Intracellular Lipid Flux and Membrane Microdomains as Organizing Principles in Inflammatory Cell Signaling

This information is current as Michael B. Fessler and John S. Parks of September 24, 2021. J Immunol 2011; 187:1529-1535; ; doi: 10.4049/jimmunol.1100253 http://www.jimmunol.org/content/187/4/1529 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Intracellular Lipid Flux and Membrane Microdomains as Organizing Principles in Inflammatory Cell Signaling Michael B. Fessler* and John S. Parks† Lipid rafts and caveolae play a pivotal role in organiza- remodeling of raft lipid is not only necessary in many sig- tion of signaling by TLR4 and several other immune naling cascades, but that primary perturbations of raft lipid receptors. Beyond the simple cataloguing of signaling (e.g., cholesterol loading or unloading, raft coalescence) can events compartmentalized by these membrane microdo- also be sufficient initiating events to trigger protein signaling. mains, recent studies have revealed the surprisingly cen- Using the macrophage and, in particular, TLR signaling in tral importance of dynamic remodeling of membrane macrophages as a primary case in point, the dependence of lipid domains to immune signaling. Simple interven- inflammatory signaling upon cholesterol-loading conditions tions upon membrane lipid, such as changes in choles- and on the regulatory proteins that control homeostatic in- Downloaded from terol loading or crosslinking of raft lipids, are sufficient tracellular trafficking of cholesterol through rafts will be to induce micrometer-scale reordering of membranes highlighted. and their protein cargo with consequent signal trans- Lipid rafts and caveolae duction. In this review, using TLR signaling in the mac- rophage as a central focus, we discuss emerging evidence Lipid rafts are thought to be highly dynamic, nanoscale (i.e., , http://www.jimmunol.org/ that environmental and genetic perturbations of mem- 200 nm), cholesterol- and sphingolipid-enriched membrane brane lipid regulate protein signaling, illustrate how ho- microdomains, likely present in all eukaryotic cells, that meostatic flow of cholesterol and other lipids through compartmentalize select signaling and functional events. rafts regulates the innate immune response, and high- Although it is difficult to place a lower limit on their size in the resting state, and evidence indeed exists for “lipid shells” light recent attempts to harness these insights toward surrounding individual proteins in biological membranes therapeutic development. The Journal of Immunology, (2), rafts can also be driven to coalesce into more stable, 2011, 187: 1529–1535. micrometer-range domains through lipid–lipid, protein–

lipid, and protein–protein interactions. The mechanisms by guest on September 24, 2021 ince the inception of the lipid raft hypothesis in 1997 underlying raft “coalescence” or “clustering,” however, in (1), a profusion of studies have reported roles for these many cases remain elusive. It is generally thought that the S cholesterol-enriched membrane microdomains in or- saturated acyl chains of raft sphingolipids and phospholipids ganization of cell signaling. As a crossroads for immunology, exhibit tight packing in a manner analogous to the liquid- biophysics, and lipid science, the raft field has suffered ordered domains observed in model membranes, and that this growing pains in terminology, technique, and interpretation. may account for their resistance to solubilization by cold Progressively refined imaging techniques continue to support nonionic detergents (e.g., Triton X-100). However, as de- the existence of lateral protein/lipid heterogeneities in bi- tergent can itself induce the formation of domains in mem- ological membranes (2, 3), but the precise nature, size, and branes (6), rafts should not be equated with “detergent- malleability of these microdomains remain a matter of debate. resistant membranes” (DRMs), nor can identification of A burgeoning field that has cataloged an increasing number of a protein in DRMs be taken as sufficient evidence for signaling events within rafts at the same time finds itself at risk assigning raft localization in vivo. Although good