RAGE-TLR4 Crosstalk Is the Key Mechanism by Which High Glucose Enhances the Lipopolysaccharide-Induced Inflammatory Response in Primary Bovine Alveolar Macrophages

Abstract

The receptor of advanced glycation end products (RAGE) and Toll-like receptor 4 (TLR4) are important receptors for inflammatory responses induced by high glucose (HG) and lipopolysaccharide (LPS) and show crosstalk phenomena in inflammatory responses. However, it is unknown whether RAGE and TLR4 can influence each other's expression through a crosstalk mechanism and whether the RAGE-TLR4 crosstalk related to the molecular mechanism of HG enhances the LPS-induced inflammatory response. In this study, the implications of LPS with multiple concentrations (0, 1, 5, and 10 μg/mL) at various treatment times (0, 3, 6, 12, and 24 h) in primary bovine alveolar macrophages (BAMs) were explored. The results showed that a 5 μg/mL LPS treatment at 12 h had the most significant increment on the pro-inflammatory cytokine interleukin 1β (IL-1β), IL-6, and tumor necrosis factor (TNF)-α levels in BAMs (p < 0.05) and that the levels of TLR4, RAGE, MyD88, and NF-κB p65 mRNA and protein expression were upregulated (p < 0.05). Then, the effect of LPS (5 μg/mL) and HG (25.5 mM) co-treatment in BAMs was explored. The results further showed that HG significantly enhanced the release of IL-1β, IL-6, and TNF-α caused by LPS in the supernatant (p < 0.01) and significantly increased the levels of RAGE, TLR4, MyD88, and NF-κB p65 mRNA and protein expression (p < 0.01). Pretreatment with FPS-ZM1 and TAK-242, the inhibitors of RAGE and TLR4, significantly alleviated the HG + LPS-induced increment of RAGE, TLR4, MyD88, and NF-κB p65 mRNA and protein expression in the presence of HG and LPS (p < 0.01). This study showed that RAGE and TLR4 affect each other's expression through crosstalk during the combined usage of HG and LPS and synergistically activate the MyD88/NF-κB signaling pathway to promote the release of pro-inflammatory cytokines in BAMs.

Keywords: TLR4–RAGE crosstalk; alveolar macrophages; glucose; inflammatory; lipopolysaccharide (LPS).

Figure 1

Effects of different LPS treatment times/doses on pro-inflammatory cytokine release in BAMs. (A–C) Effects of various LPS concentrations (0, 1, 5, and 10 μg/mL) on the release of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in BAMs. (D–F) Effects of different LPS treatment times (0, 3, 6, 12, and 24 h) on the release of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in BAMs. Each experiment was carried out at least three times, and the data were displayed using mean ± SD. *, p < 0.05; **, p < 0.01 vs. control group; ^#, p < 0.05; ^##, p < 0.01 vs. other treatment groups; One-way ANOVA.

Figure 2

TLR4, RAGE and their interaction were involved in LPS-induced inflammatory response in BAMs. (A,D) Effects of different LPS concentrations (0, 1, 5, and 10 μg/mL) on the levels of TLR4 and RAGE mRNA expression. (B,E) Effects of different LPS treatment time (0, 3, 6, 12, and 24 h) on the levels of TLR4 and RAGE mRNA expression. (C,F) Effects of pretreatment with FPS-ZM1 and TAK-242 on the levels of TLR4 and RAGE mRNA expression. (G) Comparison of gel images and grayscale for RAGE and TLR4 immunoblot detection. (H–J) The levels of IL-1β, IL-6, and TNF- α in the supernatants of each group. Each experiment was carried out at least three times, and the data were displayed using mean ± SD. *, p < 0.05; **, p < 0.01 vs. control group; ^#, p < 0.05; ^##, p < 0.01 vs. other treatment groups; One-way ANOVA.

Figure 3

RAGE and TLR4 synergistically activated the MyD88/NF-κB signaling pathway in the inflammation caused by LPS. (A,D) Effects of different LPS concentrations (0, 1, 5, and 10 μg/mL) on the levels of MyD88 and NF-κB p65 mRNA expression. (B,E) Effects of different LPS treatment times (0, 3, 6, 12, and 24 h) on the levels of MyD88 and NF-κB p65 mRNA expression. (C,F) Effects of FPS-ZM1 and TAK-242 pretreatment on the levels of MyD88 and NF-κB p65 mRNA expression. (G,H) Comparison of gel images and grayscale for MyD88 and NF-κB p65 immunoblot detection. Each experiment was carried out at least three times, and the data were displayed using mean ± SD. *, p < 0.05; **, p < 0.01 vs. control group; ^#, p < 0.05; ^##, p < 0.01 vs. other treatment groups; One-way ANOVA.

Figure 4

Effects of different glucose concentrations on LPS-induced pro-inflammatory cytokine secretion and the RAGE/TLR4/MyD88/NF-κB p65 pathway in BAMs. (A–D) The impact of NG and HG on the levels of RAGE, TLR4, MyD88, and NF-κB p65 mRNA expression in the presence of different concentrations of LPS (0, 1, 5, and 10 μg/mL). (E–G) The impact of NG and HG on the levels of IL-1β, IL-6, and TNF-α in the presence of various concentrations of LPS (0, 1, 5, and 10 μg/mL). Each experiment was carried out at least three times, and the data were displayed using mean ± SD.*, p < 0.05; **, p < 0.01 vs. control group; ^#, p < 0.05; ^##, p < 0.01 vs. other treatment groups; One-way ANOVA.

Figure 5

RAGE–TLR4 crosstalk regulated the synergism between high glucose and LPS on the inflammatory response in BAMs. (A–D) The levels of RAGE, TLR4, MyD88 and NF-κB p65 mRNA expression in each group. (E–J) The levels of RAGE, TLR4, MyD88 and NF-κB p65 protein expression in each group. (K–M) The levels of IL-1β, IL-6, and TNF-α in each group. Each experiment was carried out at least three times, and the data were displayed using mean ± SD. *, p < 0.05; **, p < 0.01 vs. control group; ^##, p < 0.01 vs. other treatment groups; One-way ANOVA.

Figure 6

Schematic representation of the TLR4–RAGE crosstalk participates in the HG-enhanced LPS-induced inflammation in BAMs (created with BioRender.com). HG firstly upregulated the levels of RAGE and TLR4 genes and protein expression in the BAM membrane and the secretion of pro-inflammatory-related mediators (such as HMGB1, S100A8/9, and AGEs) through the ROS/NF-κB pathway. Then, pro-inflammatory-related mediators bind to RAGE and TLR4, causing RAGE–TLR4 crosstalk (increasing the levels of RAGE and TLR4) to synergistically activate the downstream MyD88/NF-κB signaling pathway and promote the release of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. When HG acts together with LPS, HG upregulates the levels of RAGE and TLR4 genes and protein expression, which can provide more receptor sites for LPS binding, and the LPS also upregulates the levels of RAGE and TLR4 genes and protein expression that can offer more receptor sites for pro-inflammatory-related mediators. The combination of HG and LPS through RAGE–TLR4 crosstalk further activates the downstream MyD88/NF-κB signaling pathway and exacerbates pro-inflammatory cytokine release in BAMs.