Docosahexaenoic acid prevents dendritic cell maturation, inhibits antigen-specific Th1/Th17 differentiation and suppresses experimental autoimmune encephalomyelitis

Abstract

Docosahexaenoic acid (DHA), the most abundant essential n-3 polyunsaturated fatty acid in the CNS, emerged recently together with eicosapentaenoic acid (EPA) and DHA/EPA metabolic derivatives as a major player in the resolution of inflammation. Protective anti-inflammatory effects of DHA were reported in clinical studies and animal models of colitis, sepsis, and stroke. Here we report for the first time a beneficial effect of dietary n-3 fatty acids in experimental autoimmune encephalomyelitis (EAE), a model for human multiple sclerosis. In the present study we investigated the effects of DHA on the function of bone marrow-derived dendritic cells (DC) in CD4(+) T cell stimulation and differentiation. Pretreatment of DC with DHA prevented LPS-induced DC maturation, maintaining an immature phenotype characterized by low expression of costimulatory molecules and lack of proinflammatory cytokine production (IL-12p70, IL-6, and IL-23). DHA-treated DC were poor stimulators of antigen-specific T cells in terms of proliferation and Th1/Th17 differentiation. This was associated with an increase in p27(kip1), a cell cycle arresting agent, and with decreases in Tbet, GATA-3, and RORγt, master transcription factors for Th1, Th2, and Th17. In contrast, T cells co-cultured with DC-DHA express higher levels of TGFβ and Foxp3, without exhibiting a functional Treg phenotype. Similar to the in vitro results, the beneficial effect of DHA in EAE was associated with reduced numbers of IFNγ- and IL-17-producing CD4(+) T cells in both spleen and CNS.

Fig. 1. DHA prevents CCR7 expression and cytokine production in LPS-treated DC

(A) CD11c⁺ DC were treated with 50 µM DHA for 24h, followed by LPS (0.1 µg/ml) for 24h and CCR5 (upper panel) and CCR7 (middle panel) expression was determined by real time RT-PCR. Migration of 1×10⁵ DC toward 100 ng/ml CCL 19 was determined in a 4h Transwell chemotaxis assay as described in Methods (lower panel). (B) CD11c⁺ DC (1×10⁶/ml for IL-12p70 and IL-6, and 2×10⁶/ml for IL-23 and IL-10) were cultured in the presence or absence of 50 µM of DHA for 24h, followed by 0.1µg of LPS treatment for 12h (for IL-23) or 24h (for IL-12p70, IL-6 and IL-10). Supernatants were subjected to ELISA. Data represent the mean +/− SD of three experiments performed in triplicate. (C) CD11c⁺ DC were preincubated with 50 µM DHA for 24h, followed by treatment with 0.1 µg LPS for 24h. Cells were stained with Propidium Iodide (PI) and Annexin V and apoptosis was analyzed by flow cytometry (quadrant values represent percentage of cells). One representative experiment of three is shown. Med- medium control.

Fig. 2. DC-DHA are poor stimulators of antigen specific CD4⁺ T cells

(A and B) CD11c⁺ DC from B10.A mice (A) or C57BL/6 (B) were treated with (DC-DHA) or without (DC) 50 µM DHA for 24 h, stimulated with LPS and pulsed with 5 µM PCCF (pigeon cytochrome c fragment), 50 µg/ml MOG_35–55 (myelin oligodendrocyte glycoprotein), or PLP (proteolipid protein) (20 µg/ml; nonspecific Ag) for an additional 24h, followed by extensive washing. Various numbers of DC-DHA or DC were cultured with TCR Tg CD4⁺ T cells (2×10⁵ cells/well) (PCCF-specific in A and MOG-specific in B) in 96-well plates for 3 days. [³H]-thymidine (1µCi per well) was added and incorporation was measured after 16h. (C) DC from C57BL/6 mice were cultured with MOG_35–55 specific CD4⁺ T cells at 1/20 ratio. Three days later, supernatant was collected and IL-2 production was determined by ELISA. (D) The apoptotic status of T cells was assessed using flow cytometry (annexin V/PI staining) after 3 days of co-culture and quadrant values represent percentage of cells. One representative experiment of three is shown. (E) DC and DC-DHA were cultured with MOG-specific CD4⁺ T cells at 1/20 ratio. T cells were collected after 0 h, 24 h, 48h, and 72h and p27(kip1) expression was detected by real time RT-PCR. * P<0.05, compared with DC-T cells co-cultures. (F) Cell cycle analysis was performed on activated CD4⁺T cells after 3 days of co-culture with DC or DC-DHA pulsed with MOG. The changes in cell cycle were quantified by flow cytometry after PI staining as described in Methods. The experiment was performed three times, and the ratios of cells in G0/G1, S, and G2/M phase were expressed as mean ± SD. * P<0.05, compared with DC-T cells co-cultures.

