Witztum, Peter Libby, Matthias Nahrendorf, Ralph Weissleder and Filip K. SwirskiAndreas Zirlik, Herbert Y. Lin, Galina K. Sukhova, Jagdish Butany, Barry B. Rubin, Joseph L.Ayelet Gonen, Yoshiko Iwamoto, Norbert Degousee, Tobias A.W. Holderried, Carla Winter,
Ingo Hilgendorf, Igor Theurl, Louisa M.S. Gerhardt, Clinton S. Robbins, Georg F. Weber,Adaptive Immunity
Innate Response Activator B Cells Aggravate Atherosclerosis by Stimulating T Helper-1
is online at: Circulation Information about subscribing to Subscriptions:
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Atherosclerosis is a lipid-driven inflammatory disease that mobilizes a diverse repertoire of leukocytes. Although
macrophages accumulate in lesions in the greatest number, other leukocytes can modulate the course of disease. Over the last 20 years, many studies have explored how leukocytes influence atherosclerosis. For example, M1 macrophages, T helper-1 (T
H1) cells, and B2 B cells accelerate whereas T
regulatory (Treg
) cells and B1 B cells attenuate lesion growth by either augmenting or restraining inflammation.1−10 These observations have clinical implications because they suggest
that harnessing protective leukocyte activities and silencing those that are harmful could furnish novel treatments for ath-erosclerosis and other inflammatory diseases.
Clinical Perspective on p 1687
Innate response activator (IRA) B cells develop in the spleen during the inflammatory phase of sepsis.11 IRA B cells produce granulocyte macrophage colony-stimulating factor (GM-CSF), a pleiotropic growth factor that, although dispensable to hematopoiesis in the steady state, promotes
Background—Atherosclerotic lesions grow via the accumulation of leukocytes and oxidized lipoproteins in the vessel wall. Leukocytes can attenuate or augment atherosclerosis through the release of cytokines, chemokines, and other mediators. Deciphering how leukocytes develop, oppose, and complement each other’s function and shape the course of disease can illuminate our understanding of atherosclerosis. Innate response activator (IRA) B cells are a recently described population of granulocyte macrophage colony-stimulating factor–secreting cells of hitherto unknown function in atherosclerosis.
Methods and Results—Here, we show that IRA B cells arise during atherosclerosis in mice and humans. In response to a high-cholesterol diet, IRA B cell numbers increase preferentially in secondary lymphoid organs via Myd88-dependent signaling. Mixed chimeric mice lacking B cell–derived granulocyte macrophage colony-stimulating factor develop smaller lesions with fewer macrophages and effector T cells. Mechanistically, IRA B cells promote the expansion of classic dendritic cells, which then generate interferon γ–producing T helper-1 cells. This IRA B cell–dependent T helper-1 skewing manifests in an IgG1-to-IgG2c isotype switch in the immunoglobulin response against oxidized lipoproteins.
Conclusions—Granulocyte macrophage colony-stimulating factor–producing IRA B cells alter adaptive immune processes and shift the leukocyte response toward a T helper-1–associated milieu that aggravates atherosclerosis. (Circulation. 2014;129:1677-1687.)
Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.113.006381
Received September 19, 2013; accepted January 29, 2014.From the Center for Systems Biology, Massachusetts General Hospital, Boston (I.H., I.T., L.M.S.G., C.S.R., G.F.W., Y.I., C.W., H.Y.L., M.N., R.W.,
F.K.S.); Department of Internal Medicine VI, Infectious Diseases, Immunology Rheumatology, Pneumology, University Hospital of Innsbruck, Innsbruck, Austria (I.T.); Toronto General Research Institute, University Health Network, Toronto, ON, Canada (C.S.R., N.D.); Department of Medicine, University of California, San Diego, La Jolla (A.G., J.L.W.); Department of Gastroenterology, Hepatology and Infectious Diseases, University of Duesseldorf, Duesseldorf, Germany (T.A.W.H.); Department of Cardiology and Angiology I, University Heart Center Freiburg, Freiburg, Germany (C.W., A.Z.); Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Boston, MA (G.K.S., P.L.); Department of Pathology (J.B.) and Division of Vascular Surgery (B.B.R.), Peter Munk Cardiac Centre, Toronto General Hospital, Toronto, ON, Canada; and Department of Systems Biology, Harvard Medical School, Boston, MA (R.W.)
Guest Editor for this article was Daniel I. Simon, MD.*Drs Hilgendorf and Theurl contributed equally.The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA.
113.006381/-/DC1.Correspondence to Ingo Hilgendorf, MD, or Filip K. Swirski, PhD, Center for Systems Biology, Massachusetts General Hospital and Harvard Medical
Innate Response Activator B Cells Aggravate Atherosclerosis by Stimulating T Helper-1 Adaptive ImmunityIngo Hilgendorf, MD*; Igor Theurl, MD, PhD*; Louisa M.S. Gerhardt, MS;
Clinton S. Robbins, PhD; Georg F. Weber, MD; Ayelet Gonen, PhD; Yoshiko Iwamoto, BS; Norbert Degousee, PhD; Tobias A.W. Holderried, MD; Carla Winter, BS; Andreas Zirlik, MD;
Herbert Y. Lin, MD; Galina K. Sukhova, PhD; Jagdish Butany, MD; Barry B. Rubin, MD, PhD; Joseph L. Witztum, MD; Peter Libby, MD;
Matthias Nahrendorf, MD, PhD; Ralph Weissleder, MD, PhD; Filip K. Swirski, PhD
Vascular Medicine
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the survival, proliferation, and activity of various leuko-cytes expressing its receptor.12−14 The function and source of GM-CSF in atherosclerosis remain obscure. Even though some have reported that GM-CSF protects against athero-sclerosis,15 the weight of evidence suggests that GM-CSF is atherogenic because Ldlr−/− Csf2−/− mice develop smaller lesions,16 whereas exogenous administration of GM-CSF to atherosclerotic mice increases plaque burden17 and stimulates intimal cell proliferation.18 In Apoe−/− mice, hematopoietic stem and progenitor cells elevate the expression of the com-mon β chain of the GM-CSF receptor downstream of impaired reverse cholesterol transport, leading to proliferation, which generates leukocytosis and monocytosis.19 GM-CSF can arise from macrophages, T cells, and epithelial cells, but it remains unknown whether IRA B cells develop in atherosclerosis and, if so, whether they have functional relevance.
MethodsA detailed description of the methods is available in the online-only Data Supplement.
AnimalsC57Bl/6J (wild-type [WT]), B6.SJL-PtprcaPepcb/BoyJ (CD45.1+), B6.129P2(SJL)-Myd88tm1.1Defr/J (Myd88−/−), B10.129S2(B6)-Ighmtm1Cgn/J (μMT), B6.Cg-Tg(TcraTcrb)425Cbn/J (OT-II), B6.129S7-Ldlrtm1Her/J (Ldlr−/−), and B6.129P2-Apoetm1Unc/J (Apoe−/−) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). GM-CSF–deficient mice (Csf2−/−) were kindly pro-vided by Dr Randy Seeley, University of Cincinnati (Cincinnati, OH). GM-CSF receptor–deficient mice (Csf2rb−/−) were kindly pro-vided by Dr Jeffrey Whitsett, Cincinnati Children’s Hospital Medical Center (Cincinnati, OH). All protocols were approved by the Animal Review Committee at the Massachusetts General Hospital.
Animal ExperimentsMixed bone marrow chimeras were generated by lethally irradiating 8-week-old male Ldlr−/− mice and reconstituting with a 50:50 mix-ture of Csf2−/− with WT (controls) and μMT bone marrow cells (IRA B knockout [KO]) or with CD45.1+, Myd88−/−, and Csf2rb−/− bone marrow. For adoptive transfer studies, 25×106 CD19+ B cells from WT and Csf2−/− mice were injected intravenously twice per mouse, 4 weeks apart.
HistologyMurine aortas and spleens were embedded in Tissue-Tek optimum cutting temperature compound (Sakura Finetek) for sectioning and staining. Human spleen samples were fixed in 10% formalin and embedded in paraffin for histological sectioning and staining.
Flow CytometryAntibodies used for flow cytometry are listed in the online-only Data Supplement. Data were acquired on a BD LSRII and analyzed with FlowJo.
Reverse Transcription–Polymerase Chain ReactionRNA was isolated from sorted cells with the RNeasy Micro Kit (Qiagen) and from snap-frozen aortas and spleens with the RNeasy Mini Kit (Qiagen). Quantitative real-time TaqMan polymerase chain reaction was run on a 7500 polymerase chain reaction thermal cycler (Applied Biosystems).
Cell CultureLineage-depleted bone marrow cells were cocultured with equal numbers of IRA B cells and murine interleukin (IL)-4 (5000 U/mL)
to generate dendritic cells (DCs). Then, 1×104 IRA B cell–generated bone marrow–derived DCs were loaded with 100 μg/mL ovalbumin or BSA and cocultured with 5×104 labeled OT-II T cells over 4 days for flow cytometric assessment of proliferation. CD4+ CD25+ T
reg
cells were sorted from IRA B KO and control mice and cocultured with sorted CD45.1+ CD4+ CD25− T
conv cells at increasing dilutions
on T cell–depleted and irradiated splenocytes loaded with 1 μg/mL anti-CD3e. Suppression of T-cell proliferation was assessed by flow cytometry after 3 days.
