Is there a relationship between fatty acid composition and function of immune cells?

In vitro studies have clearly demonstrated that alterations in the phagocyte membrane fatty acid composition are associated with altered phagocytic capacity⁽Reference Calder¹⁴⁾. Dietary studies have tended to be contradictory, but it has been demonstrated that the phagocytic activity of neutrophils and monocytes is positively related to the membrane content of n-3 PUFA⁽Reference Kew, Banerjee and Minihane¹⁵⁾ and that supplementation with n-3 PUFA enhances phagocytic activity⁽Reference Kew, Banerjee and Minihane¹⁶⁾. For other functional outcomes, the picture is less clear. Fritsche⁽Reference Fritsche¹⁷⁾ recently conducted a cross-study meta-regression dose–response analysis of the effect of dietary n-3 PUFA on the AA content of human, murine and rat immune cells, citing the accumulation of AA in tissues as a key factor in the biosynthesis of eicosanoids with pro-inflammatory activity. Baseline fatty acid profiles of human peripheral blood mononuclear cells and rat splenocytes appear to be remarkably similar in terms of AA content, while n-3 PUFA content is more variable. Rodent immune cells typically contain 15–20% AA as a proportion of total fatty acids, and very little EPA or DHA. Human immune cell phospholipids, on the other hand contain about 1% EPA and 2·5% DHA in addition to 20% AA⁽Reference Calder^14, Reference Fritsche¹⁷⁾. As the long chain n-3 PUFA content of the diet increases, lymphocyte AA decreases in a curvilinear fashion⁽Reference Fritsche¹⁷⁾. In human studies, dietary n-3 PUFA never exceeded 3%, whereas in the animal studies the intake was considerably higher, but even when this is taken into account, it is apparent that rodent lymphocytes are much more responsive to the impact of n-3 PUFA on AA content than are human lymphocytes. n-3 PUFA do not appear to lower AA or enrich cell membranes with n-3 PUFA in human blood monocytes as much as in tissue macrophages from either rats or mice⁽Reference Fritsche¹⁷⁾. Furthermore, murine lymphocytes, which already contain more DHA than rat or human lymphocytes, accumulate DHA more readily in response to dietary enrichment than these species⁽Reference Fritsche¹⁷⁾. Fritsche⁽Reference Fritsche¹⁷⁾ suggests that this may help explain the discrepancies that exist between animal and human studies investigating the immunomodulatory effects of n-3 PUFA. However, it remains unclear how much n-3 PUFA is required for biological effects in human subjects.

It is apparent from the literature that fish oil has a greater impact on immune function in the elderly compared with young subjects⁽Reference Thies, Miles and Nebe-von-Caron^12, Reference Meydani, Endres and Woods^18, Reference Thies, Nebe-von-Caron and Powell¹⁹⁾. The mechanistic basis for this is not understood, but it is interesting to note that older subjects appear to incorporate EPA into plasma and peripheral blood mononuclear cells more readily than younger subjects⁽Reference Rees, Miles and Banerjee²⁰⁾. This was associated with a dose-dependent decrease in the neutrophil respiratory burst in older, but not younger subjects⁽Reference Rees, Miles and Banerjee²⁰⁾. However, PGE₂ production by peripheral blood mononuclear cells was decreased in both groups and phagocytosis and cytokine production was not affected in either group⁽Reference Rees, Miles and Banerjee²⁰⁾. This highlights the fact that age is likely to be an important factor when considering the impact of n-3 PUFA on immunity, not only because of the influence of immunosenescence but also because immune cells from older subjects appear to be more responsive to the availability of n-3 PUFA. Recent work suggests that the cholesterol content of T-lymphocytes from healthy elderly subjects is higher than that of young subjects and that membrane fluidity is subsequently decreased⁽Reference Larbi, Douziech and Dupuis²¹⁾. Furthermore, the coalescence of lipid rafts at the site of T-cell receptor engagement was impaired in elderly subjects⁽Reference Larbi, Douziech and Dupuis^21, Reference Fulop, Larbi and Douziech²²⁾. The impact of ageing on lipid raft composition and function was most evident in the CD4⁺ T-cell population and affected cytokine signalling⁽Reference Fulop, Larbi and Douziech^22, Reference Larbi, Dupuis and Khalil²³⁾. Thus, enriching T-cell membranes of older subjects with n-3 PUFA could modulate the immune function via effects on the lipid raft structure which are distinct from those in younger subjects.

