The development and persistence of atherosclerosis depends on chronic inflammation mediated by both the innate and adaptive immune responses. Several recent publications have convincingly shown elevated levels of circulating and arterial Th17 and IL-17A+
T cells in atherosclerosis-prone Apoe−/−
mice and CAD patients.3
Despite a clear correlation between elevated levels of IL-17A and atherosclerosis, to date, the functions of IL-17A-producing cells and IL-17A in atherosclerosis remain poorly defined.
In this study, we sought to delineate the role of IL-17A+
cells and the IL-17A/IL-17RA pathway in atherosclerosis through the use of newly generated Il17a−/−Apoe−/−
mice. We previously reported a reduction in atherosclerotic plaques in soluble IL-17RA blockaded Apoe−/−
In the present study, we expanded our focus and investigated the impact of IL-17A or IL-17RA deficiency on atherogenesis. We found a significant decrease in the aortic lesions of WD, but not chow diet fed Il17a−/−Apoe−/−
mice compared with Apoe−/−
mice. To further dissect the roles of the IL-17A/IL-17RA axis, several potential sites of atherogenesis including the aortic arch, thoracoabdominal aorta, and aortic roots of mice fed WD were examined. IL-17A/IL-17RA deficiency had striking inhibiting effects on atherosclerotic plaque burden in the aortic arch, but only minor effects within the thoracoabdominal compartment. The observation that IL-17A exerts limited effects on the descending aorta is in line with the findings of Madhur et.al. that IL-17A-deficient Apoe−/−
mice display slight non-significant reductions in thoracoabdominal aortic plaques in comparison to Apoe−/−
There are several examples of the site-specific development and progression of atherosclerosis.28
Deficiency of IL-4 within the bone marrow resulted in no change in aortic-root lesion area, despite reduced en face
The deficiency of p47 NADPH oxidase subunit in Apoe−/−
mice reduced aortic atherosclerosis to a greater extent than the aortic root.30
In the present study, we characterized the aortic arch-specific influence of the IL-17A/IL-17RA axis.
Histological examination of the aortic arch and thoracoabdominal aortic collagen content surprisingly revealed no differences between Il17ra−/−Apoe−/−
mice at this time point. Interestingly, we also found no difference in SMC content between Il17ra−/−Apoe−/−
mice, indicating that the IL-17A/IL-17RA axis does not significantly influence SMC proliferation and migration within the atherosclerotic aorta. While several recent studies have suggested that IL-17A may support SMC proliferation in vitro
Danzaki, et. al. demonstrated elevated aortic root SMC content in 8 week WD-fed Il17a−/−Apoe−/−
Further examination of the effects of the IL-17A/IL-17RA axis on aortic SMC and endothelial cell functions are ultimately necessary.
One of the striking observations of this study was the differences in atherosclerotic lesions between the aortic arch and thoracoabdominal aorta of 15 week WD-fed mice. To assess the potential mechanisms for this phenomenon, we analyzed the expression of IL-17A and IL-17RA at these different anatomical sites of the aorta. IL-17RA was expressed ubiquitously in the aorta, whereas IL-17A expression was preferentially found within the aortic arch. Interestingly, we detected no difference in the expression of IL-17A in the TA and AA in 5 week-old Apoe−/− mice, indicating that the specific up-regulation of IL-17A does not occur in relatively healthy aortas. Altogether, our findings suggest that the aortic expression of IL-17A may depend on the stage of lesion development, which varies at different anatomical locations. The stage of lesion development also depends on the local inflammation that accompanies atherogenesis. Indeed, elevated expression of the pro-inflammatory cytokine TNFα and several chemokines that are involved in aortic leukocyte migration were preferentially detected in the aortic arches of 15 week WD-fed Apoe−/− mice. Further temporal studies will be required to determine if IL-17A-producing cells may be similarly recruited into advanced thoracoabdominal aortas.
To date, the exact role of IL-17A+
cells in atherosclerosis is unclear due to conflicting results from neutralizing antibody and bone marrow transfer experiments.12–14,16–18,27
However, some of the discrepant results may be attributable to differences in the experimental design, diets, or other confounding factors. In addition, most of these studies have examined aortic root atherosclerosis, but only few studies directly examined whole aortic plaque burden by en face
analysis. The results reported here clearly demonstrate the aortic arch-specific effects of IL-17A/IL-17RA, and at least partially shed light on the controversy of reported effects of IL-17A/IL-17R axis blockade during atherogenesis.
