Toll-like receptor 4 plays a key role in advanced glycation end products-induced M1 macrophage polarization

Objective: This study was aimed to investigate the role of Toll-like receptor 4 (TLR4) in advanced glycation end products (AGEs)- induced macrophage polarization toward M1. Methods: Isolated primary macrophages were exposed to prepared AGEs at concentrations of 0, 2.5, 5 and 10 mmol/L. Macrophages were also exposed to hydrogen peroxide (H2O2) which provided exogenous reactive oxygen species (ROS). Receptor for AGEs (RAGE) was over-expressed by a vector. Specific siRNA silencing TLR4 and inhibitor TAK-242 were used to pre-treat the macrophages. Intracellular ROS was determined by DCFH-DA. Immunofluorescence staining was used to evaluate the expression of inducible nitric oxide synthase (iNOS) which is the marker of M1 macrophage phenotype. Real-time PCR was used to assess the mRNA expression level of TLR4 and RAGE. Protein expression levels of cytoplasmic RAGE, TLR4, nuclear signal transducers and activators of transcription 1 (STAT1) and phosphorylation levels of cytoplasmic STAT1 were evaluated by Western blotting. ELISA was used to measure concentrations of interleukin 6 (IL6), IL12 and tumor necrosis factor (TNF)alpha in supernatant of cell culture medium of macrophages. Results: AGEs significantly elevated intracellular ROS generation, expression levels of iNOS, cytoplasmic RAGE, TLR4, nuclear STAT1, phosphorylation levels of cytoplasmic STAT1, as well as IL6, IL12 and TNF alpha contents in a concentration-dependent manner. TLR4 silencing and inhibitor pre-treatment reduced expression levels of cytoplasmic RAGE, TLR4, phosphorylation of STAT1 and nuclear STAT1 in AGEsexposed macrophages without affecting RAGE expression and intracellular ROS production levels. RAGE over-expression elevated both ROS and TLR4 expression levels in macrophages. TLR4 expression elevation was also found in H2O2-treat macrophages. Conclusion: AGEs induced macrophage polarization toward M1 via activating RAGE/ROS/TLR4/STAT1 signaling pathway. (C) 2020 Elsevier Inc. All rights reserved.