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Diet-induced docosahexaenoic acid non-raft domains and lymphocyte function
ARTICLE IN PRESS Prostaglandins, Leukotrienes and Essential Fatty Acids 82 (2010) 159–164
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Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa
Diet-induced docosahexaenoic acid non-raft domains and lymphocyte function Saame Raza Shaikh a,b,n a b
Department of Biochemistry and Molecular Biology, Brody School of Medicine, East Carolina University, 600 Moye Blvd, Greenville, NC 28590, USA The Metabolic Institute for the Study of Diabetes and Obesity, Brody School of Medicine, East Carolina University, 600 Moye Blvd, Greenville, NC 28590, USA
abstract Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid (PUFA) that generally suppresses the function of T lymphocytes and antigen presenting cells (APCs). An emerging mechanism by which DHA modifies lymphocyte function is through changes in the organization of sphingolipid/cholesterol lipid raft membrane domains. Two contradictory models have been proposed to explain how DHA exerts its effects through changes in raft organization. The biophysical model, developed in model membranes, shows that DHA-containing phospholipids form unique non-raft membrane domains, that are organizationally distinct from lipid rafts, which serve to alter the conformation and/or lateral organization of lymphocyte proteins. In contrast, the cellular model on DHA and rafts shows that DHA suppresses lymphocyte function, in part, by directly incorporating into lipid rafts and altering protein activity. To reconcile opposing biophysical and cellular viewpoints, a major revision to existing models is presented herein. Based largely on quantitative microscopy data, it is proposed that DHA, consumed through the diet, modifies lymphocyte function, in part, through the formation of nanometer scale DHA-rich domains. These nano-scale domains disrupt the optimal raft-dependent clustering of proteins necessary for initial signaling. The data covered in this review highlights the importance of understanding how dietary n-3 PUFAs modify lymphocyte membranes, which is essential toward developing these fatty acids as therapeutic agents for treating inflammatory diseases. & 2010 Elsevier Ltd. All rights reserved.
1. Docosahexaenoic acid (DHA), immunosuppression, and membrane domains Docosahexaenoic acid is an n-3 polyunsaturated fatty acid (PUFA) and a major component of fish oil. The fatty acid is recognized to have therapeutic value as an immunosuppressant for the treatment of inflammation associated afflictions, such as cardiovascular disease, rheumatoid arthritis, and colitis [1–5]. At a cellular level, DHA suppresses the function of specific lymphocytes associated with inflammation [6]. However, a major limitation of using the fatty acid effectively as an adjuvant immunosuppresant has been a limited understanding of how the fatty acid exerts its effects at a molecular level. The molecular mechanisms that have been proposed by which DHA modifies lymphocyte function include changes in eicosanoid metabolism, gene transcription, post-translational modification, protein trafficking, production of D-resolvins, and alteration of
n
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plasma membrane microdomain organization. The focus of this review is on DHA and lipid raft microdomain organization. Lipid rafts are transient sphingolipid/cholesterol membrane domains that serve to compartmentalize cellular signaling events [7]. Modification of these domains with DHA has a broad impact given that dietary intake of the fatty acid results in its incorporation into membrane phospholipids of virtually all tissues that are naturally devoid of n-3 PUFAs [8]. Two contradictory models will be reviewed that explain how DHA-containing phospholipids can suppress lymphocyte function through changes in the molecular organization of lipid rafts. First, data are presented to show that DHA adopts unique molecular orientations which make it an excellent candidate for modifying membrane domain organization and protein activity. Next, a biophysical model is presented to show that DHA-containing phospholipids form non-raft domains that are organizationally distinct from lipid rafts, to modify protein activity. The biophysical studies are then contrasted with cellular data to show that DHA can suppress the function of lymphocytes through changes in plasma membrane rafts. The model that emerges from these studies is one in which DHA localizes directly into lipid rafts to
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Table 1 Summary of biophysical studies to show that DHA-containing phospholipids form organizationally distinct non-raft domains. Method
Result
References
Atomic force microscopy
A heteroacid DHA-containing phospholipid forms distinct domains on a submicron scale in the presence of sphingomyelin and cholesterol. Heteroacid DHA-containing phospholipids form distinct domains on a micron scale when mixed with phospholipids containing saturated acyl chains The rigid structure of cholesterol is sterically incompatible with the highly flexible structure of DHA. The structural incompatibility drives formation of DHA-rich domains. The size of heteroacid DHA-containing phospholipids domains is estimated to be 20 nm. Sphingomyelin and cholesterol are immiscible with heteroacid and homoacid DHA-containing phospholipids.
[25]
Sphingomyelin and cholesterol are immiscible with heteroacid and homoacid DHA-containing phospholipids. Heteroacid and homoacid DHA-containing phospholipids localize to ‘‘non-raft’’ detergent soluble fractions of liposomes DHA-containing FRET probes cluster away from phospholipids containing saturated acyl chains DHA-containing FRET probes show formation of domains in the presence of rhodopsin and phospholipids with saturated acyl chains
[22,27] [24,25]
Widefield microscopy X-ray diffraction 2
H NMR spectroscopy Differential scanning calorimetry (DSC) Pressure–area isotherms Detergent extraction of liposomes FRET FRET
modify protein activity. The review is concluded, based on very recent microscopy data, with a major revision of existing models on how DHA-rich domains modify lipid rafts. According to this model, consumption of DHA in the diet results in the formation of nanometer scale DHA-rich domains that serve to modify lipid raft and protein lateral organization and/or conformation.
