Interleukin-6 Signaling Mediates Mitochondrial Fragmentation and Mitophagy Impairment to Induce Macrophages Ferroptosis in Atherosclerosis

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.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s10753-025-02359-5.

Highlights

IL6 causes macrophages ferroptosis through excessive ROS accumulation, lipid peroxidation and suppressed GPX4 expression.

IL6/IL6R signaling induces the activation of p-Drp1^Ser616 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.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10753-025-02359-5.

Introduction

Atherosclerosis remains an underlying condition of major cerebrovascular diseases or cardiovascular diseases and the leading cause of morbidity and mortality worldwide [1]. Chronic inflammation drives plaque progression and eventual destabilization [2]. Interleukin-6 (IL6), a pleiotropic proinflammatory cytokine, has emerged as a more promising therapeutic target than C-reactive protein (CRP) in clinical cohorts [3, 4]. Mendelian randomization evidence suggests that variations in the interleukin-6 receptor (IL6R) are causally related to atherosclerosis risk [5]. IL6/IL6R signaling is one of the key mediators linking chronic inflammation to plaque progression. Previous studies have focused primarily on the classical inflammatory response pathways of IL6/IL6R signaling, such as the janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway [3, 6]. Macrophages are among the predominant immune-infiltrating cells within atherosclerotic plaques and play a pivotal role in progression of plaques [7]. However, the mechanisms of IL6/IL6R signaling regulating macrophages in atherosclerosis remains unclear.

Emerging evidence indicates that IL6 derived from macrophages could promote the degradation of glutathione peroxidase 4 (GPX4) to regulate ferroptosis and lipid accumulation [8]. Ferroptosis is characterized by GPX4 inactivation, glutathione depletion, lipid peroxidation and iron overload, which disrupts membrane integrity and ultimately leads to cell death [9–11]. Central to these pathways is the excessive generation of reactive oxygen species (ROS) [12, 13]. ROS not only drive cellular oxidative stress [14] but also further impair antioxidant defense systems, including GPX4, to amplify the cell death process [15–17]. The vicious cycle of ROS overproduction and lipid peroxidation constitutes a critical mechanism linking ferroptosis to vascular injury, macrophages death and the necrotic core in plaques [18–20].

Oxidative stress and ROS are regulated mainly by mitochondrial homeostasis [21]. Mitochondrial dynamics [22] and mitophagy [23] are critical regulators of mitochondrial homeostasis. Dynamin-related protein 1 (Drp1) is a GTPase that orchestrates mitochondrial fission by translocating from the cytoplasm to the outer mitochondrial membrane, where it assembles into ring-like structures that constrict and divide mitochondria [24]. While basal fission is necessary for mitochondrial dynamics, excessive Drp1 activation leads to mitochondrial fragmentation [25], dysfunction of oxidative phosphorylation (OXPHOS) [26], ferroptosis [27] and proinflammatory differentiation of macrophages [28]. In addition, mitophagy is one of the selective removal process of clearing damaged mitochondria and one most recent study showed mitophagy protects against ferroptosis [29]. Nonetheless, the precise mechanism linking IL6/IL6R signaling to mitochondrial homeostasis and ferroptosis in atherosclerosis remains unclear.

Therefore, in this study, we indicated that the effect of the IL6/IL6R signaling pathway on regulating macrophages ferroptosis in atherosclerosis. Our findings showed that IL6/IL6R induced macrophages ferroptosis in atherosclerosis by driving mitochondrial fragmentation and obstructed the clearance of mitochondrial fragmentation by undermining mitophagy. Mitophagy impairment and ferroptosis interacted to drive a detrimental cycle. In vivo, antagonizing IL6R facilitate the attenuation of atherosclerotic lesions, potentially through modulating mitochondrial fragmentation, macrophages ferroptosis and mitophagy.

Materials and Methods

Materials and methods are available in the Supplementary Materials.

Results

IL6 Stratified Early and Advanced Plaques During Atherosclerosis Progression

An integrative analysis of two human carotid plaque transcriptome sequencing datasets (GSE163154, n = 39; GSE28829, n = 29) revealed 446 upregulated differentially expressed genes (DEGs) between advanced plaques and early plaques (Fig. 1A and Supplementary Fig. 1A-D). Protein‒protein interaction (PPI) network of top 50 overlapping DEGs was performed in Fig. 1B. In both datasets, following median dichotomization of expression levels, logistic regression analysis was conducted after initial univariate screening (Supplementary Tables 1 and Supplementary Table 2). IL6 was identified as the central inflammatory hub gene. The risk score, defined as the mean log2 expression of IL6, distinguished advanced plaques and early plaques, and IL6 expression was consistently correlated with advanced plaque stage in both datasets (Fig. 1C, E). The receiver operating characteristic (ROC) curves revealed the high diagnostic value of IL6 in both datasets (GSE163154, AUC = 0.98; GSE28829, AUC = 0.92) (Fig. 1D, F).

