Protective role for Toll-like receptor-9 in the development of atherosclerosis in apolipoprotein E-deficient mice.

OBJECTIVE: Atherosclerosis is driven by inflammatory reactions that are shared with the innate immune system. Toll-like receptor-9 (TLR9) is an intracellular pattern recognition receptor of the innate immune system that is currently under clinical investigation as a therapeutic target in inflammatory diseases. Here, we investigated whether TLR9 has a role in the development of atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice. APPROACH AND RESULTS: Newly generated double-knockout ApoE(-/-):TLR9(-/-) mice and control ApoE(-/-) mice were fed a high-fat diet from 8 weeks and effects on lesion size, cellular composition, inflammatory status, and plasma lipids were assessed after 8, 12, 15, and 20 weeks. All 4 time points demonstrated exacerbated atherosclerotic lesion severity in ApoE(-/-):TLR9(-/-) mice, with a corresponding increase in lipid deposition and accumulation of macrophages, dendritic cells, and CD4(+) T cells. Although ApoE(-/-):TLR9(-/-) mice exhibited an increase in plasma very low-density lipoprotein/low-density-lipoprotein cholesterol, the very low-density lipoprotein/low-density lipoprotein:high-density lipoprotein ratio was unaltered because of a parallel increase in plasma high-density lipoprotein cholesterol. As a potential mechanism accounting for plaque progression in ApoE(-/-):TLR9(-/-) mice, CD4(+) T-cell accumulation was further investigated and depletion of these cells in ApoE(-/-):TLR9(-/-) mice significantly reduced lesion severity. As a final translational approach, administration of a TLR9 agonist (type B CpG oligodeoxynucleotide 1668) to ApoE(-/-) mice resulted in a reduction of lesion severity. CONCLUSIONS: Genetic deletion of the innate immune receptor TLR9 exacerbated atherosclerosis in ApoE(-/-) mice fed a high-fat diet. CD4(+) T cells were identified as potential mediators of this effect. A type B CpG oligodeoxynucleotide TLR9 agonist reduced lesion severity, thus identifying a novel therapeutic approach in atherosclerosis.

Cytokine therapy with interleukin-2/anti-interleukin-2 monoclonal antibody complexes expands CD4+CD25+Foxp3+ regulatory T cells and attenuates development and progression of atherosclerosis.

BACKGROUND: CD4+CD25+Foxp3+ regulatory T cells (Tregs) attenuate atherosclerosis, but their therapeutic application by adoptive transfer is limited by the need for their expansion in vitro and limited purity. Recently, an interleukin (IL)-2/anti-IL-2 neutralizing monoclonal antibody (IL-2/anti-IL-2 mAb) complex has been shown to expand these Tregs. We examined the capacity of a modified IL-2/anti-IL-2 mAb treatment to expand Tregs and inhibit both the progression and development of developed atherosclerosis. METHODS AND RESULTS: Six-week old apolipoprotein E-deficient mice fed a high-fat diet for 8 weeks were administered IL-2/anti-IL-2 mAb commencing 2 weeks after starting the diet. Tregs in the spleen, lymph node, and liver were selectively expanded without affecting CD4+, CD8+, or natural killer cells. Tregs were increased in lesions and lesion size reduced. CD4+ T-cells, macrophages, mature dendritic cells, proliferating cell nuclear antigen+ cells, and monocyte chemoattractant protein-1 and vascular cell adhesion molecule-1 were reduced. In anti-CD3-stimulated splenocytes, proliferation and secretion of Th1, Th2, and Th17 (IL-17) cytokines and IL-1ß were reduced. To determine whether treatment attenuated progression of developed atherosclerosis, 6-week-old apolipoprotein E-deficient mice were fed a high-fat diet for 6 weeks, followed by IL-2/anti-IL-2 mAb treatment for 6 weeks while continuing the high-fat diet. Treatment also increased Tregs without affecting CD4+, CD8+, or natural killer cells, suppressed inflammation, and greatly attenuated progression of atherosclerosis. CONCLUSIONS: IL-2/anti-IL-2 mAb treatment in vivo attenuates atherosclerosis via selective Tregs expansion. The findings suggest that cytokine-based IL-2/anti-IL-2 mAb complex therapy could represent an attractive approach for treating atherosclerosis, because it markedly attenuates progression as well as development, by modulating its immunoinflammatory component.

High-mobility group box protein 1 neutralization reduces development of diet-induced atherosclerosis in apolipoprotein e-deficient mice.

OBJECTIVE: High-mobility group box protein 1 (HMGB1) is a DNA-binding protein and cytokine highly expressed in atherosclerotic lesions, but its pathophysiological role in atherosclerosis is unknown. We investigated its role in the development of atherosclerosis in ApoE-/- mice. METHODS AND RESULTS: Apolipoprotein E-deficient (ApoE-/-) mice fed a high-fat diet were administered a monoclonal anti-HMGB1 neutralizing antibody, and the effects on lesion size, immune cell accumulation, and proinflammatory mediators were assessed using Oil Red O, immunohistochemistry, and real-time polymerase chain reaction. As with human atherosclerotic lesions, lesions in ApoE-/- mice expressed HMGB1. Treatment with the neutralizing antibody attenuated atherosclerosis by 55%. Macrophage accumulation was reduced by 43%, and vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 expression was attenuated by 48% and 72%, respectively. CD11c+ dendritic cells were reduced by 65%, and the mature (CD83+) population was reduced by 60%. Treatment also reduced CD4+ cells by nearly 50%. mRNAs in lesions encoding tumor necrosis factor-α and interleukin-1ß tended to be reduced. Mechanistically, HMGB1 stimulated macrophage migration in vitro and in vivo; in vivo, it markedly augmented the accumulation of F4/80+Gr-1(Ly-6C)+ macrophages and also increased F4/80+CD11b+ macrophage numbers. CONCLUSIONS: HMGB1 exerts proatherogenic effects augmenting lesion development by stimulating macrophage migration, modulating proinflammatory mediators, and encouraging the accumulation of immune and smooth muscle cells.