Macrophage-Specific Overexpression of Interleukin-5 Attenuates

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 descending thoracic aorta compared with 41.4 ± 3.9% in recipients medium used was collected for IL-5 measurement. Our results of control pLVCD68-transduced BMCs (Po0.05) (Figure 3b). showed that a considerable amount of IL-5 was produced by We next determined the size of atherosclerotic lesions at the macrophages from mice that received pLVCD68-IL5-transfected aortic root. Analysis of histological cross sections proximal to the bone marrow (Figure 2a). Importantly, IL-5 mRNA levels were not aortic valve revealed that the intimae isolated from mice significantly different in the liver, lung and heart between mice overexpressing macrophage-specific IL-5 were markedly thin overexpressing macrophage-specific IL-5 and those that received (Figure 3c). Further quantification revealed that these lesions

8 ∗ 8

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2 IL-5 (ng/mL) IL-5 (ng/mL) 2

0 0 untransduced pLVCD68 pLVCD68-IL5 10 30 50 100 200 multiplicity of infection (MOI)

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3 2 IL-5 (ng/mL) IL-5 (ng/mL)

0 0 1 5 10 15 20 25 30 untransduced pLVCD68 pLVCD68-IL5 Days after transduction Figure 1. Expression of IL-5 in RAW264.7 cells and primary macrophages assessed by ELISA. (a) IL-5 secretion by RAW264.7 cells. The cells were transduced with either pLVCD68 or pLVCD68-IL5 lentiviruses at multiplicity of infection (MOI) of 100. In pLVCD68-IL5, IL-5 expression was driven by a macrophage-speciﬁc CD68 promoter. The pLVCD68 transduced cells and untransduced cells were used as controls. (b) IL-5 secretion levels following various MOI of LVCD68-IL5 lentiviruses. The cells were transduced with increasing MOI ranging from 10 to 200. (c) Time course of secretion of IL-5 by RAW264.7 cells transduced with pLVCD68-IL5 lentiviruses (MOI = 100). (d) IL-5 expression in primary macrophages transduced with pLVCD68-IL5 or pLVCD68, or untransduced cells. Data are expressed as mean ± s.e.m. (n = 12 cell clones/group). *Po0.05, compared with pLVCD68 transduced or untransduced control cells.

