OxLDL-mediated immunologic memory in endothelial cells

Highlights

• oxLDL induces a proinflammatory memory in human aortic endothelial cells.

• Memory formation is dependent on metabolic and epigenetic reprogramming.

• mTOR-HIF1α-signaling regulates cellular reprogramming.

• TLR2-signaling is selectively activated and necessary for oxLDL priming.

• ICAM-1 expression as well as monocyte adhesion, migration and transmigration are elevated and regulated through corresponing mechanisms in oxLDL-primed cells.

Abstract

Trained innate immunity describes the metabolic reprogramming and long-term proinflammatory activation of innate immune cells in response to different pathogen or damage associated molecular patterns, such as oxidized low-density lipoprotein (oxLDL). Here, we have investigated whether the regulatory networks of trained innate immunity also control endothelial cell activation following oxLDL treatment. Human aortic endothelial cells (HAECs) were primed with oxLDL for 24 h. After a resting time of 4 days, cells were restimulated with the TLR2-agonist PAM3cys4. OxLDL priming induced a proinflammatory memory with increased production of inflammatory cytokines such as IL-6, IL-8 and MCP-1 in response to PAM3cys4 restimulation. This memory formation was dependent on TLR2 activation. Furthermore, oxLDL priming of HAECs caused characteristic metabolic and epigenetic reprogramming, including activation of mTOR-HIF1α-signaling with increases in glucose consumption and lactate production, as well as epigenetic modifications in inflammatory gene promoters. Inhibition of mTOR-HIF1α-signaling or histone methyltransferases blocked the observed phenotype. Furthermore, primed HAECs showed epigenetic activation of ICAM-1 and increased ICAM-1 expression in a HIF1α-dependent manner. Accordingly, live cell imaging revealed increased monocyte adhesion and transmigration following oxLDL priming. In summary, we demonstrate that oxLDL-mediated endothelial cell activation represents an immunologic event, which triggers metabolic and epigenetic reprogramming. Molecular mechanisms regulating trained innate immunity in innate immune cells also regulate this sustained proinflammatory phenotype in HAECs with enhanced atheroprone cell functions. Further research is necessary to elucidate the detailed metabolic regulation and the functional relevance for atherosclerosis formation in vivo.

Introduction

In innate immune cells, the term immunometabolism describes the regulation of immune cell functions through adjustments of intracellular metabolic pathways. Complex metabolic modulation allows for a rapid or sustained response to extracellular cues and provides the necessary energy and metabolites to fulfill the required functions [1]. Recently, Netea et al. have established the immunometabolic concept of trained innate immunity. Trained innate immunity describes a long-term proinflammatory activation of innate immune cells through metabolic reprogramming in response to different pathogen or damage associated molecular patterns (PAMPS and DAMPs) [2,3]. Specific metabolic reprogramming during trained innate immunity alters the epigenetic landscape, thereby shifting promoters of inflammatory genes towards facilitated activation [4]. Accumulating evidence also points to a role of trained innate immunity in atherosclerosis. Pro-atherogenic DAMPs such as oxidized low-density lipoprotein (oxLDL), lipoprotein (a) or aldosterone can induce trained innate immunity in human monocytes [[5], [6], [7], [8], [9]] and human Smooth Muscle Cells (SMCs) [10] in vitro. In vivo, high cholesterol feeding of LDL-receptor knockout mice leads to sustained proinflammatory priming of hematopoietic stem cells with epigenetic and metabolic reprogramming [11]. Furthermore, monocytes of patients with familial hypercholesterolemia show corresponding metabolic and epigenetic changes and proinflammatory activation [12]. Endothelial cells form the inner layer of blood vessels and are crucially involved in initiation and progression of atherosclerosis. Interestingly, endothelial cells are capable of performing functions that are commonly ascribed to immune cells [13]. This immunologic function is dependent on the expression of pattern recognition receptors, which enable the detection of PAMPs and DAMPs in the blood stream. Activated endothelial cells express immune regulatory cytokines and are able to acquire antigen presenting capabilities [13,14]. In fact, Li et al. have described an innate immune transdifferentiation of endothelial cells in response to lysophospholipids [15]. Similarly to monocytes, endothelial cell phenotypes and cellular metabolic processes are closely intertwined. Environmental cues such as angiogenic growth factors or disturbed flow patterns initiate specific metabolic programs. While it has been shown that proatherogenic DAMPs such as oxLDL induce a proinflammatory activation of endothelial cells [16,17], the role of immunologic signaling pathways as well as metabolic and epigenetic programs in this context remain largely elusive [18]. Recently, we demonstrated that human coronary smooth muscle cells can be primed with oxLDL to show characteristic features of trained immunity, such as epigenetic and metabolic reprogramming with increased lactate production and glucose consumption [10]. Therefore, we hypothesized that oxLDL can induce a corresponding innate immune memory phenotype in human aortic endothelial cells (HAECs) through metabolic and epigenetic reprogramming.