Th17 cells play an important role in the immune response, and are major contributors to autoimmune diseases such as multiple sclerosis, inflammatory bowel disease and arthritis.5
There are several pathways by which IL-17A-producing T cells might affect local inflammation. IL-17A supports the production of IL-6 and IL-8, and the chemokines CCL5, CCL2, CXCL1 and CXCL10 in several cell types, including endothelial and vascular smooth muscle cells (VSMCs),33,34
fibroblasts and epithelial cells.6,20
The results reported here clearly demonstrate that the IL-17A/IL-17RA axis affects the expression of multiple aortic chemokines, including Ccl2, Ccl20, Cx3cl1, Cxcl1, Cxcl12, Cxcl2
, and Cxcl5
, thus accelerating leukocyte recruitment to atherosclerotic vessels.
In support of this notion, we detected reduced numbers of T cells, CD68+
myeloid cells, and CD68−
neutrophils in WD fed Il17ra−/−Apoe−/−
aortas at steady-state conditions. Interestingly, the phenotype of reduced aortic MΦ content was also reported when anti-IL-17A Abs were used to block IL-17A functions in vivo.12,16
Importantly, additional separate examination of the aortic arch and thoracoabdominal segments of Il17ra−/−Apoe−/−
, and Apoe−/−
aortas by flow cytometry revealed diminished numbers of T cells, MΦ, and neutrophils specifically within the aortic arches of Il17ra−/−Apoe−/−
mice. Thus, these results clearly emphasize a distinct role for the IL-17A/IL-17RA axis in the regulation of the number of T and myeloid cells within the aortic arch, and to a lesser extent, the thoracoabdominal aorta. It is interesting to note that the reduction in leukocyte cellularity within the aortic arch was relatively smaller compared with the reduction in plaque burden, suggesting that other IL-17/IL-17RA-dependent factors in addition to the total cellularity are involved in atherogenesis.
Monocytes express IL-17RA, and recent data suggests that IL-17A can also directly affect monocyte chemotaxis in vivo
and in vitro
Antibody blockade of IL-17A in the synovial fluid of rheumatoid arthritis patients inhibited in vitro
monocyte chemotaxis, and MΦ accumulation in the bronchoalveolar lavage fluid during allergic airway inflammation.25
Lethally irradiated low density lipoprotein receptor-deficient (Ldlr−/−
) mice reconstituted with IL-17RA-deficient bone marrow resulted in reduced aortic root lesions, neutrophil, and mast cell content in comparison to Il17ra+/+
recipients suggesting a potential role of IL-17RA on hematopoetic populations during atherogenesis.27
Evidence have also demonstrated that vascular ECs21
express IL-17RA, and are able to respond to IL-17A. To address the extent to which IL-17RA expression by vascular cells impacts IL-17A-induced adhesion to atherosclerotic aortas, we performed ex vivo
adhesion assays. Supplementation of explanted aortas with rIL-17A strongly supported monocyte adherence in a manner that depended on vascular IL-17RA.
The recruitment of leukocytes into the aortas during the initial and established stages of atherosclerosis is one of the key components of the progression of atherosclerosis. To further establish the role of IL-17A in the regulation of leukocyte content in the aortas, we determined whether the IL-17A/IL-17RA axis affects the migration of monocytes and neutrophils to the aortas in short-term adoptive transfer experiments. We detected significant reductions of CFSE+ emigrated Apoe−/− monocytes and neutrophils within the aortas of Il17ra−/−Apoe−/− recipients highlighting an essential role of vascular IL-17RA in supporting myeloid cell migration. Of note, we found no difference in the migration of Il17ra−/−Apoe−/− and Apoe−/− monocytes and neutrophils to Apoe−/− aortas in the short-term homing experiments. These findings suggest that the reduction of monocyte and neutrophil content of the aorta in steady-state conditions is due to impaired capacity of myeloid cells to migrate into atherosclerotic aortas in the absence of IL-17A or vascular IL-17RA. Interestingly, as studies involving chemokine or chemokine receptor-deficient mice display distinct anatomical alterations in atherosclerotic plaque formation, the overt phenotype of Il17a−/−Apoe−/− and Il17ra−/−Apoe−/− mice can be partially attributed to the regulation of multiple aortic chemokines by IL-17A.
