The IL-17A/IL-17RA axis plays a proatherogenic role via the regulation of aortic myeloid cell recruitment

Abstract

Rationale: Atherosclerosis is a disease of large- and medium-sized arteries that is characterized by chronic vascular inflammation. While the role of Th1, Th2, and T-regulatory subsets in atherogenesis is established, the involvement of IL-17A-producing cells remains unclear.

Objective: To investigate the role of the IL-17A/IL-17RA axis in atherosclerosis.

Methods and results: We bred apolipoprotein-E-deficient (Apoe(-/-)) mice with IL-17A-deficient and IL-17 receptor A-deficient mice to generate Il17a(-/-)Apoe(-/-) and Il17ra(-/-)Apoe(-/-) mice. Western diet fed Il17a(-/-)Apoe(-/-) and Il17ra(-/-)Apoe(-/-) mice had smaller atherosclerotic plaques in the aortic arch and aortic roots, but showed little difference in plaque burden in the thoracoabdominal aorta in comparison with Apoe(-/-) controls. Flow cytometric analysis of Il17a(-/-)Apoe(-/-) and Il17ra(-/-)Apoe(-/-) aortas revealed that deficiency of IL-17A/IL-17RA preferentially reduced aortic arch, but not thoracoabdominal aortic T cell, neutrophil, and macrophage content in comparison with Apoe(-/-) aortic segments. In contrast to ubiquitous IL-17RA expression throughout the aorta, IL-17A was preferentially expressed within the aortic arch of WD-fed Apoe(-/-) mice. Deficiency of IL-17A or IL-17RA reduced aortic arch, but not thoracoabdominal aortic TNFα and CXCL2 expression. Aortic vascular IL-17RA supports monocyte adherence to explanted aortas in ex vivo adhesion assays. Short-term homing experiments revealed that the recruitment of adoptively transferred monocytes and neutrophils to the aortas of Il17ra(-/-)Apoe(-/-) mice is impaired in comparison with Apoe(-/-) recipients.

Conclusions: The IL-17A/IL-17RA axis increases aortic arch inflammation during atherogenesis through the induction of aortic chemokines, and the acceleration of neutrophil and monocyte recruitment to this site.

Fig.1. Deficiency of IL-17A attenuates atherosclerosis in Apoe^−/− mice

(A–C) Il17a^−/−Apoe^−/− and Apoe^−/− mice were fed a WD for 15 weeks and assessed for atherosclerotic plaques (n=18 mice/group). (A) Representative en face Oil Red O staining and lesion sizes (% of whole aorta) from Il17a^−/−Apoe^−/− and Apoe^−/− mice. (B) Representative en face staining and lesion sizes (% of whole aorta) of the aortic arch and (C) thoracoabdominal aortic segments of Il17a^−/−Apoe^−/− and Apoe^−/− mice. (D) Representative Movat staining of Il17a^−/−Apoe^−/− and Apoe^−/− aortic root sections (n=8–12). The data depicts the mean±SEM. Each symbol represents 1 animal; horizontal bars represent means. ***-P<0.001, **-P<0.01, *-P<0.05.

Fig.2. Deficiency of IL-17RA attenuates atherosclerosis in Apoe^−/− mice

(A–C) Il17ra^−/−Apoe^−/− and Apoe^−/− mice (n=16 mice) were fed a WD for 12 weeks. (A) Representative Oil Red O staining and lesion sizes (% of whole aortas) of Il17ra^−/−Apoe^−/− and Apoe^−/− aortas. (B) Representative aortic arch and (C) thoracoabdominal aortic segements from Il17ra^−/−Apoe^−/− and Apoe^−/− mice. (D) Representative Movat staining of Il17a^−/−Apoe^−/− and Apoe^−/− aortic root sections. The data depicts the mean±SEM. Each symbol represents 1 animal; horizontal bars represent means. ***-P<0.001, **-P<0.01, *-P<0.05.

Fig.3. IL-17RA promotes lesion development in the aortic arch without affecting thoracoabdominal aortic lesions or collagen content

(A) Average fold difference in aortic arch (AA) and thoracoabdominal aortic (TA) lesion area of 12 week WD Il17ra^−/−Apoe^−/− and Apoe^−/− mice (% of aorta, n=6–8 mice/genotype). (B) Average fold difference of collagen maturation of Il17ra^−/−Apoe^−/− and Apoe^−/− mice (% of aorta section, n=6–9). (C) Representative picrosirious red staining from the AA and TA segments of Il17ra^−/−Apoe^−/− and Apoe^−/− mice (mature: red/orange, immature: yellow/green). The data depicts the mean±SEM. ***-P<0.001.

Fig.4. Disruption of the IL-17A/IL-17RA axis decreases the overall T cell content of the aortic arch, but not the thoracoabdominal aorta