Are lipid rafts responsible for the immunomodulatory effects of n-3 PUFA?

The lipid raft hypothesis suggests that there is a degree of self-organization within the cell membranes such that dynamic microenvironments are created within the exoplasmic leaflets of the phospholipid bilayer of plasma membranes to preferentially group transmembrane proteins according to their function⁽Reference Katagiri, Kiyokawa and Fujimoto²⁴⁾. These rafts have been proposed to serve as platforms to facilitate apical sorting, the association of signalling molecules and interactions between cell types⁽Reference Katagiri, Kiyokawa and Fujimoto^24, Reference Harder, Scheiffele and Verkade²⁵⁾. Despite a large body of work, some doubts still persist regarding the existence and nature of lipid rafts⁽Reference Heerklotz^26–Reference Shaw²⁹⁾. These doubts have arisen mainly due to limitations in the interpretation of the methods available to study rafts. The most widely used technique is the preparation of detergent-resistant membranes which are suggested to represent raft domains, since they contain the glycosylphosphatidylinositol-anchored proteins, cholesterol and sphingolipids characteristic of lipid rafts. In support of this idea, the liquid-ordered phases rich in cholesterol and sphingolipids in artificial membranes are resistant to detergent extraction⁽Reference Schroeder, Ahmed and Zhu³⁰⁾. However, there is a possibility that detergent solubilization could induce non-physiological rearrangements in the bilayer structure, and in particular that the detergent could induce the formation of holes in the membrane, which allow mixing of the inner and outer leaflet and the appearance of cell-signalling proteins in detergent-resistant membrane fractions⁽Reference Heerklotz^26, Reference Munro²⁷⁾. Thus, while in vitro studies using the detergent extraction procedure for isolation of lipid rafts have demonstrated that treatment with EPA at a concentration of 50 μm results in marked enrichment of both EPA and docosapentaenoic acid in lipids isolated from rafts and the subsequent displacement of acylated proteins⁽Reference Stulnig, Huber and Leitinger³¹⁾. If raft formation and stability are based on interactions between saturated acyl chains and cholesterol, then the observations that PUFA incorporated into these detergent-resistant fractions is somewhat contrary to expectations. Although a limited number of animal studies support the role of lipid rafts in mediating the effects of PUFA on immune function, it is still largely unclear whether the displacement of key signalling proteins from putative lipid rafts and the down-regulation of signalling pathways by n-3 PUFA are physiological phenomena that could explain the immunomodulatory properties of fish oil in human subjects⁽Reference Calder³²⁾. Clearly, direct visualization of rafts would resolve uncertainties about their existence and structure, but fluorescence microscopy studies have tended to produce mixed results⁽Reference Munro²⁷⁾ and it is argued that rafts are too small to be resolved by conventional microscopy techniques⁽Reference Shaw²⁹⁾. The size, stability and functionality of putative membrane microdomains including rafts are therefore still very much in debate. It has been suggested that although some domains are macroscopic and stable for extended periods, others are tiny and unstable, existing only momentarily and as a result are very poorly understood⁽Reference Pike^33, Reference Siddiqui, Harvey and Zaloga³⁴⁾. Kenworthy⁽Reference Kenworthy³⁵⁾ suggests that mechanistic models linking the microdomain structure and function are required to systematically evaluate how the structural and dynamic features of lipid rafts influence protein diffusion and reaction kinetics.

