Polyunsaturated Fatty Acid-Derived Lipid Mediators and T Cell Function
1Manchester Pharmacy School, Faculty of Medical and Human Sciences, The University of Manchester, Manchester, UK
2Centre for Biochemical Pharmacology, William Harvey Research Institute, Queen Mary University of London, London, UK
*Correspondence: Anna Nicolaou, Manchester Pharmacy School, Faculty of Medical and Human Sciences, The University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK e-mail: anna.nicolaou@manchester.ac.ukAbstract
Fatty acids are involved in T cell biology both as nutrients important for energy production as well as signaling molecules. In particular, polyunsaturated fatty acids are known to exhibit a range of immunomodulatory properties that progress through T cell mediated events, although the molecular mechanisms of these actions have not yet been fully elucidated. Some of these immune activities are linked to polyunsaturated fatty acid-induced alteration of the composition of cellular membranes and the consequent changes in signaling pathways linked to membrane raft-associated proteins. However, significant aspects of the polyunsaturated fatty acid bioactivities are mediated through their transformation to specific lipid mediators, products of cyclooxygenase, lipoxygenase, or cytochrome P450 enzymatic reactions. Resulting bioactive metabolites including prostaglandins, leukotrienes, and endocannabinoids are produced by and/or act upon T leukocytes through cell surface receptors and have been shown to alter T cell activation and differentiation, proliferation, cytokine production, motility, and homing events. Detailed appreciation of the mode of action of these lipids presents opportunities for the design and development of therapeutic strategies aimed at regulating T cell function.
Introduction
The regulation of energy metabolism is crucial to T cell-mediated immunity including activation, proliferation, and differentiation (1). Following recognition of antigen in the lymph nodes, naïve T lymphocytes undergo massive clonal expansion and differentiation, followed by a contraction or death phase, and the establishment and maintenance of immunological memory (2, 3). Before undergoing division, T cells activate biosynthetic pathways for the production of proteins, nucleic acids, lipids, carbohydrates, and other “building blocks” necessary for the generation of new cells. Following this stage, the metabolic machinery of T cells is reprogramed, switching from the β-oxidation of fatty acids in naive T cells to the glycolytic pathways in activated T cells (4–6).
Downstream of T cell receptor (TCR) signaling, phosphatidylinositol 3′-kinase (PI3K) leads to the activation of the serine–threonine kinase AKT, which promotes glucose metabolism by stimulating the localization of the glucose transporter Glut1 to the plasma membrane, and the activity of hexokinase and phosphofructokinase, two rate-limiting enzymes of the glycolytic pathway. Increased glycolytic flux enables activated T cells to generate ATP and, at the same time, efficiently utilize carbon sources in the form of amino acids and lipids for the biosynthesis of proteins and membranes necessary for the expansion phase that characterizes the immune response (7–11). AKT also controls the activation state of mammalian target of rapamycin (mTOR), a sensor of nutritional and energetic status in cells that promotes protein synthesis.
T cell activation also initiates distinct transcriptional programs, which determine their differentiation into functional subsets depending on the context [cytokines, prostaglandins (PG), and other extracellular signals] in which they were activated (12–14). These subsets define the characteristics of the immune response. Whereas CD8+ T cells differentiate into cytotoxic T lymphocytes that kill infected host cells, CD4+ T lymphocytes differentiate into either the Th1, Th2, or Th17 subset of helper T cells (effector T cells) that mediate appropriate immune responses or into induced regulatory T cells (iTreg cells) that suppress uncontrolled immune responses (12). There is evidence that the cytokine milieu in which T cells differentiate can influence their metabolic programing. A comparison of activated T cells responding to related cytokines IL-2 and IL-15 illustrates the differential regulation of T lymphocyte metabolism by distinct cytokine environments: IL-2 promotes elevated glucose metabolism and glycolysis, while IL-15 does not maintain this metabolic state and T cells responding to IL-15 are smaller with reduced nutrient uptake and glycolysis (15, 16).
After clearance of the infection, most clonally expanded and differentiated T cells undergo apoptosis (contraction phase). The surviving antigen-specific T cells (memory T cells) are responsible for enhanced immunity after re-exposure to the same pathogen. Of these various T cell subsets, the iTreg cells and memory T cells rely on lipid oxidation as a major source of energy, whereas cytotoxic T lymphocytes and effector T cells are characterized by high glycolytic activity (17–19).