Fig. 1.

Bioinformatic analysis revealed an IL6-mediated inflammatory module stratifying human atherosclerotic plaques. (A) Venn diagram of DEGs from GSE163154 and GSE28829. A total of 446 overlapping DEGs were identified. (B) PPI network constructed from the overlapping DEGs. Node color denotes connectivity (degree) from high (red) to low (yellow). (C,E) Box plots showing the IL6 risk score between early and advanced atherosclerotic plaques. Statistical differences were assessed by a two-sided Wilcoxon rank-sum test. (D,F) ROC curves for discriminating the IL6 risk score in early and advanced plaques. In GSE28829, the AUC= 0.923, and in GSE163154, the AUC= 0.976. (G, H) WGCNA module trait heatmap. The rows represent module eigengenes, and the columns represent the relative abundances of 22 immune-cell subsets estimated via CIBERSORT. The cell color depicts the Pearson correlation coefficient (red = positive, blue = negative); the first number is r, and the second is the P value (FDR-adjusted). The blue module eigengenes had the greatest correlation with M0 macrophages and with IL6. The scatter plot within the blue module illustrates the strong relationship between the blue module and gene significance for the trait “Macrophage M0” (cor = 0.8, p< 2 × 10⁻¹⁰). Abbreviations: DEGs, differentially expressed genes; PPIs, protein–protein interactions; WGCNA, weighted gene coexpression network analysis; FDR, false discovery rate; ROC, receiver operating characteristic; AUC, area under the curve

IL6 was Correlated with Macrophages Ferroptosis in Atherosclerosis

Weighted gene coexpression network analysis (WGCNA) was used to gain deeper insights into the association between IL6 and immune infiltration. Module trait mapping revealed that the blue module was most positively correlated with the M0 macrophages subset in GSE163154 (r = 0.80, p < 2e-10; Fig. 1G, H). A soft threshold power of 16 was chosen to ensure a scale-free topology (R²> 0.85) and stabilize the mean connectivity (Supplementary Fig. 2A-C). In the validation dataset GSE28829, the brown module was most positively correlated with the M0 macrophages subset (r = 0.69, p < 4e-05; Supplementary Fig. 3A-C). Therefore, we speculated that IL6 is correlated with the macrophage-centered immune system in atherosclerosis. The heatmaps displayed Spearman correlation coefficients between the expression of the hub genes and the relative abundance of 22 immune cell types (Supplementary Fig. 4A). The functional enrichment analysis of the 446 DEGs highlighted pathways at the intersection of inflammation, oxidative stress, macrophage activation and autophagy, including response to oxidative stress (GO:0006979, p.adj = 2.11e-03), macrophage activation (GO:0042116, p.adj = 2.11e-10), superoxide anion generation (GO:0042554, p.adj = 4.50e-06), lipid and atherosclerosis (hsa05417, p.adj = 1.14e-07), phagosome (hsa04145, p.adj = 1.20e-08), lysosome function (hsa04142, p.adj = 1.47e-08), and cytokine/chemokine signaling (hsa04060, p.adj = 1.47e-04) (Supplementary Fig. 4B, C). On the basis of the above analysis, we further identified 15 differentially expressed ferroptosis genes (FEGs) by mapping the identified DEGs to the ferroptosis gene set (Fig. 2A). FEG-based functional enrichment analysis revealed potential correlations among macrophages, oxidative stress and ferroptosis (Fig. 2B).

Fig. 2.

IL6 mediated excessive ROS accumulation, lipid peroxidation and suppressed GPX4 expression in macrophages. (A) Venn diagram of DEGs from the GSE163154, GSE28829 and ferroptosis datasets. A total of 15 overlapping FEGs were identified. (B) KEGG pathway enrichment of the overlapping FEGs. Bars indicate enriched gene counts. The color depicts the scale of p values. (C) Cell viability assays of macrophages treated with increasing concentrations of IL6. (D) Flow cytometry overlay histograms of ROS fluorescence (FITC channel) in macrophages after 24 h of treatment with the vehicle control, IL6 (30 ng/mL) or IL6 + Fer-1 (30 ng/mL+2 µM). “Positive” is the H₂O₂ reference; “Blank” is the unstained reference. (E) Quantification of the mean fluorescence intensity and (F) percentage of ROS-positive cells. The data are the means ± SDs (n = 3). ns, not significant,*p< 0.05,**p< 0.01,***p< 0.001 (ANOVA). (G) Lipid peroxidation levels of macrophages. Confocal images of lipid peroxidation staining with Lipifluro and nuclei stained. Scale bar= 40 µm. (H) Quantification of the relative MDA concentration (n = 4). *p< 0.05,**p< 0.01 (ANOVA). (I) Quantification of relative GSH activity (n = 4). ****p< 0.0001 (ANOVA). (J-K) Representative western blots and quantification of GPX4 protein levels (mean ± SD, n=3) in macrophages. *p< 0.05 (ANOVA). Abbreviations: IL6, interleukin-6; Fer-1, ferrostatin-1; FEGs, differentially expressed ferroptosis genes; GPX4, glutathione peroxidase 4; KEGG, Kyoto Encyclopedia of Genes and Genomes; FITC, fluorescein isothiocyanate; ROS, reactive oxygen species; ANOVA, one-way analysis of variance; GSH, reduced glutathione; MDA, malondialdehyde; ns, not significant

