Gene Therapy (2015) 22, 645–652 © 2015 Macmillan Publishers Limited All rights reserved 0969-7128/15 www.nature.com/gt ORIGINAL ARTICLE Macrophage-specific overexpression of interleukin-5 attenuates atherosclerosis in LDL receptor-deficient mice W Zhao1,TLei2,HLi2, D Sun2,XMo2, Z Wang2, K Zhang2 and H Ou2 Interleukin-5 (IL-5) increases the secretion of natural T15/EO6 IgM antibodies that inhibit the uptake of oxidized low-density lipoprotein (LDL) by macrophages. This study aimed to determine whether macrophage-specific expression of IL-5 in LDL receptor- deficient mice (Ldlr−/−) could improve cholesterol metabolism and reduce atherosclerosis. To induce macrophage-specific IL-5 expression, the pLVCD68-IL5 lentivirus was delivered into Ldlr−/− mice via bone marrow transplantation. The recipient mice were fed a Western-type diet for 12 weeks to induce lesion formation. We found that IL-5 was efficiently and specifically overexpressed in macrophages in recipients of pLVCD68-IL5-transduced bone marrow cells (BMC). Plasma titers of T15/EO6 IgM antibodies were significantly elevated by 58% compared with control mice transplanted with pLVCD68 lacking the IL-5 coding sequence. Plaque areas of aortas in IL-5-overexpressing mice were reduced by 43% and associated with a 2.4-fold decrease in lesion size at the aortic roots when compared with mice receiving pLVCD68-transduced BMCs. The study showed that macrophage-specific overexpression of IL-5 inhibited the progression of atherosclerotic lesions. These findings suggest that modulation of IL-5 cytokine expression represents a potential strategy for intervention of familial hypercholesterolemia and other cardiovascular diseases. Gene Therapy (2015) 22, 645–652; doi:10.1038/gt.2015.33 INTRODUCTION can stimulate innate B-1 cells, a subset of B lymphocytes, to Familial hypercholesterolemia (FH) is an inherited metabolic produce and secrete natural T15/EO6 IgM antibodies.9 These T15/ disorder caused by mutations in the low-density lipoprotein EO6 IgM antibodies recognize oxidation-specific epitopes and receptor gene (Ldlr). As a result of the Ldlr gene dysfunction, show high affinity toward OxLDL, as well as other forms of excess cholesterol in the circulation cannot be cleared through the modified LDL such as malondialdehyde-LDL and acrolein-LDL but LDL receptor pathway, and is subsequently oxidized by endothe- not native LDL.10,11 They can bind to the phosphatidylcholine lial and smooth muscle cells after transport across the endothe- moiety of the oxidized phospholipids in OxLDL, thus inhibiting the lium. Macrophages take up the accumulated oxidized LDL uptake of OxLDL by the macrophage scavenger receptor CD36 (OxLDL), transform into foam cells and contribute to the and the scavenger receptor class B type I (SR-BI), which prevent development of premature atherosclerosis. Current clinical man- foam cell formation in vivo.9 This combined evidence suggests agement of FH includes lifelong treatment with statins or non- that IL-5 and T15/EO6 have beneficial effects in protection against fi statin drugs such as bile acid resin, niacin or brate. Surgical atherosclerosis. interventions may include orthotopic liver transplantation or LDL It is widely recognized that monocyte-derived macrophages apheresis, in which the plasma LDL is purged to control high 1,2 play a pivotal role in the initiation and progression of plasma cholesterol. Evidently, restoration of cholesterol home- atherosclerosis, as described above. The lipid-laden macrophages ostasis in vivo and prevention of atherosclerosis through are the predominant cell type found in fatty streaks and represent molecular intervention represents a more ideal and radical a substantial fraction of the cells found in fibrous plaques and strategy for FH treatment. complex lesions. These macrophages generate an extraordinary Although atherosclerosis is a known lipid disorder, accumulat- number of secretory products, some of which may have the ing evidence demonstrates that inflammation is implicated in all — potential to exhibit multiple effects in atherogenesis, including stages of atherosclerosis from foam cell formation and fatty 3 streak development to plaque establishment and ultimate atheroprotective effects. On the basis of these facts, we rupture. A wide array of cytokines, including interferon-γ, investigated whether overexpression of IL-5 exclusively in macro- fi −/− interleukins (ILs) and tumor necrosis factor-α that are produced phages in LDL receptor-de cient mice (Ldlr ) could have by macrophages; activated T-lymphocytes; and other leukocytes therapeutic effects on atherosclerosis. We used a macrophage- −/− within atherosclerotic lesions have been reported to have either specific CD68 promoter to direct IL-5 expression in Ldlr mice pro- or anti-atherosclerotic properties.3 IL-5 is a pleiotropic through transplantation of lentiviral-transduced bone marrow cytokine that is mainly expressed by T helper-2 lymphocytes cells (BMCs). Our results showed that the treatment greatly and mast cells. It was originally discovered as an eosinophil reduced atherosclerosis while considerably improving T15/EO6 differentiation and maturation factor and has been widely studied IgM titers, thus suggesting that it might be used to mitigate FH for its role in asthma and other allergic diseases.4–8 Moreover, IL-5 and other cardiovascular diseases. 