OBJECTIVEIndividuals with type 2 diabetes have a myriad of metabolic aberrations including increased inflammation, increasing their cardiovascular risk. Toll-like receptors (TLRs) and their ligands play a key role in insulin resistance and atherosclerosis. However, there is a paucity of data examining the expression and activity of TLRs in type 2 diabetes. Thus, in the present study, we examined TLR2 and TLR4 mRNA and protein expression, their ligands, and signaling in monocytes of recently diagnosed type 2 diabetic patients.RESEARCH DESIGN AND METHODSTLR mRNA, protein expression, TLR ligands, and TLR signaling were measured in freshly isolated monocytes from healthy human control subjects (n = 23) and type 2 diabetic subjects (n = 23) using real-time RT-PCR, Western blot, and flow cytometric assays.RESULTSType 2 diabetic subjects had significantly increased TLR2, TLR4 mRNA, and protein in monocytes compared with control subjects (P < 0.05). Increased TLR2 and TLR4 expression correlated with BMI, homeostasis model assessment–insulin resistance (HOMA-IR), glucose, A1C, Nε-(carboxymethyl) lysine (CML), and free fatty acid (FFA). Ligands of TLR2 and TLR4, namely, HSP60, HSP70, HMGB1, endotoxin, and hyaluronan levels, were elevated in type 2 diabetic subjects and positively correlated with TLR2 and TLR4. Type 2 diabetic subjects showed increased MyD88, phosphorylated IRAK-1, Trif, TICAM-1, IRF-3, and NF-κB p65 expression in monocytes compared with control subjects. Furthermore, TLR-MyD88-NF-κB signaling resulted in elevated levels of cytokines (P < 0.05), but increased interleukin (IL)-1β, interferon (IFN)-γ, and endotoxin were not significant when adjusted for BMI.CONCLUSIONSIn this comprehensive study, we make the novel observation that TLR2 and TLR4 expression and their ligands, signaling, and functional activation are increased in recently diagnosed type 2 diabetes and contribute to the proinflammatory state.

OBJECTIVE-Hyperglycemia-induced inflammation is central in diabetes complications, and monocytes are important in orchestrating these effects. Toll-like receptors (TLRs) play a key role in innate immune responses and inflammation. However, there is a paucity of data examining the expression and activity of TLRs in hyperglycemic conditions. Thus, in the present study, we examined TLR2 and TLR4 mRNA and protein expression and mechanism of their induction in monocytic cells under high-glucose conditions. RESEARCH DESIGN AND METHODS-High glucose (15 mmol/l) significantly induced TLR2 and TLR4 expression in THP-1 cells in a time-and dose-dependent manner (P Ͻ 0.05). High glucose increased TLR expression, myeloid differentiation factor 88, interleukin-1 receptor-associated kinase-1, and nuclear factor-B (NF-B) p65-dependent activation in THP-1 cells. THP-1 cell data were further confirmed using freshly isolated monocytes from healthy human volunteers (n ϭ 10). RESULTS-Pharmacological inhibition of protein kinase C (PKC) activity and NADPH oxidase significantly decreased TLR2and TLR4 mRNA and protein (P Ͻ 0.05). Knocking down both TLR2 and TLR4 in the cells resulted in a 76% (P Ͻ 0.05) decrease in high-glucose-induced NF-B activity, suggesting an additive effect. Furthermore, PKC-␣ knockdown decreased TLR2 by 61% (P Ͻ 0.05), whereas inhibition of PKC-␦ decreased TLR4 under high glucose by 63% (P Ͻ 0.05). Small inhibitory RNA to p47Phox in THP-1 cells abrogated high-glucose-induced TLR2 and TLR4 expression. Additional studies revealed that PKC-␣, PKC-␦, and p47Phox knockdown significantly abrogated high-glucose-induced NF-B activation and inflammatory cytokine secretion.CONCLUSIONS-Collectively, these data suggest that high glucose induces TLR2 and -4 expression via PKC-␣ and PKC-␦, respectively, by stimulating NADPH oxidase in human monocytes.