StatisticsResults are shown as mean±SEM. The unpaired Student t test was applied to evaluate differences between 2 study groups. One-way ANOVA with post hoc Dunnett multiple-comparisons test was per-formed to compare >2 groups. Values of P≤0.05 denote significant changes.
ResultsGM-CSF–Producing IRA B Cells Expand in AtherosclerosisWe asked whether IRA B cells develop in atherosclerosis. The spleens of both Ldlr−/− and Apoe−/− mice consuming a diet high in fat and cholesterol (HCD) contained a popu-lation of GM-CSF–producing cells. Most of these were IgMhigh B220+ CD23low CD21low CD138high CD43high VLA4high IRA B cells, resembling IRA B cells generated by lipo-polysaccharide stimulation (Figure 1A and Figure IA in the online-only Data Supplement), as originally described.11 Immunofluorescence staining for GM-CSF revealed a popu-lation of GM-CSF–producing IgM+ B cells in the marginal zone and red pulp of the spleen (Figure 1B), a location where IRA B cells typically reside. Analysis of various organs showed preferential IRA B cell accumulation in the spleen, although the bone marrow and lymph nodes also harbored smaller IRA B-cell populations (Figure 1C). IRA B-cell numbers rose most dramatically and progressively in Apoe−/− mice (Figure 1D), a finding that agrees with the prevailing notion that Apoe−/− mice display more severe inflammation and atherosclerosis than Ldlr−/− mice. B cells accumulate in the aortic adventitia,20 but we did not detect IRA B cells in the aorta, indicating that IRA B cells do not furnish lesional GM-CSF21 and suggesting that IRA B cells do not affect lesions locally. Humans with severe coronary and peripheral artery disease contained more GM-CSF+ IgM+ IRA B cells in the spleen compared with humans with-out atherosclerotic disease (Figure 1E and Figure IB and IC in the online-only Data Supplement). Thus, IRA B cells accumulate in secondary lymphoid organs in humans and mice with atherosclerosis. Future studies will need to deter-mine the exact triggers and risk factors responsible for IRA B-cell production.
During experimental sepsis, IRA B cells arise by engag-ing Myd88-dependent Toll-like receptors.11 To test whether IRA B cells require Myd88 in atherosclerosis, we gener-ated mixed chimeras by lethally irradiating Ldlr−/− mice and reconstituting with a mixture of WT CD45.1+ and Myd88−/− CD45.2+ bone marrow cells (Figure ID in the online-only Data Supplement). After reconstitution, mice consumed HCD for 12 weeks. CD45.1+ WT but not CD45.2+ Myd88−/− cells developed into IRA B cells, indicating a requirement for direct
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Hilgendorf et al IRA B Cells Aggravate Atherosclerosis 1679
Myd88 engagement in B cells (Figure IE in the online-only Data Supplement).
IRA B Cells Aggravate AtherosclerosisDetermining the impact of IRA B cells on atherosclero-sis required selective depletion of GM-CSF from B cells. To achieve this, we adapted the mixed chimeric strategy (Figure 2A). Ldlr−/− mice were lethally irradiated and recon-stituted with a mixture of bone marrow cells from Csf2−/− (ie, GM-CSF−/−) and μMT mice (ie, B cell deficient). In the recon-stituted animals, B cells were the only population completely lacking the capacity to produce GM-CSF because only Csf2−/− cells could give rise to B cells. As controls, we reconstituted Ldlr−/− mice with bone marrow from Csf2−/− and WT mice,
thus ensuring that differences between the groups, should any arise, would exclusively reflect B cell–derived GM-CSF defi-ciency while preserving GM-CSF production by other sources. Reverse transcription–polymerase chain reaction analysis on sorted B cells, T cells, and myeloid cells confirmed the selec-tive deletion of GM-CSF from B cells (Figure 2B). After 6 weeks of reconstitution, we profiled the leukocyte repertoires in the Csf2−/−/μMT (henceforth simply referred to as IRA B KO mice) and the Csf2−/−/WT (control) mice. The blood, spleen, bone marrow, and peritoneal cavity contained similar numbers of leukocyte subsets in both groups (Figure IIA–IID in the online-only Data Supplement), indicating successful reconstitution and a similar leukocyte basal population before the triggering of disease.
Figure 1. Granulocyte macrophage colony-stimulating factor (GM-CSF)–producing innate response activator (IRA) B cells expand in atherosclerosis. A, Identification of GM-CSF+ B220+ IgMhigh IRA B cells in spleens of Ldlr−/− and Apoe−/− mice after 3 months of a high-fat and -cholesterol diet (HCD) by flow cytometry and (B) immunofluorescence histochemistry. C, Flow cytometry–based enumeration of IRA B cells in peripheral blood (per 1 mL), total bone marrow, spleen, peritoneal lavage, 4 para-aortic lymph nodes, and aorta in aged-matched Ldlr−/−, C57Bl/6 wild-type (WT), and Apoe−/− mice after 3 months of normal chow diet (gray) and HCD (white; n≥3 mice per group). Cell counts are presented as mean±SEM. *P≤0.05, **P≤0.01, chow vs HCD per organ. D, Kinetics of IRA B-cell development in spleens of Apoe−/− mice. The 8-week-old Apoe−/− mice were placed on an HCD and euthanized after 4, 8, 12, and 24 weeks on HCD to quantify IRA B-cell numbers (n≥3 mice per time point). Cell counts are presented as mean±SEM. E, Left, Identification of IgM+ GM-CSF+ IRA B cells in the spleen of a patient with atherosclerosis. Right, Quantification of IRA B cells in spleen sections of patients with (white) or without (gray) symptomatic cardiovascular disease (CVD). Cells were counted in 12 randomly selected visual fields of 0.1 mm2 per sample. The combined number of IRA B cells in all 12 visual fields per patient was divided by the total area analyzed (12×0.1 mm2). Results are presented as mean±SEM. **P≤0.01; n=4 per group. For all flow cytometric plots, the ticks represent 0, 102, 103, 104, and 105 fluorescence units, except axes labeled side scatter (SSC), for which the ticks represent 0, 50 000, 100 000, 150 000, 200 000, and 250 000 fluorescence units.
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After reconstitution, the animals consumed an HCD for 10 weeks. GM-CSF expression in the spleen of IRA B KO mice was reduced by 70%, which shows a dominant role for IRA B cells as a source of GM-CSF in the spleen during atherosclero-sis (Figure IIE in the online-only Data Supplement). GM-CSF production by other leukocytes was similar between the groups (Figure IIF in the online-only Data Supplement), as were body weights and plasma cholesterol levels (Figure IIIA in the online-only Data Supplement). The absence of IRA B cells, however, yielded smaller atherosclerotic lesions in the aorta,
particularly the aortic root (Figure 2C and 2D), diminished the lipid- and macrophage-rich areas, reduced the number of CD4 T cells, but did not change the smooth muscle cell and collagen content. The changes in lesion size and macrophage content did not depend on circulating monocyte and neutrophil number (Figure IIIB in the online-only Data Supplement), as might be expected.9,14 Moreover, blood Ly-6Chigh monocytes expressed similar levels of CCR2, VLA4, and CD62L (Figure IIIC in the online-only Data Supplement), which argued against a defect in the capacity of the monocytes to accumulate.
Figure 2. Innate response activator (IRA) B cells promote atherosclerosis. A, For generation of mixed bone marrow chimeras with B cell–restricted granulocyte macrophage colony-stimulating factor (GM-CSF) deficiency (IRA B KO), lethally irradiated 8-week-old Ldlr−/− mice were reconstituted with a 50:50 mixture of GM-CSF–deficient (Csf2−/−) and B cell–deficient (μMT) bone marrow (white). Control mice were reconstituted with a 50:50 mixture of GM-CSF–deficient (Csf2−/−) and WT bone marrow (gray). After 6 weeks of reconstitution, mice were placed on high-fat and -cholesterol diet (HCD) for another 10 weeks. B, Validation of B cell–restricted GM-CSF deficiency in IRA B KO mice after reconstitution and 10 weeks of HCD. Identification of GM-CSF (Csf2) mRNA expression by semiquantitative reverse transcription–polymerase chain reaction in sorted CD3+ (T cells), CD19+ (B cells), and CD11b+ (myeloid cells) splenocytes from control and IRA B KO mice. Rpl19 serves as the housekeeping gene. C, En face Oil Red O (ORO) staining of excised aortas from control and IRA B KO mice after 10 weeks of HCD (left) and quantification of lesion area (right; n=7 per group). Results are presented as mean±SEM. **P≤0.01, gray for control, white for IRA B KO mice. D, Representative hematoxylin and eosin (H&E) staining of aortic root sections from control (gray) and IRA B KO (white) mice after 10 weeks of an HCD with quantification of lesion size in 2 independent experiments (n≥20 per group). Results are presented as mean±SEM. In addition, immunohistology depicting ORO-, Mac3-, smooth muscle actin (SMA)–, Masson trichrome–, and CD4-positive staining of aortic root lesions representative of both groups with quantification of n≥10 samples per group. Results are presented as mean±SEM. *P≤0.05, **P≤0.01.
