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

Introduction: 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.

Objectives: 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.

Methods: 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.

Results: 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.

Conclusion: 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.