[21] [28,29] [26] [21,22,25–27]
[20] [30]
Recent NMR and MD simulation studies show that DHA acyl chains can bind in the grooves between the helices of rhodopsin [14–17] which in turn disrupts the interhelical packing of rhodopsin and thereby its conformation and function [11,18].
2.3. Biophysical model of DHA and lipid rafts 2. Biophysical studies of DHA and membrane structure 2.1. Molecular orientation of DHA The molecular structure of DHA, due to its 22 carbons and 6 double bonds, is highly disordered relative to other unsaturated and saturated fatty acids [9]. A series of spectroscopic studies in model membranes, supported by molecular dynamic (MD) simulations, show that DHA, due its highly flexible structure, rapidly converts between various conformational states on a subnanosecond time scale [10,11]. The flexibility of the acyl chain is conferred by low potential energy barriers to rotation about the single carbon–carbon bonds that compensate for the rigidity of the double bonds [11]. The increased flexibility results in a higher density of DHA, relative to other unsaturated fatty acids, toward the water interface of the membrane [12]. This is highly unusual given that the presence of any fatty acid near the water interface would appear to be thermodynamically unfavorable. As a result of high conformational flexibility of DHA, various physical properties of the membrane are modified. These include changes in membrane elasticity, hydrophobic mismatch, curvature stress, vesicle fusion, permeability, microviscosity, phospholipid flipflop, and membrane lateral organization [9]. 2.2. DHA and protein activity The unique physical properties of DHA can alter protein conformation or lateral organization, which in turn would modify cellular activity. One example of a protein that undergoes changes in conformation in response to DHA’s effects on the physical properties of the membrane is rhodopsin, the photo-inducible G protein coupled receptor of the rod outer segment (ROS). Functional studies of ROS membranes isolated from animals fed with n-3 PUFA deficient diets showed a drastic reduction in G protein coupled receptor signaling as a consequence of replacing DHA with docosapentaenoic acid (DPA, 22:5n-6) [13]. The change in signaling was a consequence of changes in acyl chain order [13].
One hypothesis that emerged nearly a decade ago is that DHA, due to its high conformational flexibility, modified cellular activity by altering the lateral organization of both lipid and protein molecules [9]. In support of this view, a series of studies with fluorescence resonance energy transfer (FRET), pressure– area isotherms, and differential scanning calorimetry (DSC) showed that heteroacid and homoacid DHA-containing phosphatidylcholine (PC) and phosphatidylethanolamine (PE) phospholipids did not mix with homoacid phospholipids containing saturated acyl chains (Table 1) [19–21]. This led to the hypothesis that DHA-containing phospholipids could form unique membrane domains that excluded saturated acyl chains. However, a major limitation of these studies was that the phospholipids used as controls contained saturated acyl chains with little physiologically relevance. More recent membrane studies, using a physiologically relevant model system, have led to a model in which DHAcontaining phospholipids form novel organizationally distinct domains (Fig. 1). Atomic force microscopy, DSC, and 2H NMR studies show that a heteroacid PE containing palmitic acid in the sn-1 acyl chain position and DHA in the sn-2 acyl chain forms membrane domains distinct from the lipid raft molecules sphingomyelin and cholesterol [22–27]. In contrast, a heteroacid PE containing palmitic acid in the sn-1 position and oleic acid in the sn-2 position does not form membrane domains in the presence of lipid raft molecules. The molecular mechanism for the formation of DHA-rich domains came from X-ray diffraction measurements that demonstrated that the highly flexible DHA acyl chain is sterically incompatible with the rigid structure of cholesterol [28,29]. In addition, very recent data also show unfavorable molecular interactions between the DHA acyl chain and the saturated acyl chains of sphingomyelin (Shaikh, Wassall, and Stillwell, unpublished results). Therefore, steric incompatibility of DHA with cholesterol and limited immiscibility with sphingomyelin appears to be the trigger for the formation of DHArich domains. These studies are in agreement with the work of others to show that cholesterol preferentially interacts with the sn-1 saturated acyl chains of heteroacid phospholipids with an
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Fig. 1. Contradictory models on how DHA-containing phospholipids modify lipid rafts and raft associated proteins. (Left) According to the biophysical model, n-3 PUFAs cannot sterically interact with cholesterol and therefore do not incorporate into rafts. Instead, n-3 PUFAs form their own organizationally distinct domains. (Right) According to the cell based model, n-3 PUFAs incorporate into both raft and non-raft regions of the membrane. Both models predict that incorporation of n-3 PUFAs into rafts will modify the lateral organization of proteins (purple) from the raft to non-raft phase, or vice versa. Note that lipid and protein molecules are not drawn to scale. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
sn-2 polyunsaturated acyl chain [19,30]. A very recent study in liposomes defines the size of DHA-rich domains to be 20 nm or less [26]. The major prediction of the biophysical model on DHA and lipid rafts is that lymphocyte function will be modified in response to the formation of DHA-rich domains that serve to alter protein activity (Fig. 1, Left). The model states that the formation of DHA-rich domains, as a consequence of dietary consumption of fish oil, will displace proteins from lipid rafts to non-rafts or vice versa. In addition, formation of DHA-rich domains could modify the conformation of membrane proteins. In support of this view, liposomes of DHA-containing phospholipids altered the conformation of the major histocompatibility complex (MHC) class I protein, which has a central role in antigen presentation to cognate CD8 + T lymphocytes (described below) [31]. The same laboratory also showed that fusion of lymphocytes with DHA-containing phospholipids modified the conformation of the CD8 co-receptor on the surface of CD8 + T cells [32]. A major limitation of the biophysical DHA-lipid raft model is that it is largely based on studies with liposomes and requires validation at the cellular and whole animal levels.