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Hilgendorf et al IRA B Cells Aggravate Atherosclerosis 1681
IRA B Cells Promote the Expansion of TH1 Effector CellsNumerous studies have identified a role for CD4 T cells in atherosclerosis. Naive CD4 T cells can differentiate into vari-ous helper subsets, exhibiting either protective or atherogenic properties.1−4,7,10,22 The observation that lesions in IRA B KO mice accumulated fewer CD4 T cells prompted us to inves-tigate this leukocyte population in more detail. The blood of IRA B KO mice contained both effector CD44high CD62Llow CD4 T cells and regulatory Foxp3+ T
reg T cells (Figure 3A). At
the onset of the experiment, both groups contained equal num-bers of these subsets in the blood and spleen (Figure 3B), but after 10 weeks of an HCD, IRA B KO mice developed fewer effector T cells in the blood, spleen, and para-aortic lymph nodes compared with controls. T
reg cells, on the other hand,
developed similarly in both groups in terms of number and suppressive function (Figure 3B and Figure IVA and IVB in the online-only Data Supplement).
Among effector T cells, interferon-γ (IFNγ)–producing T
H1 cells augment atherosclerosis.2,10,22,23 We detected fewer
IFNγ-producing TH1
cells in blood, spleen, and lymph nodes in IRA B KO mice compared with controls after 10 weeks of HCD feeding (Figure 3C and 3D). Neither group differed in the number of splenic T
H17 or IL-4–producing T
H2 cells
(Figure IVC in the online-only Data Supplement). Do IRA B cells shape an antigen-specific T
H1 milieu? In atheroscle-
rosis, it is thought that low-density lipoproteins generate an adaptive immune response, presumably as a result of a break in peripheral tolerance against self-antigens.24 Low-density lipoprotein also undergoes oxidation, which can mobilize T
H1 responses via antigen presentation in the context of oxi-
dative stress–related danger signals.25,26 TH1
cells contribute to isotype switching and thus influence antigen-specific humoral immunity.27−29 T cell–derived IFNγ, for example, induces IgG2a/c and dampens IgG1 production, whereas IL-4 has the opposite effect.30 In these experiments, IRA B KO mice were impaired in generating T
H1-dependent IgG2c
antibodies against copper-oxidized and malondialdehyde-modified low-density lipoprotein, even though total IgG and IgM levels increased similarly in both groups (Figure 3E–3G and Figure IVD–IVG in the online-only Data Supplement). Titers of the atheroprotective IgM natural antibody E06 against oxidation-specific epitopes remained unaffected (Figure IIIC in the online-only Data Supplement). Hence, as the IRA B-cell number rose in secondary lymphoid organs, so did the number of effector IFNγ-producing T
H1 cells
and the concentration of antigen-specific, IFNγ-dependent IgG2c. That said, we reasoned that IRA B cells did not aug-ment T-cell numbers directly because T cells do not express the GM-CSF receptor.31
IRA B Cells Promote the Generation of Classic DCsEffector T cells arise in lymphoid organs when their T-cell receptor recognizes antigen on DCs. In the context of specific secondary signals, antigen presented on major histocompat-ibility complex class II (MHCII) can give rise to effector T
H1
cells that expand, enter the circulation and tissue, and partici-pate in immunity.32 Unlike T cells, DCs and their precursors express the GM-CSF receptor and therefore might be directly
influenced by IRA B cells. To test this hypothesis, we enu-merated DCs in the spleen, where IRA B cells expand most prominently. Three populations of CD11c+ MHCII+ classic (c)DC were identifiable: CD11b+ CD8− CD103−, CD11b− CD8+ CD103−, and CD11b− CD8+ CD103+ (Figure 4A). Before the onset of atherosclerosis, both mouse groups contained similar numbers of all 3 subsets, in agreement with the observation
Figure 3. Innate response activator (IRA) B cells promote the generation of T helper-1 (TH1) effector cells in atherosclerosis. A, Representative dot plots showing gating for CD3+ CD4+ CD44high CD62Llow T effector (Teff) cells and CD3+ CD4+ Foxp3+ regulatory T cells (Treg) in blood. B, Kinetics of Teff and Treg cell development in blood and spleen, and the proportion of Teff cells in para-aortic lymph nodes during 10 weeks of high-fat and -cholesterol diet (HCD) feeding of IRA B knockout (KO; white) and control (gray) mice. Results are presented as mean±SEM. *P≤0.05, **P≤0.01, IRA B KO vs control mice at 10 weeks; n≥6 per group. C, Representative dot plots showing gating for CD3+ CD4+ interferon-γ–positive (IFNγ+) T cells in blood. D, Quantification of IFNγ-producing T cells in blood, spleen, and para-aortic lymph nodes after 10 weeks of HCD feeding of IRA B KO (white) and control (gray) mice. Results are presented as mean±SEM. *P≤0.05; n≥10 per group. E, Kinetics of total IgG and IgM serum levels during 10 weeks of HCD feeding of IRA B KO (white) and control (gray) mice. Results are presented as mean±SEM; n≥6 per group and time point. F, Quantification of IgG2c antibody titers against malondialdehyde (MDA)–low-density lipoprotein (LDL) and copper-oxidized LDL (CuOxLDL) in 1:25 diluted individual serum samples (n≥10 per group). Results are presented as mean±SEM. *P≤0.05, **P≤0.01. G, Quantitative ratio of IgG2c and IgG1 titers against MDA-LDL and CuOxLDL in 1:25 diluted individual serum samples (n≥10 per group). Results are presented as mean±SEM-fold changes of the IgG2c:IgG1 ratio to illustrate shifts in isotype switching between control and IRA B KO mice. *P≤0.05, **P≤0.01.
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that GM-CSF does not affect the generation of splenic DCs in the steady state.33,34 During inflammation and with increased GM-CSF, DCs expand.33,35 Consequently, over the course of HCD consumption, control mice selectively increased the number of CD11b+ cDCs (Figure 4B). In contrast, IRA B
KO mice maintained their cDC numbers at steady-state lev-els in the spleen (Figure 4B) and lymph nodes (Figure 4C). Remarkably, not only did IRA B KO mice generate fewer cDCs, but these cDC also expressed less T
H1-priming IL-12p40
(Figure 4D). Although there were no differences in CD86
Figure 4. Innate response activator (IRA) B cells promote the generation of classic dendritic cells (cDCs) in atherosclerosis. A, Representative dot plots showing gating for CD19− MHCIIhigh CD11chigh cDCs, CD8− CD11b+, CD8+ CD11b−, and CD8+ CD103+ subsets in the spleen. B, Kinetics of the splenic cDC subset development during 10 weeks of feeding a high- and -cholesterol diet (HCD) of IRA B knockout (KO; white) and control (gray) mice. Results are presented as mean±SEM. *P≤0.05, **P≤0.01, IRA B KO vs control mice at 10 weeks (n≥6 per group). C, Identification and quantification of the proportion of cDCs in para-aortic lymph nodes. Results are presented as mean±SEM. *P≤0.05; n≥10 per group. D, Quantification of interleukin (IL)-12p40 expression in splenic cDCs sorted from IRA B KO (white) and control (gray) mice by real-time polymerase chain reaction (PCR). Results are presented as mean±SEM-fold change in 2∆Ct. *P≤0.05; n≥10 per group. E, Ldlr−/− mice were lethally irradiated, reconstituted with a 50:50 mixture of CD45.1+ wild-type (WT; black) and CD45.2+ Csf2rb−/− (white) bone marrow, and placed on and HCD for 3 months. F, Assessment of chimerism for CD45.1 (WT in black) and CD45.2 (Csf2rb−/− in white) in CD11b+ and CD8+ splenic cDCs. Results are presented as mean±SEM. *P≤0.05; n=5 per group. G, Quantification of IL-12p40 expression in sorted CD45.1 (WT in black) and CD45.2 (Csf2rb−/− in white) splenic cDCs by real-time PCR. Results are presented as mean±SEM-fold change in 2∆Ct. *P≤0.05; n=5 per group. H, Flow-assisted cell sorting of CD23low IgMhigh CD43high CD138high cells from WT and Csf2−/− mice after 4×25 mg/d lipopolysaccharide (LPS) IP. Representative dot plot showing enrichment for granulocyte macrophage colony-stimulating factor (GM-CSF)–positive IRA B cells in WT mice. Dashed lines represent isotype controls. I, Representative dot plot showing major histocompatibility complex class II (MHCII) and CD11c expression in lineage-depleted (Lin=CD3, CD90.2, CD19, B220, NK1.1, Ly6G) CD45.1+ bone marrow cells before in vitro culture. Dashed lines represent isotype controls. J, Representative dot plot showing high MHCII, CD11c, CD86, and CD40 expression on bone marrow–derived DCs (BMDCs) generated through coculture with IRA B cells and IL-4 over 8 days. Dashed lines represent isotype controls. K, Enumeration of MHCII+ CD11c+ BMDCs after coculture with medium alone (dark gray), medium plus IL-4 (black), IRA B cells and IL-4 (gray), or corresponding B cells from LPS-challenged GM-CSF−/− mice with IL-4 (white). Results are presented as mean±SEM. *P≤0.05, WT vs all other groups by ANOVA; n≥3 per group. L, Evaluation of DC morphology of BMDCs generated with IRA B cells or Csf2−/− B cells. Left, Representative phase-contrast microscopy images. Right, Quantification of cells with typical dendritiform protrusions per visual field. Results are presented as mean±SEM analyzed in 6 visual fields per well and group. *P≤0.05, **P≤0.01. M, CD4+ CD25− B6.Cg-Tg(TcraTcrb)425Cbn/J (OT-II) cells were cocultured with IRA B cell–generated BMDCs loaded with ovalbumin (OVA; 100 μg/mL) or BSA (100 μg/mL) for 4 days. Representative histograms show cell divisions of OT-II T cells labeled with a cell tracer dye.
