Itaconate inhibits ferroptosis of macrophage via Nrf2 pathways against sepsis-induced acute lung injury

Abstract

Itaconate, a metabolite produced during inflammatory macrophage activation, has been extensively described to be involved in immunoregulation, oxidative stress, and lipid peroxidation. As a form of iron and lipid hydroperoxide-dependent regulated cell death, ferroptosis plays a critical role in sepsis-induced acute lung injury (ALI). However, the relationship between itaconate and ferroptosis remains unclear. This study aims to explore the regulatory role of itaconate on ferroptosis in sepsis-induced ALI. In in vivo experiments, mice were injected with LPS (10 mg/kg) for 12 h to generate experimental sepsis models. Differential gene expression analysis indicated that genes associated with ferroptosis existed significant differences after itaconate pretreatment. 4-octyl itaconate (4-OI), a cell-permeable derivative of endogenous itaconate, can significantly alleviate lung injury, increase LPS-induced levels of glutathione peroxidase 4 (GPX4) and reduce prostaglandin-endoperoxide synthase 2 (PTGS2), malonaldehyde (MDA), and lipid ROS. In vitro experiments showed that both 4-OI and ferrostatin-1 inhibited LPS-induced lipid peroxidation and injury of THP-1 macrophage. Mechanistically, we identified that 4-OI inhibited the GPX4-dependent lipid peroxidation through increased accumulation and activation of Nrf2. The silence of Nrf2 abolished the inhibition of ferroptosis from 4-OI in THP-1 cells. Additionally, the protection of 4-OI for ALI was abolished in Nrf2-knockout mice. We concluded that ferroptosis was one of the critical mechanisms contributing to sepsis-induced ALI. Itaconate is promising as a therapeutic candidate against ALI through inhibiting ferroptosis.

Fig. 1. 4-OI significantly alleviates sepsis-induced ALI in vivo.

A Representative images of H&E and Masson staining of lung tissue. Morphology was examined using light microscopy. B Semiquantitative histological scores of lung injury in groups described in panel (n = 6). C Lung wet-to-dry weight ratio and was determined in all groups (n = 6). D ELISA for TNF-α, IL-1β and IL-6 in murine lung tissue (n = 6). E–G Relative levels of TNF-α, IL-1β and IL-6 mRNAs in murine lung tissue (n = 6). H Representative images of immunofluorescence staining for CD68 and DAPI in lung tissue. (Data are presented as Mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 2. 4-OI inhibits ferroptosis in murine lung during sepsis-induced ALI.

A Heatmap of selected ferroptosis associated genes in macrophage between LPS and Itaconate + LPS (4 h) group. B Western blots for GPX4 and PTGS2 in murine lung tissue. C, D Relative mRNA levels of GPX4 and PTGS2 in murine lung tissue (n = 6). E, F Relative iron and MDA levels of murine lung tissue (n = 6). G Representative images of immunohistochemistry staining for 4-HNE in murine lung tissue. (Data are presented as Mean ± SD *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 3. 4-OI inhibits ferroptosis through increasing and activating Nrf2 in vivo during ALI.

A Western blots for Nrf2, GCLM, HO-1 and SLC7A11 in murine lung tissue. B–E Relative mRNA levels of Nrf2, GCLM, HO-1 and SLC7A11 in murine lung tissue (n = 6). F, G Relative levels of GSH and GSH/GSSG ratio in murine lung tissue (n = 6). H, I Representative images of fluorescence probe for ROS and its statistical results (n = 6) in lung tissue. (Data are presented as Mean ± SD *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 4. 4-OI inhibits LPS induced ferroptosis through Nrf2-dependent pathways in THP-1 cell.

A After induced into macrophage-like state through 100 ng/ml PMA (6 h), THP-1 cells were harvested following 4-OI pretreatment (12 h) and LPS (3 h), immunoblotted for the GPX4, Nrf2, GCLM and SLC7A11 level. B, C Relative level of GSH and GSH/GSSG ratio in THP-1 cell lysates. D Relative MDA level in THP-1 cell lysates. E After 4-OI pretreatment (12 h) and LPS (3 h), Trypan Blue staining was used to determine THP-1 cell viability (n = 6). F, G Representative images of fluorescence probe for ROS and its quantitative results in THP-1 cell (n = 6). H, I Western blots for GPX4, Nrf2, GCLM and HO-1 in THP-1 cells transfected with siRNA against Nrf2. J-M Relative mRNA levels of GPX4, Nrf2, GCLM and HO-1 in THP-1 cells with or without si-Nrf2 transfection (n = 6). (Data are presented as Mean ± SD *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 5. The protection of 4-OI for sepsis-induced ALI was abolished in Nrf2-KO mice.

A Representative images of H&E and Masson staining of murine lung tissue. B Representative images of immunohistochemistry staining for 4-HNE in murine lung tissue. C, D Representative images of fluorescence probe for ROS and its quantitative results in murine lung tissue (n = 6). (Data are presented as Mean ± SD *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 6. The inhibition of 4-OI for ferroptosis was abolished in Nrf2-KO mice.

A–C Relative GSH, GSH/GSSG ratio and MDA levels in murine lung tissue (n = 6). D, E Western blots for GPX4, PTGS2, Nrf2, GCLM and SLC7A11 in murine lung tissue. F–I Relative mRNA levels of GPX4, GCLM, SLC7A11 and HO-1 in murine lung tissue (n = 6). J Relative iron levels of murine lung tissue (n = 6). (Data are presented as Mean ± SD *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 7. Graphical abstract of 4-OI alleviate sepsis-induced ALI.

In macrophages, 4-OI inhibited Nrf2 degradation and promoted the transcription of target genes, including SLC7A11, GCLM and GPX4. They contributed to inhibiting the ferroptosis induced by LPS and alleviated sepsis-induced ALI.