CD80⁺ Macrophage-Induced IL-17⁺CD8⁺ T cells accumulate in hyperlipidemic patients and murine vascular lesions to promote atherosclerotic progression

Abstract

Background

Tc17 cells (IL-17 ⁺ CD8⁺T) are found in many inflammatory diseases. However, the distribution, regulation, and function of Tc17 cells in ApoE deficient-induced atherosclerosis are currently largely unexplored.

Methods

The percentage of Tc17 cells, monocytes and IL-1β⁺ monocytes in blood of hyperlipidemic patients or healthy donors were analyzed by flow cytometry. The content of IL-1β in plasma was detected by ELISA. Correlations between parameters of clinical samples were assessed using Spearman r correlation analysis or linear regression analysis as appropriate. Aorta, blood, spleen, and bone marrow of atherosclerotic mice with different stages were collected for flow cytometry to analyze the percentage of Tc17 cells and monocytes/macrophages. Hearts were collected for Oil Red O staining, Masson’s staining, immunofluorescence staining or immunohistochemistry staining. GEO database (GSE43292) was used to analyze the expression of Tc17 cell-related inducers in plaques of AS patients. CD8⁺T cells and macrophages were isolated for ex vivo function assays. Adoptive transfer and IL-1β neutralization experiments were utilized for in vivo mechanism assays.

Results

Higher populations of Tc17 cells, IL-1β⁺monocytes or CD80⁺ monocytes were found in the peripheral blood of hyperlipidemic patients compared to that in healthy donors. In addition, positive correlation between Tc17 cells and CD80⁺ monocytes was observed in hyperlipidemic patients. Furthermore, Tc17 cells and CD80⁺ monocytes/macrophages increased in the aorta, blood, spleen, and bone marrow of atherosclerotic mice, reaching the highest level on week 12 post feeding. Based on the analysis of GSE43292, we found increased IL-1β expression in AS plaques, not the other inducing differentiation factors of Tc17 cells, compared to normal tissues. Finally, ex vivo co-culture and transwell experiment and in vivo adoptive transfer and IL-1β neutralization experiments confirmed that CD80⁺ macrophages promoted Tc17 cells expansion by IL-1β in vascular lesions and facilitated murine atherosclerotic progression.

Conclusion

The present results show that suppressing IL-1β expression by preventing CD80⁺ macrophage polarization may alleviate atherosclerosis through the reduction of Tc17 cells in atherosclerotic lesions. Our study demonstrates that decreasing Tc17 cells population by the inhibition of CD80⁺ macrophage-derived IL-1β is a potentially promising therapeutic strategy for the treatment of atherosclerosis.

Graphical Abstract

IL-17⁺CD8⁺ T cells (Tc17 cells) accumulate in atherosclerosis due to CD80⁺ macrophage-derived IL-1β; releasing of IL-17 by Tc17 cells exacerbates atherosclerosis progression, which is characterized by the accumulation of lipids in blood vessel walls and the formation of atherosclerotic plaques. This figure was drawn by Figdraw.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12964-026-02785-4.

Introduction

Atherosclerosis stands as the primary cause of coronary heart disease, cerebral infarction, and peripheral vascular disease [1]. The precise pathogenesis of atherosclerosis remains poorly understood, with several risk factors contributing to its development [2], including abnormal lipid metabolism, hypertension [3], diabetes [4], obesity [5], and smoking [6]. Current knowledge defines atherosclerosis as a chronic inflammatory disease of the arterial wall, characterized by arterial thickening, hardening, and narrowing of the vascular lumen [7].

Atherosclerosis is a multifactorial disease, driven by the immune system’s involvement in both systemic circulation and localized vascular lesions. Single-cell transcriptomic and proteomic analyses of human atherosclerotic plaques have revealed immune cell dysregulation, with a particular emphasis on the activation of T cells and macrophages [8]. T lymphocytes have been observed in both early and advanced atherosclerotic lesions [9] and were shown to contribute to lesion development and progression [10]. As one of the major T cells populations, CD8⁺T cells are abundant in atherosclerotic plaques in both humans and mice [11], where they exhibit activated, cytotoxic, dysfunctional, or depleted cell phenotype [12]. The role of CD8⁺T cells in atherosclerosis remains controversial, possibly due to their diverse phenotypes. Our previous study revealed that Th17 cells recruit monocytes/macrophages and neutrophils to atherosclerotic lesions in ApoE^−/− mice, promoting inflammation and playing a pro-atherosclerotic role in ApoE deficiency-induced atherosclerosis [13]. In addition to Th17 cells, IL-17⁺CD8⁺T (Tc17) cells are also important sources of IL-17. However, the distribution, regulation, and function of Tc17 cells at different stages of ApoE deficiency-induced atherosclerosis remain unclear.

Enhanced monocyte production in the bone marrow has been observed in both mouse models of atherosclerosis and patients with hypercholesterolemia [14, 15]. Circulating monocytes, originating from bone marrow hematopoietic progenitors [16], are recruited and transmigrate into the subendothelial space, where their differentiation into macrophages represents a critical step in early atherogenesis [17]. Moreover, extensive macrophage infiltration has been observed in ruptured plaques in both atherosclerotic patients and mouse models [18, 19], underscoring the pivotal role of macrophages in disease progression. Macrophages, with their diverse functional phenotypes, play distinct roles in the progression of atherosclerosis [20]. Classical pro-inflammatory macrophages secret pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β, express CD80, CD86, iNOS, etc., which sustain the chronic inflammatory state of blood vessels and promote atherosclerotic progression [21]. However, the anti-inflammatory phenotype expressing CD163, CD206, Arginase 1, etc., promotes the resolution of local inflammation and tissue repair through the secretion of IL-10, TGF-β, etc [22]. Macrophages interact synergistically with other immune cells, with their plasticity playing a pivotal role in sustaining chronic inflammation [23]. Interestingly, exposure CD8⁺T cells to the cytokines such as IL-1β, IL-6, IL-21, and transforming growth factor-β (TGF-β), results in upregulation of RAR-associated orphan nuclear receptor γt (RORγt), driving their differentiation into Tc17 cells [24, 25]. Whether macrophage-derived cytokines interact with Tc17 cells in the atherosclerotic microenvironment remains to be further explored.

Herein, we established a dynamic profile of Tc17 cells and macrophages during the progression of atherosclerosis, revealing a gradual increase in their proportions from the early to advanced stages, followed by a decline in the late stage. Clinical data reveal increased Tc17 cells in hyperlipidemic patients, with a positive correlation to CD80⁺ monocytes, suggesting their role in the early stage of human atherosclerosis. By adoptive transfer of cells and antibody blockade in vivo, these findings finally emphasize CD80⁺ macrophage-IL-1β-Tc17 cell interactions and their contribution to the inflammatory microenvironment, proposing Tc17 cells or IL-1β as potential therapeutic targets for atherosclerosis.

