n-3 polyunsaturated fatty acids suppress CD4(+) T cell proliferation by altering phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] organization

Abstract

The mechanisms by which n-3 polyunsaturated fatty acids (n-3 PUFA), abundant in fish oil, exert their anti-inflammatory effects have not been rigorously defined. We have previously demonstrated that n-3 PUFA decrease the amount of phosphatidylinositol-(4,5)-bisphosphate, [PI(4,5)P2], in CD4(+) T cells, leading to suppressed actin remodeling upon activation. Since discrete pools of PI(4,5)P2 exist in the plasma membrane, we determined whether n-3 PUFA modulate spatial organization of PI(4,5)P2 relative to raft and non-raft domains. We used Förster resonance energy transfer (FRET) to demonstrate that lipid raft mesodomains in the plasma membrane of CD4(+) T cells enriched in n-3 PUFA display increased co-clustering of Lck(N10) and LAT(ΔCP), markers of lipid rafts. CD4(+) T cells enriched in n-3 PUFA also exhibited a depleted plasma membrane non-raft PI(4,5)P2 pool as detected by decreased co-clustering of Src(N15), a non-raft marker, and PH(PLC-δ), a PI(4,5)P2 reporter. Incubation with exogenous PI(4,5)P2 rescued the effects on the non-raft PI(4,5)P2 pool, and reversed the suppression of T cell proliferation in CD4(+) T cells enriched with n-3 PUFA. Furthermore, CD4(+) T cells isolated from mice fed a 4% docosahexaenoic acid (DHA)-enriched diet exhibited a decrease in the non-raft pool of PI(4,5)P2, and exogenous PI(4,5)P2 reversed the suppression of T cell proliferation. Finally, these effects were not due to changes to post-translational lipidation, since n-3 PUFA did not alter the palmitoylation status of signaling proteins. These data demonstrate that n-3 PUFA suppress T cell proliferation by altering plasma membrane topography and the spatial organization of PI(4,5)P2.

Keywords: Fluorescence resonance energy transfer; Membrane; Phosphatidylinositol; Polyunsaturated fatty acids; T cell.

Fig 1. n-3 PUFA perturb the biophysical properties of the plasma membrane detected by increased interaction of fluorescent lipid raft markers

(A, B, and C) Schematic diagrams of the interaction between neighboring fluorescent probes targeted to the raft or the non-raft membrane fraction of the plasma membrane. Lck(N10) and Src(N15) consist of the first 10 and 15 N-terminal amino acids of Lck and Src, respectively. LAT(ΔCP) consists of the first 36 amino acids, representing the extracellular, transmembrane, and the membrane anchoring portions of LAT. Splenic CD4⁺ T cells were incubated with lentivirus containing (D) Lck(N10)/Lck(N10), (E) Lck(N10)/LAT(ΔCP), or (F) Lck(N10)/Src(N15) before FRET by acceptor photobleaching (n = 4 mice per genotype). FRET efficiency (E%) was determined by comparing the CFP intensity before and after acceptor photobleaching, and corrected as described in the Materials and Methods (*P < 0.05 between genotypes, two-tailed Student’s t-test). The rate of recovery after photobleaching was also determined for CD4⁺ T cells expressing (G) Lck(N10)-YPet and (H) LAT(ΔCP)-YPet (*P < 0.05 between genotypes, two-tailed Student’s t-test).

Fig 2. n-3 PUFA do not affect the interaction of fluorescent raft and PI(4,5)P₂ markers detected using FRET in Fat-1 CD4⁺ T cells

(A and B) Schematic diagrams of the interaction between neighboring fluorescent probes targeted to the raft membrane fraction of the plasma membrane, and PH(PLC-δ), a PI(4,5)P₂ reporter. (C) A control PH_R40L(PLC-δ) was also expressed to test the specificity of the PH(PLC-δ) reporter. CD4⁺ T cells were incubated with lentivirus containing (D) Lck(N10)/PH(PLC-δ) (n = 8 per genotype), (E) LAT(ΔCP)/PH(PLC-δ) (n = 6 per genotype), or (F) Lck(N10)/PH_R40L(PLC-δ) (n = 4 per genotype) before FRET by acceptor photobleaching. FRET efficiency (E%) was determined as described in Fig 1 (*P < 0.05 between genotypes, two-tailed Student’s t-test). (G) CD4⁺ T cells were isolated and transduced with Lck(N10)-ECFP and PH(PLC-δ)-YPet for 48 hrs prior to incubation with 1.25 μM PI(4,5)P₂ or PBS (0 μM) for one hr. Cells were analyzed as described in Fig 1. (H) Colocalization between the lipid raft marker GM1 and PI(4,5)P₂ is not altered in the presence of n-3 PUFA. Splenic CD4⁺ T cells were positively selected and incubated with lentivirus containing pLenti-PH(PLC-δ)-GFP for 48 hrs (n = 4 mice per genotype). Cells were collected, washed, labeled with Alexa 647 Cholera Toxin B (final concentration 10 μg/mL) and crosslinked with anti-Cholera Toxin B [Vybrant Lipid Raft Labeling Kit (Life Technologies)] according to the manufacturer’s protocol. Cells were imaged using a Zeiss 510 LSM confocal microscope. The plasma membrane region of the cell was drawn and Pearson’s coefficient was determined using NIS-Elements (Nikon).

