NR4A1 Deletion in Marginal Zone B Cells Exacerbates Atherosclerosis in Mice-Brief Report

Abstract

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.

Keywords: animals; atherosclerosis; cholesterol; diet, Western; mice.

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.

Figure 2.

Nr4a1 (nerve growth factor IB) deletion in marginal zone B (MZB) cells increases atherosclerosis development. Ldlr^−/−; Cd79a^Cre/+; Rbpjk^flox/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.