Remodelling of primary human CD4+ T cell plasma membrane order by n-3 PUFA

Abstract

Cell membrane fatty acids influence fundamental properties of the plasma membrane, including membrane fluidity, protein functionality, and lipid raft signalling. Evidence suggests that dietary n-3 PUFA may target the plasma membrane of immune cells by altering plasma membrane lipid dynamics, thereby regulating the attenuation of immune cell activation and suppression of inflammation. As lipid-based immunotherapy might be a promising new clinical strategy for the treatment of inflammatory disorders, we conducted in vitro and in vivo experiments to examine the effects of n-3 PUFA on CD4+ T cell membrane order, mitochondrial bioenergetics and lymphoproliferation. n-3 PUFA were incorporated into human primary CD4+ T cells phospholipids in vitro in a dose-dependent manner, resulting in a reduction in whole cell membrane order, oxidative phosphorylation and proliferation. At higher doses, n-3 PUFA induced unique phase separation in T cell-derived giant plasma membrane vesicles. Similarly, in a short-term human pilot study, supplementation of fish oil (4 g n-3 PUFA/d) for 6 weeks in healthy subjects significantly elevated EPA (20 : 5n-3) levels in CD4+ T cell membrane phospholipids, and reduced membrane lipid order. These results demonstrate that the dynamic reshaping of human CD4+ T cell plasma membrane organisation by n-3 PUFA may modulate down-stream clonal expansion.

Keywords: n-3 PUFA; n-3 PUFA n-3 PUFA; ECAR extracellular acidification rate; FO fish oil; GP generalised polarisation; GPMV giant plasma membrane vesicle; LA linoleic acid; OCR VO2 rate; OO olive oil; UT untreated; CD4+ T cells; Generalised polarisation; Giant plasma membrane vesicles; Phase separation.

Fig. 1

Exogenous fatty acids dose-dependently alter human CD4⁺ T cell membrane order and giant plasma membrane vesicle (GPMV) phase separation. Human pan CD4⁺ T cells isolated from buffy-coat leucocytes were incubated with various doses (0–50 μM) of linoleic acid (LA), EPA and DHA conjugated with bovine serum albumin for 2 d. Cultures were further stimulated for 3 d with Dynabeads Human T-Activator CD3/CD28 at the bead-to-cell ratio of 1:1, in the presence of their respective FA. Membrane order was measured following 5 d of total incubation. Data are expressed as the change in generalised polarisation (ΔGP) compared with the untreated (UT) group in (A) whole cells and (B) GPMV. (C) n-3 PUFA significantly increased numbers of GPMV with phase separation. (D) Representative images of GPMV showing no phase separation (UT and LA 50 μM) and phase separation (DHA 50 μM and EPA 50 μM). White dashed line represents region of interest identifying GPMV and used to determine membrane order. Note that GPMV rarely detach fully from the cell. Scale bar equals 2 μm. (E) Average histogram of normalised GP values obtained from non-phase separated ( ) and phase separated ( ) GPMV from LA 50 μM and EPA 50 μM treated cells, respectively. Values are as means (n 50–183, pooled from three separate experiments), with their standard errors. ^a,b,c,d Mean values with unlike letters are significantly different (P < 0·05).

Fig. 2

Exogenous fatty acids alter human CD4⁺ T cell bioenergetic profiles and proliferation. See Fig. 1 legend for in vitro culture details. Cellular bioenergetic profiles in basal and activated states were measured. (A) VO₂ rate (OCR):extracellular acidification rate (ECAR) ratio under basal conditions (following 2 d FA incubation). (B) OCR:ECAR ratio after additional 3 d activation (5 d FA incubation). (C) Mitochondrial respiration-associated proton leak after activation. (D) Cell proliferation 3 d after activation. Values are means (n 7–21, pooled from two separate experiments for bioenergetic assays; n 14–24, pooled from four separate experiments for cell proliferation assays), with their standard errors. The proliferation index was calculated as the ratio of live cell fluorescense in activated:basal states. ^a,b,c,d,e,f Mean values with unlike letters are significantly different (P < 0·05).

Fig. 3

Weight percentage of the major n-9, n-6 and n-3 PUFA in CD4⁺ T cell membrane total phospholipids over 6 weeks of oral fish oil supplementation in elderly humans. Senior (age 60–87 years) volunteers were provided fish oil (FO, ) capsules (4 g n-3 PUFA daily) or placebo olive oil (OO, ) capsules for 6 weeks. Fresh whole blood was collected at time 0, 3 and 6 weeks. CD4⁺ T cells were isolated and membrane total phospholipids were extracted, separated by TLC, and the FA mass determined. Values are means (n 5–7), with their standard errors. * Significantly different from time 0 (P < 0·05).

Fig. 4

Activated CD4⁺ T cell membrane order was decreased by dietary fish oil and olive oil supplementation in elderly humans. See Fig. 3 legend for dietary intervention details. CD4⁺ T cells isolated from volunteers were activated using Human T-Activator CD3/CD28 Dynabeads at a bead-to-cell ratio of 1:1 for 3 d. (A, B) Whole cell and (C, D) giant plasma membrane vesicle membrane order under both basal and activated conditions were measured. Data are expressed as change in generalised polarization (ΔGP) compared with time 0, and reported as means (n 5–7), with their standard errors. ■, Fish oil; □, olive oil. ^a,b Mean values with unlike letters are significantly different (P < 0·05).