Modulation of atherosclerosis in mice by Toll-like receptor 2

Abstract

Epidemiologic evidence has established a relationship between microbial infection and atherosclerosis. Mammalian TLRs provide clues on the mechanism of this inflammatory cascade. TLR2 has a large ligand repertoire that includes bacterial-derived exogenous and possibly host-derived endogenous ligands. In atherosclerosis-susceptible low-density lipoprotein receptor-deficient (Ldlr-/-) mice, complete deficiency of TLR2 led to a reduction in atherosclerosis. However, with BM transplantation, loss of TLR2 expression from BM-derived cells had no effect on disease progression. This suggested that an unknown endogenous TLR2 agonist influenced lesion progression by activating TLR2 in cells that were not of BM cell origin. Moreover, with intraperitoneal administration of a synthetic TLR2/TLR1 agonist, Pam3CSK4, disease burden was dramatically increased in Ldlr-/- mice. A complete deficiency of TLR2 in Ldlr-/- mice, as well as a deficiency of TLR2 only in BM-derived cells in Ldlr-/- mice, led to striking protection against Pam3CSK4-mediated atherosclerosis, suggesting a role for BM-derived cell expression of TLR2 in transducing the effects of an exogenous TLR2 agonist. These studies support the concept that chronic or recurrent microbial infections may contribute to atherosclerotic disease. Additionally, these data suggest the presence of host-derived endogenous TLR2 agonists.

Figure 1

Measurement of atherosclerosis in male Ldlr^–/– and Ldlr–/–Tlr2^–/– mice after 10 or 14 weeks of consuming an HFD. (A) Aortic lesion area was calculated as fraction of total aorta covered by lesion. (B) Aortic sinus lesion volume was calculated from an integration of the cross-sectional areas of the lesion in the proximal 500 μm of the sinus. Aortic lesion area and aortic valve lesion volume were significantly decreased in Ldlr–/–Tlr2^–/– mice.

Figure 2

Measurement of heart aortic sinus atherosclerosis volume after 16 weeks of HFD feeding in female Ldlr^–/– and Ldlr–/–Tlr2^–/– mice that underwent BM reconstitution with BM from Tlr2+/+ or Tlr2^–/– donors. Regardless of BM cell genotype, a loss of TLR2 on non-BM–derived cells significantly reduced disease.

Figure 3

ELISA measurement of plasma SAA 24 hours following i.p. injections of vehicle, 25 μg of Pam3, or 50 μg of Pam3 demonstrated a dose-dependent systemic inflammatory response. Double-mutant mice exhibited baseline (time = 0) SAA values similar to those of Ldlr^–/– mice. Error bars indicate SEM.

Figure 4

Measurement of aortic atherosclerosis in Pam3-exposed mice. (A) Aortic atherosclerosis expressed as a fraction of total area after 12 weeks of an HFD and weekly i.p. injections of vehicle (veh) or Pam3 in female Ldlr^–/– and Ldlr–/–Tlr2^–/– mice. (B–D) Representative aortae from Ldlr^–/– mice in the following groups: vehicle (B), 25 μg Pam3 (C), and 50 μg Pam3 (D). A dose-response effect of Pam3 administration and lesion development was observed. Profuse abdominal aortic atherosclerosis was observed in mice exposed to 50 μg Pam3. Lesion severity in TLR2-deficient animals was not affected by Pam3. Scale bar: 0.5 cm.

Figure 5

Measurement of heart aortic sinus atherosclerosis volume in Pam3-exposed mice. (A) Heart aortic sinus atherosclerosis volume after 12 weeks of an HFD and weekly i.p. injections of Pam3 in female Ldlr^–/– and Ldlr–/–Tlr2^–/– mice. (B–D) Representative cross-sections of the aortic sinus from Ldlr^–/– mice of the following groups: vehicle (B), 25 μg Pam3 (C), and 50 μg Pam3 (D). A dose-response effect of Pam3 administration and lesion development was observed. Lesion severity in TLR2-deficient animals was not affected by Pam3. Scale bar: 0.5 mm.

Figure 6

Measurement of aortic atherosclerosis in Pam3-exposed TLR2 chimeric mice. (A) Aortic atherosclerosis expressed as a percentage of total area after 12 weeks of an HFD and weekly i.p. injections of Pam3 in female Ldlr^–/– mice that underwent BM reconstitution from Tlr+/+ or Tlr2^–/– donor mice. (B–E) Representative aortae from Ldlr^–/– recipient mice of the following groups: Tlr2+/+ BM donor, vehicle injection (B); Tlr2+/+ BM donor, 50 μg Pam3 per week (C); Tlr2^–/– BM donor, vehicle injection (D); and Tlr2^–/– BM donor, 50 μg Pam3 per week (E). Pam3 administration increased lesion severity in animals expressing TLR2 in their BM-derived cells resulting in profuse abdominal aortic atherosclerosis. Slight increases in lesion severity were observed in Tlr2^–/– recipient animals receiving Pam3. Scale bar: 0.5 cm.

Figure 7

Measurement of heart aortic sinus atherosclerosis volume in Pam3-exposed TLR2 chimeric mice. (A) Heart aortic sinus atherosclerosis after 12 weeks of an HFD and weekly i.p. injections of Pam3 in female Ldlr^–/– mice that underwent BM reconstitution from Tlr+/+ or Tlr2^–/– donor mice. (B–E) Representative aortic sinus cross-sections from Ldlr^–/– mice of the following groups: Tlr2+/+ BM donor, vehicle injections (B); Tlr2+/+ BM donor, 50 μg Pam3 injections (C); Tlr2^–/– BM donor, vehicle injections (D); and Tlr2^–/– BM donor, 50 μg Pam3 injections (E). Pam3 administration increased lesion severity only in animals expressing TLR2 in their BM-derived cells. Scale bar: 0.5 mm.

Figure 8

Plasma lipoprotein cholesterol distribution. Pooled plasma samples taken from representative mice after 8 weeks of consumption of the HFD were fractionated by FPLC, and total cholesterol was measured. (A) Complete or BM cell–derived loss of TLR2 did not alter the distribution of cholesterol relative to control. (B) Administration of 50 μg Pam3 also did not have an impact on the distribution of cholesterol relative to control mice given the vehicle injections.