B cells promote inflammation in obesity and type 2 diabetes through regulation of T-cell function and an inflammatory cytokine profile

Abstract

Patients with type 2 diabetes (T2D) have disease-associated changes in B-cell function, but the role these changes play in disease pathogenesis is not well established. Data herein show B cells from obese mice produce a proinflammatory cytokine profile compared with B cells from lean mice. Complementary in vivo studies show that obese B cell-null mice have decreased systemic inflammation, inflammatory B- and T-cell cytokines, adipose tissue inflammation, and insulin resistance (IR) compared with obese WT mice. Reduced inflammation in obese/insulin resistant B cell-null mice associates with an increased percentage of anti-inflammatory regulatory T cells (Tregs). This increase contrasts with the sharply decreased percentage of Tregs in obese compared with lean WT mice and suggests that B cells may be critical regulators of T-cell functions previously shown to play important roles in IR. We demonstrate that B cells from T2D (but not non-T2D) subjects support proinflammatory T-cell function in obesity/T2D through contact-dependent mechanisms. In contrast, human monocytes increase proinflammatory T-cell cytokines in both T2D and non-T2D analyses. These data support the conclusion that B cells are critical regulators of inflammation in T2D due to their direct ability to promote proinflammatory T-cell function and secrete a proinflammatory cytokine profile. Thus, B cells are potential therapeutic targets for T2D.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

B cells secrete a proinflammatory cytokine profile in response to obesity. (A) Cytokine production by total splenocytes from lean (low-fat diet–fed) or obese (high-fat diet–fed) mice with cells stimulated as indicated. LPS, purified Escherichia coli lipopolysaccharide (TLR4 ligand); Pam3, Pam3CSK4 (TLR2 ligand); BCR, anti-IgM; CD40, α-CD40 antibody. (B) Cytokine production by purified splenic B cells from lean and obese mice with cells stimulated as indicated. n = 6–8 per group. Bars show mean and SEM. Significantly different groups are indicated by *P < 0.05 in comparison of obese and lean group for the same treatment; #P < 0.05, ##P < 0.01, stimulated compared with respective unstimulated (media) control with the same diet group, calculated by two-way ANOVA.

Fig. 2.

Absence of B cells associates with protection from adipose tissue (AT) hypertrophy, insulin resistance, and impaired fasting glucose. (A) Weight gain in lean WT mice (black triangle); lean B cell–null μMT mice (gray triangle); obese WT mice (black circle); obese μMT mice (gray circle). Error bars are obscured by symbols at most points. All lean mice were fed a low-fat diet (LFD), and all obese mice were fed a high-fat diet (HFD). (B) Cell size distribution (by analysis of H&E-stained slides) of epididymal AT adipocytes as indicated. On average, adipocytes were 15% smaller in µMT compared with WT tissue. (C) Leptin secretion from ex vivo epididymal AT organ cultures or (D) in 6-h fasting serum. B–D are results from 16-wk HFD/obese mouse samples. (E) Six-hour fasting blood glucose in obese (circles) or lean (triangles) WT (black) or μMT (gray) mice at 15 wk of diet. (F) Insulin tolerance tests (ITTs) of obese WT (black) and μMT (gray) mice after 10 wk on HFD (circles) or LFD (triangles). Analysis after 15-wk HFD feeding gave similar results. Shown are absolute blood glucose levels. n = 6–8 for each panel, and when appropriate, mean and SEM are shown. *WT and μMT data are significantly different (P < 0.05) by Student t test (C–E) or two-way repeated measures ANOVA (F). In F, area under the curve analysis confirmed differences (P < 0.05).

Fig. 3.

Absence of B cells associates with lower systemic inflammation and an increased percentage of regulatory T cells (Tregs) in response to obesity. (A) Serum cytokines in lean and obese WT and μMT mice as indicated. (B) Cytokines in supernatants from total splenocytes prepared from obese WT and μMT mice and cultured for 40 h either unstimulated (media) or stimulated with α-CD3/α-CD28. For A and B, *μMT are significantly different from WT (P < 0.05); #significant difference between stimulated and unstimulated (P < 0.05). Differences were determined by two-way ANOVA. In μMT mice, increases in T-cell percentages compensate for lack of B cells, indicating that T-cell cytokine production in μMT mice is much lower than shown when calculated on a “per cell” basis. (C) Flow cytometric analysis of Tregs from spleen of the indicated mice. Tregs were identified as CD3+CD4+CD25+Foxp3+ with gating strategy shown in Fig. S6. *Difference (P < 0.05) between WT and μMT mice under same diet conditions; #difference between lean and obese mice of same genotype. (D) Treg analysis from lean (white) or obese (gray) μMT mice from indicated tissues. P values for differences calculated by a two-tailed Student t test are as shown. (E) Relative mRNA expression of the indicated T cell–associated or (F) pro-/anti-inflammatory genes in epididymal AT from obese WT (white) or μMT (gray) mice. *Difference (P < 0.05) between WT and μMT as determined by a two-tailed Student t test. n = 6–8 for all panels.

Fig. 4.

Human B cells, but not monocytes, support T2D-associated Th17 function and thus inflammation. (A) Supernatants from PBMCs (Left); cocultured monocytes, B cells, and T cells (M, B, and T, respectively; Center); or purified CD4+ T cells (T; Right) stimulated as indicated and assayed for IL-17 by ELISA. (B) IL-17 in supernatants from MBT (Left; regraphed from A) or BT cocultures of cells purified from non-T2D or T2D subjects and then stimulated as indicated. (C) IL-17 in supernatants from MBT cocultures (Left; regraphed from A), B cell–depleted PBMCs (Center; CD19−), or B cell–depleted PBMCs with recombinant IL-10 supplementation (Right; CD19−+IL-10). (D) IL-17 in supernatants from MBT (Left; regraphed from A) or MT cocultures of cells from non-T2D or T2D subjects. (E) IL-17 production by MT cultures in the presence of B cells (MBT) or in the presence of B cells physically separated from MT cells by a cytokine-permeable, cell-impermable transwell membrane (MT/B). MBT and MT/B cultures in E used a subset of the samples analyzed in A–C. For all panels, *difference (P < 0.05) between T2D (white bars) and non-T2D (black bars) under the same treatment condition; †difference between purified T cells and both MBT and PBMC results (A); @difference between MBT and BT (B) or CD19−+/− IL-10 PBMCs (C) or MT/B (E) within non-T2D or T2D cohort; $CD19−+/− IL-10 PBMCs results differ within non-T2D or T2D cohort (C). All differences were calculated by three-way ANOVA. IL-17 in all stimulated cultures was elevated compared with relevant unstimulated (media) controls. N for each culture type is indicated below x axis. Differences in MT cocultures from non-T2D and T2D subjects were insignificant (NS; D).