Original article
Effect of hyperlipidemia on Foxp3 expression in apolipoprotein E-knockout mice Zhixiao Wang, Shan Mao, Zhongqun Zhan, Kefei Yu, Chaorong He and Chongquan Wang Background The transcription factor forkhead box P3 (Foxp3) plays an essential role in the development and function of regulatory T cells. Aims To examine the effect of hyperlipidemia on the expression of Foxp3 in mice. Methods Twenty-four 8-week-old male apolipoprotein E (ApoE)S/S mice on a C57BL/6 background were randomly divided into control group and high fat diet group, 12 mice per group. The blood-lipid levels, the number of Foxp3RCD4R CD25R T cells, and the size of the atherosclerotic lesions in every group were measured. The expression levels of Foxp3 in different tissues were detected. Results Compared with the control group, the level of plasma lipids was significantly higher in the high fat-fed group, but the number and function of Foxp3RCD4R CD25R T cells, the levels of Foxp3 protein expression, and Foxp3
Introduction Atherosclerosis is a multifactorial process that involves interactions among endothelial cells, macrophages, smooth muscle cells and lymphocytes.1–3 A subtype of T cells, called regulatory T cells (Tregs), play a critical role in eliminating autoimmune processes and inflammatory responses. Previous studies show Tregs play a role in negatively regulating the development and progression of atherosclerosis. A recent study showed the number of Tregs was decreased and the function of the cells diminished in atherosclerotic patients,4,5 with no exact mechanisms elucidated yet. The transcription factor forkhead box P3 (Foxp3), a key regulatory gene of Tregs, plays an essential role in the development and function of Tregs. Further studies showed an antiatherogenic role of Foxp3.6,7 However, the mechanisms for Foxp3 in the pathogenesis of atherosclerosis remain unclear, and the expression of Foxp3 in vivo has not been previously examined. In this study, we examined the effect of an important risk factor of atherosclerosis, hyperlipidemia, on the expression of Foxp3, in order to further explore the potential mechanism for hyperlipidemia causing atherosclerosis.
Materials and methods All procedures in the present study conformed to the principles outlined in the Guide for the Care and Use of 1558-2027 ß 2014 Italian Federation of Cardiology
gene transcript in selected tissues were lower in the high fat-fed group. Conclusion Hyperlipidemia inhibits the expression and function of Foxp3 in various immune organs, which may be one of the mechanisms by which hyperlipidemia aggravates the formation of atherosclerosis. J Cardiovasc Med 2014, 15:273–279 Keywords: atherosclerosis, Foxp3, Foxp3RCD4R CD25R T cells, hyperlipidemia Department of Cardiology, Taihe Hospital, Hubei University of Medicine, Shiyan, China Correspondence to Zhixiao Wang, Department of Cardiology, Taihe Hospital, Hubei University of Medicine, No. 32 South People’s Road, Shiyan, China, 442000 E-mail: [email protected] Received 12 January 2012 Revised 1 June 2013 Accepted 4 June 2013
Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). Male ApoE/ mice on the background of C57BL/ 6 were purchased from Peking University and kept under standard laboratory conditions in Tongji Medical College, Huazhong University of Science and Technology, Wuhan. The C57BL/6 mouse is one of the most commonly used strains of mice for biomedical research. It is an inbred line of mice, which have the characteristics of high precision, good comparability and uniform stress response. At 8 weeks of age, 12 male ApoE/ mice were fed with a high fat diet. And the other half of the mice were used as a control group, which were fed with a normal chow diet with the following ingredients per kg: 140 g casein, 715 g corn flour, 50 g solkafloc, 40 g corn oil, 50 g vitamin and mineral mixture, 2 g choline bitartrate, 2 g L-cystine, and 1 g DL-methionine. In the high fat diet, 180 g/kg corn flour in the basal diet was replaced with 157.5 g/kg of fat and 12.5 g/kg of cholesterol for 12 weeks. Plasma lipid analysis
Plasma from the high fat-fed ApoE/ mice was diluted with buffer containing 150 mmol/l NaCl, 1 mmol/l EDTA (pH 7.4) to adjust the OD measurement into the range of the standard curve. Total cholesterol (TC) and triacylglycerol were assayed enzymatically using commercial DOI:10.2459/JCM.0b013e3283641b9c
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274 Journal of Cardiovascular Medicine 2014, Vol 15 No 4
kits (Merck, Darmstadt, Germany). High-density lipoprotein-cholesterol (HDL-C) and low-density lipoprotein-cholesterol (LDL-C) were determined by precipitation with phosphotungstic acid/magnesium chloride or with heparin/sodium citrate, respectively, using reagents supplied by Merck. Very low-density lipoprotein (VLDL) and intermediate-density lipoproteincholesterol (IDL-C) (VLDLþIDL-C) were determined by subtracting HDL-C and LDL-C from TC. Quantification of atherosclerotic lesions in the aortic root
Atherosclerotic lesion size in the ascending aorta was determined based on eight hematoxylin–eosin-stained serial sections, 8 mm thick, starting from the region where the aortic sinus becomes the ascending aorta, as described previously.8,9 Morphometric analysis of plaques was carried out by computerized morphometry (Tonx-432, JVC, Japan). Flow cytometry
Splenocytes were costained with monoclonal antibodies fluorescein isothiocyanate-labeled anti-CD4 (7D4; Miltenyi Biotec, Cologne, Germany) and phycoerythrinlabeled anti-CD25 (GK1.5; Miltenyi Biotec). For fluorescence-activated cell sorting (FACS) analysis of intracellular Foxp3 expression, fluorescein isothiocyanate antimouse Foxp3 staining set was used (clone FJK-16s, e-Bioscience, Shanghai, China). Stained cells were analyzed on a FACS with Cell Quest software (BectonDickinson, New York, USA).