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Hilgendorf et al IRA B Cells Aggravate Atherosclerosis 1683
and CD40 expression, MHCII decreased slightly on CD11b+ cDCs in IRA B KO mice (Figure VA in the online-only Data Supplement). IRA B-cell deficiency affected neither the gen-eration of granulocyte-macrophage and common dendritic cell progenitors in the bone marrow nor the number of pre-DCs in the bone marrow and spleen, which argues against IRA B cell–dependent mobilization and expansion of DC progenitors (Figure VB in the online-only Data Supplement).
To determine whether the changes in cDC subset and func-tion depended on the direct interaction of GM-CSF with cDCs, we reconstituted lethally irradiated Ldlr−/− mice with a 50:50 mixture of bone marrow cells from CD45.1+ WT mice and CD45.2+ mice deficient in the common β chain of the GM-CSF-receptor (Csf2rb−/−) and placed them on an HCD for 3 months (Figure 4E). Whereas the CD45.1/CD45.2 splenic cDC chimerism was ≈50:50 among the CD8+ subset, chimerism was skewed toward the CD45.1+ WT cells (60:40) among CD11b+ cDCs, suggesting that Csf2rb−/− cells were impaired in generating CD11b+ cDCs (Figure 4F). This obser-vation agrees with an earlier report that exogenous GM-CSF administration augmented the number of CD11b+ but not CD8+ cDCs in the spleen.36 Moreover, IL-12p40 expres-sion was lower in sorted Csf2rb−/− cDCs compared with WT cDCs (Figure 4G), thereby reproducing the main effects we observed in IRA B KO mice and suggesting that IRA B cells influence cDCs directly via GM-CSF.
Because DCs can differentiate from bone marrow precur-sors through culture with recombinant GM-CSF and IL-4, we wondered whether IRA B cells can act as GM-CSF sources capable of generating functionally active DC. We sorted IRA B cells from lipopolysaccharide-stimulated WT mice (Figure 4H) and placed them in culture with lineage-depleted (ie, enriched for hematopoietic stem and progenitor cells) bone marrow cells and IL-4 (Figure 4I). After 8 days, MHCII+ CD11c+ CD40+ CD86+ DCs appeared (Figure 4J). As controls, we cultured bone marrow cells with IL-4 alone or with B cells from Csf2−/− mice plus IL-4 and enumerated fewer DCs (Figure 4K). The group cultured with IRA B cells yielded more cells with characteristic DC morphology (Figure 4L), thus complementing the surface marker characteristics. To determine the functionality of IRA B cell–generated DCs, we pulsed them with ovalbumin and cocultured with ovalbumin- specific transgenic OT-II CD4+ cells that had been labeled with a tracer. In the absence of ovalbumin, OT-II cells did not proliferate, but when ovalbumin was added, T cells prolifer-ated robustly, as determined by the progressive loss of their tracer dye (Figure 4M). Likewise, IRA B cells sorted from spleens of atherosclerotic Ldlr−/− mice generated functional bone marrow–derived DCs capable of processing ovalbumin for effective antigen presentation and OT-II cell proliferation (Figure VC and VD in the online-only Data Supplement). These experiments indicate that IRA B cells indeed stimulate the generation of mature DCs, which can promote antigen-specific T-cell expansion.
Transfer of GM-CSF–Competent B Cells Aggravates AtherosclerosisIf lesions are smaller in the absence of IRA B cells, could the adoptive transfer of GM-CSF–competent B cells into IRA B
KO mice give rise to IRA B cells and reverse the phenotype by promoting IFNγ-producing T
H1 cells and atherogenesis?
To test this conjecture, we adoptively transferred WT B cells (ie, B cells capable of producing GM-CSF) and Csf2−/− B cells into IRA B KO mice on an HCD twice, 4 weeks apart (Figure 5A). To establish whether IRA B cells develop in recipient animals, we transferred CD45.1+ WT B cells into CD45.2+ IRA B KO mice and profiled their phenotype after 8 weeks of an HCD. Remarkably, a population of CD45.1+ GM-CSF+ IRA B cells appeared in the spleen (Figure 5B), thus allowing us to determine the impact of IRA B-cell deliv-ery on the development of atherosclerosis. The transfer of WT but not Csf2−/− B cells increased GM-CSF production in the spleen by >50% and gave rise to a larger number of splenic cDCs and blood effector T cells, including IFNγ- producing T
H1 cells (Figure 5C and 5D). Moreover, we found
augmented expression of the TH1
transcription factor Tbet and T
H1-associated cytokine IFNγ in atherosclerotic lesions
of mice receiving WT but not Csf2−/− B cells or vehicle only (Figure 5E). However, expression of the T
-associated RORγt and IL-17 remained unaffected (Figure 5F and 5G). Quantification of lesion size and mor-phology, as determined by Oil Red O, Mac3, CD4, smooth muscle actin, and Masson trichrome staining, correlated these findings with those reported in Figure 2: Mice receiving WT B cells had larger lesions with more macrophages and T cells compared with mice receiving Csf2−/− or no B cells (Figure 5H–5L). Together, these data indicate that IRA B cells aggravate atherosclerosis by stimulating DC production and shifting the host response toward T
H1-associated immu-
nity (Figure 6).
DiscussionDCs are professional antigen-presenting cells that, in the context of secondary signals such as IL-12, can generate IFNγ-producing T
H1 cells capable of activating macrophages
and promoting isotype switching in B cells. In atherosclero-sis, T
H1-type immunity promotes disease, but the orchestrat-
ing pathways remain poorly understood.37 This study reveals that IRA B cells can shape immunity in atherosclerosis. By expressing GM-CSF in microenvironments that support the production of mature DCs, IRA B cells operate in a strategic location and deliver a potent signal that instructs the host to mount an adaptive-like immune response.
In murine disease models that depend on antigen sensiti-zation and challenge such as rheumatoid arthritis, multiple sclerosis, and myocarditis, GM-CSF deficiency ameliorates disease,38−41 suggesting a major role for the growth factor in DC-mediated generation of adaptive immunity. Our data expand on these observations by showing that, in athero-sclerosis, IRA B cells are major sources of DC-promoting GM-CSF. In the steady state when IRA B cells are exceed-ingly rare or when they are absent altogether, splenic DCs develop normally, indicating that cellular sources other than IRA B cells maintain this population. In response to danger, however, mobilization of IRA B cells stimulates the devel-opmental expansion of mature CD11b+ cDCs, a subset with
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a dependence on GM-CSF in atherosclerosis that was previ-ously unrecognized. These changes translate to the increase in IFNγ-producing T cells and oxidized low-density lipoprotein–specific and T
H1-dependent IgG2c antibodies.
Assessing the role of B cells in atherosclerosis is complex, in part because of the difficulty in separating the intrinsic biological effects of B cells from the effects of the antibod-ies they secrete and because of the increasing evidence for a variety of functionally distinct B-cell subsets.42 Considerable data support an atheroprotective role of B1 cells, particularly B1a cells, which are believed to protect against atherosclerosis
by secreting natural oxidized lipoprotein–scavenging IgM antibodies. Controversy surrounds the role of B2 cells, how-ever, which are the main producers of adaptive IgG antibodies. Clinically, high antioxidized low-density lipoprotein IgG lev-els in cardiovascular patients positively correlate with disease burden.43,44 But how do they function? On the one hand, IgG- mediated antigen scavenging may provide protection, similar to natural IgM antibodies. On the other hand, IgG isotypes bind variably to different Fc receptors, which can either activate or inhibit target cells such as macrophages, regardless of antigen binding. Signaling via different Fc receptors can thus have
Figure 5. Transfer of granulocyte macrophage colony-stimulating factor (GM-CSF)–competent B cells aggravates atherosclerosis. A, Experimental strategy for B-cell adoptive transfer. Naive innate response activator (IRA) B knockout (KO) mice were divided into 3 groups receiving either 2.5×107 CD19+ B cells from wild-type (WT; gray) or Csf2−/− (white) mice (n=7 per group) or vehicle (Dulbecco PBS) alone (black; n=5) at weeks 0 and 4 of a 8-week period of high-fat and -cholesterol diet (HCD) feeding. B, Representative dot plots showing identification of IRA B cells in the spleen of a CD45.2+ IRA B KO recipient on an HCD 8 weeks after transfer of 25×106 CD45.1+ WT B cells twice, 4 weeks apart. C, Quantification of GM-CSF (Csf2) expression in whole-spleen tissue of IRA B KO mice 8 weeks after transfer of WT (gray), Csf2−/− (white; n=7 per group), or no B cells (black; n=5) by real-time polymerase chain reaction (PCR). Results are presented as mean±SEM-fold change in 2∆Ct. *P≤0.05, WT vs the other groups by ANOVA. D, Enumeration of spleen classic dendritic cells (cDCs), blood T effector cells, and blood interferon-γ (IFNγ)–producing T cells in recipients of WT (gray) cells compared with those receiving Csf2−/− (white) B cells (n=7 per group) or vehicle (black; n=5) after 8 weeks of HCD feeding. Results are presented as mean±SEM. *P≤0.05, WT vs the other groups by ANOVA. E through G, Quantification of T helper-1 (TH1)–associated Tbet and IFNγ, Treg-associated Foxp3, transforming growth factor-β1 (TGFβ1) and interleukin (IL)-10, and TH2- and TH17-associated GATA3, IL-4, RORγT, and IL-17 expression in aortic tissue of WT (gray) vs Csf2−/− (white) B-cell recipients (n=7 per group) and vehicle group (black; n=5) by real-time PCR. Results are presented as mean±SEM-fold change in 2∆Ct. *P≤0.05, WT vs the other groups by ANOVA. H, Quantification of Oil Red O (ORO)–rich areas in aortic root sections of recipients of WT (gray) vs Csf2−/− (white) B cells (n=7 per group) and vehicle group (black; n=5) on the right and representative images on the left. Results are presented as mean±SEM. *P≤0.05, WT vs the other groups by ANOVA. I through L, Representative images and quantification of Mac3-, CD4-, and smooth muscle actin (SMA)–positive and Masson trichrome staining in aortic root sections of the 3 groups. Results are presented as mean±SEM. *P≤0.05, WT vs the other groups by ANOVA.