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

Type 2 diabetes (T2DM) is characterized by hyperglycemia, dyslipidemia, and increased inflammation. Previously, we showed that high glucose (HG) induces Toll-like receptor (TLR) expression, activity, and inflammation via NF-B followed by cytokine release in vitro and in vivo. Here, we determined how HG-induced inflammation is affected by free fatty acids (FFA) in human monocytes. THP-1 monocytic cells, CD14 ϩ human monocytes, and transiently transfected HEK293 cells were exposed to various FFA (0 -500 M) and glucose (5-20 mM) for evaluation of TLR2, TLR4, NF-B, IL-1␤, monocyte chemoattractant protein-1 (MCP-1), and superoxide release. In THP-1 cells, palmitate increased cellular TLR2 and TLR4 expression, generated reactive oxygen species (ROS), and increased NF-B activity, IL-1␤, and MCP-1 release in a dose-and time-dependent manner. Similar data were observed with stearate and FFA mixture but not with oleate. Conversely, NADPH oxidase inhibitor treatment repressed glucoseand palmitate-stimulated ROS generation and NF-B activity and decreased IL-1␤ and MCP-1 expression. Silencing TLR2, TLR4, and p47phox with small inhibitory RNAs (siRNAs) significantly reduced superoxide release, NF-B activity, IL-1␤, and MCP-1 secretion in HG and palmitate-treated THP-1 cells. Moreover, data from transient transfection experiments suggest that TLR6 is required for TLR2 and MD2 for TLR4 to augment inflammation in FFA-and glucoseexposed cells. These findings were confirmed with human monocytes. We conclude that FFA exacerbates HG-induced TLR expression and activity in monocytic cells with excess superoxide release, enhanced NF-B activity, and induced proinflammatory factor release.palmitate; hyperglycemia; Toll-like receptor 2; Toll-like receptor 4 THE LEVELS OF PLASMA free fatty acids (FFA) and glucose are elevated in obesity and type 2 diabetes (T2DM) and are implicated in the excess cardiovascular disease (CVD) risk associated with diabetes (15). Cell culture and animal studies have established chronic inflammation in the development and progression of insulin resistance (IR) and T2DM (29). In parallel, epidemiologic studies showed elevated plasma levels of C-reactive protein (CRP), serum amyloid A, cytokines, and chemokines as potential mediators of inflammation and predictors of CVD in T2DM patients (28). Monocytes and macrophages are pivotal cells orchestrating the development of IR and CVD (4). They are closely linked to the chronic inflammatory processes that underline IR, T2DM, and atherosclerosis (27). Proinflammatory factors derived from them have deleterious effects in the development and progression of IR and diabetes (27). Thus the mechanisms by which these metabolic abnormalities (dyslipidemia and hyperglycemia) contribute to systemic inflammation are essential for understanding of the pathophysiology of IR, T2DM, and CVD.Activation of the innate immune system via Toll-like receptors (TLRs) is implicated in a plethora of inflammatory diseases (8,33,36,41). TLRs are evolutionarily conserved pattern recognition recep...

C-reactive protein (CRP) is present in the atherosclerotic plaques and appears to promote atherogenesis. Intraplaque CRP colocalizes with oxidized low density lipoprotein (OxLDL) and macrophages in human atherosclerotic lesions. Matrix metalloproteinase-9 (MMP-9) has been implicated in plaque rupture. CRP promotes OxLDL uptake and MMP induction in vitro; however, these have not been investigated in vivo. We examined the effect of CRP on OxLDL uptake and MMP-9 production in vivo in Wistar rats. CRP significantly increased OxLDL uptake in the peritoneal and sterile pouch macrophages compared with human serum albumin (huSA). CRP also significantly increased intracellular cholesteryl ester accumulation compared with huSA. The increased uptake of OxLDL by CRP was inhibited by pretreatment with antibodies to CD32, CD64, CD36, and fucoidin, suggesting uptake by both scavenger receptors and Fc-g receptors. Furthermore, CRP treatment increased MMP-9 activity in macrophages compared with huSA, which was abrogated by inhibitors to p38 mitogen-activated protein kinase, extracellular signalregulated kinase (ERK), and nuclear factor (NF)-kB but not Jun N-terminal kinase ( JNK ) before human CRP treatment. Because OxLDL uptake by macrophages contributes to foam cell formation and MMP release contributes to plaque instability, this study provides novel in vivo evidence for the role of CRP in atherosclerosis.-Singh, U., M. R. Dasu, P. G. Yancey, A. Afify, S. Devaraj, and I. Jialal. Human C-reactive protein promotes oxidized low density lipoprotein uptake and matrix metalloproteinase-9 release in Wistar rats.