Immune and inflammatory mechanisms of atherosclerosis

Cardiovascular disease is the biggest killer globally and the principal contributing factor to the pathology is atherosclerosis; a chronic, inflammatory disorder characterized by lipid and cholesterol accumulation and the development of fibrotic plaques within the walls of large and medium arteries. Macrophages are fundamental to the immune response directed to the site of inflammation and their normal, protective function is harnessed, detrimentally, in atherosclerosis. Macrophages contribute to plaque development by internalizing native and modified lipoproteins to convert them into cholesterol-rich foam cells. Foam cells not only help to bridge the innate and adaptive immune response to atherosclerosis but also accumulate to create fatty streaks, which help shape the architecture of advanced plaques. Foam cell formation involves the disruption of normal macrophage cholesterol metabolism, which is governed by a homeostatic mechanism that controls the uptake, intracellular metabolism, and efflux of cholesterol.

Atherosclerosis is an inflammatory disease of the wall of large- and medium-sized arteries that is precipitated by elevated levels of low-density lipoprotein (LDL) cholesterol in the blood. The ubiquitous monocyte, the precursor of macrophages in all tissues, is present in every phase of atherogenesis. Monocyte-derived macrophages are scavenging and antigen-presenting cells, and they secrete cytokines, chemokines, growth-regulating molecules, and metalloproteinases and other hydrolytic enzymes.

Vascular inflammation is associated with and in large part driven by changes in the leukocyte compartment of the vessel wall. Although dendritic cells (DCs) and lymphocytes are found in the adventitia of normal arteries, their number is greatly expanded and their distribution changed in human and mouse atherosclerotic arteries. Macrophages, DCs, foam cells, lymphocytes, and other inflammatory cells are found in the intimal atherosclerotic lesions. Beneath these lesions, adventitial leukocytes organize in clusters that resemble tertiary lymphoid tissues. Experimental interventions can reduce the number of available blood monocytes, from which macrophages and most DCs and foam cells are derived, and reduce atherosclerotic lesion burden without altering blood lipids. Under proatherogenic conditions, nitric oxide production from endothelial cells is reduced and the burden of reactive oxygen species (ROS) and advanced glycation end products (AGE) is increased. Incapacitating ROS-generating NADPH oxidase or the receptor for AGE (RAGE) has beneficial effects. Targeting inflammatory adhesion molecules also reduces atherosclerosis. Conversely, removing or blocking IL-10 or TGF-beta accelerates atherosclerosis. Regulatory T-cells and B1-cells secreting natural antibodies are atheroprotective.

Pro-inflammatory cytokines can affect intracellular lipid metabolism. A variety of effects have been described for different cell types; hepatocyte lipid turnover pathways are inhibited during inflammation, whereas interleukin-1beta (IL-1beta) reduces intracellular cholesterol levels in fibroblasts. Levels of the pro-inflammatory cytokines IL-1beta and tumour necrosis factor-alpha (TNF-alpha) are up-regulated at sites of formation of atherosclerotic plaques. Plaque formation is though to begin with infiltration of monocytes to the intimal layer of the vascular wall, followed by differentiation to macrophages and macrophage uptake of modified lipoproteins, resulting in accumulation of intracellular lipids. The lipid-filled cells are referred to as macrophage foam cells, a key feature of atherosclerotic plaques.

Macrophage foam cell formation is a prominent feature of human atherosclerotic plaques, usually considered to be correlated to uptake of and inflammatory response to oxidized low density lipoproteins (OxLDL). However, there are alternative pathways for formation of macrophage foam cells and the effect of such lipid loading on macrophage function remains to be fully characterized.

Investigated the effects of IL-1beta and TNF-alpha on macrophage foam cells in order to assess whether presence of the pro-inflammatory cytokines improves or aggravates macrophage foam cell formation by affecting lipid accumulation and lipid turn-over in the cells. Investigated basal and inducible cytokine expression in primary human macrophages either loaded with triglycerides through incubation with very low density lipoproteins (VLDL) or with cholesterol through incubation with aggregated low density lipoproteins (AgLDL). Analyzed how foam cell lipid content affected secretion of three pro-inflammatory cytokines: IL-1beta, IL-6 and TNF-alpha, and of one chemokine: IL-8, all of which are considered pro-inflammatory, pro-atherosclerotic, and are expressed by cells in atherosclerotic tissue. Differentiated primary human macrophages or THP-1 cells were lipid loaded by uptake of aggregated low density lipoproteins (AgLDL) or very low density lipoproteins (VLDL), and then incubated with IL-1beta (0 - 5000 pg/ml) in lipoprotein-free media for 24 h. Cells incubated in absence of cytokine utilized accumulated neutral lipids, in particular triglycerides. Addition of exogenous IL-1beta resulted in a dose-dependent retention of intracellular cholesterol and triglycerides. Exchanging IL-1beta with TNF-alpha gave a similar response. Analysis of fatty acid efflux and intracellular fatty acid activation revealed a pattern of decreased lipid utilization in cytokine-stimulated cells.

Formation of triglyceride-loaded foam cells resulted in a four-fold increase in basal IL-1beta secretion, whereas cholesterol loading lacked significant effect on IL-1beta secretion. In contrast, secretion of TNF-alpha and IL-6 decreased significantly following both cholesterol and triglyceride loading, with a similar trend for secretion of IL-8. Lipid loading did not affect cell viability or expression of caspase-3, and did not significantly affect macrophage ability to respond to stimulation with exogenous TNF-alpha. IL-1beta and TNF-alpha enhance macrophage foam cell formation, in part by inhibition of macrophage intracellular lipid catabolism. If present in vivo, these mechanisms will further augment the pro-atherogenic properties of the two cytokines.

Lipid loading of primary human macrophages resulted in altered cytokine secretion from cells, where effects were similar regardless of neutral lipid composition of cells. The exception was IL-1beta, where triglyceride, but not cholesterol, lipid loading resulted in a stimulation of basal secretion of the cytokine. It is apparent that macrophage cytokine secretion is affected by lipid loading by lipoproteins other than OxLDL. As both VLDL and AgLDL have been found in the vessel wall, macrophage cytokine response to uptake of these lipoproteins may have a direct effect on atherosclerotic development in vivo. However, macrophage neutral lipid amount and composition did not affect cellular activation by exogenous TNF-alpha, making it likely that lipoprotein lipid loading can affect foam cell cytokine secretion during basal conditions but that the effects can be overruled by TNF-alpha during acute inflammation.