The FASEB Journal article fj.201500131. Published online February 18, 2016. THE

JOURNAL

• RESEARCH •

www.fasebj.org

Specific autoantigens in experimental autoimmunityassociated atherosclerosis Aksam J. Merched,*,†,‡,1 Dani`ele Daret,§ Lan Li,‡ Nathalie Franzl,§ and Maria Sauvage-Merched{

*Department of Pharmaceutical Sciences, and †INSERM U1053, University of Bordeaux, Bordeaux University, Bordeaux, France; ‡Department of Cell Biology, Baylor College of Medicine, Houston, Texas, USA; §INSERM U1034, Pessac, France; and {Children's Hospital, University Hospital of Bordeaux, Bordeaux, France

Higher cardiovascular morbidity in patients with a wide range of autoimmune diseases highlights the importance of autoimmunity in promoting atherosclerosis. Our purpose was to investigate the mechanisms of accelerated atherosclerosis and identified vascular autoantigens targeted by autoimmunity. We created a mouse model of autoimmunity-associated atherosclerosis by transplanting bone marrow (BM) from FcgRIIB knockout (FcRIIB2/2) mice into LDL receptor knockout (LDLR2/2) mice. We characterized the cellular and molecular mechanisms of atherogenesis and identified specific aortic autoantigens using serologic proteomic studies. En face lesion area analysis showed more aggressive atherosclerosis in autoimmune mice compared with control mice (0.64 6 0.12 mm2 vs. 0.32 6 0.05 mm2; P < 0.05, respectively). At the cellular level, FcRIIB2/2 macrophages showed significant reduction (46–72%) in phagocytic capabilities. Proteomic analysis revealed circulating autoantibodies in autoimmune mice that targeted 25 atherosclerotic lesion proteins, including essential components of adhesion complex, cytoskeleton, and extracellular matrix (ECM), and proteins involved in critical functions and pathways. Microscopic examination of atherosclerotic plaques revealed essential colocalization of autoantibodies with endothelial cells (ECs), their adherence to basement membranes, the internal elastica lamina, and necrotic cores. The new vascular autoimmunosome may be a useful target for diagnostic and immunotherapeutic interventions in autoimmunity-associated diseases that have accelerated atherosclerosis.—Merched, A. J., Daret, D., Li, L., Franzl, N., Sauvage-Merched, M. Specific autoantigens in experimental autoimmunity-associated atherosclerosis. FASEB J. 30, 000–000 (2016). www.fasebj.org

ABSTRACT:

KEY WORDS:

phagocytosis

•

extracellular matrix

•

cytoskeleton

Atherosclerosis is an immunoinflammatory disease affected by innate and adaptive immune responses (1, 2). Cardiovascular diseases and accelerated atherosclerosis may be a feature of several autoantibody-associated diseases, such as systemic lupus erythematosus (SLE), 2/2 , knockout/deficient; 2/3D, 2/3-dimensional; Actn, alpha actinin; ANA, antinuclear antibody; Anxa1, Annexin A1; apoE, apolipoprotein E; Arp2, actin-related protein 2; BM, bone marrow; Col6, collagen VI; EC, endothelial cell; ECM, extracellular matrix; Eef2, eukaryotic translation elongation factor 2; Fbln5, fibulin 5; FcRIIB, FcgRIIB; Fgg, fibrinogen g; Flna, filamin a; Fn1, fibronectin 1; Ganab, a glucosidase 2 a neutral subunit; GM-CSF, granulocyte macrophage-colony-stimulating factor; Gsn, gelsolin; Hsp70, heat shock protein 70; KC, keratinocytederived chemokine; Kif5b, kinesin 5B; LC, liquid chromatography; LDLR, LDL receptor; LRP, LDL receptor-related protein; MHC, major histocompatibility complex; Mif, macrophage migration inhibitory factor; MS, mass spectrometry; Mvp, major vault protein; PAS, periodic acid Schiff; PSR1, phosphatidylserine receptor 1; Sec13, SEC13-like 1; SLE, systemic lupus erythematosus; Tln2, Talin2; UFD1, ubiquitin fusion degradation protein 1; Vcl, vinculin; VCP, valosin-containing protein

ABBREVIATIONS:

1

Correspondence: UFR Pharmacie, Laboratoire de Biologie Cellulaire, 146 rue L´eo Saignat, 33076 Bordeaux cedex, France. E-mail: aksam. [email protected]

doi: 10.1096/fj.201500131 This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

0892-6638/16/0030-0001 © FASEB

•

cell junctions

antiphospholipid syndrome, and rheumatoid arthritis (3–5). This suggests that autoimmune processes may contribute to the progression of atherosclerosis, and it may be important to identify early diagnostic markers and immunotherapeutic targets for treatment. The presence of Ig in atherosclerotic plaques is a hallmark of atherosclerosis. However, the exact features of the targeted antigens are unknown. Several self-antigens have been proposed as candidate targets of both cell-mediated and humoral immune responses, including epitopes from heat shock proteins (Hsps) (6), ECs (7), b2-glycoprotein I (8, 9), and apolipoprotein A-I (10). Autoantigens in atherosclerosis include oxidized LDL (11). However, the contribution of these autoantigens to the immune complexes in lesions and to overall autoimmune responses is not fully understood. The central function of the immune system includes degradation and elimination of foreign antigens. Tolerance to self-antigens is important in preventing autoimmune disorders. FcRs are cell-surface molecules that bind to the Fc domain of IgG and initiate reactions, including the elimination of IgG-containing immune complexes. The FcRs trigger the internalization of captured 1

immune complexes, degradation of antigen–antibody complexes, and delivery of antigenic peptides to the major histocompatibility complex (MHC) class I or class II antigen-presentation pathway (12). The FcRs may be activating (FcRs, FcRI, and FcRIII) or inhibitory (FcRIIB), depending on the component motifs, and the balance between activation and inhibition may determine the effector response (13, 14). Inhibition of FcRIIB may increase autoantibody production, initiate autoimmune diseases, and promote atherogenesis in atherosclerosissusceptible mice (15–19). The present study was designed to evaluate the autoimmune component of atherogenesis and identify putative antigens that may trigger the autoimmune reactions. We created a mouse model of autoimmunityassociated atherosclerosis by transplanting BM from FcRIIB2/2 mice to LDLR2/2 mice. This approach combined 2 genetic backgrounds associated with lupus and atherosclerosis. We characterized the cellular and molecular mechanisms of atherogenesis and identified specific autoantigens in the vascular wall using serologic proteomic studies. In this approach, aortic atherosclerotic protein lysate was separated by 2-dimensional (2D) gel electrophoresis, and specific antigen spots were determined by Western blot analysis using autoimmune and control sera. Differential and higher immunoreactive protein spots to autoimmune sera were identified by mass spectrometry (MS). MATERIALS AND METHODS Animals, BM transplant, quantitative morphometry, and histology The C57BL/6J mice used were FcRIIB2/2 (Taconic Biosciences, Hudson, NY, USA) and LDLR2/2 (The Jackson Laboratory, Bar Harbor, ME, USA). Female LDLR2/2 mice (n = 32 mice; age 8–10 wk) were transplanted with BM from wild-type (FcRIIB+/+) mice or FcRIIB2/2 mice, and the extent of atherosclerosis was evaluated after 14 wk of high-fat feeding by quantitative morphometry, as described previously (20). Proteinuria was assessed from 16 h urine samples that were collected in metabolic cages. Urinary protein concentrations were determined by the Bradford method, adapted to a microtiter plate assay. Coomassie reagent (USB Corp., Cleveland, OH, USA) was added to the diluted urine samples. After 10 min, the absorbance at 595 nm was determined (ELx800 microplate reader; BioTek, Winooski, VT, USA). The protein concentrations were calculated by reference to bovine serum albumin standards (Sigma-Aldrich, St. Louis, MO, USA). Serum and urine creatinine were analyzed using a creatinine kit (SigmaAldrich). For histology, kidneys were fixed in 4% buffered formalin, and paraffin sections (4 mm) were stained with hematoxylin-eosin or periodic acid Schiff (PAS) reagent. Gene expression and macrophage function Peritoneal macrophages were used to study gene expression using quantitative real-time PCR (20). For the phagocytosis assay, we used 2 labeled substrates [pHrodo Escherichia coli BioParticle (Thermo Fisher Scientific, Grand Island, NY, USA) and FluoroSpheres (Molecular Probes, Thermo Fisher Scientific)] in a protocol described previously (20). The RAW macrophage cell 2

