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. 2010 Apr 20;121(15):1746-55.
doi: 10.1161/CIRCULATIONAHA.109.924886. Epub 2010 Apr 5.

Blockade of interleukin-17A results in reduced atherosclerosis in apolipoprotein E-deficient mice

Emily Smith  1 , Konkal-Matt R Prasad, Matthew Butcher, Anca Dobrian, Jay K Kolls, Klaus Ley, Elena Galkina
Affiliations

Blockade of interleukin-17A results in reduced atherosclerosis in apolipoprotein E-deficient mice

Emily Smith et al. Circulation. .

Abstract

Background: T cells play an important role during the immune response that accompanies atherosclerosis. To date, the role for interleukin (IL)-17A in atherogenesis is not well defined. Here, we tested the hypothesis that atherosclerosis-prone conditions induce the differentiation of IL-17A-producing T cells, which in turn promote atherosclerosis.

Methods and results: IL-17A was found to be elevated in the plasma and tissues of apolipoprotein E-deficient (Apoe(-/-)) mice. IL-17A-expressing T cells were significantly increased in the aortas, spleen, and lamina propria of aged Apoe(-/-) mice compared with age-matched C57BL/6 mice. IL-17A(+) T cells resided in both adventitia and aortas of aged Apoe(-/-) mice fed a chow diet. Elevated levels of IL-17A(+) T cells were also detected in the aortas of 21-week-old Apoe(-/-) mice fed a Western diet for 15 weeks. IL-17A(+) T cells were characterized as predominantly CD4(+) T helper 17 (Th17) cells and gammadelta(+) T cells. Blockade of IL-17A in Apoe(-/-) mice by use of adenovirus-produced IL-17 receptor A reduced plaque burden in Apoe(-/-) mice fed a Western diet for 15 weeks. In addition, the treatment diminished circulating IL-6 and granulocyte colony-stimulating factor levels and limited CXCL1 expression and macrophage content within the aortas. Conversely, IL-17A treatment of whole aorta isolated from Apoe(-/-) mice promoted aortic CXCL1 expression and monocyte adhesion in an ex vivo adhesion assay.

Conclusions: These results demonstrate that atherosclerosis-prone conditions induce the differentiation of IL-17A-producing T cells. IL-17A plays a proatherogenic inflammatory role during atherogenesis by promoting monocyte/macrophage recruitment into the aortic wall.

