Identification of Naturally Occurring Fatty Acids of the Myelin Sheath That Resolve Neuroinflammation

RESEARCH ARTICLE

MULTIPLE SCLEROSIS Identification of Naturally Occurring Fatty Acids of the Myelin Sheath That Resolve Neuroinflammation

Peggy P. Ho,1* Jennifer L. Kanter,1,2* Amanda M. Johnson,1,2 Hrishikesh K. Srinagesh,1 Eun-Ju Chang,2,3 Timothy M. Purdy,2,3 Keith van Haren,1,2,3 William R. Wikoff,4 Tobias Kind,4 Mohsen Khademi,5 Laura Y. Matloff,1 Sirisha Narayana,1,2 Eun Mi Hur,1 Tamsin M. Lindstrom,2,3 Zhigang He,6 Oliver Fiehn,4 Tomas Olsson,5 Xianlin Han,7 May H. Han,1 Lawrence Steinman,1*† William H. Robinson2,3*†

Lipids constitute 70% of the myelin sheath, and autoantibodies against lipids may contribute to the demyelination that characterizes multiple sclerosis (MS). We used lipid antigen microarrays and lipid mass spectrometry to identify bona fide lipid targets of the autoimmune response in MS brain, and an animal model of MS to explore the role of the identified lipids in autoimmune demyelination. We found that autoantibodies in MS target a phosphate group

in phosphatidylserine and oxidized phosphatidylcholine derivatives. Administration of these lipids ameliorated Downloaded from experimental autoimmune encephalomyelitis by suppressing activation and inducing apoptosis of autoreactive T cells, effects mediated by the lipids’ saturated fatty acid side chains. Thus, phospholipids represent a natural anti-inflammatory class of compounds that have potential as therapeutics for MS.

INTRODUCTION tory responses in the CNS and that this protective mechanism is com- http://stm.sciencemag.org/ In multiple sclerosis (MS), aberrant adaptive immune responses target promised in MS, because these guardian lipids are attacked by the and destroy the myelin sheath. Although MS is classically considered a adaptive arm of the immune system. These naturally occurring myelin disease driven by T cells, it is now known that autoantibodies also lipids may have therapeutic potential in MS and other inflammatory contribute to its pathogenesis (1, 2). Several studies on MS demon- brain diseases. strate T cell and antibody reactivity to lipids (3–6), which constitute more than 70% of the myelin sheath. Synthesis of anti-lipid antibodies within the central nervous system (CNS) is associated with an aggres- RESULTS sive disease course in MS (7), and, in an experimental model of MS, by guest on March 14, 2019 anti-lipid antibodies both induced demyelination and prevented re- Anti-lipid antibody reactivity differentiates between MS myelination (8). Despite recent interest in the potential pathogenicity patients and controls of antibodies directed against brain lipids, the specificities of the anti- We printed lipid antigen arrays containing more than 50 brain lipids lipid antibody responses in MS remain undefined. and used these arrays to profile autoantibodies in cerebrospinal fluid Here, we report a “functional lipidomics” approach to discovering (CSF) samples derived from MS and control patients. An anti–IgG (im- autoimmune targets and developing therapeutic strategies for MS. We munoglobulin G) + IgM secondary antibody was used to detect anti- used lipid autoantigen microarrays and lipid mass spectrometry to lipid antibody binding. The Significance Analysis of Microarrays (SAM) identify targets of the adaptive autoimmune response in MS patients. (9) algorithm identified 17 lipids that had significantly greater reac- We then explored these results in an animal model of MS, experi- tivity with autoantibodies in CSF from the 33 individuals with MS mental autoimmune encephalomyelitis (EAE), to define the biological [18 with relapsing-remitting MS (RRMS), 14 with secondary progres- role of the autoantibody-targeted lipids in the pathogenesis of auto- sive MS (SPMS), and 1 with primary progressive MS (PPMS)] versus immune demyelination. Unexpectedly, we found that several of the the 26 controls [21 with other (noninflammatory) neurological diseases autoantibody-targeted lipids—phospholipids naturally present in the (ONDs) and 5 healthy controls (HCs)] [false discovery rate (FDR) = brain—could attenuate EAE. Our findings suggest that phosphatidyl- 0.048]; patient demographics and clinical characteristics are listed in serine and oxidized phosphatidylcholine derivatives containing table S1. We used a hierarchical cluster algorithm (10)todiscernrela- saturated fatty acid side chains serve as natural brakes on inflamma- tionships between patient samples and SAM-identified lipids. Most MS samples clustered together on the basis of the similarity of their 1 Department of Neurology and Neurological Sciences, Stanford University School of anti-lipid autoantibody profiles (Fig. 1A). Specifically, the PPMS sam- Medicine, Stanford, CA 94305, USA. 2Division of Immunology and Rheumatology, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, ple, and half of the RRMS and SPMS samples, clustered in the group USA. 3Geriatric Research, Education and Clinical Center, Veterans Affairs Palo Alto Health with the highest anti-lipid autoantibody reactivity, whereas only 3 of Care System, Palo Alto, CA 94304, USA. 4Department of Molecular and Cellular Biology the 21 ONDs and none of the HC samples were represented in this and Genome Center, Metabolomics Group, University of California, Davis, Davis, CA 95616, USA. 5Department of Clinical Neuroscience, Karolinska Institute, 171 76 Stock- group. Most of the controls (15 of 21 ONDs and 3 of 5 HCs) clus- holm, Sweden. 6F.M. Kirby Neurobiology Center, Harvard Medical School, Boston, MA tered in the group with the lowest anti-lipid autoantibody reactivity, 02115, USA. 7Diabetes and Obesity Research Center, Sanford-Burnham Medical Re- whereas only 1 SPMS sample and 4 of the 18 RRMS samples clus- search Institute, Orlando, FL 32827, USA. tered in this group. Enzyme-linked immunosorbent assay (ELISA) *These authors contributed equally to this work. †To whom correspondence should be addressed. E-mail: [email protected] (W.H.R.); analysis showed that levels of total IgG were higher than those of total [email protected] (L.S.) IgM in both RRMS and OND CSF (fig. S1, A and B), and, as expected,

www.ScienceTranslationalMedicine.org 6 June 2012 Vol 4 Issue 137 137ra73 1 RESEARCH ARTICLE levels of total IgG were significantly high- A er in RRMS and SPMS CSF than in OND CSF (fig. S1C).

1-Palmitoyl-2-glutaroyl-sn- glycero-3-phosphocholine OND 02-100 reduces EAE severity OND 02-099 OND 02-001 OND 02-096 OND 02-095 OND 02-091 RRMS 02-024 RRMS 02-017 OND 01-085 To determine whether the autoantibody- targeted lipids have a role in autoimmune demyelination, we tested the effect of se- lect lipids on EAE, a mouse model of MS. In our initial antigen array experi- ment(Fig.1A),wescreenedlipidsthat OND 02-099 fell into four categories: (1) brain and myelin lipids (for example, cerebrosides, sulfatides, and gangliosides), (2) mem- B brane lipids (for example, cholesterol, 5 Lipid co-immunization Downloaded from phosphatidylcholine, and sphingomyelin), (3) oxidized lipids [for example, 1-palmitoyl- 4 2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and its derivatives], and (4) micro- bial lipids [for example, lipopolysaccharide 3