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Hilgendorf et al IRA B Cells Aggravate Atherosclerosis 1685
opposing effects on atherosclerosis, as studies in mice deficient for either the activating Fcγ receptors or the inhibitory Fcγ receptor IIb have shown.42 Adding to the complexity, IgG sub-classes exhibit different activation-to-inhibition ratios.45 IRA B cell–dependent production of the IgG2c isotype, which has the highest ratio, may therefore be consistent with the observation that IRA B cells aggravate atherosclerosis. Beyond antibod-ies, B cells are also sources of cytokines and chemokines such as regulatory IL-106 and monocyte-mobilizing Monocyte che-motactic protein-3/Chemokine ligand 7.46 Although the role of these subsets in atherosclerosis is still unknown, our study on another class of mediator, a growth factor, reveals that B cells can aggravate atherosclerosis by generating T
H1-priming cDCs.
One somewhat puzzling observation is that a B cell should be a major source of GM-CSF in the first place. After all, B cells participate fundamentally in humoral immunity, so it may be counterintuitive that B cells should also act in the generation of DCs, the cells specialized in the afferent limb of the T-cell response. Yet, IRA B cells may be ideally suited for a senti-nel role in adaptive immunity. The spleen screens blood for pathogens, and B cells are the most numerous occupants in the spleen. B cells physically interact with DCs in the spleen to ini-tiate T cell–independent immunity.47 Moreover, recent studies have shown that signaling via Myd88 in B cells is important to DC function in lupus and T
H1 priming.48,49 The finding that IRA
B cells require signaling via Myd88 raises the possibility that the link between Myd88 signaling in B cells and DC function in lupus likewise involves B cell–derived GM-CSF. Strategically located and equipped with a plethora of receptors capable of recognizing molecular patterns, IRA B cells may indeed repre-sent a cellular node that bridges innate and adaptive immunity.
The function of the pleiotropic cytokine GM-CSF depends on concentration, location, and timing of expression. Although
it is remarkable that a small population of B cells secreting GM-CSF elicited a significant difference in lesion size, Oil Red O area, and macrophage and T-cell content, it should be noted that other leukocytes, including macrophages and T cells, can also produce GM-CSF.21,50 Therefore, attention to the cellular source may be critical to understanding the pleiotro-pic behavior of a cytokine. This study focused on the interplay of GM-CSF–producing IRA B cells with DCs partly because IRA B cells selectively increased in secondary lymphoid organs where DC reside. Even though we did not observe effects of IRA B cells on monocytosis and neutrophilia, IRA B cells could elicit other effects on myeloid cells as disease progresses. Similarly, the absence of IRA B cells in the aorta in our setting does not preclude their accumulation and local influence in more advanced disease. Future studies will need to determine how alternative cellular sources of GM-CSF at various stages of disease influence atherosclerosis.
Statins have proven benefit in reducing cardiovascular events in individuals in broad categories of risk, in part through direct anti-inflammatory actions. Yet, despite treat-ment with the best available therapeutics, a considerable burden of residual events threatens individuals prone to complications of atherosclerosis. This challenge has ener-gized efforts at targeting inflammation. Understanding the complex, redundant, and interlinked networks of innate and adaptive immunity implicated in atherogenesis is essential to the development of effective but nuanced immune-targeting approaches. An integrated, systems-wide model that charts how the immune system recognizes harmful atherosclero-sis-promoting molecular patterns, how it incorporates and propagates this information, and how it ultimately affects disease should aid the development of specific and finely tuned treatments. The function of IRA B cells described here illuminates a previously unknown regulatory node operating in atherosclerosis and is worthy of consideration as a candi-date for therapeutic intervention.
Acknowledgments We thank Michael Waring and Adam Chicoine (Harvard Medical School) for sorting cells. We also thank Eugenia Shvartz, Thibaut Quillard (Brigham and Women’s Hospital), and Benjamin G. Chousterman (Massachusetts General Hospital) for technical sup-port, as well as thank Andrew H. Lichtman (Brigham and Women’s Hospital) for fruitful discussion.
Sources of FundingThis work was supported in part by National Institutes of Health grants 1R01HL095612 (to Dr Swirski), P01 HL 088093 (to Dr Witztum), and R01HL080472 (to Dr Libby) and Canadian Institutes of Health Research grant MOP 126205 (to Dr Rubin). Dr Hilgendorf was supported by the German Research Foundation and the Society of Thrombosis and Haemostasis Research. Dr Theurl was supported by the Max Kade Foundation. L.M.S. Gerhardt was supported by the Boehringer Ingelheim Funds. Dr Weber was supported by the German Research Foundation. Dr Robbins was supported by an American Heart Association postdoctoral fellowship and the Massachusetts General Hospital Executive Committee on Research Postdoctoral Award.
DisclosuresNone.
Figure 6. Model of innate response activator (IRA) B cell–dependent T helper-1 (TH1) skewing during atherosclerosis. During atherosclerosis, IRA B cells arise in secondary lymphoid organs via Myd88-dependent signaling and promote the generation of interleukin (IL)-12–producing classical dendritic cells (cDCs). CD4+ T helper cells that recognize disease-related antigens (ie, possibly oxidation-specific epitopes) presented by these cDCs differentiate into interferon-γ (IFNγ)–producing TH1 cells. TH1 cells infiltrate atherosclerotic lesions and stimulate macrophages. Antigen-specific interaction between TH1 cells and B cells leads to IFNγ-dependent isotype switching from IgG1 to IgG2a/c, which carry the highest Fcγ receptor–mediated activation capacity. By instructing TH1-priming cDCs, IRA B cells aid in bridging innate and adaptive immunity. Solid arrows depict functional relationship; dashed arrows, spatial relationship. TLR indicates Toll-like receptor.
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CLINICAL PERSPECTIVEAtherosclerosis is a lipid-driven inflammatory disease, yet treatment regimens currently lack genuinely anti-inflammatory approaches. The growth of human and mouse atherosclerotic lesions is characterized by the influx of functionally diverse leukocytes to the vessel wall. Because leukocytosis is a risk factor for complications of atherosclerosis in humans, under-standing how leukocytes affect the course of disease is important and may lead to new strategies that either augment or attenuate particular leukocyte function. This study shows that a previously unknown leukocyte population plays an impor-tant role in atherosclerosis. Innate response activator B cells develop in lymphoid organs in humans with atherosclerosis at high numbers and in the 2 major mouse models. Functionally, innate response activator B cells aggravate atherosclerosis by shaping Th1-type adaptive immunity. The study identifies an upstream node of leukocyte communication and suggests that targeting innate response activator B cell function might be a strategy for the treatment of cardiovascular disease.
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Overarching aims and description of the study. Aim 1: To quantify IRA B cells in two mouse models of atherosclerosis and in human atherosclerosis. These data are shown in Figure 1 and Supplemental Figure 1. Aim 2: To determine whether IRA B cells are important in mouse atherosclerosis and to profile key features such as lesion size, cell accumulation, etc. To achieve this, mixed chimeric mice were generated (described below). These data are shown in Figure 2 and Supplemental Figures 2 and 3. Aim 3: To profile the observed differences in T cells more rigorously. These data are shown in Figure 3 and Supplemental Figure 4. Aim 4: To link IRA B cells with the observed differences mechanistically. These data are shown in Figure 4 and Supplemental Figure 5 describing the interaction between IRA B cells and Dendritic cells. Aim 5: To rescue the phenotype and thus link IRA B cells to the phenotype functionally. These data are shown in Figure 5. Overall, the study used 235 mice. After generation of mixed chimeric mice, the blood leukocyte profile was analyzed and only animals that were reconstituted successfully were placed in the study. Typically ~93% of mixed chimeras were reconstituted and used in the study. The number of mice used in each experiment is indicated in the Figure legends. All mice admitted to the study survived the intended duration of the study.