IL6 Mediated Excessive ROS Accumulation, Lipid Peroxidation To Induce Macrophages Ferroptosis

In order to explore the effects of IL6 signaling on macrophages ferroptosis, the cells were cultivated with different concentrations of IL6 (0, 10, 20, 30, 40 and 50 ng/mL) for 24 h, and the cell viability was measured via cell counting kit-8 (CCK8) assays. The viability of macrophages was reduced in a dose-dependent manner, and the half maximal inhibitory concentration (IC50) of IL6 was calculated to be 30 ng/mL, 95% CI [27.9–32.3 ng/mL] (Fig. 2C). To clarify the impact of IL6 on macrophages ferroptosis, we measured ROS levels in macrophages via flow cytometry (measured via DCFH-DA). As shown in Fig. 2D-F, IL6 increased both the mean fluorescence intensity (FITC channel) of cellular ROS and the percentage of positively stained cells. Ferrostatin-1 (Fer-1) can offset the impact of IL6 on ROS levels. In addition, IL6 increased the intensity of oxidized Liperfluo-positive staining (Fig. 2G) and the concentration of malondialdehyde (MDA) (Fig. 2H) in macrophages, which reflected the level of lipid peroxidation in the cells. Compared with those in the control group, the relative glutathione (GSH) activity (Fig. 2I) and relative GPX4 expression in the IL6-treated macrophages were lower (Fig. 2J, K). Similarly, Fer-1 could offset the impact of IL6 on lipid peroxidation and GPX4 expression. These results indicated that IL6 induced macrophages ferroptosis through promoting ROS accumulation and lipid peroxidation.

IL6/IL6R Signaling Mediated Mitochondrial Fragmentation and Dysfunction by Increasing the Phosphorylation of Drp1 Serine 616

ROS accumulation and lipid peroxidation are the important performances of redox homeostasis in cells. Mitochondrial fragmentation, one of the changes in mitochondrial dynamics, could aggravates redox homeostasis imbalance [27]. To explore the potential mechanisms of IL6-induced mitochondrial fragmentation, macrophages were treated with culture medium, IL6 (30 ng/mL) or IL6 + ziltivekimab (ZVM, an IL6 receptor inhibitor, 20 µg/mL). Compared with control treatment, the phosphorylation of Drp1 serine 637 (p-Drp1^Ser637), an inhibitor of mitochondrial fission, was decreased by IL6 treatment, although the difference was not significant (Fig. 3A and B). Conversely, the phosphorylation of Drp1 serine 616 (p-Drp1^ser616), a promoter of mitochondrial fission, was significant increased by IL6 treatment (Fig. 3A and C, p < 0.05). ZVM treatment effectively reversed these effects (Fig. 3A-C). Morphological alterations were confirmed by MitoTracker staining and transmission electron microscopy (TEM) images. The morphology of the mitochondria displayed a filamentous or tubular pattern, forming interconnected networks in normal cells. In contrast, the mitochondria were shortened, and more punctate staining was observed in the IL6-treated macrophages than in the control macrophages, which indicates mitochondrial fragmentation. ZVM treatment partially restored normal mitochondrial morphology and reduced fragmentation (Fig. 3D). TEM images and quantification of mitochondrial length further validated these findings and revealed shorter and fragmented mitochondria in IL6-treated macrophages than in control cells (Fig. 3E, F, p < 0.0001). Compared with IL6 treatment, ZVM treatment consistently attenuated these structural alterations (Fig. 3E, F, p < 0.01).

Fig. 3.