(LPS) and lipoteichoic acid]. From our http://stm.sciencemag.org/ set of 17 lipid hits, we selected several lipids 2 from categories 1 to 3 that exhibited higher Vehicle autoantibody reactivity in MS samples than Mean clinical EAE score PGPC in OND or HC samples. We tested the 1 Sphingomyelin effects of these lipids in T cell proliferation 0 01020 30 40 50 60 Fig. 1. Autoantibody targeting of lipids Days after immunization is higher in MS CSF than in OND CSF by guest on March 14, 2019 and normal CSF, and the autoantibody- targeted lipid PGPC attenuates EAE. (A) Lipid array profiling of IgG + IgM anti- C body reactivity in CSF samples from MS 4 Lipid treatment patients, HCs, and OND controls. Lipid hits with the lowest FDR (q = 0.048) were clustered according to their reactivity 3 profiles. Sample type and ID number are shown above the heatmap, and the lipids targeted are shown to the right ’ 2 of the heatmap. The patients demo- Sphingomyelin graphics and clinical characteristics are presented in table S1. (B) Clinical EAE Vehicle PGPC scores of mice coinjected subcutaneously 1 Mean clinical EAE score with PLP139–151 and 6 mg per injection of PGPC or sphingomyelin (day 0); on days 4 and 7 after immunization, the lipids sol- ubilized in 0.05% Tween 20 in PBS (vehicle) 0 0 10 20 30 40 50 were injected intraperitoneally. Arrows indicate when injections were adminis- Days after immunization tered. Each point represents the mean ± SEM, and results are representative of four independent experiments. +P <0.05 by Mann-Whitney test comparing vehicle-treated (n = 5) and PGPC- vehicle alone, for a total of five intravenous injections. Arrows indi- treated (n =5)mice;*P <0.05byMann-Whitneytestcomparing cate when injections were administered. Each point represents the vehicle-treated (n = 5) and sphingomyelin-treated (n = 5) mice. (C) Clin- mean ± SEM. +P < 0.05 by Mann-Whitney test comparing vehicle-treated ical EAE scores of mice administered PGPC, sphingomyelin, or vehicle (n = 10) and PGPC-treated (n =9)mice;*P < 0.05 by Mann-Whitney at the onset of disease. Upon developing clinical signs of EAE, mice test comparing vehicle-treated (n = 10) and sphingomyelin-treated (n = were intravenously administered 100 mg of PGPC or sphingomyelin, or 9) mice.

www.ScienceTranslationalMedicine.org 6 June 2012 Vol 4 Issue 137 137ra73 2 RESEARCH ARTICLE assays and in EAE. We found that cerebrosides and gangliosides were ALDO, azPC ester, and azPC). All eight of the lipids identified as tar- unable to suppress myelin basic protein (MBP)Ac1–11–specific T cell gets with the lowest FDR (0.029) had a phosphate group linked to a proliferation, that administration of cerebrosides did not affect EAE, nitrogen moiety through two carbons (Fig. 2B). The nonpolar portion and that oxidized cholesterol had only a minimal effect on EAE (fig. S2). of the targeted lipids contained either one or two side chains; the sec- We previously tested sulfatides, which worsened EAE (3). ond side chain in some cases contained a terminal carboxyl group PGPC, one of the oxidized lipids tested in this screen, was of par- (Fig. 2B). This suggests that autoantibodies present in RRMS CSF tar- ticular interest because it is a derivative of oxidized phosphatidyl- get the phospholipids’ phosphate head group and that the affinity of choline, and antibodies to oxidized phosphatidylcholine are present in antibody-lipid binding is not specific to a particular phospholipid. MS brain lesions (11). Indeed, phosphatidylcholine makes up 30.1% of In support of this idea, autoantibodies in MS consistently targeted the lipids in the gray matter and 15.0% of the lipids in the white mat- sphingomyelin (3)(Figs.1Aand2,AandB)butdidnottargetcer- ter of the adult human brain (12), lipid peroxidation occurs in MS amide, which is sphingomyelin without the phosphate polar head brain lesions (13), and oxidized phosphatidylcholine is present in group (fig. S4). MS brain lesions (11). We used two treatment regimens to test the To further investigate the structural basis of the lipids’ antigenicity, effect of exogenous PGPC on EAE. In the first regimen, we sub- we examined autoantibody reactivity to an additional 14 lipids that cutaneously administered PGPC together with the proteolipid protein contain various head group and side-chain modifications of PGPC. (PLP)139–151 that we use to induce EAE in SJL/J mice, and then we The lipids in this Mini Array II are listed in table S2. We probed Mini intraperitoneally administered PGPC on its own 4 and 7 days after Array II with CSF samples from RRMS and OND control patients Downloaded from the immunization. In contrast to sulfatide, a myelin glycosphingolipid using a sample set similar to that used in the previous array exper- that exacerbated EAE when delivered in this prophylactic regimen (3), iments and identified autoantibody reactivity to many PGPC-related PGPC unexpectedly reduced the severity of EAE throughout the dis- lipids using an anti-IgG secondary antibody(Fig.2C).Wenotedthat ease course (Fig. 1B). In the second regimen, we started administering six of the seven targeted lipids had a phosphate group, in most cases PGPC 10 days after the immunization, that is, at the time of disease attached through two carbons to another polar moiety such as nitrogen onset (Fig. 1C), and found that PGPC could also attenuate EAE that or oxygen (Fig. 2D). One such lipid, 1-palmitoyl-2-oleoyl-sn-glycerol, http://stm.sciencemag.org/ was already established. Sphingomyelin makes up 6.9 and 7.7% of the contained only a hydroxyl group at this position. Although several of lipids in the gray and white matter, respectively, of an adult human the targeted lipids are endogenously synthesized in human brain, 1,2- brain (12) but exhibited only moderate reactivity to antibodies in MS dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), a synthetic cationic CSF (Fig. 1A). Sphingomyelin also attenuated EAE when administered surfactant that has the same head group as PGPC, was also targeted. during the immunization (Fig. 1B). However, when administered 10 days Of the lipids that were not targeted, 1,2-dipropionoyl-sn-glycero-3- after the immunization, sphingomyelin exacerbated EAE (Fig. 1C). Thus, phosphocholine (DGPC, right column, fifth lipid) had a phosphate unlike the other lipids tested, PGPC attenuated the development of head group with a structure similar to that of the targeted lipids. Un- EAE and ameliorated EAE after onset. like the targeted lipids, however, DGPC did not contain long side by guest on March 14, 2019 A reduction in T cell activation, a process important in MS patho- chains, suggesting that the lipid side chain may also facilitate antibody genesis, accompanied the PGPC-induced attenuation of EAE. The pro- binding. Despite having only short side chains, DGPC bound to the portion of CD4+ T cells expressing the early activation marker CD69 polyvinylidene difluoride (PVDF) membrane on our array (fig. S5). was reduced among lymph node cells isolated from EAE mice treated The other six lipids that were not targeted either lacked a phosphate prophylactically with PGPC. Specifically, 14.2% of the cells isolated group or contained a phosphate group connected to a bulky group, for from vehicle-treated mice were CD4+CD69+ compared to 7.96% of the example, a ring structure or a phosphate group linked to four carbons cells isolated from PGPC-treated mice (fig. S3A). Moreover, compared (Fig. 2, C and D). Using ELISA analysis to validate our array results, we to cells from vehicle-treated mice, lymph node cells from PGPC-treated found that PGPC, 1-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine mice proliferated less (fig. S3B), and splenocytes from PGPC-treated (azPC), 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (azPC mice secreted less interferon-g (IFN-g) and less tumor necrosis factor ester), and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (POPS) (TNF) (fig. S3C) upon stimulation with the encephalitogenic PLP139–151 are indeed targeted by autoantibodies in CSF and that the levels of these peptide. autoantibodies are significantly higher in CSF from patients with RRMS than in CSF from patients with ONDs (fig. S6). Phosphocholine head group confers antigenicity Thus, binding of RRMS CSF autoantibodies to these lipids is de- The commonality of the phosphatidylcholine backbone in lipids tar- pendent on the presence of (i) a nonbulky polar head group such as a geted by autoantibodies in MS prompted us to explore this structure phosphate group and (ii) at least one long hydrocarbon side chain. as a potential determinant of antigenicity. We investigated autoantibody targeting of seven lipids that have a glycero-3-phosphocholine back- Antibody targets are natural brain lipids bone in common with PGPC, as well as targeting of other structurally To determine whether the lipids identified as targets of the auto- similar lipids from the lipid array used in the previous experiments antibody response (Figs. 1A and 2 and fig. S6) are present in MS brain (Fig. 1A), such as those containing features in common with PGPC, lesions, we performed lipidomic mass spectrometric analysis of path- including a phosphate head group with one or two nonpolar side ological specimens taken at autopsyfromthebrainsofMSpatients,as chains. previously described (14). Lipids detected in MS brain lesions included We used Mini Array I, comprising 17 lipids (table S2), to profile phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, autoantibody responses in CSF samples from RRMS patients and phosphatidylglycerols, phosphatidylserines, phosphatidic acids, sphin- OND controls. SAM analysis revealed autoantibody reactivity to three gomyelins, sulfatides, cerebrosides, ceramides, and lysophosphatidyl- of the seven glycerol-3-phosphocholine–containing lipids (Fig. 2A, cholines (table S3).