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line was used to overexpress FcRIIB after transfection by a plasmid containing the FcRIIB gene under control of the scavenger receptor promoter (21). Proteomic studies To select panels of antigens in aortic lesions and to eliminate immunoreactivity bias of serum toward aortic components from the same mice, we used aortic extracts from syngeneic apolipoprotein E (apoE)2/2 mice (age 20–24 mo), which are considered as prototypes of atherosclerostic lesions. We extracted proteins from 2–3 pooled frozen aortas of these mice. Panels of antigens from these aortas were created with 2D SDS-PAGE. Proteins were transferred onto PVDF membranes. Sera from autoimmune mice (BM FcRIIB2/2, LDLR2/2) and control mice (BM FcRIIB+/+, LDLR2/2) were screened for antibodies that reacted against the separated proteins by Western blot analysis. Total aortic protein extracts were labeled with cyanine dye Cy3 and resolved on 2D analytical or preparative gels (Applied Biomics, Hayward, CA, USA). Immunoblots from analytical gels were performed on the same membranes (first with control sera; then with autoimmune sera after stripping the membrane). Highly immunoreactive spots were localized on a preparative gel and selected for identification by matrix-assisted laser desorption/ ionization time-of-flight MS. Immunofluorescence and confocal microscopy To evaluate colocalization of specific antigens with lesion mouse IgG, we used commercial antibodies (Supplemental Table 1). Tissue samples were analyzed with a confocal microscope (FV1000; Olympus, Tokyo, Japan) and an oil-immersion objective (360/1.4 ApoPlan, Olympus). The 2-axis scans had an optical slice thickness of 0.4 mm. Visualization was obtained after digitized image multichannel deconvolution (AutoDeblur; MediaCybernetics, Rockville, MD, USA), and 3D projections were digitally reconstituted from stacks of confocal optical slices (Imaris software; Bitplane AG, Zurich, Switzerland). Statistics Data were expressed as means 6 SEM and analyzed with MannWhitney U test or Student’s t test. Statistical significance was defined by P # 0.05.

RESULTS Inactivation of BM FcRIIB causes autoimmune phenotype and accelerates atherosclerosis LDLR2/2 mice lacking leukocyte FcRIIB (BM FcRIIB2/2) had high titers of antinuclear antibodies (ANAs) and anticardiolipin antibodies, the prototypic autoantibodies in SLE; high levels of immune complexes; and total IgG, indicating polyclonal B cell activation (Fig. 1A). Histologic analysis of the spleen and the kidneys showed splenomegaly and marked renal inflammation and damage in BM FcRIIB2/2 mice, including larger glomeruli with more cells and crescent formations than control mice (Fig. 1B, C). Proteinuria was observed in the autoimmune mice after 13 wk, consistent with renal functional impairment (Fig. 1C). BM FcRIIB2/2 was associated with 46% lower mean

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MERCHED ET AL.

Figure 1. Evidence of autoimmune phenotype, splenomegaly, and renal dysfunction in the FcRIIB2/2 transplanted mice (2/2) vs. control mice (+/+). A) Mean ANA titer (n = 8 and 7), immune complex titer (n = 8 and 7), anticardiolipin titer (n = 7 and 7), and IgG level (n = 8 and 7) were significantly increased in the absence of FcRIIB. B) Representative spleens from LDLR2/2 mice transplanted with BM, with (Bii, n = 16) or without (Biii, n = 16) expression of FcRIIB. For comparison, a spleen is shown from an LDLR2/2 mouse fed a normal chow diet (Bi). C ) Proteinuria in FcRIIB2/2 mice was significantly increased compared with control mice at 13 wk (n = 7 and 10). No difference was noted at 8 wk (n = 8 and 11). Histology of a kidney from an autoimmune (lower right) mouse revealed marked renal inflammation and damage, including enlarged glomeruli with hypercellularity and crescent formation, compared with normal renal morphology in a control mouse (upper right; PAS staining). D) Mean total plasma cholesterol (n = 9 and 10) and triglyceride concentrations (n = 7 and 9) were significantly lower in the absence of FcRIIB (n = 7) compared with controls (n = 5). E ) Analysis of lipoprotein by fast protein liquid chromatography (LC) showed that all lipoprotein fractions were altered [high-density lipoprotein (HDL), intermediate density lipoprotein (IDL), LDL, and very LDL (VLDL)]. *P , 0.05; **P , 0.01; ***P , 0.001.

plasma triglyceride level (106 6 18 vs. 196 6 14 mg/dl; P , 0.001) and 41% lower mean cholesterol level (472 6 65 vs. 796 6 66 mg/dl; P , 0.05; Fig. 1D). Lipid parameters are different compared with previous studies using the FcRIIB background (17, 19), probably because of variability in experimental designs and dietary conditions. VASCULAR TARGETS OF AUTOIMMUNITY IN ATHEROGENESIS

Lipoprotein examination by fast protein LC showed that all lipoprotein profiles were affected (Fig. 1E). BM FcRIIB inactivation and the associated autoimmunity were significantly associated with more aggressive atherosclerosis occurring in ascending aortas (Fig. 2A and Supplemental Fig. 1), despite significantly lower plasma 3

Figure 2. FcRIIB2/2 significantly accelerated atherosclerosis and modulated inflammation. A) Analysis of the aortas showed significantly more plaques in aortas of FcRIIB2/2 mice than FcRIIB+/+ mice [0.64 6 0.12 mm2 (n = 17) vs. 0.32 6 0.05 mm2 (n = 15); P = 0.03]. B) Thickness of the aortic sinus was similar in FcRIIB2/2 and FcRIIB+/+ mice [54 782 6 5018 m2 (n = 15) vs. 41 679 6 4545 m2 (n = 15); P = 0.06]. C ) Cytokine array analysis (n = 5–7) showed disrupted systemic inflammatory balance with significant reduction of IL-1a, IL-10, keratinocyte-derived cytokine (KC), and granulocyte macrophage-colony-stimulating factor (GM-CSF) and increase of IL-12p40. CCL5, Chemokine ligand 5; MIP-1a, macrophage inflammatory protein 1a. D) Immunohistochemical staining of mouse IgG in an aortic atherosclerotic lesion revealed a distribution of 2 layers of IgG located at the ECs (CD31 marker), necrotic core, and the internal elastic lamina.3D Z-projection view of confocal sections (upper right). Atheromas are delineated by discontinuous lines. Merged images are superimposed images showing all stains. L, Lumen; nc, necrotic cores; Smc, smooth muscle cell. *P , 0.05; **P , 0.01.

