b-cell aortic homing and atheroprotection depend on id3...

Tsimikas, Timothy P. Bender and Coleen A. McNamaraGlover, Kimberly A. Kelly, Elena V. Galkina, Klaus Ley, Joseph L. Witztum, Sotirios

Kirsti A. Campbell, Marcus D. Skaflen, Alexis Cutchins, Daniel J. Lee, David K. Amanda C. Doran, Michael J. Lipinski, Stephanie N. Oldham, James C. Garmey,

B-Cell Aortic Homing and Atheroprotection Depend on Id3

ISSN: 1524-4571 Copyright © 2011 American Heart Association. All rights reserved. Print ISSN: 0009-7330. Online

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Original Research

B-Cell Aortic Homing and Atheroprotection Depend on Id3Amanda C. Doran,* Michael J. Lipinski,* Stephanie N. Oldham, James C. Garmey,

Kirsti A. Campbell, Marcus D. Skaflen, Alexis Cutchins, Daniel J. Lee, David K. Glover,Kimberly A. Kelly, Elena V. Galkina, Klaus Ley, Joseph L. Witztum, Sotirios Tsimikas,

Timothy P. Bender, Coleen A. McNamara

Rationale: B cells are abundant in the adventitia of normal and diseased vessels. Yet, the molecular and cellularmechanisms mediating homing of B cells to the vessel wall and B-cell effects on atherosclerosis are poorlyunderstood. Inhibitor of differentiation-3 (Id3) is important for atheroprotection in mice and polymorphism inthe human ID3 gene has been implicated as a potential risk marker of atherosclerosis in humans. Yet, the roleof Id3 in B-cell regulation of atherosclerosis is unknown.

Objective: To determine if Id3 regulates B-cell homing to the aorta and atheroprotection and identify molecularand cellular mechanisms mediating this effect.

Methods and Results: Loss of Id3 in Apoe�/� mice resulted in early and increased atherosclerosis. Flowcytometry revealed a defect in Id3�/� Apoe�/� mice in the number of B cells in the aorta but not the spleen, lymphnodes, and circulation. Similarly, B cells transferred from Id3�/� Apoe�/� mice into B-cell–deficient micereconstituted spleen, lymph node, and blood similarly to B cells from Id3�/� Apoe�/� mice, but aorticreconstitution and B-cell–mediated inhibition of diet-induced atherosclerosis was significantly impaired. Inaddition to retarding initiation of atherosclerosis, B cells homed to regions of existing atherosclerosis, reducedmacrophage content in plaque, and attenuated progression of disease. The chemokine receptor CCR6 wasidentified as an important Id3 target mediating aortic homing and atheroprotection.

Conclusions: Together, these results are the first to identify the Id3-CCR6 pathway in B cells anddemonstrate its role in aortic B-cell homing and B-cell–mediated protection from early atherosclerosis. (CircRes. 2011;109:00-00.)

Key Words: atherosclerosis � B lymphocytes � transcription factors � helix-loop-helix � homing

Atherosclerosis is a progressive, chronic inflammatorydisease resulting in plaque formation in large arter-

ies1,2 that can lead to heart attack, stroke, and death.Targeting the immune cells that participate in atherogen-esis is a promising novel approaches for atherosclerosistreatment and prevention.3 Much is known about therecruitment of macrophages and T cells to the vessel walland their role in plaque progression4; however, B cells inatherosclerosis are incompletely understood. As early asthe 1960s, studies have identified B cells, plasma cells, andimmunoglobulins in association with atheroscleroticplaques of mice and humans.5–14 More recent studies inmice identified B-cell– containing aortic tertiary lymphoidorgans (ATLO) in the adventitia adjacent to atherosclerotic

plaques, raising the possibility that adventitial B cells mayregulate a local immune response within the vesselwall.5,6,14 In addition, although ATLOs are not present inthe normal/noninflamed aorta before Western feeding,5 asignificant population of leukocytes, including B cells, arepresent in the aortic adventitia of normal vessels.14 GlobalB-cell deficiency established before Western diet feed-ing15,16 results in increased atherosclerosis in mice, sug-gesting that B cells function in early atheroprotection. Yet,the molecular mechanisms that regulate homeostatic traf-ficking of B cells to the aorta and their impact on thedevelopment of atherosclerosis are unknown.

Inhibitor of differentiation-3 (Id3), a member of the helix-loop-helix (HLH) family of transcription factors, is a domi-

Original received September 8, 2011; revision received October 17, 2011; accepted October 20, 2011. In September 2011, the average time fromsubmission to first decision for all original research papers submitted to Circulation Research was 16 days.

From the Cardiovascular Research Center (A.C.D., M.J.L., S.N.O., J.C.G., K.A.C., M.D.S., A.C., D.J.L., D.K.G., K.A.K., C.A.M.), the Departmentof Medicine, Cardiovascular Division (A.C.D., M.J.L., S.N.O., J.C.G., K.A.C., M.D.S., A.C., D.K.G., C.A.M.), the Department of Molecular Physiologyand Biological Physics (A.C.D., M.J.L., C.A.M.), the Department of Biomedical Engineering (K.A.K.), and the Department of Microbiology (T.P.B.),University of Virginia, Charlottesville, VA; the Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, VA(E.V.G.); the Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA (K.L.); and the Department of Medicine,University of California, San Diego, La Jolla, CA (J.L.W., S.T.).

*A.C.D. and M.J.L. contributed equally to this work.Correspondence to Coleen A. McNamara, MD, University of Virginia, PO Box 801394, 415 Lane Rd, Charlottesville, VA 22908. E-mail

[email protected]© 2011 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.111.256438

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nant negative inhibitor of bHLH protein-DNA binding andgene expression in B cells.17 The Id3�/� mouse has normalnumbers and maturity of B cells but develops a Sjogren-likesyndrome with lachrymal and salivary glands lymphocyticinfiltrates,18,19 raising the interesting possibility that Id3 mayregulate B-cell homing to sites of disease. That hyperlipemiaincreased Id3 expression in vitro and in the vessel wall in aporcine model suggested a link between Id3 and atheroscle-rosis.20 This link was recently confirmed by studies demon-strating that aged and Western diet–fed Apoe�/� mice null forId3 had significantly increased atherosclerosis compared withApoe�/� mice wild-type for Id3.21 Moreover, Id3 may beinvolved in atheroprotection in humans as the human ID3gene contains a single nucleotide polymorphism (SNP) thatalters Id3 protein function and is associated with increasedcarotid intima-media thickness in humans.21

The present study demonstrates a new function for Id3, asa critical regulator of B-cell aortic trafficking and B-cell–mediated atheroprotection and identifies CCR6 as an Id3target gene mediating these effects. In addition, we providethe first evidence that B cells home to regions prone to andwith existing atherosclerosis leading to reduced macrophageaccumulation and attenuation of lesion progression.

MethodsDetailed experimental procedures and associated references are in theOnline Supplemental Material available at http://circres.ahajournals.org.All procedures using animals were carried out according to protocolsapproved by the Animal Care and Use Committee at the Universityof Virginia. For B-cell adoptive transfer studies, spleens wereharvested from 10- to 12-week-old mice and B cells were isolated bynegative selection using MACS anti-CD43 microbeads or a combi-nation of MACS anti-CD43, anti-CD4, and anti-CD11b microbeads(Miltenyi Biotec). Serum cholesterol levels were determined usingan Archtect 8000 series analyzer. Antibody titers were determined asdetailed in the Online Supplemental Material.

Analysis of AtherosclerosisAfter euthanizing the mice, the aorta was harvested from the heart tothe iliac bifurcation. Both en face Sudan IV staining of the aorta andcross-sectional analysis of the root was used in this study to quantifyatherosclerosis. Fluorescent imaging at the UVA Advanced Micros-copy Core enabled identification of CFDA-SE-labeled B cells withinthe aorta. Immunohistochemical analysis of atherosclerosis wasachieved through staining for MCP-1 (Santa Cruz BiotechnologyInc) and macrophage content was determined with Mac-2 staining(Cedarlane Laboratories). These protocols are detailed in the OnlineSupplemental Material.

Bone Marrow TransplantationId3�/�Apoe�/� and Id3�/�Apoe�/� mice were subjected to asublethal dose of radiation (500 rads�2 irradiations) and subse-quently reconstituted with 5�106 bone marrow cells harvested fromthe femurs and tibias of Id3�/�Apoe�/� and Id3�/�Apoe�/� donormice, as detailed in the Online Supplemental Material.

Flow CytometryLymph nodes, spleens, blood, and aortas, including the adventitia,were harvested under a dissection microscope and processed for flowcytometry as previously described14 and detailed in the OnlineSupplemental Material.

Ex Vivo Imaging of Radiolabeled B CellsB cells were radiolabeled by incubation in indium-111 oxinesolution. After adoptive transfer of radiolabeled B cells, aortas wereharvested and exposed to a high-sensitivity, medium-resolutionphosphor imaging screen (PerkinElmer) overnight. The phosphorimaging screen was scanned using a PerkinElmer Cyclone PlusPhosphor Imaging System. This protocol is detailed in the OnlineSupplemental Material.