3. DHA suppresses lymphocyte function through changes in membrane domain organization Antigen presenting cells (APCs), which have a central role in adaptive immunity, present peptides to cognate T cells in the context of MHC class I or class II molecules. MHC class I molecules, found on the surface of all nucleated cells, present self or viral peptides from the cytosol to CD8 + T lymphocytes. MHC class II molecules, found on the surface of dendritic cells (DCs), macrophages, and B cells present peptides from pathogens that either reside within, or were endocytosed into, intracellular compartments. T cells are activated on encountering antigen,
which results in either lysis of target cells by CD8 + T lymphocytes or recruitment of other effector cells by CD4 + T cells. Both APC and T cell function are sensitive to treatment with n-3 PUFAs at the cellular and whole animal levels, as described below.
3.1. The n-3 PUFA-lipid raft model in T cells Several in vitro and ex vivo animal and human studies have shown that n-3 PUFAs suppress CD4 + T cell activation [33–42]. This is highly significant given that CD4 + T cells have a role in the production of inflammatory cytokines. One emerging model to explain the suppression of T cell activation by n-3 PUFAs is through changes in the organization of lipid rafts [43]. According to this model, (Fig. 1, Right), DHA and EPA incorporate directly into lipid raft domains to modify the lateral organization of plasma membrane proteins from rafts to non-rafts, or vice versa. The change in protein lateral organization suppresses downstream signaling events necessary for activation of genes involved in cytokine production and T cell proliferation. One major limitation of this model is that it is heavily based on the use of detergent extraction at cold temperatures. It is now accepted that detergent can induce artifacts and does not report on changes in lipid or protein lateral organization on the micro to nanometer length scales on which domains form [44,45]. In addition, the model is contradictory to the aforementioned biophysical model, which states that n-3 PUFA-containing phospholipids cannot incorporate into rafts [46]. One approach to overcome the limitations of detergent extraction has been the use of quantitative microscopy to address changes in lipid raft organization in response to n-3 PUFAs. Two recent microscopy studies, which are contradictory, have attempted to explain how n-3 PUFAs modify lipid rafts. Both studies have tested the effects of n-3 PUFAs on lipid raft order in the T cell immunological synapse, a highly dynamic interface between T
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cells and APCs that displays spatio-temporal resolution [47]. The first study showed that CD4 + T cells from the fat-1 transgenic mouse, which have higher levels of n-3 PUFAs in their membranes compared with those from wild type mice, displayed increased order within the immunological synapse compared to cells isolated from wild type mice [38]. This study did not address whether the increase in lipid raft order was due to EPA or DHA. In contrast, it has been very recently shown that EPA treatment of Jurkat T cells disrupted the acyl chain order in the immunological synapse [48]. This correlated with a significant remodeling of the acyl chain composition of membrane raft associated lipids isolated selectively form the synapse. Both studies demonstrate that n-3 PUFAs modify lipid raft organization in the synapse, albeit differently, but the exact mechanism remains to be determined. The differences between the two studies could be in the sensitivity of the probes, or differences in synapse stability between human Jurkat T cells and mouse T cells and in the methods used to modify plasma membrane lipid composition.
