Crosstalk Imuno-Estromal em Feridas Crônicas: Papel do Inflamassoma NLRP3 e da Gasdermina D na Indução de Senescência em Fibroblastos.

The chronicity of cutaneous wounds, associated with diabetes mellitus and "Staphylococcus aureus" biofilm colonization, represents a global clinical challenge. Biofilm persistence blocks the resolution of inflammation and traps macrophages in a non-resolving M1 profile. However, the molecular crosstalk linking immune dysfunction to stromal repair failure remains unelucidated. This postdoctoral project aims to investigate whether hyperactivation of the NLRP3 inflammasome pathway and Gasdermin D (GSDMD) pores in M1 macrophages acts as a paracrine trigger for Stress-Induced Premature Senescence (SIPS) in fibroblasts, and whether pharmacological blockade restores tissue repair. To this end, *S. aureus* biofilm-conditioned medium (BCM) will be obtained and applied to RAW 264.7 macrophages. Activation of the NLRP3/Caspase-1/GSDMD axis and the M1/M2 polarization profile will be characterized via Western Blot, confocal microscopy, flow cytometry, qPCR, and ELISA (IL-1β, IL-18, and HMGB1). The paracrine effect on fibroblasts (NIH-3T3) will be mapped using a Transwell system, evaluating senescence markers (SA-β-Gal, p16INK4a, p21CIP1, γ-H2AX), the SASP profile, and cell migration (scratch wound assay). In vitro causality tests and in vivo validation in diabetic (STZ-induced) C57BL/6J mice with infected excisional wounds will involve treatment with the selective NLRP3 inhibitor (MCC950), the GSDMD oligomerization inhibitor (disulfiram), and vancomycin. In this context, we hypothesize that the release of IL-1β and DAMPs via N-GSDMD pores in macrophages induces SIPS in fibroblasts, and that blocking this axis disrupts the inflammatory circuit, thereby promoting re-epithelialization and extracellular matrix deposition. The proposal is notable for its innovative approach to stromal mapping and the potential for the future clinical use of Disulfiram in treating refractory chronic wounds.