Toll-like receptors regulate B cell cytokine production in patients with diabetes
- First Online:
- Cite this article as:
- Jagannathan, M., McDonnell, M., Liang, Y. et al. Diabetologia (2010) 53: 1461. doi:10.1007/s00125-010-1730-z
- 911 Downloads
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.
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.
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.
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.
KeywordsB lymphocytes Cytokines Diabetes mellitus Human Toll-like receptors
Granulocyte/macrophage colony stimulating factor
Mean fluorescence intensity
Rhodobacter sphaeroides lipopolysaccharide
Chronic inflammation characterises a wide range of disorders, including diabetes mellitus, and associates with diabetes mellitus aetiology and co-morbidities such as cardiovascular disease [1, 2, 3]. Inflammation and insulin resistance in diabetes mellitus patients is thought to stem, at least in part, from an ongoing immune response to ligands that include endogenous NEFAs and endotoxin [4, 5, 6].
Cells of the innate immune system sense many endogenous ligands and bacterial products through Toll-like receptors (TLRs). TLRs play important roles in diabetes mellitus, as shown both in humans and in animal models of diabetes mellitus. For example, mice with an inactive Tlr4 gene were significantly less prone to diet-induced insulin resistance [7, 8]. Likewise, inhibition of TLR2 function in mice exposed to a high-fat diet led to improved insulin sensitivity and decreased activation of pro-inflammatory pathways . Furthermore, polymorphisms in TLRs and in members of TLR-downstream signalling pathways that encode hyper- or hypoactive responses predict the development of type 1 and type 2 diabetes [10, 11, 12]. Overall, these studies support the idea that TLR2 and TLR4 activities promote diabetes mellitus. It is generally assumed that cells of the myeloid lineage are predominantly responsible for the demonstrated effect of TLRs in diabetes mellitus. However, B cell TLRs have also been recognised as important mediators of innate immune responses in inflammatory diseases [13, 14].
Human B cells express multiple TLRs and can produce both pro- and anti-inflammatory cytokines in response to TLR ligands [15, 16, 17, 18]. The most commonly studied B cell TLR is TLR9, which mediates B cell response to CpG. B cells from healthy humans, in contrast to mice, generally express little to no surface TLR2 and TLR4 [15, 16, 19, 20]. However, our recent work demonstrated that B cells from chronic inflammatory disease patients, specifically periodontal disease (PD) patients, have elevated responses to TLR2 and TLR4 ligands compared with B cells from healthy donors [13, 19]. These studies also showed that cross-talk between TLRs differentially regulates cytokine production by B cells from patients compared with healthy donors. Overall, this work highlighted the complexity and elegant specificity of the B cell response to TLR ligands, and suggested that B cell TLR expression and downstream cytokine production influences the overall milieu in chronic inflammation. Our studies introduced B cell TLRs and TLR-downstream cytokine production as potential players in chronic non-autoimmune inflammatory disease for the first time. Based on high incidence of PD in diabetes mellitus patients, these studies raised the possibility that B cells may also play roles in the systemic inflammation characterising diabetes mellitus.
The B cell cytokine most commonly implicated in chronic inflammatory diseases is IL-10. IL-10 is generally an anti-inflammatory cytokine that promotes inflammation resolution. B cell IL-10 has specifically been implicated in inflammatory autoimmune diseases characterised by B cell dysfunction. For example, B cells from multiple sclerosis (MS) patients secrete lower levels of IL-10 than B cells from healthy donors, and secretion increases in patients treated palliatively with mitoxantrone. These data suggest that B cell IL-10 production decreases clinical symptoms . IL-10-secreting B cells from healthy donors, but not lupus patients, also block Th1 differentiation . Furthermore, B cell IL-10 is critical for recovery from arthritis and a mouse version of MS (experimental autoimmune encephalitis [23, 24]). Finally, genetic studies have linked elevated IL-10 levels to protection from metabolic syndrome and diabetes mellitus in humans , and thus indicate IL-10 can protect against a wide array of chronic inflammatory diseases. Importantly, recent studies demonstrated that B cells specifically require TLRs to produce the IL-10 that blocks T cell-mediated inflammation . Whether similar B cell TLR responses regulate IL-10 production by B cells from diabetes mellitus patients is untested.
Differences between TLR expression and function in B cells from chronic inflammatory disease patients [13, 27] led us to question how B cell TLRs function in diabetes mellitus patients. We demonstrate that TLR ligands activate B cell cytokine production, most significantly IL-8, in diabetes mellitus vs non-diabetic (ND) donors. Pro-inflammatory cytokine production is likely to be physiologically bolstered by the complete inability of B cells from diabetes mellitus patients to upregulate IL-10 secretion in response to TLR ligands. Surprisingly, the B cell response to combinations of TLR ligands uncovered further disease-associated changes in TLR function. These results indicate that altered TLR function rather than surface expression levels regulate the B cell contribution to diabetes mellitus. The unexpected anti-inflammatory function of TLR4 coupled with signatures more characteristic of pro-inflammatory cells indicates that fundamental alterations in B cell responses play complex, overall pro-inflammatory roles in systemic inflammation in diabetes mellitus.
