Obesity Accelerates Age-Associated Defects in Human B Cells Through a Metabolic Reprogramming Induced by the Fatty Acid Palmitate

Abstract

We have measured the secretion of autoimmune antibodies in plasma samples and in culture supernatants of blood-derived B cells from four groups of individuals: young lean (Y_L), elderly lean (E_L), young obese (Y_O) and elderly obese (E_O). We found secretion comparable in Y_O and E_L individuals, suggesting that obesity accelerates age-associated defects in circulating B cells. To define at least one possible molecular pathway involved, we used an in vitro model in which B cells from Y_L and E_L individuals have been stimulated with the Fatty Acid (FA) palmitate, the most common saturated FA in the human body. The rationale to use palmitate is that there is a chronic increase in circulating levels of palmitate, due to increased spontaneous lipolysis occurring during aging and obesity, and this may induce autoimmune B cells. Results herein show that in vitro incubation of B cells from Y_L and E_L individuals with the FA palmitate induces mRNA expression of T-bet, the transcription factor for autoimmune antibodies, as well as secretion of autoimmune IgG antibodies, with B cells from Y_L individuals looking similar to B cells from E_L individuals, confirming our initial hypothesis. The generation of autoimmune B cells in the presence of the FA palmitate was found to be associated with a metabolic reprogramming of B cells from both Y_L and E_L individuals. These results altogether show the critical role of the FA palmitate in inducing human B cell immunosenescence and show for the first time the importance of metabolic pathways in this process.

Keywords: B cells; aging; autoimmunity; metabolism; obesity.

FIGURE 1

Effect of aging and obesity on the secretion of autoimmune IgG antibodies. Plasma samples were isolated from Y_L, E_L, Y_O and E_O individuals and analyzed by ELISA for the presence of AD-specific (A) or MDA-specific (D) IgG. B cells, isolated from the same individuals in A using magnetic beads, were stimulated for 8 days with CpG, supernatants were collected after 8 days and AD-specific (B) or MDA-specific (E) IgG were measured by ELISA. Correlations of AD-specific (C) and MDA-specific (F) IgG in plasma and culture supernatants. Mean comparisons between groups were performed by two-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Correlations were calculated by bivariate Pearson’s correlation analyses.

FIGURE 2

Effect of aging and obesity on lipid accumulation by B cells. PBMC were isolated from Y_L, E_L, Y_O and E_O individuals and stained first with the Deep Red Neutral Lipid Stain LipidTOX and then with anti-CD45, anti-CD19, and the Live/Dead detection kit. (A) MFI profiles of one representative donor/group. MFI values for negative controls (without the addition of LipidTOX are, respectively: 915 (Y_L), 1214 (Y_O), 837 (E_L), 1025 (E_O). (B) MFI data from all donors. Mean comparisons between groups were performed by two-way ANOVA: **p < 0.01, ****p < 0.0001.

FIGURE 3

Palmitate in vitro stimulates the secretion of AD-specific autoimmune antibodies in B cells from both Y_L and E_L individuals. B cells, isolated from Y_L and E_L individuals using magnetic beads, were stimulated with CpG, alone or in the presence of palmitate, for 8 days. Then supernatants were collected and analyzed by ELISA for the presence of AD-specific (A) or MDA-specific (C) IgG. Naïve B cells, sorted from the blood of tha same Y_L and E_L individuals, were stimulated with CpG/anti-Ig, alone or in the presence of palmitate, for 8 days. Culture supernatants were tested by ELISA for the presence of AD-specific (B) or MDA-specific (D) IgG. Mean comparisons between groups were performed by two-way ANOVA: **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 4

Palmitate in vitro increases mRNA expression of T-bet, the transcription factor for autoimmune antibody production, in B cells from both Y_L and E_L individuals. Total B cells (A) and naïve B cells (B), isolated from the same Y_L and E_L individuals in Figure 3, were stimulated with CpG or CpG/anti-Ig respectively, alone or in the presence of palmitate, for 1 day. Then the mRNA was extracted and qPCR reactions run to evaluate the expression of tbx21. Results show qPCR values (2^−ΔCt) of tbx21, normalized to GAPDH. Mean comparisons between groups were performed by two-way ANOVA: ***p < 0.001, ****p < 0.0001.

FIGURE 5

Palmitate in vitro induces a hyper-metabolic phenotype in B cells from both Y_L and E_L individuals. Total B cells from the same Y_L and E_L individuals in Figures 3, 4 were stimulated with CpG, alone or in the presence of palmitate for 6 h, and evaluated for the mRNA expression of HK-2, LDHA, PDHX and ACACB. Heatmap shows qPCR values (2^−ΔCt) of metabolic markers, normalized to GAPDH, from four individuals/group.

FIGURE 6

Palmitate in vitro induces higher ECAR in B cells from both Y_L and E_L individuals. Total B cells from the same Y_L and E_L individuals in Figures 3, 4 were stimulated with CpG, alone or in the presence of palmitate for 6 h, and seeded into the wells of an extracellular flux analyzer at the concentration of 2 × 10⁵/well in triplicate and run in a mitostress test. Bottom two groups refer to: B cells from Y_L individuals. Top two groups refer to: B cells from E_L individuals.