While several studies have suggested a pro-atherogenic role of IL-17A based on neutralizing strategies,12,13,16
and bone marrow transfers,27
Taleb, et al.4
and Danzaki, et al.32
recently proposed an atheroprotective role for IL-17A+
T cells through cross-regulation of Th1 cells and IFNγ. Danzaki, et. al. demonstrated elevated Th1 cell content and IFNγ production in 8 week WD-fed Il17a−/−Apoe−/−
Although initial in vitro
differentiation assays have implicated cross-regulation of Th17 cell differentiation by Th1 cells,5
recent data have shown that Th17 cells are induced in parallel to Th1 cells in some pathological conditions.3
Indeed, while Th1 cells represent the major T helper subset in atherosclerosis, both Th17, Th1, and Th1/Th17 cells are present in both murine and human atherosclerotic arteries.3,11–13,27
Therefore, we tested if the deficiency of IL-17A or IL-17RA would alter the Th1 response during atherogenesis. While the percentage and number of splenic and peripheral blood Th1 cells were unaltered, the numbers of aortic Th1 cells were diminished within Il17a−/−Apoe−/−
mice. These data indicate that the deficiency of IL-17A or IL-17RA has no effect on the generation and maintenance of Th1 cells in atherosclerosis. Altogether, our data suggest that while the IL-17A/IL-17RA axis does not affect the percentage of Th1 cells, it does influence the number of aortic T cells and therefore, the total levels of T cell-derived IFNγ.
In summary, using IL-17A and IL-17RA deficient Apoe−/− mice, we demonstrate that the IL-17A/IL-17RA pathway plays a pro-inflammatory role during atherogenesis preferentially within the aortic arch. Upon atherogenesis, Th17 and other IL-17A-producing cells accumulate within the aortas and release IL-17A, which in turn, induces the production of TNFα and various chemokines - resulting in accelerated monocyte and neutrophil homing and further development of atherosclerosis preferentially within the aortic arch of the aorta.
Novelty and Significance
What is known?
- Elevated levels of Interleukin-17A (IL-17A), a hallmark cytokine of T helper 17 and IL-17A+ γδ T cells, are detectable within murine and human atherosclerotic plaques as well as the plasma of patients with coronary artery disease and artery disease (post-endarterectomy).
- Attempts to neutralize IL-17A via neutralizing antibodies, decoy receptors, and bone marrow transfer have yielded inconsistent results, suggesting both pro- and anti-atherogenic roles for IL-17A.
- Recent studies with Il17a−/−Apoe−/− mice revealed slight changes in aortic root and thoracoabdominal aortic lesions, despite a favorable trend towards decreased aortic leukocyte infiltration.
What new information does this article contribute?
- We found that in mice placed on Western diet for 15 weeks, IL-17A plays a pro-inflammatory role by affecting aortic arch lesions, pro-inflammatory cytokines, and chemokine production.
- IL-17A supports aortic inflammation by promoting vascular-IL-17RA dependent chemokine expression thereby stimulating monocyte and neutrophil recruitment to atherosclerotic plaques.
- Deficiency of IL-17A reduces the number, but not the percentage, of aortic IFNγ+ T cells.
The role of IL-17A in atherosclerosis is currently unclear due to conflicting data obtained from IL-17A neutralization studies and differences in measurements of atherosclerosis. In other pathological states, IL-17A promotes leukocyte recruitment to sites of inflammation by supporting the production of stromal chemokines. In the present study, we sought to clarify the role of IL-17A in atherosclerosis using both IL-17A- and IL-17RA- deficient atherosclerosis-prone Apoe−/− mice. We demonstrate that IL-17A and IL-17RA deficient mice display reductions in aortic arch but not in extent of thoraco-abdominal aortic plaques, numbers of leukocytes, nor levels of chemokines. Additionally, examination of aortic arch and thoraco-abdominal aortic expression of Il17ra and Il17a within WD-fed mice revealed the exclusive expression of IL-17A within the aortic arch, which corresponded with elevated levels of pro-inflammatory TNFα, CXCL2, and CXCL1. Adoptive transfer experiments demonstrate reduced recruitment of monocytes and neutrophils to the aortas of IL-17RA-deficient Apoe−/− mice. These findings suggest that IL-17A plays a site-specific pro-inflammatory role and that it specifically stimulates chemokine and cytokine production, as well as monocyte and neutrophil recruitment in the aortic arch.