Dysregulated Toll-like receptor-induced interleukin-1β and interleukin-6 responses in subjects at risk for the development of type 1 diabetes

Abstract

We tested the hypothesis that altered Toll-like receptor (TLR) signaling may be involved in early stages of type 1 diabetes (T1D). To do so, we analyzed TLR-induced interleukin (IL)-1β and IL-6 responses in freshly isolated peripheral blood mononuclear cells (PBMNCs) from seropositive compared with seronegative subjects. Similar frequencies of myeloid dendritic cells (mDCs), plasmacytoid DCs (pDCs), and monocytes were observed in seropositive and seronegative subjects. Subjects with autoantibodies had increased proportions of monocytes expressing IL-1β ex vivo. Activating PBMNCs with TLR3, TLR4, or TLR7/8 agonists in vitro led to increased percentages of IL-1β-expressing monocytes and mDCs from seropositive versus seronegative subjects. TLR ligation also resulted in a diminished IL-6 response in seropositive individuals as lower frequencies of IL-6-expressing monocytes and mDCs were induced. The dysregulated TLR-induced IL-1β and IL-6 pathways were more readily detectable in children aged <11 years and from 11 to <21 years, respectively, and did not involve altered HbA(1c) or the presence of one or more autoantibodies. Finally, subjects with autoantibodies had lower amounts of serum chemokine (C-X-C motif) ligand 10 compared with autoantibody-negative subjects. Our data may imply that alterations in innate immune pathways are detectable in genetically susceptible individuals and could be linked with the early course of T1D.

FIG. 1.

Frequencies of monocytes and DC subsets in the peripheral blood. PBMNCs isolated from the blood of autoantibody-positive subjects enrolled in the Type 1 Diabetes TrialNet and DAISY studies and autoantibody-negative control subjects stained for surface markers characteristic of monocytes, mDCs, pDCs, or the appropriate isotype control. A: Representative flow images show stained monocytes and DCs from autoantibody-negative vs. autoantibody-positive subjects. B: Frequencies are shown of monocytes and DC subsets from seronegative vs. seropositive subjects. The results are expressed as percentages of cell subtypes from the total PBMNCs. The bars indicate the mean values. (A high-quality color representation of this figure is available in the online issue.)

FIG. 2.

TLR-induced IL-1β expression in monocytes and mDCs. PBMNCs were unactivated or activated with purified LPS, Poly(I:C), and R848 in the presence of brefeldin A, followed by staining with fluorochrome-conjugated mAbs directed against surface markers for monocytes (CD14⁺) and mDCs (CD1C⁺CD19⁻). The cells were fixed, permeabilized, and labeled with fluorochrome-conjugated mAbs against IL-1β or the appropriate isotype controls. Each symbol represents an individual percentage of monocytes (A), or mDCs (B) expressing IL-1β from the total monocytes and mDCs, respectively. The bars indicate mean values. P values were evaluated using the nonparametric Mann-Whitney U test.

FIG. 3.

Correlation between TLR-induced IL-1β in monocytes and mDCs and age. IL-1β responses were evaluated in subjects stratified into three age groups: <11 years (young children), from 11 to <21 years (adolescents), and ≥21 years (adults). Each symbol represents an individual frequency of IL-1β–expressing monocytes (A) and mDCs (B) after activation with LPS. The bars represent the mean values. P values were evaluated using the nonparametric Mann-Whitney U test.

FIG. 4.

TLR-induced IL-6 in monocytes and mDCs. PBMNCs were unactivated or stimulated with purified TLR agonists, followed by staining for intracellular IL-6. Each symbol represents an individual percentage of monocytes (A) or mDCs (B) expressing IL-6 of the total monocytes and mDCs, respectively. The bars indicate the mean values. Significant differences were evaluated using the nonparametric Mann-Whitney U test.

FIG. 5.

Correlation between TLR-induced IL-6 in monocytes and mDCs vs. age. IL-6 responses were evaluated in subjects grouped by age: <11 years (young children), from 11 to <21 years (adolescents), and ≥21 years (adults). Each symbol represents an individual frequency of IL-6–expressing monocytes (upper panel) and mDCs (lower panel) after stimulation with R848. The bars represent the mean values. P values were evaluated using the nonparametric Mann-Whitney U test.

FIG. 6.

Serum cytokine and chemokine expression levels and correlation with autoantibody number. A: Each symbol represents an individual level of the indicated cytokine or chemokine. B: The serum expression level of the indicated cytokine or chemokine was stratified into two groups based on the expression of one and more than one anti-islet autoantibodies. The bars indicate mean values. For statistical comparisons, cytokine concentrations were log-transformed to obtain approximately normally distributed measures and then analyzed using ANOVA.