To the Editor: Atherosclerosis is a chronic inflammatory disease that involves multiple types of immune cells. Adaptive immunity exerts diverse and profound effects on atherosclerosis. The effector phenotype of lesional T cells may affect lesion growth and stability. Regulatory T cells with anti-inflammatory properties have been reported in atherosclerotic plaques. The targets of suppression of regulatory T cells include naive and effector CD4+ and CD8+ T cells, dendritic cells, and macrophages. A new sub-set of B cells, with CD19+CD24hiCD38hi phenotype in humans, is becoming a hot issue in the field of autoimmunity such as systemic lupus erythematosus and arthritis. These regulatory B cells (Bregs) play important roles in immune system by regulating functions of effector and regulatory T cells. Bregs exhibit protective role by attenuating the neointimal formation of atherosclerosis through an interleukin 10 (IL-10) mediated mechanism in a experimental study. There have been few studies on human Bregs in atherosclerosis disease until now. In this study we aimed to investigate CD19+CD24hiCD38hi B cells in coronary artery disease (CAD) patients requiring coronary artery bypass graft (CABG) surgery.
We enrolled 73 patients with CAD who required CABG surgery. A control group of 36 blood donors as healthy volunteers was included in this study. Coronary angiography was performed via transradial approach and by a standard technique. The severity of coronary atherosclerosis was assessed by one experienced independent observer in the projection with the greatest degree of stenosis. The severity of CAD was assessed by the modified Gensini stenosis scoring system. Peripheral blood mononuclear cells were processed within 2 h after collection. For surface antigen staining, the following antibodies and reagents were used: CD19-APC (BioLegend, San Diego, California, USA), CD19-APC-Cy7 (BioLegend), CD20-PerCP-Cy7 (BioLegend), CD24-PE-Cy7 (BD Biosciences, Franklin Lakes, New Jersey, USA), CD27-V450 (BD Biosciences), CD38-APC (BD Biosciences), and CD38-FITC (BD Biosciences). Intracellular antigens analysis was performed in mononuclear leukocytes. For cytokine detection the mononuclear cells were additionally cultivated in the presence of phorbol myristate acetate, ionomycin, and Brefeldin A for 5 h. Cell staining was performed with IL-10-BV605 and transforming growth factor (TGF)-β-BV421 (all reagents from BD Biosciences).
CD19+CD24hiCD38hi phenotypic Bregs were observed in both CAD patients and healthy controls. We observed significantly lower percentages of CD19+CD24hiCD38hi B cells in patients with CAD than in healthy individuals (P = 0.006) [Figure 1A]. Upon cytosine-phosphate-guanine (CpG) stimulation, all B cell sub-sets could produce IL-10 and TGF-β. The levels of both IL-10 and TGF-β producing CD19+CD24hiCD38hi B cells were significantly lower in CAD patients than in healthy controls (P < 0.001 and P = 0.001, respectively) [Figure 1B and 1C]. The average Gensini score was 66 ± 28. Spearman correlation coefficient showed a significant moderate opposite association between the frequencies of Bregs and Gensini scores (r = 0.283, P = 0.015).
We observed a numerical deficit of CD19+CD24hiCD38hi B cells in CAD and decreased secretion of anti-inflammatory cytokines IL-10 and TGF-β by Bregs. CD19+CD24hiCD38hi B cells deficient in CAD is negatively correlated with the severity of atherosclerosis.
Atherosclerosis is a chronic inflammatory disease characterized by a lipid-initiated chronic inflammation of vascular walls involving both innate and adaptive immune cells in the pathophysiologic process. Patients with both rheumatoid arthritis and systemic lupus erythematosus are characterized by an increased risk of cardiovascular complications, mainly ischemic heart disease, which is associated with the development of pre-mature atherosclerosis. The innate response starts with the activation of endothelial cells in vessel walls, and it is followed by an adaptive immune response to an array of potential antigens presented to effector T cells. Regulatory T cells are a minor sub-population of T cells that exert atheroprotective effects by suppressing the activity of proatherogenic effector T cells.
While the role of T cells in atherosclerosis has been studied extensively, the role of B cells has only recently begun to gain attention. The first investigations into the role of B cells in atherosclerosis tended to show that they had atheroprotective effects. B cells are divided into two main families: B1 and B2 cells. It is B1 cells, rather than B2 that exert the atheroprotective effects mainly via the production of natural IgM antibodies that bind oxidized low-density lipoprotein and apoptotic cells. The Bregs have the similar functional and phenotypic features with B-1a cells. Although the body of knowledge regarding the correlation between atherosclerosis and immunity in animal models is growing rapidly, studies carried out on cardiovascular disease in humans have been scarce. CD19+CD24hiCD38hi and CD19+CD24hiCD27+ have been identified as phenotypic Bregs in humans. CD19+CD24hiCD38hi B cells with regulatory function may fail to prevent the development of autoreactive responses and inflammation, leading to graft-versus-host disease and autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. Numerical deficit of Bregs in CAD patients was observed in this study, as we expected. Two anti-inflammatory cytokines, IL-10 and TGF-β, have been shown to have important atheroprotective effects by inhibiting T-cell activation. Overexpression of IL-10 by activated T-cells inhibits atherosclerosis in low-density lipoprotein-receptor-deficient mice, whereas deficiency of IL-10 increases atherosclerosis in apoE knockout mice. Disruption of TGF-β signaling in apoE knockout mice causes rapid development of large, unstable atherosclerotic lesions. Strom identified a specific lymph nodes-derived B2-Breg sub-set that confers IL-10 mediated protection from neointima formation. In our study, we found that IL-10 and TGF-β expressing CD19+CD24hiCD38hi B cells were significantly lower in CAD patients than in healthy controls. That means the atheroprotective effect of Bregs in CAD may be IL-10- or TGF-β-dependent.
We conclude that CD19+CD24hiCD38hi B cells are numerically and functionally deficient in CAD patients. Bregs may be a biomarker of the severity of atherosclerosis.
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Conflicts of interest
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