Materials and methods

Research participants

Male ApoE^−/− and C57BL/6 (C57) mice, aged 6 weeks, were purchased from Beijing Huafukang Biotechnology Co. and bred at the Animal Center of Chongqing Medical University. The mice were randomly assigned to different groups and maintained under a 12-hour light/dark cycle in a standard pathogen-free animal facility.

Fresh peripheral blood samples were collected from 153 hyperlipidemic patients and 157 healthy donors at the University-Town Hospital of Chongqing Medical University. None of the donors had any other metabolic diseases, such as hypertension or diabetes.

Atherosclerotic mice

For the establishment of atherosclerosis mouse models at different stages, ApoE^−/− mice were fed with western diet (WD) to induce atherosclerotic mice. C57 mice fed with a normal diet (ND) were designated as healthy group (WT). The feed components used for WD and ND are shown in Table S1, S2. After 4, 8, 12, 16, and 20 weeks (w) of feeding, mice were anesthetized via intraperitoneal injection of 200 mg/kg sodium pentobarbital. Blood was collected, and the mice were euthanized by cervical dislocation under anesthesia. For certain experiment as indicated, ApoE^−/− mice were fed ND to induce spontaneous atherosclerotic plaque (ApoE^−/−+ND group), or WD to accelerate plaque formation (ApoE^−/−+WD group); C57 mice fed similar diets, were designated as control groups (C57 + ND group and C57 + WD group). After 12w of feeding, the mice were anesthetized and euthanized as described above. The vasculature was perfused with sterile phosphate-buffered saline (PBS)-heparin sodium solution by cardiac puncture. Hearts were excised and either frozen or fixed in 4% paraformaldehyde for further use. Aorta was digested for 1 h at 37℃ using an enzyme cocktail containing 450 U/mL collagenase I, 125 U/mL collagenase XI, 60 U/mL DNase I, and 60 U/mL hyaluronidase, as previously reported [26]. Bone marrow was extracted from the femurs and tibias. Aorta, blood, spleen, and bone marrow were processed into single-cell suspension for further use.

Adoptive transfer experiment

Bone marrow-derived macrophages were isolated as previously reported [27]. Briefly, cells were collected from femurs and tibias of C57 mice and cultured in 50 ng/mL recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) for 5 days. Floating cells were then discarded, and the adherent macrophages were cultured for an additional 2 days without GM-CSF treatment. For further investigation, macrophages were treated with 100 ng/mL LPS for additional 2 days to generate CD80⁺ macrophages. Starting from the third week, ApoE^−/− mice feeding a WD were intraperitoneally injected with 1 × 10⁶ CD80⁺ macrophages once a week for 4 weeks. Mice injected with 1 × 10⁶ M0 macrophages or 100 µL sterile PBS solution were used as control. The mice were then anesthetized and sacrificed as above. Aorta, blood, spleen, and bone marrow were collected for flow cytometry. Hearts were either frozen or fixed in 4% paraformaldehyde for histological analysis.

In vivo IL-17 or IL-1β neutralization

ApoE^−/− mice were fed a WD. From the third week, 50 µg of purified Rat Monoclonal IgG anti-mIL-17 (Clone: 50104) [13] or anti-mouse/rat IL-1β antibody (Clone: B122) was injected intraperitoneally, twice a week for 4 weeks. Mice that received 50 µg rat IgG2α isotype, polyclonal Armenian hamster IgG or 100 µL sterile PBS were served as control groups. Then, the mice were anesthetized and sacrificed, and samples were collected as above.

Histological analysis

Frozen heart sections were stained with Oil Red O. Lesion areas and lipid accumulation in lesions were measured by ImageJ. Paraffin-embedded hearts sections were prepared for Masson’s staining to quantify collagen contents in atherosclerotic lesions.

Immunofluorescent staining

Immunofluorescent analysis was conducted on aortic root sections from ApoE^−/− mice using various antibody combinations for dual staining, including anti-CD8, anti-F4/80, anti-IL-17, anti-IL-1β, and antibodies against iNOS or CD206. Different signal amplification methods (e.g., CY3-Tyramide and FITC-Tyramide) and secondary antibodies (e.g., Alexa Fluor 488, CY3 Goat anti-rabbit IgG) were used for visualization. The details of the antibodies are shown in Table S3. Slides were examined using a fluorescent microscope (ECLIPSE C1, NIKON) or a confocal laser scanning microscope (DMi 8, Leica).

Immunohistochemistry

Immunohistochemistry was performed according to standard protocols. The paraformaldehyde-fixed, paraffin-embedded heart was sectioned and incubated overnight at 4 °C, protected from light, with rabbit anti-IL-1β antibody (Service Bio), followed by staining with horseradish peroxidase (HRP)-conjugated goat anti-rabbit fluorescent secondary antibody. The sections were then stained with DAB chromogenic reagent (Service Bio) and subsequently counterstained with hematoxylin. The slides were examined using a fluorescent microscope (ECLIPSE C1, NIKON).

Blood lipids measurement and IL-1β measurement

Murine plasma was collected. Blood lipid levels, including total cholesterol (TC), total triglycerides (TG), low-density lipoprotein-cholesterol (LDL-C), and high-density lipoprotein-cholesterol (HDL-C), were measured using corresponding assay kits with a biochemical analyzer (Mindray, China).

Peripheral blood from patients with hyperlipidemia and healthy donors was centrifuged, and the plasma was collected. Human IL-1β levels in plasma were quantified according to the instructions of the human IL-1β ELISA kit (Absin, China).

Ex vivo cell culture

CD8⁺T cells were isolated from splenocytes of C57 mice using the EasySep™ Mouse CD8⁺T Cell Isolation Kit, following the manufacturer’s protocol. The purity of the isolated CD8⁺T cells was over 98% (Fig. S1a). Bone marrow-derived macrophages were treated with recombinant mouse IL-17 at concentrations of 20, 40, and 80 ng/mL to investigate the role of IL-17 in macrophage differentiation. In the CD8⁺ T cell-macrophage co-culture system, bead-purified splenic CD8⁺T cells (1 × 10⁵ cells/well) were co-cultured in 96-well plates with M0 or CD80⁺ macrophages at 1:1 ratio in complete RPMI 1640 medium, along with mouse recombinant IL-2 (40 ng/mL), mrIL-23 (40 ng/mL), anti-CD3 (5 µg/mL), and anti-CD28 (2 µg/mL) antibodies.

