Arteriosclerosis, Thrombosis, and Vascular Biology BASIC SCIENCES NR4A1 Deletion in Marginal Zone B Cells Exacerbates Atherosclerosis in Mice—Brief Report Meritxell Nus , Gemma Basatemur , Maria Galan , Laia Cros-Brunsó , Tian X. Zhao , Leanne Masters, James Harrison, Nichola Figg, Dimitrios Tsiantoulas , Frederic Geissmann, Christoph J. Binder , Andrew P. Sage , Ziad Mallat OBJECTIVE: NR4A orphan receptors have been well studied in vascular and myeloid cells where they play important roles in the regulation of inflammation in atherosclerosis. NR4A1 (nerve growth factor IB) is among the most highly induced transcription factors in B cells following BCR (B-cell receptor) stimulation. Given that B cells substantially contribute to the development of atherosclerosis, we examined whether NR4A1 regulates B-cell function during atherogenesis. APPROACH AND RESULTS: We found that feeding Ldlr−/− mice a Western diet substantially increased Nr4a1 expression in marginal zone B (MZB) cells compared with follicular B cells. We then generated Ldlr−/− mice with complete B- or specific MZB-cell deletion of Nr4a1. Complete B-cell deletion of Nr4a1 led to increased atherosclerosis, which was accompanied by increased T follicular helper cell–germinal center axis response, as well as increased serum total cholesterol and triglycerides levels. Interestingly, specific MZB-cell deletion of Nr4a1 increased atherosclerosis in association with an increased T follicular helper–germinal center response but without any impact on serum cholesterol or triglyceride levels. Nr4a1−/− MZB cells showed decreased PDL1 (programmed death ligand-1) expression, which may have contributed to the enhanced T follicular helper response. CONCLUSIONS: Our findings reveal a previously unsuspected role for NR4A1 in the atheroprotective role of MZB cells. GRAPHIC ABSTRACT: A graphic abstract is available for this article. Downloaded from http://ahajournals.org by on November 2, 2023 Key Words: animals ◼ atherosclerosis ◼ cholesterol ◼ diet, Western ◼ mice A therosclerosis is a chronic inflammatory disease to a high-fat/high-cholesterol (HF/HC) diet, MZB cells involving interactions between vascular, circulating, activate an atheroprotective programme, limiting T follicu- and immune cells. B cells play an important role in the lar helper (Tfh) cell motility in a PDL1 (programmed death chronic immunoinflammatory response, producing antibod- ligand-1)-dependent manner.5 ies and regulating T-cell and natural killer–cell activation. NR4A1 (nerve growth factor IB; also called Nur77) The role of B cells in atherosclerosis is complex, with ath- is a member of the NR4A orphan nuclear receptor sub- erogenic and protective roles assigned for distinct B-cell family. NR4A1 has been involved in negative selection subsets.1 B2 cells comprise the phenotypically distinct fol- of thymocytes, differentiation of regulatory T cells, and licular B (FOB) cells of the spleen and the lymph nodes development of Ly6C− monocytes.6 However, little is and the marginal zone B (MZB) cell population of the known about its role in B cells besides the fact that it spleen. While depletion of all B2 cells reduces atheroscle- is rapidly induced upon BCR (B-cell receptor) signal- rosis,2–4 we have recently discovered that selective deple- ing activation7 and that it may regulate the survival of tion of MZB cells promotes atherosclerosis.5 In response self-reactive B cells.8 NR4A1 also plays important but Correspondence to: Meritxell Nus, PhD, Department of Medicine, Addenbrooke’s Hospital, Level 5, Box 157, CB2 0QQ Cambridge, United Kingdom. Email mn421@cam.ac.uk This manuscript was sent to William C. Sessa, Senior Consulting Editor, for review by expert referees, editorial decision, and final