Optical Imaging of Aortic B CellsEx vivo fluorescence-mediated tomography (FMT) quantitative im-aging (FMT 2500, VisEn Medical) was performed after incubationof aortas with Cy5.5-labeled anti-B220 antibodies (eBiosciences andRockland Immunochemicals). This protocol is detailed in the OnlineSupplemental Material.

Real-Time PCRTotal cellular RNA was collected from B cells using an RNeasy kit(Qiagen) as per the manufacturer’s instructions, cDNA was thensynthesized using an iScript cDNA synthesis kit (BioRad), andreal-time PCR reaction using a Bio-Rad iCycler and iQ SYBR GreenSupermix (BioRad) was performed as detailed in the OnlineSupplemental Material.

Cellular Migration StudiesSplenic B cells were purified from Id3�/� Apoe�/� and Id3�/�

Apoe�/� mice and placed in the upper chamber of 5-�m pore sizetranswells containing either 1000 ng/mL of CXCL13 or 500 ng/mLof CCL20 in the bottom of the transwell. After incubating for 6hours, the number of cells that had migrated through the transwellwas counted, using a flow cytometer as detailed in the OnlineSupplemental Material.

Statistical AnalysisA probability value �0.05 was considered statistically significant.All statistical analyses were performed using NCSS 2001 (NumberCrunching Statistical Software, Kaysville, UT) and GraphPadPrism5 (La Jolla, CA). Statistical analysis is detailed in the OnlineSupplemental Material.

ResultsId3 Is Necessary for Early AtheroprotectionTo determine if Id3�/� Apoe�/� mice developed prematureatherosclerosis, Id3�/� Apoe�/� and Id3�/� Apoe�/� micewere fed a Western diet for 4 or 8 weeks, then harvested foren face analysis of lesion area (Figure 1A). After 4 weeks ofWestern feeding, Id3�/� Apoe�/� but not Id3�/� Apoe�/�

mice developed a discernable amount of lesion(0.053�0.009% versus 1.16�0.483%; P�0.004). After 8weeks of Western feeding, both genotypes developed detect-able atherosclerosis, with significantly more lesion seen in theId3�/� Apoe�/� mouse (0.347�0.111% versus4.03�0.859%; P�0.002) (Figure 1B). There were no differ-

Non-standard Abbreviations and Acronyms

ApoB apolipoprotein B

ATLO aortic tertiary lymphoid organs

CFDA-SE carboxyfluorescein diacetate succinimidyl ester

HLH helix-loop-helix

Id3 inhibitor of differentiation-3

MDA malondialdehyde

OxPL oxidized phospholipids

2 Circulation Research December 9, 2011


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ences in body weight, serum lipids, or the content ofimmunochemically detected oxidized phospholipids (OxPL) ormalondialdehyde (MDA) epitopes on apoB lipoproteins (oxPL/ApoB or MDA/apoB), glucose, or insulin in Id3�/� Apoe�/�

compared with Id3�/� Apoe�/� mice (Online Table I).

Id3 Atheroprotection Is Predominantly Mediatedby a Bone Marrow–Derived CellBone marrow chimeras fed a Western diet for 16 weeksconfirmed that Id3 deletion increased atherosclerosis andrevealed that Id3�/� Apoe�/� mice reconstituted with bonemarrow from Id3�/� Apoe�/� mice had a significant attenu-ation of atherosclerosis compared with those reconstitutedwith Id3�/� Apoe�/� marrow (3.1-fold reduction). Similarly,Id3�/� Apoe�/� mice developed significantly less atheroscle-rosis when reconstituted with bone marrow from Id3�/�

Apoe�/� compared with Id3�/� Apoe�/� mice (5.2-foldreduction) (Figure 1E). There was a significant increase inatherosclerosis when Id3�/� Apoe�/� compared with controlId3�/� Apoe�/� recipient mice were reconstituted with bonemarrow from Id3�/� Apoe�/� mice, suggesting some contri-bution of loss of Id3 in cells not derived from bone marrow,

although this effect was much less marked than when thebone marrow cells lacked Id3. No differences in weight, lipidparameters, glucose or insulin were noted in the bone marrowchimeras (Online Table II).

Id3�/� Apoe�/� Mice Have Significantly Fewer BCells in the Aorta Compared With Id3�/�

Apoe�/� MiceThe early onset atherosclerosis in the Id3�/� Apoe�/� miceraised the interesting hypothesis that Id3 may regulate im-mune cells resident in the aorta before lesion development.Flow cytometry of aortas from chow-fed Id3�/� Apoe�/� andId3�/� Apoe�/� mice demonstrated equivalent numbers oftotal leukocytes (CD45�) (Figure 2A), T cells (Figure 2B),and dendritic cells (Figure 2C). The number of macrophageswas higher in the aortas of Id3�/� Apoe�/� mice (Figure 2D).In contrast, the number of B cells in the aorta was signifi-cantly lower in Id3�/� Apoe�/� mice (Figure 2E and 2F).This finding cannot be explained by a global reduction in thenumber of B cells in the Id3�/� Apoe�/� mouse, as consistentwith previous findings in Id3�/� mice,18 the number of Bcells in the spleen, peri-aortic lymph nodes, and whole blood

Figure 1. Loss of Id3 accelerates atherosclerosis development in Apoe�/� mice fed Western diet: an effect mediated by a bonemarrow-derived cell type. A, Schematic of the experiment. Starting at 8 weeks of age, Id3�/� Apoe�/� or Id3�/� Apoe�/� mice wereplaced on a Western diet for 4 or 8 weeks. Aortas were perfused with paraformaldehyde, harvested, opened longitudinally, and stainedwith Sudan IV. B, En face lesion area was quantified using Image-Pro 5.0 software. *P�0.004, **P�0.002. C, Representative en faceimages are shown. D, Schematic of the experiment. At 4 weeks of age, Id3�/� Apoe�/� or Id3�/� Apoe�/� mice were irradiated andtransplanted with donor bone marrow, allowed to recover for 4 weeks, and then placed on a Western diet for 16 weeks. E, Aortas werethen harvested and en face lesion area was quantified as above. Each point represents a single animal. *P�0.01, **P�0.0005.

Doran et al Id3-Dependent B-Cell Homing and Atheroprotection 3



were similar between Id3�/� Apoe�/� and Id3�/� Apoe�/�

mice (Table 1).

Optical Imaging Confirms Fewer B Cells in Aortaof Id3�/� Apoe�/� Mice and Reveals That the BCells Present Predominantly Localize toAtheroprone Regions of the AortaOptical imaging using a Cy5.5-labeled anti-B220 antibodyprovided a second method to demonstrate B cells in the aorta

of young chow-fed Id3�/� Apoe�/� and Id3�/� Apoe�/�

mice. As a control, �MT mice, which lack peripheral B cellsdue to deletion of genomic DNA sequences that encode thetransmembrane domain of the B-cell receptor � heavychain,22 were bred to Apoe�/� mice to generate B-cell–deficient Apoe�/� mice (�MT Apoe�/�). B-cell–deficient�MT Apoe�/� aortas were found to have a minimal amountof background signal, which probably represents the nonspe-cific retention of the Cy5.5-labeled anti-B220 antibody.Although a significant increase in fluorescence over back-ground was observed in both Id3�/� Apoe�/� and Id3�/�

Apoe�/� aortas (Figure 2G), consistent with our previousflow cytometric data, the increase in fluorescence overcontrols in the aortas of Id3�/� Apoe�/� mice was signifi-cantly lower than that in the aortas of Id3�/� Apoe�/� mice(15% versus 42%; P�0.0006) (Figure 2G and 2H). Notably,peak fluorescence was greatest within the aortic arch and theabdominal aorta (Figure 2G), regions that are particularlyprone to the development of atherosclerosis in this animalmodel.23

Figure 2. B-cell number in the aortas ofId3�/� Apoe�/�

mice is significantlydecreased compared with the aortas ofId3�/� Apoe�/� mice. Aortas, spleens,lymph nodes, and whole blood were har-vested from chow-fed Id3�/� Apoe�/� orId3�/� Apoe�/� mice at 8 weeks of age.Each point represents an aorta from a sin-gle animal. Quantification of aortic A, leuko-cytes (CD45�). B, T cells (CD45� CD3e�).C, dendritic cells (CD45� CD11c�), and D,macrophages (CD45� CD68�). *P�0.003.E, Representative plots, and F, quantifica-tion of aortic B cells (CD45� CD19�). G,Representative 2-dimensional fluorescentreflectance images of aortas after incuba-tion with Cy5.5-labeled anti-B220 antibody.H, Quantification of mean fluorescence ofId3�/�Apoe�/� and Id3�/� Apoe�/� mice.Data are represented as increase abovemean fluorescence in the �MT Apoe�/�

control group. Error bars reflect SEM.*P�0.0006.