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A Luminescence units We next asked whether PGPC, azPC, azPC ester, > 20000 10000 and POPS, specific lipid targets of the autoantibody 5000 2500 response in MS (Figs. 1A and 2 and fig. S6), are 1000 500 present in MS brain lesions. Mass spectrometric 250 100 analysis demonstrated the presence of POPS at 0.5 OND 02-041 OND 02-028 OND 01-180 OND 02-033 OND 01-184 OND 02-030 OND 02-016 RRMS 01-168 OND 01-185 RRMS 01-001 RRMS 02-036 RRMS 01-008 OND 01-181 OND 02-001 RRMS 02-040 RRMS 02-027 RRMS 01-182 OND 02-010 RRMS 02-012 RRMS 02-013 RRMS 02-023 RRMS 02-025 RRMS 01-190 RRMS 01-189 <10 L-α-phosphatidylcholine (PC) to 1.3 nmol per milligram of protein (Fig. 3, A and B) L-α-phosphatidylserine (PS) 1-Palmitoyl-2-9’-oxo-nonanoyl-sn-glycero-3-phosphocholine (ALDO) in MS brain lesions. Because PGPC, azPC, and azPC 1-Palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (azPC ester) Sphingomyelin ester were not detected in our initial mass spectro- 1-O-Hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine (azPC) L-α-lysophosphatidylserine (Lyso PS) metric analysis, we used single reaction monitoring L-α-phosphatidylethanolamine (SRM) to test for the presence of these specific lipids B in healthy brain tissue and MS brain lesions. Using Polar head group Nonpolar SRM, we detected azPC, azPC ester, and PGPC at O side chains PC O Sphingomyelin H OH O P 5to20pmolpermilligramofprotein(Fig.3B).All O O P O O O + N+ – N four lipids were detected both in MS samples and O H O– NH H O O O O O O PS O – azPC in age-matched control samples, and levels of azPC, H O P O O P O O O O + + NH HO – N azPC ester, PGPC, and POPS were significantly O H O– 3 O H O Na+ lower in MS samples (Fig. 3B and table S4), con- O O O Downloaded from O O ALDO O O Lyso PS O – H O sistent with the observed decrease in phosphatidyl- P O O P O O O O + + N – NH3 H O H O– HO H O cholinelevels(tableS3).Wefurtherconfirmedthe Na+ presence of PGPC (fig. S7), azPC (fig. S8), and O O azPC ester O O PE O O P P azPC ester (fig. S9) in human brain extract by liquid O O O + O O O + HO – N – NH3 chromatography–high-resolution mass spectrome- O H O O H O O O O try (LC-HRMS), using three criteria: accurate mass http://stm.sciencemag.org/ agreement with calculated mass (figs. S7A, S8A, and C S9A), retention time on the column in comparison to authentic standards (figs. S7B, S8B, and S9B), and tandem mass spectrometry (MS/MS) fragmentation

patterns in comparison to authentic standards (figs. OND 02-041 OND 02-016 OND 02-022 OND 02-019 OND 02-035 OND 01-185 OND 01-184 RRMS 01-190 OND 01-180 RRMS 02-025 OND 02-030 OND 02-001 OND 02-028 RRMS 02-023 OND 01-183 OND 01-181 RRMS 02-027 RRMS 02-036 RRMS 02-013 RRMS 01-188 RRMS 01-182 RRMS 02-012 RRMS 02-015 RRMS 02-014 RRMS 01-189 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA) S7C, S8C, and S9C). 1-Palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) 1-Palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-1-glycerol] (POPG) 1-Palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (POPS) 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) POPS and oxidized phosphatidylcholine 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) by guest on March 14, 2019 derivatives treat established EAE 1-Palmitoyl-2-oleoyl-sn-glycerol

Because PGPC attenuated both T cell proliferation D Reactive Nonreactive (fig.S3B)andEAE(Fig.1,BandC),weperformed 1,2-Dipalmitoyl-3- POPA O O trimethylammonium-propane O a second T cell proliferation screen to determine P O O OH O N+ O H O– whether any other oxidized phosphatidylcholine O H Cl– O O derivatives from our Mini Arrays (table S2) could Na+ PGPC O O 1,2-Dipalmitoyl ethylene glycol O O P O O + O HO – N O H O O

O O O Fig. 2. PGPC-related lipids with nonbulky polar O O 1,2-Dipalmitoyl-sn-glycero- POPG OH O 3-galloyl O head groups are targeted by antibodies in CSF of O O P O OH OH O O H O– O MS patients. (A) Mini Array I: IgG + IgM antibody Na+ O H O OH O reactivity to various glycero-3-phosphocholine lipids O O OH POPS O – 1,2-Dipalmitoyl-sn-glycero- H O O P O in CSF samples from patients with RRMS and from O O O + 3-phosphobutanol – NH3 P O H O O O O CH3 control patients with OND. Lipid hits with the lowest Na+ O H O– O Na+ FDR (q = 0.029) were clustered according to their re- O POPE O O 1,2-Dipropionoyl-sn-glycero- O O activity profiles. Sample type and ID number are P O O O + 3-phosphocholine – NH3 O O P O O H O N+ shown above the heatmap, and the lipids targeted O H O– O are shown to the right of the heatmap. (B) Structures sn O O O 1-Palmitoyl- -glycero- of lipid hits in (A). (C) Mini Array II: IgG antibody reac- DPPC 2,3-cyclic-phosphate O O O P O N+ O tivity to lipids constituting polar head group and side- O H O– O O P O chain modifications of PGPC in CSF samples from O O– + NH4 RRMS patients and OND controls. Lipid hits with the 1-Palmitoyl-2-oleoyl-sn-glycerol O 1-Palmitoyl-2-oleoyl-sn-glycero- 3-ethylphosphocholine O O lowest FDR (q = 0.016) were clustered according to O OH O H O O P O N+ their reactivity profiles. All of the lipids screened in O H O O Cl– O Mini Array I and II are listed in table S2. (D)Leftcol- H3C 1-Palmitoyl-2-oleoyl-sn-glycero- umn, structures of the lipid targets identified in (C), O 3-phospho(TEMPO)choline O CH3 CH3 with green boxes around the polar head group; right O O P O – + – O H O N N O H3C CH3 column, structures of the lipids that were not tar- CH3 geted, with red boxes around the polar head group. O CH3