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TABLE 1. Isotyping of plasma Igs FcR2b genotype

+/+ 2/2 P value

IgG1

IgG2a

IgG2b

IgG3

IgA

IgE

IgM

217 6 56 8 6 2 589 6 245 133 6 48 17 6 8 14 6 3 550 6 360 282 6 79 56 6 25 1465 6 970 585 6 217 14 6 4 18 6 3 457 6 164 0.13 0.001 0.06 0.0005 0.18 0.03 0.6

Mann-Whitney rank sum test, n = 7–8.

lipid levels (Fig. 1). Comparison of similar size lesions from both groups of mice showed a more prominent necrotic core in BM FcRIIB 2/2 (Supplemental Fig. 1). Ig isotyping showed higher levels of IgG2 and IgG3 in BM FcRIIB 2/2 than BM FcRIIB+ /+ mice (Table 1). In addition, cytokine array analysis showed lower IL-10, IL-1a, GM-CSF, and KC in BM FcRIIB2/2 than BM FcRIIB+/+ mice, indicating important changes in immune balances in these mice (Fig. 2C). We immunostained lesions for IgG, because the presence of plaque-localized immune complexes is an important hallmark of atherosclerosis. Autoantibodies were identified in 4 major areas: on ECs covering the fibrous caps, at the neointima-media interface, in necrotic cores, and in adventitia (Fig. 2D and Supplemental Fig. 1). Interestingly, levels of circulating immune complexes are correlated with the extent of lesions (R = 0.76, P = 0.0028, n = 13; data not shown).

analysis of the autoimmunosome attributed these protein spots to interconnected structures and pathways (see Table 3).

Macrophages lacking FcRIIB are defective in phagocytosis Defective apoptosis and clearance of cellular debris and immune complexes are responsible for generating SLE autoantigens, such as antinucleoprotein antibodies (22). Phagocytosis by macrophages lacking FcRIIB was defective in the uptake of both E. coli and immune complexes of microspheres coated with IgG (Fig. 3A). The macrophage cell line (RAW) overexpressing FcRIIB (Fig. 3B) had higher phagocytic capabilities compared with normal cells. In the absence of FcRIIB, defective phagocytosis was associated with low expression of different receptors involved in phagocytosis, including LRP1 and -5 and PSR1 (Fig. 3C).

Autoantibody-based proteomic approach reveals 25 vascular autoantigen targets A serologic proteomic approach was used to characterize IgG further in atherosclerotic lesions and to identify the targets of these autoantibodies. We created panels of aortic antigens by extracted proteins from aortas isolated from old apoE2/2 mice with very advanced lesions. The extract had a very distinctive 2D electrophoresis pattern (Fig. 4A). This proteomic approach led to the discovery of 25 protein spots that were differentially recognized or highly immunoreactive to autoimmune sera. These spots were related to 19 different proteins and protein isoforms (Table 2), which we coined “vascular autoimmunosome.” Functional VASCULAR TARGETS OF AUTOIMMUNITY IN ATHEROGENESIS

Figure 3. Evaluation of phagocytosis modulated by FcRIIB. A) Mean uptake of fluorospheres coated with IgG and mean uptake of fluorescentE.coliparticlesweresignificantlydefectiveinmacrophages (Mf) lacking FcRIIB (n = 5 and 6). B) Macrophage RAW cells transfected with the FcRIIB gene (++) showed higher phagocytic capability of fluorospheres than cells transfected with empty plasmid (+). Right panels show RAW cells incorporating fluorospheres (small dots)andcounter-stainedwithDAPI.C)Geneexpressionbyreal-time PCR showed down-regulated expression of LDLR-related proteins 1 and 5 (LRP1 and -5) and phosphatidylserine receptor 1 (PSR1) in FcRIIB2/2 macrophages; n = 6. **P , 0.01; ***P , 0.001. 5

Figure 4. Autoantibody-based proteomic studies revealed strong autoreactivity in plasma of autoimmune mice against 25 different epitopes in aortic atherosclerotic lesions. A) The 2D electrophoresis patterns of Cy3-dyed protein extracted from aortic lesions (left) and silver stained (right) showed well-separated and resolved protein spots between 10 and 200 kD of molecular weights and 4–9 isoelectric points (left to right). B) Visualization of spots after the gel was transferred to a PVDF membrane was used as a reference for Western blotting. C ) Preparative gel run in the same condition as in A and kept for selection of spots to be excised and identified. D) Immunoreactivity of control sera (FcRIIB+/+). The membrane (B) was incubated with control sera followed by a secondary CF647-labeled goat antimouse IgG. E ) Immunoreactivity of autoimmune serum (FcRIIB2/2). The membrane (B) was stripped and reused for immunoblotting with autoimmune sera. There were 5 regions (R1–R5) outlined, representing specific or highly immunoreactive spots recognized by autoimmune sera. F ) The autoimmune immunoblot image was superimposed on the image of the initially visualized membrane (B) to help localize specific spots to be excised and identified. G) Highlights of 5 immunospecific regions superimposed onto the preparative gel in C, from which 25 immunoreactive spots were excised for identification by MS. The protein spots identified by MS were labeled with circles, indicated by numbers, and listed in Table 2.

Immunohistochemical analysis shows colocalization of autoantibodies with their targets in atherosclerotic lesions Proteomic findings highlight the presence of serum immunoreactivity toward components of very advanced lesions. To extend these findings and to confirm that circulating autoantibodies colocalized with their targets in plaques, we analyzed lesion distribution of some of the targets and autoantibodies. Immunohistochemical staining of filamin 1, actinin, Vcl, and VCP showed colocalization with mouse IgG, primarily at the basement membrane of ECs covering fibrous caps in necrotic cores, and at both basement membrane and internal elastic lamina in advanced lesions (Fig. 5). Analysis in unrelated LDLR2/2 6

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mice that had various stages of lesions showed costaining in all stages of atherogenesis, including the very early stage of lesions (Supplemental Fig. 2). The colocalization of autoantibodies and their targets in aortic lesions is a strong validation of proteomic-based immunoreactivity. The use of very advanced lesions from 2-yr-old mice helped identify neoepitopes that may be recognized earlier by circulating IgG levels in unrelated and much younger mice, and this is consistent with the presence of autoimmune responses at different stages of atherogenesis. This was confirmed with the presence of plaque-bound autoantibodies at all stages of atherosclerotic lesions (Supplemental Fig. 3). These findings underscore the fact that that autoantigens are present in every lesion, as they are real hallmarks of atherosclerosis and part of the building blocks of atherosclerotic plaques.

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TABLE 2. Identification of target protein spots Spot

Accession

Name (gene)

MW, Da

pI

1 2 3 4 5 6 7 8 9

6753484 26006145 148669535 148669535 148669535 13277753 82659196 18079351 148701451

108,421.9 154,737.9 123,780.8 123,780.8 123,780.8 94,021.3 102,655.4 95,893.2 108,032.2

5.2 5.1 5.54 5.54 5.54 5.13 5.3 5.43 5.79

10 11 12 13 14 15

215406565 19343834 148670554 11230802 28916693 38511951

274,456.7a 89,173.1 90,990.6 104,911.4 85,888.1 93,495

5.64 5.60 5.26 5.25 5.83 6.42

16 17 18 19 20

33859482 33859482 6753824 148683478 5031571

95,252.9 95,252.9 50,159.6 49,345.8 44,732.2

6.41 6.41 4.55 5.54 6.30

21

2501439

34,462.3

6.97

22 23 24 25

61657921 196168728 12805321 6754696

Collagen, type VI, a 1 (Col6a1) Talin 2 (Tln2) Vinculin, isoform CRA_b (Vcl) Vcl, isoform CRA_b Vcl, isoform CRA_b Hspa70 a Actinin 1a (Actn1) Major vault protein (Mvp) a Glucosidase 2 a neutral subunit (Ganab) Filamin a (Flna) Fibronectin 1 (Fn1) Valosin-containing protein (VCP) Actn4 Gelsolin (Gsn) Eukaryotic translation elongation factor 2 (Eef2) Eef2 Eef2 Fibulin 5 (Fbln5) Fibrinogen g polypeptide (Fgg) Actin-related protein 2 isoform b (Arp2) Ubiquitin fusion degradation protein 1 (Ufd1l) Kinesin family member 5B (Kif5b) Annexin A1 (Anxa1) SEC13 protein (Sec13) Macrophage migration inhibitory factor (Mif)

109,483.7a 38,726 35,501 12,496

6.06 6.97 5.15 6.79

a Fragment, calculated molecular weight (MW), and isoelectric point (pI) may differ from obtained values.