1 : 4, and 0 : 1), in a 96-well microplate at 378C with 5%CO2 air. The final volume was 200 ml per well. On the 2nd, 3rd, 4th and 5th days, 25 ml diluted 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added into each well and incubated for 4 h, followed by addition of 100 ml dimethyl sulphoxide into each well. The microplates were incubated at 378C overnight to dissolve MTT crystals before optical density values were determined by ELISA reader at a wavelength of 490 nm. Interferon-g (IFN-g), interleukin-10 (IL-10) and transforming growth factor-b (TGF-b) in the supernatants were determined by ELISA according to the manufacturer’s protocol (Jingmei Biotech Co. Ltd, Shenzhen, China). Real-time quantitative reverse transcription-PCR for the expression of Foxp3 mRNA in various tissues and blood cells
Quantitative reverse transcription-PCR (RT-PCR) was used to assess the transcriptional level of Foxp3 mRNA in spleen, thymus gland, atherosclerosis plaque and circulating blood of the mice. Western blot analysis of Foxp3 protein
Western blotting was performed with mouse serum antifoxp3 (monoclonal antibody; Abcam, Cambridge, UK) at a dilution of 1 : 100 and a secondary antibody, peroxidase-conjugated AffiniPure goat antimouse IgG (Abcam). b-Actin served as a control protein.
CD4RCD25R T-cell purification
Spleen and lymph node cell suspensions were concentrated with monoclonal antibodies fluorescein isothiocyanate-labeled anti-CD4 (7D4; Miltenyi Biotec) and phycoerythrin-labeled anti-CD25 (GK1.5; Miltenyi Biotec). CD25þ T cells were isolated from total splenocytes by a first step of negative sorting of CD4 with a cocktail of hapten-conjugated CD8, CD11b, CD16, CD19, CD36 and CD56 antibodies, and microbeads coupled with an antihapten monoclonal antibody (CD4 T-cell isolation kit; Miltenyi Biotec). This was followed by a selection of CD25-positive cells by microbead separation (CD25 microbeads; Miltenyi Biotech), providing more than 90% purity assessed by FACS analysis. Function of CD4RCD25R regulatory T cells and cytokine assay
To verify the dysfunction of Tregs, an in-vitro assay was performed. For this assay, different ratios of CD4þ CD25þ T cells (suppressor) and freshly purified CD4þCD25 T cells (responder; 1 105 cells per well), with the addition of splenocytes devoid of red blood corpuscles as antigen-presenting cells (2 104 cells per well) and ox-LDL (0.5 mg) were cocultured (1 : 1, 1 : 2,
Statistics
Statistical analyses were performed with SPSS 17.0 software (SPSS Inc., Chicago, Illinois, USA). Data are presented as mean SD. Statistical differences in the amounts of continuous variables were compared by t-test. All tests were two-sided and the differences were considered statistically significant if P was less than 0.05.
Experimental results Body weight and plasma lipid levels in apolipoprotein ES/S mice
The body weight of the 12 mice was 20 1 g in each group prior to the study (8 weeks of age). At the end of the study at 20 weeks of age, the body weight was 28 2 g for control group and 29 3 g for the high fat diet-fed group. There was no statistical difference in body weight between the two groups. High fat diet resulted in a significant increase in plasma triacylglycerol, TC, LDL and VLDLþIDL-C. In contrast, there was no statistical difference in plasma HDL-C between control mice and high fat diet-fed mice (Table 1).