3.2. DHA and antigen presenting cells Several studies have established that n-3 PUFAs can suppress antigen presentation through the MHC class I and class II pathways. The implications of this are not clear. On the one hand, suppression of such pathways could render individuals more susceptible to infection [49–51]. On the other hand, suppression of these pathways has potential therapeutic value for the treatment of autoimmune and inflammatory disorders. For instance, autoimmune myositis is characterized by high levels of MHC I molecules and type I diabetics have increased autoantigens that are recognized by cytotoxic T lymphocytes [52,53]. Similarly, MHC class II surface levels are elevated in various inflammatory diseases [54]. In vitro and ex vivo studies have shown that n-3 PUFAs can suppress MHC class II mediated antigen presentation [55–57]. For example, feeding rats fish oil for 6 weeks suppressed antigen presentation by DCs [58]. Treating human B lymphoblast APCs with DHA and arachidonic acid (AA) rendered them resistant to lysis by cytotoxic CD8 + T cells [59]. We sought to determine the mechanism by which DHA and AA made the APCs less susceptible to T cell lysis. We initially hypothesized that the PUFAs exerted their effects by modifying MHC class I lateral organization. This hypothesis was based on existing data which showed that depleting cholesterol from the APC membrane increased MHC class I clustering and enhanced lysis by CTLs [60,61]. However, when we tested changes in MHC class I lateral organization with fluorescence recovery after photobleaching (FRAP) microscopy, we did not find a change in MHC I membrane organization. Instead, we found that PUFAs lowered APC susceptibility to T cell lysis by inhibiting the forward trafficking rate of MHC class I and suppressing the ability of APCs to conjugate with CTLs [59]. The change in conjugation suggested that the APC side of the immunological synapse is sensitive to the lipid environment. In collaboration with the Edidin laboratory, we have indeed found that the APC side of the immunological synapse is sensitive to membrane lipid composition. By concentrating the lipid raft associated lipid phosphatidylinositol 4,5bisphosphate (PIP2) on the APC side of the immunological synapse, we increase APC resistance to CTL lysis [62]. Very recently, we have started to address how n-3 PUFAs can modify lipid raft organization of EL4 cells, which are often used as target APCs, using a quantitative microscopy approach. We have found that DHA-, but not EPA-, treatment diminishes the clustering of cholera toxin subunit B crosslinked lipid rafts and increases the size of rafts on the micron scale [63]. Co-localization studies showed that as a consequence of the increase in lipid raft
Fig. 2. Proposed model by which DHA-containing phospholipids modify the organization of lipid rafts. According to this model, which reconciles contradictory data from biophysical studies with liposomes and from studies utilizing cellular detergent extraction, DHA-containing phospholipids incorporate into both nonrafts and rafts. Within the rafts, DHA-containing phospholipids form their own organizationally distinct domains, driven by steric incompatibility between cholesterol and DHA. The formation of DHA-rich domains in rafts appears to increase the size of lipid rafts on a micron scale. It is predicted that the micron scale domains are actually comprised of nanometer scale lipid rafts and nanometer scale DHA-rich domains. Formation of DHA-domains would then drive changes in protein lateral organization and conformation (depicted by the change in width of the protein). Figure adapted from Ref. [63].
size, we observed an increase in MHC class I co-localizing with rafts. The increase in lipid raft size with DHA-treatment is consistent with the recent data, described above, showing that n-3 PUFAs increase raft order. However, it is unclear how n-3 PUFAs increase raft size. In the next section, we conclude by proposing a model that can explain how n-3 PUFAs modify lipid raft size and distribution to modify protein activity.
4. Reconciling biophysical and cellular studies Based on studies from our laboratory and others, we propose a major revision to existing models to reconcile the contradiction between the biophysical and cellular studies (Fig. 2). The contradiction is that biophysical studies show that DHAcontaining phospholipids cannot incorporate into lipid rafts whereas cellular studies show that DHA does incorporate into rafts. Our proposed model is largely based on quantitative microscopy studies that report on the micro- to nanometer size scale on which membrane domains form [38,63,64]. According to this model, DHA-containing phospholipids would incorporate into both lipid raft and non-raft domains. In the non-rafts, DHAcontaining phospholipids would form their own organizationally distinct domains and modify protein conformation and/or lateral organization, as predicted by the biophysical liposome studies [22–28]. The model also states that the DHA-containing phospholipids will incorporate into lipid rafts, consistent with cellular data. Within lipid rafts, DHA-containing phospholipids would form organizationally distinct domains on a nanometer scale. This would explain the observed increase in lipid raft size
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upon the incorporation of DHA or fish oil in either cell culture or ex vivo [38,63]. The larger rafts would then be composed of nanometer scale rafts and nanometer scale DHA-rich domains. These domains would then serve to disrupt the optimal size and distribution of lipid rafts, which is essential for protein clustering. There are also two other possibilities to explain how DHA and other n-3 PUFAs could increase lipid raft size. The first possibility is that DHA-containing phospholipids would localize only into non-rafts, as predicted by biophysical studies. This would then drive cholesterol into lipid rafts and make them larger. Preliminary data from our laboratory does not favor this model [63]. As a first approximation, we treated mouse EL4 cells with DHA and assayed for changes in localization of radiolabeled cholesterol in raft versus non-raft phases using detergent extraction. We found no effect of DHA on the distribution of cholesterol between rafts and non-rafts. However, further work is required in this area given that detergent can induce artifacts [44]. A second possibility is that incorporation of DHA into the membrane could modify the steady state levels of cholesterol. This would again be driven by steric incompatibility between cholesterol and DHA. For example, DHA could lower the levels of plasma membrane cholesterol; this would drive the remaining cholesterol into rafts and make them larger. Indeed, detergent extraction studies show that DHA can lower the levels of plasma membrane cholesterol [65]. However, testing the alternative models will require the development of methods that do not rely on detergent. In summary, we present a major revision to existing models on DHA and lipid rafts. The proposed model predicts that DHAcontaining phospholipids will form nanometer scale domains in response to dietary intake of fatty acid [63]. These domains will then modify the optimal size and distribution of lipid rafts that serve to compartmentalize protein clusters. This can explain, in part, how DHA-containing phospholipids can suppress T cell and APC function. To further develop this model, we need to initiate studies using more sophisticated microscopy methods. This will require developing multi-scale DHA probes for testing changes in protein organization and conformation. By understanding how DHA modifies plasma membrane organization, we can start developing new strategies for using the fatty acid in the clinic, especially for the treatment of inflammatory diseases. In addition, we can make better dietary recommendations for the general public on the potential benefits and drawbacks of dietary consumption of DHA.