(A) Aortic cell suspensions from 15 week WD fed Il17a^−/−Apoe^−/−, Il17ra^−/−Apoe^−/−, and Apoe^−/− mice were stained and analyzed by flow cytometry (n=7 mice/genotype, five independent experiments; white bars-Il17a^−/−Apoe^−/−, gray bars-Il17ra^−/−Apoe^−/−, and black bars-Apoe^−/− mice). (B) mRNA expression of Il6, Ifnγ, and Tnf from the aortas Il17a^−/−Apoe^−/−, Il17ra^−/−Apoe^−/−, and Apoe^−/− mice (n=12 aortas/genotype, three independent experiments). (C) Representative flow cytometry plots of IFNγ⁺CD3⁺ and IL-17A⁺CD3⁺ cells in the aortas of 15 week WD fed Il17a^−/−Apoe^−/−, Il17ra^−/−Apoe^−/−, and Apoe^−/− mice. Plots are gated on CD45⁺CD3⁺ T cells (n=5 mice/genotype, four experiments). (D) Total number of IFNγ⁺CD3⁺ and IL-17A⁺CD3⁺ cells in the aortas of Il17a^−/−Apoe^−/−, Il17ra^−/−Apoe^−/−, and Apoe^−/− mice. (E–F) Aortic arch and thoracoabdominal aortic cell suspensions from Apoe^−/−, Il17a^−/−Apoe^−/−, and Il17ra^−/−Apoe^−/− mice were stained with anti-CD45 and TCRαβ Abs (n=9 aortas/genotype, four independent experiments). (E) Representative flow cytometry plots from Apoe^−/−, Il17a^−/−Apoe^−/−, and Il17ra^−/−Apoe^−/− aortic arch and thoracoabdominal aortas. (F) Total number of aortic arch and thoracoabdominal aortic TCRαβ T cells from Apoe^−/−, Il17a^−/−Apoe^−/−, and Il17ra^−/−Apoe^−/− mice. The data depicts means±SEM. ***-P<0.001, **-P<0.01, *-P<0.05.

Fig.5. Disruption of the IL-17A/IL-17RA axis decreases MΦ and neutrophil cellularity in the aortic arch, but not in the thoracoabdominal aorta

(A–B) 15 week WD Il17a^−/−Apoe^−/−, Il17ra^−/−Apoe^−/−, and Apoe^−/− aortic cell suspensions were stained and analyzed by flow cytometry (n=9 mice/genotype). (A) Representative flow cytometry plots are gated on CD45⁺CD11b⁺ cells (n=9 mice/genotype, seven independent experiments). (B) The total number of CD68⁺ MΦ, CD68⁺Gr-1^high leukocytes and Gr-1⁺CD68⁻ neutrophils were decreased in the aortas of WD fed Il17ra^−/−Apoe^−/− (grey bars) compared with Apoe^−/− (black bars) mice. (C) Representative flow cytometry plots are gated on CD45⁺CD11b⁺ cells (n=9 mice/genotype, four independent experiments). (D) Total numbers of aortic arch (left) and thoracoabdominal aortic (right) CD68⁺Gr1⁻ MΦ, CD68⁺Gr1⁺ leukocytes, and CD68⁻Gr1⁺ neutrophils (white bars-Il17a^−/−Apoe^−/−, gray bars-Il17ra^−/−Apoe^−/−, and black bars-Apoe^−/− mice). The data depicts means±SEM. ***-P<0.001, **-P<0.01, *-P<0.05.

Fig.6. Vascular IL-17RA supports monocyte recruitment in an IL-17A dependent manner

(A) mRNA expression of chemokines in 15 week WD fed Il17a^−/−Apoe^−/−, Il17ra^−/−Apoe^−/−, and Apoe^−/− aortas (n=12 aortas/genotype, three independent experiments). (B) Aortas from Apoe^−/− and Il17ra^−/−Apoe^−/− mice were treated with 100 ng/ml IL-17A (black bars) or a vehicle (white bars). CFSE-labeled Apoe^−/− monocytes (green) were added to aortas for an adhesion assay, and adherent monocytes were counted (n=5 experiments/condition).

Figure 7. Aortic arch IL-17A affects the regional production of pro-inflammatory TNF-α, Cxcl1, and Cxcl2

mRNA expression levels (δCt) of Il17ra (A) and Il17a (B) within the aortic arch (AA) and thoracoabdominal (TA) aortas of 12 week WD-fed Apoe^−/− and Il17ra^−/−Apoe^−/− mice (A) or Apoe^−/− and Il17ra^−/−Apoe^−/− mice (B), (n=9 mice/genotype, 3 independent experiments). (C–E) Combined aortic arch (AA) and thoracoabdominal (TA) aortic mRNA expression and protein levels of TNFα (C), CXCL2 (D), CXCL1 (E) within 12 week WD-fed Apoe^−/−, Il17a^−/−Apoe^−/−, and Il17ra^−/−Apoe^−/− mice. RT-PCR: n=9 mice/genotype, 3 independent experiments; Flow cytomix: n=6 mice/genotype, two independent experiments.

Fig. 8. Reduced recruitment of neutrophils and monocytes to atherosclerotic plaques of Il17a^−/−Apoe^−/− and Il17ra^−/−Apoe^−/− mice

(A–B) Apoe^−/− peripheral blood leukocytes were adoptively transferred to 15 week WD-fed recipient Apoe^−/− and Il17ra^−/−Apoe^−/− mice. After 12 hours, recipient aortas, blood and spleens and PLNs (not shown) were stained for CD45, CD11b, CD68, Gr-1 and evaluated by flow cytometry. (A) Representative flow cytometry plots of emigrated myeloid cells: MΦ/monocytes (CD68⁺Gr-1⁻), CD68⁺Gr-1⁺ cells, and neutrophils (CD68⁻Gr-1⁺). Numbers in quadrants are a percentage of positive cells. All plots are gated on CD45⁺CD11b⁺CFSE⁺ cells. (B) Homing of Apoe^−/− leukocytes (as numbers of CD45⁺CD11b⁺CFSE⁺ cells) in blood and the aortas of Il17ra^−/−Apoe^−/− (gray bars, n=11) and Apoe^−/− (back bars, n=11) recipients from 5 independent experiments. The data depicts means±SEM. ***-P<0.001, **-P<0.01, *-P<0.05.