Dietary n-3 polyunsaturated fatty acids promote activation-induced cell death in Th1-polarized murine CD4+ T-cells - PubMed (original) (raw)

Dietary n-3 polyunsaturated fatty acids promote activation-induced cell death in Th1-polarized murine CD4+ T-cells

Kirsten C Switzer et al. J Lipid Res. 2004 Aug.

Abstract

Dietary n-3 PUFAs have been shown to attenuate T-cell-mediated inflammation. To investigate whether dietary n-3 PUFAs promote activation-induced cell death (AICD) in CD4+ T-cells induced in vitro to a polarized T-helper1 (Th1) phenotype, C57BL/6 mice were fed diets containing either 5% corn oil (CO; n-6 PUFA control) or 4% fish oil (FO) plus 1% CO (n-3 PUFA) for 2 weeks. Splenic CD4+ T-cells were cultured with alpha-interleukin-4 (alphaIL-4), IL-12, and IL-2 for 2 days and then with recombinant (r) IL-12 and rIL-2 for 3 days in the presence of diet-matched homologous mouse serum (HMS) to prevent loss of cell membrane fatty acids, or with fetal bovine serum. After polarization, Th1 cells were reactivated and analyzed for interferon-gamma and IL-4 by intracellular cytokine staining and for apoptosis by Annexin V/propidium iodide. Dietary FO enhanced Th1 polarization by 49% (P = 0.0001) and AICD by 24% (P = 0.0001) only in cells cultured in the presence of HMS. FO enhancement of Th1 polarization and AICD after culture was associated with the maintenance of eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) in plasma membrane lipid rafts. In conclusion, n-3 PUFAs enhance the polarization and deletion of proinflammatory Th1 cells, possibly as a result of alterations in membrane microdomain fatty acid composition.

Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.

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Figures

Fig. 1

Fig. 1

Diet-induced alteration of T-helper1 (Th1) cell polarization is observed only in the presence of homologous mouse serum [HMS (MS)]. Splenic CD4+ T-cells were isolated from mice fed corn oil (CO)- or fish oil (FO)-containing diets for 2 weeks followed by polarization toward Th1 in the presence of HMS or FBS. After 5 days in culture, cells were reactivated with phorbol-12-myristate-13-acetate (PMA)/Ionomycin in the presence of monensin and assessed for the coexpression of IFNγ and interleukin-4 (IL-4) by intracellular cytokine staining as described in Experimental Procedures. A: Representative two parameter flow cytometric histograms of HMS- versus FBS-cultured cells. Numbers in each quadrant represent the percentage of cells positive for IFNγ and/or IL-4. B: Bars with different letters denote significant differences (P < 0.05). Data represent means ± SEM; n = 6 replicates per diet group, and 4 mice were pooled per analysis.

Fig. 2

Fig. 2

Dietary n-3 PUFAs promote CD4+ T-cell activation-induced cell death (AICD) only in the presence of HMS (MS). Splenic CD4+ T-cells were isolated from mice fed CO- or FO-containing diets for 2 weeks followed by polarization toward Th1 in the presence of HMS or FBS. After 5 days in culture, cells were reactivated with PMA/Ionomycin and assessed for apoptosis by Annexin V-FITC and propidium iodide (PI) staining as described in Experimental Procedures. A: Representative two parameter flow cytometric histograms of HMS- versus FBS-cultured cells. Numbers in each quadrant represent the percentage of cells positive for Annexin V-FITC and/or PI. B: Bars with different letters denote significant differences (P < 0.05). Data represent means ± SEM; n = 6 replicates per diet group, and 4 mice were pooled per analysis.

Fig. 3

Fig. 3

AICD and Th1 differentiation are highly correlated in mouse serum-cultured CD4+ T-cells. The relationship between AICD and intracellular IFNγ levels was examined by both Spearman and Pearson tests of IFNγ+ cells versus apoptotic cells. Closed circles represent HMS-cultured cells isolated from FO-fed animals, and open circles represent HMS-cultured cells isolated from CO-fed animals. Data represent means ± SEM; n = 6 replicates per diet group, and 4 mice were pooled per analysis (P < 0.05).

Fig. 4

Fig. 4

Membrane microdomain distribution of eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) in CO- and FO-fed mouse CD4+ T-cells. Splenic CD4+ T-cell membrane fractions were isolated from mice fed CO- or FO-containing diets for 2 weeks either immediately (day 0) or after culture in the presence of either 5% FBS complete medium or HMS for 5 days. Raft and soluble membrane fractions were isolated, and EPA and DHA were analyzed. Data represent means ± SEM; n = 3 replicates per diet group, and 10 mice were pooled per analysis. Letters indicate significant differences between diets for the same membrane fraction and fatty acid (P < 0.05).

Fig. 5

Fig. 5

CD4+ T-cells from n-3 PUFA-fed mice cultured in the presence of homologous HMS maintain the cholesterol content of lipid rafts. Splenic CD4+ T-cells were isolated from mice fed CO- or FO-containing diets for 2 weeks followed by membrane isolation (day 0) or after culture in the presence of either 5% FBS complete medium (FBS) or HMS (MS) for 5 days. Raft and soluble membrane fractions were isolated, and total phospholipid (PL) and cholesterol (chol) levels were expressed as a mol% ratio. Data represent means ± SEM; n = 3 replicates per diet group, and 10 mice were pooled per analysis. Letters indicate significant differences between culture conditions for the same membrane fraction and diet (P < 0.05).

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