disposition. The Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/ATVBAHA.120.314607. For Sources of Funding and Disclosures, see page 2604. © 2020 The Authors. Arteriosclerosis, Thrombosis, and Vascular Biology is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited. Arterioscler Thromb Vasc Biol is available at www.ahajournals.org/journal/atvb 2598   November 2020 Arterioscler Thromb Vasc Biol. 2020;40:2598–2604. DOI: 10.1161/ATVBAHA.120.314607 Nus et al B-Cell NR4A1 in Atherosclerosis Nonstandard Abbreviations and Acronyms Highlights Basic Sciences - AL BCR B-cell receptor • NR4A1 is a new target that modulates B-cell func- BM bone marrow tions in atherosclerosis. FOB follicular B • Nr4a1 deletion in B2 cells increases atherosclerosis and is associated with increased Tfh-GC response GC germinal center and circulating lipid levels. LDL low-density lipoprotein • NR4A1 expression in MZB cells regulates PDL1 LDLR low-density lipoprotein receptor expression, limiting the Tfh-GC response and pro- MZB marginal zone B tecting from atherosclerosis. Tfh T follicular helper WT wild type dose of 4 Gy resulted in reconstitution of the majority of MZB cells (>99%) with donor CD45.2−; CD45.1+ cells, while the majority of the FOB cells and the rest of the BM-derived cells complex roles in atherosclerosis, at least in part, due to (including other lymphocytes [FSClow] and myeloid [FSChi] cells; its impact on vascular cell,9 monocyte,6,10,11 and T-cell biol- 90%) were from the CD45.2+; CD45.1− recipient (Figure I in ogy.6,12 We have now addressed the role of NR4A1 spe- the Data Supplement). cifically in B cells in the development of atherosclerosis. For atherosclerotic experiments, all recipient mice were ran- domly assigned to different experimental groups based on their weight at the beginning of the experiment. After 6 weeks of MATERIALS AND METHODS recovery, mice were started on HF/HC (21% fat, 0.15% cho- The data that support the findings of this study are available lesterol) for 8 weeks. from the corresponding author upon reasonable request. Extent and Composition of Atherosclerotic Animals Lesions All experiments were approved by the Home Office, United The lesions in the root of the aorta beneath all 3-valve leaf- Kingdom, and were performed under Personal Project Licence lets were analyzed. To measure atherosclerotic plaque size, 80/2426. All mice were on C57Bl/6J background. Cd79aCre/+, we stained paraffin-embeded sections with Masson trichrome. Downloaded from http://ahajournals.org by on November 2, 2023 µMT, Nr4a1−/−, and Ldlr–/– mice were originally from The Jackson To characterize atherosclerotic lesion composition, we stained Laboratory. Rbpjkflox/flox mice were kindly provided by Tasuku sections for macrophages (Mac-3 [lysosome-associated mem- Honjo.13 To generate an atherosclerotic mouse model that lacks brane protein 2], 1:200, clone M3/84; Santa Cruz), T cells (poly- MZB cells, Cd79aCre/+; Rbpjkflox/flox were crossed with Ldlr–/– to clonal CD3, 1:100; Dako), and neutrophils (Ly6G [lymphocyte generate Ldlr−/−; Cd79aCre/+; RBPflox/flox mice. For atherosclerosis antigen 6 complex locus G6D], 1:100, clone 1AB; BD). We experiments, we generated 2 different mice models. For dele- performed each staining on a single slide (3 sections for each tion of Nr4a1 in all B cells, 6- to 8-week-old Ldlr–/– mice were staining) from approximately the same level of the aortic root for lethally irradiated (two 5.5 Gy fractioned doses) and injected all experimental and control mice. Computer-assisted analysis with a mixed bone marrow (BM) chimera containing 80% uMT (Image-J) was used to determine lesion size and areas or cell plus either 20% WT (wild type) C57Bl/6J (to reconstitute with counts for specific cell types. All slides for each experiment