n-3 Polyunsaturated Fatty Acids Alter Mouse CD4+ T Cell Activation by Modifying the Lipid Bilayer Properties

Epidemiological and clinical studies have shown that very long chain n-3 polyunsaturated fatty acids (n-3 PUFA) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) possess anti-inflammatory properties. The mechanisms by which n-3 PUFA exert their anti-inflammatory effects remain undefined. Extending earlier observations that actin remodeling at the immunological synapse (IS) is suppressed in CD4+ T cells supplemented with n-3 PUFA, we hypothesized that the lipid mediator phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], which modulates actin remodeling, is perturbed by n-3 PUFA. Utilizing the transgenic Fat-1 mouse model that synthesizes n-3 PUFA de novo and enriches the plasma membrane with n-3 PUFA, and wild type (WT) mice fed either a 5% corn oil (CO, control) or a 4% DHA triglyceride-enriched diet, we found that splenic CD4+ T cells from Fat-1 mice and WT mice fed a DHA-enriched diet, when compared to WT and CO-fed CD4+ T cells, exhibited i) decreased PI(4,5)P2; ii) unchanged PI(4,5)P2 upon activation; and iii) suppressed actin remodeling, as assessed by immunofluorescence, upon activation, which was rescued in Fat-1 CD4+ T cells by incubation with exogenous PI(4,5)P2. Mechanistically, recruitment of the Wiskott-Aldrich syndrome protein (WASP), an actin-remodeling protein regulated by PI(4,5)P2, to the IS upon anti-CD3/anti-CD28 coated bead stimulation was inhibited in Fat-1 CD4+ T cells.

Since discrete pools of PI(4,5)P2 may exist in the plasma membrane, we also determined whether n-3 PUFA modulate the spatial organization of PI(4,5)P2 relative to raft and non-raft domains by transducing CD4+ T cells from WT and Fat-1 mice with fluorescence resonance energy transfer (FRET) lipid raft probes Lck(N10) and LAT(?CP), and the non-raft probe Src(N15). Co-clustering of PH(PLC-?1), a PI(4,5)P2 probe, and Lck(N10) or LAT(?CP), was not affected by n-3 PUFA; however, co-clustering of PH(PLC-?1) and Src(N15) exhibited a decrease in Fat-1 CD4+ T cells, suggesting that n-3 PUFA alter the spatial organization of PI(4,5)P2. Incubation with exogenous PI(4,5)P2 rescued the effects on the non-raft PI(4,5)P2 pool, and reversed the corresponding suppression of T cell proliferation in Fat-1 CD4+ T cells.

Previous research has shown that n-3 PUFA can inhibit Fyn palmitoylation. Therefore, we proposed an alternative hypothesis that n-3 PUFA affect the palmitoylation, and hence activation, of signaling proteins upon T cells activation. Using a newly developed technique in which palmitoylated proteins are coupled to biotin via Click chemistry, we demonstrated that n-3 PUFA do not affect the palmitoylation of signaling proteins, such as LCK, in Fat-1 CD4+ T cells.