evidence of losing sight of the implications of this localization. In this supports the coexistence within cell membranes of heteroge- review, rather than focus on definitions of rafts/caveolae (for neous populations of lipid rafts, isolation of DRMs of discrete this, the reader is referred to recent scholarly reviews in Refs. composition with the use of different detergents should not 2, 4, 5), the objective is to synthesize and interpret emerging be considered as evidence for discrete raft domains in vivo. insights on how genetic and environmental modification of Caveolae are ∼60- to 80-nm cholesterol-enriched mem- raft lipid plays a fundamental role in determining immune brane invaginations whose flask-shaped morphology derives signaling and disease. The case will be made that dynamic from caveolin proteins, the expression of which suffices to

*Laboratory of Respiratory Biology, National Institute of Environmental Health Scien- Address correspondence and reprint requests to Dr. Michael B. Fessler, National Institute ces, National Institutes of Health, Research Triangle Park, NC 27709; and †Section on of Environmental Health Sciences, 111 T.W. Alexander Drive, P.O. Box 12233, MD D2- Lipid Sciences, Department of Pathology and Biochemistry, Wake Forest School of 01, Research Triangle Park, NC 27709. E-mail address: [email protected] Medicine, Winston-Salem, NC 27157 Abbreviations used in this article: ABC, ATP-binding cassette; apo, apolipoprotein; Received for publication April 1, 2011. Accepted for publication May 10, 2011. DRM, detergent-resistant membrane; ER, endoplasmic reticulum; HDL, high-density lipoprotein; HMG-CoA, 3-hydroxy-3-methyl-glutaryl-CoA; mbCD, methyl-b-cyclo- This work was supported in part by the Intramural Research Program of the National dextrin; NPC1, Niemann–Pick C1; oxLDL, oxidized low-density lipoprotein; PUFA, Institutes of Health, National Institute of Environmental Health Sciences (Z01 polyunsaturated fatty acid; SR, scavenger receptor. ES102005) and by Grants HL094525 and HL049373 (to J.S.P.). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100253 1530 BRIEF REVIEWS: MEMBRANE ORGANIZATION OF SIGNALING confer caveolar morphology (7). Caveolae are thought to re- in neutrophils (24). Imaging techniques with higher resolu- present a discrete, specialized subpopulation of membrane mi- tion than fluorescence microscopy will almost certainly be re- crodomains, and thus should not be simply equated with quired to properly characterize raft colocalization and co- lipid rafts. The caveolin proteins, through direct regulatory alescence. Nonetheless, taken together, these findings confirm interactions with other proteins (e.g., TLR4) (8), are in par- that membrane lipid remodeling is sufficient to drive cell sig- ticular thought to play a central role in signal regulation naling by reorganizing protein cargo, and they also demon- within caveolae. Of interest, although caveolae are well strate, perhaps paradoxically, that cholesterol depletion can studied in certain cell types (e.g., endothelial cells, fibroblasts) increase membrane order and coalescence of raft-like domains, and thought to be absent in others (e.g., lymphocytes), their a topic to which we return below. presence in macrophages is less well defined and indeed con- Notably, Ab-mediated crosslinking of several GPI-linked troversial, varying by macrophage type (reviewed in Ref. 9). proteins can also coalesce/remodel rafts and induce signaling Although rafts and/or caveolae promote immune receptor by copatching proteins within rafts. Crosslinking of external signaling in several pathways by serving as platforms for dy- leaflet raft proteins induces copatching and activation of inner namic assembly of signaling complexes, in other cases, raft leaflet raft proteins such as H-ras (25), whereas crosslinking of localization suppresses signaling (e.g., TGF-b and epidermal GM1 can interestingly induce its copatching with TLR4 (26) growth factor receptors) (10–12). Moreover, in addition to and CD18 (27). As oligomeric cholesterol-binding cytolysins concentrating signaling proteins, the lipid microenvironment such as listeriolysin O both cluster CD14-rich rafts (28) and of