An Interleukin-6 Receptor Antibody Suppresses Atherosclerosis in Atherogenic Mice

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.

inTrODUcTiOn Nuclear factor-κB (NF-κB) plays essential roles in mediating immune systems. Cytoplasmic IκB family proteins regulate the transcriptional activity of NF-κB, because excessive activation is detrimental to the host (1). After translocation of NF-κB from the cytoplasm to the nucleus, nuclear proteins that are structurally similar to cytoplasmic IκBs take part in the mediating of NF-κB transcriptional activity, as activators or inhibitors, by associating with NF-κB subunits (2). Thus, the regulatory IκBlike nuclear molecules are described as nuclear IκB proteins. Although IκBNS is one of nuclear IκB Anti-IL-6 Therapy for Atherosclerosis Frontiers in Cardiovascular Medicine | www.frontiersin.org December 2017 | Volume 4 | Article 84 proteins, the role of IκBNS in the development of atherosclerosis is poorly understood. Our previous study demonstrated that deficiency of IκBNS induces atherogenesis in LDL receptor-deficient (LDLr −/− ) mice fed a high-fat diet and increases in interleukin (IL)-6 production by macrophages, indicating that IκBNS plays an important role in the suppression of atherosclerotic lesions in vivo (3). However, whether or not increased IL-6 production in IκBNS-deficient LDLr −/− mice accelerates atherogenesis remains undetermined.
Several previous reports indicate that IL-6 could accelerate atherosclerosis. Increased plasma levels of IL-6 were associated with cardiovascular mortality over 5-year independent of other risk factor of atherosclerosis (4,5). Injection of IL-6 itself accelerated atherosclerosis in apolipoprotein E-null mice and C57Bl/6 mice either (4). The genetic polymorphism in the IL-6 signaling pathway concordantly associates with lifetime lower risks of coronary heart disease (6). However, whether inhibition of IL-6 might be effective for the suppression of atherogenesis remains to be elucidated.
We demonstrated here that treatment of an anti-mouse IL-6 receptor antibody (MR16-1) suppressed atherosclerosis lesion in atherogenic mice via the inhibition of IL-6-STAT3 signaling pathway.

Mice
The generation of LDLr −/− mice that lacked IκBNS (IκBNS −/− / LDLr −/− ) used in this study has been described previously (3). Details of IκBNS-deficient mice were described in the previous report (7). We investigated atherosclerotic lesions in LDLr −/− and IκBNS −/− /LDLr −/− mice after 16 weeks consumption of a high-fat diet (MF diet containing 0.5% cholesterol, Oriental Yeast Co.). This study was performed according to the protocols approved by the Juntendo University Board for Studies in Experimental Animals.

Plasma lipid Measurement
Plasma total cholesterol, triglyceride, high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol levels were measured by high-performance liquid chromatography at Skylight Biotech Inc. (Akita, Japan).

Quantification of atherosclerotic lesions
After blood collection, the animals were euthanized by pentobarbital injection, and the heart and aorta were flushed with 0.9% NaCl followed by 4% paraformaldehyde. After perfusion procedure, the aorta was harvested and fixed with 10% neutralbuffered formalin for 48 h, embedded in paraffin, and sectioned from just above the aortic valve throughout the aortic sinus (each 6-µm thickness). We used equally spaced 10 cross sections (100-µm interval) to qualify arteriosclerotic lesions in the aortic sinus for each mouse. The samples were stained with Elastica van Gieson, and then photographed using a BX53 microscope (OLYMPUS, Tokyo, Japan). The luminal, arteriosclerotic lesions and medial areas were calculated using NIH Image J 1.42 (National Institutes of Health, public domain software). Quantification of the atherosclerotic lesions was performed by two blinded observers.
The whole aortas were also stained with Sudan IV. The surface atherosclerotic lesions were expressed as the percent of the lesion area extending from the ascending aorta to the iliac bifurcation.

statistical analysis
Results are shown as mean ± SEM. The two groups were compared using Student's t-test. Differences between groups were analyzed using one-way ANOVA test followed by Bonferroni's t-test to determine statistical differences after multiple comparisons. p < 0.05 was considered statistically significant.
These results show that an anti-mouse IL-6 receptor antibody significantly suppresses IκBNS in both LDLr −/− and IκBNS −/− /LDLr −/− mice without major confounding effects on LDL-cholesterol or HDL-cholesterol, suggesting that inhibition of IL-6 might be significantly effective to prevent atherogenesis.  (Figure 5A).