IL6/IL6R signaling mediated mitochondrial fission resulting in impaired OXPHOS and ROS accumulation by promoting Drp1^Ser616 phosphorylation in macrophages. (A) Representative western blots of p-Drp1^Ser616 (pro-fission), p-Drp1^Ser637 (anti-fission) and total Drp1 in macrophages after 24 h of treatment with vehicle control, IL6 (30 ng/mL) or IL6 + ZVM (30 ng/mL + 20 µg/mL). The total Drp1 expression served as the control. (B-C) Quantification of p-Drp1^Ser637 (B) and p-Drp1^Ser616 (C) relative expression (mean± SD, n = 3). ns, not significant, *p< 0.05, **p< 0.01 (ANOVA). (D) Confocal micrographs stained with MitoTracker Red (mitochondria, red) and Hoechst 33342 (nuclei, cyan). The enlarged drawing highlights the morphology of the mitochondrial network. Scale bar = 200 µm. (E) TEM images of the macrophages ultrastructure (upper images) and enlarged mitochondrial regions (lower images) under the three conditions. Scale bars = 2 µm (upper) and 500 nm (lower). (F) Quantification of mitochondrial length from TEM images (n = 6). **p< 0.01, ****p< 0.0001 (ANOVA). (G) OCR profile measured with the Seahorse experiment. Arrows indicate sequential injections of oligomycin (ATP synthase inhibitor), FCCP (uncoupler), and AA + ROT (complex III/I inhibitors). (H) Quantification of the following OCR parameters: basal respiration, ATP production, maximal respiration, proton leakage, spare capacity, and coupling efficiency (mean ± SD, n = 3). ns, not significant, *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001 (ANOVA). Abbreviations: Drp1, dynamin-related protein 1; IL6, interleukin-6; ZVM, ziltivekimab; ANOVA, one-way analysis of variance; TEM, transmission electron microscopy; OCR, oxygen consumption rate; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; AA+ROT, antimycin A + rotenone

Recent study revealed that mitochondrial dynamics, including oxidative phosphorylation, OXPHOS and the tricarboxylic acid cycle, are involved in mitochondrial bioenergetics [30, 31]. In addition, changes in energy metabolism induced by OXPHOS are correlated with the regulation of ferroptosis [32]. Seahorse experiment was conducted to measure the mitochondrial function of OXPHOS. Compared with those in control cells, the oxygen consumption rate (OCR) analysis revealed impaired mitochondrial respiration in IL6-treated cells, as evidenced by reduced basal respiration (p < 0.0001), ATP production (p < 0.0001), maximal respiration (p < 0.0001), proton leakage (p < 0.01) and spare respiratory capacity (p < 0.01) (Fig. 3G, H). The coupling efficiency showed minor changes and no significant differences among the three groups (Fig. 3G, H). Treatment with ZVM improved these parameters, highlighting the role of anti-IL6R therapy in improving mitochondrial function. Taken together, these results indicate that IL6/IL6R signaling increases the phosphorylation of Drp1^Ser616 and mitochondrial fragmentation to impair mitochondrial morphology and OXPHOS.

Drp1 Silencing Rescued IL6-Induced Mitochondrial Dysfunction and Excessive ROS Accumulation

WB was used to demonstrate the silencing efficiency of Drp1 in macrophages, and quantification analysis revealed that, compared with siNC treatment, siDrp1 treatment significantly reduced Drp1 protein expression (Fig. 4A, B; p < 0.001). The fluorescence of ROS staining and relative fluorescence intensity demonstrated that Drp1 silencing attenuated IL6-induced ROS accumulation in macrophages (Figure C, D). TEM images and quantitative analysis revealed that Drp1 silencing alleviated mitochondrial fragmentation and increased mitochondrial length in IL6-exposed macrophages (Fig. 4E, F). OCR analysis further revealed that Drp1 silencing significantly rescued basal respiration (p < 0.001) and ATP production (p < 0.0001) in IL6-exposed macrophages (Fig. 4G, H). However, the maximal respiration, proton leakage, spare capacity, and coupling efficiency were not significantly rescued by Drp1 silencing (Fig. 4G, H). Overall, these findings indicate that IL6 induces excessive ROS production and oxidative stress via Drp1-dependent mitochondrial fragmentation.

Fig. 4.

Drp1 silencing obstructed IL6-induced mitochondrial fragmentation and respiratory dysfunction in macrophages. (A) Representative western blots of Drp1 in macrophages transfected with either a negative control (si-NC) or siRNA targeting Drp1 (si-Drp1). β-Actin served as the control. (B) Quantification of relative Drp1 expression (mean ± SD, n = 3).***p<0.001 (unpaired two-tailed t test). (C) Bright-field (upper) and ROS fluorescence (green; lower) micrographs. Scale bar = 40 µm. (D) Relative fluorescence intensity of ROS in macrophages. (E) TEM image of the mitochondrial ultrastructure. The yellow arrowheads in the upper panels represent the mitophagy structure, and the lower panels show enlarged images of the mitochondrial morphology. Scale bars: upper = 2 µm, lower = 500 nm. (F) Quantification of mitochondrial length from TEM images (n = 6). ns, not significant, *p< 0.05, **p< 0.01 (ANOVA). (G) OCR profile measured with the Seahorse experiment in THP-1-derived macrophages treated with the above three interventions. Arrows denote sequential injections of oligomycin, FCCP, and AA + ROT. (H) Quantification of the following OCR parameters: basal respiration, ATP production, maximal respiration, proton leakage, spare capacity, and coupling efficiency (mean ± SD, n = 3). ns, not significant, *p< 0.05; **p< 0.01, ***p< 0.001, ****p< 0.0001 (ANOVA). Abbreviations: IL6, interleukin-6; Drp1, dynamin-related protein 1; ROS, reactive oxygen species; ANOVA, one-way analysis of variance; TEM, transmission electron microscopy; OCR, oxygen consumption rate; FCCP, carbonyl cyanide-p-trifluoromethoxy-phenylhydrazone; AA+ROT, antimycin A + rotenone; ns, not significant

