Lupeol Attenuates Oxysterol-Induced Dendritic Cell Activation Through NRF2-Mediated Antioxidant and Anti-Inflammatory Effects

Oxysterols such as 7-ketocholesterol (7KCh) contribute to the pathogenesis of autoimmune and chronic inflammatory diseases by inducing oxidative stress and promoting pro-inflammatory immune cell activation. Dendritic cells (DCs) play a central role in maintaining immune tolerance, and their dysregulation is a key driver of autoimmunity. Targeting DCs by using natural compounds offers a promising strategy to restore redox balance and suppress aberrant immune responses. This study investigated the immunomodulatory and antioxidant properties of Lupeol, a natural triterpenoid, in human monocyte-derived DCs exposed to 7KCh. Flow cytometry and cytokine profiling demonstrated that Lupeol preserved the immature, tolerogenic phenotype of DCs by promoting a dose-dependent increase in the anti-inflammatory cytokine IL-10. Lupeol also inhibited the 7KCh-induced upregulation of maturation markers (CD83, CD86) and suppressed the release of pro-inflammatory cytokines IL-1β and IL-12p70. Functionally, Lupeol-treated DCs directed T cell polarization toward an anti-inflammatory and regulatory profile while dampening the inflammatory responses triggered by 7KCh. This immunoregulatory effect was further supported by the decreased secretion of the pro-inflammatory cytokines IL-1β and IL-12p70 in DC culture supernatants. Mechanistic analyses using immunofluorescence showed that Lupeol alone significantly increased nuclear NRF2 levels and upregulated HO-1 expression. Western blot analysis further confirmed Lupeol's ability to activate the KEAP1-NRF2 signaling pathway, as evidenced by increased expression of NRF2 and its downstream target, NQO1. The use of ML385, a selective NRF2 inhibitor, in ROS and cytokine assays supported the involvement of NRF2 in mediating the Lupeol antioxidant and anti-inflammatory effects in DCs. Notably, the oxidative burden induced by 7KCh limited the full activation of NRF2 signaling triggered by Lupeol. Furthermore, docking and MM/PBSA analyses revealed the specific interactions of Lupeol with the kelch domain of KEAP1. These findings suggest that Lupeol may serve as a promising orally available immunomodulatory agent capable of promoting tolerogenic DCs, offering potential applications in autoimmune and other chronic inflammatory diseases.