Both linoleic acid and DHA increased the clustering of a lipid raft probe compared with oleic acid and untreated cells, demonstrating that PUFA appear to specifically increase the clustering of proteins in cholesterol-dependent microdomains⁽Reference Chapkin, Seo and McMurray³⁶⁾. The authors suggest that the poor affinity of long-chain PUFA for cholesterol provides a lipid-driven mechanism for lateral phase separation of cholesterol-rich microdomains and alters the dynamic partitioning of acylated proteins⁽Reference Chapkin, Seo and McMurray³⁶⁾. Similarly, Shaikh and Edidin⁽Reference Shaikh and Edidin^37, Reference Shaikh and Edidin³⁸⁾ suggest that phospholipids containing highly disordered polyunsaturated acyl chains that exhibit low affinity to cholesterol would be expected to phase separate from rafts. They further demonstrated that an oleic acid-containing phosphatidylethanolamine (PE) and a DHA-containing PE phase separated differently from the lipid raft molecules, sphingomyelin and cholesterol in monolayer and bilayer membranes⁽Reference Shaikh, Brzustowicz and Gustafson^39, Reference Shaikh, Dumaual and Castillo⁴⁰⁾. The interactions between DHA-containing PE and cholesterol were less favourable, and as a result, these PE species were less likely to be found in detergent-resistant membrane fractions than oleic acid-containing PE⁽Reference Shaikh, Dumaual and Castillo⁴⁰⁾.

Membrane microdomains have been studied extensively with respect to T-lymphocyte responses to activation⁽Reference Katagiri, Kiyokawa and Fujimoto^24, Reference Harder, Scheiffele and Verkade^25, Reference Razzaq, Ozegbe and Jury⁴¹⁾. There is visual evidence of clustering of signalling components at T-lymphocyte synapses using the non-toxic B subunit of cholera toxin, which binds the glycosphingolipid, GM1, a putative raft reporter⁽Reference Harder, Scheiffele and Verkade²⁵⁾. A novel approach whereby anti-T-cell receptor-coated microbeads were attached to T-cells and then stripped away, along with patches of membrane, has demonstrated the presence of the T-cell receptor and associated signalling molecules including linker for activation of T-cells, but there did not appear to be an increase in cholesterol, as might be expected on the basis of its presence in putative lipid rafts⁽Reference Harder⁴²⁾. The authors concluded that protein–protein interactions, rather than protein–lipid interactions, and subsequent clustering are the key features of signalling assemblies⁽Reference Harder and Kuhn⁴³⁾. This view is supported by Douglas and Vale⁽Reference Douglas and Vale⁴⁴⁾, who used sophisticated imaging techniques to track individual fluorescent proteins involved in T-cell receptor signalling to show that linker of activated T-cells mutants lacking residues specifically required for protein–protein interactions did not cluster. Other studies by the Harder group have used fluorescence techniques to show that condensation of the plasma membrane occurs at the T-cell receptor activation site, suggesting the formation of ordered lipid phases⁽Reference Gaus, Chklovskaia and Fazekas de St Groth^45, Reference Gaus, Zech and Harder⁴⁶⁾. These studies used a dye, Laurdan, which is incorporated into the membrane and undergoes changes in its emission spectrum, corresponding to the level of lipid ordering, allowing quantification of the condensation⁽Reference Gaus, Chklovskaia and Fazekas de St Groth^45, Reference Gaus, Zech and Harder⁴⁶⁾. In vitro treatment of Jurkat T-cells with EPA impaired membrane condensation of T-cell receptor activation sites, which was directly related to perturbation of the fatty acid composition of phospholipid species in the immuno-isolated membrane fractions⁽Reference Zech, Ejsing and Gaus⁴⁷⁾. This is the first study relating to PUFA modification of membrane domains which is not based on detergent extraction. Recently, fat-1 transgenic mice have been used to gain further insight into the impact of n-3 PUFA on lipid rafts in T-cells. These mice bear the Caenorhabditis elegans desaturase gene capable of converting n-6 PUFA into n-3 PUFA, resulting in substantially elevated levels of n-3 PUFA in tissues including T-cells. Kim et al.⁽Reference Kim, Fan and Barhoumi⁴⁸⁾ investigated the effect of this n-3 PUFA enrichment of lipid raft formation at the immunological synapse by co-culturing Laurdan-labelled CD4⁺ T-cells with anti-CD3 hybridoma cells (serving as antigen-presenting cells). They reported that raft formation was enhanced in the fat-1 cells compared to wild-type, but the relocalization of several signalling molecules into the immunological synapse and cell proliferation were suppressed⁽Reference Kim, Fan and Barhoumi⁴⁸⁾. The enhanced raft formation can be explained by the low affinity of DHA for cholesterol which effectively causes the coalescence of cholesterol-rich domains, consistent with immunogold data showing increased lipid raft clustering in response to DHA enrichment⁽Reference Kim, Fan and Barhoumi^48, Reference Chapkin, McMurray and Dvaidson⁴⁹⁾. There is no information regarding the extent to which membrane microdomains in human lymphocytes are modulated by dietary supplementation with n-3 PUFA, although lymphocyte lipids (in a whole-cell extract) are readily modified by fish oil supplementation⁽Reference Kew, Mesa and Tricon⁵⁰⁾ and murine T-lymphocyte rafts have been shown to be responsive to dietary fish oil⁽Reference Geyeregger, Zeyda and Zlabinger^51, Reference Switzer, Fan and Wang⁵²⁾. Interestingly, peripheral blood T-cells from patients with systemic lupus erythamatosus, an autoimmune disorder, contain higher levels of the ganglioside, GM1 and cholesterol, which may alter membrane organization and potentially create an impact on signalling homeostasis⁽Reference Kabouridis and Jury⁵³⁾. Higher levels of CD45 in lipid rafts in autoimmune lymphocytes have also been documented⁽Reference Kabouridis and Jury⁵³⁾. In an experimental model of colitis, n-3 PUFA prevented inflammation-induced exit of tight junction proteins from lipid rafts and decreased disruption of tight junctions in the intestinal mucosa⁽Reference Li, Zhang and Wang⁵⁴⁾. This suggests that the reported beneficial effects of n-3 PUFA in inflammatory bowel disease (and perhaps other inflammatory disorders), may be mediated, at least in part, through the modulation of membrane microdomains. Clearly, there is some compelling evidence from in vitro and animal experiments indicating that n-3 PUFA modulate the immune function by effects on membrane microdomains, yet direct evidence that this is physiologically relevant in human subjects is still lacking and should be considered for future research.

