Increased toll-like receptor (TLR) 2 and TLR4 expression in monocytes from patients with type 1 diabetes: further evidence of a proinflammatory state

Abstract

Context: Type 1 diabetes (T1DM) is associated with increased cardiovascular mortality. It is a pro-inflammatory state as evidenced by increased circulating biomarkers and monocyte activity. The toll-like receptors (TLRs) are pattern recognition receptors, expressed abundantly on monocytes. TLR2 and TLR4 are important in atherosclerosis. However, there is a paucity of data examining TLR2 and TLR4 expression in T1DM and examining its contribution to the proinflammatory state.

Objective: Thus, we examined TLR2 and TLR4 expression in monocytes from T1DM patients compared with controls (n = 31 per group).

Setting: The study was performed at the University of California Davis Medical Center.

Patients: Healthy controls (n = 31) and T1DM patients (n = 31) were included in the study.

Results: TLR2 and TLR4 surface expression and mRNA were significantly increased in T1DM monocytes compared with controls. Downstream targets of TLR, nuclear factor kappaB, myeloid differentiation factor 88, Trif, and phosphorylated IL-1 receptor-associated kinase were significantly up-regulated in T1DM. Finally, the release of IL-1beta and TNF-alpha was significantly increased in monocytes from T1DM compared with controls and correlated with TLR2 and TLR4 expression (P < 0.005). In addition, TLR2 and TLR4 expression was significantly correlated to glycosylated hemoglobin, carboxymethyllysine, and nuclear factor kappaB (P < 0.02).

Conclusion: Thus, we make the novel observation that TLR2 and TLR4 expression and signaling are increased in T1DM and contribute to the proinflammatory state.

Figure 1

TLR2 and TLR4 expression in T1DM. Surface expression of TLR2 (resting and Pam3CSK4 activated) (A) and surface expression of TLR4 (resting and LPS activated) (B) on human monocytes isolated from control and T1DM patients (n = 31 per group) were assessed by flow cytometry as described in Subjects and Methods. Data are expressed as mean fluorescence intensity units (mfi). *, P < 0.05 vs. control basal; #, P < 0.05 vs. control activated. C, TLR2 and TLR4 mRNA in T1DM. Representative RT-PCR results for TLR2 and TLR mRNA from three different controls and three T1DM patients were assessed as described in Subjects and Methods using GAPDH as control. Lower panel, The TLR2 and TLR4 to GAPDH ratio, respectively, for all control and T1DM patients (n = 31 per group). *, P < 0.05 vs. control.

Figure 2

TLR signaling proteins in T1DM. A, NFKβ activity in control (C) and T1DM monocytes was performed using TransAM reagents as described in Subjects and Methods. *, P < 0.001 compared with controls. B, Representative Western blotting results of TLR downstream signaling proteins MyD88, pIRAK-1, and Trif was performed using specific rabbit antibodies to the respective (phospho)proteins as described in Subjects and Methods using β-actin as loading and internal control for MyD88 and Trif and IRAK for pIRAK-1. C, Control. Densitometric ratios. *, P < 0.05 vs. control. Controls and T1DM, n = 31 per group.