Table 1. Number of B Cells in the Spleen, Peri-Aortic LymphNodes, and Whole Blood

Id3�/� Apoe�/� Id3�/� Apoe�/� P Value

Peripheral B lymphocytes,�106 CD19� CD45� cells

Spleen 56.5�10.4 56.3�9.31 NS

Lymph nodes 0.89�0.11 0.88�0.14 NS

Blood, per mL 1.96�0.21 1.91�0.29 NS

Values are mean�SD. NS indicates not significant.




Id3 Is Necessary for B-Cell Homing tothe AortaTo determine if Id3 regulated homeostatic trafficking of Bcells to the aorta, adoptive transfer studies were conducted.Splenic B cells from Id3�/� Apoe�/� or Id3�/� Apoe�/�

mice were adoptively transferred to �MT Apoe�/� mice.Control animals received an equal volume of vehicle. Six,24, or 72 hours after the transfer of purified B cells, theaorta, spleen, lymph nodes, and whole blood were har-vested (Figure 3A) and analyzed by flow cytometry asdescribed above. Apoe�/� B cells wild-type for Id3 appearin the aorta 6 hours after tail vein injection and continue toaccumulate in the aorta up to 72 hours later. In contrast, noappreciable numbers of Id3�/� Apoe�/� B cells appear inthe aorta at any of the time points tested (Figure 3B).Analysis of the spleens, lymph nodes, and whole blood ofthe same animals revealed that B-cell reconstitution ofthese compartments was similar between the two geno-types at each time point, providing evidence that reducedaortic B-cell number was not due to a reduction in the totalB-cell pool (Table 2).

To confirm Id3-dependent trafficking of B cells to theaorta, imaging of radiolabeled B cells injected into �MT

Apoe�/� mice was performed. Splenic B cells were isolatedfrom Id3�/� Apoe�/� and Id3�/� Apoe�/� mice and radio-labeled with indium-111 (In-111) oxine. Recipient �MTApoe�/� mice were injected with 1�107 radiolabeled B cellsor with In-111 oxine in normal saline as control. Aortas wereharvested 20 hours later, opened en face, and phosphorimaging was performed. Significantly more radioactive signalwas present in the aortas of mice receiving Id3�/� Apoe�/�

compared with Id3�/� Apoe�/� B cells (Figure 3C and 3D).Consistent with optical imaging data (Figure 2G), radiola-beled B cells traffic to regions of the aorta that are prone toatherosclerosis (Figure 3C and 3E). To determine the layer ofthe vessel wall to which B cells traffic in these experiments,splenic B cells were purified from Id3�/� Apoe�/� mice,incubated with CFDA-SE, and adoptively transferred to �MTApoe�/� mice. Seventy-two hours after transfer, aortas wereharvested from mice, sectioned and analyzed for the presenceof these labeled B cells. Although the media and scatteredadventitial cells revealed autofluorescence, CFDA-SE labeledB cells were only detected within the adventitia (Figure 3F).B cells in the spleen served as a positive control and spleenfrom mice receiving vehicle injection served as negativecontrol.

Figure 3. Id3 is necessary for B-cell homing tothe aorta. A, Schematic of the experiment. B,Quantification of aortic B cells after adoptive trans-fer. Plotted values indicate the average B-cellnumber obtained from 6 animals, �SEM for eachtime point. *P�0.03, **P�0.002. C, Representativeen face images of aortas and corresponding exvivo phosphor images of chow-fed �MT Apoe�/�

recipient mice 20 hours after tail vein injection ofIn-111 control or 1�107 In-111 radiolabeled Bcells from Id3�/� Apoe�/� or Id3�/� Apoe�/�

mice. D, Quantification of aortic radioactivity bygamma well counting (counts per minute/grams ofaortic tissue/injected radioactive dose in �Ci).Error bars represent SEM. *P�0.05. E, Regionalsignal intensity analysis comparing the aortic archand the abdominal aorta relative to the descendingthoracic aorta for ex vivo phosphor images ofId3�/� Apoe�/� B-cell recipient aortas. Error barrepresents SEM. *P�0.05. F, Left panel: Repre-sentative immunofluorescent images showing thelocation of CFDA-SE labeled Id3�/� Apoe�/� Bcells within the adventitia 72 hours after adoptivetransfer to chow-fed �MT Apoe�/� mice. Centerpanel: Spleen negative control from �MT Apoe�/�

mice that received PBS vehicle. Right panel:Spleen positive control, demonstrating CFDA-SE–labeled B cells.




Reconstitution of �MT Apoe�/� Mice WithId3�/� Apoe�/� But Not Id3�/� Apoe�/� B CellsInhibited Western Diet–Induced AtherosclerosisTo determine whether Id3 is essential for B-cell–mediatedattenuation of atherosclerosis, adoptive transfer studies in�MT Apoe�/� mice were performed. Splenic B cells fromId3�/� Apoe�/� or Id3�/� Apoe�/� mice were transferredthrough tail vein injection to B-cell–deficient �MT Apoe�/�

mice. Control mice received an equal volume of PBS vehicle.All recipient mice were then fed a Western diet for 16 weeks,

after which atherosclerosis was assessed by en face analysis(Figure 4A). Consistent with previous studies of B-cell–deficient mice,15,16 �MT Apoe�/� mice fed 16 weeks of aWestern diet developed significant atherosclerosis. A singleinjection of 30�106 Id3�/�Apoe�/� B cells to �MT Apoe�/

�mice significantly reduced atherosclerotic lesion area(9.69�0.568% versus 5.10�0.836%; P�0.002). B-cell inhi-bition of atherosclerosis was dose-dependent, as there was aneven greater reduction in atherosclerosis with the delivery of60�106 Id3�/�Apoe�/� B cells (9.69�0.568% versus2.74�0.263%; P�0.001). In contrast, lesion area was notsignificantly changed in animals receiving either 30 or60�106 Id3�/� Apoe�/� B cells (9.69�0.568% versus8.18�0.735% or 7.82�1.16%) (Figure 4B and 4C). Flowcytometry confirmed a dose dependent increase in aorticB-cell number with increasing numbers of Id3�/�Apoe�/� Bcells injected. Injection of 30�106 Id3�/�Apoe�/� B cellsresulted in an average of 2034 aortic B cells (data not shown)and 60�106 Id3�/�Apoe�/� B cells resulted in �5000 aorticB cells (Figure 3B), measured 72 hours after tail veininjection. For B cells from Id3�/� Apoe�/� mice, there were�200 B cells in the aorta regardless of the number of B cellsinjected (data not shown and Figure 3B). There were nodifferences in the numbers of peripheral B cells (Table 3).These data demonstrate that B cell delivery before Westerndiet feeding protects �MT Apoe�/� mice from diet-inducedatherosclerosis and that Id3 is essential for this B-cell–mediated attenuation of atherosclerosis.

CCR6 Is a Novel Id3 Target Regulating B-CellHoming to the Aorta and AtheroprotectionTo determine the chemokine receptors on B cells that may beregulating homing to the aorta in an Id3-dependent manner,

Table 2. Number of B Cells in the Spleen, Peri-AorticLymph Nodes, and Whole Blood After Adoptive Transfer to�MT Apoe�/� Mice

VehicleId3�/�

Apoe�/�Id3�/�

Apoe�/�P

Value

Peripheral B lymphocytes,�105 CD19� CD45� cells

6 Hours after transfer

Spleen 39.9�0.59 37.6�0.22 NS

Lymph node 1.37�0.64 1.02�0.41 NS

Blood, per mL 1.50�0.40 1.60�0.30 NS


Spleen 0.006�0.001 72.1�1.42 76.7�1.37 NS

Lymph node 0.005�0.001 1.11�0.12 1.22�0.19 NS

Blood, per mL 0.006�0.001 1.20�0.30 1.00�0.20 NS


Spleen 94.5�0.50 93.4�0.58 NS

Lymph node 1.01�0.10 1.12�0.08 NS

Blood, per mL 1.40�0.70 1.50�0.40 NS

Values are mean�SEM. NS indicates not significant.