DISCUSSION In the present study, we created a mouse model combining susceptibilities to both lupus and atherosclerosis, investigated the mechanisms of autoimmunity-associated atherosclerosis, and identified vascular autoantigens targeted by autoimmune reactions. The autoimmune phenotype was more pronounced, and the progression of atherosclerosis was more aggressive in these mice despite significantly lower plasma lipid levels than unaffected mice (Fig. 1). Macrophages associated with autoimmunity showed defective clearance capabilities, probably related to abnormal expression of specific receptors (e.g., LRP1, LRP5, and PSR1) (20). Serologic proteomic studies showed circulating autoantibodies targeting structurally and functionally related intracellular proteins involved in vital functions of cellular adherence and phagocytosis and as components of ECM (see Table 3). There were 4 targets (Col6, fibronectin, fibrinogen, and Fbln5) that are major components of the vascular ECM. Col6 is present primarily in fibrous caps of advanced lesions (23). Fibronectin and fibrinogen are incorporated into the subendothelial matrix during inflammation (24). Fbln5 is involved in adherence of ECs to the ECM and the organization of the internal elastic lamina, which is located beneath the endothelium of blood vessels (25). The second group of targets (actinins 1 and 4, VASCULAR TARGETS OF AUTOIMMUNITY IN ATHEROGENESIS

Arp2, filamin, Gsn, Tln2, and Vcl) are intracellular actinassociated proteins that mediate different types of cellular adhesion (26). The third set of intracellular neoepitopes are associated with autophagosomes and proteasomal ubiquitin-dependent pathways and includes 2 partners [VCP (p97) and UFD1] of the proteasome ubiquitin complex p97-UFD1-nuclear protein localization protein 4, Saccharomyces cerevisiae homolog SEC13-like 1 (SEC13) protein, MVP, Hsp70, Eef2, and Kif5b. Mif, a pluripotent cytokine, was implicated in autophagy (27). Many of these autophagic factors are associated with phagocytosis, suggesting a close interaction of these 2 major pathways (28). Many partners in cell adhesion, which dictate actin filament structure and cytoskeleton organization, also are important for phagocytosis (29). Some of these biomarkers have been associated with atherosclerosis and cardiovascular disease, such as 70 kD Hsp and Mif (Table 3) (6, 30–33). Recent genome-wide association and proteomic studies associated 4 of these targets with autoimmune and cardiovascular diseases or related traits, including actinin 4 with lupus (83), fibrinogen with cardiovascular disease (54), and Fbln5 with HDL particle size (57). In addition, the lipoma-preferred partner, a member of the zyxin proteins that interact with actinin 1 in focal adhesions, was implicated in SLE by a genome-wide association study (84). Furthermore, Gsn plasma levels were significantly down-regulated in type 1 and 2 diabetes (60). 7

TABLE 3. Functional and pathologic implications of target proteins Specific targets

Functional implicationa

Anxa1 Actn1 and -4

HSP70

Phagocytosis, anti-inflammation Focal adhesion, cell adhesion and motility, phagocytosis Focal adhesion, cell adhesion and motility, autophagy, intracellular transport ECM, autophagy Protein translation, autophagy N-Glycosylation, protein folding, cell migration ECM, thrombosis ECM ECM, elastic fiber assembly Focal adhesion, cell adhesion and motility Focal adhesion, cell adhesion and motility, phagocytosis Autophagy, phagocytosis, endosomal traffic Autophagy, phagocytosis

MIF

Phagocytosis

Sec13 Tln2 UFD1

Endosome transport, autophagy Focal adhesion Autophagy, ubiquitin proteasome system Autophagy, ubiquitin proteasome system Focal adhesion, cell adhesion and motility, phagocytosis

Arp2 Col6 Eef2 Ganab Fgg Fn1 Fbln5 Flna Gsn Kif5b

VCP Vcl

Pathologic associationa

References

SLE, idiopathic pulmonary fibrosis SLE, lupus nephritis, autoimmune hepatitis, multiple sclerosis Cancer

(34–38) (39–42)

CVD, cancer, muscular dystrophy Melanoma Cancer, polycystic liver disease

(45–47) (48–51) (52, 53)

CVD CVD CVD Periodontitis

(54, 55) (56) (57, 58) (59)

Atherosclerosis, diabetes, cancer, amyloidosis Cancer

(60–63)

Atherosclerosis, various rheumatic and autoimmune disorders Septic shock, rheumatoid arthritis, SLE and atherosclerosis

(39, 43, 44)

(64–66) (33, 51, 67, 68) (27, 30–32, 69, 70)

Muscular dystrophy DiGeorge syndrome

(71, 72) (39, 73) (74–76)

Autoimmune hepatitis, cancer

(76–82)

Cancer

(39, 80)

a

Nonexhaustive listing, functions relevant to inflammation, and atherosclerosis. CVD, Cardiovascular disease.

Most of the autoimmunosome identified in the present study included membrane-associated intracellular proteins. These proteins may have originated from ruptured cell membranes and disintegrating intracellular structures because of postnecrotic events in lupus-associated autoimmunity. In normal immunity, postapoptotic necrosis may be prevented by effective phagocytosis of dead cells. However, the defective clearance machinery of phagocytosis demonstrated in the present study may cause secondary necrosis and postnecrotic neoantigens; necrotic cells may lose membrane integrity and expose intracellular antigens and structurally altered selfcomponents to autoreactive lymphocytes (22). In addition, walling and denudation of ECs may expose modified structures of the ECM and internal elastic lamina to autoimmunity, such as Col6, fibronectin, fibrinogen, and Fbln5. In the absence of effective phagocytosis, cell death [induced by apoptosis, necroptosis (85), or autophagy] may cause abnormal antigenic availability and immunogenic presentation by antigen-presenting cells and autoreactive lymphocytes, and this process may generate autoantibodies (86, 87). It is unknown how intracellular proteins become autoantigens, but post-translational modifications may enable immune recognition of neoself epitopes. These effects may include increased affinity of the MHC or T cell receptor binding or subtle effects on the activity of proteolytic 8

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enzymes involved in antigen processing (88). Autoimmunity may also occur by accident during immune responses to pathogens by ways of molecular mimicry or cross-reactivity of antibodies with self and nonself (89). Finally, basal autoimmunity may be enhanced because of failure in the control process of normally occurring autoantibodies, which is a physiologic feature of the autoimmune system and part of a control process of the autoreactivity itself through the idiotypic network (90). Previous studies showed adherence of IgG to intracellular structures and the presence of IgG along the cell membrane associated with extracellular collagen and elastic fibers (91). The current results include molecular and immunohistochemical evidence that these IgGs are autoantibodies that are triggered by specific components of the ECM, the focal adhesion complex at the membrane, and probably intracellular aggresomes associated with autophagy and phagocytosis. This is evidence against the long-standing theory that IgG is nonspecifically deposited or trapped inside atherosclerotic plaques. We should emphasize that not all reactivity to a particular autoantigen causes pathogenic autoimmunity. Some subsets of autoreactive T and B lymphocytes within the repertoire of lymphocytes that recognize this autoantigen may be more relevant than others to the disease process (92). Therefore, it is important to characterize these

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Figure 5. Autoantigens colocalized with IgG in atherosclerotic lesions. Triple or double immunofluorescence analysis of aortic cross-sections was performed to validate the proteomic findings and to colocalize mouse IgG with some autoantigens identified by proteomic studies. The atheromas are delineated by discontinuous lines. A) Immunostaining of mouse IgG (red), filamin (green), and ECs (CD31+, blue). B) Triple immunostaining of mouse IgG (red), Actn (green), and smooth muscle cells [SMC; a-actin, magenta (upper) and green (lower)] showed colocalization at the fibrous cap, the adventitia, and the internal elastic lamina. C ) Colocalization of Vcl (green) with mouse IgG (red) at EC layer (blue), necrotic cores, and internal elastic lamina. D) Double immunofluorescence of VCP (blue) and mouse IgG (red) at the endothelium. DAPI, 49,6-diamidino-2-phenylindole stain (blue color).