IL6/IL6R Signaling Obstructed Clearance of Mitochondrial Fragmentation by Mediating the Interaction Between Mitophagy and Ferroptosis

Excessive fragmented mitochondria are removed by mitophagy in normal tissues, while mitophagy impairment exacerbates the pathological accumulation of fragmented mitochondria [33]. Mitophagy impairment also exacerbates ferroptosis [34, 35], in turn, ferroptosis can further compromise mitophagy function [36]. To examine how ferroptosis and mitophagy interact in IL6-induced macrophages, IL6, mitoquinone (MitoQ) (a mitophagy activator) + IL6 and MitoQ + erastin (an activator of ferroptosis) + IL6 were respectively added to the cells. The treatment with MitoQ efficiently increased the mitophagy related protein expression and the GPX4 expression in IL6 treated macrophages (Fig. 5A-F). This result indicated that activation of mitophagy could mitigate IL6 induced macrophages ferroptosis. Additionally, the activation of ferroptosis by erastin eliminated part of the upregulation of P62, Pink1, Parkin and LC3II/I by MitoQ in IL6 treated macrophages. This implied that the worsening of ferroptosis may further compromise mitophagy, contributing to a detrimental cycle in IL6-induced macrophages. Consistently, liperfluo staining revealed that the treatment with MitoQ efficiently inhibited the lipid peroxidation in IL6 treated macrophages, but the activation of ferroptosis by erastin aggravated intracellular lipid peroxidation (Fig. 5G). These results indicated that an intracellular interaction between mitophagy and ferroptosis in in IL6-induced macrophages.

Fig. 5.

IL6/IL6R signaling obstructed clearance of mitochondrial fragmentation by mediating the interaction between mitophagy and ferroptosis. (A) Western blots of P62, Pink1, Parkin, GPX4 and LC3BI/II in macrophages treated with IL6, MitoQ+ IL6 (1 μM+30 ng/mL) or MitoQ+ erastin+ IL6 (1 μM+10 μM+30 ng/mL). β-Actin served as the loading control. (B–F) Quantification of P62 (B), Pink1 (C), Parkin (D), GPX4 (E) relative expression and the LC3B II/I ratio (F) (mean ± SD, n = 3). ns, not significant, *p< 0.05; **p< 0.01 (ANOVA). (G) Lipid peroxidation levels of macrophages. Confocal images of lipid peroxidation staining with Lipifluro and nuclei stained. Scale bars = 40 µm. (H) Oil Red O staining of lipid droplets in macrophages treated with culture medium, IL6 (30 ng/mL) or IL6 + ZVM (30 ng/mL+ 20 µg/mL). Scale bars = 40 µm. (I) Transwell assay and representative micrographs of macrophages treated with culture medium, IL6 (30 ng/mL) or IL6 + ZVM (30 ng/mL+ 20 µg/mL). Scale bars =100 µm. (J–M) Western blots and quantification of Parkin (J, K), Pink1 (J, L) and Tom20 (J, M) in macrophages treated with culture medium, IL6 (30 ng/mL) or IL6 + ZVM (30 ng/mL+ 20 µg/mL). β-Actin served as the loading control (mean ± SD, n = 3). ns, not significant, *p< 0.05, **p< 0.01, ****p< 0.0001 (ANOVA). Abbreviations: ANOVA, one-way analysis of variance; P62, sequestosome 1; LC3B, light chain 3B; Pink1, PTEN-induced kinase 1; Parkin, RBR E3 ubiquitin-protein ligase; GPX4, glutathione peroxidase 4; MitoQ, mitochondrion-targeted ubiquinone; ZVM, ziltivekimab

We further investigated the effects of antagonizing IL6/IL6R signaling on macrophages function and mitophagy. Oil Red O staining revealed increased lipid droplet accumulation in the IL6-treated cells compared with the control cells, which was alleviated by ZVM (Fig. 5H). Transwell assays revealed that ZVM treatment reduced macrophages migration induced by IL6 (Fig. 5I). Compared with the control, IL6 treatment markedly suppressed the relative expression of Parkin (Fig. 5J, K; p < 0.001), Pink1 (Fig. 5J, L; p < 0.05) and Tom20 (Fig. 5J, M; p < 0.05), whereas ZVM treatment reversed the reduction of Parkin and Pink1 expression. Theses results indicated that antagonizing IL6R could facilitate the activation of mitophagy and amelioration of macrophages function.