were WT B cells) or 20% Nr4a1−/− (to reconstitute with Nr4a1−/− B analyzed by the same blinded observer to minimize variations. All cells). We only used male mice to limit the number of animals measurements were performed by manual selection of stained used in our experiments. In subsequent experiments, we used pixel thresholds and presented as percent positive (stained) both males and females. For deletion of Nr4a1 selectively in area relative to the entire intimal area. CD4+ T and Ly6G+ cells MZB cells, we developed a partial irradiation model in athero- were counted, and the data are presented as the number of T sclerotic mice lacking MZB cells. Recipient Ldlr−/−; Cd79aCre/+; cells per total number of cells or as the total number of cells RBPflox/flox male and female mice were partially irradiated (1 per section, respectively, in atherosclerotic plaques and adventi- single dose of 4 Gy) and injected intravenously with BM from tia. Representative images of immunofluorescence staining are Nr4a1−/− or WT C57Bl/6J male and female mice, respectively, included in Figure II in the Data Supplement. Whole aortas were which we expected to result in reconstitution of MZB cell com- prepared en face for Oil Red O staining to measure lipid deposi- partment with cells coming from the donors (Nr4a1−/− or WT), tion and quantification of lesion size using J-Microvision. while the rest of immune cells (including FOB, T, myeloid, etc) would be mostly from the recipient. To validate this partial irra- diation model, we used the CD45 (cluster of differentiation Flow Cytometry 45) congenic lineage tracing system. We exposed recipient Single-cell suspensions of homogenized spleen and inguinal CD45.2+; CD45.1− male and female mice lacking MZB cells lymph nodes were stained with fluorophore-conjugated antibod- (LDLr−/−; Cd79a Cre/+; RBPflox/flox) to different doses of irradia- ies and analyzed using an LSRII Fortessa (Becton, Dickinson and tion (from 4 to 7 Gy), then injected CD45.2−; CD45.1+ male Company) or FACSCantoII (BD) flow cytometer as described pre- and female BM donor cells, respectively, to allow us to track the viously.5 Representative plots of gating strategies can be found in reconstitution of B-cell subsets using flow cytometry. A single Figure II in the Data Supplement. Arterioscler Thromb Vasc Biol. 2020;40:2598–2604. DOI: 10.1161/ATVBAHA.120.314607 November 2020   2599 Nus et al B-Cell NR4A1 in Atherosclerosis Determination of Circulating Antibodies male mice (which cannot produce B cells) supplemented with either 20% BM from WT C57Bl/6J mice (referred Basic Sciences - AL Specific antibody titers to given antigens in plasma were deter- mined by chemiluminescent ELISA as described previously.14,15 to as the WT B-cell group) or 20% of BM cells from Nr4a1−/− mice (referred to as the Nr4a1−/− B-cell group). MZB and FOB cells were reconstituted equally in both Determination of Serum Lipid Levels groups (Figure III in the Data Supplement). After recov- Total cholesterol, triglycerides, and HDL-C were measured by an enzymatic method in a Siemens Dimensions RxL analyzer. ery, mice were put on a high-fat/high-cholesterol diet for 8 weeks. Specific Nr4a1 deletion in B cells mark- edly accelerated the development of atherosclerosis in Purification of MZB and FOB the aortic sinus (Figure 1A) and the aortic arch (Figure Single-cell suspensions were prepared from spleens by dis- III in the Data Supplement). Serum levels of total choles- sociation through 70-µm filters using a syringe plunger. For terol and triglycerides were also significantly increased MZB and FOB cell purification, B cell–enriched populations in the Nr4a1−/− B-cell group (Figure 1B through 1D), were separated by Automacs using a B-cell purification kit (Miltenyi Biotech), and