For transwell experiments, CD8⁺T cells (1 × 10⁵ cells/well) were seeded in the lower chamber of a Transwell^® cell culture (3-µm pore size) containing complete RPMI 1640 medium with mouse recombinant IL-2 (40 ng/mL) and mrIL-23 (40 ng/mL). CD80⁺ macrophages were plated either in the upper or the lower chamber. For IL-1β neutralization assays ex vivo, CD8⁺ T cells (1 × 10⁵ cells/well) were co-cultured with CD80⁺ macrophages at 1:1 ratio in complete RPMI 1640 medium, with or without neutralizing antibodies against IL-1β (10 µg/mL) or in the presence of recombinant mouse IL-1β (40 ng/mL). After five days, cells were harvested for intracellular IL-17 staining.

Flow Cytometry (FCM)

Flow cytometric analysis was conducted according to standard protocols. To block Fc receptors during the analysis of monocytes and macrophages in atherosclerotic mouse models after12w-feeding, cells were pre-incubated with TruStain FcX™ PLUS (anti-mouse CD16/32) antibody on ice, protected from light, prior to antibody staining. For surface marker analysis, cells were stained with fluorescence-labeled antibodies against CD45, CD3, CD8, F4/80, CD14, CD80, and CD163 at 4 °C for 30 min.

For intracellular staining, cells were stimulated with cell activation cocktail containing Brefeldin A for 6 h. Intracellular cytokines, including IL-17, RORγt, and IL-1β, were stained using fluorescence-labeled antibodies following fixation, permeabilization, and serum blockade. To define live cell, single-cell suspensions from 12-week HFD atherosclerotic mouse models were treated with the Zombie Aqua™ Fixable Viability Kit (1:1000 dilution) at room temperature, protected from light.

Details of the antibodies used are provided in Table S3. Detailed staining protocols and gating strategies for mouse Tc17 cells and monocytes/macrophages are illustrated in Fig.S1b and S1c.

GEO database analysis

Samples from the GEO database (GSE43292) were obtained from 32 hypertensive patients undergoing carotid endarterectomy, including AS plaques (stages IV and above) and distant intact tissues (stages I and II). The expression of differentiation-inducing factors for Tc17 cells in human AS plaques was analyzed using a volcano plot.

Statistical analysis

Results are presented as mean ± standard error of the mean (SEM). The normality of data distributions was assessed using the Shapiro-Wilk test. For normally distributed data, comparisons were made using an unpaired t-test, one-way ANOVA, or two-way ANOVA. Non-normally distributed data were analyzed using the Mann-Whitney test or Kruskal-Wallis test. Correlations between parameters were determined by Spearman’s rank correlation or linear regression analysis, as appropriate. Statistical analyses were conducted with GraphPad Prism 8.4. All statistical tests were two-tailed, and a p-value of < 0.05 was considered statistically significant. The sample size (n) and specific statistical tests used are provided in the figure legends.

Results

The abundance of Tc17 cells, monocytes, and CD80⁺monocytes is significantly increased in hyperlipidemic patients

Hyperlipidemia is the initiation stage of most atherosclerosis cases. To investigate the infiltration of Tc17 cells and monocytes in hyperlipidemic patients, we collected fresh peripheral blood from 153 hyperlipidemic patients and 157 healthy donors. Clinical information for both groups is provided in Table S4. As shown in Fig. 1a, b, CD8⁺T cells in the peripheral blood of hyperlipidemic patients secreted significantly higher levels of IL-17 compared to healthy donors. Furthermore, phenotypic analysis of monocytes revealed a significantly higher proportion of monocytes in hyperlipidemic patients compared to healthy donors (Fig. 1c, d). Additionally, the percentage of CD80⁺ monocytes was significantly elevated in hyperlipidemic patients (Fig. 1e, f), while no significant difference was observed in the percentage of CD163⁺ monocytes (Fig. 1g, h). To control for potential confounding factors, we performed a re-analysis using a multiple linear regression model (Analysis of Covariance, ANCOVA), with age and gender included as covariates. This analysis confirmed that the differences between the hyperlipidemic patients and healthy donors remained statistically significant after adjusting for age and gender for Tc17 cells, monocytes, and CD80⁺ monocytes. The difference in CD163⁺ monocyte percentage remained non-significant (Table S5). These findings suggest that both Tc17 cells and monocytes may play a crucial role in the pathogenesis of human atherosclerosis.

Fig. 1.

Distribution of Tc17 cells and monocytes in blood of hyperlipidemic patients and healthy donors. Representative dot plots and statistical analysis of (a, b) Tc17 cells (IL-17⁺CD8⁺T cells), (c, d) CD14⁺ monocytes (CD45⁺CD14⁺ cells), Representative histograms and statistical analysis of (e, f) CD80⁺ monocytes (CD45⁺CD14⁺CD80⁺ cells), (g, h) CD163⁺ monocytes (CD45⁺CD14⁺CD163⁺ cells) in peripheral blood of hyperlipidemic patients and healthy donors. Each dot represents an individual participant. Horizontal bars in panels b, d, f, and h indicate mean values. Sample number: 157 healthy donors (n = 157) and 153 hyperlipidemic patients (n = 153). Statistical analysis was performed using the Mann-Whitney test. *p˂0.05, ***p˂0.001

Tc17 cells positively correlate with monocytes and CD80⁺Monocytes in hyperlipidemic patients

To assess the clinical significance of Tc17 cells and monocytes in hyperlipidemic patients, we stratified the cohort into elderly and younger groups based on the median age (50 years). Elderly patients exhibited a significantly higher proportion of Tc17 cells and monocytes compared to younger patients (Fig. 2a and Fig.S2a), whereas the proportions of CD80⁺ and CD163⁺ monocytes showed no notable differences between the groups. Regarding gender, no significant differences were observed in the percentages of Tc17 cells (Fig. 2b), monocytes, CD80⁺ monocytes, or CD163⁺ monocytes (Fig.S2b).

Fig. 2.

