In the present study we have demonstrated that genetic deficiency of Ccr6 significantly reduces lesion development in the ApoE−/− mouse model of atherosclerosis. The reduction was apparent throughout the aorta and the aortic root, was large at both of two time points measured over 6 months of age, and was not attributable to changes in cholesterol or triglyceride levels. At the cellular level, protection was associated with a major reduction in macrophage accumulation in plaque in Ccr6−/− ApoE−/− mice compared to Ccr6+/+ApoE−/− mice. Transplantation studies indicated that protection was mediated by a Ccr6+ bone marrow-derived cell(s).
Consistent with a direct effect of monocyte/macrophage Ccr6 signaling on macrophage accumulation in the vessel wall in the model, we found that 1) both Ccr6 and its ligand Ccl20 were expressed in atherosclerotic aorta in this model; 2) Ccr6 is expressed on a subset of primary mouse monocytes and the mouse monocyte/macrophage cell line RAW 264.7; 3) Ccr6 mediates chemotactic responses of both primary mouse monocytes and RAW 264.7 cells to Ccl20 in vitro; 4) Ccr6−/− ApoE−/− mice were monocytopenic compared to Ccr6+/+ApoE−/− mice; and 5) Ccl20 could induce monocytosis when injected into Ccr6+/+ApoE−/− mice. Taken together, we propose that the mechanism by which Ccr6 deficiency protects against atherogenesis in this model involves Ccr6-dependent monocyte trafficking into the vessel wall due to reduced monocyte levels in the blood and/or reduced migration capacity into atherosclerotic lesions.
The magnitude of protection in the absence of Ccr6 (40%) was comparable to what has been reported previously for Ccr2−/−
mice in the same ApoE−/−
mouse model that we used. Importantly, Ccr6 deficiency in ApoE−/−
mice did not affect the expression of other chemokine receptors (e.g. Ccr2, Cx3cr1, Ccr5) or chemokines in the atherosclerotic aorta (Supplemental Figure VIII
), suggesting that these receptors may have non-redundant roles in atherogenesis. This could reflect action at different stages of atherogenesis or on different subsets in the monocyte migration process. For example, although all of these receptors are known to mediate monocyte/macrophage migration, Cx3cr1 may be more important as an adhesion receptor fostering interactions of foam cells with each other and with smooth muscle cells once they migrate into the vessel wall.5
The fact that Ccr6 deficiency results in a level of protection similar to these other monocyte/macrophage receptors may at first seem surprising since it is expressed on only a small subset of monocytes. As a potential mechanism, Ccr6 appears to operate at two steps important in macrophage accumulation: control of blood monocyte levels and direct recruitment of the cells into vessel wall. Dual action could greatly amplify the overall effect on pathogenesis beyond what might be expected based simply on the frequency of Ccr6+
cells. Additional work will be needed to define temporal and spatial expression of these ligands and receptors in the model to gain further insight into different mechanisms of monocyte recruitment. It is important to note that all of these receptors (Ccr2, Ccr5, Cx3cr1 and Ccr6) are also expressed on other leukocyte subsets represented in lesions in the model, which could also be contributing to pathogenesis through their recruitment and function. In particular, Ccr6 is expressed on neutrophils, dendritic cells, NKT cells, B cells and subsets of CD4+
T lymphocytes, and it is known that depletion of any of these cell types results in reduced atherogenesis in the model.2, 10, 22
However, the major leukocyte subtype by far present in atherosclerotic lesions is the foam cell, derived from blood monocytes.2
We found that Ccr6 deletion in ApoE−/−
mice not only significantly reduced the percentage of monocytes among peripheral blood leukocytes but also the total absolute monocyte counts in the blood, while other cell subsets were unaffected (). Monocyte levels did not appear to depend on Ccr6 in ApoE+/+
mice, which were maintained under the same conditions as ApoE−/−
mice (Supplemental Figure II
). The monocytopenia in Ccr6−/− ApoE−/−