Supplemental MethodsAnimal models and in vivo interventions. Mixed bone marrow chimeras: 8 weeks old male Ldlr–/– mice were lethally irradiated (950cGy) and reconstituted with a 50:50 mixture of Csf2–/– with WT (Controls) and µMT bone marrow cells (IRA B KO), respectively. In analogy male Ldlr–/– were reconstituted with a 50:50 mixture of CD45.1+ and Myd88–/– or Csf2rb–/– bone marrow. To induce atherosclerosis Ldlr–/– mice consumed a high-fat/cholesterol diet (HCD; D12108C, Research Diets, New Brunswick, NJ, USA) and Apoe–/– were placed on a Western diet (TD.88137, Harlan Teklad, Indianapolis, IN, USA) at 8 weeks of age for different time periods as indicated in the text. Bone marrow chimeras were switched to HCD 6 weeks after reconstitution. Adoptive transfer: B cells from WT and Csf2–/– mice were isolated by magnetic cell separation. Pooled cell suspensions from spleens and peritoneal lavages were incubated with 4 µl anti-CD19-PE Ab (Biolegend, San Diego, CA, USA) per 1 x 108 cells in sterile 2% FBS (fetal bovine serum, Atlanta Biologicals, Lawrenceville, GA, USA), 0.5% BSA (bovine serum albumin, MP Biomedicals, Solon, OH, USA) in PBS for 30 min on ice, washed and incubated with 100 µl anti-PE MACS beads (Miltenyi Biotec, Auburn, CA, USA) per 1 x 108 cells in 0.5% BSA, 2mM EDTA in PBS for another 30 min on ice. Labeled cells were positively selected in a Midi MACS separator and LS column according to the manufacturer’s instructions. Cells were manually counted in a Neubauer chamber, a purity of 93% and viability of over 95% were confirmed by flow cytometric analysis and Trypan blue staining, respectively. At 6 weeks after reconstitution IRA B KO mice received either 25 x 106 B cells/mouse from WT or Csf2–/– mice by tail vein injection. Mice were placed on HCD and received another 25 x 106 B cells/mouse after 4 weeks. Mice were sacrificed after 8 weeks on HCD. When 25 x 106 CD45.1+ CD19+ cells were transferred, twice, 4 weeks apart into CD45.2+ IRA B KO mice, 66,542 ± 24,556 CD45.1+ IRA B cells were still retrieved after 8 weeks (n = 3, mean ± SEM).
Cell isolation. Peripheral blood was collected by retroorbital bleeding with heparinized capillaries, and erythrocytes were lysed in RBC Lysis buffer (Biolegend). Peritoneal lavages and organs were harvested at day of sacrifice. Spleens, femurs, aortas and paraaortic lymph nodes (1 proximal, 1 abdominal, 2 distal lymph nodes at aortic bifurcation per mouse) were excised after vascular perfusion with 10 ml sterile PBS. Minced spleens and flushed bone marrow were strained through a 40 µm-nylon mesh (BD Biosciences, San Jose, CA, USA). Spleen cell suspensions were further subjected to RBC lysis. Aortas were minced and digested in 450 U/ml collagenase I, 125 U/ml collagenase XI, 60 U/ml DNase I and 60 U/ml hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) PBS for 1 h at 37°C while shaking. Lymph nodes were minced and digested in 168 U/ml collagenase III (Worthington Biochemicals, Lakewood, NJ, USA), 60 U/ml DNase I (Sigma-Aldrich), 2% FBS RPMI-1640 (Cellgro, Manassas, VA, USA) for 1h at 37°C while shaking. Cells were counted in a Neubauer chamber. One femur contains ∼5% of all bone marrow cells 1. Bone marrow cell counts were extrapolated accordingly. Cell sorting: IRA B cells were expanded in vivo by 4 daily intraperitoneal injections of 25mg LPS per mouse or by 3 months of HCD in
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Ldlr–/– mice. Spleens from LPS stimulated WT and Csf2–/– mice and from atherosclerotic Ldlr–/– mice were homogenized and incubated with 4 µl anti-CD138-Biotin Ab (clone 281-2, BD Biosciences) per 1 x 108 cells in sterile 2% FBS, 0.5% BSA in PBS for 30 min on ice, followed by incubation with 100 µl anti-Biotin MACS beads (Miltenyi Biotec) per 1 x 108 cells in 0.5% BSA, 2mM EDTA in PBS for another 30 min on ice. Cells were positively selected with a Midi MACS separator and LS column and stained for anti-IgM-Fitc (BD Biosciences), anti-CD45.2-PerCp-Cy5.5 (BD Biosciences), anti-CD23-PECy7 (Biolegend), anti-CD43-APC (BD Biosciences), Streptavidin-Alexa Fluor 700 (Life Technologies, Carlsbad, CA, USA), anti-CD19-APCCy7 (Biolegend). CD45.2+, CD23low, IgMhigh, CD43+, CD138high cells were sorted on a FACS Aria II cell sorter (BD Biosciences). Splenocytes from OT-II mice were stained with anti-CD45.2-Fitc (BD Biosciences), anti-CD4-PE (BD Biosciences), anti-CD25-APC (BD Biosciences), and transgenic CD4+ CD25– T cells were sorted on a FACS Aria II cell sorter. Splenocytes from IRA B KO and controls and from CD45.1+ C57Bl/6J mice were incubated with 4 µl anti-CD19-Biotin Ab (BD Biosciences) and anti-CD11b-Biotin (BD Biosciences) per 1 x 108 cells in sterile 2% FBS, 0.5% BSA in PBS for 30 min on ice, followed by incubation with 100 µl anti-Biotin MACS beads (Miltenyi Biotec) per 1 x 108 cells in 0.5% BSA, 2mM EDTA in PBS for another 30 min on ice. Cells were negatively selected with a Midi MACS separator and LS column enriching for T cells and stained for anti-CD45.2-Fitc (BD Biosciences), anti-CD4-PE (BD Biosciences), anti-CD25-APC (BD Biosciences), anti-CD45.1-Alexa700 (Biolegend). Regulatory T cells (Treg) from IRA B KO and control mice were sorted as CD45.2+ CD4+ CD25+ cells, while conventional T cells (Tconv) were sorted as CD45.1+ CD4+ CD25– cells on a FACS Aria II cell sorter (BD Biosciences). TCRβ– B220– MHCIIhigh, CD11chigh cDC were sorted from splenocytes directly into RLT buffer for subsequent RNA isolation.
Serum analysis. Cholesterol measurement: Serum was collected after overnight fasting. Total cholesterol levels were measured with the Cholesterol E colorimetric assay (Wako Chemicals, Richmond, VA, USA) in a Safire2 microplate reader (Tecan, Maennedorf, Switzerland) according to the manufacturer’s instructions. VLDL, LDL and HDL cholesterol were determined by Skylight Biotech (Skylight Biotech, Inc., Japan) Immunoglobulin (Ig) measurement: Total serum IgG and IgM were measured by ELISA (Bethyl Laboratories, Montgomery, TX, USA) according to the manufacturer’s instructions. Isotype- and antigen-specific antibody titers were determined by chemiluminescent enzyme immunoassays as previously described 2. In brief, antigens were coated at 5 µg/mL PBS overnight at 4°C (IgG and IgG2c (goat anti-ms-IgG (Pierce 31160)), IgG1 (Rat anti-ms-IgG1 (BD 553445)), AB-12, CuOxLDL, MDA-LDL). The plates were blocked with 1% BSA in TBS, serially diluted antisera from individual mice were added, and the plates incubated for 1.5h at room temperature. Bound plasma immunoglobulin (Ig) isotype levels were detected with various anti-mouse Ig isotype-specific alkaline phosphatase (AP) conjugates (Abcam) using LumiPhos 530 (Lumigen, Southfield, MI, USA) solution, and a Dynex Luminometer (Dynex Technologies, Chantilly, VA, USA). Data are expressed as relative light units counted per 100 milliseconds (RLU/100 ms). GM-CSF ELISA: GM-CSF was measured in undiluted serum with the Mouse GM-CSF Quantikine ELISA Kit (assay range 7.8-500pg/ml) according to the manufacturer’s instructions.
Histology. Spleens were embedded in Tissue-Tek O.C.T compound (Sakura Finetek, Torrance, CA, USA), frozen in ice-cold 2-Methylbutane (Fisher Scientific, Fair Lawn, NJ, USA) and sectioned into 6 µm slices yielding 30-40 sections per mouse. The following antibodies were used for immunofluorescence staining: Anti-GM-CSF (clone MP1-31G6; Abcam, Cambridge, MA, USA), secondary biotinylated anti-rat IgG (Vector Laboratories, Burlingame, CA, USA), streptavidin-Alexa Flour 594 (Life technologies), and FITC anti-IgM (clone II/41; BD Biosciences). Images were recorded using a BX63 motorized microscope (Olympus, Center Valley, PA, USA). Murine aortic roots were embedded in Tissue-Tek O.C.T compound, frozen and sectioned into 5 µm slices yielding 30-40 sections per root. Sections that capture the maximum lesion area were used to compare lesion sizes between study groups. Adjacent sections were used for additional immunohistochemical staining. Following stains were performed to assess lesions size and composition: Hematoxylin and eosin (H&E), Oil-Red-O (ORO; Sigma-Aldrich) for lipids, anti-Mac3 (clone M3/84; BD Biosciences) for macrophages, anti-α-smooth muscle actin (ab5694; Abcam) for smooth muscle cells (SMA), Masson’s Trichrome staining for collagen (Masson), anti-CD4 (clone RM4-5; BD Biosciences) for T helper cells. Biotinylated secondary antibodies
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and avidin-complex were used, and all sections counterstained with hematoxylin. Images were recorded using a Nanozoomer 2.0RS (Hamamatsu Photonics, Hamamatsu City, Japan). En-face ORO staining was performed on pinned aortas after fixation in 10% formalin. Human spleen samples were obtained from surgical specimens and autopsy at the Department of Pathology, Toronto General Hospital, Toronto, ON, Canada. All immunohistochemistry studies on human patients samples were approved by the research ethics board at University Health Network. Spleens from 4 patients without a history or signs of cardiovascular disease upon examination were compared to those from 4 patients with symptomatic cardiovascular disease. Samples were fixed in 10% formalin and embedded in paraffin for histologic sectioning (4 µm thick slices). Following dewaxing and heat-induced antigen retrieval sections were blocked with donkey serum for 10min and stained with primary antibodies rabbit anti-human GM-CSF (bs-3790R, Bioss Inc. MA, USA) and goat anti-human IgM (NB7436, Novus Biologicals, CO, USA) over night. Donkey anti-rabbit Cy3 and donkey anti-goat Cy5 were used as secondary antibodies (Millipore, Billerica, MA, USA) for immunohistochemical staining. Images were recorded with an Olympus Fluo View 1000 confocal laser scanning microscope (Olympus, Tokoyo, Japan).
