Deficiency of HIF1alpha in antigen-presenting cells aggravates atherosclerosis and type 1 T helper cell responses in mice

Introduction

Atherosclerosis is a chronic and systemic inflammatory disease characterized by the accumulation of immune cells in the vessel wall.^{1, 2} Dendritic cells (DCs) localize to the intima and adventitia in healthy arteries in regions predisposed to atherosclerosis and accumulate in atherosclerotic lesions.^{3, 4} DCs are increasingly regarded to play important roles in immune mechanisms governing atherogenesis.^4–6 Both local and systemic adaptive immune responses control atherogenesis, and pro- and anti-atherogenic CD4⁺ T helper cell subsets and their cytokines have been defined⁷. In particular, CD4⁺ type 1 T helper cells (Th1) and their cytokine IFN-γ promote atherosclerosis, whereas regulatory T cells (Tregs) inhibit vascular inflammation.^7,8 The function of the Th17 subtype is still unclear, as contradicting reports have been published.⁷

The Hypoxia-inducible factor (HIF)-1α is among the primary transcription factors induced under hypoxic conditions, but can also be upregulated by inflammatory stimuli, such as oxLDL and TNFα in normoxia.⁹ In addition to regulating cell responses to hypoxia, e.g. glycolysis, and angiogenesis, HIF1α was identified to modulate adaptive and innate immune responses.¹⁰

Due to the high metabolic activity of inflammatory cells within lesions, and the reduced availability of oxygen in deeper plaque areas, atherosclerotic lesions harbor areas of hypoxia¹¹, and HIF-1α can be detected in atherosclerotic lesions in both mice and humans.^11–14 The direct cell-specific role of HIF1α in atherosclerosis in vivo, however, has not been addressed previously.

We here investigated the function of HIF1α in atherosclerosis in antigen-presenting cells (APCs). By deleting Hif1a specifically in CD11c⁺ cells, we here for the first time reveal a critical role of HIF1α in balancing APC-driven Th1-polarization during atherogenesis in Ldlr^−/− mice, and to attenuate the progression of atherosclerosis in low-density lipoprotein receptor-deficient (Ldlr^−/−) mice.

Materials and Methods

Materials and Methods are available online.

Results

Discussion

Although T cell responses and in particular Th1-mediated immunity drive atherosclerotic lesion formation⁸, the pathways in APCs that control T cell activation remain largely elusive. The transcription factor HIF1α is known to modulate immune responses¹⁰ and to be found in atherosclerotic plaques.^11–14 We here unveil that HIF-1α expression is upregulated in CD11c⁺ APCs in atherosclerotic Ldlr^−/− mice. In order to assess the (patho-) physiological relevance of HIF-1α in this cell type, we used CKO mice with a deletion of Hif1a in CD11c⁺ APCs in Ldlr^−/− mice. Importantly, an accelerated atherosclerotic lesion formation was observed in mice deficient in HIF-1α in APCs, together with an expansion of pro-inflammatory Th1 cells both locally within lesions, but also systemically, indicating that APC-expressed HIF-1α is of paramount importance in balancing uncontrolled Th1 cell responses and atherosclerosis in Ldlr^−/− mice. Mechanistically, we could demonstrate that HIF1α directly binds the Stat3 promoter to control its transcription. Overexpression of Stat3 in Hif1a-deficient APCs in bone marrow reversed enhanced atherosclerotic lesion formation and reduced Th1 cell-expansion in chimeric Ldlr^−/− mice. These findings offer unique insight into the regulatory function of HIF1α in APCs (Supplementary Figure XI), and substantiate the critical role of APCs in controlling immune mechanisms that drive atherogenesis.

In humans and apolipoprotein E-deficient mice, HIF1α expression was detected in atherosclerotic lesions and to increase from early to stable lesions.^{11,13, 14} In line, we were able to detect an upregulation of HIF1α expression in atherosclerotic aortae and aortic roots of Ldlr^−/− mice when compared to healthy controls, and in advanced versus early human atherosclerotic lesions. Expressed in many cell types, HIF1α was also found to co-localize with hypoxic CD11c⁺ cells within lesions in both mice and humans.

Limited evidence on the role of HIF1α in atherosclerotic lesion formation exists. Mice lacking HIF1α in CD4⁺ T cells were previously shown to display increased T cell activation, associated with an augmented neointimal femoral artery hyperplasia after cuff placement.²⁸ Systemic hydrodynamic injection of plasmids encoding constitutively active Hif1a into Apoe^−/− mice, resulting in HIF1α overexpression predominantly in CD4⁺ T cells, lead to a reduction in lesion formation, associated with a shift towards an anti-inflammatory cytokine expression profile in CD4⁺ T cells.²⁹ However, in contrast to these studies, which would be consistent with an induction of Foxp3 and Tregs by HIF1α³⁰ and the demonstration of a protective function of Tregs in atherosclerosis,⁸ deficiency in HIF1α was more recently shown to diminish Th17 but to enhance Treg development in CD4⁺ T cells.³¹

