Plasma membrane lipid rafts are heterogeneous cholesterol and glycosphingolipid (GSL)-enriched microdomains, within which the tight packing of cholesterol with the saturated-acyl chains of GSLs creates a region of liquid-order relative to the surrounding disordered membrane. Thus lipid rafts govern the lateral mobility and interaction of membrane proteins and regulate a plethora of signal transduction events, including T-cell antigen receptor (TCR) signalling. The pathways regulating homoeostasis of membrane cholesterol and GSLs are tightly controlled and alteration of these metabolic processes coincides with immune cell dysfunction as is evident in atherosclerosis, cancer and autoimmunity. Indeed, membrane lipid composition is emerging as an important factor influencing the ability of cells to respond appropriately to microenvironmental stimuli. Consequently, there is increasing interest in targeting membrane lipids or their metabolic control as a novel therapeutic approach to modulate immune cell behaviour and our recent work demonstrates that this is a promising strategy in T-cells from patients with the autoimmune disease systemic lupus erythematosus (SLE).
Lipid raft-mediated T-cell signalling
Cluster of differentiation (CD)4+ T-cells are activated in response to T-cell antigen receptor (TCR) recognition of antigen peptide–major histocompatibility complex (MHC)II complexes on antigen-presenting cells and binding of co-stimulatory receptors, such as CD28. TCR ligation triggers phosphorylation of the TCR, which instigates signalling complex assembly and initiation of downstream signalling cascades that ultimately mediate CD4+ T-cell effector functions; proliferation and cytokine production.
The involvement of lipid rafts in TCR signalling was recognized soon after the lipid raft hypothesis was first proposed [1,2] and remains an area of active research. Upon TCR ligation, small, dynamic, rafts converge to form larger, more stable microdomains with an increased dependence on protein–lipid and protein–protein interactions [3,4]. These microdomains serve as signalling platforms and selectively incorporate signalling proteins to maintain a balance between activating proteins, including lymphocyte cell-specific protein tyrosine kinase (Lck) and adaptor molecule linker for T-cell activation (LAT) and inhibitory proteins, such as cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), to modulate the propagation and duration of TCR signalling.
The clustering of rafts can be visualized as membrane condensation at the immune synapse (IS) using polarity-sensitive fluorescent probes LAURDEN and di-4-ANEPPDHQ (ANE), which integrate into the membrane and emit different wavelengths of light in liquid-ordered raft and disordered non-raft regions [5,6]. Culture of Jurkat T-cells with 7-ketocholesterol (7-KC), an oxidized form of cholesterol, reduces membrane lipid order, prevents raft condensation in response to activation and disrupts downstream signalling . Furthermore 7-KC profoundly inhibits IS formation between Jurkat T-cells and antigen loaded Raji B-cells (Figure 1), clearly demonstrating the importance of membrane lipid organization in TCR-mediated signalling and T-cell function.
Cholesterol and oxysterol levels influence IS formation
As advances in microscopy and spectroscopy techniques provide new insight into the complexity and dynamic nature of this phenomenon, it is apparent there is still much to learn about the organization of the membrane and sequence of events required for appropriate assembly and dissolution of the IS [8,9].
Membrane raft heterogeneity: endogenous regulation of raft composition
Variations in membrane lipid composition can influence the properties and function of lipid rafts . Culture of Jurkat T-cells with polyunsaturated fatty acids (PUFAs) alters the lipid composition of immuno-isolated membrane fragments associated with activated TCR domains and prevents clustering of rafts in live cells . In primary human CD4+ T-cells, high membrane lipid order is associated with stable IS formation, robust proliferation and release of anti-inflammatory T-helper (TH) 2 cytokines interleukin (IL)-4 and -10. In comparison, intermediate order cells form an unstable IS and favour production of pro-inflammatory TH1 cytokines interferon (IFN)-γ and IL-6. T-cells with low lipid order do not respond to TCR stimulation and are more prone to apoptosis .
Membrane lipid order positively correlates with cholesterol content and negatively correlates with ganglioside M1 (GM1) levels, whereas GM3 is not differentially expressed . This demonstrates that the composition of lipid rafts contributes to relative membrane lipid order. Thus pathways that regulate GSL and cholesterol content of membrane rafts, can influence cell function.