Flow Cytometry. Cell suspensions were stained in PBS supplemented with sterile 2% FBS and 0.5% BSA. The following monoclonal antibodies were used for flow cytometric analysis:anti-Ly6C (clone AL-21, BD Biosciences), anti-CD34 (clone RAM34, BD Biosciences), anti-IgM (clone II/41, BD Biosciences), anti-CD45.1 (clone A20, Biolegend), anti-CD45.2 (clone 104, BD Biosciences), anti-CD86 (clone GL1, BD Biosciences), anti-CD3e (clone 145-2C11, ebioscience), anti-CD4 (clone GK1.5, Biolegend) , anti-CD8 (clone 53-6.7, BD Biosciences), anti-CD90.2 (clone 53-2.1, BD Biosciences), anti-TCRβ (clone H57-597, Biolegend), anti-CD19 (clone 6D5, Biolegend), anti-B220 (clone RA3-6B2, BD Biosciences), anti-CD25 (clone PC61, BD Biosciences), anti-MHCII (clone AF6-120.1, BD Biosciences), anti-F4/80 (clone BM8, Biolegend), anti-CD49b (clone DX5, BD Biosciences), anti-NK1.1 (clone PK136, BD Biosciences), anti-Ly6G (clone 1A8, BD Biosciences), anti-Gr-1 (clone RB6-8C5, ebioscience), anti-CD11b (clone M1/70, BD Biosciences), anti-CD11c (clone HL3, BD Biosciences), anti-CD115 (clone AFS98, ebioscience), anti-CD21 (clone 7E6, Biolegend), anti-CD23 (clone B3B4, Biolegend), anti-CD40 (clone 3/23, Biolegend), anti-CD43 (clone S7, BD Biosciences), anti-CD93(clone AA4.1, BD Biosciences), anti-CD49d/VLA4(R1-2, BD Biosciences), anti-CD44 (clone IM7, Biolegend), anti-CD117/ckit (clone 2B8, BD Biosciences), anti-Ly6A/Sca1 (clone D7, ebioscience) , anti-CD127/ILRα (clone A7R34, ebioscience), anti-CD138 (clone 281-2, BD Biosciences), anti-CD103 (clone 2E7, Biolegend), anti-Ter119 (clone Ter-119, BD Biosciences), anti-GM-CSF (MP1-22E9, BD Biosciences), anti-Foxp3 (clone FJK-16s, ebioscience), anti-IFNγ (clone XMG1.2, BD Biosciences). For intracellular staining cells were fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) according to the manufacturer’s instructions. For intracellular staining of IFNγ, IL-4, and IL-17, cell suspensions were stimulated in 2% FBS RPMI-1640 medium with 20 ng/ml PMA and 1 µg/ml ionomycin (Sigma-Aldrich) in the presence of GolgiStop and GolgiPlug (BD Biosciences) for 3 hours at 37°C, 5% CO2 prior to fixation and permeabilization. For proliferation assays, target cells were labeled with Cell Tracer Violet (Life Technologies) and live cells were identified with Fixable Viability Dye eFluor 780 (ebioscience). Data were acquired on a LSRII and analyzed with FlowJo (Tree Star, Ashland, OR, USA). Specifically, monocytes were identified as CD45+, Lin1– (Lin1 = Ter119, CD3, CD90.2, CD19, B220, NK1.1, CD49b, Ly6G), CD11b+, MHCIIlow, CD11clow, CD115+ cells, subdivided into Ly6Chigh and Ly6Clow cells. Neutrophils were identified as CD45+, Lin1+, CD11b+, MHCIIlow, CD11clow, SSChigh, Ly6Cint cells. Unless otherwise noted, B cells were identified as CD45+, CD19+ cells. CD4+ and CD8+ T cells were identified within the CD45+, CD19– CD3+ population. Classical Dendritic cells (DC) were identified as CD45+, CD19–, MHCIIhigh, CD11chigh cells. PreDC were identified as CD45+, Lin1–, F4/80–, CD11b–, CD115–, MHCIIlow, CD11c+ cells. Lin2– (Lin2 = Ter119, CD3, CD90.2, CD19, B220, NK1.1, CD49b, Gr-1, CD11b, CD11c, IL7Rα), ckit+, Sca1–, CD34high, CD16/32high myeloid progenitors were identified as GMP when CD115– and as ckithigh MDP and ckitlow CDP when CD115+.
Reverse transcription PCR. Cells: 1 x 105 sorted CD3+, CD19+, CD11b+ splenic cells were lysed in RLT buffer with 1% β-mercaptoethanol. RNA was isolated with the RNeasy Micro Kit (Qiagen, Venlo, Netherlands) followed by cDNA transcription with the iScript Select cDNA Synthesis Kit (Bio Rad,
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Hercules, CA, USA) according to the manufacturers’ instructions. Semi-quantitative PCR for GM-CSF (csf2, primers 5’-TCAAAGAAGCCCTGAACCTCC-3’ and 5’-AATATCTTCAGGCGGGTCTGC-3’) and housekeeping gene rpl19 (primers: 5’-AGGCATATGGGCATAGGGAAG-3’ and 5’-TTGACCTTCAGGTACAGGCTGT-3’) was performed in a 7300 PCR thermal cycler (Applied Biosystems, Carlsbad, CA, USA). PCR products were loaded on a 2% agarose gel (Lonza, Basel, Switzerland). Tissue: Snap-frozen aortas were homogenized in QIAzol (Qiagen, Venlo, Netherlands) and spleens were homogenized in RLT buffer with 1% β-mercaptoethanol followed by RNA extraction with the RNeasy Mini Kit (Qiagen, Venlo, Netherlands). Quantitative real-time TaqMan PCR for Tbet/Tbx21 (Mm00450960_m1; Applied Biosystems), Foxp3 (Mm00475162_m1; Applied Biosystems), GATA3 (Mm00484683_m1; Applied Biosystems), RORγt (Mm01261022_m1; Applied Biosystems), IFNγ (Mm01168134_m1; Applied Biosystems), IL-4 (Mm00445259_m1; Applied Biosystems), IL-10 (Mm00439614_m1; Applied Biosystems), IL-12b (Mm00434174_m1; Applied Biosystems), IL-17a (Mm00439618_m1; Applied Biosystems), TGFβ1 (Mm01178820_m1; Applied Biosystems), Csf2 (Mm01290062_m1; Applied Biosystems) and housekeeping gene β-actin (4352341E; Applied Biosystems) was run on a 7500 PCR thermal cycler (Applied Biosystems).
Cell culture. Bone marrow cells from CD45.1+ mice were incubated with anti-CD3-Biotin (clone 145-2C11, Biolegend), anti-CD90.2-Biotin (clone 53-2.1, BD Biosciences), anti-CD19-Biotin (clone 6D5, Biolegend), anti-B220-Biotin (clone RA3-6B2, BD Biosciences), anti-NK1.1-Biotin (clone PK136, BD Biosciences), anti-Ly6G-Biotin (clone 1A8, Biolegend) Ab at 4 µl/1 x 108 cells followed by incubation with 100 µl anti-Biotin MACS beads (Miltenyi Biotec) per 1 x 108 cells. After passing through a Midi MACS separator and LS column negatively selected cells were counted in the lineage depleted flow-through. 7 x 105 Lin– bone marrow cells were cultured in 1 ml RPMI-1640 supplemented with 10% FBS, 25 mM HEPES, 2mM L- glutamine, 50 µM β-mercaptoethanol, 100 U/ml penicillin, 100 U/ml streptomycin (complete medium) in a 24 well plate (Cellgro). 7 x105 LPS induced IRA B cells and corresponding Csf2–/– B cells, respectively, were added to the culture together with 5000 U/ml murine IL-4 (Peprotech, Rocky Hill, NJ, USA) at day 0 and day 5 with replacing medium. Adherent cells were harvested after 8 days of culture, counted and stained for Dendritic cell (DC) marker expression with flow cytometric antibodies. In analogy 1 x105 IRA B cells (IgMhigh, CD23low, CD43high, CD138high) isolated from atherosclerotic Ldlr–/– mice (3 months on HCD) were co-culture with 1 x105 Lin– bone marrow cells in a flat-bottom 96-well plate. For T cell proliferation assays 1 x 104 WT IRA B cell generated bone marrow derived DC (BMDC) were transferred into U-shaped wells of a 96-well plate and loaded with 100 µg/ml ovalbumin (OVA; Sigma-Aldrich A7641) or 100 µg/ml BSA in a final volume of 200 µl/well. Ldlr–/– IRA B cell generated BMDC were kept in the flat-bottom 96-well plate and loaded with 100 µg/ml OVA or BSA. Sorted CD4+ CD25– T cells from OT-II mice were stained with Cell Tracer Violet (Life Technologies) according to the manufacturer’s instructions. Thereafter 5 x 104 OT-II CD4 T cells were added to each well and harvested after 4 days of co-culturing for flow cytometric assessment of proliferation cycles. For Treg suppression assay C57Bl/6J splenocytes were incubated with anti-CD90.2-Biotin Ab (clone 53-2.1, BD Bioscience) at 4 µl/1 x 108 cells followed by incubation with 100 µl anti-Biotin MACS beads (Miltenyi Biotec) per 1 x 108 cells. After passing through a Midi MACS separator and LS column negatively selected cells were counted in the flow-through and resuspended in complete medium. T cell-depleted splenocytes were transferred to a U-bottom 96-well plate (3 x 105 cells/well) and irradiated with 30Gy prior to loading with 1 µg/ml anti-CD3e (clone 145-2C11, ebioscience) where indicated. Sorted CD45.1+ CD4+ CD25– Tconv cells were labeled with Cell Tracer Violet (Life Technologies) according to the manufacturer’s instructions and 3 x 104 Tconv cells were added to 3 x 105
irradiated T cell depleted splenocytes in a total volume of 200µl/well. Sorted CD45.2+ CD4+ CD25+ Treg cells were added to Tconv cells at varying ratios (Tconv : Treg = 1:0, 27:1, 9:1, 3:1, 1:1, 1:2). Cells were harvested after 3 days of co-culturing for flow cytometric assessment of proliferation cycles in CD45.1+ Tconv cells.