Clinical significance of Tc17 cells in hyperlipidemic patients. (a) Tc17 cells percentage in elderly group and younger group of hyperlipidemic patients. Younger group (≤ 50, n = 73); elderly group (> 50, n = 80). Mann-Whitney test. (b) Tc17 cells percentage in male group (n = 72) and female group (n = 81) of hyperlipidemic patients. Mann-Whitney test. Correlation analyses between Tc17 cell percentages and blood lipid levels including (c) TC, (d) TG, (e) LDL-C, and (f) HDL-C. Correlation analyses between Tc17 cells and (g) monocytes, (h) CD80⁺ monocytes, and (i) CD163⁺ monocytes in the peripheral blood of hyperlipidemic patients. Correlations were evaluated using Spearman’s rank correlation or linear regression analysis, as appropriate. Each dot represents an individual patient (n = 153). Horizontal bars in panels a and b indicate mean values. *p˂0.05

Furthermore, no significant correlations were detected between Tc17 cells and serum lipid levels, including total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) (Fig. 2c-f). Similarly, monocytes, CD80⁺ monocytes, and CD163⁺ monocytes showed no significant associations with serum lipid levels (Fig.S2c-f). These findings suggest that the infiltration of Tc17 cells or monocytes in hyperlipidemic patients is not directly regulated by lipid levels.

Importantly, we analyzed the relationships between Tc17 cells and monocytes, CD80⁺ monocytes, or CD163⁺ monocytes, and the results revealed that Tc17 cells were positively correlated with monocytes (Fig. 2g) and CD80⁺ monocytes (Fig. 2h), but no significant correlation was observed between Tc17 cells and M2 monocytes (Fig. 2i). These results highlight the potential interaction between Tc17 cells and CD80⁺ monocytes during the early stages of human atherosclerosis.

Elevated Tc17 cell infiltration in atherosclerotic mice

Building on clinical findings from hyperlipidemic patients, we assessed the distribution of Tc17 cells within the inflammatory microenvironment of ApoE deficiency-induced atherosclerosis. An experimental atherosclerotic mouse model was established by feeding ApoE^−/− mice with WD, while C57BL/6 mice fed with ND served as controls. Blood lipid analysis revealed significantly elevated levels of TC, TG, LDL-C, and HDL-C in atherosclerotic mice compared to controls (Fig.S3a). Oil Red O staining demonstrated plaque formation in the aortic root of atherosclerotic mice after 4 weeks of WD feeding, with progressive enlargement over time (Fig.S3b, c). Collagen content in the aortic root exhibited a similar pattern (Fig.S3d, e), confirming successful model establishment.

To investigate Tc17 cell infiltration, we analyzed their distribution in various tissues and organs of atherosclerotic mice at 4, 8, 12, 16, and 20 weeks of WD feeding. To ensure the accuracy of the analysis, dead/alive staining was performed to exclude false positives from dead cells, confirming that more than 95% of live cells were included in the flow cytometric analysis of T cells stimulated with a cell activation cocktail (with Brefeldin A) (Fig.S1d). Compared to control mice, atherosclerotic mice exhibited a significant increase in the proportion of Tc17 cells in the aorta, which peaked at 12 weeks of WD feeding. Consistently, the absolute number of Tc17 cells per million cells in the aorta was markedly higher in atherosclerotic mice than in controls (Fig. 3a, b). Similar Tc17 cell infiltration patterns were observed in the blood (Fig. 3c, d), spleen (Fig.S4a, b), and bone marrow (Fig.S4c, d), reflecting the trends seen in the aorta.

Fig. 3.

Tc17 cells and Monocyte/macrophage accumulation in aorta and blood of atherosclerotic mice. 6-week-old male ApoE^−/− mice were fed with WD for 4 weeks (4w), 8 weeks (8w), 12 weeks (12w), 16 weeks (16w), or 20 weeks (20w) to generate different stage of atherosclerotic mice. Age-matched male C57 mice fed with ND were set as wild type group (WT). Representative dot plots of Tc17 cells (gating on CD45⁺CD3⁺CD8⁺IL-17⁺ cells) in the (a) aorta and (c) blood of ApoE^−/− mice fed with WD or WT mice fed with ND for 12w. The percentage of Tc17 cells in the (b) aorta and (d) blood of ApoE^−/− mice fed with WD or WT mice fed with ND for 4w, 8w, 12w, 16w, or 20w and the corresponding number per million cells in the aorta of ApoE^−/− mice fed with WD or WT mice fed with ND for 12w. Representative dot plots of (e) macrophages (gating on CD45⁺F4/80⁺cells), (i) CD80⁺ macrophages (gating on CD45⁺F4/80⁺CD80⁺ cells) and (m) CD163⁺ macrophages (gating on CD45⁺F4/80⁺CD163⁺ cells) in the aorta or (g) monocytes (gating on CD45⁺CD14⁺cells), (k) CD80⁺ monocytes (gating on CD45⁺CD14⁺CD80⁺ cells) and (o) CD163⁺ monocytes (gating on CD45⁺CD14⁺CD163⁺ cells) in the blood of ApoE^−/− mice fed with WD or WT mice fed with ND for 12w. The percentage of (f) macrophages, (j) CD80⁺ macrophages, or (n) CD163⁺ macrophages in the aorta and (h) monocytes, (l) CD80⁺ monocytes, or (p) CD163⁺ monocytes of ApoE^−/− mice fed with WD or WT mice fed with ND for 4w, 8w, 12w, 16w, or 20w and the corresponding number per million cells in the aorta of ApoE^−/− mice fed with WD or WT mice fed with ND for 12w. b, f, j, n, n = 3, two-way ANOVA, or unpaired t test. d, WT-4w, n = 5, ApoE^−/−-4w, n = 5, WT-8w, n = 5, ApoE^−/−-8w, n = 6, WT-12w, n = 6, ApoE^−/−-12w, n = 6, WT-16w, n = 6, ApoE^−/−-16w, n = 4, WT-20w, n = 5, ApoE^−/−-20w, n = 4. h, n = 6, except for ApoE^−/−-16w, n = 5. l, WT-4w, n = 5, ApoE^−/−-4w, n = 5, WT-8w, n = 5, ApoE^−/−-8w, n = 6, WT-12w, n = 3, ApoE^−/−-12w, n = 5, WT-16w, n = 5, ApoE^−/−-16w, n = 6, WT-20w, n = 6, ApoE^−/−-20w, n = 4. p, WT-4w, n = 5, ApoE^−/−-4w, n = 5, WT-8w, n = 4, ApoE^−/−-8w, n = 6, WT-12w, n = 5, ApoE^−/−-12w, n = 5, WT-16w, n = 6, ApoE^−/−-16w, n = 6, WT-20w, n = 5, ApoE^−/−-20w, n = 6, two-way ANOVA. ^*p˂0.05, ^**p˂0.01, ^***p˂0.001

These findings indicate a sustained increase in Tc17 cell infiltration throughout the progression of atherosclerosis. Nevertheless, further studies are required to clarify the mechanisms driving Tc17 cell infiltration in the atherosclerotic microenvironment.