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A determined whether the lipids could directly inhibit the T cell–mediated inflammatory responses that underpin EAE. We assessed the effect of these lipids on MBPAc1–11–induced production of cytokines by naïve splenocytes from mice transgenic for the MBPAc1–11–specific T cell receptor (15). azPC reduced MBPAc1–11–induced T cell production of the inflammatory cytokines IFN-g,TNF,andinterleukin-6(IL-6). POPS also reduced TNF and IL-6 levels butdidnotsignificantlyreduceIFN-g levels. PGPC and azPC ester reduced levels of some of the cytokines, but these effects were less marked than those of POPS or azPC (Fig. 4B). Neither POPS nor the

oxidized phosphatidylcholine derivatives Downloaded from B affected production of IL-12p40. These PGPC azPC ester azPC POPS lipids had similar inhibitory effects on 25 12 25 2.5 – * * * PLP139–151 induced production of IL-17, * 20 20 2.0 IL-6, IFN-g, and TNF by splenocytes from 9 PLP139–151–immunized mice (fig. S12A). 15 15 1.5 To determine whether the lipids af- http://stm.sciencemag.org/ 6 fect T cell proliferation, we measured 3 10 10 1.0 [ H]thymidine incorporation in splenocytes stimulated with MBPAc1–11 in the 3 5 5 0.5 presence of the different lipids. Sulfatide, pmol per mgprotein pmol per mgprotein pmol perprotein mg nmol per mgprotein which worsened EAE, and sphingomyelin, 0 0 0 0.0 which could not treat established EAE [(3) Control MS Control MS Control MS Control MS andFig.1C],wereusedascontrolsinthese Brain tissue Brain tissue Brain tissue Brain tissue assays. POPS, PGPC, azPC, and azPC ester by guest on March 14, 2019 Fig. 3. Levels of POPS, PGPC, azPC, and azPC ester are higher in MS brain than in healthy brain. (A)Nega- reduced T cell proliferation in response to tive ion electrospray ionization mass spectrometry analyses of POPS in lipid extracts of normal-appearing MBPAc1–11, whereas sulfatide and sphingo- white matter from an age-matched healthy control brain (left panel) and of an active lesion from a brain myelin did not (Fig. 4C). We obtained afflicted with RRMS (right panel). DMPS, dimyristoyl phosphatidylserine; IS, internal standard. (B)SRManal- similar results with PLP139–151–stimulated ysis of PGPC azPC ester, azPC, and POPS levels in samples of MS brain lesions (n = 6) and healthy brain tissue splenocytes from mice immunized with (n = 6). Controls: age-matched individuals with no signs of neurological disease; MS: three patients with PLP – (fig. S12B), as well as with T cells RRMS, one patient with SPMS, and two patients with chronic MS. *P < 0.05 by unpaired Student’s t test. 139 151 stimulated with anti-CD3 and anti-CD28 antibodies (fig. S13). Studies using T cells suppress T cell proliferation as effectively as PGPC. On the basis of the from mice deficient in CD1d showed that the lipid-mediated suppres- results from this screen (figs. S10 and S11), we selected the oxidized sion of T cell responses occurred through a CD1d-independent mech- phosphatidylcholine derivatives (azPC and azPC ester) and the phos- anism (fig. S13). phatidylserine (POPS) for testing in EAE. To investigate the therapeutic potential of these autoantibody- POPS and oxidized phosphatidylcholine derivatives induce targeted lipids in EAE, we administered the initial dose of POPS, PGPC, apoptosis of immune cells azPC, or azPC ester to mice with established EAE. We administered the To assess whether programmed cell death contributed to the lipid- lipids systemically to establish a delivery modality appropriate for transla- mediated reduction in proliferation of activated T cells, we performed tional studies in humans. The first dose of 100 mg of lipid was injected annexin V staining and 7-amino-actinomycin D (7AAD) uptake as- − intravenously into the tail of mice once they developed tail or hindlimb says, which enable identification of early apoptotic (annexin V+,7AAD ) paralysis, and the lipid treatment wasrepeatedeveryotherday,such and late apoptotic (annexin V+, 7AAD+)cells(Fig.5A).At48hours, that 10 injections were administered intravenously during the course of azPC ester or PGPC increased T cell apoptosis two- to threefold, azPC EAE. POPS, PGPC, azPC, and azPC ester were all able to ameliorate increased apoptosis more than fourfold, and POPS increased apoptosis established EAE (Fig. 4A). more than fivefold. The lipids had differential effects on other cell types, however. azPC and azPC ester suppressed proliferation (fig. POPS and oxidized phosphatidylcholine derivatives directly S14A), whereas only azPC increased apoptosis (fig. S14B), of a mouse suppress T cell activity macrophage cell line (RAW 264.7) stimulated with LPS. In contrast, To investigate the mechanisms by which the autoantibody-targeted azPC, azPC ester, and POPS modestly suppressed proliferation of pu- lipids provide therapeutic benefit in EAE (Figs. 1B and 4A), we rified B cells stimulated with anti-IgM F(ab′)2 fragment and anti-CD40

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A 4 antibodies (fig. S15A), and only POPS increased apoptosis of the B cells (fig. S15B). Vehicle To determine whether lipid treatment induced 3 POPS apoptosis in vivo in the context of EAE, we performed PGPC azPC ester TUNEL (terminal deoxynucleotidyl transferase–mediated 2 azPC deoxyuridine triphosphate nick end labeling) staining on sections of brains and spinal cords from EAE mice P < 0.05 for POPS treated with azPC or POPS. POPS and azPC increased 1 P < 0.05 for PGPC

Mean clinical EAE score the number of TUNEL-positive cells with lymphocyte P < 0.05 for azPC ester P < 0.05 for azPC morphology in the brain and spinal cord of mice with 0 EAE (Fig. 5, B and C). 0 20 40 60 Days following immunization POPS and oxidized phosphatidylcholine B derivatives induce proapoptotic and 800 1000 proinflammatory pathways *# * 800 To investigate the molecular mechanisms underlying 600 # #* * Downloaded from 600 the lipid-mediated modulation of T cells, we examined 400 the effects of these lipids on inflammatory, survival, 400

IL-6 (pg/ml) and apoptotic signaling pathways. The transcription 200 k k IL-12 p40 (pg/ml) 200 factor nuclear factor B (NF- B) plays a crucial role in the activation, survival, and proliferation of T cells 0 0 (16) by driving the transcription of proinflammatory

IFN- TNF IL-6 17–19 http://stm.sciencemag.org/ Media Media POPS PGPC azPC Media Media POPS PGPC azPC cytokine genes [including g, ,and ( )], azPC ester azPC ester anti-apoptotic genes (20), and genes involved in cell Control MBP Control MBP cycle progression (21). Deficiency in NF-kBsignaling 22 800 500 suppresses the expansion of autoreactive T cells ( ). Likewise, extracellular signal–regulated kinase (ERK) ac- 400 23 600 * tivity is integral to cell proliferation and survival ( ). Costimulation of T cells with anti-CD3 and anti-CD28 300 * * 400 antibodies (CD3/CD28) activated the canonical NF-kB 200 IFN- γ ( pg/ml)

TNF (pg/ml) pathway, as indicated by an increase in phosphorylation 200 by guest on March 14, 2019 100 of the activating kinases IKKa [inhibitor of NF-kB(IkB) # * kinase a]andIKKb, a decrease in levels of IkBa,and 0 0 536 PC an increase in Ser phosphorylation of p65, the major G Media Media POPS PGPC azPC Media Media POPS P azPC transactivating subunit of NF-kB(Fig.5D).Italsoin- azPC ester azPC ester duced ERK activity. POPS, PGPC, azPC, and azPC Control MBP Control MBP ester each suppressed the CD3/CD28-induced activation C of the NF-kB and ERK pathways (Fig. 5D). These lip- 20000 ids also suppressed the CD3/CD28-induced activity (phosphorylation) of B cell lymphoma protein–2 15000 * 10000 *# # *# Fig. 4. Administration of lipids that are targeted by auto- * antibodies and whose levels are decreased in MS attenuate 5000 # ongoing EAE and T cell activation. (A) Clinical scores of

H]Thymidine incorporation * 3 PLP139–151-immunized SJL mice treated at the peak of [ (c.p.m.) 0 EAE with 100 mg per injection of POPS (n =10),PGPC(n = 10), azPC ester (n =10),azPC(n = 10), or vehicle alone (n = azPC Media Media POPS PGPC ulfatide 10). Arrows indicate injections of lipid or vehicle. Each point S azPC ester represents the mean clinical score ± SEM (*P < 0.05 by Control MBP Sphingomyelin Mann-Whitney test comparing vehicle treatment versus lipid treatment). (B to D) Cytokine production by (B) and D proliferation of (C) naïve MBPAc1–11-TCR (T cell receptor) O O O O – azPC ester O POPS H O P P transgenic splenocytes stimulated with MBPAc1–11 (2 mg/ml) O O O + O O O + NH3 N O H O– HO O H O– in the presence of lipid (30 mg/ml) [structures shown in (D)], Na+ O O O as indicated. Values are the means ± SEM of triplicate ex- PGPC O O azPC O periments. Results are representative of three independent O O P O P + O O O + – N N HO O H O HO O H O– experiments. *P <0.05byStudent’s t test comparing each O O O O lipid plus MBPAc1–11 to MBPAc1–11 alone.