Further to oxidation for energy production, fatty acids are involved in many other aspects of T cell biology. In particular, omega-3 polyunsaturated fatty acids (n-3 PUFA) are recognized as modulators of inflammation and immunity mediating their pleiotropic activity through regulation of gene expression, influencing signaling cascades, and altering the composition of the cellular membranes (20, 21). The latter has implications for the structure and function of the membrane, as well as a direct impact on the production of n-6 and n-3 PUFA-derived bioactive lipids including PG, leukotrienes (LT), resolvins (Rv), protectins (PD), endocannabinoids, and related congeners.
Although the immunomodulatory properties of PUFA have been known for many years, the molecular mechanisms underlying these properties are not fully understood. It has been shown that n-3 PUFA suppress antigen presentation, T cell activation and proliferation, and lower the expression of signature cytokines (21–27). Disappointingly, early studies using daily supplementation with foods rich in n-3 PUFA failed to show significant improvement in organ transplantation rejection (28, 29). However, recent reports indicate that administration of purified eicosapentaenoic acid (EPA; 20:5n-3) induces the differentiation of regulatory T cells through upregulation of peroxisome proliferator-activated receptor γ (PPARγ), a ligand-activated nuclear receptor that regulates lipid and glucose metabolism, leading to increased allograft survival (30, 31).
Following this direction, studies have explored the effect of cellular incorporation of the main n-3 PUFA, EPA, and docosahexaenoic acid (DHA; 22:6n-3). These fatty acids can alter the composition and molecular organization of membrane rafts with a consequent impact on the activity of raft-associated signaling proteins and related events. Examples include recruitment and activation of PLCγ and F-actin, impairing mitochondrial translocation necessary to maintain Ca+ signaling for NFκB and AP-1 activation and IL-2 secretion, and suppression of phosphatidylinositol-dependent actin remodeling, all linked to reduced CT4+ T cell activation [recently reviewed in Ref. (20)]. Importantly, many of the PUFA mediated activities are conveyed through their metabolites that tend to be produced and metabolized upon request, can act near the site of their synthesis or transported via circulation and in this way mediate systemic effects (autacoids). These families of potent mediators are intimately involved in inflammation and immunity, with pro- and/or anti-inflammatory, proliferative, and chemoattractive activities (21).
Overall, these new findings suggest that a better understanding of the molecular mechanism of action of PUFA may lead to the development of effective therapeutics. In this article, we will overview the current knowledge of the function and impact of eicosanoids and related metabolites, as well as that of endocannabinoids and their congeners on T cell function, and examine potential applications in biomedical research.
PUFA-Derived Lipid Mediators: Biosynthesis and Metabolism
The cellular membrane serves as a pool of PUFA available for further metabolism to various bioactive lipids. These potent autacoids act as local hormones and are produced upon request following the activation of signaling pathways or effect of environmental and other stimuli. The arachidonic acid (AA; C20:4n-3)-derived eicosanoids are some of the best known and studied bioactive lipids. The term “eicosanoids” is used to describe the bioactive derivatives of three fatty acids with 20-carbon acyl chains, namely: AA, EPA, and dihomo-gamma linolenic (DGLA; 20:3n-6). These metabolites, although mostly linked to inflammation, are also involved in cell migration, proliferation, chemotaxis, and immune reactions (32–34). Eicosanoids and related mediators derive from the activities of cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 (CYP) epoxygenases and mono-oxygenases (Figures 1 and 2) [reviewed in Ref. (35)]. The term “endocannabinoids” refers to endogenous lipids ligands of the cannabinoid receptors CB1 and CB2. These are also derivatives of AA, while other PUFA ethanolamides are now recognized as members of this family (36). Although endocannabinoids can be metabolized by COX and LOX, their precursor phospholipids and metabolism are different to eicosanoids (Figure 3).
Cyclooxygenase-mediated formation of prostanoids
The eicosanoid cascade starts with the activation of phospholipases (PL), predominantly PLA2 but also PLD and diacylglycerol (DAG) lipase that release AA and other PUFA from the cellular membrane (35). The family of PLA2 comprises a large number of enzymes with distinct characteristics in terms of their activation, cellular localization, and substrate specificity (37). There is evidence for the presence of various PLA2 isoforms in primary T cells and the Jurkat T cell line, including cPLA2, sPLA2, and iPLA2 (38–42). Inducible isoforms of PLC and DAG lipase have also been identified in tumor and peripheral T lymphocytes (42, 43).