Distribution of CD80⁺Monocytes/Macrophages correlates with Tc17 cells in atherosclerotic mice

Monocytes/macrophages, as key immune cells in atherosclerotic lesions, play critical roles in the initiation, progression, and rupture of arterial plaques. To investigate their distribution, we assessed monocytes/macrophages in various tissues and organs of atherosclerotic mice. To minimize false positives from dead cells and nonspecific staining, we performed dead/alive staining and Fc blocking. As shown in Fig.S1e, over 95% of macrophages in the aorta were viable cells. Furthermore, Fc receptor blocking did not significantly affect the proportions of macrophages in the aorta or spleen, as shown in Fig.S1f, g.

Our results revealed a significant elevation in the proportion of monocytes/macrophages in the aorta (Fig. 3e, f) and blood (Fig. 3g, h) of atherosclerotic mice, compared to healthy controls. In contrast, no such differences were observed in the spleen and bone marrow (Fig.S5a-d). Additionally, the number of infiltrating macrophages in the aorta of atherosclerotic mice was higher than in control mice (Fig. 3f). Given the distinct roles of CD80⁺ and CD163⁺ monocytes/macrophages in atherosclerosis, we further characterized their phenotypes in atherosclerotic mice. Notably, CD80⁺ monocytes/macrophages were markedly increased in the aorta (Fig. 3i, j), blood (Fig. 3k, l), spleen (Fig.S5e, f), and bone marrow (Fig.S5g, h) of atherosclerotic mice, peaking at the 12th week of WD feeding (Fig. 3i-l, Fig.S5). The distribution of CD80⁺ macrophages closely paralleled the infiltration patterns of Tc17 cells. Conversely, CD163⁺ macrophages exhibited significantly reduced proportions and numbers in the aorta, reaching their lowest levels at the 12th week of WD feeding (Fig. 3m, n). No significant differences in CD163⁺ monocytes/macrophages were observed in the blood (Fig. 3o, p), spleen (Fig.S5i, j), or bone marrow (Fig.S5k, l) between atherosclerotic and control mice.

To minimize the influence of genetic background, ApoE^−/− mice were fed with either a ND to induce spontaneous atherosclerotic plaque formation (ApoE^−/− +ND group), or a WD to accelerate plaque formation (ApoE^−/−+WD group). C57 mice fed the same diets were used as control groups (C57 + ND group and C57 + WD group). Body and organ weights of mice in each group were shown in the Fig.S6a, b. Blood lipid levels were significantly elevated in atherosclerotic mice compared to controls (Fig.S6c). Oil red O staining demonstrated plaques in the aortic arch and root of atherosclerotic mice (Fig.S6d, e), with collagen content in the aortic root following a similar trend (Fig.S6f). These results confirmed the successful establishment of atherosclerotic mouse model.

Further analysis revealed significantly higher proportions of Tc17 cells and RORγt⁺CD8⁺ T cells in the aorta of ApoE^−/− mice fed with WD compared to those fed with ND or C57 mice fed with WD (Fig. 4a-d). Although macrophage infiltration pattern did not completely match Tc17 cell trends, pro-inflammatory macrophages (F4/80⁺ iNOS⁺) were more abundant in the aorta of ApoE^−/− mice fed with WD than in ApoE^−/− mice fed with ND or C57 mice fed with WD. Conversely, anti-inflammatory macrophages (F4/80⁺ CD206⁺) were reduced (Fig. 4e-g).

Fig. 4.

Infiltration of Tc17 Cells and Macrophages in Atherosclerotic Mice After 12 Weeks of Feeding. ApoE^−/− mice fed with ND or WD and C57 mice fed with the same diets for 12w were anesthetized and sacrificed, separately. Representative dot plots and statistical analysis of (a, b) Tc17 cells (gating on CD45⁺CD3⁺CD8⁺IL-17⁺ cells), (c, d) RORγt⁺CD8⁺cells (gating on CD45⁺CD3⁺CD8⁺RORγt⁺ cells), and (e, f) macrophages (gating on CD45⁺F4/80⁺cells) in aorta of mice at 12w of feeding. b, n = 4, except for C57 + WD (n = 3), d, n = 4, one-way ANOVA. f, n = 4, Kruskal-Wallis test. (g) Dual immunofluorescent staining of F4/80 (red) and iNOS (green) or CD206 (green) in the aortic root of mice at 12w of feeding. Blue: DAPI. Scale bar, 100 μm. (h) Dual immunofluorescent staining of CD8 (red) and F4/80 (green) in the aortic root of ApoE^−/− mice fed with WD for 12w. Blue: DAPI. Scale bar, 100 μm. *p˂0.05, **p˂0.01, ***p˂0.001

Immunofluorescent staining revealed the adjacent spatial positioning of macrophages and CD8⁺T cells in the aortic root of atherosclerotic mice (Fig. 4h). Collectively, the similar distribution patterns, spatial proximity, and positive correlation between these two cell types in clinical samples suggest potential interactions between CD8⁺T cells and macrophages within the atherosclerotic microenvironment.

CD80⁺Macrophages Stimulate Tc17 cell expansion and accelerate disease progression

The above findings indicate a potential interaction between Tc17 cells and CD80⁺ macrophages, though the precise regulatory mechanism remains to be elucidated. To address this, we first investigated the impact of CD80⁺ macrophages on Tc17 cell differentiation ex vivo. CD8⁺T cells were stimulated with either M0 macrophages, CD80⁺ macrophages, or left unstimulated, and IL-17 production was subsequently measured. It showed that CD80⁺ macrophages markedly enhanced IL-17 production, compared to M0 macrophages or CD8⁺T cells alone (Fig. 5a, b), suggesting that CD80⁺ macrophages promote Tc17 cell differentiation.

Fig. 5.