B cells were stained for 15 minutes at which could account, at least in part, for the increased 4°C with anti-CD23-phycoerythrine and anti-CD21-fluores- atherosclerosis seen in these mice. Analysis of plaque cein isothiocyanate. After washing and staining with 7-AAD for composition showed a trend toward increased %necrotic cell viability, MZB (CD21hiCD23low) and FOB (CD23hiCD21low) core area but a similar percentage of macrophages and were sorted using an AriaIII Cell, Influx, or FACSJazz sorter CD3+ T cells in lesions of Nr4a1−/− compared with WT (BD). Purity of both populations was higher than 95%. B-cell mice (Figure III in the Data Supplement). In the adventitia, we found similar percentages of CD3+ T cells BCR Stimulation and a trend toward increased percentages of neutrophils Purified sorted MZB and FOB from WT or Nr4a1−/− mice were (Ly6G+) in the Nr4a1−/− compared with WT B-cell mice stimulated for 30 minutes, 3 hours, or 6 hours with F(ab)2 frag- (Figure III in the Data Supplement). We did not find any ment of the anti-IgM (eBioscience; 10 ug/mL). difference in blood immune cell subsets (including B cells, T cells, neutrophils, and monocytes) between both groups (Figure III in the Data Supplement). Quantitative Real-Time Polymerase Chain We then characterized the innate and the adaptive Reaction immune responses in the spleen. Splenic dendritic cell, For gene expression analysis, RNA from sorted MZB and monocyte, and neutrophil levels were similar between Downloaded from http://ahajournals.org by on November 2, 2023 FOB cells was isolated using RNAeasy mini kit (Qiagen). qRT- the 2 groups of mice (Figure III in the Data Supplement). PCR was performed using Quantitect whole transcriptome kit. qRT-PCR was performed with SYBR-green primers. Primer Nr4a1−/− B-cell mice showed a trend toward increased Tfh sequences are listed in Table I in the Data Supplement. cell numbers (P=0.07) and had significantly increased ger- minal center (GC) B cells (P<0.05) compared with mice with WT B cells (Figure 1E and 1F). All other T-cell subsets Statistical Analysis including T effector memory, T regulatory cells, and T follic- Values are expressed as means±SEM. Based on pilot experi- ular regulatory cells were not significantly altered between ments, we found that MZB deficiency increases lesion size by the two groups (Figure IV in the Data Supplement). 2-fold. Use of the equation for Student t test for the difference of 2 means gives us an n=6 to detect a 50% increase in lesion We have previously demonstrated that B2 cells play size with α=0.05 and 90% power. Where data sets passed nor- different roles in atherosclerosis, with MZB cells being mality tests, differences between values were examined using atheroprotective through limitation of Tfh cell formation parametric 2-tailed unpaired Student t test, other data sets by (MZB cell deletion leads to increased Tfh)5—a pheno- using nonparametric Mann-Whitney U test; and were considered type that is similar to the present phenotype observed significant at P<0.05 for the B-cell model where only males were for B cell–specific depletion of Nr4a1−/−. Furthermore, used. In the partial irradiation model, we used both males and expression of Nr4a1 was significantly upregulated in females and performed 2-way ANOVA to determine comparisons purified MZB cells but downregulated in FOB cells from between genotypes. When sex did not have a significant effect on LDLr−/− mice consuming a high-fat/high-cholesterol diet, the results, we pooled the male and female data. When sex had a in comparison with mice fed a standard laboratory diet significant effect, data were not pooled, and Bonferroni post hoc (Figure 1G). Therefore, we addressed the role of NR4A1 analysis was performed to find differences