Fig. 3. DC-DHA affects CD4⁺ T cell differentiation

DC from C57BL/6 mice were treated with or without 50 µM DHA for 24h, followed by 0.1 µg LPS and pulsing with MOG_35–55 for another 24h and extensive washing. DC and DC treated with DHA (DC-DHA) were cultured with MOG_35–55-specific CD4⁺ T cells at 1/20 ratio. 24 and 48h later, T cells were collected, the expression of Tbet (A), RORC (B), GATA-3 (C) and Foxp3 (D, left panel) were assessed by real time RT-PCR. Three days later, supernatants were collected and the production of IFNγ and IL-17 was determined by ELISA (A and B). T cells were collected and subjected to real time RT-PCR to detect expression of TGFβ (D, middle panel). Foxp3⁺ expression was analyzed by flow cytometry in gated CD4⁺CD25⁺ T cells. (D, right panels). One representative experiment of three is shown.

Fig. 4. DC-DHA activated CD4⁺T cells are not anergic or suppressive

(A) Activated T cells (T_DC or T_DC-DHA) were generated from MOG_35–55-specific CD4⁺ T cells cultured with DC or DC treated with docosahexaenoic acid (DC-DHA) pulsed with MOG_35–55 and activated with LPS. T_DC or T_DC-DHA (2×10⁵ cells/well) were cultured with different number of regular DC stimulated with LPS and pulsed with MOG_35–55. Three days later, proliferation was measured. (B) Activated T cells were generated from MOG-specific CD4⁺ T cells cultured with DC or DC-DHA in the presence or absence of 2 ng/ml TGFβ plus 50U IL-2. The Foxp3⁺ expression was analyzed in the same way as in Fig 3 (D). (C) 1×10⁵ cells/well activated T cells (T_DC, T_DC-DHA, T_DC+TGFβ and T_DC-DHA+TGFβ generated from naïve MOG-specific CD4+ T cells cultured with DC, DC-DHA, DC+TGFβ and DC-DHA+TGFβ, respectively) were co-cultured with CFSE-labeled syngeneic MOG-specific naïve CD4⁺ T cells (Tn) (1×10⁵ cells/well) in the presence of DC (1×10⁴ cells/well) pulsed with 50 µg/ml MOG_35–55. T cell proliferation was determined by CFSE dilution using FACS. One representative experiment of three is shown.

Fig. 5. Dietary DHA suppresses experimental autoimmune encephalomyelitis

C57BL/6 mice were fed a control (n=13) or DHA diet (n=13) for 5 wks. Mice were immunized with MOG _35–55 as described in Methods. Clinical scores and weight were followed daily for 60 days. At the end of the observation period three mice had died in the control group and one in the DHA group. (A) Kinetics of mean clinical score; (B) Percentage change in weight. * p<0.05, compared with control group.

Fig. 6. Dietary DHA inhibits Th1 and Th17 differentiation and reduces the numbers of CNS infiltrating Th1, Th17 and total CD4⁺ T cells

C57BL/6 mice fed a control or DHA diet were immunized with MOG_35–55 as described in Methods. (A) Splenocytes (2×10⁶/ml) isolated from spleen on day 11 post immunization (before disease onset) were cultured ex vivo in the presence of 50 µg MOG_35–55 for 3 days. Proliferation was determined by [³H]-thymidine incorporation. Supernatants were subjected to ELISA for IL-17 and IFNγ. One representative experiment out of three is shown. (B–C) Intracellular cytokine staining of FITC-gated CD4⁺ T cells isolated from brain (B) or spinal cord (C) of mice at peak of disease (day 18) (cells pooled from 3 animals) (left panels show flow cytometric analysis; right panels show calculated numbers of cytokine-producing cells). Data are representative of two independent experiments.