n-3 polyunsaturated fatty acids suppress phosphatidylinositol 4,5-bisphosphate-dependent actin remodelling during CD4+ T-cell activation

Abstract

n-3 PUFA (polyunsaturated fatty acids), i.e. DHA (docosahexaenoic acid), found in fish oil, exhibit anti-inflammatory properties; however, the molecular mechanisms remain unclear. Since PtdIns(4,5)P2 resides in raft domains and DHA can alter the size of rafts, we hypothesized that PtdIns(4,5)P2 and downstream actin remodelling are perturbed by the incorporation of n-3 PUFA into membranes, resulting in suppressed T-cell activation. CD4+ T-cells isolated from Fat-1 transgenic mice (membranes enriched in n-3 PUFA) exhibited a 50% decrease in PtdIns(4,5)P2. Upon activation by plate-bound anti-CD3/anti-CD28 or PMA/ionomycin, Fat-1 CD4+ T-cells failed to metabolize PtdIns(4,5)P2. Furthermore, actin remodelling failed to initiate in Fat-1 CD4+ T-cells upon stimulation; however, the defect was reversed by incubation with exogenous PtdIns(4,5)P2. When Fat-1 CD4+ T-cells were stimulated with anti-CD3/anti-CD28-coated beads, WASP (Wiskott-Aldrich syndrome protein) failed to translocate to the immunological synapse. The suppressive phenotype, consisting of defects in PtdIns(4,5)P2 metabolism and actin remodelling, were recapitulated in CD4+ T-cells isolated from mice fed on a 4% DHA triacylglycerol-enriched diet. Collectively, these data demonstrate that n-3 PUFA, such as DHA, alter PtdIns(4,5)P2 in CD4+ T-cells, thereby suppressing the recruitment of WASP to the immunological synapse, and impairing actin remodelling in CD4+ T-cells.

Figure 1. Total PtdIns(4,5)P₂ is decreased in Fat-1 CD4⁺ T-cells

(A) Relative PtdIns(4,5)P₂ was decreased by 50% in Fat-1 CD4⁺ T-cells. CD4⁺ T-cells were isolated from wild-type and Fat-1 spleens (n = 8 for Fat-1; n = 12 for wild-type). CD4⁺ T-cell populations from two spleens were pooled for analysis using MS as described in the Experimental section. Total PtdIns(4,5)P₂ was quantified following comparison with an internal standard and normalized relative to total CD4⁺ T-cells counted using a Coulter Counter. Relative PtdIns(4,5)P₂ levels were compared by normalizing to wild-type PtdIns(4,5)P₂. (B) PtdIns(4,5)P₂ molecular species were altered in Fat-1 CD4⁺ T-cells. PIP₂, PtdIns(4,5)P₂.

Figure 2. Metabolism of PtdIns(4,5)P₂ upon (A) anti-CD3/anti-CD28 and (B) PMA/ionomycin stimulation was suppressed in Fat-1 CD4⁺ T-cells

Splenic CD4⁺ T-cells were isolated from wild-type and Fat-1 mice (n = 4). CD4⁺ T-cells (5×10⁵) were seeded in 96-well plates containing plated anti-CD3 and anti-CD28 for the indicated times. PtdIns(4,5)P₂ was subsequently extracted using acidic organic solvents, dried under nitrogen and dissolved in 1×PBS containing 0.0025% protein stabilizer. PtdIns(4,5)P₂ was detected using indirect anti-PtdIns(4,5)P₂ ELISA as described in the Experimental section. Absolute values were determined using a standard curve and normalized relative to wild-type PtdIns(4,5)P₂. A two-tailed Student’st test was used to compare individual times points between genotypes (*P < 0.05 between genotypes at specific time points).

Figure 3. Actin remodelling upon anti-CD3/anti-CD28 stimulation is suppressed in Fat-1 CD4⁺ T-cells

(A) Representative Alexa Fluor® 568-conjugated phalloidin immunofluorescence images of wild-type and Fat-1 CD4⁺ T-cells seeded on to slides containing only poly-L-lysine (unstimulated) or on slides containing anti-CD3/anti-CD28 (stimulated) for 30 min. Images were pseudo-coloured with increasing intensity from blue to red. (B) Quantification of actin remodelling as assessed by Alexa Fluor® 568-conjugated phalloidin fluorescence. Purified splenic CD4⁺ T-cells (n = 4 per genotype) were seeded, fixed, permeabilized and labelled as described in the Experimental section. ROIs were drawn around individual cells, and background was subtracted by drawing ROIs not occupied by cells. Fluorescence values were divided by the number of cells present in each field. Six fields were obtained per group, with each field containing between three and seven cells. Four independent experiments were conducted. Different letters denote a significant difference at P < 0.05. A.U., arbitrary unit; DIC, differential interference contrast.

Figure 4. WASP localization to the IS and co-localization with actin at the IS are decreased in Fat-1 CD4⁺ T-cells

(A) Actin remodelling at the IS was suppressed in Fat-1 CD4⁺ T-cells. Purified splenic CD4⁺ T-cells (n 3 per genotype) were seeded, stimulated with anti-CD3/anti-CD28-coated beads, fixed, permeabilized and labelled as described in the Experimental section. ROIs were drawn and fluorescence=values divided by the area of the IS. In total, 10-15 cells were analysed per genotype. Three independent experiments were conducted. A two-tailed Student’s t test was used to compare between genotypes. (B) WASP recruitment to the IS was suppressed in Fat-1 CD4⁺ T-cells. Experimental details are given above. (C) Representative immunofluorescence images of purified splenic CD4⁺ T-cells isolated from wild-type and Fat-1 mice. Cells were seeded on to poly-L-lysine and stimulated with anti-CD3/anti-CD28-coated beads. The ROI was drawn as an oval or polygon (white broken line) as shown in the wild-type panels. (D) WASP recruitment to the IS as assessed by immunoisolation. IS fractions from wild-type (WT) (n = 3) and Fat-1 (n = 4) mice were prepared as described in the Experimental section. WASP (66 kDa) was probed in both the IS and input fractions. The enrichment factor was determined by calculating the ratio of the band intensities from the IS divided by the input. A two-tailed Student’s t test was used to compare between genotypes. AU, arbitrary unit; DIC, differential interference contrast.

