Title: HIGH GLUCOSE INDUCES TOLL-LIKE RECEPTOR EXPRESSION IN HUMAN MONOCYTES: MECHANISM OF ACTIVATION Authors
|Dasu, Mohan - UCDMC|
|Devaraj, Sridevi - UCDMC|
|Ling, Zhao - UCD & USDA, ARS, WHNRC|
|Jialal, Ishwarlal - UCDMC|
Submitted to: Diabetes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 25, 2008
Publication Date: July 23, 2008
Repository URL: http://diabetes.diabetesjournals.org/content/early/2008/07/23/db08-0564.full.pdf+html
Citation: Dasu, M.R., Devaraj, S., Ling, Z., Hwang, D.H., Jialal, I. 2008. HIGH GLUCOSE INDUCES TOLL-LIKE RECEPTOR EXPRESSION IN HUMAN MONOCYTES: MECHANISM OF ACTIVATION. Diabetes 57(11):3090-3098, 2008. Interpretive Summary: The major cause of death in type 1 and type 2 diabetes patients is atherosclerosis. The pathogenesis of the accelerated atherosclerosis is multifactorial. Inflammation is pivotal in the development of atherosclerosis. Recent studies have shown that diabetes is a proinflammatory state. We and others have shown that the pro-inflammatory phenotype in diabetes is characterized by elevated plasma C-reactive protein (CRP), cytokines, chemokines, adhesion molecules, monocytic activity etc. Hyperglycemia contributes to vascular complications of diabetes. High glucose has been shown to induce inflammatory cytokines, chemokines, p38 MAPKinase, ROS, PKC, and NF-kB activity in both clinical and experimental systems. Several lines of evidence support a role for oxidative stress in the development of diabetic complications. Diabetics have increased O2- production in monocytes and neutrophils, however, the mechanism of the interactions among these mediators remains unclear. Toll-like receptors (TLRs) recognize conserved pathogen associated molecular patterns (PAMPs) and induce innate immune responses that are essential for host defenses. TLRs are activated by both endogenous and exogenous agonists of microbial and non microbial origin. TLR activation by their agonists triggers a signaling cascade leading to cytokine production and initiation of an adaptive immune response. TLR expression is increased in a plethora of inflammatory disorders including atherosclerosis and diabetes. Some of the endogenous ligands for TLR2 and TLR4 include HMGB1 (high-mobility group B1), biglycan, hyaluronic acid fragments, necrotic cells and SAA (serum amyloid A), AGE (Advanced glycation end products), and extracellular matrix components. Among the TLRs, TLR2 and TLR4 play an important role in atherosclerosis. TLR2 and TLR4 bind to components of the gram positive and gram negative bacteria, respectively. They are expressed in multiple cells and tissues, primarily in monocytes. TLR2 and TLR4 expression is increased in atherosclerotic plaque macrophages and in animal models of atherosclerosis. Plaques of TLR4 knockout mice on a high fat diet show reduced lesion size, lipid content, and macrophage infiltration. TLR2/LDLR-/- and TLR2/ApoE-/- double knock out mice are protected from the development of atherosclerosis. In addition, total loss of MyD88 (Myeloid differentiation factor 88), a common adapter molecule of TLR2 and TLR4 in the cell, results in reduced plaque size, lipid content, inflammation, and plasma IL-1 and TNF-a. The interactions among inflammation, hyperglycemia, and diabetes have clear implications for the immune system. Mohammad et al reported increased TLR2 and TLR4 expression in Type 1 diabetic NOD mice, correlating with increased NF-_B activation in response to endotoxin, and increased pro-inflammatory cytokines. Kim et al using TLR2-/- , TLR4-/- knock outs, and NOD mice have demonstrated that TLR2 senses beta cell death and contributes to the instigation of autoimmune diabetes. Devaraj et al showed increased TLR2 and TLR4 expression, intracellular signaling, and TLR mediated inflammation in monocytes with significant correlation to HbA1c levels in type 1 diabetes patients. Also, Song et al reported increased TLR4 mRNA expression in differentiating adipose tissue of db/db mice. Creely et al showed increased TLR2 expression in the adipose tissue of Type 2 diabetes patients with strong correlates to endotoxin levels. These observations taken together suggest a potential role for TLR2 and TLR4 in the pathology of diabetes with limited mechanistic details. However, data examining the mechanism of increased TLR2 and TLR4 expression in diabetes is unknown. Therefore, this study aimed to test the ability of high glucose, one of the key components of diabetic condition, to induce TLR expression in human monocytes.
Technical Abstract: 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 as well as 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. Methods & Results: High glucose (HG-[15mM]) significantly induced TLR2 and TLR4 expression in THP-1 cells in a time and dose dependent manner (P<0.05). HG increased TLR expression, MyD88, IRAK-1, and NF-kB p65 dependent activation in THP-1 cells. THP-1 cell data was further confirmed using freshly isolated monocytes from healthy human volunteers (n=10). Pharmacological inhibition of PKC activity and NADPH oxidase significantly decreased TLR2 and TLR4 mRNA and protein (P<0.05). Furthermore, PKC-alpha knock down decreased TLR2 by 61% (P<0.05), while inhibition of PKC-delta decreased TLR4 under HG by 63% (P<0.05). Also, knock down of NADPH oxidase subunit p47Phox in THP-1 cells abrogated HG induced TLR2 and TLR4 expression. Additional studies revealed that, knock down of PKC-alpha, PKC-delta, or p47Phox also significantly abrogated HG induced NF-kB activation and inflammatory cytokine secretion, consistent with decreased TLR4 and TLR2 expression. Conclusions: Collectively, these data suggests that HG induces toll like receptor 2 and 4 expression via PKC-alpha and PKC-delta, respectively, by stimulating NADPH oxidase subunit p47Phox in human monocytes.