Phenotypic and Functional Characterization of Double Negative B Cells in the Blood of Individuals With Obesity

Abstract

We have previously shown that obesity is associated with increased secretion of IgG antibodies with anti-self-reactivity. In this paper, we confirm and extend our previous findings. We show that the plasma of individuals with obesity is enriched in autoimmune antibodies whose levels are positively associated with blood frequencies of the subset of Double Negative (DN) B cells, which is the most pro-inflammatory B cell subset. We also show that DN B cells, significantly increased in the blood of obese versus lean individuals, are characterized by higher expression of immune activation markers and of the transcription factor T-bet, both associated with autoimmunity. The removal of DN B cells from the peripheral B cell pool significantly decreases in vitro secretion of anti-self IgG antibodies. These results altogether confirm the crucial role of DN B cells in the secretion of anti-self IgG antibodies in individuals with obesity.

Keywords: B cells; aging; antibody responses; inflammation; obesity.

Figure 1

The plasma of individuals with obesity is enriched in IgG antibodies specific for dsDNA, MDA and adipocyte-derived antigens. Plasma samples were isolated from individuals with obesity and from lean controls. Mean comparisons between groups were performed by Student’s t test (two-tailed). ***p < 0.001, ****p < 0.0001.

Figure 2

The frequencies of DN B cells significantly increase in the blood of obese versus lean individuals. Top. Gating strategies and a representative dot plot from one lean and one obese individual. Bottom. Results show frequencies of the four B cell subsets. Mean comparisons between groups were performed by Student’s t test (two-tailed). ***p < 0.001, ****p < 0.0001.

Figure 3

IgG antibodies specific for dsDNA, MDA, adipocyte-derived antigens are positively associated with blood frequencies of DN B cells. IgG antibodies were measured in plasma as indicated in Figure 1 . DN B cell frequencies were measured by flow cytometry as indicated in Figure 2 . Correlation coefficients and p values are shown for each antibody specificity.

Figure 4

DN B cells are characterized by higher expression of IA markers associated with autoimmunity. (A) Cells were stained to evaluate the expression of several markers of IA on DN B cells from individuals with obesity and from lean controls. Results show mean fluorescence intensity (MFI)± SE for each marker in DN B cells from lean (black line) and obese (red line) individuals (18 individuals/group). (B) PCA analysis with the axes showing the percentage of variation explained by PC1 and PC2. Each symbol indicates an individual. White symbols: lean individuals. Red symbols: obese individuals.

Figure 5

DN B cells are characterized by higher expression of transcription factors associated with autoimmunity. DN B cells were sorted from the peripheral blood of individuals with obesity and of lean controls and left unstimulated. Total RNA was extracted to evaluate by qPCR the expression of transcription factors. Heatmap shows qPCR values (2^-ΔΔCt) of several transcription factors, normalized to GAPDH. Results show average qPCR values from 18 individuals/group.

Figure 6

The removal of DN B cells significantly reduces the secretion of IgG autoimmune antibodies. B cells were isolated with magnetic beads from the blood of four individuals with obesity. Top. Gating strategies to remove DN B cells. B cells were stained with anti-CD27 and anti-IgD antibodies. DN B cells were sorted out using a Sony SH800 cell sorter. Bottom. After stimulation of total B cells and total B cells without DN B cells for 10 days with CpG, supernatants were collected and analyzed for the presence of anti-dsDNA, anti-MDA, and anti-adipocyte IgG by ELISA. **p < 0.01, ***p < 0.001.