2.0 0.5

* 0.4 1.5

0.3 1.0 0.2

IL-5 (ng/mL) 0.5 0.1 IL-5 mRNA abundance

0.0 0 Control pLVCD68-IL5 Liver lung Heart

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* Control pLVCD68-IL5 400

300 IL-5 200 GAPDH 100 IL5/GAPDH (% of control) 0 Control pLVCD68-IL5 Figure 2. IL-5 effectively and macrophage-specifically overexpressed in pLVCD68-IL5 bone marrow-transplanted Ldlr−/− mice. The mice transplanted with pLVCD68-transduced BMCs were used as controls. (a) Levels of IL-5 secreted by peritoneal macrophages assessed by ELISA. (b) IL-5 mRNA abundance measured by qRT-PCR in the liver, lung and heart. (c) Western blot determined the IL-5 expression in macrophages residing within atherosclerotic lesions in Ldlr−/− mice transplanted with pLVCD68-IL5 transduced BMCs and control mice transplanted with pLVCD68 BMCs. Values for each treatment represent a mean ± s.e.m. of triplicate determination (n = 12 cell clones/group). *Po0.05 versus pLVCD68-transduced BMC-recipient mice. were significantly (2.4-fold) smaller than those in control affected the production of these antibodies. After collection of mice (205.0 × 103 ± 19.3 × 103 μm2 vs 495.5 × 103 ±25.3×103 μm2) macrophages and blood samples from Ldlr−/− mice transplanted (Po0.05) (Figure 3d), which was similar to the en face results. with pLVCD68-IL5 or pLVCD68-transfected BMCs, the macrophage Additionally, both the data from cross-section staining and en face T15/EO6 IgM mRNA levels and plasma titers were measured. The analysis in IL-5-receiving mice showed slightly higher but not IL-5 macrophage overexpression group demonstrated a fourfold significant compared with baseline. These findings demonstrate increase in T15/EO6 mRNA relative to the control group that macrophage-specific IL-5 overexpression attenuates the (Figure 4a). Consistent with this finding, T15/EO6 IgM titers in progression of diet-induced atherosclerosis but does not result the plasma were significantly elevated by 58% compared with in the lesion regression in Ldlr−/− mice. those in the control mice and 60% in baseline group (Po0.05) (Figure 4b). We further tested the effect of increased T15/EO6 IgM Effect of macrophage IL-5 overexpression on plasma lipid levels natural antibody on OxLDL level in serum measured by As hypercholesterolemia is a major causative factor and a strong immunology assay. The result showed a 32% less than those in the control mice (Po0.05) (Figure 4c), which indicated that the determinant of atherogenesis, we verified whether the ameliora- OxLDL was dramatically reduced when T15/EO6 IgM increased. tion of atherosclerosis was due to modulation of plasma lipid Our results suggested the oxidation-specific epitopes of OxLDL levels after macrophage-specific delivery of IL-5. We performed were blocked by secreting T15/EO6 IgM, which lowered the detailed analyses of plasma lipid and lipoprotein levels in IL-5- detectable OxLDL. overexpressing and control mice. No significant differences were observed in serum total cholesterol and triglycerides between the control and experimental groups over the course of the diet DISCUSSION fi (Table 1). Serum lipid levels and lipoprotein pro les in the mice Here, we reported that macrophage-specific overexpression of IL-5 receiving pLVCD68-IL5-transduced BMCs were similar to those in −/− −/− in Ldlr mice stimulated the secretion of T15/EO6 IgM and untreated Ldlr mice and pLVCD68-transduced BMC recipients. caused a reduction in atherosclerotic lesion size and plaque area. Thus, the overexpression of IL-5 in macrophages did not impact fi In agreement with these results, the atheroprotective function of serum lipid and lipoprotein pro les. These results suggested that IL-5 has been further confirmed in a human population.12 the decrease in atherosclerotic lesion surface area observed in Specifically, clinical data demonstrated an inverse association IL-5-overexpressing mice is not caused by lowering circulating between plasma IL-5 levels and carotid intima-media thickness cholesterol and triglyceride levels. and revealed that the IL-5 level was positively correlated to the secretion of IgM antibodies specific for OxLDL.12 Therefore, the Macrophage-specific IL-5 expression increased T15/EO6 titers increase of OxLDL antibodies may explain how IL-5 protects in plasma vessels from atherogenesis. A recent report indicated that IL-5 A possible mechanism of IL-5 protection from atherosclerosis is by expression by macrophages can be enhanced by liver X receptor increasing T15/EO6 IgM natural antibody secretion. Therefore, we (LXR).13 The LXR is an important regulator of cholesterol and fatty next determined whether macrophage-specific IL-5 expression acid homeostasis, and regulates the expression of genes critical in

Figure 3. Specific expression of IL-5 in macrophages ameliorates atherosclerosis in Ldlr−/− mice. (a and b) Oil red O-stained en face aorta from the aortic arch to descending thoracic aorta and quantitative assessment of lesion area, expressed as percentage of atherosclerotic area/total area of the aorta. (c and d) Representative examples of cross sections from the aortic sinus stained with hematoxylin and eosin. Quantification of atheroma area is shown (magnification ×40). In baseline group, the Ldlr−/− mice were fed chow diet for 4 weeks and then challenged with Western-type diet for 12 weeks. The mice transplanted with either pLVCD68 or pLVCD68-IL5 transduced BMCs were fed Western-type diet for additional 12 weeks. Mice receiving pLVCD68-transduced BMCs were used as controls. Data represent the average values of six sections per mouse. The values are expressed as mean ± s.e.m., n = 10 mice in each group. *Po0.05, compared with pLVCD68-transduced BMC-recipient control mice.