Figure 4. Id3�/� Apoe�/� but not Id3�/�

Apoe�/� B cells confer atheroprotec-tion to B-cell–deficient �MT ApoE�/�

mice. A, Schematic of the experiment. At8 weeks of age, 30�106 or 60�106

splenic B cells from either Id3�/�

Apoe�/� or Id3�/� Apoe�/� donors wereadoptively transferred into �MT Apoe�/�

recipients by tail vein injection. Controlanimals received an equal volume of PBSvehicle. Animals were allowed to recoverfor 72 hours, then fed a Western diet for16 weeks. Aortas were harvested and an-alyzed by en face as described in Figure1. B, Representative photos of aorticSudan IV staining. C, Quantification ofaortic lesion size. Each point represents asingle animal. *P�0.05, **P�0.002,***P�0.001, ****P�0.005.




we performed a murine PCR chemokines and receptor array(SA Biosciences) using RNA from B cells derived fromId3�/� Apoe�/� or Id3�/� Apoe�/� mice. Notably, thechemokine receptor, CCR6, implicated in cell homing to sitesof disease was significantly reduced. Real-time PCR con-firmed the array results, identifying a 3-fold reduction inCCR6 mRNA expression in Id3�/� Apoe�/� as comparedwith Id3�/� Apoe�/� B cells (Figure 5A). No change wasnoted in the levels of CXCR5, CXCR6, and L-selectin, whichhave previously been demonstrated to play a role in T-cellhoming to the aorta.14,24 Differences in surface expression ofCCR6 protein was confirmed by flow cytometry (Figure 5B)and impaired migration of Id3�/� Apoe�/� B cells in re-sponse to the cognate ligand for CCR6 (CCL20) was dem-onstrated using transwell assays (Figure 5C). Id3 regulatestarget gene expression through dimerization with E2A geneproducts, such as E12, antagonizing E12 DNA binding andtranscription regulatory effects. Transient cotransfection stud-ies in BJAB cells (a human B-cell lymphoma line), demon-strated that E12 inhibits CCR6 promoter activation, an effectantagonized by cotransfection with an expression plasmidencoding Id3 (Figure 5D). Flow cytometry of aorta fromC57BL/6 (B6) and CCR6�/� mice revealed fewer aortic Bcells in CCR6�/� mice than those of age-matched littermateB6 control mice (Figure 5E and 5F). Adoptive transfer ofCCR6�/� B cells to �MT Apoe�/� mice yielded significantlyfewer B cells within the aortas of mice receiving B cells fromCCR6�/� compared with B6 mice (Figure 5G and 5H).Moreover, adoptive transfer of 60�106 Ccr6�/�Apoe�/� Bcells to �MT Apoe�/�mice significantly reduced atheroscle-rotic lesion area in response to 16 weeks of Western dietfeeding (16.1�0.992% versus 9.28�0.783%; P�0.0002). Incontrast, lesion area was not significantly changed in animalsreceiving Ccr6�/� Apoe�/� B cells (16.1�0.992% versus13.4�0.823%; P�NS) (Figure 5I). These data identify CCR6as an Id3 target involved in the aortic “address” for B-cellhoming and B-cell–mediated atheroprotection.

B Cells Traffic to Regions of the Aorta WithExisting AtherosclerosisOptical and phosphor imaging demonstrated that B cellstraffic to regions of the aorta known to be prone to athero-

sclerosis (Figures 2 and 3). To directly determine if B cellstraffic to regions of atherosclerosis, �MT Apoe�/� mice werefed 8 weeks of Western diet to develop atherosclerosis.Animals were then injected with 2�107 radiolabeledApoe�/� B cells or vehicle control. En face staining withSudan IV demonstrated lipid deposition predominantly in thearch and abdominal aorta. Ex vivo phosphor imaging ofaortas revealed the highest signal intensity in the regions ofthe aorta positive for Sudan IV staining (Figure 6A). Injectionof an equal number of radiolabeled Id3�/� Apoe�/� B cellsserved as a control as we previously demonstrated an aortichoming defect in B cells null for Id3 (Figure 6A and B).

Delivery of Id3�/� Apoe�/� B Cells InhibitedProgression of Western Diet–InducedAtherosclerosis: An Effect Attenuated by Lossof Id3B-cell injection significantly inhibited atherosclerosis devel-opment when the B cells were delivered before Western dietfeeding15,16 (Figure 4). To determine whether B cells impactthe progression of existing atherosclerosis, Apoe�/� B cellswere adoptively transferred into �MT Apoe�/� mice withexisting atherosclerosis. The �MT Apoe�/� mice fed 8 weeksof a Western diet underwent baseline atherosclerosis quanti-fication (n�10) or received 45�106 Apoe�/� B cells (n�9)or vehicle control (n�13). Injected animals received anadditional 8 weeks of Western diet and were then assessed byen face staining of the descending aorta and cross-sectionalanalysis of the aortic arch. Movat staining of cross sectionsconfirm that en face lipid staining was accompanied by thehistomorphologic components of atherosclerotic plaque (ne-crotic core and cellular infiltration). Consistent with wholeaorta en face findings (Figures 4 and 6), there was significantatherosclerosis in the abdominal aorta by en face stainingand in the arch by cross-sectional analysis in the vehicle-treated group. In contrast, adoptive transfer of B cells fromApoe�/� mice significantly attenuated the progression ofatherosclerosis when compared with vehicle recipients asmeasured by en face (4.9�0.7% versus 9.7�1.3% respec-tively, P�0.011) (Figure 6C) and cross-sectional analysis(35.6�3.2% versus 45.3�3.1%, P�0.046) (Figure 6D andE). Serum collected at the time of aorta harvest wasanalyzed for lipids. There were no significant differencesin plasma cholesterol, triglycerides, or OxPL/apoB levelsbetween the groups (data not shown).

B Cells Attenuate Aortic Macrophage ContentThe mechanisms whereby aortic B cells attenuate plaquedevelopment are unknown. As Id3�/� mice have increasedtotal IgM in serum,25 we investigated the serum levels ofantibodies known to be correlated with atherosclerosis inId3�/� Apoe�/� and Id3�/� Apoe�/� chow-fed mice. Para-doxically, IgM MDA-LDL and IgM Cu-OxLDL, which areassociated with lower levels of atherosclerosis,26 were foundto be increased in Id3�/� Apoe�/� mice compared withId3�/� Apoe�/� mice. Levels of IgG MDA-LDL and IgGCu-OxLDL were not significantly different between groups(Online Table I). Macrophages are present in the aorta beforeWestern diet feeding14 (Figure 2D) and infiltrate the vessel

Table 3. Number of B Cells in the Spleen, Peri-Aortic LymphNodes, and Whole Blood After Adoptive Transfer to �MTApoe�/� Mice and 16 Weeks of Western Diet Feeding

Id3�/�

Apoe�/�Id3�/�

Apoe�/�P

Value

Peripheral B lymphocytes, �105

CD19� CD45� cells

30�106 B cells transferred

Spleen 11.1�1.31 10.6�1.23 NS

Lymph nodes 1.86�0.12 1.75�0.17 NS

Blood, per mL 0.29�0.10 0.20�0.10 NS

60�106 B cells transferred

Spleen 16.9�1.15 16.3�0.81 NS

Lymph nodes 2.70�0.16 2.60�0.10 NS

Blood, per mL 0.44�0.20 0.29�0.10 NS

Values are mean�SEM. NS indicates not significant.




wall early in the disease process.3 To determine if B-celldelivery to �MT Apoe�/� mice alters plaque macrophagecontent, immunohistochemical staining of plaques from ve-hicle and B-cell injected �MT Apoe�/� mice was performed.Compared with control, adoptive transfer of B cells resultedin a significant reduction in Mac2 (Figure 6F and 6G,0.34�0.02 versus 0.42�0.02, respectively, P�0.024), andMCP-1 staining (Figure 6F and 6H, 0.074�0.009 versus0.121�0.012, P�0.007). Flow cytometry analysis of �MTApoe�/� mice injected with Apoe�/� B cells to beforeWestern diet feeding also revealed a significant decrease inaortic macrophage number (Figure 6I and 6J).

DiscussionThe present study clearly demonstrates that B cells canattenuate atherosclerosis and provides the first evidencelinking resident aortic B cells with this atheroprotection.Previous studies using transplantation of bone marrow fromB-cell–deficient �MT to Ldlr�/� mice16 or splenectomy ofApoe�/� mice15 had suggested an atheroprotective role for Bcells. However, more recent studies using a strategy ofreducing circulating B cells with CD20 monoclonal antibodytreatment resulted in attenuation of atherosclerosis,27,28 sug-gesting that the impact of B cells on atherosclerosis may besubset and context-dependent. Indeed, recent studies have

Figure 5. Id3�/� Apoe�/� B cells have decreased expression of CCR6 and expression of CCR6 on B cells mediates homing tothe aorta and atheroprotection. A, Real-time PCR (see Methods for sequences) of B-cell mRNA from Id3�/� Apoe�/� mice expressedrelative to Id3�/� Apoe�/� values. B, CCR6 staining of B cells from Id3�/� Apoe�/� or Id3�/� Apoe�/� mice. C, Percent specific migra-tion of B cells in response to 500 ng/mL of CCL20 or 1000 ng/mL of CXCL13. *P�0.006. D, Luciferase activity from BJAB cellscotransfected with 2.3-kb human CCR6 promoter-luciferase construct and a pEF4 E12, empty pEF4, or pEF4 Id3 expression vectors.Experiments performed in triplicate were repeated 3 times. E, Representative flow plot, and F, quantification of B-cell staining withinaortas harvested from 10- to 12-week-old C57BL/6 (B6) or Ccr6�/� mice (n�5) *P�0.032. G, Representative flow plot, and H, quantifi-cation of B-cell staining within aortas of �MT Apoe�/� mice 24 hours after adoptive transfer of 60�106 splenic B cells from B6 orCcr6�/� mice or vehicle control. Each point represents a single animal. *P�0.009. I, Quantification of en face lesion area of �MTApoe�/� mice after adoptive transfer of 60�106 B cells from either Ccr6�/� Apoe�/� or Ccr6�/� Apoe�/� mice or vehicle control. Ani-mals were fed a Western diet for 16 weeks starting 72 hours after adoptive transfer. Each point represents a single animal. *P�0.0002,**P�0.002. NS indicates not significant.




addressed the subset question and provide evidence that B2cells promote27,28 whereas B1a cells attenuate Western diet-induced atherosclerosis.29 Results herein, address the ques-tion of context, providing the first evidence that mice with Bcells resident in the aorta at baseline have less atherosclerosis

in response to Western diet feeding compared with those withfew aortic B cells. Id3�/� Apoe�/� mice, which had pre-served numbers of circulating B cells but lacked sufficientaortic B cells (Figure 2F and 2G), developed significantlymore atherosclerosis than Id3�/� Apoe�/� mice (Figure 1).