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Supplemental Figures and Figure Legends
Supplemental Figure 1: IRA B cell phenotype and ontogeny. (A) Surface marker expression profile of IRA B cells isolated from spleens of LPS challenged C57Bl/6 (4 x 25mg/day LPS i.p.) and atherosclerotic Ldlr–/– and Apoe–/– mice (3 months on HCD). GM-CSF+ leukocytes are depicted in red and GM-CSF– leukocytes in grey. (B) Isotype staining controls on human spleen sections. (C) Characteristics of patients with and without cardiovascular disease (CVD). (D) Ldlr–/– mice were lethally irradiated, reconstituted with a 50:50 mixture of CD45.1+ WT and CD45.2+ Myd88–/– bone marrow and placed on HCD for 3 months. (E) Discrimination of Myd88–/– and Myd88+/+ (WT) splenocytes based on CD45.2 and CD45.1 staining and flow cytometry. Further staining for GM-CSF, B220 and IgM allowed for identification of GM-CSF+ IRA B cells in the CD45.1+ WT but not the CD45.2+ Myd88–/– B cell population. Representative dot plots are shown for one of three mixed chimeras).
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Supplemental Figure 2: Leukocyte subsets in reconstituted mixed bone marrow chimeras. (A) Enumeration of monocytes (Mono), neutrophils (Neutro), CD4+ and CD8+ T cells (CD4 Tc, CD8 Tc), CD19+ B cells (CD19 B) in the spleen of control (gray) and IRA B KO (white) mice 6 weeks after reconstitution. Results are presented as mean ± SEM, n = 6 per group., (B) Identification and quantification of splenic B cell subsets in both groups 6 weeks after reconstitution. The CD21high CD23low population contains marginal zone B cells, CD21low CD23low cells encompass B1 and plasma cells, and B2/T2 cells are CD23high. Results are presented as mean ± SEM, n = 6 per group, controls in gray and IRA B KO mice in white. (C) Splenic B cell expression of antigen presenting and costimulatory molecules MHCII, CD86 and CD40 as determined by flow cytometry. Results are presented as mean fluorescence intensity (MFI) ± SEM, n = 6 per group. (D) Leukocyte subsets in blood, spleen, bone marrow and peritoneum of control (gray) and IRA B KO (white) mice 6 weeks after reconstitution. Results are presented as mean ± SEM, n = 6 per group. (E) Quantification of GM-CSF (Csf2) expression in whole spleen tissue of IRA B KO (white) and control (gray) mice by real-time PCR. Results are presented as mean ± SEM fold change of 2∆Ct, ** p ≤ 0.01, n ≥ 20 per group. (F) Relative contribution of non-B cells to leukocyte derived GM-CSF production as assessed by flow cytometry. Results are presented as mean ± SEM, n = 7 per group.
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Supplemental Figure 3: IRA B cell deficiency does not affect monocytosis and hypercholesterol-emia. (A) Measurement of total body weight, serum levels of total cholesterol (n ≥ 10 per group) and VLDL, LDL and HDL cholesterol (3 pooled samples of n ≥ 6 mice per group) after 10 week HCD feeding. Results are presented as mean ± SEM. (B) Enumeration of total monocytes, Ly6Chigh monocytes, neutrophils and CD11blow F4/80high red pulp macrophages in blood, spleen or bone marrow after 10 week HCD feeding. Results are presented as mean ± SEM, n ≥ 10 per group, * p ≤ 0.05, controls in gray and IRA B KO mice in white. (C) Expression of CCR2, VLA4 and CD62L on Ly6Chigh blood monocytes after 10 week HCD feeding as determined by flow cytometry. Quantification of MFI presented as mean ± SEM fold change between control (gray) and IRA B KO (white) mice, n ≥ 10 per group.
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Supplemental Figure 4: IRA B cells influence TH1 cell-dependent anti-OxLDL isotype switching but not Treg suppressive function. (A) Regulatory T cell (Treg) suppression assay. WT CD4+ CD25– conventional T cell (Tconv) were co-cultured with CD4+ CD25+ Treg cells sorted from IRA B KO and control mice after 10 week HCD feeding at increasing dilutions as indicated. Representative histograms show proliferation of cell tracer dye-labeled Tconv cells in response to soluble anti-CD3e (1 µg/ml) and T cell-depleted, irradiated spleen stimulator cells under the suppressive influence of Treg cells. (B) Dose-dependent quantification of Treg-induced suppression of Tconv proliferation. Results are presented as mean ± SEM, n = 4 per group, controls in gray circles and IRA B KO mice in white squares. (C) On the left representative dot plot showing intracellular staining of CD3+ CD4+ T cells for IL-4 and IL-17. On the right quantification of IL-4-producing TH2 and IL-17-producing TH17 cells in spleens of IRA B KO (white) and control (gray) mice after 10 week HCD feeding. Results are presented as mean ± SEM, n = 7 per group. (D) Antibody binding dilution curves for total serum IgG2c and IgG1 antibodies. Results are presented as mean for triplicates of pooled samples, n ≥ 6 per group, controls in gray circles and IRA B KO mice in white squares. (E, F) Quantification of IgG1 and total IgG antibody titers against MDA-LDL and copper-oxidized LDL (CuOxLDL) in 1:25 diluted individual serum samples, n ≥ 10 per group. Results are presented as mean ± SEM, * p ≤ 0.05, controls in gray and IRA B KO mice in white. (G) Quantification of anti-AB1-2-IgM (EO6) antibody titers in 1:25 diluted individual serum samples, n ≥ 10 per group. Results are presented as mean ± SEM, controls in gray and IRA B KO mice in white.
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Supplemental Figure 5: IRA B cell deficiency does not affect generation of cDC progenitors.(A) Expression of MHCII, CD86, and CD40 on CD8– CD11b+ splenic cDC as determined by flow cytometry in IRA B KO (white) and control (gray) mice after 10 week HCD feeding. On the left representative histograms show mean fluorescence intensities (MFI) for MHCII, CD86 and CD40 compared to isotype controls (dashed line). On the right quantification of MFI presented as mean ± SEM fold change between control (gray) and IRA B KO (white) mice, n ≥ 10 per group, * p ≤ 0.05. (B) Enumeration of granulocyte-macrophage progenitors (GMP), common dendritic cell progenitor (CDP) and preDC precursor in bone marrow and spleen after 10 week HCD feeding. Results are presented as mean ± SEM, n ≥ 10 per group, controls in gray and IRA B KO mice in white. (C) Flow assisted cell sorting of CD23low IgMhigh CD43high CD138high (IRABLdlr–/– HCD) cells from atherosclerotic Ldlr–/– mice (3 months on HCD). Dashed lines represent isotype controls. (D) CD4+ CD25– OT-II cells were co-cultured
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with IRA BLdlr–/– HCD cell-generated BMDC loaded with chicken ovalbumin (100µg/ml) or BSA (100µg/ml) for 4 days. Representative histograms show cell divisions of CD4+ OT-II cells labeled with a cell tracer dye.
Supplemental References:1. Colvin GA, Lambert JF, Abedi M, Hsieh CC, Carlson JE, Stewart FM, Quesenberry PJ. Murine marrow cellularity and the concept of stem cell competition: geographic and quantitative determinants in stem cell biology. Leukemia. 2004;18:575-583.2. Binder CJ, Horkko S, Dewan A, Chang MK, Kieu EP, Goodyear CS, Shaw PX, Palinski W, Witztum JL, Silverman GJ. Pneumococcal vaccination decreases atherosclerotic lesion formation: molecular mimicry between Streptococcus pneumoniae and oxidized LDL. Nat Med. 2003;9:736-743.