mice was caused by a significant reduction of Ly6Chigh
inflammatory monocytes in the blood. At the same time, there was a significant increase of Ly6Chigh
inflammatory monocytes in the bone marrow of Ccr6−/− ApoE−/−
mice, indicating that Ccr6 likely affects the bone marrow egress of these cells. This is reminiscent of a previously identified role of Ccr2 in controlling monocyte mobilization from the bone marrow.23
It has been reported that Ccr6−/−
mice did not exhibit gross abnormalities in any major organ but they have increased numbers of T cells in the intestinal mucosa.24, 25
Also, it has been shown that Ccr6−/−
mice have impaired development of M cells and underdeveloped Peyer’s patches with a two-fold decrease of total leukocytes in the intestinal mucosa,26
However, this is the first report of Ccr6 regulation of blood monocytosis. Monocytosis is an independent predictor of subclinical carotid atherosclerosis and is a predictor of atherosclerotic plaque progression in acute myocardial infarction.27, 28
In the mouse model of atherosclerosis it has also been reported that monocytes accumulate continuously in the aorta during atheroma formation.29
In particular, total blood Ly6Chigh
monocytes increase dramatically in hypercholesterolemic ApoE−/−
mice fed a high-fat diet compared with wild type mice,29
which is consistent with our findings in the present study (Supplemental Figure II
). We found that both Ly6Chigh
monocytes express low levels of Ccr6 but only Ly6Chigh
monocytes were dysregulated in Ccr6−/− ApoE−/−
mice, which may reflect the different characteristics of these two subsets, e.g. Ly6Chigh
monocytes have been shown to preferentially adhere to activated endothelium and accumulate in atherosclerotic plaques when compared with Ly6Clow
Thus, we propose that the significant reduction of circulating monocyte counts in Ccr6−/− ApoE−/−
mice may directly reduce the monocytes available for recruitment to atherosclerotic sites, as an explanation for reduced macrophage content and atherosclerotic lesion size in these mice. Future studies comparing effects of transferring Ccr6+
monocytes or effects resulting from monocyte-specific deletion of Ccr6 will be needed to further refine this conclusion.
We found that Ccr6−/− ApoE−/−
mice had an 80% reduction of Ccl20 expression in the aorta compared with Ccr6+/+ApoE−/−
mice, whereas the systemic serum level of Ccl20 in these two groups was similar (). This suggests that Ccr6 and Ccl20 may form a local positive feedback loop in the vessel wall. A CCL20/CCR6 positive feedback loop has been previously described for Th17 cells, which are both CCR6 positive and able to produce CCL20.30
We found very few Th17 cells in atheroma, but they could contribute to macrophage accumulation. Previous studies showed that IL-17A may reduce, increase or have no effect on atherosclerosis development31–33
and recently Madhur et al.
reported that IL-17A deficiency does not alter plaque burden in ApoE−/−
indicating that more studies will be needed to clarify the role of IL-17 and Th17 cells in atherogenesis.
Our data on Ccr6 and the previously reported results connecting other chemokines and chemokine receptors to outcome in ApoE−/−
mice are consistent with the inflammation theory of atherogenesis. In this regard, Ccr6 and Ccl20 have been linked to multiple other mouse models of chronic inflammatory disease, including psoriasis, inflammatory bowel disease, rheumatoid arthritis and experimental autoimmune encephalomyelitis.16, 35, 36, 37
Recently, a triallelic dinucleotide polymorphism of CCR6 was correlated with the expression level of CCR6 and was associated with susceptibility to rheumatoid arthritis, Grave’s disease and Crohn’s disease;38
however, its association with atherosclerosis has not been defined yet.
In conclusion, Ccr6 deficiency in ApoE−/− mice causes significant reduction of circulating blood monocytes and reduces progression of atherosclerosis. Ccr6−/− ApoE−/− mice had markedly less Ccl20 in the aorta, suggesting a local positive feedback loop. Considering the atherogenic effect of Ccr6 in this mouse model, Ccr6 should be further considered in the molecular pathogenesis and therapeutic targeting of this disease.