Table 1. Effects of IL-5 overexpression speciﬁcally in macrophages on serum lipid levels and lipoprotein proﬁles

Mice n Diet TC (mmol l − 1) TG (mmol l − 1) LDL-C (mmol l − 1) HDL-C (mmol l − 1)

Untreated Ldlr−/− mice 10 ND 10.98 ± 0.66 1.61 ± 0.19 8.96 ± 1.15 0.87 ± 0.42 10 WTD 24.23 ± 1.13 2.91 ± 0.41 12.11 ± 1.37 3.03 ± 0.71 pLVCD68-transplanted mice 9 WTD 26.51 ± 0.97 3.40 ± 0.58 10.46 ± 1.11 3.24 ± 1.08 pLVCD68-IL5-transplanted mice 8 WTD 27.12 ± 1.85 3.08 ± 0.62 13.31 ± 1.32 3.94 ± 0.81 Abbreviations: HDL-C, High-density lipoprotein cholesterol; LDL-C, Low-density lipoprotein cholesterol; ND, Normal diet; TC, Total cholesterol; TG, Triglyceride; WTD, Western-type diet (21% fat and 0.15% cholesterol). Data are expressed as mean ± s.e.m. No statistically signiﬁcant differences were observed in mice receiving pLVCD68-IL5-transduced bone marrow cells (BMCs) compared with mice that received pLVCD68-transduced BMCs or non-transplanted Ldlr−/− mice fed a Western-type diet.

various steps of cholesterol metabolism such as cholesterol involved in cholesterol metabolism.14,15 They induce ATP binding biosynthesis and uptake, reverse cholesterol transport, and cassette transporter A1 (ABCA1) expression and enhance the cholesterol absorption and excretion. The findings suggested cholesterol efflux from macrophages to apoAI receptor, that, in addition to increasing OxLDL-specific antibodies, IL-5 also a process that facilitates the elimination of excess peripheral reduces atherosclerosis by directly modulating cholesterol meta- cholesterol in serum.14,15 Moreover, during this study, we bolism. However, the detailed mechanisms remain unclear. In observed that liver sections collected from pLVCD68-IL5- addition to IL-5, IL-6 and IL-10 are also T helper-2-associated recipient mice appeared smoother and showed less lipid cytokines that have known atheroprotective functions3 and are accumulation by oil red O staining (data not shown). Therefore,