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Figures

Figure 1
Figure 1. Basal levels of IL-17A are elevated in female and male Apoe−/− mice
(A) IL-17A mRNA was found elevated in the aorta, spleen, LP and PLN of Apoe−/− mice (white bars, n=6) compared to C57BL/6 mice (black bars, n=6). (B) mRNA expression of IL-17C, D, and IL-17F was normalized compared to IL-17A (set as 100%) in the aortas of Apoe−/− mice (n=8). (C) Serum IL-17A, TNF-α, IL-2 were analysed in the plasma of Apoe−/− (white bars, n=10–11) and C57BL/6 mice (black bars, n=10–11), (D) Neutrophil (PMN) counts are elevated in Apoe−/− mice (white bars, n=8) compared to C57BL/6 mice (black bars, n=5); white blood cells (WBC). *P<0.05, **P<0.01 by unpaired Students t test.
Figure 2
Figure 2. IL-17A+CD3+ and IL-17A+CD3int T cells are found elevated in female and male Apoe−/− mice
To examine leukocyte population within the analyzed tissues, a gate was set on CD45+ cells. (A) Representative FACS plot showing an elevation of IL-17A+CD45+CD3+ and IL-17A+CD45+ CD3int T cells in the aortas of aged Apoe−/− mice compared to C57BL/6 mice. (B) IL-17A+CD3+ T cells were found elevated in the aortas, spleen and LP of aged Apoe−/− (black bars, n=12) mice and 21 week old Apoe−/− mice fed WD (grey bars, n=4) compared to C57BL/6 mice (white bars, n=10). *p<0.05, **p<0.01 by unpaired Students t test with Bonferroni-Holm correction for multiple comparison (aorta and spleen). (C, D) Representative FACS plot of an aorta (C) and spleen (D) showing elevated IL-17A+ cells. The phenotype of (E) IL-17A-expressing cells in the aorta and spleen of C57BL/6 (grey bars) and Apoe−/− (black bars) mice. Results show mean ± SEM, n=6 mice from at least 3 independent experiments. (F) Representative image of isolated murine aortic adventitia and aorta. (G) Representative FACS plot showing the presence of IL-17A+ T cells in the adventitia and aortic wall of 45 week old Apoe−/− mice.
Figure 2
Figure 2. IL-17A+CD3+ and IL-17A+CD3int T cells are found elevated in female and male Apoe−/− mice
To examine leukocyte population within the analyzed tissues, a gate was set on CD45+ cells. (A) Representative FACS plot showing an elevation of IL-17A+CD45+CD3+ and IL-17A+CD45+ CD3int T cells in the aortas of aged Apoe−/− mice compared to C57BL/6 mice. (B) IL-17A+CD3+ T cells were found elevated in the aortas, spleen and LP of aged Apoe−/− (black bars, n=12) mice and 21 week old Apoe−/− mice fed WD (grey bars, n=4) compared to C57BL/6 mice (white bars, n=10). *p<0.05, **p<0.01 by unpaired Students t test with Bonferroni-Holm correction for multiple comparison (aorta and spleen). (C, D) Representative FACS plot of an aorta (C) and spleen (D) showing elevated IL-17A+ cells. The phenotype of (E) IL-17A-expressing cells in the aorta and spleen of C57BL/6 (grey bars) and Apoe−/− (black bars) mice. Results show mean ± SEM, n=6 mice from at least 3 independent experiments. (F) Representative image of isolated murine aortic adventitia and aorta. (G) Representative FACS plot showing the presence of IL-17A+ T cells in the adventitia and aortic wall of 45 week old Apoe−/− mice.
Figure 3
Figure 3. Apoe−/− mice treated with Ad-IL-17RA:Fc show a reduction in plaque burden over the whole aorta
(A) Lane 1–3, Detection of soluble IL-17RA in the plasma of an Apoe−/− mouse 7 days after Ad-IL-17RA:Fc treatment by immunoprecipitaion and western blot; lane-4, KO, (plasma from Il17ra−/− mice). (B) Western blot demonstrating the detection of sIL-17RA in the plasma of Apoe−/− mice injected with Ad-IL-17RA:Fc on the day of termination (21 week old Apoe−/− mice). Lane 1, KO-negative control (Il17ra−/− plasma), lane 2–6-experimental Apoe−/− mice. (C) Representative en face staining of aortas excised from 21 week-old (15 weeks on WD) Apoe−/− mice treated with Ad-IL-17RA:Fc (right) or Ad-Luc (left). (D) Lesion sizes for Apoe−/− mice treated with Ad-IL-17RA:Fc (n=7) or Ad-Luc (n=11), (% of whole aorta). Each symbol represents one animal, horizontal bars represent means. **P<0.01.
Figure 4
Figure 4. Decreased plasma levels of IL-6 and G-CSF in Apoe−/− mice treated with Ad-IL-17RA:Fc
Collected plasma supernatants from Apoe−/− mice treated with Ad-IL-17RA:Fc or with control Ad-Lu were analyzed for the presence of (A) IL-6, (B) IFN-γ, (C) G-CSF and (D) IL-17F. Results show mean±SE from 5 to 10 mice. *, P<0.05, **, P<0.01.
Figure 5
Figure 5. IL-17A increases monocyte adhesion to aortic wall via CXCL1
(A) Paraffin-embedded sections of aortic roots from 21 week old Apoe−/− mice that received either Ad-IL-17RA:Fc or Ad-Lu adenoviruses were stained with Abs against CXCL1 (A, brown) and Mac-2 (B, brown), and counterstained with hematoxylin (blue). Representative staining from one mouse is shown. Percentage of (C) CXCL1 and (D) Mac-2 positive staining relative to the total area of the aortic root. Values represent mean±SEM of 5 to 10 mice per group. (E) IL-17A-treated aortas show increased monocyte adhesion. Aortas were harvested from 25–30 week old Apoe−/− mice and treated with IL-17A as described in Materials and Methods. Fluorescently labeled monocytes (green) were added to aortas for an adhesion assay and adherent monocytes were counted by blinded observers using a fluorescent microscope. (F) Data represent the mean±SE of 3–4 counted aorta per group from 4 independent experiments. (G) Aortic mRNA expression. Total cellular RNA from vehicle-treated (white bars), IL-17A-treated (grey bars) and unwashed IL-17A-treated aortas (black bars) were examined for CXCL1 using quantitative real-time PCR. * P<0.05, ** P<0.01 by Student’s t test with Bonferroni-Holm correction for multiple comparison (for Fig. 5F and G). Data are from 3 independent experiments using 3–4 aortas per group.
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