ROS-TXNIP-NLRP3 inflammasome axis-driven macrophage activation contributes to endothelial dysfunction in Kawasaki...

The role of the reactive oxygen species (ROS)-TXNIP-NLRP3 inflammasome axis in mediating macrophage-endothelial crosstalk during Kawasaki disease (KD) vasculitis remains unclear. This study investigated its involvement using a lipopolysaccharide/adenosine triphosphate (LPS/ATP)-induced tandem model of THP-1 macrophages and human umbilical vein endothelial cells (HUVECs).

Phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 macrophages were stimulated with LPS and ATP to establish an NLRP3 inflammasome activation model. Control, LPS/ATP, LPS/ATP+N-acetylcysteine (NAC), and LPS/ATP+MCC950 groups were enrolled. Macrophage mitochondrial ROS (mtROS), TXNIP protein abundance, TXNIP-NLRP3 association by co-immunoprecipitation, apoptosis-associated speck-like protein containing a CARD (ASC) speck formation, cleaved caspase-1 (p20), and interleukin (IL)-1β/IL-18 secretion were assessed. Conditioned media from each group were subsequently transferred to HUVEC monolayers, and endothelial barrier function (fluorescein isothiocyanate (FITC)-dextran permeability), angiogenic capacity (tube formation assay), and adhesion molecule expression (intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin) were evaluated. IL-1β neutralization was further performed to determine the contribution of macrophage-derived IL-1β to endothelial dysfunction.

LPS/ATP stimulation significantly elevated macrophage mtROS levels and TXNIP expression, enhanced TXNIP-NLRP3 association, promoted NLRP3 inflammasome activation as evidenced by increased ASC speck formation and cleaved caspase-1, and markedly enhanced IL-1β/IL-18 secretion. Consequently, LPS/ATP-conditioned medium impaired HUVEC barrier integrity, suppressed tube formation, and upregulated ICAM-1, VCAM-1, and E-selectin mRNA expression. NAC pretreatment attenuated inflammasome activation by scavenging mtROS, reducing TXNIP expression, and weakening TXNIP-NLRP3 association, whereas MCC950 directly inhibited downstream NLRP3 inflammasome assembly without altering total NLRP3 or TXNIP levels. Both interventions significantly ameliorated macrophage-induced endothelial dysfunction across all measured parameters. Moreover, IL-1β neutralization attenuated LPS/ATP-conditioned medium-induced endothelial inflammatory activation, barrier disruption, VE-cadherin discontinuity, and impaired tube formation.

The ROS-TXNIP-NLRP3 axis represents a critical mechanism underlying macrophage activation-mediated endothelial injury in vitro. NAC and MCC950 attenuated endothelial dysfunction through upstream antioxidant activity and downstream NLRP3 inhibition, respectively. In addition, macrophage-derived IL-1β acted as a key soluble mediator linking inflammasome activation to endothelial barrier disruption and angiogenic impairment. These findings provide mechanistic insight into macrophage-endothelial inflammatory crosstalk in vitro and suggest that the ROS-TXNIP-NLRP3/IL-1β axis warrants further validation in KD animal models before its therapeutic relevance can be established.