Free AA is then metabolized via the constitutive and inducible COX isoforms (COX-1 and -2, respectively) to the unstable endoperoxide PGH2 that is then transformed to PG, thromboxanes (TX), and prostacyclin (PGI2) via tissue specific terminal prostaglandin synthases (Figure 1); these COX-derived mediators belong to the family of eicosanoids and are collectively known as prostanoids. Apart from AA, prostanoids are formed from the other two 20-carbon containing PUFA, DGLA, and EPA, with the resulting metabolites having different activities and being considered less-inflammatory than the AA-derived ones (35, 44).
The exact profile of prostanoids is determined by the prevalence of specific synthases in the cell type or tissue of interest. PGE2 is produced by prostaglandin E synthase (PGES) that is found as membrane bound (mPGES-1 and -2) or cytosolic (cPGES). mPGES-1 is an inducible isoform and is frequently found co-expressed with COX-2 (45, 46). PGD2 is produced by the hematopoietic-type (H-PGDS) or the lipocalin-type (L-PGDS) synthases (47), while further non-enzymatic hydrolysis of PGD2 gives rise to the anti-inflammatory cyclopentanone PGs PGJ2 and 15d-PGJ2 (48, 49). PGF2α is produced either directly from PGH2 via the prostaglandin F synthase (PGFS) or through further metabolism of PGE2 and PGD2 by PGE 9-ketoreductase and PGD 11-ketoreductase, respectively (50). Prostacyclin (PGI2) is produced via the prostacyclin synthase (PGIS) and is usually detected as its stable but inactive metabolite 6-keto-PGF1α (51). Finally, thromboxane synthase (TXS) converts PGH2 to TXA2, an unstable prostanoid that is quickly hydrolyzed to the stable but inert metabolite TXB2 (51). The bioactivity of prostanoids is mediated through G protein-coupled receptors for PGE2, PGD2, PGF2α, PGI2, and TXA2, designated EP, DP, FP, IP, and TP, respectively. Pharmacological studies into their ligand-binding profiles and signal transduction pathways, and genetic analysis led to their classification into eight groups (EP1, EP2, EP3, EP4, DP1, FP, IP, and TP) although new developments have revealed the presence of a second PGD receptor, DP2, and the presence of heterodimers (52, 53). Overall, prostanoids are potent autacoids and their levels are controlled through enzymatic catabolism via dehydrogenations and reductions resulting in the formation of metabolites with significantly reduced bioactivities (e.g., 15-keto- and 13,14-dihydro-15-keto PGs) (54).
Lipoxygenase-mediated production of leukotrienes and other hydroxy fatty acids
Lipoxygenases mediate the oxygenation of free fatty acids including AA and other PUFA. Their activities are commonly defined by their positional selectivity when they oxygenate AA and, following this system, the main mammalian LOX enzymes are defined as 5-, 12-, and 15-LOX. They catalyze the stereoselective insertion of OH in the S configuration, with the exception of a mammalian skin-specific enzyme 12R-LOX. The products of LOX reactions are unstable hydroperoxides that are then reduced to hydroxy acids (55–57) (Figure 2). 5-LOX acts in concert with 5-lipoxygenase activating protein (FLAP) to metabolize AA to 5S-hydroperoxyeicosatetraenoic acid (HPETE) that is further reduced to 5S-HETE or dehydrated to LTA4, an unstable epoxide containing a conjugated triene system characteristic of all LT. LTA4 can be metabolized to LTB4 or form the cysteinyl LT, LTC4, LTD4, LTE4 following conjugation with reduced glutathione (58). 5S-HETE can be also enzymatically reduced to the 5-oxo-eicosatetraenoic acid (5-oxo-ETE), a chemoattractant mediator (59). Mammalian 12- and 15-LOX isozymes oxygenate a range of PUFA, both free and esterified in membrane phospholipids and lipoproteins (57), forming a multitude of mono- and poly-hydroxy fatty acids: e.g., AA produces hydroxyeicosatetraenoic acids (HETE), EPA generates hydroxyeicosapentaenoic acids (HEPE), DHA produces docosanoids including hydroxydocosahexaenoic acids (HDHA), linoleic acid (LA; 18:2n-6) forms octadecanoids such as hydroxy octadecadienoic acids (HODE), DGLA forms hydroxyeicosatrienoic acids (HETrE), etc.