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aggressive clones that drive and propagate the atherogenic process. It will be also interesting to identify the structural alterations of the neoantigens and the specific epitopes that trigger autoimmune responses. Some limitations of the present study are related to the proteomic approach itself, which by definition, does not deal with nonprotein epitopes and consequently misses lipid- and DNA-derived antigens. Moreover, as the denaturating conditions related to the detergent present in the proteomic protocol, antibodies directed against nondenatured conformational structures will not be detected. Finally, it is important to know that autoreactivity is also present but to a lesser extent in control mice (e.g., BM FcRIIB2/2), as demonstrated by the immunoreactivity of sera from these mice toward vascular lesional components and by the presence of autoantigens in lesions from these mice. This autoimmunity is associated with hyperlipidemia, which is responsible for the appearance and progression of lesions in these mice, probably by interfering with proresolution of inflammation and protective functions, such as phagocytosis (93). Notwithstanding, the above many questions concerning whether to consider these autoantibodies as culprits or passive bystanders remain unanswered. Nonetheless, evidence supports an active role of some of these autoantibodies in the pathologic process rather than being passive bystanders. At the endothelial levels, even if antibodies do arise as a secondary event to endothelial damage, these autoantibodies may contribute to the amplification of endothelial damage by different processes, including cell-surface binding with cytolysis, immune complex-mediated damage, penetration into living cells, and binding to cross-reactive extracellular molecules (94). Likewise, autoantibodies against mediators of phagocytosis and autophagy may amplify the disruption of these vital functions. For instance, antibodies against the Anxa1 may block the cell-surfaceexposed Anxa1 presented on apoptotic cell surfaces, likely preventing efficient removal of apoptotic cells, which leads to the release of cellular constituents into the bloodstream and tissues and exacerbates autoimmune responses (95).

CONCLUSIONS Our approach in this study was to exacerbate autoimmune responses (through FcRIIB2/2) to be able to detect their targets in the vasculature and to study in this context the progression of atherosclerosis. The autoantibody-based proteomic study in this mouse model of autoimmunityassociated atherosclerosis identified antigens from 19 different, conserved proteins that elicited the immune reaction in the vascular wall. Most of the autoimmunosome included intracellular components of vascular wall cells (mostly located at the basement membrane of ECs, necrotic cores, and internal elastic lamina), probably exposed or released after cell membrane damage or secondary necrosis. Therefore, autoantibodies to damaged or modified ECs structures may provide a foundation for the build-up of atherosclerotic lesions, and continuous challenges by 10

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the immune system may contribute to the start of a growing, dynamic plaque. The present study supports the concept that inflammation and autoimmunity are primary causes of atherogenesis in many autoimmunity-associated diseases. The new vascular autoimmunosome may be a useful target for diagnostic and immunotherapeutic interventions in autoimmunity-associated diseases that have accelerated atherosclerosis. This work received grant support from the American Heart Association (SDG 0730172N) and Marie Curie Actions (FP7 Intra-European Fellowship PIEF-GA-2009-237028; both to A.J.M.). The authors declare no conflicts of interest.

REFERENCES 1. Hansson, G. K., and Hermansson, A. (2011) The immune system in atherosclerosis. Nat. Immunol. 12, 204–212 2. Libby, P. (2012) Inflammation in atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 32, 2045–2051 3. Frieri, M., and Stampfl, H. (2016) Systemic lupus erythematosus and atherosclerosis: review of the literature. Autoimmun. Rev. 15, 16–21 4. Skaggs, B. J., Hahn, B. H., and McMahon, M. (2012) Accelerated atherosclerosis in patients with SLE—mechanisms and management. Nat. Rev. Rheumatol. 8, 214–223 5. Symmons, D. P., and Gabriel, S. E. (2011) Epidemiology of CVD in rheumatic disease, with a focus on RA and SLE. Nat. Rev. Rheumatol. 7, 399–408 6. Xu, Q., Metzler, B., Jahangiri, M., and Mandal, K. (2012) Molecular chaperones and heat shock proteins in atherosclerosis. Am. J. Physiol. Heart Circ. Physiol. 302, H506–H514 7. Narshi, C. B., Giles, I. P., and Rahman, A. (2011) The endothelium: an interface between autoimmunity and atherosclerosis in systemic lupus erythematosus? Lupus 20, 5–13 8. Denas, G., Jose, S. P., Bracco, A., Zoppellaro, G., and Pengo, V. (2015) Antiphospholipid syndrome and the heart: a case series and literature review. Autoimmun. Rev. 14, 214–222 9. Hollan, I., Meroni, P. L., Ahearn, J. M., Cohen Tervaert, J. W., Curran, S., Goodyear, C. S., Hestad, K. A., Kahaleh, B., Riggio, M., Shields, K., and Wasko, M. C. (2013) Cardiovascular disease in autoimmune rheumatic diseases. Autoimmun. Rev. 12, 1004–1015 10. Teixeira, P. C., Cutler, P., and Vuilleumier, N. (2012) Autoantibodies to apolipoprotein A-1 in cardiovascular diseases: current perspectives. Clin. Dev. Immunol. 2012, 868251 11. Binder, C. J. (2012) Naturally occurring IgM antibodies to oxidationspecific epitopes. Adv. Exp. Med. Biol. 750, 2–13 12. Amigorena, S., and Bonnerot, C. (1999) Fc receptor signaling and trafficking: a connection for antigen processing. Immunol. Rev. 172, 279–284 13. Ravetch, J. V., and Lanier, L. L. (2000) Immune inhibitory receptors. Science 290, 84–89 14. Takai, T. (2002) Roles of Fc receptors in autoimmunity. Nat. Rev. Immunol. 2, 580–592 15. Bolland, S., and Ravetch, J. V. (2000) Spontaneous autoimmune disease in Fc(gamma)RIIB-deficient mice results from strain-specific epistasis. Immunity 13, 277–285 16. Jiang, Y., Hirose, S., Abe, M., Sanokawa-Akakura, R., Ohtsuji, M., Mi, X., Li, N., Xiu, Y., Zhang, D., Shirai, J., Hamano, Y., Fujii, H., and Shirai, T. (2000) Polymorphisms in IgG Fc receptor IIB regulatory regions associated with autoimmune susceptibility. Immunogenetics 51, 429–435 17. Mendez-Fernandez, Y. V., Stevenson, B. G., Diehl, C. J., Braun, N. A., Wade, N. S., Covarrubias, R., van Leuven, S., Witztum, J. L., and Major, A. S. (2011) The inhibitory FcgRIIb modulates the inflammatory response and influences atherosclerosis in male apoE(2/2) mice. Atherosclerosis 214, 73–80 18. Pritchard, N. R., Cutler, A. J., Uribe, S., Chadban, S. J., Morley, B. J., and Smith, K. G. (2000) Autoimmune-prone mice share a promoter haplotype associated with reduced expression and function of the Fc receptor FcgammaRII. Curr. Biol. 10, 227–230 19. Zhao, M., Wigren, M., Dun´er, P., Kolbus, D., Olofsson, K. E., Bj¨orkbacka, H., Nilsson, J., and Fredrikson, G. N. (2010)

The FASEB Journal x www.fasebj.org

MERCHED ET AL.