In macrophages, HIF1α has been described to be critical for maintaining intracellular energy homeostasis, and Hif1a-deficient LysM-cre⁺ macrophages were shown to display normal cytokine production but an abrogated migratory capacity, preventing skin infiltration and inflammation.¹⁷ However, a reduced production of pro-inflammatory cytokines was demonstrated in Hif1a-deficient LysM-cre⁺ macrophages in response to LPS, together with a protection from LPS-induced sepsis.³² In the context of atherosclerosis, HIF1α was suggested to exert pro-atherogenic functions in cultured macrophages by promoting cholesterol accumulation.¹⁴ Variable effects of hypoxia-induced HIF1α expression have also been shown in DCs. For instance, a reduction in costimulatory molecule expression and of the stimulatory capacity for T-cell functions was observed in one study whereas increased expression of costimulatory molecules and an induction of allogeneic lymphocyte proliferation in response to LPS was noted in another report in vitro, whereas both studies described an up-regulated production of proinflammatory cytokines^{33, 34}. Prior to our study, the direct in vivo role of HIF1α in APCs in atherosclerosis had not been addressed.

We here deleted Hif1a specifically in CD11c⁺ APCs, allowing a definite assessment of its role under physiological conditions and in atherosclerosis in vivo. APCs differentiated normally with no differences in their numbers or maturation in Hif1a-deficient mice. Moreover, no differences in APC phenotype and T cell activation were noted in young, healthy mice, indicating that HIF1α plays a subordinate role in maintaining homeostatic APC functions. In atherosclerotic Ldlr^−/− mice, however, a significant increase in IL-12 was observed in Hif1a-deficient APCs, whereas other cytokines and the expression of MHC-II and co-stimulatory molecules were unaltered. Moreover, an enhanced activation of CD4⁺ T cells and increased frequencies of Th1 cells were observed in Hif1a-CKO Ldlr^−/− versus Hif1a-WT Ldlr^−/− mice in vivo in the aorta and spleen, and in co-cultures with Hif1a-deficient APCs isolated from atherosclerotic Ldlr^−/− mice and loaded with OVA as a model antigen in vitro. These data suggest that Hif1a-deficiency in APCs drives T cell activation and Th1-differentiation, and that the effects of HIF1α deficiency are systemic.

Hypoxia can frequently be detected in atherosclerotic plaques. Hypoxyprobe (pimonidazole) is metabolized in living cells experiencing oxygen levels below 10mmHg (~1% O2). Cells positive for pimonidazole are thus viable and hypoxic, but do not experience a total lack of oxygen (anoxia). Both the thickness of the plaque exceeding the maximum oxygen diffusion distance, and more importantly, the high metabolic demand of cells within chronically inflamed tissue contribute to plaque hypoxia also within the oxygen-diffusion limit in symptomatic patients, rabbits and mice.^{11, 12, 35}. In line, we detected hypoxic regions in atherosclerotic lesions of Ldlr^−/− mice in luminal and intramural plaque cells. In addition to hypoxia, however, HIF1α expression can also be triggered and potentiated by oxLDL, lipopolysaccharides and pro-inflammatory cytokines^{9, 36, 37}. Hence, HIF1α expression in hypoxic vascular APCs, known to ingest lipids and to be exposed to cytokines⁴, may arise from a combination of these factors. Likewise, increased HIF1α in splenic APCs may have been activated by systemically increased lipid mediators or atherogenic cytokines, possibly in combination with relative hypoxia due to higher oxygen consumption under conditions of splenic inflammation. In this regard it is interesting that similar changes in Th1-polarization were observed upon systemic immunization with OVA in otherwise healthy CKO mice, providing further evidence that HIF1α controls APC-driven T cell responses in inflammation also unrelated to atherosclerosis.

Increased atherosclerotic lesion size in Hif1a-CKO Ldlr^−/− mice was accompanied by an increased necrotic core area. It was recently shown that silencing of HIF1α provokes a loss in viability with increased rates of apoptosis and necrosis in cultured human macrophages, potentiated in the presence of oxLDL or under hypoxic conditions.³⁸ Although the potential impact of reduced monocyte/macrophage viability in atherosclerotic plaques is unclear and may depend on plaque stage, an increased apoptosis/necrosis of HIF1α-deficient APCs may have contributed to the expansion of the necrotic core in our model, warranting further investigations of this mechanism and its impact on atherogenesis in the future.

Notably, Ldlr^−/− mice carrying LysM-cre⁺ Hif1a^flox/flox bone marrow did not display any differences in plaque size or T cell activation. This may appear counterintuitive as a substantial proportion of CD11c⁺ APCs, e.g. monocyte-derived DCs and CD11c⁺ macrophages, would also lose expression of HIF1α in this model. However, HIF1α may have pro-atherogenic functions in CD11c⁻ myeloid cell subsets that promote atherosclerosis, such as Ly6C^high monocytes², macrophages and neutrophils³⁹ that contrast with its protective role in CD11c⁺ APCs. For instance, several reports have described a pro-inflammatory function of HIF1α in neutrophils^{17, 40} or macrophages.³⁸ Hence, the loss of protective HIF1α signaling in some CD11c⁺ cells may have been counterbalanced by the loss of its pro-atherogenic functions in other cell types in LysM-cre⁺ Hif1a^flox/flox mice. Alternatively, the phenotype observed in CD11c-cre⁺ Hif1a^flox/flox mice may be preferentially related to atheroprotective functions of HIF1α in classical DCs. In the future, lineage-specific deletion of HIF1α in novel models may provide a clearer picture of its role in these various cell populations.