IL6R Deficiency Attenuated Atherosclerotic Plaques by Mitigating Mitochondrial Fragmentation, Suppressing Macrophages Ferroptosis and Activating Macrophages Mitophagy

To investigate the role of anti-IL6/IL6R signaling in atherosclerotic plaques, an IL6R-knockdown mouse model was generated via intravenous tail injection of AAV-shIL6R into ApoE^−/− mice (Fig. 6A). Considering IL6 is associated with cognitive impairment in vivo [37], we first assessed the cognitive abilities using the novel object recognition (NOR) test (Fig. 6B). Compared with the control, shIL6R improved cognitive function in high fat diet (HFD) fed ApoE^−/− mice (Fig. 6B, p < 0.01). IL6R immunohistochemical staining within aortic plaques also revealed a significant reduction trend in shIL6R groups (Fig. 6C; p < 0.01). Besides, we also assessed the four plasma lipid levels, including triglyceride (TG), cholesterol (CHO), low density lipoprotein (LDL) and high density lipoprotein (HDL). As the results showed, shIL6R-treated could reduce the plasma TG (Fig. 6D, p < 0.0001), CHO (Fig. 6D, p < 0.05) and LDL (Fig. 6D, p < 0.01), but shIL6R-treated only slightly increased the plasma HDL and the difference was not statistically significant (Fig. 6D, ns). Moreover, the plaque area (Fig. 6E, F; p < 0.01), lipid deposition area (Fig. 6E, G; p < 0.01), necrotic core area (Fig. 6E, H; p < 0.001), and collagen content area (Fig. 6E, I; p < 0.0001) were suppressed by IL6R knockdown. The α-SMA + staining area presented an increasing trend, but the difference was not significant in shIL6R-treated mice (Fig. 6E, J). These results indicate that shIL6R can attenuate atherosclerotic plaque burden, reduce lipid accumulation and the number of necrotic cores and increase fibrous cap stability. Furthermore, TEM were used to assessed the mitochondrial morphology and length of macrophages in the atherosclerotic plaque of mice arteries (Fig. 6K). The results demonstrated that the HFD-fed induced macrophages ferroptosis and mitochondrial fragmentation, characterized by small size, shrinkage of mitochondria, loss or increase density of mitochondrial cristae and membrane, which are the morphological feature of ferroptosis. Whereas, both mitochondrial morphology (Fig. 6K) and length (Fig. 6L, p < 0.001) were also restored by shIL6R treatment. Immunofluorescence staining revealed that, compared with shNC, shIL6R-treated reduced CD68⁺ macrophages infiltration (red channel) and increased GPX4 expression (green channel) within aortic plaques (Fig. 6M). Quantitative colocalization of fluorescence profiling revealed greater overlap between CD68 and GPX4 in the shIL6R group than in the control group, indicating the inhibition of ferroptosis (Fig. 6N). IL6R deficiency also promoted the mitophagy related proteins expression (Fig. 6O, P). These findings suggest that the IL6R deficiency could inhibit macrophages ferroptosis and improved mitophagy in atherosclerosis.

Fig. 6.

Targeting IL6R attenuated atherosclerotic plaques by suppressing macrophages ferroptosis and enhancing macrophages mitophagy. (A) Experimental design. Six-week-old male ApoE^-/- mice received a tail-vein injection of AAV-shNC (control) or AAV-shIl6R before being fed a HFD. After 12 weeks HFD-fed, the mice were sacrificed for analysis (n = 6). (B) Trajectory and heatmap plots of the novel object recognition test and quantification of the discrimination index in the novel object recognition test.**p< 0.01 (unpaired two-tailed t test). (C) Immunohistochemistry of IL6R in aortic root lesions and quantification of the IL6R positive staining area. The results showed the efficiency of knockdown with shIL6R.**p< 0.01 (unpaired two-tailed t test). (D) Quantification of four plasma lipid levels, including TG, CHO, LDL and HDL. *p< 0.05, **p< 0.01, ****p< 0.0001 (unpaired two-tailed t test). (E-J) H&E, Oil Red O, Masson’s trichrome, and α-SMA staining of aortic root plaques (E). IL6R knockdown reduced the plaque area (F), lipid deposition (G), necrotic core area (H) and collagen content (I). The α-SMA+ positive area (J) presented an increasing trend, but the difference was not significant (mean± SD, n = 3). ns, not significant, **p< 0.01, ***p< 0.001, ****p< 0.0001 (unpaired two-tailed t test). (K) TEM image of the mitochondrial ultrastructure in the atherosclerotic plaque of mice arteries. The yellow arrowheads represent the mitochondria structure in macrophages of mice arteries. Scale bars: 1 μm. (L) Quantification of mitochondrial length from TEM images (n = 6). ns, not significant, ***p< 0.001 (unpaired two-tailed t test). (M) Immunofluorescence of aortic-root lesions. Sections were stained for macrophages (CD68, red), ferroptosis markers (GPX4, green) and nuclei (DAPI, blue). Scale bars: left: 100 μm; right: 40 μm. (N) The fluorescence intensity profiles of aortic root lesions in the shNC and shIL6R groups. (O) Representative western blots of mitophagy-related proteins (Pink1, Parkin, P62, and LC3B II/I) in aortic root lesions. β-Actin served as the loading control. (P) Quantification of Pink1, Parkin, and P62 relative expression and the LC3B-II/LC3B-I ratio (mean ± SD, n = 3). *p< 0.05, **p< 0.01, ****p< 0.0001. (unpaired two-tailed t test). Abbreviations: AAV, adeno-associated virus; HFD, high-fat diet; TG, triglyceride; CHO, cholesterol; LDL, low density lipoprotein; HDL, high density lipoprotein; P62, sequestosome 1; LC3B, light chain 3B; Pink1, PTEN-induced kinase 1; Parkin, RBR E3 ubiquitin-protein ligase; GPX4, glutathione peroxidase 4