20.

21.

22. 23.

24.

25.

26. 27.

28. 29. 30.

31.

32. 33. 34.

35.

36.

37.

38. 39.

FcgammaRIIB inhibits the development of atherosclerosis in low-density lipoprotein receptor-deficient mice. J. Immunol. 184, 2253–2260 Merched, A., Tollefson, K., and Chan, L. (2010) Beta2 integrins modulate the initiation and progression of atherosclerosis in lowdensity lipoprotein receptor knockout mice. Cardiovasc. Res. 85, 853–863 Merched, A. J., Ko, K., Gotlinger, K. H., Serhan, C. N., and Chan, L. (2008) Atherosclerosis: evidence for impairment of resolution of vascular inflammation governed by specific lipid mediators. FASEB J. 22, 3595–3606 Rullo, O. J., and Tsao, B. P. (2013) Recent insights into the genetic basis of systemic lupus erythematosus. Ann. Rheum. Dis. 72 (Suppl 2), ii56–ii61 Katsuda, S., Okada, Y., Minamoto, T., Oda, Y., Matsui, Y., and Nakanishi, I. (1992) Collagens in human atherosclerosis. Immunohistochemical analysis using collagen type-specific antibodies. Arterioscler. Thromb. 12, 494–502 Hahn, C., and Schwartz, M. A. (2009) Mechanotransduction in vascular physiology and atherogenesis. Nat. Rev. Mol. Cell Biol. 10, 53–62 Chapman, S. L., Sicot, F. X., Davis, E. C., Huang, J., Sasaki, T., Chu, M. L., and Yanagisawa, H. (2010) Fibulin-2 and fibulin-5 cooperatively function to form the internal elastic lamina and protect from vascular injury. Arterioscler. Thromb. Vasc. Biol. 30, 68–74 Wehrle-Haller, B. (2012) Structure and function of focal adhesions. Curr. Opin. Cell Biol. 24, 116–124 Chuang, Y. C., Su, W. H., Lei, H. Y., Lin, Y. S., Liu, H. S., Chang, C. P., and Yeh, T. M. (2012) Macrophage migration inhibitory factor induces autophagy via reactive oxygen species generation. PLoS One 7, e37613 Levine, B., Mizushima, N., and Virgin, H. W. (2011) Autophagy in immunity and inflammation. Nature 469, 323–335 Mooren, O. L., Galletta, B. J., and Cooper, J. A. (2012) Roles for actin assembly in endocytosis. Annu. Rev. Biochem. 81, 661–686 Bernhagen, J., Krohn, R., Lue, H., Gregory, J. L., Zernecke, A., Koenen, R. R., Dewor, M., Georgiev, I., Schober, A., Leng, L., Kooistra, T., Fingerle-Rowson, G., Ghezzi, P., Kleemann, R., McColl, S. R., Bucala, R., Hickey, M. J., and Weber, C. (2007) MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment. Nat. Med. 13, 587–596 Pan, J. H., Sukhova, G. K., Yang, J. T., Wang, B., Xie, T., Fu, H., Zhang, Y., Satoskar, A. R., David, J. R., Metz, C. N., Bucala, R., Fang, K., Simon, D. I., Chapman, H. A., Libby, P., and Shi, G. P. (2004) Macrophage migration inhibitory factor deficiency impairs atherosclerosis in low-density lipoprotein receptor-deficient mice. Circulation 109, 3149–3153 Santos, L. L., and Morand, E. F. (2009) Macrophage migration inhibitory factor: a key cytokine in RA, SLE and atherosclerosis. Clin. Chim. Acta 399, 1–7 Xu, Q. (2002) Role of heat shock proteins in atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 22, 1547–1559 Dalli, J., Norling, L. V., Renshaw, D., Cooper, D., Leung, K. Y., and Perretti, M. (2008) Annexin 1 mediates the rapid antiinflammatory effects of neutrophil-derived microparticles. Blood 112, 2512–2519 Goulding, N. J., Podgorski, M. R., Hall, N. D., Flower, R. J., Browning, J. L., Pepinsky, R. B., and Maddison, P. J. (1989) Autoantibodies to recombinant lipocortin-1 in rheumatoid arthritis and systemic lupus erythematosus. Ann. Rheum. Dis. 48, 843–850 Kurosu, K., Takiguchi, Y., Okada, O., Yumoto, N., Sakao, S., Tada, Y., Kasahara, Y., Tanabe, N., Tatsumi, K., Weiden, M., Rom, W. N., and Kuriyama, T. (2008) Identification of annexin 1 as a novel autoantigen in acute exacerbation of idiopathic pulmonary fibrosis. J. Immunol. 181, 756–767 Pruzanski, W., Goulding, N. J., Flower, R. J., Gladman, D. D., Urowitz, M. B., Goodman, P. J., Scott, K. F., and Vadas, P. (1994) Circulating group II phospholipase A2 activity and antilipocortin antibodies in systemic lupus erythematosus. Correlative study with disease activity. J. Rheumatol. 21, 252–257 Yona, S., Heinsbroek, S. E., Peiser, L., Gordon, S., Perretti, M., and Flower, R. J. (2006) Impaired phagocytic mechanism in annexin 1 null macrophages. Br. J. Pharmacol. 148, 469–477 Le Clainche, C., and Carlier, M. F. (2008) Regulation of actin assembly associated with protrusion and adhesion in cell migration. Physiol. Rev. 88, 489–513