8 20000 * 15

6 * 15000 10

4 10000 *

2 T15/O6 antibodies 5000 oxLDL level (μg/mL)l T15/EO6 mRNA abundance 0 0 0 Baseline Control pLVCD68-IL5 Baseline Control pLVCD68-IL5 Baseline Control pLVCD68-IL5 Figure 4. Assessment of T15/EO6 IgM antibody expression and OxLDL level. (a) The relative T15/EO6 IgM mRNA abundance measured in macrophages. (b) Plasma T15/EO6 IgM titers were determined. The values are in relative light units and expressed as mean ± s.e.m. (n = 8). (c) The OxLDL levels in the plasma. Data were obtained from baseline group (Western diet treatment for 12 weeks) and two bone marrow- receiving groups (Western diet treatment for 24 weeks). Experiments were performed in independent triplicate. Mice transplanted with vehicle pLVCD68-transduced BMCs were used as controls. *Po0.05 vs control. whether IL-5 exerts anti-atherosclerosis effects directly by sequence. Levin et al.28 reported that the 150-bp proximal region regulating the expression of genes related to cholesterol of the CD68 promoter is sufficient for maximal promoter activity metabolism (such as ABCA1, ABCG1 and SR-BI), thereby modulat- specifically in macrophages. Recently, macrophage-specific syn- ing cholesterol homeostasis, requires further investigation. thetic promoters generated by random ligation of myeloid/ In this study, primary bone marrow-derived macrophages were macrophage cis elements, including PU.1, C/EBPα, and myeloid- used as therapeutic target cells. After transduction with recombi- associated cis elements (for example, AP-1), were developed and − − nation lentiviral vectors, the cells were transplanted into Ldlr / demonstrated to provide high levels of macrophage-specific mice. Macrophages are pivotal to both innate and acquired transgene expression.29,30 However, characterization of these immune responses. They are implicated in many diseases, promoters needs to be more fully evaluated in therapeutic including infections, arthritis and cancer, as well as in the applications. development of atherosclerosis. In addition, the accessibility of It should be mentioned that the macrophage population will be hematopoietic stem cells, which differentiate into macrophages, found in multiple sites in receptors including the liver and lung and their natural tendency to migrate into lesion sites after BMT. However, we failed to observe the dramatically make macrophages an attractive vehicle for delivery of increasing level of IL-5 in such tissues. Also, we note that MMP-9 therapeutic genes. and IL-10 genes that were both directed by the CD68 promoter Unfortunately, efficient gene transfer into macrophages has displayed the same expression pattern, whereas the expression of been hampered by the nature of the cells themselves. Macro- foreign genes were not significantly elevated in tissues after BMT phages are terminally differentiated cells that barely proliferate as reported in others’ reports.31,32 A potential explanation for this under normal conditions. This precludes the use of viral vectors is that could be due to the property of human CD68 promoter, such as retroviruses that require cell division for efficient which may display stronger activity in fully differentiated and transduction. Adenoviruses have also shown to be relatively active macrophage in inflammatory tissues. This is similar to ineffective in infecting monocytes. Moreover, their transient chicken lysozyme gene promoter, which is another commonly transgene expression due to a lack of integration into the host used regulatory element to control expression of foreign genes genome makes adenoviruses less suitable for transfection of exclusively in macrophages. The expression of foreign genes such macrophages and other peripheral blood cells, as they are easily as liver X receptor-α (LXR-α) and 15-Lipoxygenase driven by the cleared from the blood stream.16 Lentiviral vectors based on HIV-1 element could not be detected in any of the other tissues such as structure show the ability to stably express therapeutic gene and liver, lung, brain, kidney, heart, muscle and small intestine other offer the most effective transfection of DNA into macrophages than monocyte-derived macrophages in transgenic rabbits.33,34 among the viral delivery systems. The lentiviral vectors have been This is because the chicken lysozyme gene promoter has been widely used to deliver transgene into such cells in a recent demonstrated to be mainly active in differentiated macrophages, – study.17 20 However, the efficiency of transduction is apparently but not in the total spleen tissue in earlier studies.35 still lower than that in other cell types (data not shown). However, it is noteworthy that, although not strikingly, a Another barrier to the overexpression of therapeutic genes in moderate elevated expression of IL-5 in plasma was detected in macrophage is the lack of desired gene regulatory sequences. mice overexpressing macrophage-specific IL-5 compared with Several promoter/enhancer elements have been characterized those in the control group in this study (data not shown). As and used to restrict gene expression to the monocyte- circulating monocytes could scarcely produce IL-5 under the CD68 macrophage lineage; these include regulatory elements of the promoter, the elevation might be caused by differentiated scavenger receptor-A (SR-A), chicken lysozyme, c-fms (CSF-1R) and macrophages, which then secreted into circulation. human CD68.21–25 Among these, the human CD68 promoter/ FH is characterized by elevated serum LDL cholesterol levels, enhancer that has been demonstrated can direct transgene which is caused by a loss-of-function mutation in Ldlr; which, in expression in macrophages with high activity and specificity. It has turn, leads to accelerated atherosclerosis and increased risk of been more widely used than other regulatory elements to drive premature coronary heart disease. With this framework, the macrophage-specific expression of various genes such as IL-10, traditional strategy for FH treatment relies on improving the transforming growth factor-β (TGF-β) and tissue inhibitor of lipoprotein profile, and extensive efforts have been invested in metalloproteinase 3 (TIMP3).22,26,27 However, the unusually large altering the expression of genes related to lipid metabolism, such size of the promoter/enhancer element, which comprises several as LDLR, VLDLR, LXRα and PPARα/γ, to maintain cholesterol hundred to 2.9 kilo base pairs of fragment upstream from first homeostasis. Our increasing knowledge regarding the role of intron, complicates viral packaging and gene delivery. To date, inflammation in atherosclerosis not only facilitates a better many efforts have been made to optimize the regulatory understanding of the pathogenesis of this disorder but also