Tc17 cell expansion stimulated by CD80⁺ macrophages ex and in vivo. (a) Representative dot plots and (b) statistical analysis of IL-17 production in CD8⁺T cells cocultured with M0 or CD80⁺ macrophages for 5 days. b, n = 6, Kruskal-Wallis test. (c) Schematic diagram of the adoptive transfer experiment: 6-week-old male ApoE^−/− mice were fed with WD for 2 weeks, followed by intraperitoneal injection of sterile PBS (PBS-group), M0 macrophages (M0-group) or CD80⁺ macrophages (CD80⁺-group) once weekly for 4 weeks, with WD feeding continued throughout the 6-week study. Representative dot plots and statistical analysis of Tc17 cells in the (d, e) aorta, (f, g) blood, (h, i) spleen, and (j, k) bone marrow of ApoE^−/− mice in PBS, M0, and M1-groups. e, PBS, n = 3, M0 and CD80⁺ , n = 4, one-way ANOVA, multiple comparisons; g, PBS, n = 5, M0 and CD80⁺, n = 4, one-way ANOVA; i, PBS, n = 5; M0, n = 4, CD80⁺, n = 6, one-way ANOVA or Kruskal-Wallis test; k, n = 5; one-way ANOVA. (l) Dual immunofluorescent staining of CD8 (green) and IL-17 (red) in the aortic root of ApoE^−/− mice in PBS, M0, and CD80⁺-groups. Blue: DAPI. Scale bar, 100 µm. (m) Oil Red O, (n) Masson’ and (o) HE staining of the aortic root and quantification of plaque area of ApoE^−/− mice across groups. n = 3, one-way ANOVA. Scale bar, 500 μm. *p˂0.05, **p˂0.01, ***p˂0.001

To further validate the role of CD80⁺ macrophages in vivo, we conducted an adoptive transfer experiment. ApoE^−/− mice fed with WD were intraperitoneally injected with M0 macrophages, CD80⁺ macrophages, or sterile PBS, and mice were sacrificed four weeks post-injection (experimental scheme shown in Fig. 5c). As confirmed in Fig.S7, both M0 and CD80⁺ macrophages were successfully transferred into the mice. Compared to the PBS-injected group, ApoE^−/− mice injected with M0 or CD80⁺ macrophages exhibited significantly increased Tc17 cell infiltration in the aorta (Fig. 5d, e), blood (Fig. 5f, g), spleen (Fig. 5h, i), and bone marrow (Fig. 5j, k). Notably, the CD80⁺ macrophage-treated group displayed the highest level of Tc17 cell infiltration (Fig. 5d-k). Interestingly, while M0 macrophages did not induce IL-17 production ex vivo, their adoptive transfer still slightly promoted Tc17 cell expansion in vivo, possibly due to the differentiation of M0 macrophages into CD80⁺ macrophages within the atherosclerotic microenvironment. Immunofluorescent staining of the aortic root further revealed increased Tc17 cell infiltration in mice treated with CD80⁺ macrophages (Fig. 5l). Assessment of atherosclerosis severity via Oil red O, Masson’s and HE staining showed that CD80⁺ macrophage-injected mice exhibited larger plaques, collagen fiber content, and necrotic core compared to PBS group (Fig. 5m-o). Collectively, our ex and in vivo findings provide supportive evidence that CD80⁺ macrophages drive the differentiation of Tc17 cells, thereby contributing to the progression of atherosclerosis.

To determine whether Tc17 cells regulate macrophage differentiation, we stimulated M0 macrophages with varying concentrations of IL-17. However, IL-17 alone did not induce differentiation of M0 macrophages into CD80⁺ or CD163⁺ macrophages (Fig.S8a, Fig.S9a). We then assessed the role of IL-17 in monocyte/macrophage distribution and phenotype in vivo by administering an anti-IL-17 antibody, isotype control antibody, or sterile PBS to ApoE^−/− mice fed with WD. As previously reported [13], IL-17 promoted macrophage migration into atherosclerotic lesions. However, IL-17 blockades had no significant effect on the differentiation of CD80⁺ macrophages (Fig.S8b-e) or CD163⁺ macrophages (Fig.S9b-e).

IL-1β derived from CD80+ macrophages drives Tc17 cell expansion and exacerbates atherosclerosis progression

To investigate the mechanism by which CD80⁺ macrophages stimulate Tc17 cell differentiation, we conducted a transwell assay. The results demonstrated that the stimulatory effect of CD80⁺ macrophages on CD8⁺T cells was independent of direct cell-to-cell contact (Fig. 6a, b), suggesting soluble cytokines secreted by CD80⁺ macrophages mediate Tc17 cell differentiation. Among the known Tc17-inducing cytokines, such as TGF-β, IL-6, IL-1β, IL-21, and IL-23 [28], analysis of the GEO database (GSE43292) revealed significantly elevated IL-1β levels in human atherosclerotic plaques (Fig. 6c). As a pro-inflammatory mediators secreted by CD80⁺ macrophages, IL-1β is known to exacerbate vascular inflammation and atherosclerosis progression [29] and to facilitate Tc17 cell differentiation in both mice and humans [28]. To validate these findings, we evaluated IL-1β expression in hyperlipidemic patients and atherosclerotic mice. Flow cytometry and ELISA revealed significantly elevated IL-1β levels in the blood monocytes and plasma of hyperlipidemic patients compared to healthy controls (Fig. 6d, e). This difference in IL-1β⁺ monocytes remained significant after adjustment for the potential confounders of age and gender. Immunohistochemistry staining further confirmed increased IL-1β expression in the aortic roots of atherosclerotic mice (Fig. 6f).

Fig. 6.

Tc17 cell expansion promoted by IL-1β ex and in vivo. (a) Schematic diagram of the Transwell assay: CD8⁺T cells were seeded in the lower chamber, while CD80⁺ macrophages were placed in either the upper or lower chamber. IL-17 production by CD8⁺T cells was assessed via FCM. (b) Representative dot plots and statistical analysis of Tc17 cells from the transwell assay. n = 6, one-way ANOVA. (c) Volcano plot of Tc17-promoting cytokines in human atherosclerotic plaque based on GEO database GSE43292. (d) Representative dot plots and statistical analysis of IL-1β⁺ monocytes (CD45⁺CD14⁺IL-1β⁺ cells) in the peripheral blood of hyperlipidemic patients and healthy donors. Healthy donors, n = 63, hyperlipidemia, n = 45. Mann-Whitney test. (e) IL-1β levels in plasma of peripheral blood from hyperlipidemic patients and healthy donors. Healthy donors, n = 38, hyperlipidemia, n = 40. Mann-Whitney test. (f) Representative images and statistical analysis of IL-1β-positive staining in the aortic root of C57 or ApoE^−/− mice fed with ND or WD for 12w. C57 + ND, n = 4; C57 + WD, n = 4; ApoE^−/−+ND, n = 4; ApoE^−/−+WD, n = 3; one-way ANOVA. Scale bar, 200 μm–100 μm. Positive IL-1β staining is shown in brown. CD8⁺T cells were co-cultured for 5 days with CD80⁺ macrophages, recombinant IL-1β, or CD80⁺ macrophages plus anti-IL-1β antibody in RPMI 1640 medium. (g) Representative dot plots and statistical analysis of Tc17 cells. n = 6; one-way ANOVA. (h) Schematic of IL-1β neutralization experiment in vivo: 6-week-old male ApoE^−/−mice were fed with WD. Two weeks later, mice were intraperitoneally injected with anti-IL-1β antibody, isotype antibody or sterile PBS twice weekly for 4 weeks. Representative dot plots and statistical analysis of Tc17 cells in the (i) aorta, (j) blood, (k) spleen, and (l) bone marrow of ApoE^−/−mice following treatments. i, PBS, n = 4; Isotype, n = 5; anti-IL-1β, n = 5; one-way ANOVA; j, PBS, n = 5; Isotype, n = 5; anti-IL-1β, n = 6; one-way ANOVA, or Kruskal-Wallis test; k, PBS, n = 6; Isotype, n = 7; anti-IL-1β, n = 6; one-way ANOVA, or Kruskal-Wallis test; l, PBS, n = 5; Isotype, n = 6; anti-IL-1β, n = 6; one-way ANOVA. m Oil Red O staining of the aortic root and plaque area in ApoE^−/− mice treated with anti-IL-1β antibody, isotype antibody, or PBS. PBS, n = 4; Isotype, n = 3; anti-IL-1β, n = 4; one-way ANOVA. Scale bar, 200 μm. *p˂0.05, **p˂0.01, ***p˂0.001