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A 4 MediaData.037 control +Data.038 PGPC 10 104 (Bcl-2) (Fig. 5D), an important antiapoptotic pro- 8% 17% Late tein. Bcl-2–interacting molecule (Bim) and Bad, 103 apoptosis 3 10 proapoptotic members of the Bcl-2 family, antago-

2 10 102 nize the antiapoptotic activity of Bcl-2. Bim plays Live 81% 10% Early 71% 12% critical roles in both activation and apoptosis of auto- cells 1 1 10 apoptosis 10 reactive T cells in EAE (24). ERK-mediated phos- 69 0 0 phorylation of Bim at Ser suppresses the apoptotic 10 10 0 1 2 3 4 100 101 102 103 104 10 10 10 10 10 25 AnnexinV AnnexinV activity of Bim ( ), whereas the NF-kB pathway

4 + Data.044POPS 4 + azPCData.039 ester 4 +Data.040 azPC 26 27 10 10 10 suppresses Bim expression ( , ). Similarly, ERK- 47% 20% 32% mediated phosphorylation of Bad at Ser112 inhibits 3 3 103 10 10 the apoptotic activity of Bad (28, 29). Collectively,

2 2 102 10 10 POPS, PGPC, azPC, and azPC ester suppressed the 32% 18% 68% 10% 46% 20% CD3/CD28-induced phosphorylation of Bim and 1 1 101 10 69 112 10 Bad at Ser and Ser , respectively. Sulfatide, which 7AAD (fluorescence intensity) 7AAD (fluorescence 3 0 0 worsens EAE ( ), neither inhibited the NF-kBand 100 10 10 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 AnnexinV AnnexinV AnnexinV ERK pathways nor suppressed the phosphorylation

Annexin V (fluorescence intensity) of the apoptotic Bcl-2 family proteins (Fig. 5D). Downloaded from Together, these results suggest that suppression B Vehicle azPC POPS of the NF-kB and ERK pathways, and the resulting derepression of apoptotic pathways, underlies the anti- inflammatory and antiproliferative effects of POPS and the oxidized phosphatidylcholine derivatives. http://stm.sciencemag.org/ Brain Saturated fatty acid side chains mediate T cell suppression Unsaturated fatty acid–derived mediators generated in resolving exudates can contribute to the ter- mination of an inflammatory response (30, 31). We asked whether the saturated fatty acid side chains within POPS, PGPC, azPC, and azPC ester could modulate T cell proliferation and EAE. Fatty acids es- Spinal cord by guest on March 14, 2019 terified to the phosphate head group at the sn-1 position, and the oxidizable fatty acids at the sn-2 position, were of particular interest in light of their C 25 Brain Spinal cord 20 Fig. 5. POPS, PGPC, azPC, and azPC ester induce apoptotic signaling pathways and T cell apoptosis. 15 (A) Annexin V and 7AAD staining of CD3+ Tcellspu- rified from wild-type B6 mice and stimulated with plate- 10 bound anti-CD3 and anti-CD28 antibodies for 48 hours with or without lipid (30 mg/ml). Cells are gated on 5 CD4+ T cells, and results are representative of three # TUNEL-positive cells # TUNEL-positive experiments. (B) TUNEL staining of brain and spinal 0 Vehicle azPC POPS cord tissue from mice immunized with PLP139–151 peptide (to induce EAE) and treated for 12 hours with ve- D hicle, azPC, or POPS (200 mgin0.2ml)onday15after immunization. Arrows indicate TUNEL-positive (bright pink/red) infiltrating cells in the perivascular cuffs of lesions from mice with active EAE. Original magnifica- tion, ×400. (C) Quantification of TUNEL-positive infiltrating cells in brain and spinal cord sections shown in (B). (D) Immunoblot analysis of phopsho-ERK1/2, phospho-IKKa/b,phospho-p65,IkBa,phospho–Bcl-2, phospho-Bad, and phospho-Bim in lysates of wild-type CD3+ T cells stimulated with plate-bound anti-CD3 and anti-CD28 antibodies for 15 min (left panel) and 24 hours (right panel) in the presence of lipid (30 mg/ml), as indicated. Blots are representative of two independent experiments.

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A O reported roles in modulating inflammation (32). We tested Palmitic acid a variety of structural analogs of these fatty acid side OH chains, including palmitic acid, ethyl palmitate, methyl B octanoate, octanoic acid, sebacic acid, and suberic acid. 300000 These molecules form part of the saturated, nonpolar 250000 side chain of our lipids of interest, which may be cleaved * 200000 * * from the phospholipids by lipases that are up-regulated * 150000 in MS brain (14, 33). Palmitic acid suppressed T cell pro- * (c.p.m.) 100000 * liferation as effectively as the phospholipids (Fig. 6B and * fig. S16); ethyl palmitate, methyl octanoate, octanoic 50000 acid, sebacic acid, and suberic acid also suppressed T cell H]Thymidine incorporation H]Thymidine

3 0 [ proliferation, albeit to a lesser extent (fig. S17). Palmitic

Media Media acid also suppressed T cell production of the inflammatory cytokines IL-6, IL-17, IFN-g,andTNF(Fig.6C) Control Anti-CD3 + anti-CD28 activation and induced T cell apoptosis (Fig. 6D). In vivo, administering palmitic acid at the time of disease onset attenu- C 500 ated EAE, and disease relapsed once the palmitic acid Downloaded from 1200 400 1000 treatment was halted (Fig. 6E), indicating that this fatty l) acid side chain is itself therapeutically efficacious. m 800 300 * 600 We identified palmitic acid as a fatty acid side chain 200 * 400 * present in phosphatidylcholines and as a free, unbound IL-6 (pg/ IL-6 *

100 (pg/ml) IL-17 200 fattyacidinbothMSandhealthybrain(tableS5).Be- * * * * http://stm.sciencemag.org/ 0 0 cause phospholipases liberate fatty acids from lipids and Media Media PGPC azPC azPC POPS ETOH PA Media Media PGPC azPC azPC POPS ETOH PA are up-regulated in MS brain (14, 33), we tested the effect ester ester Control Anti-CD3 + anti-CD28 activation Control Anti-CD3 + anti-CD28 activation of azPC on MBP-induced proliferation of T cells pre- treated with small-molecule inhibitors of the phospho- 3000 1600 1400 lipases PLC (inhibitor U73122); cPLA2a (EMD525143); 2500 1200 iPLA2 (FKGK11); sPLA2 groups IIA, IID, IIE, V, and X 2000 1000 (YM 26734); sPLA groups IIA and V (LY 311727); 800 2

(pg/ml) 1500

γ 600 sPLA2 group V (CAY10590); or sPLA2 group IIA 1000 * (pg/ml)TNF 400 *

IFN- (EMD525145). We found that preincubating T cells 500 * 200 * by guest on March 14, 2019 * * * * with any of the four sPLA inhibitors suppressed the 0 0 2 Media Media PGPC azPC azPC POPS ETOH PA Media Media PGPC azPC azPC POPS ETOH PA effects of azPC (that is, MBP-stimulated T cell prolifer- ester ester ation was partially restored), whereas preincubating the Control Anti-CD3 + anti-CD28 activation Control Anti-CD3 + anti-CD28 activation T cells with various concentrations of the other phospholipase inhibitors had no effect or, at high doses, D α α 4 -CD3/CD28Data.001 α-CD3/CD28+ETOHData.002 4 -CD3/CD28+PAData.005 10 104 10 24% 25% 39%