© 2015 Macmillan Publishers Limited Gene Therapy (2015) 645 – 652 Interleukin-5 attenuates atherosclerosis W Zhao et al 650 suggests the novel possibility that reduction of atherosclerotic procedures in animals were conducted in accordance with the National lesions can be achieved by modulating the immune response. The Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals successful amelioration of atherosclerosis as a result of macro- (NIH), and approved by the Animal Care and Use Committee of Guiyang phage overexpression of IL-5 revealed in the present study further Medical University. verifies the feasibility of this approach as an alternative, effective method for FH therapy. Bone marrow transduction and transplantation IL-5 is a pleiotropic cytokine that is known to be an essential BMCs were isolated by flushing the femurs and tibias from 5-fluorouracil- factor for terminal eosinophil differentiation as well as eosinophil treated donor female Ldlr−/− mice (8 weeks old, C57BL/6 background) with activation. Clinical observations have demonstrated that patients PBS. Single-cell suspensions were prepared by passing the cells through with asthma show decreased atherosclerosis.36 However, the 30-μm nylon gauze, and then were transduced by 24-h incubations with − / − recombinant lentiviral pLVCD68-IL5 supernatant at multiplicity of infection peripheral blood eosinophil counts in the mice receiving IL-5 − − +/+ 11 of 20 with 1 × 108 TU ml 1 titers containing 4 μgml 1 polybrene and and IL-5 bone marrow were reportedly not different. There- − 1 fore, whether the delivery of IL-5 specifically into macrophages 2000 U ml recombinant human macrophage-colony stimulating factor (R&D Systems, Minneapolis, MN, USA). The cells were cultured for can accelerate allergic diseases such as asthma requires further additional 4 days in Dulbecco’s modified Eagle’s medium (Invitrogen, investigation. Additionally, plaques contained with abundant Carlsbad, CA, USA) containing 2000 U ml − 1 recombinant human macrophages, thin smooth muscle cells and less collagen are macrophage-colony stimulating factor. After harvesting, the cells were considered to be unstable and rupture-prone, which usually washed twice in PBS and suspended in fresh medium prior to injection. caused clinical events. Therefore, a thorough pathologic char- Ten female recipient Ldlr−/− littermates at 16 weeks of age were acterization of lesion phenotype by determining the ratio of maintained on antibiotic-containing water, and were lethally irradiated (9 Gy) from a cesium gamma source 1 week later. The Ldlr−/− mice were macrophage/collagen content within the plaque is also necessary 6 before application of the strategy in practical use. then transplanted with 5 × 10 transduced BMCs via tail vein injection In conclusion, we found that the overexpression of IL-5 within 6 h of irradiation. Mice were fed regular chow for 4 weeks after BMT fi to allow for donor bone marrow reconstitution and then switched to an speci cally in macrophages resulted in a reduction in plaque atherogenic Western-type diet containing 21% fat and 0.15% cholesterol areas and lesion sizes, and a corresponding increase in the plasma for an additional 12 weeks to induce lesion formation. The mice received titers of T15/EO6 IgM antibodies. Our results suggested that IL-5 is BMCs transduced with pLVCD68 lentivirus used as control. After the an effective gene target to prevent atherosclerosis, and treatment, the two groups were killed for further analysis. macrophage-specific transfer of IL-5 may provide a novel gene therapy strategy for FH as well as other cardiovascular diseases. Atherosclerotic lesion analysis Hearts together with aortas were excised, perfused with PBS, and then fi MATERIALS AND METHODS xed in 10 ml of 4% paraformaldehyde. For en face analysis, aortas were dissected from the proximal aortic arch to the thoracic artery. After Construct generation and lentiviral particle production adventitial fat was removed, the section of aortas were cut longitudinally, A recombinant lentiviral construct that expresses IL-5 specifically in pinned en face onto flat plates and stained with oil red O solution (2% oil macrophages was generated by standard molecular cloning methods and red O, wt/vol, in 60% isopropanol, filtered through a 0.2-mm filter; Sigma- verified by sequencing. Briefly, total RNA was extracted from mitogen- Aldrich, St Louis, MO, USA) for 40 min at room temperature, followed by stimulated murine peripheral blood mononuclear cells and full-length IL-5 