4-Octyl itaconate-induced Nrf2 nuclear translocation mitigates Caspase-11-dependent noncanonical macrophage pyroptosis and acute lung injury

In this study, we demonstrated that exogenous administration of 4-OI activated the nuclear translocation of Nrf2 in macrophages. Activated Nrf2 directly bound to the promoter of Casp4 and exerted transcriptional inhibition, thereby specifically blocking the Caspase-11-dependent noncanonical pyroptosis pathway: this effect suppressed the cleavage and activation of GSDMD, the formation of pyroptotic pores, and the release of mature IL-1β p17. In in vivo experiments, 4-OI treatment significantly ameliorated CLP-induced ALI/ARDS in mice, providing a novel molecular target and intervention strategy for the treatment of sepsis-associated lung injury. • 4-OI inhibits Caspase-11-dependent non-canonical pyroptosis via Nrf2 activation, reducing ALI/ARDS severity • 4-OI suppresses macrophage pyroptosis by blocking GSDMD-NT cleavage and IL-1β release in vivo and in vitro. • Activated Nrf2 inhibits Caspase-11 transcription in LPS-stimulated macrophages. • Acod1 ⁻/⁻ mice exhibit exacerbated lung injury, rescued by 4-OI via restoring Nrf2 nuclear translocation. • First evidence linking itaconate derivatives to Caspase-11 inhibition through the Nrf2/KEAP1 axis in ALI/ARDS. Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are severe inflammatory conditions, with mortality rates reaching 40%. A key driver of their pathogenesis is macrophage pyroptosis, which results in excessive inflammation and tissue damage. The aim of this study was to investigate the regulatory effect and underlying mechanism of the itaconate derivative 4-octyl itaconate (4-OI) on macrophage pyroptosis and sepsis-induced ALI/ARDS. The study employed a cecal ligation and puncture (CLP)-induced septic mouse model and LPS-stimulated RAW264.7 cells, and bone marrow-derived macrophages. Pyroptosis was assessed using propidium iodide (PI) staining to detect membrane pore formation, as well as quantitative fluorescence analysis and immunohistochemical quantitative analysis of GSDMD-NT. Additional evaluations included the western blot analysis of pyroptosis-related proteins (GSDMD-NT, IL-1β p17). Mechanistic insights were explored using the Nrf2 inhibitor ML385, Acod1 ⁻/⁻ mice, Casp4 ⁻/⁻ mice, and Nfe2l2 ⁻/⁻ mice, along with a parallel experiment of DOTAP-transfected LPS. Based on the increase induced by LPS stimulation, 4-OI significantly reduced the proportion of PI-positive cells and also decreased the fluorescence expression of GSDMD-NT, thereby confirming its inhibition of pyroptotic pore formation. It alleviated pulmonary edema, cytokine release, and histological damage in CLP-induced septic mice. Mechanistically, Nrf2 specifically inhibited the transcription of Casp4 , thereby reducing Caspase-11-dependent non-canonical macrophage pyroptosis. Casp4 ⁻/⁻ mice and Casp4 siRNA experiments demonstrated that 4‑OI specifically attenuates Caspase‑11‑dependent noncanonical pyroptosis. Mechanistically, experiments in Nfe2l2 ⁻/⁻ mice and with ML385 revealed that this effect is mediated by Nrf2‑dependent transcriptional inhibition of Casp4 . Furthermore, Acod1 deficiency mice exacerbated Caspase‑11‑driven noncanonical pyroptosis. The results demonstrate that 4-OI effectively inhibits Caspase-11-mediated pyroptosis and subsequent inflammation in experimental ALI/ARDS. This effect is mechanistically dependent on the activation of the Nrf2-Caspase-11 axis. The study thus identifies a novel therapeutic strategy whereby 4-OI targets pyroptotic pore formation, offering a potential therapeutic intervention for ALI/ARDS.