Discussion

An increasing number of clinical studies have confirmed that treatments targeting the IL6/IL6R signaling pathway are safe and effective in treating inflammatory diseases [38], cardiovascular diseases [39] and tumors without hindering immunity [40]. In the present study, we investigated the mechanism underlying IL6/IL6R signaling-induced macrophages ferroptosis and mitochondrial dysfunction in atherosclerosis. In vitro, we indicated that IL6/IL6R signaling mediated macrophages ferroptosis by Drp1-dependent mitochondrial fragmentation and ROS accumulation.

Mitophagy impairment reduced the clearance of fragmented mitochondria and exacerbated ferroptosis in IL6-induced macrophages. Ferroptosis, in turn, aggravated mitophagy impairment. In vivo, IL6R deficiency attenuated macrophage ferroptosis, activated mitophagy and reduced atherosclerotic plaque burden. Consequently, intervention targeting IL6/IL6R signaling is crucial for exploring effective prevention and treatment modalities of atherosclerosis.

To initiate our study, we conducted the bioinformatics analysis on both early and advanced atherosclerotic plaque. We found that IL6 expression was correlated with advanced plaque and had a good diagnostic value for advanced plaque. In addition, IL6 regulates macrophages activation [41, 42], T-cell balance [43, 44], B-cell antibody production and neutrophil recruitment [45] through autocrine or paracrine pathways to drive a self-perpetuating inflammatory cascade and link innate and adaptive immunity, profoundly shaping the vascular immune microenvironment. Among these immune systems, we demonstrated that IL6 was most positively correlated with the macrophage-centered immune system in atherosclerosis. Macrophages represent the most abundant and functionally active immune cell population within atherosclerotic plaques [46]. Macrophages death contributes to the formation of necrotic cores in atherosclerosis [7]. A recent study revealed that IL6/IL6R signaling determines the inflammatory metabolic pathways of macrophages and ultimately leads to macrophages sensitivity to cell deaths [47]. Macrophages ferroptosis was been confirmed as a pivotal mechanism in the pathology of the necrotic core [48]. In this study, IL6 was identified as one of the FEGs in advanced atheroslcerotic plaque. IL6 induced the accumulation of ROS and MDA, as well as the consumption of GSH in macrophages. IL6 also promoted the generation of lipid peroxidation and disrupted the expression of GPX4 in macrophages. Consistent with our findings, W. Cai et al. observed that IL6 could promote ferroptosis via ubiquitylation of GPX4 in nonalcoholic fatty liver disease [8]. Our results revealed that IL6 may mediate the macrophages sensitivity to ferroptosis in atherosclerosis.

Ferropotosis is regulated by oxidative stress and is characterized by abnormal mitochondrial dynamics [49] or abnormal mitochondrial dysfunction [50]. Mitochondrial dynamics are driven by the continuous processes of fusion and fission. Drp1 activation is pivotal for mitochondrial division and is accompanied by the dephosphorylation of Drp1^ser637 and the phosphorylation of Drp1^ser616 [51]. Hyper-activation of Drp1 and excessive mitochondrial fission contribute to mitochondrial fragmentation [52]. Drp1-dependent mitochondrial fragmentation is a new hallmark of cardiovascular disease. Inhibiting mitochondrial fission has been reported to alleviate atherosclerosis through regulating macrophages polarization [53]. Our study revealed that IL6/IL6R signaling mediated the dephosphorylation of Drp1^ser637 and the phosphorylation of Drp1^ser616 in macrophages, which resulted in mitochondrial fragmentation, which further exacerbated ROS accumulation and impaired OXPHOS. It has been reported that macrophages clusters that exhibit low OXPHOS increase ferroptosis sensitivity in atherosclerosis [54]. We further confirmed that silencing of Drp1 could inhibit IL6-induced ROS accumulation and energy metabolism impairment. The above results collectively indicated that IL6/IL6R signaling mediated mitochondrial fragmentation and dysfunction by increasing the phosphorylation of Drp1^Ser616, ultimately contributing to macrophages ferroptosis.

