The interactions among inflammation, hyperglycemia, insulin resistance, and type 2 diabetes have clear implications for atherosclerosis via the innate immune system. Besides being activators of inflammation under hyperglycemia and insulin resistance (3
), both TLR2 and TLR4 are critical in atherosclerosis (7
). In this study, we provided key evidence for increased monocyte TLR2 and TLR4 expression, activation, cofactor expression, ligands, and downstream signaling contributing to systemic inflammation seen in type 2 diabetic subjects. Our observations significantly add to the emerging role of TLRs in atherosclerosis and diabetes and are consistent with other studies in this context (3
). Increased TLR2 and TLR4 expression is demonstrated in atherosclerotic plaque macrophages and in animal models of atherosclerosis. TLR2/4 knockout mice on C57BL6, ApoE−/−
, and LDLR−/−
background and MyD88 knockout mice show reduced lesion size, lipid content, and macrophage infiltration in plaques and reduced inflammation (22
In two recent studies, we showed increased TLR2 and TLR4 expression, TLR ligands, intracellular signaling, and TLR-mediated inflammation in monocytes with significant correlation to A1C levels in type 1 diabetic patients (11
). Moreover, TLR2/4 expression and activation is increased in human monocytes under hyperglycemia conditions (3
). Reyna et al. (15
) have shown abnormal TLR4 expression in skeletal muscle of a small number of insulin-resistant subjects, with little information on the TLR4-MyD88 signaling, levels of TLR4 cofactors (CD14/MD-2), TLR4 ligand (endotoxin), and its correlation to TLR4 expression. Moreover, the in vitro experiments in this article with myotubes and FFAs lacked appropriate endotoxin controls, and it is not clear if the study subjects were on any cholesterol-lowering medications, adding complexity to the conclusions. Creely et al. (16
) showed increased TLR2 expression in the adipose tissue of type 2 diabetes with strong correlates to endotoxin levels, with no change in TLR4 expression. This study is inconclusive in terms of the lack of TLR4 expression, even under high endotoxin levels, smaller sample size, and minimal patient medication details. Du et al. (24
) in a descriptive study showed that monocytes from LADA, (latent autoimmune diabetes in adults) type 2 diabetic patients on sulfonylurea therapy have significantly higher TLR4 and CD14 expression than healthy control subjects, with no mechanistic details. In this context, our study fills in all the above gaps sequentially: First, we provide data on both TLR2 and TLR4 mRNA/protein levels and critical downstream signaling events that follow their ligation in type 2 diabetes; second, we determined cofactors required for TLR2/4 activation; third, we demonstrated ligands in use for TLR2/4 activation; fourth, notably, we and others have shown that statins, thiazolidinediones, and angiotensin receptor blockers downregulate TLRs and our type 2 diabetic patients are not on any of these drugs; fifth, our TLR data showed significant correlation with major clinical estimates of type 2 diabetes, namely adjusted BMI, glucose, HOMA-IR, FFAs, ligands, cytokines; and finally, data are shown for both MyD88-dependent and -independent signaling pathways, which may be acting collectively.
In addition to the well-characterized microbial ligands, several molecules of the host origin (endogenous) have been proposed to act as ligands for TLR2 and TLR4, including HMGB1, HSPs, and hyaluronan (25
), and were not examined in type 2 diabetes. HMGB1 is considered as a “late” proinflammatory mediator in sepsis. It induces activation of intracellular signaling pathways via TLR2, TLR4, and the receptor for advanced glycation end products (RAGEs), thus acting as an extracellular alarmin. HSPs also act as endogenous ligands of TLR2 and TLR4. HSP60 and HSP70 induce the production of proinflammatory cytokines via activation of TLR2 and TLR4. The inflammatory effects of recombinant human HSP60 were shown to be TLR4 dependent, suggesting that HSP60 may be a TLR4 ligand. Low–molecular weight degradation products of hyaluronan elicit proinflammatory responses in murine alveolar macrophages in rheumatoid arthritis models and other chronic inflammatory conditions. Endotoxin is the most important ligand required for TLR4 activation (14
), and its levels are significantly increased in type 2 diabetic patients. In the present study, we show that type 2 diabetic patients have high circulating levels of HMGB1, HSP60, HSP70, and hyaluronan, which could trigger TLR2 and TLR4 activation, leading to a proinflammatory state by the activation of TLR2/4, synergistically with glucose, FFAs, and endotoxin. Our coimmunoprecipitation studies further suggest an association between TLR2, TLR4, HMGB1, and HSP60.
Dimerization is a major event in the functional activation of TLRs and results in cytokine production (3
). TLR2 activity requires heterodimerization with TLR1 or TLR6 to recognize ligands. Using luciferase reporter assays and real-time RT-PCR, we showed that high glucose induces TLR2 and TLR6 heterodimerization, resulting in NF-κB activation and cytokine production (3
). MD-2 is required for TLR4 ligation with endotoxin (14
). Here, we demonstrate that both TLR6 and MD-2 mRNA expression is increased in type 2 diabetic patients, suggesting their requirement for TLR ligation in type 2 diabetic patients.
TLRs mainly signal through the adapter protein MyD88 via activation of NF-κB, resulting in the increased transcription of inflammation-related genes, such as those encoding indicated cytokines (3
). In addition, a MyD88-independent pathway involving Trif is essential to TLR3 and TLR4 signaling and induces IFN-β. In two recent studies, we showed that activation of the MyD88 pathway is increased in monocytes exposed to high glucose and in type 1 diabetic patients (3
). In the present study, we examined MyD88-dependent and -independent TLR activation, by determining IRF-3 and IFN-β levels as a biological indicator of MyD88-independent activation.
Taken together, the novel findings of this comprehensive study suggest that there is significant elevation of TLR2 and TLR4 protein, mRNA, endogenous ligands, and cofactors in type 2 diabetic patients, which, in concert with hyperglycemia, contributes to the increase in TLR2 and TLR4 signaling that results in the proinflammatory state of type 2 diabetes. In future studies, we will address the mechanism of synergistic effects of hyperglycemia, FFAs, and endogenous ligands on TLR2 and TLR4 expression and signaling.