Role of interleukin 17 in inflammation, atherosclerosis, and vascular function in apolipoprotein e-deficient mice

Abstract

Objective: Interleukin 17A (IL17A) is involved in many inflammatory processes, but its role in atherosclerosis remains controversial. We examined the role of IL17A in mouse and human atherosclerosis.

Methods and results: Atherosclerosis was induced in apolipoprotein E (ApoE)(-/-) and IL17A/ApoE(-/-) mice using high-fat feeding, angiotensin II infusion, or partial carotid ligation. In ApoE(-/-) mice, 3 months of high-fat diet induced interferon-γ production by splenic lymphocytes, and this was abrogated in IL17A/ApoE(-/-) mice. IL17A/ApoE(-/-) mice had reduced aortic superoxide production, increased aortic nitric oxide levels, decreased aortic leukocyte and dendritic cell infiltration, and reduced weight gain after a high-fat diet compared with ApoE(-/-) mice. Despite these favorable effects, IL17A deficiency did not affect aortic plaque burden after a high-fat diet or angiotensin II infusion. In a partial carotid ligation model, IL17A deficiency did not affect percentage of stenosis but reduced outward remodeling. In this model, neutralization of the related isoform, IL17F, in IL17A/ApoE(-/-) mice did not alter atherosclerosis. Finally, there was no correlation between IL17A levels and carotid intima-media thickness in humans.

Conclusions: IL17 contributes to vascular and systemic inflammation in experimental atherosclerosis but does not alter plaque burden. The changes in plaque composition caused by IL17 might modulate plaque stability.

Figures

Figure 1

Cytokine production, body weight and vascular reactive oxygen species in ApoE−/− and IL17/ApoE−/− mice in response to high fat diet. Splenic lymphocytes from ApoE−/− and IL17/ApoE−/− mice fed regular (Reg) diet or high fat (HF) diet for 3 months were cultured on anti-CD3 plates and the culture supernatants were analyzed for IL17A using ELISA (A), or cytokine bead array (B–D) [n=4–7 per group]. Body weight (in grams) was measured at baseline and after 3 months of HF diet in ApoE−/− and IL17/ApoE−/− mice (panel E, n=12–15 per group). Aortic superoxide production was measured by dihydroethidium-HPLC (panel F, n=5–7 per group). Aortic nitric oxide levels after 3 months of high fat diet feeding were measured by electron spin resonance (ESR). Example ESR spectra are shown in panel G and summary data are shown in panel H [n=4–5 per group]. Data in panels A and H were analyzed using Student's t test. Data in panel E were analyzed using two way repeated measures ANOVA. Other statistical data were analyzed using one way ANOVA with Neuman-Keuls post-hoc test.

Figure 2. Effect of IL17 on atherosclerotic lesion development

ApoE−/− and IL17/ApoE−/− mice were fed a high fat diet for 3 months. Atherosclerotic plaque burden in the descending aortas was quantified using planimetry. Example aortas are shown in panel A and mean values are shown in panel B [n=14–16 per group; p=n.s.]. Atherosclerotic plaque area in the aortic root was analyzed by paraffin sectioning, hematoxylin and eosin staining, and planimetry using ImageJ software. Example sections are shown in panel C, and mean data are shown in panel D [n=7 per group; p=n.s.]. Data were analyzed using Student's t test.

Figure 3

Effect of IL17 on Plaque Composition. ApoE−/− and IL17/ApoE−/− mice were fed 3 months of high fat diet. Panel A shows Russell-Movat pentachrome staining in the aortic root at 4× and 10× magnification. Black=elastic fibers, yellow=collagen, blue/green=mucins, red=muscle, intense red=fibrinoid. Figures are representative of 4 per group. To analyze inflammatory cell content, flow cytometry of single cell suspensions of whole aortas were performed as shown in panel B. Representative scatter plots are shown on the left, and summary data of n=9–12 mice per group are shown on the right. Data were analyzed using Student's t test. CD45+ cells represent total leukocytes, CD3+ cells represent T cells, CD11b-11c+ cells represent dendritic cells, and CD11b+F4/80+ cells represent macrophages. Panel C shows plaque macrophage content in the aortic root as determined by immunostaining with a Mac3 antibody. Alkaline phosphatase (pink) was used to detect the secondary antibody. Slides were counterstained with hematoxylin. A representative of n=3 per group is shown at 4× and 10× magnification.

Figure 4

Effect of IL17 on Angiotensin II Induced Atherosclerosis and Examination of IL17F Levels. ApoE−/− and IL17/ApoE−/− mice were infused with angiotensin II for 4 weeks via osmotic minipump. Atherosclerotic lesions in the thoracic aortas were quantified by planimetry (A). Example aortas are shown on the left, and summary data are shown on the right [n=7 per group; p=n.s.]. Splenic lymphocytes from ApoE−/− and IL17/ApoE−/− mice fed regular (Reg) diet or high fat (HF) diet for 3 months were cultured on anti-CD3 plates, and IL17F released into the media was measured by ELISA [panel B, n=5–7 per group]. Data were analyzed using Student's t test. The statistical values in panel B represent a Bonferonni correction for 4 comparisons.

Figure 5

Effect of IL17A and F Isoforms on Partial Carotid Ligation Induced Atherosclerosis. The left carotid artery (LCA) of ApoE−/− and IL17A/ApoE−/− mice was partially ligated followed by 2 weeks of high fat feeding to induce accelerated atherosclerosis in the ligated artery. Mice were injected weekly with IL17F or isotype control antibody for 3 weeks starting one week before ligation. Frozen sections of carotid arteries were stained with Oil Red O, hematoxylin, and eosin. A representative LCA and right carotid artery (RCA) from each group is shown in (A). Quantification of internal elastic lamina (IEL) area of the LCA is shown in (B), and percent stenosis of the LCA is shown in (C) [n=5 per group]. Data were analyzed using one way ANOVA.