Glycosphingolipid (GSL) levels in the plasma membrane are regulated by the expression and activity of their biosynthetic enzymes and their intracellular trafficking and degradation. Different immune-cell types exhibit distinct GSL ‘fingerprints’ , suggesting that regulatory processes may be cell type and even subset specific. For example, CD4+ T-cells exclusively require a-series GSLs (e.g., GM1 and GM3) for TCR activation whereas CD8+ T-cells rely on o-series and differentiating thymocytes regulate the expression of GSL biosynthesis enzymes accordingly .
The importance of GSL homoeostasis is demonstrated by lysosomal storage diseases (LSDs), a group of pathologies associated with mutations in proteins involved in the trafficking [NPC (Niemann Pick type C)] or metabolism (Fabry's and Gaucher's diseases) of lipids in lysosomes and result in aberrant accumulation of lipid species, including GSLs and cholesterol. The severity and nature of clinical manifestations varies, the most common being progressive neurological degeneration. Indeed, there is a striking co-incidence of Gaucher's disease and Parkinson's disease .
Mice globally deficient for uridine diphosphate (UDP)-glucose ceramide glucosyltransferase (UGCG), the first enzyme involved in GSL biosynthesis (Figure 2), are embryonic lethal. Depletion of other GSL enzymes results in mild to severe abnormalities, primarily neurological . Lipid raft defects are evident in some of these models, including impaired assembly of the tripartite glial cell-line-derived neurotrophic factor (GDNF) receptor and symptoms of Parkinson's disease in a GM1-deficient mouse . In contrast, a lacto/neolacto-series knockout exhibits up-regulation of GM1 lipid rafts in B-cells, which are enriched for B-cell activation molecules, resulting in an enhanced proliferative response to stimulation .
Schematic illustrating pathways for GSL biosynthesis
Membrane cholesterol homoeostasis is regulated by de novo biosynthesis, cellular uptake and efflux. Interestingly lipid raft stability can be influenced by differential pathways of cellular cholesterol biosynthesis. The Bloch pathway produces desmosterol, which has a lower affinity than cholesterol for raft domains , whereas the Kandutsch Russel branch yields lathosterol and 7-dehydrocholesterol, which generate more stable lipid rafts than cholesterol  (Figure 3). Furthermore oxidized metabolites of cholesterol (oxysterols) can differentially affect membrane rafts depending on the nature of their oxidation and structural similarity to cholesterol. For example 25-hydroxycholesterol and 27-hydroxychoelsterol promote raft formation, but 7β-hydroxycholesterol and 7-KC inhibit rafts and have cytotoxic effects .
Cholesterol biosynthesis and oxysterol formation
Oxysterols are also endogenous ligands for the liver X receptors (LXRs), transcription factors integral to the regulation of cellular cholesterol homoeostasis. In response to oxysterol binding, they up-regulate transcription of their target genes, including cholesterol ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, which are responsible for the efflux of cholesterol to apolipoprotein-A1 and high density lipoprotein (HDL) respectively, the inducible degrader of the low density lipoprotein (LDL) receptor (IDOL) and NPC proteins 1 and 2 (NPC1 and 2)  which regulate lysosomal/late endosomal trafficking and recycling of membrane lipids. In particular, NPC1 is involved in endocytosis and hydrolysis of LDL to unesterified cholesterol, which is transported to the plasma membrane or endoplasmic reticulum. Mutation of NPC1 causes 95% of cases of the NPC LSD .
It is well established that cholesterol efflux by ABCA1 disrupts lipid rafts , a mechanism which protects immune cells from HIV infection . Incubation of antigen-presenting cells with HDL or apolipoprotein-A1 results in raft depletion and reduced capacity to present antigen and activate T-cells . Regulation of NPC1 and NPC2 expression would also influence plasma membrane cholesterol and GSL levels. LXR activation has been reported to up-regulate expression of enzymes for the biosynthesis of ceramide, the substrate of GSL biosynthesis, in keratinocytes  and our recent work has shown treatment of ex vivo human CD4+ T-cells with LXR agonists transiently increases GM1 expression in the plasma membrane . Therefore, LXRs and their endogenous ligands, the oxysterols, regulate many pathways controlling membrane lipid homoeostasis and provide a link between serum lipoproteins and cellular lipid metabolism.