rafts may itself alter protein function (13), in some cases activate TLR4 (29), it seems plausible that some TLR4 ago- Downloaded from shaping signaling much more selectively than as just a simple nists may activate this receptor through raft-mediated receptor binary switch. Thus, localization of the TNF receptor to raft clustering. In this light, it is important to remember that LPS, versus non-raft domains determines responses to TNF-a, the canonical TLR4 ligand, is itself a polymeric molecule that including cell fate as well as signaling events (14). induces receptor clustering. Protein localization to rafts, in many cases determined by

GPI linkage or palmitoylation, is also thought to be responsive The two faces of rafts: signal inhibition and activation by http://www.jimmunol.org/ to raft cholesterol levels. Indeed, lipid-induced changes in the raft-perturbing agents raft proteome likely explain reports, discussed below, that acute Perhaps the most widely used experimental tools used to or chronic changes in raft cholesterol may determine protein “disrupt” rafts are the b-cyclodextrins, mbCD and 2-hy- signaling. Conversely, some proteins (e.g., NAP-22) and droxyl-b-CD, cyclic oligosaccharides that remove cholesterol peptides (apolipoprotein [apo]A-I mimetic 4F) may them- from membranes. Although numerous papers have used selves induce phase separation of cholesterol-rich and -poor mbCD to infer that cell signals, including those induced by domains (15) or induce raft signaling by deforming mem- LPS, are raft-dependent, some caution is warranted (reviewed brane lipids (16). Importantly, note that proteins, through in Ref. 30). Thus, mbCD depletes cholesterol to varying by guest on September 24, 2021 scaffolding and other interactions, have been shown in some degrees in different cell types, may under high concentrations contexts to play dominant roles in determining membrane (i.e., .10 mM) or prolonged incubations (.30 min) also domains in immune cells (17, 18). Raft coalescence induced remove extra-raft cholesterol or even cause cell death, and may in dendritic cell membranes by physical contact of uric acid interact with nonsterol lipids or immobilize membrane pro- crystals (19), or in RAW 264.7 membranes by altered to- teins through effects on the cytoskeleton (30). Moreover, pography of the cell substratum (20), can also activate sig- mbCD and other in vitro manipulations of cell membrane k naling proteins, including Syk and NF- B. Taken together, lipid may not necessarily be physiologically relevant. These these findings suggest that protein and lipid remodeling of concerns notwithstanding, good evidence suggests that low the membrane interact to shape domains and cell signaling, concentration/short incubation usage of mbCD may be se- and that raft signaling may be profoundly influenced or in- lective for raft cholesterol (30–32). Moreover, multiple con- deed induced by “ligand-independent” interventions upon trol strategies are available, including clamping of cell plasma membrane lipid. cholesterol with mbCD–cholesterol complexes, use of the structurally dissimilar cholesterol-sequestering agents filipin Rafts as poised signaling units: signal initiation by microdomain and nystatin, cholesterol depletion by lipoprotein-deficient coalescence serum, as well as additional raft-perturbing agents that have Interestingly, recent work indicates that the resting plasma been described (Table I) (33–46). membrane may be poised at the edge of a phase boundary Although raft isolation and perturbation strategies have been such that simple membrane perturbations can drive large- used to show the requirement for raft integrity in several scale phase separation of discrete protein/lipid macrodomains, signaling pathways, a perhaps more intriguing chain of liter- thereby inducing signaling. Thus, crosslinking of the raft ature has shown that acute cholesterol depletion can itself glycosphingolipid GM1 with cholera toxin B subunit induces initiate signaling cascades in a cell type-dependent fashion. cholesterol-dependent coalescence of micrometer-scale GM1 CDs activate ERK in Rat-1 and RAW 264.7 cells (31, 47), p38 domains that recruit lipid-anchored raft proteins