To determine the role of CD80⁺ macrophage-derived IL-1β on Tc17 cell differentiation, coculture experiments were performed. CD8⁺T cells were cocultured with CD80⁺ macrophages in the presence or absence of an IL-1β neutralizing antibody. As shown in Fig. 6g, CD80⁺ macrophages significantly facilitated Tc17 cell differentiation, an effect significantly attenuated by IL-1β neutralization. Additionally, IL-1β alone was sufficient to enhance Tc17 cell differentiation, although less robustly than CD80⁺ macrophages, likely due to the presence of other cytokines secreted by CD80⁺ macrophages. These findings suggest that IL-1β partially mediates the pro-Tc17 effect of M1 macrophages.

To further explore the role of IL-1β in vivo, a neutralizing experiment was performed in ApoE^−/− mice feeding a WD (experimental scheme shown in Fig. 3h). Immunofluorescence staining confirmed effective IL-1β blockade (Fig.S10a). Although slight reduction in heart and liver weight was observed (Fig.S10b-d), no apparent adverse effects were recorded. FCM revealed a marked reduction in Tc17 cell infiltration in the aorta (Fig. 6i), blood (Fig. 6j), and spleen (Fig. 6k) of IL-1β-blocked mice compared to isotype or PBS groups, whereas no significant changes were observed in the bone marrow (Fig. 6l). Immunofluorescence staining corroborated the reduced Tc17 cell infiltration in the aortic root of IL-1β-blocked mice (Fig. S10e). This reduction in Tc17 cell infiltration was associated with decreased lipid deposition in the aortic root (Fig. 6m), indicating ameliorated atherosclerosis severity following IL-1β blockade. In summary, these findings demonstrate that IL-1β secreted by CD80⁺ macrophages promotes Tc17 cell expansion, thereby accelerating the progression of atherosclerosis.

Discussion

In this study, we identify an increased infiltration of Tc17 cells in atherosclerotic mice and the peripheral blood of hyperlipidemic patients. Using multiple experimental approaches, we map the mechanisms underlying the induction, biologic function, and clinical relevance of these cells in atherosclerosis. Our founding reveals that within atherosclerotic environment, Tc17 cells and CD80⁺ monocytes/macrophages share a similar distribution pattern and adjacent spatial localization. Further investigation demonstrate that CD80⁺ macrophages promote Tc17 cell differentiation via IL-1β, thereby facilitating disease progression in vivo. Clinical data support these findings, demonstrating positive correlations between Tc17 cells and both total monocytes and CD80⁺ monocytes in hyperlipidemic patients.

While Tc17 cells have been described in early-stage atherosclerosis [30], we characterize their dynamic changes from early to advanced stages of atherosclerosis and reveal their disease-promoting function. However, it has been reported that Tc17 cells do not contribute to early atherosclerosis development in low-density lipoprotein receptor knockout (LDLr^−/−) mice [30], which is inconsistent with our findings. The functional differences of Tc17 cells may be attributed to variations in the mouse models of atherosclerosis. As reported, LDLr^−/− mice exhibited higher plasma cholesterol levels and resembling that of humans, but required a WD to induce atherosclerotic plaque formation. In contrast, ApoE deficiency contributed to both inflammation and immune dysregulation [31, 32]. On a high-cholesterol diet, ApoE^−/− mice developed plaques more rapidly, exhibited more advanced lesion phenotypes, and presented the full spectrum of atherosclerotic lesions, making them more suitable for studying the immunopathogenesis of atherosclerosis. Differences in these models suggested that Tc17 cells may play distinct roles in LDLr^−/− and ApoE^−/− mice.

IL-17 is a critical cytokine implicated in various immune and infectious diseases [33, 34]. It is produced by multiple immune cells types, including Th17 cells, natural killer (NK) cells, NKT cells [35]. While research on IL-17 in atherosclerosis has predominantly focused on Th17 cells and their roles in disease progression and plaque stability [36], Tc17 cells also represent a significant source of IL-17 [28]. In our study, we observe a marked increase of Tc17 cells within the aorta, blood, spleen, and bone marrow throughout the whole stages of atherosclerosis. These results suggest that Tc17 cells may play a pivotal role in atherosclerosis, contributing to disease progression from early to advanced stages.

Atherosclerotic plaques contain a diverse array of immune cells, including macrophages, lymphocytes and mast cells [21]. Among these, macrophages play a central role in the initiation and progression of atherosclerosis. Histological analysis of human atherosclerotic plaques reveals that M1 macrophages are lipids-rich, while M2 macrophages exhibit lower lipid content and are typically located away from the lipid core [37]. The heterogeneity and complexity of macrophages highlight their plasticity and potential for interacting with other cells types, which may, in turn, polarize these cells within the local microenvironment [38]. Our analysis of monocytes/macrophages in murine atherosclerosis confirms that the proportions of CD80⁺ monocytes/macrophages increase in the aorta, blood, spleen, and bone marrow of atherosclerotic mice. This temporal pattern aligns with the dynamics of Tc17 cells. In situ analysis further reveals that CD8⁺T cells are located in close proximity to macrophages in the aortic root of atherosclerotic mice. These findings suggest a potential interaction between Tc17 cells and macrophages within atherosclerotic lesions.