VASCULAR TARGETS OF AUTOIMMUNITY IN ATHEROGENESIS

40. Oikonomou, K. G., Zachou, K., and Dalekos, G. N. (2011) Alphaactinin: a multidisciplinary protein with important role in B-cell driven autoimmunity. Autoimmun. Rev. 10, 389–396 41. Pandey, S., Dioni, I., Lambardi, D., Real-Fernandez, F., Peroni, E., Pacini, G., Lolli, F., Seraglia, R., Papini, A. M., and Rovero, P. (2013) Alpha actinin is specifically recognized by multiple sclerosis autoantibodies isolated using an N-glucosylated peptide epitope. Mol. Cell. Proteomics 12, 277–282 42. International Consortium on the Genetics of Systemic Erythematosus. (2011) A comprehensive analysis of shared loci between systemic lupus erythematosus (SLE) and sixteen autoimmune diseases reveals limited genetic overlap. PLoS Genet. 7, e1002406 43. Boldogh, I. R., Yang, H. C., Nowakowski, W. D., Karmon, S. L., Hays, L. G., Yates III, J. R., and Pon, L. A. (2001) Arp2/3 complex and actin dynamics are required for actin-based mitochondrial motility in yeast. Proc. Natl. Acad. Sci. USA 98, 3162–3167 44. Monastyrska, I., He, C., Geng, J., Hoppe, A. D., Li, Z., and Klionsky, D. J. (2008) Arp2 links autophagic machinery with the actin cytoskeleton. Mol. Biol. Cell 19, 1962–1975 45. De la Cuesta, F., Alvarez-Llamas, G., Maroto, A. S., Donado, A., Zubiri, I., Posada, M., Padial, L. R., Pinto, A. G., Barderas, M. G., and Vivanco, F. (2011) A proteomic focus on the alterations occurring at the human atherosclerotic coronary intima. Mol. Cell. Proteomics 10, M110 003517 46. Grumati, P., Coletto, L., Sabatelli, P., Cescon, M., Angelin, A., Bertaggia, E., Blaauw, B., Urciuolo, A., Tiepolo, T., Merlini, L., Maraldi, N. M., Bernardi, P., Sandri, M., and Bonaldo, P. (2010) Autophagy is defective in collagen VI muscular dystrophies, and its reactivation rescues myofiber degeneration. Nat. Med. 16, 1313–1320 47. Chiu, K. H., Chang, Y. H., Wu, Y. S., Lee, S. H., and Liao, P. C. (2011) Quantitative secretome analysis reveals that COL6A1 is a metastasisassociated protein using stacking gel-aided purification combined with iTRAQ labeling. J. Proteome Res. 10, 1110–1125 48. Hait, W. N., Wu, H., Jin, S., and Yang, J. M. (2006) Elongation factor-2 kinase: its role in protein synthesis and autophagy. Autophagy 2, 294–296 49. Py, B. F., Boyce, M., and Yuan, J. (2009) A critical role of eEF-2K in mediating autophagy in response to multiple cellular stresses. Autophagy 5, 393–396 50. Suzuki, A., Iizuka, A., Komiyama, M., Takikawa, M., Kume, A., Tai, S., Ohshita, C., Kurusu, A., Nakamura, Y., Yamamoto, A., Yamazaki, N., Yoshikawa, S., Kiyohara, Y., and Akiyama, Y. (2010) Identification of melanoma antigens using a serological proteome approach (SERPA). Cancer Genomics Proteomics 7, 17–23 51. Wu, T., and Tanguay, R. M. (2006) Antibodies against heat shock proteins in environmental stresses and diseases: friend or foe? Cell Stress Chaperones 11, 1–12 52. Chiu, C. C., Lin, C. Y., Lee, L. Y., Chen, Y. J., Lu, Y. C., Wang, H. M., Liao, C. T., Chang, J. T., and Cheng, A. J. (2011) Molecular chaperones as a common set of proteins that regulate the invasion phenotype of head and neck cancer. Clin. Cancer Res. 17, 4629–4641 53. Drenth, J. P., Martina, J. A., van de Kerkhof, R., Bonifacino, J. S., and Jansen, J. B. (2005) Polycystic liver disease is a disorder of cotranslational protein processing. Trends Mol. Med. 11, 37–42 54. Lovely, R. S., Yang, Q., Massaro, J. M., Wang, J., D’Agostino, Sr., R. B., O’Donnell, C. J., Shannon, J., and Farrell, D. H. (2011) Assessment of genetic determinants of the association of g9 fibrinogen in relation to cardiovascular disease. Arterioscler. Thromb. Vasc. Biol. 31, 2345–2352 55. Mannila, M. N., Lovely, R. S., Kazmierczak, S. C., Eriksson, P., Samneg˚ard, A., Farrell, D. H., Hamsten, A., and Silveira, A. (2007) Elevated plasma fibrinogen gamma9 concentration is associated with myocardial infarction: effects of variation in fibrinogen genes and environmental factors. J. Thromb. Haemost. 5, 766–773 56. Rohwedder, I., Montanez, E., Beckmann, K., Bengtsson, E., Dun´er, P., Nilsson, J., Soehnlein, O., and F¨assler, R. (2012) Plasma fibronectin deficiency impedes atherosclerosis progression and fibrous cap formation. EMBO Mol. Med. 4, 564–576 57. Kaess, B. M., Tomaszewski, M., Braund, P. S., Stark, K., Rafelt, S., Fischer, M., Hardwick, R., Nelson, C. P., Debiec, R., Huber, F., Kremer, W., Kalbitzer, H. R., Rose, L. M., Chasman, D. I., Hopewell, J., Clarke, R., Burton, P. R., Tobin, M. D., Hengstenberg, C., and Samani, N. J. (2011) Large-scale candidate gene analysis of HDL particle features. PLoS One 6, e14529 58. Nakamura, T., Lozano, P. R., Ikeda, Y., Iwanaga, Y., Hinek, A., Minamisawa, S., Cheng, C. F., Kobuke, K., Dalton, N., Takada, Y., 11

59. 60.

61.

62.

63.

64. 65.

66. 67. 68.

69. 70.

71. 72. 73.

74.

75.

76.

77. 78.

12

Tashiro, K., Ross, Jr., J., Honjo, T., and Chien, K. R. (2002) Fibulin-5/ DANCE is essential for elastogenesis in vivo. Nature 415, 171–175 Koutouzis, T., Haber, D., Shaddox, L., Aukhil, I., and Wallet, S. M. (2009) Autoreactivity of serum immunoglobulin to periodontal tissue components: a pilot study. J. Periodontol. 80, 625–633 Zhang, Q., Fillmore, T. L., Schepmoes, A. A., Clauss, T. R., Gritsenko, M. A., Mueller, P. W., Rewers, M., Atkinson, M. A., Smith, R. D., and Metz, T. O. (2013) Serum proteomics reveals systemic dysregulation of innate immunity in type 1 diabetes. J. Exp. Med. 210, 191–203 Burillo, E., Recalde, D., Jarauta, E., Fiddyment, S., Garcia-Otin, A. L., Mateo-Gallego, R., Cenarro, A., and Civeira, F. (2009) Proteomic study of macrophages exposed to oxLDL identifies a CAPG polymorphism associated with carotid atherosclerosis. Atherosclerosis 207, 32–37 Li, G. H., Shi, Y., Chen, Y., Sun, M., Sader, S., Maekawa, Y., Arab, S., Dawood, F., Chen, M., De Couto, G., Liu, Y., Fukuoka, M., Yang, S., Da Shi, M., Kirshenbaum, L. A., McCulloch, C. A., and Liu, P. (2009) Gelsolin regulates cardiac remodeling after myocardial infarction through DNase I-mediated apoptosis. Circ. Res. 104, 896–904 Serrander, L., Skarman, P., Rasmussen, B., Witke, W., Lew, D. P., Krause, K. H., Stendahl, O., and N¨usse, O. (2000) Selective inhibition of IgG-mediated phagocytosis in gelsolin-deficient murine neutrophils. J. Immunol. 165, 2451–2457 Maday, S., Wallace, K. E., and Holzbaur, E. L. (2012) Autophagosomes initiate distally and mature during transport toward the cell soma in primary neurons. J. Cell Biol. 196, 407–417 Schmidt, M. R., Maritzen, T., Kukhtina, V., Higman, V. A., Doglio, L., Barak, N. N., Strauss, H., Oschkinat, H., Dotti, C. G., and Haucke, V. (2009) Regulation of endosomal membrane traffic by a Gadkin/AP1/kinesin KIF5 complex. Proc. Natl. Acad. Sci. USA 106, 15344–15349 Silver, K. E., and Harrison, R. E. (2011) Kinesin 5B is necessary for delivery of membrane and receptors during FcgR-mediated phagocytosis. J. Immunol. 186, 816–825 Kaushik, S., and Cuervo, A. M. (2012) Chaperone-mediated autophagy: a unique way to enter the lysosome world. Trends Cell Biol. 22, 407–417 Wang, R., Kovalchin, J. T., Muhlenkamp, P., and Chandawarkar, R. Y. (2006) Exogenous heat shock protein 70 binds macrophage lipid raft microdomain and stimulates phagocytosis, processing, and MHC-II presentation of antigens. Blood 107, 1636–1642 Calandra, T., and Roger, T. (2003) Macrophage migration inhibitory factor: a regulator of innate immunity. Nat. Rev. Immunol. 3, 791–800 Wu, M. Y., Fu, J., Xu, J., O’Malley, B. W., and Wu, R. C. (2012) Steroid receptor coactivator 3 regulates autophagy in breast cancer cells through macrophage migration inhibitory factor. Cell Res. 22, 1003–1021 Bruns, C., McCaffery, J. M., Curwin, A. J., Duran, J. M., and Malhotra, V. (2011) Biogenesis of a novel compartment for autophagosomemediated unconventional protein secretion. J. Cell Biol. 195, 979–992 Gillon, A. D., Latham, C. F., and Miller, E. A. (2012) Vesicle-mediated ER export of proteins and lipids. Biochim. Biophys. Acta 1821, 1040–1049 Debrand, E., Conti, F. J., Bate, N., Spence, L., Mazzeo, D., Pritchard, C. A., Monkley, S. J., and Critchley, D. R. (2012) Mice carrying a complete deletion of the talin2 coding sequence are viable and fertile. Biochem. Biophys. Res. Commun. 426, 190–195 Lee, J. J., Park, J., Jeong, J., Jeon, H., Yoon, J. B., Kim, E. E., and Lee, K. J. (2013) Complex of Fas-associated factor 1 (FAF1) with valosincontaining protein (VCP)-Npl4-Ufd1 and polyubiquitinated proteins promotes endoplasmic reticulum-associated degradation (ERAD). J. Biol. Chem. 288, 6998–7011 Lindsay, E. A., Botta, A., Jurecic, V., Carattini-Rivera, S., Cheah, Y. C., Rosenblatt, H. M., Bradley, A., and Baldini, A. (1999) Congenital heart disease in mice deficient for the DiGeorge syndrome region. Nature 401, 379–383 Meerang, M., Ritz, D., Paliwal, S., Garajova, Z., Bosshard, M., Mailand, N., Janscak, P., H¨ubscher, U., Meyer, H., and Ramadan, K. (2011) The ubiquitin-selective segregase VCP/p97 orchestrates the response to DNA double-strand breaks. Nat. Cell Biol. 13, 1376–1382 Bug, M., and Meyer, H. (2012) Expanding into new markets—VCP/ p97 in endocytosis and autophagy. J. Struct. Biol. 179, 78–82 Dai, R. M., and Li, C. C. (2001) Valosin-containing protein is a multiubiquitin chain-targeting factor required in ubiquitin-proteasome degradation. Nat. Cell Biol. 3, 740–744