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Contents lists available at ScienceDirect
Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa
Diet-induced docosahexaenoic acid non-raft domains and lymphocyte function Saame Raza Shaikh a,b,n a b
Department of Biochemistry and Molecular Biology, Brody School of Medicine, East Carolina University, 600 Moye Blvd, Greenville, NC 28590, USA The Metabolic Institute for the Study of Diabetes and Obesity, Brody School of Medicine, East Carolina University, 600 Moye Blvd, Greenville, NC 28590, USA
abstract Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid (PUFA) that generally suppresses the function of T lymphocytes and antigen presenting cells (APCs). An emerging mechanism by which DHA modifies lymphocyte function is through changes in the organization of sphingolipid/cholesterol lipid raft membrane domains. Two contradictory models have been proposed to explain how DHA exerts its effects through changes in raft organization. The biophysical model, developed in model membranes, shows that DHA-containing phospholipids form unique non-raft membrane domains, that are organizationally distinct from lipid rafts, which serve to alter the conformation and/or lateral organization of lymphocyte proteins. In contrast, the cellular model on DHA and rafts shows that DHA suppresses lymphocyte function, in part, by directly incorporating into lipid rafts and altering protein activity. To reconcile opposing biophysical and cellular viewpoints, a major revision to existing models is presented herein. Based largely on quantitative microscopy data, it is proposed that DHA, consumed through the diet, modifies lymphocyte function, in part, through the formation of nanometer scale DHA-rich domains. These nano-scale domains disrupt the optimal raft-dependent clustering of proteins necessary for initial signaling. The data covered in this review highlights the importance of understanding how dietary n-3 PUFAs modify lymphocyte membranes, which is essential toward developing these fatty acids as therapeutic agents for treating inflammatory diseases. & 2010 Elsevier Ltd. All rights reserved.
1. Docosahexaenoic acid (DHA), immunosuppression, and membrane domains Docosahexaenoic acid is an n-3 polyunsaturated fatty acid (PUFA) and a major component of fish oil. The fatty acid is recognized to have therapeutic value as an immunosuppressant for the treatment of inflammation associated afflictions, such as cardiovascular disease, rheumatoid arthritis, and colitis [1–5]. At a cellular level, DHA suppresses the function of specific lymphocytes associated with inflammation [6]. However, a major limitation of using the fatty acid effectively as an adjuvant immunosuppresant has been a limited understanding of how the fatty acid exerts its effects at a molecular level. The molecular mechanisms that have been proposed by which DHA modifies lymphocyte function include changes in eicosanoid metabolism, gene transcription, post-translational modification, protein trafficking, production of D-resolvins, and alteration of
n
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plasma membrane microdomain organization. The focus of this review is on DHA and lipid raft microdomain organization. Lipid rafts are transient sphingolipid/cholesterol membrane domains that serve to compartmentalize cellular signaling events [7]. Modification of these domains with DHA has a broad impact given that dietary intake of the fatty acid results in its incorporation into membrane phospholipids of virtually all tissues that are naturally devoid of n-3 PUFAs [8]. Two contradictory models will be reviewed that explain how DHA-containing phospholipids can suppress lymphocyte function through changes in the molecular organization of lipid rafts. First, data are presented to show that DHA adopts unique molecular orientations which make it an excellent candidate for modifying membrane domain organization and protein activity. Next, a biophysical model is presented to show that DHA-containing phospholipids form non-raft domains that are organizationally distinct from lipid rafts, to modify protein activity. The biophysical studies are then contrasted with cellular data to show that DHA can suppress the function of lymphocytes through changes in plasma membrane rafts. The model that emerges from these studies is one in which DHA localizes directly into lipid rafts to
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Table 1 Summary of biophysical studies to show that DHA-containing phospholipids form organizationally distinct non-raft domains. Method
Result
References
Atomic force microscopy
A heteroacid DHA-containing phospholipid forms distinct domains on a submicron scale in the presence of sphingomyelin and cholesterol. Heteroacid DHA-containing phospholipids form distinct domains on a micron scale when mixed with phospholipids containing saturated acyl chains The rigid structure of cholesterol is sterically incompatible with the highly flexible structure of DHA. The structural incompatibility drives formation of DHA-rich domains. The size of heteroacid DHA-containing phospholipids domains is estimated to be 20 nm. Sphingomyelin and cholesterol are immiscible with heteroacid and homoacid DHA-containing phospholipids.
[25]
Sphingomyelin and cholesterol are immiscible with heteroacid and homoacid DHA-containing phospholipids. Heteroacid and homoacid DHA-containing phospholipids localize to ‘‘non-raft’’ detergent soluble fractions of liposomes DHA-containing FRET probes cluster away from phospholipids containing saturated acyl chains DHA-containing FRET probes show formation of domains in the presence of rhodopsin and phospholipids with saturated acyl chains
[22,27] [24,25]
Widefield microscopy X-ray diffraction 2
H NMR spectroscopy Differential scanning calorimetry (DSC) Pressure–area isotherms Detergent extraction of liposomes FRET FRET
modify protein activity. The review is concluded, based on very recent microscopy data, with a major revision of existing models on how DHA-rich domains modify lipid rafts. According to this model, consumption of DHA in the diet results in the formation of nanometer scale DHA-rich domains that serve to modify lipid raft and protein lateral organization and/or conformation.
