Toll-like receptors regulate B cell cytokine production in patients with diabetes

Abstract

Aims/hypothesis: Understanding cellular and molecular events in diabetes mellitus will identify new approaches for therapy. Immune system cells are important modulators of chronic inflammation in diabetes mellitus, but the role of B cells is not adequately studied. The aim of this work was to define the function of B cells in diabetes mellitus patients through focus on B cell responses to pattern recognition receptors.

Methods: We measured expression and function of Toll-like receptors (TLRs) on peripheral blood B cells from diabetes mellitus patients by flow cytometry and multiplexed cytokine analysis. We similarly analysed B cells from non-diabetic donors and periodontal disease patients as comparative cohorts.

Results: B cells from diabetes mellitus patients secrete multiple pro-inflammatory cytokines, and IL-8 production is significantly elevated in B cells from diabetic patients compared with those from non-diabetic individuals. These data, plus modest elevation of TLR surface expression, suggest B cell IL-8 hyperproduction is a cytokine-specific outcome of altered TLR function in B cells from diabetes mellitus patients. Altered TLR function is further evidenced by demonstration of an unexpected, albeit modest 'anti-inflammatory' function for TLR4. Importantly, B cells from diabetes mellitus patients fail to secrete IL-10, an anti-inflammatory cytokine implicated in inflammatory disease resolution, under a variety of TLR-stimulating conditions. Comparative analyses of B cells from patients with a second chronic inflammatory disease, periodontal disease, indicated that some alterations in B cell TLR function associate specifically with diabetes mellitus.

Conclusions/interpretation: Altered TLR function in B cells from diabetes mellitus patients increases inflammation by two mechanisms: elevation of pro-inflammatory IL-8 and lack of anti-inflammatory/protective IL-10 production.

Fig. 1

Purified B cells from diabetes mellitus patients constitutively secrete elevated levels of IL-8 and decreased levels of IL-10. a Representative re-analysis of B cell purity (upper left quadrant) by flow cytometry. Note monocytes were undetectable (bottom right quadrant). b Cytokine production by B cells from ND (white bars) or diabetes mellitus (black bars) donors incubated in medium for 24 h. GM-CSF, TNF-α and IL-10 are quantified on the left y-axis; IL-6 is quantified on right y-axis. c IL-8 production by B cells from ND (white bar) or diabetes mellitus (black bar) donors incubated in medium for 24 h. Shown is mean and SEM of the number of samples indicated by the value immediately below each bar. Not all supernatant fractions from all samples were assayed for every cytokine. Significance was determined by Mann–Whitney U test and p values that are significant in comparisons between diabetes mellitus and ND values are shown (b ^†p = 0.0152, ^‡p = 0.0017; c ^†p = 0.0081). d, e Intracellular cytokine staining in peripheral blood mononuclear cells from ND (white bars) or diabetes mellitus (black bars) donors. d B cells were identified by surface CD19 expression. e Monocytes were identified by surface CD14 expression (absent on B cells). Shown is mean (after subtraction of isotype control) and SD of the percentage of cytokine-positive cells from three donors. All significant differences, calculated by t test, are indicated (d ^†p = 0.0398, ^‡p = 0.0309). The difference in IL-10 production in panel e approached significance (^†p = 0.0768). B cell intracellular TNF-α and monocyte intracellular IL-8 were highly variable (p>0.07) and hence not definitive. d, e Donors had a diagnosis of type 2 diabetes mellitus and included one black female and two Hispanic males, mean age 56 years. ND donors included two white females and one white male, mean age 47 years

