Differential contribution of possible pattern‐recognition receptors to advanced glycation end product–induced cellular responses in macrophage‐like RAW264.7 cells

Abstract

Advanced glycation end products (AGEs) are considered to be related to the pathogenesis of some inflammatory diseases. AGEs were reported to stimulate the receptor for AGEs (RAGE), which causes inflammatory reactions. However, recently, toll‐like receptors (TLRs), in addition to RAGE, have been reported to be related to AGE‐mediated cellular responses, and it remains unclear which receptor is responsible for AGE recognition. To reveal the role of pattern‐recognition receptors, including TLRs and/or RAGE, in AGE‐mediated cellular responses, we generated macrophage‐like RAW264.7 knockout (KO) cells lacking these receptors by genome editing using the CRISPR/Cas9 system and assessed AGE‐stimulated changes in these cells. Comparison of the established clones suggested that RAGE partially affects the expression of TLRs. In the KO clone lacking TLR4 and TLR2, AGE‐stimulated tumor necrosis factor alpha (TNF‐α) expression and phosphorylation of IκBα, p38, and extracellular signal‐regulated kinase (ERK) were significantly attenuated, suggesting that AGE‐mediated responses are largely dependent on TLRs. On the other hand, on comparison of the AGE‐stimulated responses between the KO clone lacking TLR4 and TLR2, and the clone lacking TLR4, TLR2, and RAGE, RAGE played little role in AGE‐stimulated TNF‐α transcription and ERK phosphorylation. Taken together, this study suggested that AGE‐stimulated inflammatory responses occur mainly through TLRs rather than RAGE.

The role of RAGE in host pathology and crosstalk between RAGE and TLR4 in innate immune signal transduction pathways

“…RAGE‐deficient RAW 264.7 cells revealed that the contribution of RAGE to AGE ( ie , glyceraldehyde‐modified BSA)‐dependent TNF‐α expression was slight, but significantly decreased compared to RAGE‐sufficient control cells 82 . Recognition of AGE in this model was more dependent on TLR2 and TLR4 82 .…”

“…RAGE‐deficient RAW 264.7 cells revealed that the contribution of RAGE to AGE ( ie , glyceraldehyde‐modified BSA)‐dependent TNF‐α expression was slight, but significantly decreased compared to RAGE‐sufficient control cells 82 . Recognition of AGE in this model was more dependent on TLR2 and TLR4 82 . Since it is well established that LPS pretreatment of cells induces a transient state of LPS‐hyporesponsiveness (“tolerance”) upon restimulation (reviewed by 83 ), this partial recognition of AGE by TLR4 may explain how LPS pretreatment can tolerize human monocytes to a second stimulation with AGEs 84 .…”

“…For example, RAGE has been shown to facilitate phagocytosis of DNA into host endosomes and thereby enhance TLR9‐dependent NF‐κB activation 16 . Additionally, RAGE has been shown to regulate the expression of TLR4 and TLR2 in RAW 264.7 cells 82 and the response to TLR ligands in IFN‐γ‐stimulated alveolar macrophages 53 . However, the following section will focus on evidence that supports direct cross‐talk between RAGE ligands and TLRs.…”

Diabetes and Pancreatic Cancer—A Dangerous Liaison Relying on Carbonyl Stress

“…Also called AGER, its name obviously derives from its ability to bind AGEs. Therefore, in addition to exert direct effects brought about by RCS reactions, carbonyl stress can induce indirect biological effects through binding of AGEs to receptors of the innate immune system and induction of a chronic inflammatory response [104]. In particular, AGE binding to RAGE activates transcription factors and redox sensitive signaling pathways leading to reactive oxygen species formation, inflammation, fibrosis, autophagy, proliferation, etc.…”

Modification of proteins by reactive lipid oxidation products and biochemical effects of lipoxidation

“…Recent work has demonstrated that ALEs on albumin that involve neutralization of a positive charge surrounded by acidic residues are also binders of RAGE ( 75), unveiling a novel mechanism for influencing immune function. AGEs and ALEs are essentially damage-associated molecular patterns (DAMPs); AGEs can also interact with other pattern recognition receptors, such as Toll-like receptors (76), and ALEs are considered to be oxidation-specific epitopes with effects on innate immunity (77).…”