High glucose induces toll-like receptor expression in human monocytes: mechanism of activation

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 < 0.05). High glucose increased TLR expression, myeloid differentiation factor 88, interleukin-1 receptor-associated kinase-1, and nuclear factor-kappaB (NF-kappaB) p65-dependent activation in THP-1 cells. THP-1 cell data were further confirmed using freshly isolated monocytes from healthy human volunteers (n = 10).

Results: Pharmacological inhibition of protein kinase C (PKC) activity and NADPH oxidase significantly decreased TLR2 and TLR4 mRNA and protein (P < 0.05). Knocking down both TLR2 and TLR4 in the cells resulted in a 76% (P < 0.05) decrease in high-glucose-induced NF-kappaB activity, suggesting an additive effect. Furthermore, PKC-alpha knockdown decreased TLR2 by 61% (P < 0.05), whereas inhibition of PKC-delta decreased TLR4 under high glucose by 63% (P < 0.05). Small inhibitory RNA to p47Phox in THP-1 cells abrogated high-glucose-induced TLR2 and TLR4 expression. Additional studies revealed that PKC-alpha, PKC-delta, and p47Phox knockdown significantly abrogated high-glucose-induced NF-kappaB activation and inflammatory cytokine secretion.

Conclusions: Collectively, these data suggest that high glucose induces TLR2 and -4 expression via PKC-alpha and PKC-delta, respectively, by stimulating NADPH oxidase in human monocytes.

FIG. 1.

A: Representative RT-PCR gel of TLR2 and TLR4 mRNA expression in THP-1 cells after glucose challenge as described in research design and methods. Glucose (5.5 mmol/l) was added to mannitol control. Densitometric values are normalized to 18s rRNA and expressed as mean ratio ± SD. *P < 0.05 vs. mannitol or 5.5 mmol/l glucose; n = 6 experiments. B: TLR2 and TLR4 protein expression was measured in THP-1 after glucose challenge by flow cytometry as described in research design and methods. Glucose (5.5 mmol/l) was added to mannitol control. Values are expressed as MFI/10⁵ cells ± SD. *P < 0.05 vs. mannitol or 5.5 mmol/l glucose; n = 6 experiments.

FIG. 2.

A: TLR2 mRNA expression in THP-1 cells after glucose challenge for indicated time points by real-time RT-PCR as described in research design and methods. Glucose (5.5 mmol/l) was added to mannitol control. Values are expressed as mean ratio ± SD. *P < 0.001 vs. mannitol or 5.5 mmol/l glucose (LG) at 6 h; n = 4 experiments. B: TLR2 protein expression in THP-1 cells after glucose challenge for indicated time points by flow cytometry as described in research design and methods. Glucose (5.5 mmol/l) was added to mannitol control. Values are expressed as MFI/10⁵ cells ± SD. *P < 0.05 vs. mannitol or 5.5 mmol/l glucose (LG); n = 4 experiments. C: TLR4 mRNA expression in THP-1 cells after glucose challenge for indicated time points by real-time RT-PCR as described in research design and methods. Glucose (5.5 mmol/l) was added to mannitol control. Values are expressed as mean ratio ± SD. *P < 0.001 vs. mannitol or 5.5 mmol/l glucose (LG) at 6 h; n = 4 experiments. D: TLR4 protein expression in THP-1 cells after glucose challenge for indicated time points by flow cytometry as described in research design and methods. Glucose (5.5 mmol/l) was added to mannitol control. Values are expressed as MFI/10⁵ cells ± SD. *P < 0.05 vs. mannitol or 5.5 mmol/l glucose (LG); n = 4 experiments.

FIG. 3.

A: Representative Western blot depicting the effect of high glucose on MyD88 and IRAK-1 phosphorylation in THP-1 cells. After culturing cells with high glucose, cells were lysed, and cell lysates were blotted for MyD88, total and phospho–IRAK-1, and β-actin as described in research design and methods (n = 4). B: Representative Western blot showing enhanced expression of MyD88, IRAK-1, and pIRAK-1 in THP-1 cell lysates immunoprecipitated with TLR2 antibody after high-glucose challenge as detailed in research design and methods. β-Actin was used as a loading control, n = 4 experiments. C: Representative Western blot showing enhanced expression of MyD88, IRAK-1, and pIRAK-1 in THP-1 cell lysates immunoprecipitated with TLR4 antibody after high-glucose challenge as detailed in research design and methods. β-Actin was used as a loading control, n = 4 experiments.

FIG. 4.

