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

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.