Obesity accelerates age defects in B cells, and weight loss improves B cell function

Abstract

Background: We have previously shown that obesity accelerates age-associated defects in B cell function and antibody production leading to decreased secretion of protective antibodies and increased autoimmunity. We wanted to evaluate if obese adults enrolled in a voluntary weight reduction program had higher protective and lower autoimmune antibody responses similar to those observed in lean adults.

Methods: Experiments were performed using blood isolated from an established cohort of female lean adult and elderly individuals, as well as from the blood of female adults with obesity, before and after a voluntary weight reduction program in which their Body Mass Index (BMI) was reduced 10-34% in 12 months. All participants were vaccinated with the Trivalent Inactivated Influenza vaccine. Serum samples were evaluated for the presence of pro-inflammatory cytokines and adipokines, vaccine-specific antibodies and autoimmune antibodies. We evaluated the composition of the B cell pool by flow cytometry, the expression of RNA for class switch transcription factors and pro-inflammatory markers by qPCR, the in vitro secretion of pro- and anti-inflammatory cytokines and their capacity to induce pro-inflammatory T cells.

Results: Obesity, similar to aging, induced increased serum levels of pro-inflammatory cytokines and autoimmune antibodies, while vaccine-specific antibodies were reduced. In agreement with the serum results, the B cell pool of obese adults and elderly individuals was enriched in pro-inflammatory B cell subsets and was characterized by higher expression of markers associated with cell senescence, higher levels of T-bet, the transcription factor for autoimmune antibodies and lower levels of E47, the transcription factor associated with protective responses to the influenza vaccine. B cells from obese adults and elderly individuals were also able to secrete inflammatory cytokines and support the generation of inflammatory T cells. All these pro-inflammatory characteristics of B cells from obese individuals were significantly attenuated, but not completely reversed, by weight loss.

Conclusions: Although the results from our small observational study show that obesity-induced dysfunctional B cell responses, similar to those occurring during aging, are ameliorated in some but not all obese individuals after weight loss, the effects of body weight loss on mechanistic pathways are largely missing and deserve further investigation.

Keywords: Aging; B cells; Humoral immunity; Influenza vaccination; Obesity.

Fig. 1

Weight loss decreases the serum levels of pro-inflammatory cytokines and adipokines. IL-6 (A), CRP (B), TNF (C) and leptin (D) were detected in serum samples of the 4 groups of participants by ELISA. Mean comparisons between groups were performed by two-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 2

Weight loss is associated with increased serum levels of protective responses to the influenza vaccine and reduced autoimmune antibodies. Serum samples were collected from the 4 groups of individuals. Vaccine-specific antibodies were evaluated by HAI (A). Results show the reciprocal of the titers 4 weeks after vaccination.MDA-specific (B) and dsDNA-specific (C) autoimmune antibodies were measured by ELISA. Mean comparisons between groups were performed by two-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

Obesity and aging induce similar changes in the expression of transcription factors for class switch and these changes can be reversed, at least in part, by weight loss. B cells were isolated from PBMCs using CD19 microbeads and positive selection. B cells (10⁶ cells/ml) were cultured with CpG for 1 day. The mRNA was extracted and qPCR performed to evaluate expression of E47 (A) and T-bet (B) mRNA, respectively. Results show qPCR values which are measures of RNA expression of target genes, relative to the housekeeping gene GAPDH, calculated as 2^−ΔCts. Mean comparisons between groups were performed by two-way ANOVA: *p < 0.05, **p < 0.01, ****p < 0.0001

Fig. 4

Obesity, similar to aging, increases blood frequencies of inflammatory DN B cells, associated with reduced responses to the influenza vaccine and with increased autoimmunity, which are then decreased by weight loss. Unstimulated PBMCs were membrane stained with Live/Dead detection kit and anti-CD45/CD19/CD27/IgD antibodies. Top. Gating strategies to evaluate the major B cell subsets. Bottom. Frequencies of naïve, IgM memory, switched memory and DN B cells from all donors (means ± SE), gated on live CD45 + CD19 + , are shown. Mean comparisons between groups were performed by two-way ANOVA: *p < 0.05, **p < 0.01, ****p < 0.0001

Fig. 5

Weight loss is associated with decreased expression of markers of the senescence-associated secretory phenotype, SASP, in unstimulated B cells. B cells were isolated from PBMCs using CD19 microbeads and positive selection. B cells, left unstimulated, were resuspended in TRIzol, then the RNA was extracted and the expression of SASP markers detected by qPCR to evaluate expression of RNA for pro-inflammatory cytokines, pro-inflammatory miRs, TLRs and cell cycle regulators p16^INK4, p21^CIP1/WAF1. A. qPCR values are measures of RNA expression of target genes, relative to the housekeeping genes GAPDH or U6 (for miRs quantification), calculated as 2^−ΔCts. B. PCA analysis showing the variation among groups explained by PC1 and PC2. Each symbol indicates an individual

Fig. 6

Obesity, similar to aging, increases in vitro secretion of the pro-inflammatory cytokine IL-6 and decreases that of the anti-inflammatory IL-10, and weight loss ameliorates but does not completely reverse this secretory phenotype. B cells were isolated from PBMCs using CD19 microbeads and positive selection. B cells (10⁶ cells/ml) were cultured with CpG for 2 days. IL-6 (A) and IL-10 (B) were measured in culture supernatants by ELISA. Mean comparisons between groups were performed by two-way ANOVA: *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 7

B cells from obese adults and lean elderly individuals, but not from lean adults, support T cell inflammation and this effect is reduced by weight loss. PBMCs were stimulated for 48 h with plate-bound anti-CD3 in the presence or absence of B cells, which were removed by magnetic sorting using CD19 microbeads (< 0.1% B cells in PBMC after selection). IL-17A (top) and IFN-γ (bottom) were measured in culture supernatants by ELISA. Mean comparisons between groups were performed by Student’s t test (two-tailed). **p < 0.01, ***p < 0.001, ****p < 0.0001