Under the influence of local inflammatory factors, such as IFN-γ and lipopolysaccharide (LPS), macrophages adopt a pro-inflammatory phenotype (M1 macrophages), characterized by upregulation of CD80 expression and the release of large amounts of inflammatory mediators, including interleukin-1β (IL-1β) [21]. Our ex vivo experiment demonstrated that CD80⁺ macrophages promote the differentiation of Tc17 cells in an IL-1β-dependent manner. Adoptive transfer of CD80⁺ macrophages and IL-1β blocking experiment in ApoE^−/− mice further indicate the roles of CD80⁺ macrophages and IL-1β in Tc17 cell differentiation and disease progression in vivo. While i.p. injection of CD80⁺ macrophages increased Tc17 cell frequencies and aggravated atherosclerosis, it does not model the specific homing and integration of macrophages into plaques. Therefore, the observed effects likely result from a broad shift in the systemic inflammatory milieu rather than direct local action within the aortic wall. Future studies employing more targeted delivery models (e.g., bone marrow transplantation and cell tracing) will be essential to validate the local pathogenic role of CD80⁺ macrophages. Collectively, our data delineate a CD80⁺ macrophage/IL-1β/Tc17 axis that correlates with disease severity, implying a potential role for Tc17 cells in the progression of atherosclerosis. It has been reported that IL-17 acts on vascular cells, promoting the secretion of pro-inflammatory factors and chemokines, which in turn recruits immune cells to the plaque site, thereby exacerbating plaque instability and vascular injury [39], speculating that Tc17 cells could directly contribute to plaque progression through IL-17 production. Future studies are required to directly validate the pathogenicity of Tc17 cells through in vivo functional gain- or loss-of-experiments, combined with more refined assessments of plaque stability.

In the ex vivo experiment, where only macrophages and CD8⁺T cells were co-cultured, the induction of Tc17 cell generation was less pronounced compared to the adoptive transfer experiment in vivo. This disparity may be attributed to various factors, including metabolic reprogramming, transcriptional regulation, and histone modifications involved in T cell differentiation. Studies have shown that metabolic remodeling plays a pivotal role in determining T cell differentiation and function, which in turn influences the progression of inflammatory diseases [40]. T cell metabolism in different environments relies on distinct pathways, including lipid synthesis [41], oxidation [42], mitochondrial reactive oxygen species [42], and amino acid uptake [43]. The role of IL-1β in influencing Tc17 cell formation through metabolic remodeling in the atherosclerotic microenvironment, along with the intracellular and extracellular factors involved, remains a compelling area of ongoing research.

The distribution and clinical relevance of Tc17 cells in human atherosclerosis remain poorly understood, primarily due to the challenges in obtaining clinical samples. Hyperlipidemia, as the initiating stage of most atherosclerosis cases, represents a critical point of investigation. Given that hyperlipidemia is linked to metabolic age, achieving strict age matching posed a significant challenge in this study. Consequently, the clinical cohort primarily served as a “discovery cohort,” with its main objective being to preliminarily verify whether the frequencies of Tc17 cells and monocytes differ in human disease samples. Here, we reveal a significant increase in the proportion of Tc17 cells in hyperlipidemic patients, which remained statistically significant after adjustment for age and gender. Here, we reveal a significant increase in the proportion of Tc17 cells in hyperlipidemic patients, which remained statistically significant after adjustment for age and gender. Within the patient cohort, the percentage of Tc17 cells is positively correlated with monocytes, especially CD80⁺ monocytes (a difference that also persisted following the same adjustments), suggesting that monocytes/macrophages regulate Tc17 cell differentiation. It is important to note that potential unmeasured confounding factors (e.g., smoking, BMI, medication) could influence these observations. Therefore, our preliminary findings warrant further investigation using human plaques to fully elucidate these cellular interactions, as well as confirmation through more rigorously designed, larger cohort studies capable of controlling for a broader range of variables.

Collectively, based on ex vivo and in vivo data, our findings establish a connection between CD80⁺ monocytes/macrophages, IL-1β, and Tc17 cells within the atherosclerotic microenvironment, revealing CD80⁺ macrophages-IL-1β-Tc17 cell axis and providing new insights into the inflammatory mechanisms underlying atherosclerosis pathogenesis (as shown in the graphic abstract). These findings may pave the way for future therapeutic strategies targeting proatherogenic Tc17 cells, CD80⁺ macrophages/monocytes, or IL-1β, offering novel avenues for the treatment of atherosclerosis.

Abbreviations

ApoE

Apolipoprotein E

APC

Antigen-presenting cell

FBS

Fetal bovine serum

FCM

Flow cytometry

GM-CSF

Granulocyte-macrophage colony stimulating factor

HDL-C

High-density lipoprotein-cholesterol

HRP

Horseradish peroxidase

IFN-γ

Interferon-γ

IL

Interleukin

IL-17R

IL-17 receptor

LDL-C

Low-density lipoprotein-cholesterol

LDLr

Low-density lipoprotein receptor

LPS

Lipopolysaccharide

MAPK

Mitogen-activated protein kinase

MCP-1

Monocytes chemotactic protein 1

MHC

Major histocompatibility complex

ND

Normal diet

NF-κB

Nuclear factor kappa-B

NK

Natural killer

PBS

Phosphate-buffered saline

ROR γt

RAR-associated orphan nuclear receptor γt

TC

Total cholesterol

TG

Total triglyceride

TGF-β

Transforming growth factor-β

TNF-α

Tumor necrosis factor-α

WD

Western diet

Authors’ contributions

The author contributions are as follows: M.H., T.T.W, and C.Y.: conception and design, data analysis, and manuscript revision. Y.W and P.L.: experiment design and conduction, data analysis, and manuscript drafting. W.M.L.: blood samples and clinical data collection. Y.Z., T.R.Z, X.Y.C, and S.Y.L.: experiment conduction. Y.Z, L.M.M, and Z.Y.Y: technical support and editing. All authors reviewed and approved the final manuscript.

Funding

This work was supported by Natural Science Foundation of Chongqing (NO. CSTB2024NSCQ-MSX0470), National Natural Science Foundation of China (NO. 82104469), Science and Technology Research Program of Chongqing Municipal Education Commission Grant (NO. KJQN202200470), and Special Funding for Postdoctoral Research Projects in Chongqing (NO. 2024CQBSHTB3011).

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations

Ethics approval and consent to participate

All mouse experiments were conducted under protocols approved by Ethics Committee of Chongqing Medical University (NO.2021097). All described animal experiments procedures and animal care were carried out in accordance with the recommendations of the Chongqing Management Approach of Laboratory Animals (Chongqing government order NO.195). All described human experiments were conformed to the principles outlined in the Declaration of Helsinki, and approved by the Ethics Committee of University-town Hospital of Chongqing Medical University (NO. LL-202256). Informed consent was obtained from all individual participants included in the study.

Competing interests

The authors declare no competing interests.