Aim
This study aimed to investigate the impact of the NOD1/Rip2 signaling pathway on macrophage inflammatory activation and polarity switching in ox-LDL-induced THP-1-derived macrophages.
Conclusion
Our data show that the NOD1/RIP2 signaling pathway regulates the inflammatory activation of macrophages induced by ox-LDL and controls the macrophage polarity switch.
Methods
THP-1-derived macrophages were stimulated with various concentrations (10, 25, or 50 mg/L) of ox-LDL for different durations (8, 16, or 24 h). Quantitative real-time PCR was used to measure the mRNA expression of NOD1, Rip2, IL-10, IL-12, iNOS, and Arg-1. Western blotting was used to determine the protein levels of NOD1 and Rip2. The secretion of TNF-α and MCP-1 in the cell culture supernatants was measured via ELISA. Rip2 siRNA was used to inhibit the NOD1/Rip2 signaling pathway. Oil Red O staining was employed to visualize foam cell formation. CD86, CD80, and CD163 membrane molecules were analyzed via FACS.
Results
After exposure to ox-LDL, the expression levels of NOD1 and Rip2 mRNAs and proteins in THP-1-derived macrophages increased in a dose- and time-dependent manner. This upregulation was accompanied by increased concentrations of TNF-α and MCP-1 in the cell culture supernatants. The effects of NOD1 and Rip2 expression upregulation were mitigated by Rip2 siRNA, as evidenced by decreased concentrations of TNF-α and MCP-1. Furthermore, ox-LDL downregulated the expression of M2 macrophage markers CD163, IL-12, and Arg-1 and upregulated the expression of M1 macrophage markers CD86, CD80, IL-10, and iNOS. The inhibition of Rip2 by siRNA reversed these effects and prevented the formation of foam cells.