Vol. 30 June 2016

79. Ju, J. S., Fuentealba, R. A., Miller, S. E., Jackson, E., Piwnica-Worms, D., Baloh, R. H., and Weihl, C. C. (2009) Valosin-containing protein (VCP) is required for autophagy and is disrupted in VCP disease. J. Cell Biol. 187, 875–888 ´ Boyer, A., Pouletty, N., 80. Lagu¨e, M. N., Romieu-Mourez, R., Bonneil, E., Mes-Masson, A. M., Thibault, P., Nadeau, M. E., and Boerboom, D. (2012) Proteomic profiling of a mouse model for ovarian granulosa cell tumor identifies VCP as a highly sensitive serum tumor marker in several human cancers. PLoS One 7, e42470 81. Tahiri, F., Le Naour, F., Huguet, S., Lai-Kuen, R., Samuel, D., Johanet, C., Saubamea, B., Tricottet, V., Duclos-Vallee, J. C., and Ballot, E. (2008) Identification of plasma membrane autoantigens in autoimmune hepatitis type 1 using a proteomics tool. Hepatology 47, 937–948 82. W´ojcik, C., Yano, M., and DeMartino, G. N. (2004) RNA interference of valosin-containing protein (VCP/p97) reveals multiple cellular roles linked to ubiquitin/proteasome-dependent proteolysis. J. Cell Sci. 117, 281–292 83. Ramos, P. S., Williams, A. H., Ziegler, J. T., Comeau, M. E., Guy, R. T., Lessard, C. J., Li, H., Edberg, J. C., Zidovetzki, R., Criswell, L. A., Gaffney, P. M., Graham, D. C., Graham, R. R., Kelly, J. A., Kaufman, K. M., Brown, E. E., Alarc´on, G. S., Petri, M. A., Reveille, J. D., McGwin, G., Vil´a, L. M., Ramsey-Goldman, R., Jacob, C. O., Vyse, T. J., Tsao, B. P., Harley, J. B., Kimberly, R. P., Alarc´on-Riquelme, M. E., Langefeld, C. D., and Moser, K. L. (2011) Genetic analyses of interferon pathway-related genes reveal multiple new loci associated with systemic lupus erythematosus. Arthritis Rheum. 63, 2049–2057 84. BIOLUPUS Network; GENLES Network. (2012) Identification of IRF8, TMEM39A, and IKZF3-ZPBP2 as susceptibility loci for systemic lupus erythematosus in a large-scale multiracial replication study. Am. J. Hum. Genet. 90, 648–660 85. Han, J., Zhong, C. Q., and Zhang, D. W. (2011) Programmed necrosis: backup to and competitor with apoptosis in the immune system. Nat. Immunol. 12, 1143–1149 86. Gatto, M., Zen, M., Ghirardello, A., Bettio, S., Bassi, N., Iaccarino, L., Punzi, L., and Doria, A. (2013) Emerging and critical issues in the pathogenesis of lupus. Autoimmun. Rev. 12, 523–536 87. Liu, G., Bi, Y., Wang, R., and Wang, X. (2013) Self-eating and selfdefense: autophagy controls innate immunity and adaptive immunity. J. Leukoc. Biol. 93, 511–519 88. Anderton, S. M. (2004) Post-translational modifications of self antigens: implications for autoimmunity. Curr. Opin. Immunol. 16, 753–758 89. Binder, C. J., H¨orkko¨ , S., Dewan, A., Chang, M. K., Kieu, E. P., Goodyear, C. S., Shaw, P. X., Palinski, W., Witztum, J. L., and Silverman, G. J. (2003) Pneumococcal vaccination decreases atherosclerotic lesion formation: molecular mimicry between Streptococcus pneumoniae and oxidized LDL. Nat. Med. 9, 736–743 90. Sherer, Y., and Shoenfeld, Y. (2000) Idiotypic network dysregulation: a common etiopathogenesis of diverse autoimmune diseases. Appl. Biochem. Biotechnol. 83, 155–162, discussion 297–313 91. Hansson, G. K., Bondjers, G., Bylock, A., and Hjalmarsson, L. (1980) Ultrastructural studies on the localization of IgG in the aortic endothelium and subendothelial intima of atherosclerotic and nonatherosclerotic rabbits. Exp. Mol. Pathol. 33, 302–315 92. Menezes, J. S., van den Elzen, P., Thornes, J., Huffman, D., Droin, N. M., Maverakis, E., and Sercarz, E. E. (2007) A public T cell clonotype within a heterogeneous autoreactive repertoire is dominant in driving EAE. J. Clin. Invest. 117, 2176–2185 93. Merched, A. J., Serhan, C. N., and Chan, L. (2011) Nutrigenetic disruption of inflammation-resolution homeostasis and atherogenesis. J. Nutrigenet. Nutrigenomics 4, 12–24 94. Rekvig, O. P., Putterman, C., Casu, C., Gao, H. X., Ghirardello, A., Mortensen, E. S., Tincani, A., and Doria, A. (2012) Autoantibodies in lupus: culprits or passive bystanders? Autoimmun. Rev. 11, 596–603 95. Arur, S., Uche, U. E., Rezaul, K., Fong, M., Scranton, V., Cowan, A. E., Mohler, W., and Han, D. K. (2003) Annexin I is an endogenous ligand that mediates apoptotic cell engulfment. Dev. Cell 4, 587–598

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Received for publication December 15, 2015. Accepted for publication February 1, 2016.

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