DiscUssiOn
Persistent IL-6 production has been shown to play a critical role in chronic inflammatory disease (8). IL-6 signaling has also been linked to plaque initiation and destabilization (9) and to adverse outcomes in the setting of acute ischemia (10). IL-6 stimulates growth of promoters of macrophage and vascular smooth muscle cells, which are major components of plaque (11)(12)(13), and IL-6 expression is also detected in human atherosclerotic lesions (14). These findings indicate that IL-6 participates in the development of atherosclerosis. Thus, targeting IL-6 is a rational approach for atheroprotection. To block the effect of IL-6 on the development of atherosclerosis, we used anti-mouse IL-6 receptor antibody (MR16-1). MR16-1 has proved to be effective in experimental model of arthritis (15) and humanized anti-IL-6 receptor antibody (tocilizumab) has been approved by the Food and Drug Administration for the treatment in the patients with rheumatoid arthritis.
Our previous study demonstrates that IL-6 expression and STAT3 activation were increased in the foam cell rich-atherosclerotic lesions of IκBNS −/− /LDLr −/− mice in vivo and IκBNS −/− / LDLr −/− macrophages produced much higher level of IL-6 than LDLr −/− macrophages in vitro. These results indicate deficiency of IκBNS increases in the production of IL-6 in macrophage and an increase of IL-6 contributes to the susceptibility to atherogenesis in IκBNS −/− /LDLr −/− mice (3). Consequently, because we believe that the mice were suitable for evaluating the effect of IL-6blocking therapy in atherogenesis, we used IκBNS −/− /LDLr −/− mice in this study. As expected, MR16-1 treatment abolished the significant difference of atherosclerotic lesions between IκBNS −/− / LDLr −/− and LDLr −/− mice. Interestingly, MR16-1 treatment also significantly decreased atherosclerotic lesions in LDLr −/− mice compared with PBS treatment.
Interleukin-6 is an upstream inflammatory cytokine key player, propagating the downstream inflammatory response in atherosclerosis (16). Previous study demonstrated that inflammatory gene, such as IL-6 (17), which is known as an inducer of STAT3 was highly expressed in atherosclerotic plaques. Moreover, activation of STAT3 has been detected in the plaque (18), and its activation is involved in the progression of atherosclerotic lesions (19). In this study, we analyzed pSTAT3-positive nuclei to evaluate the inhibitory effect of MR16-1 in IL-6 signaling pathway. MR16-1 inhibited STAT3 activation and development of atherosclerotic lesions in both IκBNS −/− /LDLr −/− and LDLr −/− mice, suggesting IL-6 is a main contributor to atherogenesis, and inhibition of IL-6 might be a new method to prevent the development of atherosclerosis.
Our data provide solid evidence for a causal role of the IL-6 pathway in the development of atherosclerosis, raising expectations for ongoing trials testing anti-inflammatory strategies for cardiovascular diseases (20).
Although we could not find any adverse systemic effects including signs of infection in MR16-1-treated mice, further demonstrated intraperitoneally injection of LPS induced significantly high-serum levels of IL-6 in IκBNS −/− mice compared to wild-type (IκBNS +/+ ) mice. These findings suggest that feeding a high-fat diet could induce local increase of IL-6 produced by IκBNS −/− macrophages around the vessels, but not serum levels of IL-6. In our previous study, IκBNS expression was observed in all cell types resident to the plaque microenvironment (i.e., smooth muscle cells, macrophages, and endothelial cells) in LDLr −/− mice, but not in IκBNS −/− / LDLr −/− mice. Because main IκBNS expression cells were macrophages, we previously examined the effects of the IκBNS deficiency in macrophages. We also investigated the effect of IκBNS deficiency in smooth muscle cells using small interference RNA, because they play important roles in atherogenesis. However, IκBNS deficiency in aortic smooth muscle cell did not affect in IL-6 production in vitro. Further experiments might be needed to clarify which cells are main players in atherogenesis induced by IκBNS deficiency.
In conclusion, this study highlights the potential therapeutic benefit of anti-IL-6 therapy in preventing atherogenesis in the mice with dyslipidemia and/or inflammation.

eThics sTaTeMenT
The studies were performed according to the protocols approved by the Juntendo University Board for Studies in Experimental Animals.