Regulatory activity of polyunsaturated fatty acids in T-cell signaling

Abstract

n-3 Polyunsaturated fatty acids (PUFA) are considered to be authentic immunosuppressors and appear to exert beneficial effects with respect to certain immune-mediated diseases. In addition to promoting T-helper 1 (Th1) cell to T-helper 2 (Th2) cell effector T-cell differentiation, n-3 PUFA may also exert anti-inflammatory actions by inducing apoptosis in Th1 cells. With respect to mechanisms of action, effects range from the modulation of membrane receptors to gene transcription via perturbation of a number of second messenger cascades. In this review, the putative targets of anti-inflammatory n-3 PUFA, activated during early and late events of T-cell activation will be discussed. Studies have demonstrated that these fatty acids alter plasma membrane micro-organization (lipid rafts) at the immunological synapse, the site where T-cells and antigen-presenting cells (APC) form a physical contact for antigen initiated T-cell signaling. In addition, the production of diacylglycerol and the activation of different isoforms of protein kinase C (PKC), mitogen-activated protein kinase (MAPK), calcium signaling, and nuclear translocation/activation of transcriptional factors, can be modulated by n-3 PUFA. Advantages and limitations of diverse methodologies to study the membrane lipid raft hypothesis, as well as apparent contradictions regarding the effect of n-3 PUFA on lipid rafts will be critically presented.

Figure 1. Modulatory effects of n-3 PUFA on T-cell signalling

n-3 PUFA suppress T-cell activation by modulating lipid rafts at the immunological synapse. This is linked to the displacement of PTKs and diminishment of their phosphorylation/activation status. Alternatively, “free” n-3 PUFA are released from n-3 PUFA-enriched phospholipid domains by the action of n-3 PUFA-specific PLA₂ and, consequently, modulate PKC and pHi. n-3 PUFA mobilize Ca²⁺ from the intracellular pool (i.e., endoplasmic reticulum), which perturbs [Ca²⁺]_i, followed by the opening of CRAC/SOC channels. n-3 PUFA also modulate the translocation of PKC isoforms, and alter the phosphorylation of MAPK, thereby inhibiting translocation of transcription factors (e.g., NF-kB, NF-AT, etc.) to the nucleus. n-3 PUFA, by the action of PLD, give rise to DAG-containing n-3 PUFA (DAG-DHA/EPA) which, in turn, 1) evoke increases in [Ca²⁺]_i via TRPC channels, 2) modulate RasGRP, and/or 3) modulate PKC isoforms. Collectively, n-3 PUFA alter gene transcription, leading to immunosuppression. Dashed lines indicate the action of n-3 PUFA.

Figure 2. Dietary PUFA alter inner-leaflet raft (tH) and non-raft (tK) markers in HCT-116 cells

Ripley's K-function analysis of immuno-gold co-ordinates enables the extent of clustering within a 2D point pattern to be determined. Positive deflection from L(r)-r = 0 indicates a tendency for clustering. All cultures were treated with OA (18:1n-9), LA (18:2n-6), AA (20:4n-6), EPA (20:5n-3) or DHA (22:6n-3) (50 μM) for 96 h, control-untreated. (A) GFP-truncated H-ras (located exclusively to inner leaflet rafts); displays an increase in clustering with LA and AA pre-treatment. Refer to panel (D) for statistical differences between treatments. (B) GFP-truncated H-ras, comparative effects of EPA vs DHA treatment. (C) GFP-truncated K-ras (non-raft marker) exhibits a small decrease in cluster size (EPA) or total clustering. K-functions (8-16 lawns) are means (n>500 particles) from 2 separate experiments. Difference between (D) inner-leaflet raft (tH) or (E) inner-leaflet non-raft (tK) treatments: Pairwise comparison plots. The red solid line is the mean difference between two L(r) functions; the dashed green lines mark the 95% confidence interval. There exists a statistically significant difference between the two L(r) functions at the 0.05 level (p<0.05) when part of the solid black zero line is outside the two dashed green curves, e.g., panel (D), LA vs control and panel (E), OA vs EPA. X-axis indicates the radius r (nm) in which the L(r) function is calculated. Y-axis indicates the difference between the two L(r) functions.

Figure 3. Assessment of lipid raft formation at the immunological synapse using a T-cell culture system

Human Jurkat T-cell and superantigen Staphylococal Enterotoxin E (SEE)-pulsed Raji B-cell co-cultures were assessed by laurdan labeling and generalized polarization (GP)-values. Jurkat T-cells were pre-treated with 50 μM DHA (n-3 PUFA) or AA (n-6, control) for 72 h. Culture medium was changed every 24 h with fresh fatty acid-BSA complex to avoid lipid peroxidation and consequent oxidative stress. (A) Representative two-photon microscopy RGB image of laurdan labelled Jurkat T-cells and red cell tracker labelled Raji B-cells. Three different regions of interest, i.e., (a) the immunological synapse, (b) contact whole T-cell, or (c) non-contact whole T-cell were drawn to calculate (B) GP-values for lipid raft formation assessment over time (10-30 min co-culture period). Detailed protocols for laurdan labelling, two-photon microscopic imaging, and calculation of GP-values at the regions of interest have been previously described (140). *Significant difference between AA vs DHA at the immunological synapse (P<0.05).