Cytochrome P450 mediated fatty acid epoxides and their derivatives
Cytochrome P450 mono-oxygenases relevant to PUFA metabolism catalyze epoxygenations and mid-chain and omega-hydroxylations producing a range of LOX-like mono-hydroxy fatty acids (e.g., HETE, HEPE, HODE) although not necessarily of the S configuration [reviewed in Ref. (35)]. Interestingly, partially inhibited COX-2 (e.g., acetylated COX-2 following treatment with aspirin) can also generate LOX-like products with the OH group at R configuration, e.g., 15R-HETE from AA and 18R-HEPE from EPA (60). These metabolites are important in aspects of transcellular metabolism where sequential LOX/LOX or acetylated COX-2/LOX or CYP/LOX reactions involving more than one cell types are involved in the formation of multi-hydroxy fatty acid species. These include the lipoxins (LX) that are tri-hydroxytetraene-products of AA, and the di- and tri-hydroxy-PUFA termed Rv, PD, and maresins (MaR) that are derivatives of EPA and DHA. All these mediators are involved in inflammation and immunity exhibiting a range of protective roles (61–63).
The endocannabinoids
The endocannabinoids anandamide (arachidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2AG) are derivatives of AA and act as endogenous ligands to the cannabinoid receptors CB1 and CB2 [reviewed in Ref. (36)]. This family of bioactive lipids has been extended to include other fatty acid ethanolamides and glycerols, while recent findings regarding their metabolism suggest a wider involvement in inflammation and immunity. The biochemical precursors of AEA and its congers are various N-acylated ethanolamine phospholipids (NAPE) that found in very low concentrations in the biological membranes and are hydrolyzed by NAPE-specific PLD or PLC-type lipases. 2AG production is mediated by PLC-diacylglycerol lipase. AEA and 2-AG can be deactivated via hydrolysis mediated by fatty acid amide hydrolases (FAAH) or can be metabolized by COX-2 to generated prostaglandin ethanolamides known as prostamides (e.g., PGE2-EA) and prostaglandin glyceryl esters (e.g. PGE2-GE) (Figure 3) (52). LOX isozymes can also metabolize these lipids although the prevalence and bioactivities of the resulting mediators remain to be explored.
Concluding Remarks
While current evidence support a key role for PUFA-derived bioactive lipids in the regulation of T cell immunity (Table 1), the complexity of their biological properties and the lack of a comprehensive understanding of their exact contribution to different stages of the immune response hinders the identification of mediators of interest either as markers or as target compounds for drug development. In general, it appears that lipid mediators regulate T helper cell polarization into Th1/Th2 and Th17 cells, a key event in many immune-mediated diseases. Despite the molecular mechanisms for this effect and the regulatory role of these lipids on other T cell functions have yet to be explored, an extensive number of studies in mice and humans underscore their therapeutic potential.