between groups. specifically in MZB cells during the development of atherosclerosis. RESULTS NR4A1 Deletion in B Cells Exacerbates NR4A1 Deletion Selectively in MZB Cells Atherosclerosis Exacerbates Atherosclerosis We reconstituted lethally irradiated Ldlr−/− male mice To study the role of Nr4a1 in MZB cells and its impact with a mixed BM containing 80% of BM cells from µMT on atherosclerosis, we generated an atherosclerotic 2600   November 2020 Arterioscler Thromb Vasc Biol. 2020;40:2598–2604. DOI: 10.1161/ATVBAHA.120.314607 Nus et al B-Cell NR4A1 in Atherosclerosis Basic Sciences - AL Downloaded from http://ahajournals.org by on November 2, 2023 Figure 1. Nr4a1 (nerve growth factor IB) deletion in B cells increases atherosclerosis development. Ldlr−/− mice were transplanted with a mixed chimera containing 80% µMT +20% WT (wild type; for reconstitution with WT B cells) or 20% Nr4a1−/− (for reconstitution with Nr4a1−/− B cells) BM and fed a high-fat/high-cholesterol (HF/HC) diet for 8 wk (A–F). A, Representative Masson trichrome staining and quantification of atherosclerotic plaques in aortic roots (each symbol represents one mouse, and the horizontal bars are group mean±SEM with n=7 and n=9, respectively, in each group). Original magnification, ×10. Scale bars=500 μm. B–D, Total plasma cholesterol, triglycerides, and HDL (high-density lipoprotein) cholesterol levels. E and F, Total numbers of T follicular helper (Tfh) cells (CD3+ CD4+ CD44+ CD62L− ICOS+ CXCR5+ PD1+) and germinal center (GC) B cells (B220+ CD19+ CD95+ GL7+; n=13–15 in each group). G, Nr4a1 relative expression in sorted marginal zone B (MZB) and follicular B (FOB) cells from Ldlr−/− mice fed a standard laboratory or HF/HC diet for 8 wk (n=3–4 in each group). For A–G, 2-tailed unpaired Student t test or 2-way ANOVA followed by Bonferroni post hoc analysis, *P<0.05 and ***P<0.001. CD indicates cluster of differentiation; CXCR5, C-X-C chemokyne receptor-5; ICOS, inducible T-cell coestimulator; and PD1, programmed cell death protein 1. mouse model in which Nr4a1−/− was specifically deleted males and females, respectively, resulting in mice recon- in MZB cells. For this aim, we generated Ldlr−/− mice stituted with WT MZB cells (the WT MZB cell group) or on Cd79aCre/+; RBPflox/flox background (mice that do not with MZB cells in which Nr4a1 has been deleted (the have MZB cells, as described previously5). After partial Nr4a1−/− MZB cell group; see Materials and Methods irradiation (4 Gy), Ldlr−/−; Cd79aCre/+; RBPflox/flox male and section for a more detailed explanation of the model and female mice were injected with WT or Nr4a1−/− BM from Figure I in the Data Supplement). Both groups had the Arterioscler Thromb Vasc Biol. 2020;40:2598–2604. DOI: 10.1161/ATVBAHA.120.314607 November 2020   2601 Nus et al B-Cell NR4A1 in Atherosclerosis same numbers of MZB and FOB cells after reconstitu- in the absence of NR4A1 (Figure VII in the Data Supple- tion (Figure I in the Data Supplement). ment). However, BCR-mediated regulation of MZB cell Basic Sciences - AL After 8 weeks on a high-fat/high-cholesterol diet, surface PDL1 protein expression was not altered in the deletion of Nr4a1 specifically in MZB cells led to absence of NR4A1 (Figure VII in the Data Supplement). increased development of atherosclerosis in the aor- These data suggest that the impact of NR4A1 deletion tic sinus (Figure 2A) and increased lipid deposition in on PDL1 expression in vivo was independent of BCR the aortic arches (Figure V in the Data Supplement), activation. without altering serum levels of total cholesterol, HDL- C, or triglycerides, compared with mice with WT MZB cells (Figure 2B through 2D). Females had increased DISCUSSION atherosclerotic plaques and decreased HDL-C levels To the best of