Figure 6. Adoptively transferred B cells traffic to regions of atherosclerosis, decrease plaque macrophage content, and attenu-ate atherosclerosis progression. A, Sudan IV staining and ex vivo phosphor imaging of aorta from �MT Apoe�/� mice fed 8 weeks ofWestern diet and injected with 2�107 In-111 radiolabeled Apoe�/� or Id3�/� Apoe�/�B cells. B, Quantification (n�4) of countsadjusted for injected radioactivity and aortic weight. *P�0.0003. C, Quantification of en face lesion area of �MT Apoe�/� mice fed 8weeks of a Western diet (baseline, n�10) or injected with vehicle control (n�13) or 45�106 Apoe�/� B cells (n�9) followed by an addi-tional 8 weeks of Western diet. *P�0.011, **P�0.0005. NS indicates not significant. D, Representative images, and E, quantification ofMOVAT-stained cross sections at the aortic cusp *P�0.05, **P�0.001. F, Representative images and quantification of G, macrophage,and H, MCP-1 content in �MT Apoe�/� mice with 8 weeks of prior Western diet feeding that received injection of vehicle control orApoe�/� B cells and an additional 8 weeks of Western diet feeding. Aortic cross sections (4 per animal at 60-�m intervals) werestained using DAPI (blue) and either a Mac-2 monoclonal antibody (red) or a MCP-1 polyclonal antibody (green). *P�0.024 and 0.007,respectively. Quantification of aortic I, B-cell (CD19� CD45�), and J, macrophage (CD68� CD45�) content determined by flow cytom-etry 72 hours after adoptive transfer of either PBS vehicle injection or 60�106 Id3�/� Apoe�/� B cells to chow-fed �MT Apoe�/� mice.*P�0.006 and 0.0005, respectively.




Although it is possible that in addition to B cells, other celltypes could contribute to the increased atherosclerosis ob-served in the Id3�/� Apoe�/� mouse, the use of the �MTApoe�/� recipient mouse in B-cell adoptive transfer studiesdemonstrates a clear role for Id3 in mediating B-cell homingand atheroprotection, as in the �MT Apoe�/� model Id3 ispresent in all cell types. Adoptive transfer of splenic B cellsfrom Id3�/� Apoe�/� mice led to equal reconstitution ofperipheral lymph tissue (spleen, lymph node, and blood) as Bcells from Id3�/� Apoe�/� mice (Table 2), but aortic recon-stitution (Figure 3b) and attenuation of Western diet-inducedatherosclerosis (Figure 4C) were significantly impaired. Thatthe adoptively transferred B cells come from a hyperlipemicmouse on an atherogenic background (Apoe�/�) appears to beimportant as neither 528 or 6030 million B cells from B6 micewere atheroprotective when transferred into �MT Apoe�/�

recipient mice. Consistent with these findings, Western dietfeeding induced Id3 expression in B cells (data not shown).Further support for aortic B-cell–mediated atheroprotection isprovided by the finding that B cells from Apoe�/� mice nullfor CCR6 but wild-type for Id3 also had reduced aortic B-cellhoming and B-cell–mediated atheroprotection (Figure 5).Taken together, results provide evidence for a model ofatheroprotection whereby resident aortic adventitial B cellsserve as early responders to atherogenic signals in the vesselwall to limit atherosclerosis.

Chemokine receptors provide the “address” for leukocytehoming to tissues31 and results of the present study providethe first evidence that CCR6 is important for B-cell traffick-ing to the aorta and attenuation of Western diet–inducedatherosclerosis. Yet, mice globally deficient in CCR6 haveattenuated atherosclerosis.32 CCR6 is expressed on variouscell types involved in atherosclerosis,33 and mice globallydeficient in CCR6 have reduced circulating monocytes due toan reduced ability of these cells to leave the bone marrow,32

providing evidence that CCR6 may have opposing effects onatherosclerosis development dependent on the cell type inwhich it is modulated. CCR6 has been identified as animportant component of B-cell trafficking to form isolatedlymphoid follicle formation in the gut in inflammatorystates34 suggesting that CCR6 may be a common chemokinereceptor mediating B-cell homing to tissue sites to modulatelocal inflammation.5,6 Results of the present study demon-strate that CCR6 is an Id3 target and that CCR6 regulatesB-cell homing to the aorta and B-cell–mediated atheropro-tection. Of note, the magnitude of the aortic homing defect inB cells from Id3�/� Apoe�/� mice is greater than that seen inCCR6�/� mice, suggesting that additional chemokine recep-tors may participate with CCR6 in the aortic “address.”Previous reports have identified CXCR6 and L-selectin asimportant regulators of T cell homing to the aorta,14,24

however our results suggest that the “address” for B-cellhoming to the aorta is independent of these factors. Grabneret al have implicated vessel wall expression of CXCL13 andCCL21 in the formation of B-cell–rich ATLOs adjacent toadvanced plaques in aged Apoe�/� mice.5 Yet, in our studiesof younger mice, B cells from Id3�/� Apoe�/� and Id3�/�

Apoe�/� mice had similar expression of CXCR5 (receptor forCXCL13) and CCR7 (receptor for CCL21) and transmigra-

tion in response to CXCL13 compared with controls (Figure5 and data not shown), suggesting that unique chemokinesignals may recruit B cells to the vessel wall before lesiondevelopment and in the early stages of atherosclerosis ascompared with advanced lesions. It is also possible that Id3may regulate other downstream target genes not involved inB-cell homing but involved in B-cell–mediatedatheroprotection.

Id3 dimerizes with E-proteins such as the E2A geneproducts, E12 and E47, which have critical functions in Bcells.35 As such, B cells from Id3 null mice may have otherfunctional defects that promote atherogenesis. B cells pro-duce antibodies and cytokine that modulate innate and adap-tive immune responses. Evidence suggests that antibodiesdirected at oxidized phospholipid epitopes, such as OxLDLand MDA-LDL, may modulate atherosclerosis. In particular,IgM specific for OxLDL and MDA-LDL have been sug-gested to attenuate the development of atherosclerosis,26,36–39

whereas IgG specific for these epitopes may be athero-genic.38,40 Given that the major site of LDL oxidation is in theartery wall, it seems plausible that resident aortic B cells maybe stimulated with vessel wall antigens to produce protectiveautoantibodies in the spleen or local tissue. We thereforeexplored circulating levels of several of these antibodiesknown to be associated with atherosclerosis. Paradoxically,serum levels of IgM to MDA-LDL and OxLDL were bothincreased in Id3�/� Apoe�/� mice compared with control.Consistent with previous literature18 examining antibodylevels in young Id3�/� mice, the loss of Id3 in Apoe�/� micewas also associated with an increase in total serum IgMlevels. This raises the possibility that the increased IgMMDA-LDL and OxLDL observed in these mice may besecondary to a nonspecific global increase in all IgMs.Moreover, the increased, not decreased, levels of the puta-tively protective IgM MDA-LDL and OxLDL in the Id3�/�

Apoe�/� mice make it unlikely that production of thesespecific antibodies is the mechanism for the increasedatherosclerosis.

B cells have long been known to reside in the adventitia ofdiseased arteries of mice and humans,5–14 yet the specificlocation of these adventitial B cells in relation to atheroproneregions of the vascular tree has not been known. We demon-strate that B cells traffic to (Figure 3D) and reside (Figure 2G)in atheroprone regions of the aorta (the aortic arch and thedescending abdominal aorta). Moreover, we demonstrate thatB cells traffic to regions with existing lipid deposition (Figure6A), reduce macrophage content within lesions (Figure 6F,6G, and 6H) and retard the progression of these early lesions(Figure 6C). Interestingly, our results suggest that B cellsregulate macrophage content of the aorta in response toWestern diet feeding and even before feeding. Although�MT Apoe�/� mice do not have B cells in their aortas, theydo have abundant macrophages. Reconstitution of the aorta of�MT Apoe�/� mice with Id3�/� Apoe�/� B cells resulted ina significant reduction in aortic wall-associated macrophages(Figure 6i and j). This significant alteration of the aorticcellular milieu did not occur when Id3�/� Apoe�/� B cellswere used (data not shown), suggesting that the effect isdependent on aortic B-cell number. Notably, 8-week-old




chow-fed Id3�/� Apoe�/� mice, which have fewer aortic Bcells, were also found to have more aortic macrophages(Figure 2). The number of cells in the aortas of Id3�/�

Apoe�/� and adoptively transferred �MT Apoe�/� micecannot be directly compared because these are distinct animalmodels and there is a significant difference in the degree ofaortic B-cell deficiency as well as in the chronicity of theB-cell deficiency. However, both models independently pro-vide evidence that B cells regulate aortic macrophage content.Given the rapid rate at which these changes occur, it isappealing to hypothesize that this effect is cytokine-mediated.Functional conduits connecting B-cell–containing ATLOs inthe adventitia and the vessel wall have recently been identi-fied,5 suggesting that adventitial B cells may pass signals intothe vessel wall. Immunoglobulins are too large to passthrough these conduits,5 however these conduits do allowpassage of low-molecular-weight molecules such as cyto-kines from the ATLO to the media. Thus, adventitial B cellscould limit macrophage accumulation in the intima throughlocal production of anti-inflammatory cytokines that can passinto the vessel wall via these conduits. Future studies will benecessary to confirm these hypotheses and identify thecytokines involved.