Description of diabetes mellitus patients used for cytokine analysis (n = 11 total)
Age, years (median and range)
HbA1c, % (median and range)
Duration of diabetes mellitus, years (median and range)
BMI, kg/m2 (median and range)
CRPa, mg/l (median and range)
Insulin usage, n = 8 (percentage of total)
Type 2 diabetes, n = 9 (percentage of total)
Type 1 diabetes, n = 2 (percentage of total)
Males, n = 6 (percentage of total)
White, n = 6 (percentage of total)
African-American, n = 5 (percentage of total)
Description of PD and ND individuals
Age, years (median and range)
Males, n (%)
Race, n (%)
For surface staining, 100 μl whole blood (coded to avoid bias) was labelled with conjugated antibodies purchased from BD Pharmingen (San Diego, CA, USA: anti-CD3, -CD14, -CD19, -CD23, -CD27, -CD38, -CD69, -CD77 and -RP105) or eBioscience (San Diego, CA, USA: anti-TLR4 and isotype controls). Erythrocytes were lysed with 2 ml 1×FACS Lysing Solution for 30 min. Cells were washed with 0.2% (wt/vol.) BSA/PBS and fixed with 2% (wt/vol.) paraformaldehyde, then analysed on a FACSCaliber (BD Biosciences, San Jose, CA, USA) with WinMDI software (J. Trotter, The Scripps Institute, Palo Alto, CA, USA). Intracellular staining was completed on mononuclear fractions incubated in medium or with anti-Igμ for 24 h. Brefeldin A (eBioscience) was added at 21 h. Cells were stained with anti-CD14 or anti-CD19 for 20 min at 4°C, then washed with 0.2% (wt/vol.) BSA/PBS, and treated with Fixation buffer then 1× Permeabilisation buffer (eBioscience). Cells were then stained with anti-TNF-α, anti-IL-10 (eBioscience) or anti-IL-8 (BD Pharmingen), and washed with 1× Permeabilisation buffer. Resuspended cells were analysed on a FACScan (BD Biosciences) with CellQuest (BD Biosciences) and FloJo (Tree Star, Ashland, OR, USA) software.
B cells were negatively isolated with magnetic beads (Miltenyi, Auburn, CA, USA) and rested (1 h 37°C) before stimulation. Cultures were 250,000 cells/250 μl in U-bottom wells. For constitutive cytokine production, B cells were incubated in complete medium (RPMI, 10% [vol./vol.] heat-inactivated FCS) alone. Alternatively, TLR ligands, or anti-Igμ/anti-CD40 were each added to a concentration of 1 μg/ml. TLR ligands (Invivogen; San Diego, CA, USA) were: Pam3CSK4 (Pam3; TLR2 ligand), Rhodobacter sphaeroides lipopolysaccharide (LPS) (rLPS; TLR4 ligand ), CpG oligodeoxynucleotide (ODN) 2006 (TLR9 ligand) and ultrapure E. coli LPS 0111:B4 (TLR4 ligand). Cells were stimulated for 24 h prior to cytokine analysis with an Invitrogen (Carlsbad, CA, USA) kit that measured IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IFN-γ, TNF-α and granulocyte/macrophage colony stimulating factor (GM-CSF) on a Luminex 200 machine (Luminex, Austin, TX, USA). Chromatin accessibility by real-time PCR (CHART-PCR) was completed as published .
The Mann–Whitney U test was used to compare values between cohorts. Paired non-parametric (Wilcoxon) t tests established significance for cytokine production by variously stimulated B cells from the same individual. p < 0.05 established significance. Analyses were performed on Prism (GraphPad Software, La Jolla, CA, USA). Sample numbers varied because of the variable number of B cells isolated per donor.
B cells from diabetes mellitus patients constitutively secrete IL-8
A modestly elevated percentage of B cells from diabetes mellitus patients express surface TLR4
TLR2 and TLR4 ligands regulate B cell cytokine production
Cytokine production by TLR-stimulated B cells
To further test the possibility that IL-8 is uniquely poised for TLR-mediated activation in diabetes mellitus B cells, we expanded our analysis of TLR-responsive cytokines by first measuring cytokine production in B cells responding to the prototypic TLR4 ligand E. coli LPS. Although B cells from diabetes mellitus donors produce significant amounts of IL-8 in response to E. coli LPS (>2 ng/ml on average; Fig. 3a), B cells from both diabetes mellitus and ND donors responded similarly to E. coli LPS (Fig. 3e) as indicated by quantification of GM-CSF, IL-1β and TNF-α production. Note that high variability in values from ND donor B cells prevented direct comparison with IL-8 production by ND B cells (not shown). The overall response of all B cells to E. coli LPS (not shown) was relatively modest (i.e. <200 pg/ml cytokine, Fig. 3e), consistent with the general dogma that human B cells fail to respond vigorously to this TLR4 ligand. This lack of response is probably due to, at least in part, the lack of the LPS delivery molecule CD14 on the B cell surface (data not shown). Importantly, E. coli LPS failed to activate even modest production of IL-10 by B cells from diabetes mellitus donors, instead stimulating levels that were lower than levels produced by ND B cells (Fig. 3e). We conclude that diabetes mellitus-associated differences in B cell TLR function specifically increase IL-8 production (Fig. 3a), but are not ‘generally’ pro-inflammatory as measured by diabetogenic cytokines such as TNF-α and IL-1β . Furthermore, the data suggest that B cells from diabetes mellitus patients are deficient in anti-inflammatory IL-10 production.