3 3 10 103 10 Fig. 6. Palmitic acid, a nonpolar side chain of 1-palmitoyl phospholipids, suppresses T cell proliferation and inflam- 2 2 10 62% 11% 102 61% 12% 10 41% 16% matory cytokine production, induces T cell apoptosis, and attenuates EAE. (A) Structure of palmitic acid. (B and C) 1 1 10 101 10 Proliferation (B) and cytokine production (C) of naïve T cells purified from wild-type B6 mice and stimulated for 100 0 100 0 1 2 3 4 10 0 1 2 3 4 10 10 10 10 10 100 101 102 103 104 10 10 10 10 10 48 hours with anti-CD3 and anti-CD28 antibody (5 mg/ml) Annexin V PE Annexin V PE Annexin V PE and either lipid (30 mg/ml), ethanol (ETOH), or 0.25 mM 7AAD (fluorescence intensity) 7AAD (fluorescence Annexin V (fluorescence intensity) palmitic acid (PA). Values are the means ± SEM of triplicate E experiments. Results are representative of three indepen- 4 Vehicle dent experiments (*P <0.05byStudent’s t test compared Palmitic acid to anti-CD3/anti-CD28 alone). (D) Apoptosis (indicated by + 3 annexin V and 7AAD staining) of CD3 T cells purified from * wild-type B6 mice and stimulated with plate-bound anti- * * CD3 and anti-CD28 antibodies for 48 hours alone or with 2 * ethanol (ETOH) or 0.25 mM palmitic acid (PA). Cells are gated + on CD4 Tcells.(E) Clinical scores of PLP139–151–immunized 1 SJL mice treated at the peak of EAE with 100 mgperinjection Mean clinical EAE EAE score clinicalMean Randomize and treat of palmitic acid (n =10),orvehiclealone(n = 10). Arrows 0 indicate injections of palmitic acid or vehicle. Each point rep- 0 2 4 6 8

10 12 14 16 18 20 22 24 26 28 30 32 34 resents the mean clinical score (*P < 0.05 by Mann-Whitney Days following immunization test comparing vehicle treatment to palmitic acid treatment).

www.ScienceTranslationalMedicine.org 6 June 2012 Vol 4 Issue 137 137ra73 8 RESEARCH ARTICLE killed the T cells (fig. S18 and table S6). Together, these results suggest patients contains antibodies to oxidized phosphatidylcholine deriv- that the nonesterified fatty acid side chains of the targeted lipids are atives, and antibodies to oxidized phosphatidylcholine have been responsible for their T cell suppressive properties. detected in MS brain lesions (11), supporting the idea that the antibodies to oxidized phosphatidylcholine derivatives that we detect in CSF can bind their lipid targets in MS brain. Moreover, studies in DISCUSSION EAE indicate that antibodies to oxidized phosphatidylcholine are generated as part of the pathological process of autoimmune demyelination The aims of this study were to determine whether lipids in the myelin (11). Apart from participating in the general destruction of the myelin sheath are targeted by autoimmune responses in MS. We report the sheath, such autoantibody targeting of oxidized phosphatidylcholine de- use of two lipidomic technologies—lipid antigen arrays and lipidomic rivatives could conceivably contribute to MS pathogenesis by reducing mass spectrometry—to identify myelin lipids targeted by autoantibody the levels or blocking the immunoregulatory activity of these protec- responses in MS. We show that these autoantibody-targeted phospho- tive lipids. lipids ameliorate disease in a mouse model of MS by inhibiting the Oxidized phospholipids are generally considered proinflammatory autoaggressive T cell responses that underpin autoimmune demyelina- and may exacerbate inflammation-associated disease (37, 38). Where- tion. Whereas the polar head groups are the lipid components targeted as oxidized phosphatidylcholine was previously identified as a marker by the autoantibodies, the fatty acid side chains are the components of neuroinflammation in MS (11),ourfindingssuggestthatoxidized that mediate the lipids’ anti-inflammatory effects. Furthermore, we find phosphatidylcholine derivatives in fact function as part of an endog- Downloaded from that the levels of these autoantibody-targeted lipids are lower in lesions enous feedback mechanism that attenuates adaptive autoimmune re- from MS brain than in tissue from healthy brain. Autoantibody target- sponses in the brain. Indeed, oxidized phospholipids are emerging as ing may thus reduce the lipids’ anti-inflammatory effect by enhancing Janus-like molecules: Whereas their pathogenic role in atherosclerosis their clearance or inhibiting their activity—and thereby compromise the is well established (32), these lipids play a protective, anti-inflammatory lipid-mediated protection against neuroinflammation. Thus, our find- role in endotoxin-induced tissue damage by inhibiting Toll-like recep- ings indicate that myelin phospholipids are targeted by autoimmune re- tor 4 signaling (39). Thus, the role of oxidized phospholipids in phys- http://stm.sciencemag.org/ sponses in MS and that these myelin phospholipids represent a natural iology and pathophysiology is context-dependent. In brain, oxidized anti-inflammatory class of compounds that have potential as therapeu- phosphatidylcholine derivatives and POPS appear to be important mit- tics for MS. igators of autoimmune responses. At the molecular level, the anti-inflammatory effects of the lipids We find that the saturated, nonpolar side chains mediate the protec- were associated with the inhibition of NF-kBandERK,signalingmol- tive effects of the therapeutic lipids. Palmitic acid, representative of such ecules that promote inflammation and cell survival and the derepres- fatty acid side chains present within the lipids, was able to reproduce sion of Bim and Bad, the molecular executors of programmed cell the therapeutic effects of the lipids in vitro and in vivo. The results of death. It is possible that POPS, which contains the phosphoserine our inhibitor studies suggest that these fatty acids exert at least some by guest on March 14, 2019 moiety detected by 7AAD, could bind and/or integrate into cellular of their immunoregulatory effects after their release from lipids by membranes and thereby exaggerate the staining observed with 7AAD. sPLA2-mediated cleavage. Levels of sPLA2, and of several other phos- Another limitation of this study includes the use of higher concentra- pholipases, are abnormally high in MS brain lesions (14, 40), and tions of phospholipids compared to the levels physiologically present sPLA2 plays a key role in the excessive production of arachidonic acid in human brain. Nevertheless, the lipids are rapidly diluted in the blood in MS (40). The increase in phospholipase expression in MS brain le- and tissues after administration, and azPC and POPS administered in- sions could increase the levels of anti-inflammatory free fatty acids as travenously demonstrated increased apoptosis of infiltrating lymphocytes part of a feedback mechanism that protects against neuroinflamma- in the perivascular cuffs of brain and spinal cord lesions of azPC- and tion. Autoantibody targeting of POPS and oxidized phosphatidylcholine POPS-treated mice with EAE. Intriguingly, osteopontin, a protein that derivatives may compromise this protective feedback mechanism by induces relapse and progression of EAE, has the opposite effect: It pro- eliminating the lipids containing the anti-inflammatory fatty acids motes the survival of MBP-reactive T cells by activating the NF-kB or by preventing the release of fatty acids from the lipids. Regardless pathway and inhibiting Bim-mediated apoptosis (34). Indeed, T cell ofwhethertheyactasfreefattyacidsorasphospholipidcomponents, apoptosisisakeymechanismbywhich autoimmune attack on the CNS these saturated fatty acids appear to serve a function similar to that is kept in check, and it plays an important role in spontaneous EAE of the unsaturated fatty acid derivatives resolvins and protectins, remission (35). which mediate resolution of inflammation in tissue exudates (30, 31). In MS brain lesions, inflammation leads to an increase in nitric oxide, The use of lipidomics for drug discovery provides previously un- which can oxidize lipid components of the brain (36). Using mass spec- realized opportunities. The adaptive immune system may target mol- trometry, we demonstrated the presence of oxidized phospholipids— ecules that are aberrantly modified or expressed and contribute to the and specifically of the autoantibody-targeted therapeutic lipids—in autoimmune pathology (41). However, our results suggest that the im- normal and in MS brain. We found that levels of oxidized phosphatidyl- mune system may also drive autoimmune disease by abrogating the choline derivatives were lower in MS brain than in healthy brain. This protective effects of molecules involved in inflammatory homeostasis. may reflect the autoimmune-mediated destruction of myelin lipids Choosing the target of the antibody as a potential therapeutic, in this that occurs in MS, such that levels of oxidized phospholipids are case the lipids identified on an autoantibody array, provides a fresh diminished despite an increase in lipid peroxidation. The destruc- strategy for screening putative therapeutics. We found that the phos- tion of myelin involves anti-lipid autoantibodies, which can induce phocholine head group is an important determinant of the antigenicity demyelination and prevent remyelination in mouse models of MS of brain lipids, a discovery that enabled the identification of addi- (8). Through antigen array screening, we found that the CSF of MS tional therapeutic lipids through stringent statistical analysis of lipid