Figure 5. Incubation of Fat-1 CD4⁺ T-cells with exogenous PtdIns(4,5)P₂ can rescue defects in actin remodelling following anti-CD3/anti-CD28 stimulation

Fat-1 CD4⁺ T-cells were isolated and incubated with PtdIns(4,5)P₂ at various concentrations (0.5, 1.25 and 2.5 μM) or PBS (0 μM) for 1 h prior to stimulation with plated anti-CD3/anti-CD28 for 30 min. Cells were analysed as described in Figure 3. Images were pseudo-coloured with increasing intensity from blue to red. A two-tailed Student’s t test was used to compare stimulation within specific concentrations [*P < 0.05 at specific concentrations of PtdIns(4,5)P₂]. A.U., arbitrary unit; DIC, differential interference contrast; PIP₂, PtdIns(4,5)P₂.

Figure 6. CD4⁺ T-cells isolated from mice fed on a 4% DHA triacylglycerol-enriched diet exhibit altered PtdIns(4,5)P₂ metabolism and actin remodelling upon anti-CD3/anti-CD28 stimulation

(A) Relative PtdIns(4,5)P₂ levels in unstimulated splenic CD4⁺ T-cells isolated from mice fed on a 5% corn oil diet (control, n = 7) or 4% DHA triacylglycerol-enriched (n = 8) diet were determined by indirect anti-PtdIns(4,5)P₂ ELISA as described in the Experimental section. Relative PtdIns(4,5)P₂ levels were compared by normalizing to wild-type PtdIns(4,5)P₂. (B) PtdIns(4,5)P₂ levels upon anti-CD3/anti-CD28 stimulation in CD4⁺ T-cells isolated from mice fed on a 5% corn oil or a 4% DHA triacylglycerol-enriched diet. CD4⁺ T-cells were seeded in 96-well plates with plate-bound anti-CD3 and anti-CD28 as described in the Experimental section. A two-way ANOVA indicated statistical significance between the two diet groups (P = 0.005). (C) PtdIns(4,5)P₂ levels upon PMA/ionomycin stimulation in CD4⁺ T-cells isolated from mice fed on a 5% corn oil or 4% DHA triacylglycerol-enriched diet. A two-way ANOVA indicated statistical significance between the diet groups (P 0.01). A two-tailed Student’s t test was used to compare individual times points between=treatment groups (*P < 0.05 at specific time points). CO, corn oil; PIP2, PtdIns(4,5)P₂.

Figure 7. Actin remodelling upon anti-CD3/anti-CD28 stimulation is suppressed in CD4⁺ T-cells isolated from animals fed on a 4% DHA triacylglycerol-enriched diet

(A) Representative immunofluorescence images of purified splenic CD4⁺ T-cells isolated from (control) 5% corn oil (CO)- and 4% DHA-fed mice. Cells were unstimulated or incubated on slides with anti-CD3/anti-CD28 (stimulated) for 30 min. Images were pseudo-coloured with increasing intensity from blue to red. (B) Quantification of actin remodelling. Refer to Figure 3(B) for experimental details. Different letters denote P < 0.05. A.U., arbitrary units; DIC, differential interference contrast.

Figure 8. n – 3 PUFA alter early steps of T-cell activation

In wild-type CD4⁺ T-cells, upon stimulation with anti-CD3 (and co-stimulatory signal from CD28), the TCR signalsome composed of Lck, ZAP-70, LAT, GADS, SLP76, NCK, ITK, VAV1, PAK and PLCγ 1 is formed, recruiting additional proteins such as PLCγ 1. PLCγ 1 hydrolyses the lipid mediator PtdIns(4,5)P₂ to diacylglycerol (DAG) and Ins(1,4,5)P₃, leading to induction of Ca²⁺ signalling and the PKCθ signalling pathway. The hydrolysis of PtdIns(4,5)P₂ is also important for actin remodelling by releasing actin-regulating proteins localized at the plasma membrane. Additionally, PtdIns(4,5)P₂ is phosphorylated by class I PI3K (phosphoinositide 3-kinase) to generate PtdIns(3,4,5)P₃ which also triggers actin remodelling. WASP is recruited to the plasma membrane and is activated by PtdIns(4,5)P₂ to regulate actin remodelling. These pathways synergistically regulate actin remodelling at the plasma membrane for mitochondrial translocation and T-cell activation. We have shown previously that n – 3 PUFA suppress total and phosphorylated PLCγ 1 translocation to the IS (dotted hammerheads signify suppression) [32] and mitochondrial translocation to the IS [30]. We now show that n – 3 PUFA, such as DHA, can suppress (i) the basal level of PtdIns(4,5)P₂ in unstimulated CD4⁺ T-cells, (ii) the metabolism of PtdIns(4,5)P₂ upon anti-CD3/anti-CD28 or PMA/ionomycin stimulation, (iii) WASP recruitment to the IS and (iv) actin remodelling upon anti-CD3/anti-CD28 stimulation. The suppression of these early immediate events in T-cell activation leads to inhibition of T-cell function, and thus may suppress inflammatory responses.