The Id3�/� Apoe�/� mouse provides a unique model withwhich to explore the cellular and molecular atheroprotectivemechanisms of leukocytes resident in the aortic adventitia ofnondiseased vessels. Using this model, our findings provideevidence for a model whereby resident adventitial B cellswithin this microenvironment are poised to react to athero-genic stimuli from the vessel wall leading to production offactors that limit macrophage accumulation in plaque andprogression of lesion development. Given the previouslyidentified association between a functionally significant SNPin the human ID3 gene and carotid intima medial thick-ness, our findings in mice may also have implications forhuman disease. Thus, identification of Id3 as a criticalregulator of aortic B-cell homing and atheroprotection isnot only important for our understanding of these mecha-nisms but may also lead to novel strategies to attenuateatherosclerosis in humans.

AcknowledgmentsWe thank Hong Pei and the UVA Mouse Surgery Transplantationand Physiology Core for excellent technical assistance, JeremyGatesman from the UVA Center for Comparative Medicine forexcellent technical assistance, Dr Borna Mehrad (UVA) for gener-ously providing CCR6�/� mice, Dr Yuan Zhuang (Duke University)for generously providing Id3�/� mice and guidance, and JoanneLannigan and Mike Solga from the UVA Flow Cytometry Core forexcellent advice.

Sources of FundingThis work was supported by NIH P01 HL55798 (C.A.M.), NIH RO1HL62522 (C.A.M.), NIH R01 HL096447 (C.A.M.), NIH R01HL58108 (K.L.), R01 HL086559 (J.L.W. and S.T.), P01 HL088093(J.L.W. and S.T.), NIH ARRA HL096447-01A1 (S.T.), FondationLeducq (S.T. and J.L.W.), NIH Training Grant 5-T32 HL007355-29(M.J.L. and A.C.), American Heart Association Pre-Doctoral Fel-lowship (A.C.D.), and American Heart Association Mid-AtlanticAffiliate postdoctoral fellowship award 10POST3560000 (M.J.L.).

DisclosuresNone.

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Novelty and Significance

What Is Known?

● B cells and immunoglobulins are in associated with atheroscleroticplaques in mice and humans, yet the factors regulating traffickingof B cells to the aorta and their impact on atherosclerosis arepoorly understood.

● Inhibitor of Differentiation-3 (Id3) regulates gene expression in B cells anddeletion of Id3 in Apoe�/� mice null increases atherosclerosis.

● Humans with a functionally significant polymorphism in the Id3 genehave increased carotid intima-media thickness compared withthose homozygous for the ancestral allele.

What New Information Does This Article Contribute?

● Id3 is necessary for early atheroprotection.● Loss of Id3 reduces B-cell CCR6 expression and deficiency of both

ID3 and CCR6 reduces B-cell aortic homing and B-cell–mediatedatheroprotection.

● B cells home to sites in the aorta prone to atherosclerosis and regionswith early lipid deposition, reduce the macrophage content of thelesion, and attenuate atherosclerosis progression.

B cells have been shown to both aggravate and attenuateatherogenesis, suggesting that the effects of B cells may besubset- or context-dependent. Here, we provide evidence thatthe effects of B cell on atherosclerosis are context-dependentbased on the following findings: (1) Apoe�/� mice with reducednumber of aortic, but normal number of peripheral B cells haveincreased atherosclerosis. (2) B cells home to, and attenuateinitiation and progression of atherosclerosis in, regions of theaorta prone to atherosclerosis and attenuate initiation andprogression of atherosclerosis. Our results identify the helix-loop-helix factor, ID3 as a critical regulator of B-cell aortichoming and B-cell–mediated atheroprotection and define CCR6as one downstream target of ID3 that promotes aortic B-cellhoming and B-cell–mediated atheroprotection. Taken to-gether with previous findings of a functionally significant SNPin the human Id3 gene associated with carotid thickening inhumans, these results suggest that strategies to enhance Id3expression or function may provide immune protectionagainst atherosclerosis.




SUPPLEMENTAL MATERIALS Supplementary Table I. Average random plasma lipid, oxidized lipoprotein, immunoglobulin, glucose and insulin profiles of Id3+/+ Apoe-/- and Id3-/- Apoe-/- mice at baseline (eight weeks of age). Id3+/+ Apoe-/- Id3-/- Apoe-/- p-value

Number 10 10 Weight (g) 24.40 ± 2.19 23.10 ± 3.34 n.s. Total cholesterol (mg/dL) 353 ± 53 355 ± 32 n.s. HDL (mg/dL) 32 ± 5 34 ± 9 n.s. Triglycerides (mg/dL) 187 ± 44 179 ± 40 n.s. Non-HDL (mg/dL) 311 ± 46 315 ± 40 n.s. OxPL/ApoB 2.42 ± 0.69 2.54 ± 0.50 n.s. MDA/ApoB 4.54 ± 1.33 3.13 ± 0.47 n.s. Total IgM (μg/ml) 100 ± 25 275 ± 60 0.038 MDA LDL IgM (RLU) 7,044 ± 1207 15,924 ± 3045 0.021 Cu-OxLDL IgM (RLU) 2,124 ± 281 3,722 ± 539 0.028 MDA LDL IgG (RLU) 1,308 ± 280 2,501 ± 711 n.s. Cu-OxLDL IgG (RLU) 355 ± 42 584 ± 81 n.s. Glucose (mg/dL) 135 ± 25 139 ± 21 n.s. Insulin (ng/mL) 0.56 ± 0.21 0.57 ± 0.26 n.s.

Values are the average + SEM n.s.: not significant Supplementary Table II. Average random plasma lipid, glucose and insulin profiles of bone marrow transplant chimeric mice. Recipient: Id3+/+ Apoe-/- Id3+/+ Apoe-/- Id3-/- Apoe-/- Id3-/- Apoe-/- Donor: Id3+/+ Apoe-/- Id3-/- Apoe-/- Id3+/+ Apoe-/- Id3-/- Apoe-/-

Number 11 10 10 11 Weight (g) 25.73 + 3.32 25.00 + 4.18 23.70 + 2.67 24.64 + 2.25 Total cholesterol (mg/dL) 1,587 + 164 1,598 + 194 1,583 + 123 1,559 + 152 HDL (mg/dL) 23 + 3 26 + 4 21 + 4 28 + 3 Triglycerides (mg/dL) 211 + 34 198 + 28 204 + 41 201 + 32 Non-HDL (mg/dL) 1,523 + 148 1,532 + 134 1,526 + 145 1,499 + 118 Glucose (mg/dL) 177 + 29 160 + 9 154 + 19 162 + 25 Insulin (ng/mL)

2.81 + 0.94 3.10 + 0.81 3.04 + 0.78 2.99 + 0.74

Values are the average + SEM No significant difference in any parameter between groups.



METHODS SUPPLEMENT Animals

All animal protocols were approved by the Animal Care and Use Committee at the University of Virginia. C57BL/6, Apoe-/- and µMT mice were purchased from Jackson Laboratory (stock #000664, #002052 and #002288 respectively). Id3-/- mice were a generous gift of Dr. Yuan Zhang (Duke University). Id3-/- and µMT mice were bred to the Apoe-/- background to obtain atherogenic Id3-/- Apoe-/- and µMT Apoe-/- strains. Ccr6-/- mice were a generous gift from Dr. Borna Mehrad (University of Virginia). All mice were given water ad libitum and either a standard chow diet (Harlan Teklad catalog #7012) or Western diet (Harlan Teklad catalog #TD88137). Only male mice were used for all experiments.