Inhibition of TLR2 and TLR4 receptors using siRNA ameliorates high-glucose–induced NF-κB p65-dependent DNA binding activity in the nuclear extracts of THP-1 cells by ELISA as described in research design and methods. Values are normalized to milligrams nuclear protein and expressed as means ± SD. *P < 0.05 vs. Sc + high glucose (HG); Sc, scramble control siRNA; TLR2si, TLR2 receptor siRNA; TLR4si, TLR4 receptor siRNA. n = 4 experiments in duplicate. □, Mannitol; , LG (5.5 mmol/l); ▪, HG (15 mmol/l).

FIG. 5.

A: IL-1β concentration in supernatants of THP-1 cells after high-glucose treatment in the absence of TLR2 and TLR4 was measured using ELISA. Values are normalized to milligrams cell protein. *P < 0.05 vs. mannitol; **P < 0.05 vs. Sc + high glucose (HG); Sc, scramble control siRNA; TLR2si, TLR2 receptor siRNA; TLR4si, TLR4 receptor siRNA. n = 4 experiments in duplicate. B: IL-6 concentration in supernatants of THP-1 cells after high-glucose treatment in the absence of TLR2 and TLR4 was measured using ELISA. Values are normalized to milligrams cell protein. *P < 0.05 vs. mannitol; **P < 0.05 vs. Sc + HG; Sc, scramble control siRNA; TLR2si, TLR2 receptor siRNA; TLR4si, TLR4 receptor siRNA. n = 4 experiments in duplicate. C: MCP-1 concentration in supernatants of THP-1 cells after high-glucose treatment in the absence of TLR2 and TLR4 was measured using ELISA. Values are normalized to milligrams cell protein. *P < 0.05 vs. mannitol; **P < 0.05 vs. Sc + HG; Sc, scramble control siRNA; TLR2si, TLR2 receptor siRNA; TLR4si, TLR4 receptor siRNA. n = 4 experiments in duplicate. D: TNF-α IL-1β concentration in supernatants of THP-1 cells after high-glucose treatment in the absence of TLR2 and TLR4 was measured using ELISA. Values are normalized to milligrams cell protein. *P < 0.05 vs. mannitol; **P < 0.05 vs. Sc + HG; Sc, scramble control siRNA; TLR2si, TLR2 receptor siRNA; TLR4si, TLR4 receptor siRNA. n = 4 experiments in duplicate. □, Mannitol; , LG (5.5 mmol/l); ▪, HG (15 mmol/l).

FIG. 6.

A: Effect of PKC inhibitors (calphostin C, chelerythrine, and GF109203X) on TLR2 and TLR4 protein expression in THP-1 cells. After culturing cells with inhibitors and high glucose, cells were washed, labeled, and analyzed by flow cytometry as described in research design and methods. Values are expressed as MFI/10⁵ cells ± SD. *P < 0.05 vs. high glucose; n = 4 experiments. B: Effect of NADPH oxidase inhibitors (apocyanin and DPI) on TLR2 and TLR4 protein expression in THP-1 cells. After culturing cells with inhibitors and high glucose, cells were washed, labeled, and analyzed by flow cytometry as described in research design and methods. Values are expressed as MFI/10⁵ cells ± SD. *P < 0.05 vs. high glucose; n = 4 experiments. C: Inhibition of NADPH oxidase subunit p47Phox and PKC isoforms using siRNAs effects high-glucose–induced TLR2 and TLR4 protein expression of THP-1 cells by flow cytometry as described in research design and methods. Values are expressed as MFI/10⁵ cells ± SD. *P < 0.05 vs. scramble control siRNA + LG; **P < 0.05 vs. scramble + high glucose (HG); p47, p47Phox siRNA; P-α, PKC-α siRNA, P-β, PKC-β siRNA; P-δ, PKC-δ siRNA; +, present; −, absent. n = 4 experiments in duplicate. D: Inhibition of NADPH oxidase subunit p47Phox and PKC isoforms using siRNAs effects high-glucose–induced TLR2 and TLR4 mRNA expression of THP-1 cells by real-time RT-PCR as described in research design and methods. Values are expressed as mean ratio ± SD. *P < 0.05 vs. scramble control siRNA + LG; **P < 0.05 vs. scramble + HG; p47, p47Phox siRNA; P-α, PKC-α siRNA; P-β, PKC-β siRNA; P-δ, PKC-δ siRNA; +, present; −, absent. n = 4 experiments in duplicate. □,TLR2; ▪, TLR4.

FIG. 7.

TLR2 and TLR4 protein expression was measured in freshly isolated and pooled human monocytes from healthy volunteers (n = 3 volunteers/experiment) after glucose challenge by flow cytometry as described in research design and methods. Values are expressed as MFI/10⁵ cells ± SD. *P < 0.05 vs. mannitol or 5.5 mmol/l glucose; n = 4 experiments. □, TLR2; ▪, TLR4.