Dendritic cell activation, phagocytosis and CD69 expression on cognate T cells are suppressed by n-3 long-chain polyunsaturated fatty acids

Abstract

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are bioactive n-3 long-chain polyunsaturated fatty acids (LCPUFAs) in fish oil that exert immunosuppressive effects. A significant amount of literature shows that n-3 LCPUFAs suppress dendritic cell (DC) function in vitro; however, few studies have determined if the effects are emulated at the animal level. In this study, we first focused on the functional consequences of 5% (weight/weight) fish oil on splenic CD11c(+) DCs. Administration of n-3 LCPUFAs, modelling human pharmacological intake (2% of total kcal from EPA,1·3% from DHA), to C57BL/6 mice for 3 weeks reduced DC surface expression of CD80 by 14% and tumour necrosis factor-α secretion by 29% upon lipopolysaccharide stimulation relative to a control diet. The n-3 LCPUFAs also significantly decreased CD11c(+) surface expression and phagocytosis by 12% compared with the control diet. Antigen presentation studies revealed a 22% decrease in CD69 surface expression on transgenic CD4(+) T lymphocytes activated by DCs from mice fed fish oil. We then determined if the functional changes were mechanistically associated with changes in lipid microdomain clustering or plasma membrane microviscosity with n-3 LCPUFAs, as reported for B and T lymphocytes. Fish oil administration to mice did not influence cholera-toxin induced lipid microdomain clustering or microviscosity, even though EPA and DHA levels were significantly elevated relative to the control diet. Overall, our data show that n-3 LCPUFAs exert immunosuppressive effects on DCs, validating in vitro studies. The results also show that DC microdomain clustering and microviscosity were not changed by the n-3 LCPUFA intervention used in this study.

Figure 1

CD80 surface expression and tumour necrosis factor-α (TNF-α) secretion from lipopolysaccharide (LPS) -stimulated dendritic cells (DCs) is lowered with n-3 long-chain polyunsaturated fatty acids (LCPUFAs). DCs were isolated from C57BL/6 mice consuming a control or n-3 LCPUFA diet and stimulated for 24 hr with LPS. (a) Sample flow cytometry histograms of CD80 and MHC class II. (b) The mean fluorescence intensity (MFI) of CD80 in the absence and presence of LPS stimulation. (c) TNF-α, interleukin-10 (IL-10), IL-12p40, IL-6 and transforming growth-factor-β (TGF-β) cytokine secretion was measured after 24 hr of LPS stimulation. Data are from 6 to 10 independent experiments. Asterisks indicate significance from control: *P < 0·05, ***P < 0·001.

Figure 2

Dendritic cell (DC) CD11c⁺ surface expression and phagocytosis are suppressed by n-3 long-chain polyunsaturated fatty acids (LCPUFAs). (a)The surface expression of CD11c on DCs isolated from C57BL/6 mice consuming a control or n-3 LCPUFA diet. (b) Sample flow cytometry histograms of fluorescence uptake of Escherichia coli bioparticles. (c) The amount of E. coli bioparticles taken up as indicated by the mean fluorescence intensity (MFI), which was was normalized due to a slight variation in the number of bioparticles administered to the cells between experiments. Data are from six to eight independent experiments in (a) and four for (b) and (c). Asterisk indicates significance from control: *P < 0·05.

Figure 3

CD69 expression on CD4⁺ T cells is lowered upon stimulation with dendritic cells (DCs) isolated from mice fed n-3 long-chain polyunsaturated fatty acids (LCPUFAs). The DCs were isolated from C57BL/6 mice consuming a control or n-3 LCPUFA diet and incubated with naive CD4⁺ T cells isolated from transgenic mice for 24 hr. T-cell activation was (a) measured by CD69 and CD25 surface expression and (b) quantified in terms of mean fluorescence intensities (MFI). (c) Interleukin-2 (IL-2) and interferon-γ (IFN-γ) secretion were measured with ELISAs. Data are from seven independent experiments. Asterisk indicates significance from control: *P < 0·05.

Figure 4

Dendritic cell (DC) GM1 lipid microdomain clustering and membrane microviscosity are not influenced by n-3 long-chain polyunsaturated fatty acids (LCPUFAs). DCs were isolated from C57BL/6 mice consuming either a control or n-3 LCPUFA diet. (a) Confocal images of cholera toxin-induced lipid microdomains and (b) distribution of microdomain size measured in terms of Feret's diameter. (c) Fluorescence intensity of di-4-ANEPPDHQ measured in the 535–565 nm and 565–675 nm channels. (d) Generalized polarization (GP) of di-4-ANEPPDHQ. Data are from six to eight independent experiments. Asterisks indicate significance from control: **P < 0·01.

Figure 5

Dendritic cell (DC) eicosapentaenoic and docosahexaenoic acid levels are increased in response to a diet enriched in n-3 long-chain polyunsaturated fatty acids (LCPUFAs). (a) Fatty acid composition of DCs isolated from mice fed control and n-3 LCPUFA diets. (b) The total saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), n-3 PUFA, n-6 PUFA and n-6 : n-3 ratio. Data are from three independent experiments. Asterisks indicate significance from control: *P < 0·05, **P < 0·01, ***P < 0·001.