Determination of Serum Cholesterol Levels and Antibody Titers Whole blood was harvested from mice at the time of sacrifice by right ventricular puncture. Blood was centrifuged at 8200 rcf (10,000 rpm) for five minutes, after which the serum layer was removed to a clean tube. Cholesterol determinations were performed by the University of Virginia Medical Laboratories using an Archtect 8000 series analyzer, employing the method of Allain1 and Roeschlau2. Determination of mouse apoB levels, malondialdehyde epitopes on mouse apoB, and mouse IgG and IgM MDA-LDL and Cu-OxLDL were determined as previously described in detail3,4. Analysis of Atherosclerosis

Animals were anesthetized by intraperitoneal injection of ketamine/xylazine and the vasculature was perfused by left ventricular puncture using 4% paraformaldehyde. Aortas were opened longitudinally, pinned and stained using Sudan IV by the en face method as previously described5{Daugherty, 2003 #146}. Aortas were then imaged and lesion area was quantified using ImagePro 5.0. In the experiments in which B cells were adoptively transferred to µMT Apoe-/- mice already fed Western diet for 8 weeks, the heart and aortic arch to the left subclavian artery was embedded in paraffin and 5 μm thick serial sections were generated from the aortic valve to the bifurcation of the brachiocephalic artery in each animal. Sections were stained at 60 μm intervals using the Movat method6 and plaque volume was assessed using ImagePro Plus 7.0 software. The descending aortas from the left subclavian artery to the iliac bifurcation were opened longitudinally, pinned and stained using Sudan IV as described above.

Immunohistochemical Analysis of Atherosclerosis

Paraffin-embedded sections were selected at 60 μm intervals starting at the aortic valve, deparaffinized, rehydrated, underwent antigen retrieval, blocked with PBS with fish scale gelatin and 10% horse serum for 1 hour, incubated with the primary antibody for 1 hour, washed and then incubated with the secondary antibody for 1 hour. When staining for macrophages, the primary antibody was applied at a concentration of 1:250 (Rat anti-mouse Mac-2 monoclonal IgG antibody, Cedarlane Laboratories) and the secondary antibody was applied at a concentration of 1:100 (Dylight 594-conjugated donkey anti-rat IgG antibody, Jackson ImmunoResearch Laboratories). When staining for MCP-1, the primary antibody was applied at a concentration of 1:250 (Goat anti-mouse MCP-1 polyclonal IgG antibody, Santa Cruz Biotechnology Inc, Cat # sc-1784) and the secondary antibody was applied at a concentration of 1:250 (Dylight 488-conjugated donkey anti-goat IgG antibody, Jackson ImmunoResearch Laboratories). The slides were then washed and the coverslip was placed following application of Vectashield Mounting Medium with DAPI (Vector Laboratories). Negative controls were also performed in which the slides were processed as above with omission of the primary antibody for one control and the secondary antibody for another control. Fluorescent microscopy was



performed along with acquisition of brightfield images. Macrophage area / intima area was calculated as the macrophage area within the intima divided by the total area of the intima using ImagePro Plus 7.0 software. MCP-1 area / intima area was calculated as the MCP-1 area within the intima divided by the total area of the intima Bone Marrow Transplantation

At four to five weeks of age, Id3+/+Apoe-/- and Id3-/-Apoe-/- mice were subjected to a sub-lethal dose of radiation (500 rads x 2 irradiations) and subsequently reconstituted with 5 x 106 bone marrow cells harvested from the femurs and tibias of Id3+/+Apoe-/- and Id3-/-Apoe-/- donor mice. After four weeks of recovery (eight weeks of age), recipient animals were started on a Western diet. After 16 weeks of Western diet, the aortas were harvested and analyzed by the en face method described above. FACS Analysis of Murine Aortas

A version of this method as well as all antibodies used for these studies have been previously validated and published7. Animals were lethally anesthetized by intraperitoneal injection of ketamine/xylazine and the vasculature was perfused by left ventricular puncture using PBS with 20 U/ml heparin. The aorta was microdissected, taking great care to remove all periaortic fat and lymph nodes while sparing the adventitia as this is where B cells are predominantly found7-9. Lymph nodes, spleens, blood and aortas, including the adventitia, were harvested under a dissection microscope. Tissues were homogenized and incubated for 45 minutes at 37oC in an enzymatic cocktail (1ml per 10mg tissue) containing 488 U/ml collagenase I (Sigma cat # C0130), 230 U/ml collagenase XI (Sigma cat # C7657), 125 U/ml hyaluronidase (Sigma cat # H3506) and 60 U/ml DNase I (Sigma catalog # D4527) in an enzymatic digestion buffer containing PBS and 20mM HEPES. Tissues were then mashed through a 70-μm cell strainer to create a suspension. Red blood cells were lysed from spleen and blood samples using a lysis buffer containing 155mM NH4Cl, 10 mM KHCO3 and 0.1mM EDTA. Cells were counted, diluted to 1 x 106 cells per 100 μl and incubated with antibodies (as named above) for 45 minutes at 4oC. After washing, the cells were then fixed, permeabilized and stained with antibodies directed against specific intracellular antigens using the Caltag Fix & Perm kit as per the manufacturer’s instructions. Cells were washed again, resuspended in FACS buffer (PBS containing 0.05% NaN3 and 1% BSA) and analyzed at the University of Virgina Flow Cytometry Core using a CyAN ADP flow cytometer. Data were analyzed using FloJO software (Tree Star Inc). FACS Antibodies

Antibodies used were as follows: anti-CD3e (APC, clone 145-2C11, 0.2 µg used at 1:100), anti-CD5 (PE, clone 53-7.3, 0.1 µg used at 1:200), anti-CD11b (Pacific Blue, clone M1/70/15, 0.1 µg used at 1:100), anti-CD11c (APC AlexaFluor750, clone N418, 0.4 µg used at 1:50), anti-CD16/32 (purified Fc Block, 0.5 µg used at 1:100), anti-CD19 (PECy7, clone 1D3, 0.2 µg used at 1:100), anti-CD21 (APC, clone 7G6, 0.2 µg used at 1:100), anti-CD23 (PECy7, clone B3B4, 0.2 µg used at 1:50), anti-CD45 (PerCP, clone 30-F11, 0.2 µg used at 1:100), anti-CD45R/B220 (PE, clone RA3-6B2, 0.2 µg used at 1:100), anti-CD68 (FITC, clone FA-11, 0.5 µg used at 1:50), F4/80 (APC, clone A3-1, 0.05 µg used at 1:100), Gr-1 (APC Cy7, clone RB6-8C5, 0.2 µg used at 1:100), I-Ab (FITC, clone AF6-120.1, 0.5 µg used at 1:100), anti-IgD (FITC, clone 11-26, 0.5 µg used at 1:200), anti-IgM (APC, clone II/41, 0.2 µg used at 1:50), anti-NK1.1 (PECy7, clone PK136, 0.2 µg used at 1:100), anti-TCRβ (APC AlexaFluor750, clone H57-597, 0.2 µg used at 1:50), anti-Ter119 (FITC, 0.5 µg used at 1:100). Either 7-AAD (BD Pharmingen) or LIVE/DEAD Fixable Aqua Dead Cell Stain (Invitrogen) was included as a viability stain with every sample.



Adoptive Transfer Spleens were harvested from 10-12 week old animals and B cells were isolated using

MACS anti-CD43 microbeads (Miltenyi Biotec) as per the manufacturer’s protocol. The purity of representative samples were analyzed by flow cytometry and found to be greater than 98%. Either 30 x 106 or 60 x 106 B cells were suspended in 200 l of PBS and adoptively transferred via tail vein injection to 8-10 week old recipients. Fluorescent Microscopy of Aortic Cross Sections B lymphocytes were purified as above from the spleens of Id3+/+ Apoe-/- mice. Cells were incubated in 10 M CFDA-SE, prepared as per the manufacturer’s instructions, for 30 minutes (Invitrogen, catalog # V12883). Lymphocytes were then washed and adoptively transferred via tail vein injection to MT Apoe-/- recipients. After 72 hours, mice were euthanized, aortas were harvested and embedded into OCT medium. After freezing at -20oC for one hour, samples were cut into 50m thick sections using a cryostat and mounted onto glass slides with DAPI-containing medium. Fluorescent imaging at the UVA Advanced Microscopy Core enabled identification of B cells using standard set green channel filters to detect CFDA-SE positive cells and the blue channel for DAPI. Optical Imaging of Aortic B Lymphocytes

Animals were anesthetized by intraperitoneal injection of ketamine/xylazine, the vasculature was perfused by left ventricular puncture using phosphate buffered saline and the aorta was carefully removed by microdissection. As with dissection for flow cytometric analysis, great care was again taken to remove all periaortic fat and lymph nodes while sparing the adventitia in order to preserve aortic lymphocytes. In order to detect the endogenous presence of B lymphocytes in the aorta with near-infrared (NIR) fluorescent imaging, 1 ml of a biotinylated anti-B220 antibody (eBioscience catalog # 14-0452, used at 0.5 mg/ml) and molar excess of Cy5.5-conjugated streptavidin (Rockland Immunochemicals, catalog # S000-13) were mixed and incubated for 30 minutes to ensure adequate conjugation. Aortas were then harvested as described above from five chow-fed eight to ten week-old Id3+/+ Apoe-/- mice, five Id3-/- Apoe-/- mice, and five MT Apoe-/- mice. Each aorta was incubated overnight at 5°C in 65 μL of Cy5.5-labeled anti-B220 solution diluted in 335 μL of PBS while rotating in the dark in amber Eppendorf tubes. The aortas were then washed three times with PBS for ten minutes and the aortic lumen was flushed with PBS to minimize non-specific retention of fluorescent antibody. Ex vivo fluorescence-mediated tomography (FMT) quantitative imaging (FMT 2500, VisEn Medical) was performed for each aorta with data acquisition at 680 nm for excitation and 700 nm for emission. Regions of interest were generated for each aorta and values for mean fluorescence from 2-dimensional fluorescence reflectance images were obtained. To enable comparison between the aortas, the maximal fluorescence was set to 10.5 count/energy for the NIH Color scale bar. Radiolabeling of B Lymphocytes