[21] [28,29] [26] [21,22,25–27]
[20] [30]
Recent NMR and MD simulation studies show that DHA acyl chains can bind in the grooves between the helices of rhodopsin [14–17] which in turn disrupts the interhelical packing of rhodopsin and thereby its conformation and function [11,18].
2.3. Biophysical model of DHA and lipid rafts 2. Biophysical studies of DHA and membrane structure 2.1. Molecular orientation of DHA The molecular structure of DHA, due to its 22 carbons and 6 double bonds, is highly disordered relative to other unsaturated and saturated fatty acids [9]. A series of spectroscopic studies in model membranes, supported by molecular dynamic (MD) simulations, show that DHA, due its highly flexible structure, rapidly converts between various conformational states on a subnanosecond time scale [10,11]. The flexibility of the acyl chain is conferred by low potential energy barriers to rotation about the single carbon–carbon bonds that compensate for the rigidity of the double bonds [11]. The increased flexibility results in a higher density of DHA, relative to other unsaturated fatty acids, toward the water interface of the membrane [12]. This is highly unusual given that the presence of any fatty acid near the water interface would appear to be thermodynamically unfavorable. As a result of high conformational flexibility of DHA, various physical properties of the membrane are modified. These include changes in membrane elasticity, hydrophobic mismatch, curvature stress, vesicle fusion, permeability, microviscosity, phospholipid flipflop, and membrane lateral organization [9]. 2.2. DHA and protein activity The unique physical properties of DHA can alter protein conformation or lateral organization, which in turn would modify cellular activity. One example of a protein that undergoes changes in conformation in response to DHA’s effects on the physical properties of the membrane is rhodopsin, the photo-inducible G protein coupled receptor of the rod outer segment (ROS). Functional studies of ROS membranes isolated from animals fed with n-3 PUFA deficient diets showed a drastic reduction in G protein coupled receptor signaling as a consequence of replacing DHA with docosapentaenoic acid (DPA, 22:5n-6) [13]. The change in signaling was a consequence of changes in acyl chain order [13].
One hypothesis that emerged nearly a decade ago is that DHA, due to its high conformational flexibility, modified cellular activity by altering the lateral organization of both lipid and protein molecules [9]. In support of this view, a series of studies with fluorescence resonance energy transfer (FRET), pressure– area isotherms, and differential scanning calorimetry (DSC) showed that heteroacid and homoacid DHA-containing phosphatidylcholine (PC) and phosphatidylethanolamine (PE) phospholipids did not mix with homoacid phospholipids containing saturated acyl chains (Table 1) [19–21]. This led to the hypothesis that DHA-containing phospholipids could form unique membrane domains that excluded saturated acyl chains. However, a major limitation of these studies was that the phospholipids used as controls contained saturated acyl chains with little physiologically relevance. More recent membrane studies, using a physiologically relevant model system, have led to a model in which DHAcontaining phospholipids form novel organizationally distinct domains (Fig. 1). Atomic force microscopy, DSC, and 2H NMR studies show that a heteroacid PE containing palmitic acid in the sn-1 acyl chain position and DHA in the sn-2 acyl chain forms membrane domains distinct from the lipid raft molecules sphingomyelin and cholesterol [22–27]. In contrast, a heteroacid PE containing palmitic acid in the sn-1 position and oleic acid in the sn-2 position does not form membrane domains in the presence of lipid raft molecules. The molecular mechanism for the formation of DHA-rich domains came from X-ray diffraction measurements that demonstrated that the highly flexible DHA acyl chain is sterically incompatible with the rigid structure of cholesterol [28,29]. In addition, very recent data also show unfavorable molecular interactions between the DHA acyl chain and the saturated acyl chains of sphingomyelin (Shaikh, Wassall, and Stillwell, unpublished results). Therefore, steric incompatibility of DHA with cholesterol and limited immiscibility with sphingomyelin appears to be the trigger for the formation of DHArich domains. These studies are in agreement with the work of others to show that cholesterol preferentially interacts with the sn-1 saturated acyl chains of heteroacid phospholipids with an
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Fig. 1. Contradictory models on how DHA-containing phospholipids modify lipid rafts and raft associated proteins. (Left) According to the biophysical model, n-3 PUFAs cannot sterically interact with cholesterol and therefore do not incorporate into rafts. Instead, n-3 PUFAs form their own organizationally distinct domains. (Right) According to the cell based model, n-3 PUFAs incorporate into both raft and non-raft regions of the membrane. Both models predict that incorporation of n-3 PUFAs into rafts will modify the lateral organization of proteins (purple) from the raft to non-raft phase, or vice versa. Note that lipid and protein molecules are not drawn to scale. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
sn-2 polyunsaturated acyl chain [19,30]. A very recent study in liposomes defines the size of DHA-rich domains to be 20 nm or less [26]. The major prediction of the biophysical model on DHA and lipid rafts is that lymphocyte function will be modified in response to the formation of DHA-rich domains that serve to alter protein activity (Fig. 1, Left). The model states that the formation of DHA-rich domains, as a consequence of dietary consumption of fish oil, will displace proteins from lipid rafts to non-rafts or vice versa. In addition, formation of DHA-rich domains could modify the conformation of membrane proteins. In support of this view, liposomes of DHA-containing phospholipids altered the conformation of the major histocompatibility complex (MHC) class I protein, which has a central role in antigen presentation to cognate CD8 + T lymphocytes (described below) [31]. The same laboratory also showed that fusion of lymphocytes with DHA-containing phospholipids modified the conformation of the CD8 co-receptor on the surface of CD8 + T cells [32]. A major limitation of the biophysical DHA-lipid raft model is that it is largely based on studies with liposomes and requires validation at the cellular and whole animal levels.