our knowledge, this is the first experimen- than males—a phenotype that has been reported pre- tal study addressing the role of NR4A1 in B cells during viously in LDLr−/− mice.16 Analysis of plaque composi- atherosclerosis. Our data show that NR4A1 expression tion showed a trend toward increased %necrotic core in MZB cells regulates PDL1 expression, limiting the area in the Nr4a1−/− MZB cell group while there were Tfh-GC response and protecting from atherosclerosis. A no differences in percentages of Mac-3+ macrophages recent work performed in a cancer line has shown that and CD3+ T cells between both groups (Figure V in NR4A1 binds to the GC motif of the PDL1 promoter and the Data Supplement). No significant differences were regulates its gene expression,18 further supporting the found in the percentages of CD3+ T cells and neutro- possibility of a direct regulation of PDL1 expression by phils in the adventitia between the 2 groups (Figure V NR4A1 in MZB cells. Previous studies suggested that in the Data Supplement). PDL1 expression in antigen-presenting cells might limit Interestingly, Nr4a1−/− MZB-cell mice had signifi- proatherogenic immune responses.19 Whether NR4A1 cantly increased splenic Tfh and GC B-cell numbers also regulates PDL1 expression in immune cells, other (Figure 2E and 2F) compared with WT MZB-cell mice, than MZB cells, is worth of investigation. without detectable changes in serum antibody titers Interestingly, NR4A1 may regulate PDL1 in a BCR- against modified LDL (low-density lipoprotein; Figure independent manner, opening up an opportunity to V in the Data Supplement). Despite our limitations modulate NR4A1-dependent pathways in B cells with- to comprehensively assess the antibody response, out affecting the BCR signaling pathway. Furthermore, Downloaded from http://ahajournals.org by on November 2, 2023 we hypothesize that other Tfh cell functions may human MZB-like cells express high levels of NR4A120 account for their atherogenic role, as discussed previ- and PDL1,5 and, therefore, we speculate that NR4A1 ously.5 Nr4a1−/− MZB cell group also had significantly may have a similar role in human B cells, although further increased T regulatory cells and T follicular regulatory studies are required to determine whether this is indeed CD25pos cells, while T effector memory, T follicular reg- the case. The potential use of Bis-Indole-NR4A1 antag- ulatory CD25neg cells, dendritic cells, neutrophils, and onists as anticancer treatments is starting to emerge,18,21 monocytes were not significantly altered (Figure VI in and, therefore, we would advise to take into consider- the Data Supplement). ation their effect on B cells to avoid or monitor potential undesirable cardiovascular side effects associated to anticancer treatments. NR4A1 Regulates PDL1 Expression NR4A1 has previously been reported to play a very We found that MZB cells from the Nr4a1−/− MZB group important role in B-cell survival.8,22 Nevertheless, in both expressed lower levels of the PDL1 encoding gene of our B-cell models of Nr4a1 deletion, we did not find (Cd274) and PDL1 protein (Figure 2G and 2H) com- any difference in B-cell numbers, making it unlikely that pared with those from the WT MZB group, which could, NR4A1 regulates B-cell survival during atherosclerosis. at least in part, explain why the Nr4a1−/− MZB cell group This is in agreement with a recent finding that NR4A1 also showed increased Tfh cells.5 regulates B-cell survival in a T cell–independent response To further examine the link between NR4A1 and but not in a T cell–dependent response.23 In our models, PDL1, we performed in vitro experiments using sorted we could not study the potential contribution of NR4A1 MZB cells from total WT and Nr4a1−/− mice. MZB cells in B1 cells