1Department of Pediatrics, The First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui, China and 2Department of Biochemistry and Molecular Biology, Guiyang Medical University, Guiyang, Guizhou, China. Correspondence: Dr H Ou, Department of Biochemistry and Molecular Biology, Guiyang Medical University, 9 Beijing Road, Guiyang, Guizhou 550004, China. E-mail: [email protected] Received 12 October 2014; revised 24 March 2015; accepted 7 April 2015; accepted article preview online 14 April 2015; advance online publication, 30 April 2015 Interleukin-5 attenuates atherosclerosis W Zhao et al 646 RESULTS control pLVCD68-transfected bone marrow (Figure 2b), indicating Exogenous IL-5 is effectively and restrictively expressed that the pLVCD68-IL5 vector did not result in significantly high IL-5 by macrophages expression in other tissues. We further investigated whether the We generated a lentiviral construct encoding IL-5 driven by the CD68 promoter-directed IL-5 could overexpress locally, by CD68 promoter pLVCD68-IL5. At the same time, a pLVCD68 differentiated, resident macrophage populations within athero- lentiviral vector containing the CD68 promoter but lacking the IL-5 sclerotic lesions. Western blot was performed for aortic lesion fi coding sequence was also generated, which served as a control. samples from both control and IL-5-transduced mice. Signi cantly To verify the recombinant construct and characterize the IL-5 higher expression of IL-5 was observed in mice that received expression profile, the mouse macrophage cell line RAW264.7 pLVCD68-IL5 BMCs than in those that received control pLVCD68 was transduced with the recombinant virus, and IL-5 levels BMCs, suggesting the successful expression of IL-5 by macro- were measured in the supernatant. Our results showed that phages in the lesions (Po0.05) (Figure 2c). Together, these data the pLVCD68-IL5-transduced macrophages produced a nearly demonstrated that the murine IL-5 transduced by lentiviral vector 30-fold higher yield of IL-5 protein at 24 h after transduction than pLVCD68-IL5 was efficiently and exclusively expressed in macro- −/− the cells transduced with control pLVCD68 lentiviral vector, and phages in vitro and in Ldlr mice. the levels were varied dependent on the different multiplicity of infection (Figures 1a and b). Moreover, IL-5 was persistently Amelioration of atherosclerosis by macrophage overexpression of expressed at a considerable level for as long as 30 days following IL-5 in mouse model of FH transduction in RAW264.7 cells (Figure 1c). To examine IL-5 To assess the effects of macrophage-specific IL-5 overexpression expression in primary cells, peritoneal macrophages were isolated on the progression of atherogenesis, we transplanted wild-type and transduced. As expected, we observed significantly high IL-5 C57BL/6 BMCs that were transduced with either pLVCD68-IL5 or expression in the cells (Figure 1d). However, we failed to detect pLVCD68 into Ldlr−/− mice. After Western-type diet treatment for measurable murine IL-5 in the supernatant from several non- 12 weeks, we evaluated the extent of atherosclerotic lesions by en macrophage cell lines such as 293T cells and HeLa cells face analysis. In contrast to mice that received pLVCD68- transduced with pLVCD68-IL5 (data not shown). transduced BMCs, a sharp decrease in lesion area was observed in oil red O-stained, longitudinally cut aortas isolated from mice Exogenous IL-5 is effectively and selectively expressed by transplanted with pLVCD68-IL5-transduced BMCs (Figure 3a). − − macrophages in Ldlr / mice Quantification of stained plague accumulated on the intimal We also examined IL-5 expression in Ldlr−/− mice receiving BMCs surface showed that the lesions covered 23.7 ± 2.2% of total area transduced with either pLVCD68-IL5 or pLVCD68. Twelve weeks from the aortic arch to descending thoracic aorta in mice after bone marrow transplantation (BMT), peritoneal macrophages overexpressing macrophage-specific IL-5, resulting in a 43% from both groups were isolated to determine the extent to which reduction in plaque area of the section from aortic arch to IL-5 remained highly expressed in macrophages. The culture