but exclude and Cdc42 in human neutrophils (48), and tyrosine phos- the non-raft transferrin receptor (21). Cholesterol depletion phorylation in RBL-2H3 cells (49). mbCD and filipin initiate with methyl-b-cyclodextrin (mbCD) also induces micrometer- ligand-independent activation of epidermal growth factor scale phase separation of the plasma membrane into fluid receptor (10, 50) and Fas (51) by a mechanism involving and ordered domains in living CHO cells (22), GM1-rich displacement of these receptors from rafts. Conversely, cho- domains that concentrate Lck and LAT and signal to ERK lesterol depletion may also cause a disintegrin and metal- activation in T cells (23), and GM1- and CD11b-rich domains loproteinase domain-containing protein 10- and/or 17-de- The Journal of Immunology 1531

Table I. Agents reported to disrupt raft structure and/or function and their associated effects on the cell

Disruptor Cell Type Effect Reference PUFAs EL4 cell ↓Raft coalescence, MHC I mislocalization 33 HDL Monocyte ↓Raft chol, ↓CD11b activation 34 4F MDM ↓Rafts, altered cell differentiation 35 LXR agonists Prostate cancer cell ↓Raft size, ↓raft Akt phosphorylation 36 SQS inhibitor Prostate cancer cell ↓Raft chol, ↓cell proliferation 37 Statins NK cell ↓Membrane chol, ↓NK cell cytotoxicity 38 oxLDL Endothelial ↓Raft chol, ↓raft eNOS, ↓eNOS activation 39 oxPAPC Endothelial ↓LPS-induced raft TLR4, ↓LPS response 40 Ceramide PBMC ↓Raft Lck, ↓raft PLD1, ↑PLD1 activity 41 DPPE CD8+ T cell ↓MHC-induced raft proteins, ↓CTL activation 42 DPPC, surfactant A549 ↓LPS-induced TLR4 translocation to rafts 43 High glucose THP-1 ↓Number and size of caveolae 44 Ethanol Mf ↓LPS-induced raft CD14 and TLR4 45 ESeroS-GS Mf ↓LPS-induced raft CD14 and TLR4 46 Selected examples are shown for each agent. Measures of raft disruption differ among reports. For some agents (e.g., ceramide), both raft stabilization and destabilization have been reported. For others (e.g., ethanol), inhibitory and stimulatory effects have been reported on the TLR4 pathway. chol, cholesterol; DPPC, dipalmitoylphosphatidylcholine; DPPE, dipalmitoylphosphatidylethanolamine; 4F, apolipoprotein mimetic peptide 4F; LXR, liver X receptor; Mf, macrophage; MDM, monocyte-derived macrophage; oxPAPC, oxidized 1-palmi- Downloaded from toyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine; SQS, squalene synthase. pendent cleavage of several receptors (IL-6R, CD44, CD30, MyD88-dependent pathway to NF-kB in macrophages (47). TNFR1, TNFR2) by causing their displacement from rafts Although the full significance of these assorted findings is not (52–55). mbCD also activates NF-kB in macrophages by a yet clear, and multiple underlying mechanisms are likely in- http://www.jimmunol.org/ mechanism involving MyD88 (47). Consistent with these volved, taken together, these reports suggest that native findings with CDs, we recently reported that the physiologic microdomains of the cell membrane may serve to maintain cholesterol acceptor apoA-I activates a TLR2-, TLR4-, and signal quiescence by sequestering pathway components, and by guest on September 24, 2021

FIGURE 1. Intracellular cholesterol trafficking regulates macrophage rafts. Cholesterol synthesized in the ER by HMG-CoA reductase (HMGCR) or internalized via scavenger receptors (CD36, SR-A) or low-density lipo- protein receptor (LDLR) is assembled into nascent rafts in the Golgi appa- ratus for caveolin- and NPC1-depen- dent transfer to the plasma membrane. NPC1 together with NPC2 also reg- ulates endosomal recycling of cho- lesterol to the plasma membrane. In turn, cholesterol is effluxed either by simple diffusion or via transporters (ABCA1, ABCG1, SR-BI) to extra- cellular acceptors (apoA-I, HDL), and also likely equilibrates with non- raft regions. Cholesterol esterification is regulated in the cytosol by choles- terol ester hydrolase (CEH) and in the ER by acyl-CoA:cholesterol acyl- transferase 1 (ACAT1). Raft choles- terol/abundance and abundance of LPS recognition proteins (CD14, TLR4) are regulated by cholesterol flux through this pathway. 