Fatty acid-derived mediators

Immune cell membranes typically have a high content of AA, which acts as a precursor for the synthesis of eicosanoids, the exact nature of which depends upon the cell type. It is well documented that the enrichment of cell membranes with EPA and DHA decreases the production of AA-derived eicosanoids, such as PGE₂, in a dose-dependent fashion⁽Reference Rees, Miles and Banerjee²⁰⁾. Incorporation of n-3 PUFA also results in the generation of a wider range of eicosanoids, since these fatty acids can also act as precursors for cyclooxygenase and lipoxygenase enzymes. These n-3 PUFA-derived eicosanoids are often (but not always) biologically less active than those derived from AA⁽Reference Calder¹⁴⁾. The transgenic fat-1 mice, which have tissue greatly enriched with n-3 PUFA, generate large amounts of PGE₃ in the colonic tissue after the induction of inflammation by dextran sodium sulphate⁽Reference Hudert, Weylandt and Lu⁵⁵⁾. The full extent of the number and nature of eicosanoids has not yet been realized, as evidenced by the increasing number of EPA- and DHA-derived eicosanoids which have been shown to be produced under physiological or pathological conditions and to have anti-inflammatory activities⁽Reference Serhan⁵⁶⁾. These include the emerging family of lipoxins, resolvins, protectins and most recently maresins, which appear to have important roles in the resolution of inflammation and return to homeostasis⁽Reference Serhan, Yang and Martinod⁵⁷⁾. A recent study demonstrated that deuterium-labelled or radioactively labelled n-3 PUFA could be identified in inflammatory exudates in a murine model of peritonitis within 2 h of induction⁽Reference Kasuga, Yang and Porter⁵⁸⁾. However, since DHA-derived resolvin D1, but not DHA itself, inhibited neutrophil movement and protected lung tissue from excessive leucocyte infiltration, the authors concluded that the resolution of inflammation required conversion to resolvins⁽Reference Kasuga, Yang and Porter⁵⁸⁾.