3. DHA suppresses lymphocyte function through changes in membrane domain organization Antigen presenting cells (APCs), which have a central role in adaptive immunity, present peptides to cognate T cells in the context of MHC class I or class II molecules. MHC class I molecules, found on the surface of all nucleated cells, present self or viral peptides from the cytosol to CD8 + T lymphocytes. MHC class II molecules, found on the surface of dendritic cells (DCs), macrophages, and B cells present peptides from pathogens that either reside within, or were endocytosed into, intracellular compartments. T cells are activated on encountering antigen,
which results in either lysis of target cells by CD8 + T lymphocytes or recruitment of other effector cells by CD4 + T cells. Both APC and T cell function are sensitive to treatment with n-3 PUFAs at the cellular and whole animal levels, as described below.
3.1. The n-3 PUFA-lipid raft model in T cells Several in vitro and ex vivo animal and human studies have shown that n-3 PUFAs suppress CD4 + T cell activation [33–42]. This is highly significant given that CD4 + T cells have a role in the production of inflammatory cytokines. One emerging model to explain the suppression of T cell activation by n-3 PUFAs is through changes in the organization of lipid rafts [43]. According to this model, (Fig. 1, Right), DHA and EPA incorporate directly into lipid raft domains to modify the lateral organization of plasma membrane proteins from rafts to non-rafts, or vice versa. The change in protein lateral organization suppresses downstream signaling events necessary for activation of genes involved in cytokine production and T cell proliferation. One major limitation of this model is that it is heavily based on the use of detergent extraction at cold temperatures. It is now accepted that detergent can induce artifacts and does not report on changes in lipid or protein lateral organization on the micro to nanometer length scales on which domains form [44,45]. In addition, the model is contradictory to the aforementioned biophysical model, which states that n-3 PUFA-containing phospholipids cannot incorporate into rafts [46]. One approach to overcome the limitations of detergent extraction has been the use of quantitative microscopy to address changes in lipid raft organization in response to n-3 PUFAs. Two recent microscopy studies, which are contradictory, have attempted to explain how n-3 PUFAs modify lipid rafts. Both studies have tested the effects of n-3 PUFAs on lipid raft order in the T cell immunological synapse, a highly dynamic interface between T
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cells and APCs that displays spatio-temporal resolution [47]. The first study showed that CD4 + T cells from the fat-1 transgenic mouse, which have higher levels of n-3 PUFAs in their membranes compared with those from wild type mice, displayed increased order within the immunological synapse compared to cells isolated from wild type mice [38]. This study did not address whether the increase in lipid raft order was due to EPA or DHA. In contrast, it has been very recently shown that EPA treatment of Jurkat T cells disrupted the acyl chain order in the immunological synapse [48]. This correlated with a significant remodeling of the acyl chain composition of membrane raft associated lipids isolated selectively form the synapse. Both studies demonstrate that n-3 PUFAs modify lipid raft organization in the synapse, albeit differently, but the exact mechanism remains to be determined. The differences between the two studies could be in the sensitivity of the probes, or differences in synapse stability between human Jurkat T cells and mouse T cells and in the methods used to modify plasma membrane lipid composition.