because these cells do not reconstitute well from Nr4a1−/− mice expressed lower levels of PDL1 than after irradiation, and, therefore, further experiments are WT MZB cells at baseline (Figure VII in the Data Supple- needed to elucidate the role of NR4A1 in B1 cells during ment). As described previously, Nr4a117 and Cd2745 sig- atherosclerosis. nificantly increased upon BCR activation (Figure VII in Surprisingly, NR4A1 deletion in all B2 cells not only the Data Supplement). BCR-induced pBTK (Bruton tyro- regulates the Tfh-GC response but also the circulating sine kinase) was not altered in the absence of NR4A1 lipid levels. Thus, B2 cells other than MZB cells could (Figure VII in the Data Supplement). BCR-induced be responsible for this effect on lipid parameters. B cells Cd274 gene expression was significantly downregulated express several receptors, such as LDLR (LDL receptor), 2602   November 2020 Arterioscler Thromb Vasc Biol. 2020;40:2598–2604. DOI: 10.1161/ATVBAHA.120.314607 Nus et al B-Cell NR4A1 in Atherosclerosis Basic Sciences - AL Downloaded from http://ahajournals.org by on November 2, 2023 Figure 2. Nr4a1 (nerve growth factor IB) deletion in marginal zone B (MZB) cells increases atherosclerosis development. Ldlr−/−; Cd79aCre/+; Rbpjkflox/flox were partially irradiated (Materials and Methods in the Data Supplement) and transplanted with WT (wild type; for reconstitution with WT MZB cells) or Nr4a1−/− (for reconstitution with Nr4a1−/− MZB cells) BM and fed a high-fat/high-cholesterol diet for 8 wk (A–H). A, Representative Masson trichrome staining and quantification of atherosclerotic plaques in aortic roots (each symbol represents one mouse, and horizontal bars are group mean±SEM with n=16–18 in each group). Original magnification, ×10. Scale bars=500 μm. B–D, Total plasma cholesterol, HDL (high-density lipoprotein) cholesterol, and triglycerides levels. E and F, Total numbers of T follicular helper (Tfh) and germinal center (GC) B cells. Cd274 (Pdl1 [programmed death ligand-1] gene) expression, quantified by qRT-PCR (n=5 in each group) in sorted MZB cells (G) and PDL1 protein expression, quantified by flow cytometry in splenic MZB cells (H; n=16–18 in each group). For A–G, 2-tailed unpaired Student t test or 2-way ANOVA followed by Bonferroni post hoc analysis, *P<0.05 and **P<0.01. MFI indicates mean fluorescence intensity; and PDL1, programmed cell death ligand-1. CD1d, and scavenger receptors, that could recognize but none of the previous atherosclerotic models target- and remove lipids.24 Moreover, previous studies modulat- ing NRA1 in monocytes have shown a significant effect ing B cells have shown changes in cholesterol levels,25,26 on lipid levels.10,11 Thus, the specific role of B cells in lipid Arterioscler Thromb Vasc Biol. 2020;40:2598–2604. DOI: 10.1161/ATVBAHA.120.314607 November 2020   2603 Nus et al B-Cell NR4A1 in Atherosclerosis metabolism is yet to be clarified, and further experiments 9. You B, Jiang YY, Chen S, Yan G, Sun J. The orphan nuclear receptor Nur77 suppresses endothelial cell activation through induction of Ikap- are required. Basic Sciences - AL paBalpha expression. Circ Res. 2009;104:742–749. doi: 10.1161/ Overall, our data demonstrate that NR4A1 in B cells CIRCRESAHA.108.192286 is an attractive target to modulate the development of 10. Hamers AA, Vos M, Rassam F, Marinković G, Marincovic G, Kurakula K, van Gorp PJ, de Winther MP, Gijbels MJ, de Waard V, et al. Bone marrow- atherosclerotic disease. specific deficiency of nuclear receptor Nur77 enhances atherosclerosis. Circ Res. 2012;110:428–438. doi: 10.1161/CIRCRESAHA.111.260760 11. Chao LC, Soto E, Hong C, Ito A, Pei L, Chawla A, Conneely OM, Tangirala RK, ARTICLE INFORMATION Evans RM, Tontonoz P. Bone marrow NR4A expression is not a dominant factor in the development of atherosclerosis or macrophage polarization in Received April 30, 2020; accepted August 26, 2020. mice. J Lipid Res. 2013;54:806–815. doi: 10.1194/jlr.M034157 Affiliations 12. Hanna RN, Shaked I, Hubbeling HG, Punt JA, Wu R, Herrley E, Zaugg C, Pei H, Geissmann F, Ley K, et al. NR4A1 (Nur77) deletion polarizes macro- Division of Cardiovascular Medicine, Department of Medicine, University of phages toward an inflammatory phenotype and increases atherosclerosis. Cambridge, United Kingdom (M.N., G.B., L.C.