Low frequency of IL-10+ B cells in patients with atherosclerosis is related with inflammatory condition

Abstract

Background and aims: B cells involvement in animal models of atherosclerosis has been unequivocally established. However, the role of these cells in patients with atherosclerosis is almost unknown. Besides the production of antibodies, B cells can also exhibit regulatory functions mainly through IL-10. Here, we characterized human B cell subsets, their production of IL-10 in patients with atherosclerosis and their potential association with inflammation.

Methods: Patients with confirmed atherosclerotic events and controls with low cardiovascular risk were included. B cells subsets were determined in mononuclear cells (PBMC) using flow cytometry. PBMC were cultured ex vivo (5 h) and in vitro (48 h) to determine IL-10⁺ B cells and in some cases TNF-α⁺ and IFN-γ⁺ CD4⁺ T cells. The inflammatory state of the participants was determined through high sensitivity C reactive protein levels.

Results: Increase in percentage and number of plasmablasts was observed in patients with atherosclerosis compared with controls. A decreased frequency of IL-10⁺ B cells was observed in patients, both in ex vivo and in vitro cultures. This decrease was detected in transitional, memory, and plasmablast subsets. Interestingly, the reduction of IL-10⁺ B cells negatively and significantly correlated with the inflammatory condition of the studied subjects and associated with an increased frequency of TNF-α⁺ and IFN-γ⁺ CD4⁺ T cells. The blockade of IL-10R did not show further effect in T cells activation.

Conclusions: There is an association between the inflammatory state and a reduction of IL-10⁺ B cells that could contribute to the development of atherosclerosis.

Keywords: Atherosclerosis; B cell subset; B cells; C reactive protein; Cardiology; Cardiovascular system; Cholesterol; Health sciences; IL-10; Immune disorder; Immune system; Immunology; Inflammation; Regulatory B cell.

Figure 1

No changes in frequency and number of total B cells in patients with atherosclerosis. A. Frequency and B. number of B cells (CD19⁺) in mononuclear cells. Median and data from 15 LCVR and 20 patients are shown. Mann-Whitney test.

Figure 2

Increased frequency and number of Plasmablasts in patients with atherosclerosis. A. Strategy of analysis of PBMC: Aggregate exclusion (Left), selection of lymphocytes (Center-left), selection of B cells (CD19⁺) (Center-right), and B cell subsets (Right) according to CD24 and CD38 expression. Transitional (CD19⁺CD24^hiCD38^hi), Mature (CD19⁺CD24^low/−CD38^low/-), Memory (CD19⁺CD24^hiCD38 ^low/-) and Plasmablast (CD19⁺CD24^-CD38^hi). B. Frequency and C. number of B cell subsets. D-F. Frequency of D. CD40⁺, E. CD80⁺ and F. CD86⁺ cells on Transitional, Mature, Memory and Plasmablast subsets. Median and data from 15 LCVR and 20 patients are shown. Mann−Whitney test. *p < 0.05.

Figure 3

Decreased frequency of IL-10⁺ B cells in patients with atherosclerosis. A-B. Representative pseudocolor plots of IL-10⁺ B cells according to IL-10 and CD19 expression under A. ex vivo and B. in vitro cultures of mononuclear cells from a control. IL-10⁺ B cells in cultures in absence (Left) or with re-stimulation with CpG (Center) or LPS (Right) plus PIB in the last 5h in both type of cultures. Frequency of IL-10⁺ B cells under ex vivo and in vitro stimulations and re-stimulation with C. CpG plus PIB and D. LPS plus PIB as showed in A and B. Median and data from 8 LCVR and 10–11 patients. Two-way ANOVA with Šidák post-test. ****p < 0.0001 ***p < 0.001, **p < 0.01, and *p < 0.05.

Figure 4

Decrease in the frequency of IL-10⁺ B cell subsets in patients with atherosclerosis. A-B. Frequency of IL-10⁺ B cell subsets under ex vivo and in vitro stimulation and re-stimulation with A. CpG or B. LPS plus PIB in the last 5 h as described in Materials and Methods. Median and data from 8 LCVR and 10–11 patients. Two-way ANOVA with Šidák post-test. ****p < 0.0001, ***p < 0.001, **p < 0.01 and *p < 0.05.

Figure 5

Correlations between the frequency of IL-10⁺ B cells and hsCRP levels in serum. Spearman correlation between serum hsCRP levels and the percentage of IL-10⁺ in total B cells and each B cell subset after A. ex vivo and B. in vitro cultures with LPS. Data from 7 controls (C) and 9–10 patients (P) are shown.

Figure 6

Increase in the frequency of TNF-α⁺ CD4⁺ T cells in PBMC from patients with atherosclerosis. Culture of total PBMC from controls and patients with anti-CD3ε plus CD40L during 48 h with re-stimulation with PIB in the last 5 h, in the presence or absence of IL-10R blocking antibody (CDw210a). Frequency of A. TNF-α⁺ and B. IFN-γ⁺ CD4⁺ T cells. Median and data from 4 LCVR and 4 patients are shown. Two-way ANOVA with Šidák post-test. **p < 0.01 and *p < 0.05.