Fig. 2

A modestly elevated percentage of B cells from diabetes mellitus patients express surface TLR4. a A representative flow cytometric analysis of TLR4 expression on the surface of B cells. Isotype control on sample from a representative diabetes mellitus patient is shown in the left panel. Results from TLR4 staining of a representative ND donor and the same diabetes mellitus patient are shown in the middle or right panels, respectively. B cells were identified based on CD19 surface expression (y-axis). Percentage numbers indicate the proportion of B cells that were also TLR4-positive. b Composite data showing percentage of TLR4-positive B cells in ND donors and diabetes mellitus patients as indicated. Each point shows analysis of a single sample; horizontal lines show median percentage TLR4-positive B cells for each cohort. The difference (median = 16.45% or 27.1% for B cells from ND or diabetes mellitus donors, respectively) was significant (p<0.05). n = 56 each for ND and diabetes mellitus samples. c Accessibility of the TLR4 promoter to 2 U micrococcal nuclease in ND monocytes or B cells, or diabetes mellitus B cells as labelled. Human Jurkat T cells are a negative control. d Percentage TLR4-positive monocytes (left) or neutrophils (right) in peripheral blood of ND or diabetes mellitus donors as indicated. n = 29 for ND monocytes; n = 24 for diabetes mellitus monocytes; n = 31 for ND neutrophils; n = 26 for diabetes mellitus neutrophils. Horizontal lines shows medians. Differences were insignificant. e RP105 surface expression levels as measured by MFI on B cells from ND donors or diabetes mellitus patients. Bar shows mean (±SD) of ten samples from each cohort. Difference was insignificant (p>0.1). >95% of B cells in each cohort expressed surface RP105 (not shown). Statistical significance was determined by Mann–Whitney U test for all comparisons, except panel d, which was analysed by ANOVA

Fig. 3

IL-8 is significantly increased in B cells from diabetes mellitus patients vs ND donors responding to TLR2 ligand (Pam3). Purified B cells from diabetes mellitus (black bars) or ND (white bars) donors were incubated for 24 h in medium alone or with TLR4 ligand (rLPS or E. coli LPS) or TLR2 ligand (Pam3) as indicated. Samples were analysed for production of IL-8 (a, b), TNF-α (c), GM-CSF (d), or cytokines indicated below the x-axis (e) in B cell responding to E. coli LPS. Number of samples is indicated by the value immediately below each bar. a, c, d Cytokines measured by multiplex protein analyses. b Percentage of CD19+ B cells that were positive for intracellular IL-8, as compared with isotype control upon stimulation with anti-Igμ. Anti-Igμ induced low but somewhat variable levels of IL-10 and TNF-α in B cell parallel analyses (not shown). Bars show mean and SEM. Medium controls for panel a are shown in Fig. 1c. p values calculated by Mann–Whitney U test are shown above bars for samples that were significantly different (p<0.05; a ^†p = 0.0123; ^‡p = 0.0381; b ^†p = 0.0124). All other comparisons were statistically insignificant (p>0.05), although differences in IL-10 production in panel e approached significance (^†p = 0.0635). Comparison between medium and TLR-stimulated cytokine production indicated significant upregulation for many cytokines in B cells responding to TLR ligand; these values are shown for diabetes mellitus and ND B cell samples in Table 3

Fig. 4

TLR ligands fail to activate IL-10 production by B cells from diabetes mellitus patients. B cells from ND (white bars) or diabetes mellitus (black bars) donors were incubated for 24 h. with stimuli as indicated. a IL-10 production. b Positive control showing B cells from diabetes mellitus patients are capable of producing cytokines in response to the TLR9 ligand CpG ODN 2006. Shown is mean and SEM of the number of samples indicated immediately below each bar. p values calculated by Mann–Whitney U test are shown above bars for samples that were significantly (p<0.05) different between ND and diabetes mellitus cohorts (a ^†p = 0.0023; ^‡p = 0.0040; ^§p = 0.0012)

Fig. 5

TLR4 engagement decreases TLR2-mediated TNF-α production by B cells from diabetes mellitus but not ND donors. Cytokine production by B cells from diabetes mellitus patients (a–d) or ND donors (e–h) stimulated with the TLR2 ligand Pam3 alone or in combination with the TLR4 ligand rLPS. A line connects values from each donor sample. Significance was determined by a non-parametric paired two-tailed t test. a p = 0.0195; b NS; c p = 0.0010; d p = 0.0039; f p = 0.0098; g p = 0.0391; h NS. Difference in panel e approached significance (p = 0.0592). Results are from ten diabetes mellitus or 11 ND donor samples

Fig. 6

Disease influences TLR-mediated B cell cytokine production. Comparison of cytokine production by B cells from diabetes mellitus (white bars) or PD (black bars) patients stimulated by the single TLR ligand indicated on the x-axis. a GM-CSF; b IL-10; c IL-6; d IL-8; e TNF-α. Bars show mean and SEM. Significant differences between values for the two patients cohorts were determined by non-parametric two-tailed t tests and are indicated by p values listed above some graphs (b ^†p = 0.0076, ^‡p = 0.040, ^§p = 0.014; d ^†p = 0.034, ^‡p = 0.040; e ^†p = 0.019. All other differences between B cells from the two cohorts stimulated with the same ligand were statistically insignificant (p>0.05)