Atherosclerosis is respectively correlated with interleukin-6/interleukin-6 receptor (IL6/IL6R) mediated inflammation signaling and macrophages ferroptosis. Nonetheless, the underlying mechanism of IL6/IL6R signaling mediated macrophages ferroptosis in atherosclerosis remains unknown. This study aims to investigate whether IL6/IL6R signaling mediated macrophages ferroptosis through mitochondrial fragmentation and mitophagy impairment. Two human atherosclerotic transcriptomic datasets were used to conduct bioinformatic analysis. In vitro, counting kit-8 (CCK-8) assays, flow cytometry, immunofluorescence staining, malondialdehyde (MDA) and glutathione (GSH) assay kits were employed to evaluate reactive oxygen species (ROS) levels and macrophages ferroptosis. Transmission electron microscopy (TEM), laser confocal microscope and seahorse experiments were used to evaluate changes of mitochondrial morphology and mitochondrial function. Western blotting (WB) was used to quantify key markers of mitophagy and ferroptosis. In vivo, histological stainings and WB were used to determine the effects of IL6R deficiency on atherosclerosis, mitophagy and ferroptosis. Integrated bioinformatic analysis revealed that the IL6 expression could stratify early and advanced plaques. IL6 induced macrophages ferroptosis by increasing ROS and MDA levels, depleting GSH level, promoting lipid peroxidation and suppressing glutathione peroxidase 4 (GPX4) expression. Dynamin-related protein 1 (Drp1) mediated excessive mitochondrial fragmentation in IL6-induced macrophages, resulting in more shortened mitochondria, impaired oxidative phosphorylation (OXPHOS) and ROS accumulation. Activation of mitophagy, the process of mitochondrial fragmentation clearance, could increase GPX4 expression and attenuate the lipid peroxidation level in IL6 induced macrophages. Aggravation of ferroptosis further compromised mitophagy-related proteins expression. Targeting IL6R signaling attenuated atherosclerotic burden in ApoE−/− mice, mitigated mitochondrial fragmentation, suppressed macrophages ferroptosis and promoted mitophagy. In atherosclerosis progression, IL6/IL6R signaling induces susceptibility of macrophages to ferroptosis by exacerbating mitochondrial fragmentation and impairing mitophagy. Ferroptosis further aggravates mitophagy impairment, contributing to a detrimental cycle. Our study supports the rationale of anti-IL6/IL6R signaling interventions in atherosclerosis therapy. IL6 causes macrophages ferroptosis through excessive ROS accumulation, lipid peroxidation and suppressed GPX4 expression. IL6/IL6R signaling induces the activation of p-Drp1Ser616 resulting in mitochondrial fragmentation and mitochondrial dysfunction. Defective mitophagy sensitizes IL6-induced macrophages to ferroptosis and and ferroptosis further aggravates mitophagy impairment, contributing to a detrimental cycle. IL6R deficiency attenuates atherosclerotic plaques by mitigating mitochondrial fragmentation, suppressing macrophages ferroptosis and activating mitophagy in vivo.
Li et al. (Mon,) studied this question.