In the last decade, it has become increasingly clear that chronic inflammation is a major factor in the development and progression of atherosclerosis (66
). Components of both innate and adaptive immunity have been implicated in disease initiation and progression (1
). DCs and macrophages represent classical professional APCs, and their numbers increase significantly during atherosclerosis progression in aortas (6
We found here that CD11b+CD11c+ cells extensively infiltrated the atherosclerotic aorta, where they resided both in the plaque and adventitia, potentially giving rise to CD11b+CD11c+ foam cells in the plaque. These cells showed high surface expression levels of MHCII and the costimulatory molecules CD80 and CD86, characterizing them as specialized and mature APCs. Some of these cells also expressed F4/80, which suggests that this heterogeneous population of CD11b+CD11c+ cells includes both DCs and macrophages.
Accumulating evidence suggests the possibility of direct T cell–DC interaction and antigen presentation in the aorta during induction and progression of atherosclerosis (14
). Here, using 2-photon microscopy, we were able to visualize T cell–APC interactions in the explanted mouse aorta. We showed that aortic APCs engaged in antigen-specific interactions with CD4+
T cells, which led to T cell proliferation and production of IFN-γ and TNF-α in the aorta. Since both T cell and APC numbers dramatically expand upon atherosclerosis progression, the number of their interactions may increase, leading to enhanced production of proatherogenic cytokines. Proinflammatory cytokines resulting from these interactions promoted uptake of oxLDL and mmLDL in macrophages in the aortic wall. Thus, our data identified a plausible mechanism by which the adaptive immune system contributes to atherosclerosis. Even though our results were obtained using an ex vivo approach, the antigen-specific interaction between T cells and APCs likely takes place and is important in vivo. However, other factors, such as intact circulation, ongoing recruitment of infiltrating leukocytes, systemic inflammatory responses, and intact lymphatic drainage, could also be important contributors to atherogenesis.
The contribution of myeloid cells to atherosclerosis is often envisioned only with regard to their ability to sustain inflammation via cytokine production, to take up lipids, and to differentiate into foam cells, thereby increasing plaque size (12
). Our data suggest that myeloid cells can also be involved in processing and presentation of antigens, resulting in antigen-dependent reactivation of T lymphocytes.
Previous work has suggested that oxLDL (44
), HSP60 (60
), or ApoB100 (47
) may represent possible atherosclerosis autoantigens. Our study did not address the identity of the atherosclerosis antigens, but showed that activated CD4+
T cells from Apoe–/–
mice participated in interactions with CD11c+
APCs and induced production of IFN-γ and TNF-α when incubated with Apoe–/–
aortas. Gene-deficient mice lacking the costimulatory molecules CD80 and CD86 have previously been shown to have reduced atherosclerosis (48
). Our data suggest that this may be explained, at least in part, by defective antigen presentation in the vessel wall. The important role of DCs and macrophages is further supported by prior reports that mice lacking CX3CR1, CCR2, or CCR5 also show reduced myeloid cell content in the artery wall and decreased plaque burden (68
). Given the heterogeneity of myeloid cell populations, it is possible that different subsets of DCs play different roles. A recent study suggested that CD103+
DCs in the arterial wall could play an immunoregulatory role by supporting Treg development (17
mice lacking Flt3, a receptor important for development and expansion of some DCs, show fewer CD103+
DCs in the aorta, which correlated with a small increase in atherosclerotic lesion size (17
). On the other hand, DC subsets, including CD11c+
cells, may be pivotal for T cell activation and inflammation during atherosclerosis, similar to their well-defined role in immune responses. Depletion of DCs has also been shown to elevate circulating LDL cholesterol (71
). Thus, the observed reduction of lesion size after depletion of APCs in Cd11c-DTR+
mice in our study may represent the sum total of all these effects.
Antigen presentation to T cells by APCs in the context of atherosclerosis has been proposed to entail APCs capturing antigen in the vessel wall and then migrating to draining lymph nodes to present this antigen to T cells (72
). However, the absence of lymphatic vessels in atherosclerotic plaques (73
) and altered homing properties of APCs under hypercholesterolemic conditions (19
) have been postulated to decrease or abrogate the ability of APCs to migrate to lymph nodes. In addition, chemokines and adhesion molecules such as CCL19, CCL21, P-selectin, and VCAM-1 within the developing atherosclerotic lesion may favor recruitment and retention of APCs, rather than their egress (73
). Therefore, an alternative and largely unexplored possibility is that tissue-residing and newly recruited APCs capture and process antigens, present them, and activate T cells in the aorta’s atherosclerotic environment. Through interactions with APCs in the arterial wall, T cells support the persistence of local chronic inflammation and promote atherosclerosis development.
Our 2-photon imaging experiments showed that CD4+
T cells underwent productive interactions with CD11c+
APCs in the aortic wall in the absence of secondary lymphoid organs. These interactions were strictly antigen dependent and more frequent with CD44hi
T cells. T cells engaged in long interactions with APCs in the vessel wall showed slow migration speeds, similar to those previously observed for T cell–DC interactions in other nonlymphoid organs (22
). The productiveness of these interactions was demonstrated by subsequent T cell proliferation and induction of proinflammatory cytokine production, particularly IFN-γ and TNF-α. The functionality of these cytokines in atherosclerosis has previously been established. IFN-γ gene ablation (76
) or blockade of TNF signaling (78
) relieves atherosclerosis, which suggests that the cytokines induced by APC interactions with T cells participate in atherosclerosis progression. The function of these cytokines in atherosclerosis is multifaceted and likely includes many different cell types and functional outcomes, such as chemokine production, cell recruitment, and cell maturation (11
Previous work has suggested that distinct cytokines can influence lipid uptake and differentiation of macrophages to foam cells (79
). SR-PSOX (CXCL16), one of the receptors for oxLDL, is expressed in human and mouse atherosclerotic lesions. In a myeloid cell line (THP-1), IFN-γ induced CXCL16 expression, suggesting at least one pathway by which IFN-γ could be proatherogenic and directly affect foam cell differentiation. Here, we showed that proinflammatory cytokines produced in response to antigen presentation enhanced uptake of oxLDL and mmLDL in primary aortic macrophages. Several lines of evidence suggested that IFN-γ coming from T cells activated in the aortic wall further regulates lipid uptake and foam cell formation. First, its production was stimulated by T cell–APC interactions in a strictly antigen-dependent manner. Second, exogenous IFN-γ enhanced mmLDL uptake. Third, blockade of CD4+
T cell–APC interaction by anti-MHCII antibody decreased oxLDL and mmLDL uptake. Fourth, transient depletion of DCs in Apoe–/–Cd11c-DTR+
mice also decreased oxLDL uptake in explanted aortas.
The main cell type that we were able to visualize in the aorta by live cell imaging engaged in productive interactions with T cells and expressed both CD11b and CD11c. Since these cells increased dramatically in number during the progression of atherosclerosis and expressed high levels of MHCII and costimulatory molecules, these double-positive CD11b+CD11c+ APCs may emerge as central drivers of the atherosclerotic process. They presented antigen and induced T cell cytokine production. Since foam cell formation is regulated by cytokines, whose production is induced upon T cell activation executed by myeloid cells in the aortic wall, our findings provide a plausible link between the adaptive and innate immune systems in atherosclerosis.
In summary, we conclude that interaction of CD11b+CD11c+ APCs with CD4+ T cells results in T cell activation, proliferation, and production of IFN-γ and TNF-α, which in turn supports foam cell formation by promoting uptake oxLDL and mmLDL by macrophages. This mechanism is a likely link by which adaptive immunity promotes atherosclerosis.