Identification of naturally occurring fatty acids of the myelin sheath that resolve neuroinflammation

Abstract

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 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.

Fig. 1

Autoantibody targeting of lipids is higher in MS CSF than in OND CSF and normal CSF, and the autoantibody-targeted lipid PGPC attenuates EAE. (A) Lipid-array profiling of IgG+IgM antibody reactivity in CSF samples from MS patients (RRMS, relapsing remitting MS; SPMS, secondary progressive MS; PPMS, primary progressive MS), healthy controls (HC), and other neurological disease (OND) controls. Lipid hits with the lowest FDR (q=0.048) were clustered according to their reactivity profiles. Sample type and ID number are shown above the heatmap, and the lipids targeted are shown to the right of the heatmap. The patients’ demographics and clinical characteristics are presented in Supplementary Table 1. (B) Clinical EAE scores of mice coinjected subcutaneoulsy with PLP_139–151 and 6 µg/injection of PGPC (day 0); on days 4 and 7 after immunization, PGPC was injected intraperitoneally. Arrows indicate when PGPC was administered. Each point represents the mean ± SEM, and results are representative of 4 independent experiments. *P < 0.05 Mann-Whitney test, vehicle control (n = 5) vs PGPC (n = 5).

Fig. 2

PGPC-related lipids with non-bulky polar head groups are targeted by antibodies in CSF of MS patients. (A) Mini-Array I: IgG antibody reactivity to various glycero-3-phosphocholine lipids in CSF samples from patients with relapsing remitting MS (RRMS) and from control patients with other neurological disease (OND). Lipid hits with the lowest FDR (q=0.029) were clustered according to their reactivity profiles. Sample type and ID number are shown above the heatmap, and the lipids targeted are shown to the right of the heatmap. (B) Structures of lipid hits in (A). (C) Mini-Array II: IgG antibody reactivity to lipids constituting polar head-group and side-chain modifications of PGPC in CSF samples from RRMS patients and OND controls. Lipid hits with the lowest FDR (q=0.016) were clustered according to their reactivity profiles. All of the lipids screened in Mini Array I and II are listed in Supplementary Table 2. (D) Left column, structures of the lipid targets identified in (C), with green boxes around the polar head group; right column, structures of the lipids that were not targeted, with red boxes around the polar head group.

Fig. 3

Levels of POPS, PGPC, azPC, and azPC ester are higher in MS brain than in healthy brain. (A) Negative-ion electrospray ionization mass spectrometric analyses of palmitoyl oleoyl phosphatidylserine (POPS) in lipid extracts of normal-appearing white matter from an age-matched healthy control brain (left panel) and of an active lesion from a brain afflicted with relapsing remitting MS (right panel). DMPS, dimyristoryl phosphatidylserine; IS, internal standard. (B) Single-reaction monitoring analysis of PGPC azPC ester, azPC, and POPS levels in samples of MS brain lesions (n = 6) and healthy brain tissue (n = 6). Controls: age-matched individuals with no signs of neurological disease; MS: 3 patients with relapsing remitting MS; 1 patient with secondary progressive MS; and 2 patients with chronic MS. *P<0.05 by unpaired Student’s t-test.

Fig. 4

Administration of lipids that are targeted by autoantibodies and whose levels are decreased in MS attenuate ongoing EAE and T-cell activation. (A) Clinical scores of PLP_139–151-immunized SJL mice treated at the peak of EAE with 100 µg/injection of POPS (n = 10), PGPC (n = 10), azPC ester (n = 10), azPC (n = 10), or vehicle alone (n = 10). Arrows indicate injections of lipid or vehicle. Each point represents the mean clinical score ± SEM (* denotes time points at which P < 0.05 by Mann-Whitney test comparing vehicle treatment vs. lipid treatment). (B) Cytokine production by and (C) proliferation of naive MBP_Ac1–11-TCR transgenic splenocytes stimulated with 2 µg/ml MBP_Ac1–11 in the presence of 30 µg/ml of lipid (structures shown in D), as indicated. Values are the mean + SEM of triplicates. Results are representative of 3 independent experiments. *P < 0.05 by Student’s t-test, each lipid plus MBP_Ac1–11 vs. MBP_Ac1–11 alone.

Fig. 5

POPS, PGPC, azPC, and azPC ester induce apoptotic signaling pathways and T-cell apoptosis. (A) 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 h with or without 30 µg/ml of lipid. Cells are gated on CD4⁺ T cells, and results are representative of 3 experiments. (B) Immunoblot analysis of phopsho-ERK1/2, phospho-ΙΚΚα/β, phospho-p65, IκBα, 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 h (right panel) in the presence of 30 µg/ml of lipid, as indicated. Blots are representative of two independent experiments. (C) TUNEL staining of brain and spinal-cord tissue from mice immunized with PLP_139–151 peptide (to induce EAE) and treated for 12 h with azPC, POPS, or vehicle on day 15 after immunization. Arrows indicate TUNEL-positive (bright pink/red) infiltrating cells in the perivascular cuffs of lesions from mice with active EAE. Original magnification, ×400. (D) Quantification of TUNEL-positive infiltrating cells in brain and spinal cord sections shown in panel (C).

Fig. 6

Palmitic acid, a non-polar side chain of 1-Palmitoyl phospholipids, suppresses T-cell proliferation and inflammatory cytokine production, induces T-cell apoptosis, and attenuates EAE. (A) Structure of palmitic acid. (B) Proliferation and (C) cytokine production of naive T cells purified from wild-type B6 mice and stimulated for 48 h with 5 µg/ml of anti-CD3 and anti-CD28 antibody and either 30 µg/ml of lipid or 0.25 mM of palmitic acid. Values are the mean + s.e.m. of triplicates. Results are representative of 3 independent experiments (*P < 0.05 by Student’s t-test, compared to anti-CD3/anti-CD28 alone). (D) Apoptosis (indicated by 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 h alone or with ethanol (ETOH) or 0.25 mM of palmitic acid (PA). Cells are gated on CD4⁺ T cells. (E) Clinical scores of PLP_139–151-immunized SJL mice treated at the peak of EAE with 100 µg/injection of palmitic acid (n = 10), or vehicle alone (n = 10). Arrows indicate injections of palmitic acid or vehicle. Each point represents the mean clinical score (*P < 0.05 by Mann-Whitney test comparing vehicle treatment vs. palmitic acid treatment).