www.ScienceTranslationalMedicine.org 6 June 2012 Vol 4 Issue 137 137ra73 9 RESEARCH ARTICLE microarray data combined with lipidomic mass spectrometric anal- verized in liquid nitrogen. Lipid extracts were generated and analyzed ysis. The identification of lipids targeted by autoantibodies affords the by electrospray ionization mass spectrometry (typically within 1 week) opportunity to mine small lipid-soluble molecules as potential new with a TSQ Quantum Ultra Plus triple-quadrupole mass spectrometer drugs for autoimmune disease. (Thermo Fisher Scientific) equipped with an automated nanospray apparatus (Nanomate HD, Advion Bioscience Ltd.) and Xcalibur system software (45). MATERIALS AND METHODS EAE induction Reagents Animal experiments were approved by, and performed in compliance We obtained POPS, PGPC, sphingomyelin, sulfatide, azPC, azPC es- with, the National Institutes of Health guidelines of the Institutional ter, and all other phosphatidylcholine derivatives listed in table S2 Animal Care and Use Committee at Stanford University. For induc- from Avanti Polar Lipids. Palmitic acid was purchased from Sigma. tion of EAE, 8- to 12-week-old female SJL/J mice (Jackson Laboratory) PLP139–151 (HCLGKWLGHPDKF) and MBPAc1–11 (ASQKRPSQRHG) were immunized subcutaneously with 100 mgofPLP139–151 emulsified were synthesized and high-performance LC (HPLC)–purified (>97%) in complete Freund’s adjuvant (CFA) (Difco Laboratories). Clinical by the Stanford Protein and Nucleic Acid facility. disease was monitored daily with the following scoring system: 0, no disease; 1, limp tail; 2, hindlimb weakness; 3, hindlimb paralysis;

Patient CSF samples 4, hindlimb and forelimb paralysis; 5,death.Specificlipidtreatment Downloaded from All human samples were collected and used under protocols approved regimens are further described in the Supplementary Materials and by the Institutional Review Boards of the Karolinska Institute and Methods. Stanford University. Patient demographics and clinical characteristics are listed in table S1. Proliferation and cytokine assays Splenocytes were harvested from mice transgenic for the MBPAc1–11– Lipid array analysis specific T cell receptor (15)andstimulatedwithMBPAc1–11 (2 mg/ml) http://stm.sciencemag.org/ Lipid arrays were generated and analyzed as previously described (3). in the presence of lipid (30 mg/ml). Lymph nodes and spleens were Briefly, we used a Camag Automatic TLC Sampler 4 robot to print 10 also harvested from naïve C57BL/6 mice, and CD3+ T cell enrich- to 100 pmol of lipids on PVDF membranes affixed to the surface of ment columns (R&D Systems) were used to isolate CD3+ Tcells.The microscope slides. These lipid arrays were probed with 1:10 or 1:20 purified CD3+ T cells were stimulated with plate-bound anti-CD3 dilutions of human CSF, followed by 1:8000 dilution of either anti- antibodies and anti-CD28 antibodies (5 mg/ml) in the presence of human IgG + IgM or anti-human IgG (Jackson ImmunoResearch) POPS, PGPC, azPC ester, brain sulfatides, or azPC (30 mg/ml) (Avanti conjugated to horseradish peroxidase (HRP). Bound HRP-conjugated Polar Lipids), 0.25 mM palmitic acid (Sigma-Aldrich), or 100% etha- antibodies were visualized by chemiluminescence (ECL Plus, Amersham) nol (as the vehicle control). For assessment of proliferation, 1 mCi of by guest on March 14, 2019 and autoradiography. We used GenePix Pro 5.0 software (Molecular [3H]thymidine was added to each well for the final 18 to 24 hours Devices) to extract the net median pixel intensities for individual fea- of culture, and incorporation of radioactivity was measured with a tures from digital images of the array autoradiographs. We applied the Betaplate scintillation counter. Cytokine assays were performed on SAM (9) algorithm (version 1.21) to identify lipids with statistically culture supernatants after 24 hours (IL-12p40) or 48 hours (IFN-g, significant differences in array reactivity between groups of humans. IL-6, IL-17A, and TNF) of culture with the BD OptEIA Mouse ELISA The list of “significant lipids” with the lowest q value (FDR) is reported kits (BD Biosciences) or Mouse IL-17 DuoSet ELISA Development kit in each heatmap figure. We arranged the SAM results into relation- (R&D Systems). ships using Cluster software and displayed the results using TreeView software (10). Flow cytometry Cells were stained according to standard protocols, run on a FACScan Lipidomic analysis of brain samples flow cytometer (BD Biosciences), and analyzed with CellQuest Archived postmortem samples from MS brain and age-matched healthy software (BD Immunocytometry Systems). The antibody conjugate brain were analyzed by shotgun lipidomics as previously described used was fluorescein isothiocyanate (FITC) anti-CD4 clone GK1.5 (42–44) and as outlined in the Supplementary Materials and Methods. (BD Pharmingen). 7AAD staining was performed with the PE Annexin The six MS brain samples analyzed were as follows: MS 1, an active V Apoptosis Detection Kit I (BD Pharmingen). lesion from a 59-year-old female with RRMS; MS 2, an active lesion from a 72-year-old male with SPMS; MS 3, a chronic active lesion from Western blotting a 47-year-old female with chronic MS; MS 4, a chronic active lesion CD3+ T cells were isolated from the lymph nodes of naïve C57BL/6 from a 76-year-old male with chronic MS; MS 5, an acute active lesion mice with CD3+ T cell enrichment columns (R&D Systems). T cells from a 31-year-old female in the relapsing phase of RRMS; MS 6, a were preincubated with lipid (30 mg/ml) for 1 hour at 37°C and then chronic active lesion from the same 31-year-old female with RRMS. stimulated with plate-bound anti-CD3 and anti-CD28 antibodies Control brain samples were thoroughly examined to rule out the pres- (5 mg/ml, eBioscience) in the presence of the lipids for 15 min or ence of neurological disease. Control samples were obtained from 24 hours. Cells were washed with ice-cold phosphate-buffered saline normal-appearing white matter from the brains of the following indi- (PBS) and lysed in radioimmunoprecipitation assay (RIPA) lysis viduals: C1, 23-year-old male; C2, 52-year-old female; C3, 23-year-old buffer containing 1× Halt protease and phosphatase inhibitor cocktail male; C4, 52-year-old male; C5, 82-year-old male; and C6, 44-year-old (Pierce) with a Dounce homogenizer. Immunoblotting was performed female. Samples of healthy brain and samples of MS lesions were pul- with antibodies against phospho-Bim (Ser69), phospho-Bad (Ser112),