3.2. DHA and antigen presenting cells Several studies have established that n-3 PUFAs can suppress antigen presentation through the MHC class I and class II pathways. The implications of this are not clear. On the one hand, suppression of such pathways could render individuals more susceptible to infection [49–51]. On the other hand, suppression of these pathways has potential therapeutic value for the treatment of autoimmune and inflammatory disorders. For instance, autoimmune myositis is characterized by high levels of MHC I molecules and type I diabetics have increased autoantigens that are recognized by cytotoxic T lymphocytes [52,53]. Similarly, MHC class II surface levels are elevated in various inflammatory diseases [54]. In vitro and ex vivo studies have shown that n-3 PUFAs can suppress MHC class II mediated antigen presentation [55–57]. For example, feeding rats fish oil for 6 weeks suppressed antigen presentation by DCs [58]. Treating human B lymphoblast APCs with DHA and arachidonic acid (AA) rendered them resistant to lysis by cytotoxic CD8 + T cells [59]. We sought to determine the mechanism by which DHA and AA made the APCs less susceptible to T cell lysis. We initially hypothesized that the PUFAs exerted their effects by modifying MHC class I lateral organization. This hypothesis was based on existing data which showed that depleting cholesterol from the APC membrane increased MHC class I clustering and enhanced lysis by CTLs [60,61]. However, when we tested changes in MHC class I lateral organization with fluorescence recovery after photobleaching (FRAP) microscopy, we did not find a change in MHC I membrane organization. Instead, we found that PUFAs lowered APC susceptibility to T cell lysis by inhibiting the forward trafficking rate of MHC class I and suppressing the ability of APCs to conjugate with CTLs [59]. The change in conjugation suggested that the APC side of the immunological synapse is sensitive to the lipid environment. In collaboration with the Edidin laboratory, we have indeed found that the APC side of the immunological synapse is sensitive to membrane lipid composition. By concentrating the lipid raft associated lipid phosphatidylinositol 4,5bisphosphate (PIP2) on the APC side of the immunological synapse, we increase APC resistance to CTL lysis [62]. Very recently, we have started to address how n-3 PUFAs can modify lipid raft organization of EL4 cells, which are often used as target APCs, using a quantitative microscopy approach. We have found that DHA-, but not EPA-, treatment diminishes the clustering of cholera toxin subunit B crosslinked lipid rafts and increases the size of rafts on the micron scale [63]. Co-localization studies showed that as a consequence of the increase in lipid raft
Fig. 2. Proposed model by which DHA-containing phospholipids modify the organization of lipid rafts. According to this model, which reconciles contradictory data from biophysical studies with liposomes and from studies utilizing cellular detergent extraction, DHA-containing phospholipids incorporate into both nonrafts and rafts. Within the rafts, DHA-containing phospholipids form their own organizationally distinct domains, driven by steric incompatibility between cholesterol and DHA. The formation of DHA-rich domains in rafts appears to increase the size of lipid rafts on a micron scale. It is predicted that the micron scale domains are actually comprised of nanometer scale lipid rafts and nanometer scale DHA-rich domains. Formation of DHA-domains would then drive changes in protein lateral organization and conformation (depicted by the change in width of the protein). Figure adapted from Ref. [63].
size, we observed an increase in MHC class I co-localizing with rafts. The increase in lipid raft size with DHA-treatment is consistent with the recent data, described above, showing that n-3 PUFAs increase raft order. However, it is unclear how n-3 PUFAs increase raft size. In the next section, we conclude by proposing a model that can explain how n-3 PUFAs modify lipid raft size and distribution to modify protein activity.
4. Reconciling biophysical and cellular studies Based on studies from our laboratory and others, we propose a major revision to existing models to reconcile the contradiction between the biophysical and cellular studies (Fig. 2). The contradiction is that biophysical studies show that DHAcontaining phospholipids cannot incorporate into lipid rafts whereas cellular studies show that DHA does incorporate into rafts. Our proposed model is largely based on quantitative microscopy studies that report on the micro- to nanometer size scale on which membrane domains form [38,63,64]. According to this model, DHA-containing phospholipids would incorporate into both lipid raft and non-raft domains. In the non-rafts, DHAcontaining phospholipids would form their own organizationally distinct domains and modify protein conformation and/or lateral organization, as predicted by the biophysical liposome studies [22–28]. The model also states that the DHA-containing phospholipids will incorporate into lipid rafts, consistent with cellular data. Within lipid rafts, DHA-containing phospholipids would form organizationally distinct domains on a nanometer scale. This would explain the observed increase in lipid raft size
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upon the incorporation of DHA or fish oil in either cell culture or ex vivo [38,63]. The larger rafts would then be composed of nanometer scale rafts and nanometer scale DHA-rich domains. These domains would then serve to disrupt the optimal size and distribution of lipid rafts, which is essential for protein clustering. There are also two other possibilities to explain how DHA and other n-3 PUFAs could increase lipid raft size. The first possibility is that DHA-containing phospholipids would localize only into non-rafts, as predicted by biophysical studies. This would then drive cholesterol into lipid rafts and make them larger. Preliminary data from our laboratory does not favor this model [63]. As a first approximation, we treated mouse EL4 cells with DHA and assayed for changes in localization of radiolabeled cholesterol in raft versus non-raft phases using detergent extraction. We found no effect of DHA on the distribution of cholesterol between rafts and non-rafts. However, further work is required in this area given that detergent can induce artifacts [44]. A second possibility is that incorporation of DHA into the membrane could modify the steady state levels of cholesterol. This would again be driven by steric incompatibility between cholesterol and DHA. For example, DHA could lower the levels of plasma membrane cholesterol; this would drive the remaining cholesterol into rafts and make them larger. Indeed, detergent extraction studies show that DHA can lower the levels of plasma membrane cholesterol [65]. However, testing the alternative models will require the development of methods that do not rely on detergent. In summary, we present a major revision to existing models on DHA and lipid rafts. The proposed model predicts that DHAcontaining phospholipids will form nanometer scale domains in response to dietary intake of fatty acid [63]. These domains will then modify the optimal size and distribution of lipid rafts that serve to compartmentalize protein clusters. This can explain, in part, how DHA-containing phospholipids can suppress T cell and APC function. To further develop this model, we need to initiate studies using more sophisticated microscopy methods. This will require developing multi-scale DHA probes for testing changes in protein organization and conformation. By understanding how DHA modifies plasma membrane organization, we can start developing new strategies for using the fatty acid in the clinic, especially for the treatment of inflammatory diseases. In addition, we can make better dietary recommendations for the general public on the potential benefits and drawbacks of dietary consumption of DHA.
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