Spatiotemporal regulation of acute wound healing by the NLRP3 inflammasome: dual roles in macrophage-fibroblast chemotaxis and phenotype during wound repair

Background: The spatiotemporal regulation of inflammatory dynamics is critical for successful wound healing. However, the precise mechanistic role of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome in orchestrating these processes remains incompletely characterized. This study aimed to delineate the specific mechanisms by which NLRP3 governs cellular and molecular events during wound healing. Methods: Multi-omics sequencing data were utilized to profile NLRP3 inflammasome activation dynamics in murine and human acute wound models. Nlrp3-/- mice were generated using CRISPR-Cas9 technology. In vitro and in vivo functional assays were performed to assess NLRP3-dependent regulation of macrophage and fibroblast recruitment, polarization, and phenotype modulation. Results: NLRP3 is predominantly expressed in macrophages and neutrophils during the inflammatory phase of wound healing. Global deletion of Nlrp3 reduces IL-1β, the main downstream effector, attenuates CCL/CXCL chemokine signaling, decreases both inflammatory and pro-reparative cell infiltration, and disrupts the phenotypic switching of macrophages and fibroblasts, collectively delaying wound closure. However, the resulting low-inflammatory microenvironment in Nlrp3-/- mice may upregulate Wnt and Notch signaling early in the repair phase, curbing fibrosis and promoting appendage regeneration. Partial IL-1β blockade in WT mice recapitulates the NLRP3-null phenotype, whereas IL-1β reconstitution in knockout mice accelerates healing but increases fibrosis. Moreover, the NLRP3 protein also modulates fibroblast phenotype independently of inflammasome activation via a ROS-dependent mechanism. Conclusion: NLRP3 exerts dual-phase regulatory roles in wound healing: (i) during inflammation, it drives chemokine-mediated macrophage/fibroblast recruitment and M1 polarization while suppressing fibroblast-mediated repair via IL-1β signaling; (ii) later, NLRP3 deficiency enhances Wnt/Notch signaling, promoting structural restoration despite transiently delayed healing. Moreover, fibroblasts with high NLRP3 expression engage an inflammasome-independent NLRP3/ROS axis that augments activation of TGF-β/Smad signaling. These findings position NLRP3 as a potential therapeutic target for modulating phase-specific inflammatory and regenerative responses.