rinsing with 60% isopropanol and distilled water. Finally, the specimens cDNA was amplified by reverse transcription-polymerase chain reaction were digitally photographed. The total arterial surface area and total lesion (RT-PCR) with the following primers: forward: 5′-atgagaaggatgcttctgca-3′; area were calculated using Image-Pro Plus version 6.0 software (Media reverse: 5′tcacccgttaccttccgact-3′. The PCR product was subsequently Cybernetics Inc., Silver Spring, MD, USA). The extent of lesion development inserted into self-inactivating lentiviral vector pLVX-Puro (Clontech, was defined as percentage of the total area of oil red O-positive plaques. Mountain View, CA, USA). The macrophage-specific human CD68 promoter For analyzing aortic sinus plaque lesions, the upper half of the heart (2.9-kb) along with an 89-bp intronic enhancer, as described containing the aortic root was embedded in Optimal Cutting Temperature previously,12,13 were amplified from human genomic DNA by PCR; these medium (Sakura Finetek, Torrance, CA, USA), frozen on dry ice and stored were used to regulate exogenous IL-5 gene expression. After deleting the at − 80 °C for cryosectioning. Serial 10-μm-thick cross sections were taken cytomegalovirus promoter from the vector, the CD68 promoter were from the apex toward the base of the heart, until the aortic valve leaflets incorporated upstream of the coding sequence to generate the final IL-5- appeared. The sections were stained with hematoxylin/eosin for evaluation expressing lentiviral construct pLVCD68-IL5. of atherosclerotic lesions. Images were viewed and captured with a Viruses were produced by transient co-transfection of 293FT cells with microscope (TS100F, Nikon, Tokyo, Japan), and the lesion sizes were pLVCD68-IL5 and Lenti-X HTX Packaging Mix (Clontech) according to the determined using Image-Pro Plus. The values were obtained by the manufacturer’s instructions. At 72 h after transfection, the medium was averaging the lesion areas in at least six sections for each mouse. harvested and filtered through a 0.45-μm pore size cellulose acetate filter (Whatman, GE Healthcare, Uppsala, Sweden), and concentrated by ultracentrifugation at 70 000–90 000 g for 0.5 h. Finally, the lentiviral Peritoneal macrophage isolation particles were resuspended in phosphate-buffered saline (PBS) and stored Female C57BL/6 mice were injected intraperitoneally with 2 ml of 3% at − 80 °C for subsequent use. Similarly, a recombinant lentiviral vector Brewer’s thioglycollate broth. Four days after injection, primary macro- pLVCD68 that contains the CD68 promoter and lacks the IL-5 coding phages were collected from animals that were killed by peritoneal lavage sequence was also generated and used as a control. using 10 ml of ice-cold PBS. Cell suspension was centrifuged at 3000 r.p.m. and the precipitate was resuspended in RPMI 1640 medium plus 10% fetal calf serum. After incubation at 37 °C at 5% CO2 for 24 h, the supernatant Study animals and collection of tissue and blood samples was discarded and the precipitate was washed three times with pre- −/− Ldlr mice on the C57BL/6 background were originally obtained from the warmed PBS to remove nonadherent cells. Finally, the peritoneal Jackson Laboratory (Bar Harbor, ME, USA) and were maintained in a macrophages were grown with 10% fetal calf serum supplemented fresh pathogen-free barrier facility at 20–22 °C under 12-h light/dark cycle RPMI 1640 medium for further analysis. conditions. The female animals (23–28 g) were fed a normal chow diet (4 weeks) and accelerated atherosclerosis was induced by feeding a Western-type diet (21% fat and 0.15% cholesterol; Tengxin Bio Inc., Immunoassay Chongqing, China) for 12 weeks. After diet induction, the animals were Total cholesterol, triglycerides and lipoproteins in plasma were determined divided into three groups. The first group (baseline) was anesthetized with using an enzymatic assay (Jiancheng Bio, Nanjing, China). Plasma levels of an intraperitoneal injection of sodium pentobarbital (150 mg kg − 1 body IL-5 and OxLDL were examined using an ELISA kit (Abcam, Cambridge, UK; weight). The liver, heart and lung were isolated and blood was collected by R&D Systems). The assays were performed following the manufacturers’ retro-orbital venous plexus puncture for various analyses. The other two instructions. The titers of T15/EO6-type antibodies in plasma (1:100 groups were subjected to BMT as described below. All experimental dilution) were assayed by a chemiluminescence immunoassay. The anti-