1532 BRIEF REVIEWS: MEMBRANE ORGANIZATION OF SIGNALING that their perturbation through cholesterol removal may in- null macrophages have enlarged, cholesterol-laden lipid rafts duce pathways the nature of which is determined by the (56, 72) (Fig. 2) containing increased TLR4 (57), and they specific signaling proteins expressed in the cell under study. are hyperresponsive to LPS (56, 72–74) as well as to TLR-2, -7, and -9 ligands (56, 74). ABCG1-null macrophages display Regulation of rafts and TLR signaling by intracellular cholesterol a similar albeit perhaps more pronounced TLR-hyperre- traffic sponsive phenotype (74, 75). NPC1-null macrophages Although it is now well recognized that pharmacologic raft- display basal activation of TLR3, -7, -8, and -4 (60), the first perturbing agents can modify raft-dependent signaling by de- three of which may reflect cholesterol overloading of endo- localization of proteins, the effects of cholesterol loading on somal rafts, and the last, TLR4 accumulation in endosomes raft function under more physiologic settings are perhaps less due to blocked trafficking (76). Taken together, these re- widely appreciated. In primary murine macrophages, raft levels ports indicate an intriguing degree of overlap between the of TLR4 and TLR9, as well as cell responsiveness to TLR2, pathway for trafficking of host lipids and that for recognition TLR4, TLR7, and TLR9 ligands, are all directly associated of microbial lipids, perhaps even suggesting common evolu- with exogenously manipulated raft cholesterol levels (56, 57). tionary roots between the two. Indeed, it was recently Moreover, hypercholesterolemia increases macrophage raft reported that, in addition to regulating efflux of cholesterol cholesterol in mice and humans in vivo, increasing cell re- and phospholipid, ABCA1 also regulates efflux of LPS from sponsiveness to LPS (58, 59). Perhaps more striking are re- the macrophage (77). ports that indicate that acute cholesterol loading of mem- Downloaded from branes may suffice to activate TLRs. Thus, cholesterol load- ing of the macrophage plasma membrane induces TLR4- dependent signaling, and loading of endosomal membranes induces TLR3- and TLR4-dependent responses (60). Con- versely, it is also recognized that in other contexts (e.g., modified lipoprotein treatment) cholesterol loading can also http://www.jimmunol.org/ be associated with reduced macrophage inflammatory func- tion (61, 62). This may in part reflect the propensity of conditions to load cytosolic cholesterol ester instead of membrane cholesterol, as well as to activate nuclear receptors (e.g., liver X receptors, peroxisome proliferator-activated receptors). Physiologically, raft/caveolar cholesterol content is regulated by homeostatic trafficking of cholesterol through the cell (Fig. by guest on September 24, 2021 1), a topic covered in depth by recent comprehensive reviews (63). In brief, following cholesterol synthesis in the endo- plasmic reticulum (ER) or endosomal recycling of internalized cholesterol to the ER/Golgi by Niemann–Pick C1 (NPC1) protein, it is thought that caveolae are assembled in the Golgi and transported to the plasma membrane in a caveolin- and NPC1-dependent fashion (7, 64). Thus, NPC-deficient fibroblasts have reduced plasma membrane caveolar choles- terol (64) and late endosomal cholesterol overload with raft overcrowding (65). Raft/caveolar cholesterol is, in turn, reg- ulated by transporter-mediated (ATP-binding cassette [ABC] FIGURE 2. ABCA1-deficient macrophages have enlarged lipid rafts. A, A1, ABCG1, and scavenger receptor [SR]-BI) efflux of plasma Rafts were imaged in peritoneal macrophages from wild-type (WT) and Abca1-null mice with the use of two raft cholesterol probes, BCu toxin (red) membrane cholesterol to extracellular acceptors, including and fluorescent polyethylene glycol cholesteryl ether (fPEG-chol) (green). lipid-free apoA-I and high-density lipoprotein (HDL), as well Nuclei were stained with DAPI (blue). This research was originally pub- as by aqueous diffusion. Overexpression of ABCA1 (66) and lished in the Journal of Lipid Research. Koseki, M., K. Hirano, D. Masuda, treatment with HDL or apoA-I (67, 68) all disrupt/deplete C. Ikegami, M. Tanaka, A. Ota, J. C. Sandoval, Y. Nakagawa-Toyama, S. B. raft domains, inhibiting raft-dependent