-B., T.X.Z., L.M., J.H., N.F., A.P.S., Circ Res. 2012;110:416–427. doi: 10.1161/CIRCRESAHA.111.253377 Z.M.). CIBER de Enfermedades Cardiovasculares, Spain (M.N., M.G.). Biomedi- 13. Tanigaki K, Han H, Yamamoto N, Tashiro K, Ikegawa M, Kuroda K, Suzuki A, cal Research Institute, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain Nakano T, Honjo T. Notch-RBP-J signaling is involved in cell fate deter- (M.G.). Department of Laboratory Medicine, Medical University of Vienna, Aus- mination of marginal zone B cells. Nat Immunol. 2002;3:443–450. doi: tria (D.T., C.J.B.). Memorial Sloan Kettering Cancer Centre, New York (F.G.). 10.1038/ni793 Institut National de la Santé et de la Recherche Médicale, Unit 970, Paris 14. Binder CJ, Hörkkö S, Dewan A, Chang MK, Kieu EP, Goodyear CS, Cardiovascular Research Center, France (Z.M.). Shaw PX, Palinski W, Witztum JL, Silverman GJ. Pneumococcal vaccina- Acknowledgments tion decreases atherosclerotic lesion formation: molecular mimicry between Streptococcus pneumoniae and oxidized LDL. Nat Med. 2003;9:736–743. We acknowledge Prof Anna Petrunkina, Natalia Savinykh, Nika Romashova, doi: 10.1038/nm876 and Simon McCallum, in the Phenotyping Hub of the Department of Medicine 15. Chou MY, Fogelstrand L, Hartvigsen K, Hansen LF, Woelkers D, Shaw PX, (University of Cambridge), for their help in flow cytometry and sorting and Maria Choi J, Perkmann T, Bäckhed F, Miller YI, et al. Oxidation-specific epitopes Ozsvar Kozma (Department of Laboratory Medicine, Medical University of Vienna, are dominant targets of innate natural antibodies in mice and humans. J Clin Austria) for help in antibody measurements. We also acknowledge Keith Burling and all the Core Biomedical Assay Laboratory in Addenbrooke Hospital for lipid Invest. 2009;119:1335–1349. doi: 10.1172/JCI36800 measurements. 16. Rinninger F, Heine M, Singaraja R, Hayden M, Brundert M, Ramakrishnan R, Heeren J. High density lipoprotein metabolism in low density lipoprotein Sources of Funding receptor-deficient mice. J Lipid Res. 2014;55:1914–1924. doi: 10.1194/jlr. M048819 This work was supported by the British Heart Foundation (BHF) Project grant PG/17/73/33251 to M. Nus and Z. Mallat and by the European Union grant 17. Tsiantoulas D, Kiss M, Bartolini-Gritti B, Bergthaler A, Mallat Z, Jumaa H, RG71687 to Z. Mallat. We were also supported by the Cambridge BHF Centre of Binder CJ. Secreted IgM deficiency leads to increased BCR signaling that Research Excellence and NIHR Cambridge Biomedical Research Centre. results in abnormal splenic B cell development. Sci Rep. 2017;7:3540. doi: 10.1038/s41598-017-03688-8 Disclosures Downloaded from http://ahajournals.org by on November 2, 2023 18. Karki K, Wright GA, Mohankumar K, Jin UH, Zhang XH, Safe S. A bis- None. indole-derived NR4A1 antagonist induces PD-L1 degradation and enhances anti-tumor immunity. Cancer Res. 2020;80:1011–1023. doi: 10.1158/0008-5472.CAN-19-2314 19. Gotsman I, Grabie N, Dacosta R, Sukhova G, Sharpe A, Lichtman AH. Pro- REFERENCES atherogenic immune responses are regulated by the PD-1/PD-L pathway 1. Nus M, Tsiantoulas D, Mallat Z. Plan B (-cell) in atherosclerosis. Eur J Phar- in mice. 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