We did not detect any alterations in NF-κB, IKKα or IκBα expression in APCs deficient in HIF1α, similar to Hif1α-deficient LysM-cre⁺ macrophages,³² indicating that the deletion of Hif1a does not directly affect the NF-κB pathway per se. In agreement with the identification of binding sites in silico, ChIP analyses demonstrated direct binding of HIF1α to the Stat3 promoter, and Hif1a-deficient APCs to display a reduction in Stat3 mRNA and protein expression. These findings for the first time reveal HIF1α as an important regulator of STAT3 expression. Interestingly, STAT3 is known to exert immune-suppressive and anti-inflammatory functions in myeloid cells,⁴¹ and mice with Stat3–deficient APCs were previously shown to produce significantly more IL-12 in response to LPS, associated with an increased capacity to stimulate T cell proliferation and IFNγ secretion.²² Accordingly, overexpression of STAT3 reduced Il12 transcript levels in BMDCs, whereas a dominant negative mutant of STAT3 elevated Il12 expression, corroborating evidence that STAT3 interferes with Il12 transcription.^{22, 42} Importantly, these effects occurred down-stream of HIF1α, as also evidenced in Hif1a-deficient APCs. Lentiviral transduction of Hif1a-CKO BM with overexpression of STAT3 in APCs reversed the enhanced atherosclerotic lesion formation, decreased T cell infiltrates and reduced Th1-cell polarization in chimeric Ldlr^−/− mice, confirming that increased levels of HIF1α and STAT3 in APCs are pivotal in controlling atherosclerotic plaque formation. In line with a clear but non-significant trend towards increased STAT3 expression in splenic APCs, marginal effects on plaque size and unaffected aortic T cell accumulation and Th1 cell responses were observed in WT-BM+Ctrl-virus→Ldlr^−/− vs. WT-BM+STAT3-virus→Ldlr^−/− mice. This may indicate that reduced STAT3 availability in CKO APCs rather than its additional supplementation in WT APCs that already inherently display increased HIF1α and STAT3 levels in atherosclerosis determines disease development in this setting.

Interestingly, human APCs exposed to hypoxia that display increased levels of HIF1α showed a reduced secretion of IL-12⁴³ and induced lower T cell IFNγ-production,^{34, 43} suggesting an HIF1α-triggered pathway restraining Th1 responses in human APCs. Notably, extending previous findings describing the presence of hypoxia and HIF1α in human atherosclerotic lesions,^11–14 we here demonstrate that APCs express HIF1α, STAT3 and IL-12 in human atherosclerotic lesions. Furthermore, an increased HIF1α and STAT3 but a decreased IL-12 mRNA expression was observed in advanced versus early carotid artery plaques. These data suggest that the regulatory signaling axis revealed in our study in APCs in mice may also be operative in human disease. Interestingly, IL-12 expression in plasma and plaque tissue was previously shown to correlate with IFN-γ expression, with T cells being the principal source of IFN-γ in the arterial wall in humans,⁴⁴ in line with the notion that IL-12-controlled Th1 T cell responses are of primary importance during plaque development.

The bidirectional effects of HIF1α-deficiency in APCs on other lesional cell types and their contribution to lesion formation remains to be addressed. For instance, mast cells are present in atherosclerotic plaques and are considered to promote lesion growth and plaque destabilization.⁴⁵ Mast cell-derived cytokines, via an induction of HIF1α in APCs, may have led to an attenuated pro-atherogenic APC phenotype balancing overshooting inflammation in atherosclerosis, and be in line with mast cells often ensuing Th2-type inflammatory responses.^{45, 46} However, pro-inflammatory mediators released by mast cells, as induced by the contact with T cells, which showed an increased activation in CKO Ldlr^−/− mice, may have also contributed to enhanced inflammation^{45, 46} and plaque progression in our study.

Although it is widely acknowledged that Th1-mediated immune responses drive atherosclerotic lesion formation,⁶ still little is known about the pathways and transcription factors in APCs that drive T cell polarization in atherosclerosis. Moreover, although HIF-1α can be detected in atherosclerotic lesions in both mice and humans^11–14 and can modulate immune responses,¹⁰ its cell-specific role in atherosclerosis had not been addressed previously. While dispensable under homeostatic conditions, our findings for the first time demonstrate that HIF1α balances APC activation and Th1-polarization during atherogenesis in Ldlr^−/− mice and attenuates disease progression.

Supplementary Material