signaling. The effect Sato, T. Kobayashi, Y. Shimada, Y. Ohno-Iwashita, F. Matsuura, I. Shimo- mura, and S. Yamashita. Increased lipid rafts and accelerated lipopolysac- of stimulated cholesterol efflux is quite complex, however, as a apoA-I, similar to mbCD, can enhance responses to some charide-induced tumor necrosis factor- secretion in Abca1-deficient macrophages. J. Lipid Res. 2007; 48:299–306. Ó the American Society for stimuli such as platelet-derived growth factor (68) by re- Biochemistry and Molecular Biology. B, Peritoneal macrophages from wild moving cholesterol from rafts (68, 69). Similarly, SR-BI– type (+/+) or macrophage-specific Abca1-null (-M/-M) mice were cholesterol mediated cholesterol efflux to HDL activates endothelial NO depleted with mbCD or cholesterol loaded with mbCD-cholesterol, stained synthase (eNOS) (70). Moreover, apoA-I may increase cav- with fPEG-chol, and then quantified by flow cytometry. Data are means 6 eolar cholesterol by stimulating its transfer from intracellular SEM. *p , 0.05, **p , 0.01. This research was originally published in the compartments faster than its efflux (71). Journal of Lipid Research. Zhu, X., J. S. Owen, M. D. Wilson, H. Li, G. L. Griffiths, M. J. Thomas, E. M. Hiltbold, M. B. Fessler, and J. S. Parks. Building on earlier reports that TLR4 signaling occurs in Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor traffick- lipid rafts (48), an exciting chain of literature has recently ing to lipid rafts by reduction of lipid raft cholesterol. J. Lipid Res. 2010; demonstrated profound effects of cholesterol trafficking 51:3196–3206. Ó the American Society for Biochemistry and Molecular through rafts on TLR signaling in the macrophage. ABCA1- Biology. The Journal of Immunology 1533

Modification of rafts and their signaling by nonsterol lipids Emerging opportunities for targeting rafts in disease Complex effects upon raft remodeling and signaling in TLR Several strategies have shown early promise as potential ther- and other pathways have also been described for sphingo- apeutic measures to modify raft signaling through inter- myelin and the product of its breakdown by sphingomyelinase, vening upon raft lipids. These include dietary polyunsaturated ceramide. Sphingomyelin and cholesterol promote raft for- fatty acids (PUFAs), statins (i.e., 3-hydroxy-3-methyl-glutaryl- mation in the Golgi via strong physical interactions (78), and CoA [HMG-CoA] reductase inhibitors), squalene synthase ceramide indeed stabilizes rafts more effectively than does inhibitors, raft-targeting lipids, and edelfosine. PUFAs such cholesterol (79). In contrast, good evidence indicates that as docosahexaenoic acid and eicosapentaenoic acid inhibit sphingomyelinase treatment and ceramide itself both displace signaling in Jurkat T cells by incorporating into rafts and cholesterol from rafts (80, 81) in a manner that could re- displacing acylated signaling proteins (Lck, Fyn, LAT) alistically occur in vivo during inflammation. Indeed, acid (89). Alternatively, it has been proposed that PUFAs do not sphingomyelinase-induced remodeling of the plasma mem- incorporate into rafts due to their unsaturation, but rather brane into enlarged ceramide-rich rafts during Pseudomonas form extraraft domains that interfere indirectly with raft- aeruginosa infection is critical for bacterial internalization and dependent protein clustering (33, 90). Statins, increasingly for successful host defense (82). Interestingly, local acid studied for their anti-inflammatory actions, attenuate leuko- sphingomyelinase-mediated ceramide production in rafts cyte function in part through membrane raft depletion (38). has also been reported to be required for LPS-induced re- Inhibitors of squalene synthase, an enzyme downstream of cruitment of TLR4 to rafts (83), and ceramide itself elicits HMG-CoA reductase in the cholesterol biosynthetic pathway, Downloaded from TLR4-dependent signaling (84). Although the full implica- selectively reduce raft cholesterol in cancer cells and induce tions of these findings are not yet clear, it appears plausible cell death (37). In an additional raft-centric strategy for cancer that ceramide and cholesterol may “compete” to form