Lupus, lipid rafts and LXRs
LXRs also modulate a variety of immune responses, through transcriptional control of immune related genes and indirectly through regulation of cellular lipid metabolism. In T-cells LXR, activation reduces proliferation  and cytokine production  in response to activation, chemotaxis  and differentiation of the TH17 lineage . Whether regulation of membrane lipid composition contributes to these effects is currently unclear. However, our recent work has linked LXRs, membrane lipid rafts and dysfunction of T-cells in the prototypical autoimmune disease systemic lupus erythematosus (SLE).
T-cells from patients with SLE have an altered plasma membrane lipid raft profile, exhibiting increased cholesterol  and GSLs, in particular lastosylceramide, Gb3 and GM1 . Moreover a greater proportion has intermediate lipid order compared with healthy controls, as measured using ANE . This is accompanied by increased localization of tyrosine phosphatase CD45 to lipid rafts, where it dephosphorylates an inhibitory residue on Lck, increasing it propensity for activation, but also dephosphorylates the TCR . In contrast, when CD45 is excluded from rafts, it promotes extracellular signal-regulated kinase (ERK)-mediated raft clustering and IL-2 production . Expression of Lck is decreased whereas its ubiquitination is increased and although CTLA-4 expression is increased, it is excluded from lipid rafts and internalization is increased, preventing it from restraining TCR signalling . Therefore, in SLE patients, the T-cell membrane is characterized by altered lipid profile and altered distribution and turnover of crucial membrane signalling proteins. The net effect of this is a lowered threshold for activation, altered TCR signalling and an aberrant immune response.
In comparison with healthy donors, T-cells from SLE patients express elevated levels of LXRβ and its target genes NPC1 and NPC2 and of sterol response element-binding protein 2 (SREBP2), a transcription factor which promotes cholesterol biosynthesis, indicating pathways regulating plasma membrane lipid composition are altered in SLE. Furthermore, culture of healthy cells with SLE serum for 1 week was sufficient to induce an SLE membrane profile and this could be inhibited by addition of an LXR antagonist . However, treatment of a lupus prone mouse with synthetic LXR agonist GW3965 ameliorates symptoms  and, therefore, the role of LXR in disease pathogenesis remains uncertain.
Interestingly, further investigation of the increased expression of NPC1 and NPC2 revealed T-cells from SLE patients exhibit accelerated and sustained internalization and recycling of plasma membrane GSL and expression of lysosomal-associated membrane protein-1 (LAMP1) is also increased. Intriguingly a Fli1 (friend leukemia virus integration 1) heterozygote mouse on a lupus prone background exhibits a significantly reduced disease phenotype, which corresponds with lower expression and activity of lysosomal neuraminidase 1 during early disease , an enzyme which negatively regulates lysosomal exocytosis by modifying LAMP1 . Neurmainidases also breakdown gangliosides and levels of lactosylceramide and glucosylceramide were reduced in Fli1+/− in late disease . This further supports the hypothesis elevated GSL levels and dysregulated recycling of membrane lipids and proteins may be involved in SLE pathogenesis.
An alternative viewpoint is presented in a recent study which reports that glycolysis and oxidative phosphorylation are elevated in T-cells from murine models of SLE and SLE patients. Treatment of 7-month-old lupus-prone mice beginning to show clinical signs of disease with inhibitors of glycolysis and metabolism was able to normalize metabolic rate and effector function of T-cells and reversed immune complex deposition and renal pathology. Furthermore, inhibition of ox-phos in T-cells from SLE patients reduced IFN-γ production in response to TH1 polarization to a level comparable with healthy controls . This suggests that dysregulation of cellular glucose metabolism also participates in T-cell dysfunction in SLE. It would be interesting to investigate the interaction of glucose metabolism, membrane turnover and lipid rafts in this system.
Targeting T-cell lipid rafts to restore immune function
Manipulation of lipid rafts in vitro is able to reverse some of the defects in T-cells from SLE patients. Our recent work demonstrates in vitro treatment of CD4+ T-cells from SLE patients with N-butyldeoxynolirimycin (NB-DNJ), an inhibitor of glucosylceramide synthase used to treat patients with Gaucher's disease, is able to normalize GSL metabolism and reverses defects in T-cell signalling and function . Chemical or genetic reduction of GSL synthesis has previously been shown to inhibit proximal and downstream signalling in response to T-cell activation, including phosphorylation of Lck and LAT and reduced IL-2 secretion and proliferation. Interestingly, T-cells from a GM3 synthase-deficient mouse model also show reduced propensity to differentiate into TH17 cells .