Gene Therapy (2015) 645 – 652 © 2015 Macmillan Publishers Limited Interleukin-5 attenuates atherosclerosis W Zhao et al 651 T15 antibody AB1–2 (BioXcell, West Lebanon, NH, USA) was used as the 10 Palinski W, Hörkkö S, Miller E, Steinbrecher UP, Powell HC, Curtiss LK et al. capture antibody and plasma antibodies were detected by alkaline- Cloning of monoclonal autoantibodies to epitopes of oxidized lipoproteins from phosphatase-conjugated goat anti-mouse IgM (Sigma) followed by apolipoprotein E-deficient mice. Demonstration of epitopes of oxidized low incubation with LumiPhos 530 (Lumigen, Southfield, MI, USA). Finally, density lipoprotein in human plasma. J Clin Invest 1996; 98: 800–814. the mixture was incubated for 5 min at room temperature, and the relative 11 Shaw PX, Hörkkö S, Chang MK, Curtiss LK, Palinski W, Silverman GJ et al. light units were measured on luminometer. Natural antibodies with the T15 idiotype may act in atherosclerosis, apoptotic clearance, and protective immunity. J Clin Invest 2000; 105:1731–1740. Western blot 12 Sämpi M, Ukkola O, Päivänsalo M, Kesäniemi YA, Binder CJ, Hörkkö S. Plasma interleukin-5 levels are related to antibodies binding to oxidized low- Atherosclerotic plaques were isolated and protein extracts were prepared density lipoprotein and to decreased subclinical atherosclerosis. J Am Coll Cardiol for analysis. After loading on a 12% SDS-PAGE gel and separation by 2008; 52:1370–1378. electrophoresis, the samples were transferred to an Immobilon-Nc 13 Chen Y, Duan Y, Kang Y, Yang X, Jiang M, Zhang L et al. Activation of liver X membrane and incubated with primary anti-IL-5 antibody (1:200 dilution; receptor induces macrophage interleukin-5 expression. J Biol Chem 2012; 287: Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by horseradish 43340–43350. peroxidase-conjugated secondary antibodies (1:5000 dilution; Santa Cruz 14 Frisdal E, Lesnik P, Olivier M, Robillard P, Chapman MJ, Huby T et al. Interleukin-6 fi Biotechnology). The proteins were visualized and quanti ed using a protects human macrophages from cellular cholesterol accumulation and chemiluminescence method (Beyotime Bio, Nantong, Jiangsu, China) and attenuates the proinflammatory response. J Biol Chem 2011; 286: 30926–30936. Quantity One (Bio-Rad, Hercules, CA, USA) software program. 15 Han X, Kitamoto S, Lian Q, Boisvert WA. Interleukin-10 facilitates both cholesterol uptake and efflux in macrophages. J Biol Chem 2009; 284: 32950–32958. Real-time quantitative RT-PCR 16 Huang S, Endo RI, Nemerow GR. Upregulation of integrins alpha v beta 3 and alpha v beta 5 on human monocytes and T lymphocytes facilitates adenovirus- Total RNA was extracted from the liver, lung and heart. Following reverse – transcription into cDNA using Superscript II reverse transcriptase (Invitro- mediated gene delivery. J Virol 1995; 69: 2257 2263. 17 Zeng L, Planelles V, Sui Z, Gartner S, Maggirwar SB, Dewhurst S et al. HIV-1-based gen) and random primers, real-time quantitative RT-PCR was performed fi using brilliant SYBR green PCR master mixture (Stratagene, La Jolla, CA, defective lentiviral vectors ef ciently transduce human monocytes-derived macro- fi ′ ′ phages and suppress replication of wild-type HIV-1. JGeneMed2006; 8:18–28. USA) with IL-5-speci c primers: forward: 5 -gctcaccgagctctgttgac-3 ; fi reverse: 5′-aatgacaggttttggaatag-3′ on the Applied Biosystem 7500 Real 18 Leyva FJ, Anzinger JJ, McCoy Jr JP, Kruth HS. Evaluation of transduction ef ciency Time PCR System (Applied Biosystems, Foster City, CA, USA). All samples in macrophage colony-stimulating factor differentiated human macrophages using HIV-1 based lentiviral vectors. BMC Biotechnol 2011; 11:13. were analyzed at least in triplicate and the results were normalized to fi mouse Gapdh expression (GenBank ID: NM_008084). The relative T15/EO6 19 Bobadilla S, Sunseri N, Landau NR. Ef cient transduction of myeloid cells by an HIV-1-derived lentiviral vector that packages the Vpx accessory protein. Gene Ther IgM mRNA abundance in macrophages was conducted as similar – procedures with specific primers, forward: 5′-cagagacacttcccaaagca-3′; 2013; 20:514 520. reverse: 5′-cccagacatcgaagtaccag-3′.37 20 Tong J, Buch S, Yao H, Wu C, Tong HI, Wang Y et al. Monocytes-derived macrophages mediated stable expression of human brain-derived neurotrophic factor, a novel therapeutic strategy for neuro AIDS. PLoS One 2014; 9: e82030. 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