Lack of Toll-like receptor 4 or myeloid differentiation factor 88 reduces atherosclerosis and alters plaque phenotype in mice deficient in apolipoprotein E

Abstract

Toll-like receptors (TLRs) and the downstream adaptor molecule myeloid differentiation factor 88 (MyD88) play an essential role in the innate immune responses. Here, we demonstrate that genetic deficiency of TLR4 or MyD88 is associated with a significant reduction of aortic plaque areas in atherosclerosis-prone apolipoprotein E-deficient mice, despite persistent hypercholesterolemia, implying an important role for the innate immune system in atherogenesis. Apolipoprotein E-deficient mice that also lacked TLR4 or MyD88 demonstrated reduced aortic atherosclerosis that was associated with reductions in circulating levels of proinflammatory cytokines IL-12 or monocyte chemoattractant protein 1, plaque lipid content, numbers of macrophage, and cyclooxygenase 2 immunoreactivity in their plaques. Endothelial-leukocyte adhesion in response to minimally modified low-density lipoprotein was reduced in aortic endothelial cells derived from MyD88-deficient mice. Taken together, our results suggest an important role for TLR4 and MyD88 signaling in atherosclerosis in a hypercholesterolemic mouse model, providing a pathophysiologic link between innate immunity, inflammation, and atherogenesis.

Fig. 1.

MyD88 and TLR4 deficiency reduces the extent of aortic atherosclerosis. (A) Aortas of Apoe^–/–/MyD88^–/–, Apoe^–/–/MyD88^+/–, and Apoe^–/–/MyD88^+/+ mice fed with a high-cholesterol diet for 6 months were isolated and stained for lipid deposition with Oil red O. Representative specimens from the three groups are shown. (B) Quantification of plaque areas in whole aortas in Apoe^–/–/MyD88^–/– (Left) and Apoe^–/–/TLR4^–/– (Right) mice. Aortas of Apoe^–/–/MyD88^–/–, Apoe^–/–/MyD88^+/–, Apoe^–/–/MyD88^+/+, Apoe^–/–/TLR4^–/–, and Apoe^–/–/TLR4^+/+ mice stained for lipid deposition with Oil red O. Means and SD (n = 13 for Apoe^–/–/MyD88^–/–, Apoe^–/–/MyD88^+/–, Apoe^–/–/MyD88^+/+; n = 8 for Apoe^–/–/TLR4^+/+; and n = 12 for Apoe^–/–/TLR4^–/–) of plaque areas are shown. Total plaque area in MyD88^–/– or TLR4^–/– was significantly reduced compared with corresponding wild-type mice (*, P < 0.01).

Fig. 2.

Lipid content, macrophage infiltration, and COX-2 expression in aortic sinus plaques is reduced in Apoe^–/–/MyD88^–/– mice. (A) Quantification of the lipid content in aortic plaques from Apoe^–/–/MyD88^–/–, Apoe^–/–/MyD88^+/–, and Apoe^–/–/MyD88^+/+ (Left) and Apoe^–/–/TLR4^–/– and Apoe^–/–/TLR4^+/+ (Right) mice. Shown are means and SD of the percentage of lipid content relative to total plaque areas (n = 10 for Apoe^–/–/MyD88^–/–, Apoe^–/–/MyD88^+/–, and Apoe^–/–/MyD88^+/+; n = 7 for Apoe^–/–/TLR4^+/+; and n = 8 for Apoe^–/–/TLR4^–/–). Relative lipid content in Apoe^–/–/MyD88^–/– or Apoe^–/–/TLR4^–/– is significantly reduced compared with corresponding wild-type mice (**, P < 0.01; *, P < 0.05). (B) Representative MOMA-2 staining of aortic sinus plaques from Apoe^–/–/MyD88^–/–, Apoe^–/–/MyD88^+/–, and Apoe^–/–/MyD88^+/+ mice. (C) Quantitative analysis of macrophage immunoreactivity in aortic sinus plaques of Apoe^–/–/MyD88^–/–, Apoe^–/–/MyD88^+/–, and Apoe^–/–/MyD88^+/+ mice, expressed as a proportion of the total plaque areas (n = 10 in each group). Means and SD are shown (*, P < 0.01). (D) Quantitative analysis of COX-2 immunofluorescent staining in sclerotic plaques of Apoe^–/–/MyD88^–/–, Apoe^–/–/MyD88^+/–, and Apoe^–/–/MyD88^+/+ mice (n = 7 in each group). Means and SD are shown (*, P < 0.01).

Fig. 3.

Serum concentration of IL-12p40 and MCP-1 are reduced in Apoe^–/–/MyD88^–/– mice. (A) IL-12p40 serum concentrations of Apoe^–/–/MyD88^–/–, Apoe^–/–/MyD88^+/–, and Apoe^–/–/MyD88^+/+ mice fed with a high-cholesterol diet for 6 months (n = 19 in each group). (B) MCP-1 serum concentrations of Apoe^–/–/MyD88^–/–, Apoe^–/–/MyD88^+/–, and Apoe^–/–/MyD88^+/+ mice (n = 24 in each group). Means and SD are shown (*, P < 0.01).

Fig. 4.

ECs from MyD88-deficient mice show reduced MM-LDL-induced adhesion of leukocytes. Adhesion of human peripheral blood mononuclear cells to murine aortic endothelial cells derived from aortas of MyD88^–/– or MyD88^+/+ mice stimulated with MM-LDL, LDL, or TNF-α. Data from one of three representative experiments with similar results are shown as fold increase in leukocyte adhesion over background (*, P < 0.05).

Fig. 5.

TLR4 deficiency reduces the extent of macrophage infiltration and MCP-1 secretion. (A) Quantitative analysis of macrophage immunoreactivity in aortic sinus plaques of Apoe^–/–/TLR4^–/– and Apoe^–/–/TLR4^+/+ mice, expressed as a proportion of the total plaque areas (n = 7 per group). Means and SD are shown (*, P < 0.01). (B) MCP-1 serum concentrations of Apoe^–/–/TLR4^–/– and Apoe^–/–/TLR4^+/+ mice (n = 8 for Apoe^–/–/TLR4^+/+ and n = 14 for Apoe^–/–/TLR4^–/–). Means and SD are shown (*, P < 0.01).