Culture of T-cells from SLE patients with atorvastatin also normalizes plasma membrane GM1 expression, phosphorylation of Lck and ERK and production of IL-10 and IL-6, implicated in the pathogenesis of SLE . Statins, such as atorvastatin, inhibit the enzyme 3-hydroxy-3-methylglutaryl-coenxyme A (HMG-CoA), the rate limiting enzyme of the mevalonate pathway for biosynthesis of cholesterol and isoprenoids (Figure 3). Consequently, they alter the cholesterol content of the membrane, but it has also been shown isoprenoid modification of proteins (protein prenylation) can mediate membrane association. In an in vivo model of experimental autoimmune encephalomyelitis statin treatment ameliorated disease and it was shown that inhibition of isoprenoid synthesis disrupted recruitment of rat sarcoma (Ras) and RhoA to the plasma membrane, which was responsible for a shift from pro-inflammatory TH1 responses towards a TH2 phenotype .
Therefore, pharmacological depletion of GSL and cholesterol levels is able to normalize membrane lipid profile and immune function of SLE CD4+ T-cells. Indeed, there is increasing evidence that other anti-inflammatory mediators exert their effects by altering lipid and protein composition of lipid rafts.
PUFAs can integrate into the cytoplasmic leaflet of the plasma membrane, altering raft composition and structure. In particular omega-3 PUFAs, are known to exert a variety of anti-inflammatory effects  and in T-cells they can suppress activation and TH1 responses  and inhibit TH17 cell differentiation by displacing the IL-6 receptor glycoprotein 130 (GP130) from lipid rafts . Furthermore, PUFAs can alter lipid modification of proteins which mediate their recruitment to raft domains, for example, in Jurkat T-cells arachidonic acid and eicosapentaenoic acid inhibit palmitoylation of fybroblast endothelial kinase (Fyn), a tyrosine kinase involved in activation of ERK–MAPK (mitogen-activated protein kinase) signalling, blocking its association with membrane rafts at the TCR immune IS . Interestingly, LXR activation has recently been shown to increase synthesis of PUFAs in human macrophages .
Pantethine, a natural thiol known to have hypolipidaemic and hypocholesterolaemic effects, has been demonstrated to alter lipid raft composition in the Jurkat human T-cell cell-line, increasing levels of monounsaturated fatty acids and PUFAs, whereas lowering saturated fatty acid and cholesterol content. This inhibits in vitro CXCL12-mediated chemotaxis and transendothelial migration of effector T-cells isolated from human blood. Additionally, pantethine treatment decreases palmitoylation of LAT, which displaces it from detergent resistant to detergent soluble membrane fractions, an indication of dissociation from lipid rafts .
Overall, modulation of plasma membrane lipids or their regulatory pathways is a promising therapeutic strategy for restoration of T-cell function in autoimmunity. In particular, the hypothesis that defective membrane recycling could drive pathogenic alterations in membrane lipid profiles warrants further investigation.
Lipid rafts have been implicated in a wide range of infectious diseases and inflammatory disorders, including HIV, malaria, cancer, cardiovascular disease and neurodegenerative diseases. Ideas of ‘membrane lipid therapy’ , the targeting of specific membrane lipids; or ‘lipid replacement therapy’ , the administration of membrane lipid constituents to replenish and restore function in the plasma membrane or organelles, are certainly attractive and underexplored approaches.
In conclusion, further investigation of mechanisms facilitating lipid and lipid-raft mediated modulation of immune cell behaviour in health and disease could identify novel therapeutic targets to restore healthy immune responses.
This work was supported by the Arthritis Research UK [grant numbers 18106 and 19607 (to E.C.J.)]; and the Lupus UK (to E.C.J.); and the British Heart Foundation PhD studentship (to K.E.W.).
cluster of differentiation
cytotoxic T-lymphocyte associated protein 4
extracellular signal-regulated kinase
friend leukemia virus integration 1
high density lipoprotein
lysosomal-associated membrane protein-1
linker for T-cell activation
lymphocyte cell-specific protein tyrosine kinase
low density lipoprotein
lysosomal storage disease
liver X receptor
Niemman Pick type C
polyunsaturated fatty acid
systemic lupus erythematosus
T-cell antigen receptor
uridine diphosphate (UDP)-glucose ceramide glucosyltransferase
Metabolic Drivers of Immunity: Held at Aston University, UK, 25 Mar 2015.