An Interleukin-6 Receptor Antibody Suppresses Atherosclerosis in Atherogenic Mice

Abstract

IκBNS is a nuclear IκB protein which negatively regulates nuclear factor-κB activity. We demonstrated that IκBNS deficiency accelerates atherosclerosis in LDL receptor-deficient (LDLr^-/-) mice via increased interleukin (IL)-6 production by macrophages. Previous studies showed that the increase in IL-6 might contribute to the development of atherosclerotic lesions. However, whether an anti-mouse IL-6 receptor antibody (MR16-1) can protect atherosclerotic lesions in atherogenic mice remains to be elucidated. We investigated atherosclerotic lesions in LDLr^-/- and IκBNS^-/-/LDLr^-/- mice after 16 weeks consumption of a high-fat diet. All mice received intraperitoneal injections of MR16-1 or phosphate-buffered saline (PBS) (control) once a week during a high-fat diet consumption. Treatment of MR16-1 yielded no adverse systemic effects, and we detected no significant differences in serum cholesterol levels in either group. The atherosclerotic lesions were significantly increased in IκBNS^-/-/LDLr^-/- compared with LDLr^-/- mice (p < 0.01) under treatment of PBS. However, MR16-1 treatment abolished the significant difference of atherosclerotic lesions between IκBNS^-/-/LDLr^-/- and LDLr^-/- mice. Interestingly, MR16-1 also significantly decreased atherosclerotic lesions in LDLr^-/- mice compared with PBS treatment (p < 0.05). Immunostaining revealed percent phospho-STAT3-positive cell were significantly decreased in the atherosclerotic lesions of MR16-1 treated both IκBNS^-/-/LDLr^-/- and LDLr^-/- mice compared with PBS-treated mice, indicating MR16-1 could suppress atherosclerotic lesions via the inhibition of IL-6-STAT3 signaling pathway. This study highlights the potential therapeutic benefit of anti-IL-6 therapy in preventing atherogenesis induced by dyslipidemia and/or inflammation.

Keywords: IκBNS; atherosclerosis; dyslipidemia; inflammation; interleukin-6.

Figure 1

Treatment with MR16-1 did not change blood pressure or body weight in both IκBNS^−/−/LDLr^−/− and LDLr^−/− mice. Both mice were received injection of phosphate-buffered saline (PBS) or MR16-1 (2 mg) once a week during a high-fat diet consumption of 16 weeks. Systolic blood pressure (A) and body weight (B) were similar among four groups. IκBNS/LDLr KO PBS: PBS-treated IκBNS^−/−/LDLr^−/− mice, LDLr KO PBS: PBS-treated LDLr^−/− mice. IκBNS/LDLr KO MR16-1: MR16-1-treated IκBNS^−/−/LDLr^−/− mice, LDLr KO MR16-1: MR16-1-treated LDLr^−/− mice. Data are expressed as mean ± SEM. NS, not significant.

Figure 2

Treatment with MR16-1 did not change cholesterol profiles in both IκBNS^−/−/LDLr^−/− and LDLr^−/− mice. The levels of total cholesterol (T-cho) (A), low-density lipoprotein (LDL)-cholesterol (B), triglyceride (TG) (C), and high-density lipoprotein (HDL)-cholesterol (D) after treatment of phosphate-buffered saline (PBS) or MR16-1 (2 mg) once a week during a high-fat diet consumption of 16 weeks. IκBNS/LDLr KO PBS: PBS-treated IκBNS^−/−/LDLr^−/− mice, LDLr KO PBS: PBS-treated LDLr^−/− mice. IκBNS/LDLr KO MR16-1: MR16-1-treated IκBNS^−/−/LDLr^−/− mice, LDLr KO MR16-1: MR16-1-treated LDLr^−/− mice. Data are expressed as mean ± SEM. NS, not significant.

Figure 3

Treatment with MR16-1 decreased in the extent of atherosclerotic lesions in the mice aortas (en face) of both IκBNS^−/−/LDLr^−/− and LDLr^−/− mice. (A) Sudan IV staining of aortas from four groups after treatment of phosphate-buffered saline (PBS) or MR16-1 (2 mg) once a week during a high-fat diet consumption of 16 weeks (bars = 10 mm). (B) Quantitative analysis of the relative surface area of the atherosclerotic lesion in aortas. IκBNS/LDLr KO PBS: PBS-treated IκBNS^−/−/LDLr^−/− mice, LDLr KO PBS: PBS-treated LDLr^−/− mice. IκBNS/LDLr KO MR16-1: MR16-1-treated IκBNS^−/−/LDLr^−/− mice, LDLr KO MR16-1: MR16-1-treated LDLr^−/− mice. Data are expressed as mean ± SEM. *p < 0.05 and **p < 0.01. NS, not significant.

Figure 4

Treatment with MR16-1 decreased in aortic root atherosclerotic lesions of both IκBNS^−/−/LDLr^−/− and LDLr^−/− mice. (A) Representative photomicrographs of sections of aortic sinus plaque from four groups after treatment of phosphate-buffered saline (PBS) or MR16-1 (2 mg) once a week during a high-fat diet consumption of 16 weeks. Adjacent sections were processed for Elastica van Gieson staining (upper panels) and H&E staining (lower panels) (bars = 500 µm). (B) Quantitative comparison of the atherosclerotic lesion in the aortic sinus. IκBNS/LDLr KO PBS: PBS-treated IκBNS^−/−/LDLr^−/− mice, LDLr KO PBS: PBS-treated LDLr^−/− mice. IκBNS/LDLr KO MR16-1: MR16-1-treated IκBNS^−/−/LDLr^−/− mice, LDLr KO MR16-1: MR16-1-treated LDLr^−/− mice. Data are expressed as mean ± SEM. *p < 0.05 and **p < 0.01. NS, not significant.

Figure 5

MR16-1 treatment significantly inhibited STAT3 activation in the atherosclerotic lesions of both IκBNS^−/−/LDLr^−/− and LDLr^−/− mice. (A) Representative photomicrographs of sections of aortic sinus plaque from four groups after treatment of phosphate-buffered saline (PBS) or MR16-1 (2 mg) once a week during a high-fat diet consumption of 16 weeks. Sections were processed for pSTAT3 staining (bars = 25 µm). (B) Quantitative analysis of pSTAT3-positive cell in vascular wall in sections from four groups. IκBNS/LDLr KO PBS: PBS-treated IκBNS^−/−/LDLr^−/− mice, LDLr KO PBS: PBS-treated LDLr^−/− mice. IκBNS/LDLr KO MR16-1: MR16-1-treated IκBNS^−/−/LDLr^−/− mice, LDLr KO MR16-1: MR16-1-treated LDLr^−/− mice. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. NS, not significant.