Fig 3. n-3 PUFA decrease the interaction of fluorescent non-raft and PI(4,5)P₂ markers detected using FRET in Fat-1 CD4⁺ T cells

(A and B) Schematic diagrams of the interaction between neighboring fluorescent probes targeted to the non-raft membrane fraction of the plasma membrane, and PH(PLC-δ), a PI(4,5)P₂ reporter, or the control PH_R40L(PLC-δ). CD4⁺ T cells were incubated with lentivirus containing (C) Src(N15)/PH(PLC-δ) (n = 7 per genotype), or (D) Src(N15)/PH_R40L(PLC-δ) (n = 4 per genotype) before FRET by acceptor photobleaching. FRET efficiency (E%) was determined as described in Fig 1 (*P < 0.05 between genotypes). (E) CD4⁺ T cells were isolated and transduced with Src(N15)-ECFP and PH(PLC-δ)-YPet for 48 hrs prior to incubation with 1.25 μM PI(4,5)P₂ or PBS (0 μM) for one hr. Cells were analyzed as described in Fig 1. A two-tailed t-test was used to compare FRET efficiencies within specific concentrations (*P < 0.05 at specific concentrations of PI(4,5)P₂).

Fig 4. Exogenous PI(4,5)P₂ rescues the n-3 PUFA-induced suppression of T cell proliferation

Representative scatterplots and CFSE profiles of (A) unstimulated, and (B) anti-CD3/anti-CD28 stimulated CD4⁺ T cell cultures. Splenic CD4⁺ T cells were stained with CFSE, then cultured (n = 4 mice per genotype) either in unstimulated or stimulated conditions for 72 hrs in triplicate. Cells were collected and gated by propidium iodide staining to exclude dead cells. (C) Cell proliferation was analyzed using the Proliferation Wizard in ModFitLT3.2 to determine the proliferation index. Different letters represent statistically significant differences between the groups after one-way ANOVA followed by Tukey post-hoc test (P < 0.05).

Fig 5. Dietary DHA reduces membrane a rachidonic acid levels

Levels of DHA (22:6, n-3) and arachidonic acid (20:4, n-6) in CD4⁺ T cell phospholipids isolated from mice fed 5% corn oil (CO), 0.95% DHA, or 4% DHA-enriched diet are shown. Fatty acids from CD4⁺ T cell total phospholipids were extracted, isolated, and characterized as previously described [19, 21]. Data represent mean ± sem (n = 4 per diet), and different letters represent statistically significant differences between the diet groups per fatty acid (P < 0.05). Note that other n-3 PUFA such as EPA (20:5, n-3) and DPA (22:5, n-3) were not detected in CD4⁺ T cell total phospholipids.

Fig 6. Dietary DHA decreases the interaction of fluorescent raft and non-raft probes with PI(4,5)P₂ marker detected using FRET, resulting in suppressed lymphoproliferation that can be rescued by exogenous PI(4,5)P₂ in CD4⁺ T cells

(A and B) Schematic diagrams of the interaction between neighboring fluorescent probes targeted to the raft or the non-raft membrane fraction of the plasma membrane, and PH(PLC-δ), a PI(4,5)P₂ reporter. CD4⁺ T cells were incubated with lentivirus containing (C) Lck(N10)/PH(PLC-δ) (n = 4 per diet), or (D) Src(N15)/PH(PLC-δ) (n = 4 per diet) before FRET by acceptor photobleaching. FRET efficiency (E%) was determined as described in Fig 1 (*P < 0.05 between genotypes). (E) Splenic CD4⁺ T cells were stained with CFSE, then cultured (n = 6 mice per diet) either in unstimulated or stimulated conditions for 72 hrs in triplicate. Cells were collected and gated by propidium iodide staining to exclude dead cells. Proliferation Index was calculated using ModFitLT 3.2 [37]. Different letters represent statistically significant differences between the groups after one-way ANOVA followed by Tukey post-hoc test (P < 0.05).

Fig 7. Palmitoylation status of membrane proteins is not altered in Fat-1 CD4⁺ T cells

(A) 1.5 × 10⁷ splenic CD4⁺ T cells were positively selected and seeded into a 35 mm culture dish with 4 mL of RPMI media supplemented with 2% FBS and rIL-2 overnight. Cells were treated with 50 μM of azido-palmitic acid for 24 h and 2 wells were pooled per sample (n = 2 mice per sample, n = 4 samples per genotype). Subsequently, 100 μg of protein lysate was used for Click chemistry and palmitoylation was detected by immunoblotting. Numbers on the left side represent molecular weight markers. (B) Palmitoylation of LCK is not affected in Fat-1 CD4⁺ T cells. Cells were selected and processed as in (A), and samples were subsequently immunoprecipitated (IP) using polyclonal rabbit IgG anti-LCK. Palmitoylated LCK was then detected by immunoblotting (IB) using an anti-biotin antibody and subsequently quantified.