therapy, adhesion and cell cycle progression of breast cancer somewhat distinct rafts, and that dynamic remodeling of raft cells were recently shown to be more effectively inhibited by lipid and protein composition by local ceramide induction targeting a Src family kinase inhibitor to rafts through pal- may be a critical step in TLR signaling and perhaps other mitoylation (91). Finally, a recent study has shown that http://www.jimmunol.org/ pathways. the phospholipid ether edelfosine may be an effective ther- Phospholipids have also been shown to modulate raft struc- apeutic for multiple myeloma by accumulating in myeloma ture and function. Dipalmitoylphosphatidylethanolamine par- cell rafts, thereby inducing apoptosis through coclustering of titions into lipid rafts, inhibiting MHC peptide-induced rafts and death receptors (92). Taken together, these reports raft recruitment of acylated proteins in CD8+ T cells (85) indicate the exciting potential to manipulate disease cells and TNF-induced recruitment of its receptor to rafts in through several independent interventions that target raft HT1080 cells (14), without displaying overt effects on raft lipids. integrity. Similarly, the surfactant phospholipid dipalmitoyl- by guest on September 24, 2021 phosphatidylcholine and surfactant itself both attenuate LPS signaling by inhibiting TLR4 recruitment to rafts (43), per- Conclusions haps suggesting that the lipid environment of the alveolus Many basic questions about the nature of rafts and caveolae may dampen innate immune responses through effects on remain unanswered, and many of these questions will almost rafts. A role for phospholipid metabolism in cell-intrinsic certainly require high-resolution imaging techniques. None- TLR4 responses is also suggested by a report that lysophos- theless, a convergence of independent approaches including phatidylcholine acyltransferase is required for LPS-induced biophysics, immunology, and lipid science has begun to in- translocation of TLR4 to rafts (85). dicate the fundamental importance of dynamic and chronic Oxidized lipids present during disease have been shown to changes in membrane lipid to signaling in immune and other modify rafts with associated effects on signaling. Thus, oxi- cells. Pathways for trafficking of cholesterol and other lipids dized low-density lipoprotein (oxLDL) reduces caveolar through the macrophage, initially studied for their relevance to cholesterol, displacing eNOS and caveolin (39). The major cell biology and metabolism, are now understood to be critical oxysterol in oxLDL, 7-ketocholesterol, partitions into rafts, determinants of TLR signaling. At a basic level, these studies depleting them of cholesterol (69) and activating Src within have also challenged the traditional paradigm of hierarchical them (86). Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero- protein signaling, showing that lateral changes in lipid domain 3-phosphorylcholine, a major oxidized phospholipid present segregation in the plasma membrane are not only necessary, in oxLDL, has also been shown to disrupt rafts through a but also sufficient to initiate protein signaling. A challenge for mechanism involving ceramide induction, thereby inhibit- the field as it looks now to apply these insights to better ing LPS-induced translocation of TLR4 to rafts (40, 87). understand “old” diseases and to develop new therapeutics Reports that reactive oxygen species are not only necessary but will be to avoid conceptual constraint by the raft hypothesis also sufficient to induce assembly of TCR-associated proteins itself. in T cell rafts (88) and TLR4 trafficking to rafts in macro- phages (26) suggest that oxidation of membrane lipids may Acknowledgments also serve as a critical step in innate and adaptive immune We thank Sue Edelstein for assistance with figure design. signaling through raft remodeling. Indeed, an antioxidant a-tocopherol derivative has been shown to attenuate LPS signaling by interfering with CD14 and TLR4 recruitment to Disclosures rafts (46). The authors have no financial conflicts of interest. 1534 BRIEF REVIEWS: MEMBRANE ORGANIZATION OF SIGNALING

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