| Lipid mediator | Receptor | Effect on T cells | Reference |
|---|---|---|---|
| PGE2 | EP1, EP2/EP4 | Differentiation | (73, 78, 79, 81, 88) |
| EP2/EP4 | Proliferation | (72, 81, 83, 85) | |
| EP2/EP4 | Cytokine production | (72, 77–80) | |
| EP2/EP4 | Apoptosis | (77, 91) | |
| EP2/EP4 | Motility of T cells | (93–95) | |
| EP2/EP4 | Treg recruitment | (102) | |
| EP2/EP4 | Th1, Th2, Th17 balance | (14, 74–76, 78, 79) | |
| PGD2 | DP1 | Differentiation of Th2; T cell apoptosis | (110, 120, 121) |
| DP1, DP2 | Recruitment, proliferation of Th2 | (111) | |
| DP2 | Activation, cytokine production, trafficking, and motility of Th2 | (106, 107, 113–119) | |
| 15d-PGJ2 | DP2 | Suppression of Th1 and promotion of Th2 | (103, 112) |
| DC–T cell interaction | (120, 121) | ||
| PGF2α | FP | Th17 differentiation | (123) |
| PGI2 | IP | Th1/Th2 balance | (13) |
| Th1, Th17 differentiation | (125, 127, 129) | ||
| TXA2 | TP | Inhibition of T cell proliferation | (134) |
| Mediation of DC-T cell interactions | (130) | ||
| LTB4 | BLT1 | Homing | (141, 144) |
| Differentiation, proliferation, and cytokine production | (146–149) | ||
| CysLTD4 | CysLT1 | Th2 differentiation | (151–153) |
| CysLTE4 | |||
| 12-HETE | Weak T cell chemotaxis | (158, 159) | |
| Metabolic changes | (137) | ||
| 15-HETE | Proliferation | (160–162) | |
| Th1 responses | (164) | ||
| LXA4 | BLT1 | Cytokine production | (167) |
| AEA | CB2 | Suppression of Th1 and Th17 proliferation and cytokine release | (185, 186) |
| – | Inhibition of proliferation; apoptosis via membrane rafts | (187, 188) | |
| Increased Tregs | (184) | ||
| 2AG | CB2 | T cell migration | (179) |
| Suppression of cytokine production via PPAR-γ | (189) |
This concept is supported by the large number of studies using their precursor fatty acids. Of particular importance is the focus on n-3 PUFA that have been explored as anti-inflammatory and immune-protective agents for a range of diseases and relevant experimental models including psoriasis, rheumatoid arthritis, and atherosclerosis (32, 33, 200). A recent study has shown that dietary n-3 PUFA favorably modulate intestinal inflammation in part by downregulating pathogenic T cell responses (201). The Fat-1 mouse, a genetic model that synthesizes long-chain n-3 PUFA de novo, was shown to be relatively resistant to colitis induction due to a reduced differentiation of Th17 cells and related cytokines (202). The immunoregulatory potential of a number of fatty acids has been reported over the years including that of DGLA and GLA (203), stearidonic acid (204) as well as various CLA mixtures used for inflammatory bowel syndrome and human Crohn’s disease (205). Parenteral administration of fatty acids has been shown to ameliorate disease via immunomodulatory effect in a model of rat sepsis (206), A randomized study in patients awaiting carotid endarterectomy showed that n-3 PUFA ethyl esters are incorporated into advanced atherosclerotic plaques and higher plaque EPA is associated with decreased plaque inflammation and T cell infiltration, and increased stability following dietary supplementation with EPA (207).
Furthermore, altering the profile of lipid mediators to strengthen the responses of T cells may be of value to cancer immunotherapy and could result in the development of potent and/or less toxic therapeutics. For example, it is well-documented that most tumors express PGE2 and this can contribute to immune suppression (103, 208). Pharmacological inhibition of PGE2 via non-steroidal anti-inflammatory drugs or EP receptor agonists could be supported or even replaced by systemic administration of EPA, precursor of the less potent eicosanoid PGE3 and the anti-inflammatory resolving series E (RvE) that can tone down the PGE2-mediated effects. Finally, a large number of other investigations have reported that immunonutrition with fatty acids leads to amelioration of a variety of immune-mediated disease by targeting T cell function. Examples include studies showing that the use of n-3 PUFA can improve lung injury and sepsis in animal models, and reduce infectious complications in patients undergoing major surgery and following severe trauma (209–211), while other reports draw attention to the contribution of fatty acids and their mediators in vaccine-induced immunity in infants, the prevention of experimental autoimmune encephalomyelitis through inhibition of Th1/Th17 differentiation by DHA, EPA-mediated protection of cardiac allografts, and amelioration of contact dermatitis following DHA and AA supplements (212–215).
Overall, there is a strong case for further developing therapeutic approaches based on the use of bioactive lipids as immunomodulators. The unmet challenge to fully exploit their therapeutic potential will be to unravel the circuits and molecular mechanisms by which these powerful mediators impact on T cell-mediated immunity.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Acknowledgements
Research support provided by the Wellcome Trust (Anna Nicolaou: WT094028) and British Heart Foundation (Claudio Mauro: FS/12/38/29640 and Federica Marelli-Berg: RG/09/002/2642) is gratefully acknowledged. We thank Dr. Sharon A. Murphy for her contribution to the figures shown in the manuscript.