www.ScienceTranslationalMedicine.org 6 June 2012 Vol 4 Issue 137 137ra73 10 RESEARCH ARTICLE phospho–Bcl-2 (Ser70), phospho-IKKa/b (Ser180/181), phospho-p65 6. A. Shamshiev, A. Donda, I. Carena, L. Mori, L. Kappos, G. De Libero, Self glycolipids as T-cell – (Ser536), phospho-ERK1/2 (Thr202/Tyr204), and IkBa from Cell autoantigens. Eur. J. Immunol. 29, 1667 1675 (1999). 7. L. M. Villar, M. C. Sádaba, E. Roldan, J. Masjuan, P. González-Porqué, N. Villarrubia, M. Espiño, Signaling Technology. J. A. García-Trujillo, A. Bootello, J. C. Alvarez-Cermeño, Intrathecal synthesis of oligoclonal IgM against myelin lipids predicts an aggressive disease course in MS. J. Clin. Invest. 115, TUNEL assay 187–194 (2005). Mice with EAE were treated intravenously with 200 mg of azPC, 200 mg 8. J. Rosenbluth, R. Schiff, W. L. Liang, W. Dou, Antibody-mediated CNS demyelination II. Focal spinal cord lesions induced by implantation of an IgM antisulfatide-secreting hybridoma. of POPS, or vehicle (0.05% Tween 20 in PBS) on day 15 after immuni- J. Neurocytol. 32,265–276 (2003). zation with CFA and PLP139–151 peptide. Mice were treated with lipids 9. V. G. Tusher, R. Tibshirani, G. Chu, Significance analysis of microarrays applied to the ionizing for 12 hours and then killed and perfused with 4% paraformaldehyde. radiation response. Proc. Natl. Acad. Sci. U.S.A. 98, 5116–5121 (2001). Brains and spinal cords were embedded in paraffin and sectioned. 10. M. B. Eisen, P. T. Spellman, P. O. Brown, D. Botstein, Cluster analysis and display of genome- – TUNEL-positive cells were detected with the In Situ Cell Death Kit, wide expression patterns. Proc. Natl. Acad. Sci. U.S.A. 95, 14863 14868 (1998). ’ 11. J. Qin, R. Goswami, R. Balabanov, G. Dawson, Oxidized phosphatidylcholine is a marker for AP (Roche) according to the manufacturer s instructions. neuroinflammation in multiple sclerosis brain. J. Neurosci. Res. 85, 977–984 (2007). 12.B.W.Agranoff,J.A.Benjamins,A.K.Hajra,Lipids,inBasic Neurochemistry: Molecular, Cellular and Medical Aspects, G. J. Siegel, B. W. Agranoff, R. W. Albers, S. K. Fisher, M. D. Fisher, SUPPLEMENTARY MATERIALS Eds. (Lippincott-Raven, Philadelphia, 1999). 13. J. van Horssen, G. Schreibelt, J. Drexhage, T. Hazes, C. D. Dijkstra, P. van der Valk, H. E. de Vries, www.sciencetranslationalmedicine.org/cgi/content/full/4/137/137ra73/DC1 Severe oxidative damage in multiple sclerosis lesions coincides with enhanced antioxidant – Downloaded from Materials and Methods enzyme expression. Free Radic. Biol. Med. 45,1729 1737 (2008). Fig. S1. Cerebrospinal fluid levels of IgG and IgM are elevated in MS. 14. M. H. Han, S. I. Hwang, D. B. Roy, D. H. Lundgren, J. V. Price, S. S. Ousman, G. H. Fernald, Fig. S2. Oxidized cholesterol, but not brain cerebrosides, modulates EAE. B. Gerlitz, W. H. Robinson, S. E. Baranzini, B. W. Grinnell, C. S. Raine, R. A. Sobel, D. K. Han, Fig. S3. PGPC treatment of mice with EAE suppresses T cell activation. L. Steinman, Proteomic analysis of active multiple sclerosis lesions reveals therapeutic targets. – Fig. S4. Ceramide is sphingomyelin without the phosphate polar head group. Nature 451,1076 1081 (2008). Fig. S5. DGPC binds to the PVDF membrane used in the lipid arrays. 15. J. J. Lafaille, K. Nagashima, M. Katsuki, S. Tonegawa, High incidence of spontaneous auto- Fig. S6. Levels of antibodies against PGPC, POPS, azPC ester, and azPC are higher in RRMS than immune encephalomyelitis in immunodeficient anti-myelin basic protein T cell receptor

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42. H. Cheng, X. Jiang, X. Han, Alterations in lipid homeostasis of mouse dorsal root ganglia Accepted 17 April 2012 http://stm.sciencemag.org/ induced by apolipoprotein E deficiency: A shotgun lipidomics study. J. Neurochem. 101, Published 6 June 2012 57–76 (2007). 10.1126/scitranslmed.3003831 43. X. Han, R. W. Gross, Shotgun lipidomics: Multidimensional MS analysis of cellular lipidomes. Expert Rev. Proteomics 2,253–264 (2005). Citation: P. P. Ho, J. L. Kanter, A. M. Johnson, H. K. Srinagesh, E.-J. Chang, T. M. Purdy, K. van Haren, 44. X. Han, R. W. Gross, Shotgun lipidomics: Electrospray ionization mass spectrometric anal- W. R. Wikoff, T. Kind, M. Khademi, L. Y. Matloff, S. Narayana, E. M. Hur, T. M. Lindstrom, Z. He, O. Fiehn, ysis and quantitation of cellular lipidomes directly from crude extracts of biological samples. T. Olsson, X. Han, M. H. Han, L. Steinman, W. H. Robinson, Identification of naturally occurring fatty Mass Spectrom. Rev. 24, 367–412 (2005). acids of the myelin sheath that resolve neuroinflammation. Sci. Transl. Med. 4, 137ra73 (2012). by guest on March 14, 2019

www.ScienceTranslationalMedicine.org 6 June 2012 Vol 4 Issue 137 137ra73 12 Identification of Naturally Occurring Fatty Acids of the Myelin Sheath That Resolve Neuroinflammation Peggy P. Ho, Jennifer L. Kanter, Amanda M. Johnson, Hrishikesh K. Srinagesh, Eun-Ju Chang, Timothy M. Purdy, Keith van Haren, William R. Wikoff, Tobias Kind, Mohsen Khademi, Laura Y. Matloff, Sirisha Narayana, Eun Mi Hur, Tamsin M. Lindstrom, Zhigang He, Oliver Fiehn, Tomas Olsson, Xianlin Han, May H. Han, Lawrence Steinman and William H. Robinson

Sci Transl Med 4, 137ra73137ra73. DOI: 10.1126/scitranslmed.3003831 Downloaded from Eliminating Excess In tough economic times, businesses trim the fat by cutting superfluous spending. The immune system also has ways to regulate excess, such as feedback mechanisms that prevent autoimmunity. In patients with multiple s clerosis (MS), these protective mechanisms fail: The immune system attacks peripheral nerves, resulting in demyelination and a decrease in the ability of axons to carry physiological signals. Researchers have identified autoantibodies to many of the lipids that make up the myelin sheath of nerve cells, and these target lipids are believed to serve as pathogenic antigens for autoimmune encephalomyelitis−−an inflammation of the brain and

spinal cord seen in MS patients. However, new work by Ho et al. shows that some fat shouldn't be trimmed; http://stm.sciencemag.org/ indeed, certain fatty acids of the myelin sheath actually serve to resolve neuroinflammation in a therapeutic mouse model of MS, experimental autoimmune encephalomyelitis (EAE). With lipid antigen microarrays and mass spectrometry, the authors identified targets of the autoimmune response in the brains of patients with MS and then used these lipid antigens to immunize mice with clinical EAE symptoms. Some of the phospholipids tested suppressed activation of and induced apoptosis in autoreactive T cells, ameliorating disease in the EAE mice, an effect that was mediated by the lipids' fatty acid side chains. These data suggest that some phospholipids are natural anti-inflammatory compounds composed of fatty acids that trim t he fat−−in this case, excessive behavior by the immune system. by guest on March 14, 2019

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