Delayed disulfiram targeting GSDMD-NETs axis rescues sepsis by limiting bacterial spread and lung injury

Sepsis therapy faces a critical paradox: Gasdermin D (GSDMD)-mediated neutrophil extracellular traps (NETs) exert protective host defense early but drive tissue injury late. Defining the temporal dynamics of intervening in this axis is central to overcoming therapeutic limitations.

In a cecal ligation and puncture (CLP) model recapitulating human sepsis progression, disulfiram (DSF, 80 mg/kg) or vehicle was administered intraperitoneally at 0 h, 4 h, or 8 h postoperatively. A comprehensive multidimensional analysis assessed survival, bacterial burden (lung/blood/peritoneum), cytokines (TNF-α/IL-6/IL-1β), lung injury (histopathology/wet-dry ratio/Evans Blue extravasation), GSDMD activation, NET formation (Sytox-green/CitH3-MPO), and neutrophil function (LY6G⁺ infiltration/viability).

Immediate DSF (0 h) proved detrimental, exacerbating bacterial dissemination (lung↑, P = 0.0036; blood↑, P = 0.0488), hyperinflammation (TNF-α↑, P = 0.0165; IL-6↑, P = 0.0140), and shortening survival (P = 0.0417). Conversely, delayed DSF (8 h) yielded significant benefits, reducing bacterial load (lung↓, P = 0.0002; blood↓, P = 0.0309), suppressing cytokines (TNF-α↓, P = 0.0121; IL-6↓, P = 0.0022), and extending survival (P < 0.0001). Mechanistically, while DSF consistently inhibited GSDMD cleavage and NETs, only delayed intervention attenuated NET-driven microthrombosis, reduced LY6G⁺ infiltration (P = 0.0112), and enhanced neutrophil viability (P = 0.0007).

Targeting the GSDMD-NETs axis is fundamentally timing-dependent. Immediate inhibition disrupts essential host defenses, whereas delayed treatment (8 h post-CLP) preserves bacterial clearance, reduces lung injury, and improves survival. By establishing a therapeutic window, our study provides the first systematic evidence that the success of disulfiram in sepsis depends on when it is administered, offering a clinically actionable framework to guide future biomarker-driven trials and bedside decision-making in critical care.