Ten to 12 week-old Id3-/- Apoe-/- and Id3+/+ Apoe-/- mice were euthanized with carbon dioxide, the spleens were removed and a single cell suspension was obtained by pressing the spleen through a 70-m strainer. Red blood cells were lysed from the cell suspension using a lysis buffer containing 155mM NH4Cl, 10 mM KHCO3 and 0.1mM EDTA. B lymphocytes were isolated using MACS anti-CD43, anti-CD11b, and anti-CD4 microbeads (Miltenyi Biotec) as per the manufacturer’s protocol. This combination of beads resulted in a B cell purity of 99% on flow cytometry. Molar excess of oxyquinoline (oxine) was incubated with indium-111 HCl (PerkinElmer) for 30 minutes at room temperature in HEPES Buffer with 10% dimethyl sulfoxide, enabling formation of indium-111 oxine. Indium-111 has a half-life of 2.8 days with



photon energies of 171 and 245 keV. Isolated B lymphocytes were centrifuged and resuspended with indium-111 oxine solution and allowed to incubate at room temperature for one hour. Cells were washed three times with normal saline after which B lymphocyte number and percent cellular viability was then determined by counting cells following Trypan Blue staining.

The radiolabelled B lymphocytes from either Id3+/+ Apoe-/- or Id3-/- Apoe-/- mice were then adoptively transferred via tail vein injection into MT Apoe-/- recipient mouse. The μCi of indium-111 per injected dose was determined by measuring the radioactivity of the syringe before and following injection into the mouse using a Radioisotope Dose Calibrator (Searle Radiographics). Additionally, control MT Apoe-/- recipient mice received an equal total radioactive dose of indium-111 oxine diluted in normal saline, thus lacking radiolabeled cells.

Ex Vivo Nuclear Imaging of Radiolabeled B Lymphocytes

Approximately 20 hours following adoptive transfer of radiolabeled B lymphocytes or control injection, the recipient mice were euthanized by intraperitoneal injection of ketamine/xylazine, the vasculature was perfused with PBS, the aortas were harvested whole from just above the heart to the iliac bifurcation as previously described, cut longitudinally and opened en face, covered with plastic saran wrap to minimize dehydration, and exposed to a high sensitivity, medium resolution phosphor imaging screen (PerkinElmer). After 18 hours, the phosphor imaging screen was scanned using a PerkinElmer Cyclone Plus Phosphor Imaging System. Signal intensity analysis for each aorta was performed using Sante DICOM software and was adjusted for injected radioactive dose per animal and background using the formula: (Mean Signal Intensity of Aorta - Background Signal Intensity) / Injected Radioactive Dose in μCi. Regions of interest within the aorta were defined by anatomical landmarks. The aortic arch was defined as the region from the aortic cusp to the left subclavian artery. The descending thoracic aorta was defined as the region from the left subclavian artery to the diaphragm. The descending abdominal aorta was defined as the region from the diaphragm to the bifurcation of the iliac arteries. Comparison of aortic indium-111 content for recipient aortas was then determined using a Minaxi 5500 Gamma Counter using an indium-111 window setting (160 to 485 keV). Gamma counting for each sample occurred over a five minute period to generate an average number of counts per minute (CPM) and the adjusted gamma counts for each aorta are determined by the formula: ((CPM of aorta - Background CPM) / Weight of the aorta in grams / Injected radioactive dose in μCi). Real Time PCR

Total cellular RNA was collected from B cells using an RNeasy kit (Qiagen) as per the manufacturer’s instructions. cDNA was then synthesized using an iScript cDNA synthesis kit (BioRad). Total cDNA was diluted 1:5 in water and 2 μl were used for each real-time PCR reaction using a Bio-Rad iCycler and iQ SYBR Green Supermix (BioRad). Analysis was performed by normalizing to cyclophilin using the standard curve method to approximate the amount of starting material. Primers used for the detection of CCR6 were: 5’ - CCC AGC ACA

TCA TAG CAT TG - 3’ and 3’ - AGT CCG AAA GAC CCA GGA TT - 5’. Primers for the detection of CXCR5 were: 5’ - AGG AAA ACG AAG CGG AAA CT - 3’ and 3’ - CGA CCA GCA GAG GAA

GAA AA - 5’. Primers for the detection of CXCR6 were: 5’ - AAC AGC CAG GAG AAC AAA CG - 3’ and 3’ - ACA CAC AGG GCA AAA AGA CC - 5’. Primers for the detection of L-selectin were: 5’ - AGC CCA ACA ACA AGA AGT CC - 3’ and 3’ - TCG TTC CAT TTC CCA GAG TC - 5’. Primers for the detection of cyclophilin were: 5’ - TGC CGG AGT CGA CAA TGA T - 3’ and 3’ - TGG AGA GCA CCA AGA CAG ACA - 5’.



B cell Migration Splenic B cells were purified from Id3+/+ Apoe-/- and Id3-/- Apoe-/- mice as described

above and resuspended in RPMI 1640 (Invitrogen) with 10% FBS (HyClone). 1 x 106 cells were placed in the upper chamber of 5 μm pore size transwells containing either 1000 ng/ml of CXCL13 or 500 ng/ml of CCL20 in the bottom of the transwell. After incubating for 6 hours, the number of cells which had migrated through the transwell were counted using a flow cytometer. The percent specific migration was determined by subtracting the spontaneous migration observed in transwells with no chemokine from the migration observed under experimental conditions. Statistical Methods Data are presented as the mean ± standard error of the mean. Unpaired Student’s t test, Mann Whitney U-test, or analysis of variance was performed to compare continuous variables between groups (presented as mean ± standard error of the mean (SEM)). Bonferroni’s correction was applied to the analysis of variance when comparisons between multiple groups were performed to provide comparison between all groups. A p-value <0.05 was considered statistically significant. All statistical analyses were performed using NCSS 2001 (Number Crunching Statistical Software, Kaysville, Utah) and GraphPad Prism5 (La Jolla, California). References 1. Allain CC, Poon LS, Chan CS, Richmond W, Fu PC. Enzymatic determination of total

serum cholesterol. Clin Chem. 1974;20:470-5. 2. Roeschlau P, Bernt E, Gruber W. Enzymatic determination of total cholesterol in serum.

Z Klin Chem Klin Biochem. 1974;12:226. 3. Tsimikas S, Palinski W, Witztum JL. Circulating autoantibodies to oxidized LDL correlate

with arterial accumulation and depletion of oxidized LDL in LDL receptor-deficient mice. Arterioscler Thromb Vasc Biol. 2001;21:95-100.

4. Merki E, Graham MJ, Mullick AE, Miller ER, Crooke RM, Pitas RE, Witztum JL, Tsimikas S. Antisense oligonucleotide directed to human apolipoprotein B-100 reduces lipoprotein(a) levels and oxidized phospholipids on human apolipoprotein B-100 particles in lipoprotein(a) transgenic mice. Circulation. 2008;118:743-53.

5. Daugherty A, Whitman SC. Quantification of atherosclerosis in mice. Methods Mol Biol. 2003;209:293-309.

6. Movat HZ. Demonstration of all connective tissue elements in a single section; pentachrome stains. AMA Arch Pathol. 1955;60:289-95.

7. Galkina E, Kadl A, Sanders J, Varughese D, Sarembock IJ, Ley K. Lymphocyte recruitment into the aortic wall before and during development of atherosclerosis is partially L-selectin dependent. J Exp Med. 2006;203:1273-82.

8. Grabner R, Lotzer K, Dopping S, Hildner M, Radke D, Beer M, Spanbroek R, Lippert B, Reardon CA, Getz GS, Fu YX, Hehlgans T, Mebius RE, van der Wall M, Kruspe D, Englert C, Lovas A, Hu D, Randolph GJ, Weih F, Habenicht AJ. Lymphotoxin beta receptor signaling promotes tertiary lymphoid organogenesis in the aorta adventitia of aged ApoE-/- mice. J Exp Med. 2009;206:233-48.

9. Moos MP, John N, Grabner R, Nossmann S, Gunther B, Vollandt R, Funk CD, Kaiser B, Habenicht AJ. The lamina adventitia is the major site of immune cell accumulation in standard chow-fed apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2005;25:2386-91.



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