High Glucose Induces Toll-Like Receptor Expression in Human Monocytes

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 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.

TLR2 expression and signaling-dependent inflammation impair wound healing in diabetic mice

“…All statistical analyses were performed using GraphPad Prism software (San Diego, CA, USA). 25,26 RESULTS Baseline characteristics of the mice are provided in Table 1. Following STZ injection, as expected, there was significant elevation in glucose levels and decrease in insulin levels when compared with C57BL/6J mice.…”

“…cDNA (50 ng) was amplified using primer probe sets for TLR1, TLR2, TLR4, TLR6, and 18 s (SA Biosciences) following the manufacturer's cycling parameters. Data were calculated using the 2 ÀDDCt method and are presented as ratio of transcripts for TLR gene normalized to 18 s. 25,26 Enzyme-Linked Immunosorbent Assay The release of cytokines, interleukin-1b (IL-1b) and TNF-a were measured in wound tissue lysates and serum using an enzyme-linked immunosorbent assay (ELISA) assay (Millipore, St Charles, MO, USA). Intra-and interassay coefficient of variation (CV) were determined to be o10%.…”

“…Intra-and interassay coefficient of variation (CV) were determined to be o10%. 26 Nuclear/Cytoplasmic Extracts and Transcription Factor Assays Nuclear and cytoplasmic extracts were prepared from wound tissues and normal skin as described previously. 25,26 Nuclear extracts were used to perform NF-kB (cat.…”

“…We used SDS-PAGE to separate bands, and detected TLR2, TLR2-myeloid differentiation factor-88 (MyD88), TRIF, and TRAF-6 using specific antibodies and ECL detection system. 26 Appropriate antibody controls were used for each experiment and no signal was observed. Densitometric ratios were determined as reported earlier from four independent assays.…”

The global diabetes epidemic as a consequence of lifestyle-induced low-grade inflammation

“…Second, TLR4 is the primary receptor on Kupffer cells for LPS leaking from the gut into circulation, which occurs in response to a highfat diet [35]. Third, TLR4 has also been found to be critical in the delivery of pro-inflammatory signals of high glucose concentrations, although it is improbable that glucose directly binds to TLR4 [96]. Direct binding of palmitate to TLR4 could also not be demonstrated [97].…”