiment. The limit of detection (LoD) is shown with dashed black lines. p-values: * <0.05, ** <0.01, *** <0.001.
Fig 6.
Activation of T cells by fatty-acid supplemented dendritic cells (DCs).
T cells were co-cultured for 6 days with DCs previously supplemented with fatty acids (50 μM); α-linolenic acid (ALA), arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA), oleic acid (OA) or ethanol only (Ctrl); and thereafter analyzed by flow cytometry. (A) Gating strategy for lymphocytes (upper row), DO11.10+ T cells (middle row) and divided DO11.10+ T cells; CellTrace™ Violetlow (bottom row). (B) Representative dot plots showing dividing and activated (CD25+ and CD69+) DO11.10+ cells for arachidonic acid (AA), DHA and control samples. (C) Proportion divided, (D) CD25+ and (E) CD69+ of DO11.10+ T cells. Each dot represents one individual. Black dots denote samples supplemented with fatty acid while white dots with black borders denote control (ethanol only). Horizontal solid black lines show median value. The median from the control group has been extended with a dotted line for easy comparison to the other groups. Statistical mean difference was compared to the control group. p-values: ** <0.01, *** <0.001, **** <0.0001.
Fig 7.
Viability of T cells after co-culture.
T cells were analyzed with 7AAD and Annexin V after 6 days of co-culture with dendritic cells (DCs) previously supplemented with fatty acids (50 μM); α-linolenic acid (ALA), arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA), oleic acid (OA) or ethanol only (Ctrl). (A) Stepwise gating procedure to eliminate debris. (B) Representative dot plots showing gating of live cells (Annexin V- 7AAD-), apoptotic cells (Annexin V+ 7AAD-) and necrotic and late apoptotic cells (Annexin V+ 7AAD+). (C) Proportion of live, (D) necrotic and late apoptotic and (E) apoptotic CD4+ T cells. Each dot represents one individual. Black dots denote samples supplemented with fatty acid while white dots with black borders denote control (ethanol only). Horizontal solid black lines show median value. The median from the control group has been extended with a dotted line for easy comparison to the other groups. Statistical mean difference was compared to the control group. Data are representative of two independent experiments. p-values: ** <0.01, *** <0.001, **** <0.0001.
Fig 8.
Co-expression of FoxP3 with Helios, CTLA-4 and PD-1.
T cells were co-cultured for 6 days with dendritic cells (DCs) previously supplemented with fatty acids (50 μM); arachidonic acid (AA), docosahexaenoic acid (DHA), oleic acid (OA) or ethanol only (Ctrl); and thereafter analyzed by flow cytometry. (A) Representative dot plots from control samples showing staining of FoxP3+ and indicated markers (left column). In each plot double-positive-cells are found in the upper right quadrant and double-negative cells in the lower left quadrant. FMO samples are shown in the right column. (B) Proportion of double-positive (right column), FoxP3+ samples (middle column) and FoxP3- samples (left column) in CD4+ cells. Each dot represents one individual. Black dots denote samples supplemented with fatty acid while white dots with black borders denote control (ethanol only). Horizontal black solid lines show median value. The median from the control group has been extended with a dotted line for easy comparison to the other groups. Statistical mean difference was compared to the control group. Data are representative of two independent experiments. p-values: * <0.05, ** <0.01, *** <0.001, **** <0.0001.
Fig 9.
Correlation between proliferation and expression of regulatory markers on T cells.
T cells were co-cultured for 6 days with dendritic cells (DCs) previously supplemented with fatty acids (50 μM); arachidonic acid (AA), docosahexaenoic acid (DHA), oleic acid (OA) or ethanol only (Ctrl); and thereafter analyzed by flow cytometry. Proportion of divided cells was correlated to proportion of T cells double-positive for FoxP3 and (A) Helios, (B) CTLA-4, and (C) PD-1. All samples, regardless of stimuli, were used in the same correlation analysis. The proportion of divided cells is different in (C) compared to (A) and (B) since this staining was performed in another experiment. r value: non-parametric Spearman correlation.
Fig 10.
Cytokines in co-culture supernatant.
Levels of (A) IL-10 and (B) IFNγ in DC: T cell co-culture supernatants. T cells were co-cultured for 6 days with dendritic cells (DCs) previously supplemented with fatty acids (50 μM); arachidonic acid (AA), docosahexaenoic acid (DHA), oleic acid (OA) or ethanol only (Ctrl). Each dot represents one individual. Horizontal black solid lines show median value. The median from the control group has been extended with a dotted line for easy comparison to the other groups. Statistical mean difference was compared to the control group. Data are representative of two independent experiments. p-values: * <0.05, ** <0.01, *** <0.001, **** <0.0001.
Fig 11.
Proliferation of CD4+ T cells with and without blocking of CD83 or PD-1 during DC: T cell co-culture.
T cells were co-cultured for 6 days with DCs previously supplemented with fatty acids (50 μM); arachidonic acid (AA), docosahexaenoic acid (DHA), oleic acid (OA) or ethanol only (Ctrl) without (-, No blocking) or with 10 μg/ml purified CD83-antibody (α-CD83) or PD-1 antibody (α-PD-1); and thereafter analyzed by flow cytometry. (A) Proportion of T cells that has proliferated (CellTrace™ CFSElow), with (+) or without (-) PD-1 blocking. (B) Proportion of T cells that has proliferated (CellTrace™ CFSElow), with (+) or without (-) CD83 blocking. Each dot represents one individual. Black dots denote samples supplemented with fatty acid while white dots with black borders denote control (ethanol only). Horizontal black solid lines show median value. Statistical mean difference, for each fatty acid or control, was compared between no blocking and blocking. Data are representative of two experiment. p-values: * <0.05, ** <0.01, *** <0.001, **** <0.0001.