TNF-α is uniquely regulated by TLR4 engagement in B cells from diabetes mellitus patients
The TLR ligand response of B cells from diabetes mellitus patients is disease-influenced
Taken together, our data indicate that altered TLR function in B cells from diabetes mellitus patients affects cytokine production, and highlights the elegant specificity of TLR-mediated outcomes of B cell activation in disease. Importantly, surface TLR expression may not be the best predictor of B cell cytokine production in diabetes mellitus. The final outcomes of TLR-activated cytokine production by diabetes mellitus B cells are, on balance, pro-inflammatory: increased IL-8 and decreased IL-10, which may override quantitatively small decreases in TNF-α. Although the precise ratio of pro- to anti-inflammatory cytokines required to promote chronic inflammation is unknown, our data clearly indicate that the B cell contribution to the overall ratio is significantly altered in diabetes mellitus and may, for at least IL-8 and IL-10, rival the importance of monocytes as cytokine producers. The complex contribution of B cells and B cell TLRs to diabetes mellitus inflammation is reminiscent of the mixed contribution of T cells to inflammation in diet-induced obesity, in which CD8 T cells promote pathology, but regulatory T cells block pathology [38, 39, 40].
Elevated IL-8 production by B cells from diabetes mellitus patients was unexpected, but may link B cells to disease pathogenesis based on several studies implicating IL-8 in diabetes mellitus. IL-8 levels are elevated in serum and in the adipose-associated stromal vascular fraction of diabetes mellitus patients, but the cellular source of IL-8 was not identified by these studies [41, 42]. B cell IL-8 may also play a role in IL-8-associated complications of diabetes mellitus, such as vascular disease . This possibility is further supported by data showing palliative treatments that decrease IL-8 levels in vivo appear to have benefits for insulin resistance and cardiovascular risk factors . Taken together, these studies suggest that elevated IL-8 production by B cells from diabetes mellitus patients may have important implications in diabetes pathogenesis. The concomitant decrease in B cell IL-10 production undoubtedly skews the pro- to anti-inflammatory cytokine ratio even further to promote an inflammatory milieu. Although B cells produce lower levels of TNF-α either constitutively or in response to TLR4 ligand (following TLR2 stimulation), the absolute changes in TNF-α production are modest in both assays. Therefore we predict these results hold value as indicators of altered TLR function in diabetes mellitus B cells, but may not identify critical quantitative changes in this diabetes-linked cytokine . The simplistic dogma that TLR-mediated nuclear factor kappa B translocation activates cytokines must be significantly refined to facilitate the design of new diabetes mellitus treatments that exploit knowledge of the highly specific mechanisms that control TLR-activated cytokine production.
Clinical treatments that attempt to decrease inflammation in diabetes mellitus patients have had variable results [46, 47, 48], perhaps caused in part by a lack of appreciation of non-conventional functions of B cells and B cell TLRs defined herein. Apart from the role B cells may play in diabetes mellitus, B cells certainly play significant roles in vaccine responses. Therefore, the current push to exploit TLR ligands in vaccine adjuvants, some of which will inevitably be used in the growing population of diabetes mellitus patients, makes understanding the responses of these patient B cells and changes in B cell TLR function more broadly important. Both treatment and vaccine strategies must take into account the net effect of TLR action, including unexpected sources of TLR activity, to most effectively harness the promise of immune system modulation in chronic inflammatory disease patients.
We thank A. Marshak-Rothstein, R. Corley, M. Clare-Salzler and W. Harnett for manuscript critiques. C. Apovian and A. Bourland generously provided samples for intracellular cytokine analyses. We thank M. Rarick and P. Skolnik from the Center for HIV/AIDS at Boston University Medical Center for use and technical expertise with the multiplex analyser, and G. Denis at the flow core facility at Boston University School of Medicine for expertise in intracellular staining. This work was supported by R01 AI54611 and a Research Grant from the American Diabetes Association (B. S. Nikolajczyk), the Evans Medical Foundation and The Broad Medical Foundation (L. M. Ganley-Leal), DE018917 (H. Hasturk) and DE15566 (T. E. Van Dyke, A. Kantarci and H. Hasturk).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.