Effective mitophagy can clear fragmented mitochondria to maintain normal cellular redox homeostasis and protect against cell deaths [55, 56]. Conversely, mitophagy impairment results in the defective clearance of damaged mitochondria and further amplifies cell death, such as ferroptosis [34]. Similarly, in this study, it was found that IL6 induced mitophagy impairment of macrophages. Meanwhile, the worsening of ferroptosis may further compromise mitophagy, contributing to a detrimental cycle in IL6-induced macrophages. Correspondingly, mitophagy activation could mitigate IL6 induced macrophages ferroptosis. These alterations emphasized that defective mitophagy sensitizes IL6-induced macrophages to ferroptosis. IL6R is a cross-disease immune signatures in multi cardiovascular diseases [57]. Additionally, it has been reported that IL6R antagonism ameliorates atherosclerosis linked to Tet2-related clonal haematopoiesis [58]. Consistent with our in vitro experiments, IL6R deficiency also attenuated macrophage ferroptosis, activated mitophagy and reduced atherosclerotic plaque burden in vivo. Our findings provided additional support for the therapeutic potential of IL6R antagonism in atherosclerosis.

Limitations

This study also presents certain limitations. First, incorporating macrophage-specific knockout mice models or human pluripotent stem cells (hIPS)-derived macrophages will be essential to more comprehensively dissect the regulatory complexity of atherosclerosis. Second, while IL6R deficiency showed therapeutic benefits in our mouse model, the 12-week experimental period may not be sufficient to evaluate potential long-term immunosuppressive consequences. Finally, it remains to be further comprehensively investigated whether Drp1 inhibition or mitophagy activation could mitigate susceptibility of macrophages to ferroptosis in IL6R deficiency mice.

Conclusions

Based on the above results observed in vivo and in vitro, we elucidates that IL6/IL6R signaling induces susceptibility of macrophages to ferroptosis by exacerbating mitochondrial fragmentation. There are crosstalk between mitophagy and ferroptosis in IL6-induced macrophages. Moreover, targeting IL6R effectively attenuated the progression of atherosclerosis. These results further support the potential clinical utility of anti-IL6R agents, such as ziltivekimab and tocilizumab.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

IL6

Interleukin-6

IL6R

Interleukin-6 receptor

GPX4

Glutathione peroxidase 4

Drp1

Dynamin-related protein 1

ROS

Reactive oxygen species

DEGs

Differentially expressed genes

PPI

Protein‒protein interaction

ROC

Receiver operating characteristic

FEGs

Differentially expressed ferroptosis genes

WGCNA

Weighted gene coexpression network analysis

PMA

Phorbol 12-myristate 13-acetate

CCK8

Cell counting kit-8

IC50

Half maximal inhibitory concentration

Fer-1

Ferrostatin-1

MDA

Malondialdehyde

GSH

Glutathione

ZVM

Ziltivekimab

WB

Western blot analysis

p-Drp1^ser616

Phospho-Drp1 serine 616

P-Drp1^ser637

Phospho-Drp1 serine 637

TEM

Transmission electron microscopy

OXPHOS

Oxidative phosphorylation

OCR

Oxygen consumption rate

P62

Sequestosome 1

LC3B

Light chain 3B

Pink1

PTEN-induced kinase 1

Parkin

RBR E3 ubiquitin-protein ligase

α-SMA

α-smooth muscle actin

AAV

Adeno-associated virus

HFD

High-fat diet

HE

Hematoxylineosin

Author Contributions

Minhui Li: Writing original draft, Investigation, Visualization, Validation, Project administration, Methodology, Formal analysis, Data curation, Conceptualization, Writing - review & editing. Lin Zheng: Writing original draft, Visualization, Validation, Software, Resources, Methodology. Xiao Tang: Review & editing, Investigation, Validation, Resources, Methodology, Data curation. Haifeng Liu: Review & editing, Investigation, Validation, Resources, Methodology, Data curation. Xiaotong Qi: Visualization, Software, Formal analysis. Zhenyu Zhou: Methodology, Investigation, Data curation. Jiaqi Zhu: Methodology, Investigation, Data curation. Changpo Lin: Methodology, Investigation, Data curation. Yongbin Shi: Review & editing, Conceptualization.Tonglei Han: Review & editing, Supervision, Data curation, Conceptualization. Hanfei Tang: Review & editing, Supervision, Project administration, Investigation, Conceptualization. Daqiao Guo: Review & editing, Validation, Supervision, Project administration, Funding acquisition, Data curation, Conceptualization.

Funding

This work was supported by the National Natural Science Foundation of China (GuoDaqiao: 82270517; Tang Hanfei: 82100516; Changpo Lin:82100505).

Data Availability

The RNA-Seq data supporting the conclusions of this study are available in the GEO database: GSE28829 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE28829);GSE163154 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE163154);

Declarations

Ethics Approval 

All animal experiments were conducted in compliance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and were approved by the Animal Experiment Center of Zhongshan Hospital, Fudan University.

Clinical Trial Number

Not applicable.

Competing Interests

The authors declare no competing interests.