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Effect of hyperlipidemia on Foxp3 expression Wang et al. 275
Table 1 Plasma lipid profiles in apolipoprotein E-knockout mice fed control or high fat diets
a
TG (mmol/l) TCb (mmol/l) HDL-Cb (mmol/l) LDL-Cb (mmol/l) VLDL þ IDL-Cb (mmol/l)
Control
High fat diet
1.0 0.13 10.6 0.12 1.0 0.1 2.2 0.5 8.0 1.6
2.8 0.19 28.2 1.2 1.1 0.3 5.8 0.2 20 0.2
Values are mean SD (n ¼ 12 for each group). TC, total cholesterol; TG, triacylglycerol; HDL, high-density lipoprotein; IDL-C, intermediate-density lipoprotein-cholesterol; LDL, low-density lipoprotein; VLDL, very low-density P < 0.01. P < 0.001. a For TG, 1 mmol/l equals 88.0 g/dl. lipoprotein. b For cholesterol (C), 1 mmol/l is 25.91 g/dl.
Morphometric analysis of plaques
Aortic roots of mice were stained by hematoxylin–eosin (Fig. 1) and the size of plaques was analyzed using computerized morphometry. The plaque area of mice in the high fat diet group (25 044.32 4920.42 mm2) was significantly larger than the control group (15 292.78 3309.10 mm2, P 0.01; and at a 0 : 1 ratio, P > 0.01, respectively; P values indicate the average of five separate experiments; Table 3). No significant differences were found in responder cell proliferation in the control mice when the assay was conducted in the presence of activated protein C (APC) from the high fat-fed mice or the control mice. When CD4þCD25þ cells from the high fat diet mice were used as responders in the described suppressive assay, the ability to inhibit the proliferation of Tregs in the high fat-fed mice was reduced, compared with the cells from the control mice (the mean value of reduction was 32.%, P < 0.01). Assessment of Foxp3 transcript levels
Foxp3 mRNA expression (Fig. 3a) was quantified using a SYBR Green I and then analyzed using real-time quantitative RT-PCR with the listed primers (Table 4). The Foxp3 mRNA expression level in various tissues was significantly lower in high fat diet group than the control group (P < 0.05) (Fig. 3b).
Fig. 1
(a)
(b)
100 µm
100 µm
Representative photomicrographs from aortic sinus sections of the control group (a) and the high fat diet group (b), stained with hematoxylin–eosin (magnification is 40).
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276 Journal of Cardiovascular Medicine 2014, Vol 15 No 4
Fig. 2
3 2
10
CD25-PE
3
10 10
10
2
CD25-PE
10
10
4
4
10
10
5
5
(a)
2
3
10
4
10
5
10
10
10
(b)
2
10
3
10
4
10
5
(c) CD4-FITC
CD4-FITC 10
22 20 P < 0.05
P < 0.05
8
16 %Foxp3/CD25I
%CD4/CD25/CD4
18 14 12 10 8
6
4
6 2
2 4
0
0 Control
High fat diet
Control
High fat diet
Comparative analysis of the number and function of regulatory T cells (Tregs) between the two groups of mice. (b) Spleen cells were obtained from 3-month-old mice and stained with antibodies to CD4 and CD25 as outlined in Methods. Data are presented from eight mice in each group. (c) Staining of splenocytes from the two groups of mice was performed for detection of CD25þFoxp3þ cells. Representative fluorescence-activated cell sorting (FACS) sheets are provided in (a).
Discussion
Quantitative assessment of the expression of Foxp3 protein
Foxp3 expression was quantified by the specific binding recorded on X-ray film. NIH Image Version 1.61 was used to quantify the intensities of the bands (Fig. 4a). Foxp3 expression (as assessed by the mean intensity of bands on the X-ray film) in the spleen, thymus, circulating blood and plaque of high fat diet group was significantly lower than that in the control group (P < 0.01) (Fig. 4b).
In the current study, we investigated the effect of hyperlipidemia on the expression and function of Foxp3 in experimental atherosclerosis. Atherosclerosis is a chronic disease of the arterial wall,1 a multifactorial process that involves interactions among endothelial cells, macrophages, smooth muscle cells and lymphocytes. Inflammation, oxidative stress and endothelial dysfunction are thought to play an active role in the initiation and progression of atherosclerosis.10 Activated Comparison of value A in every group in lymphocyte proliferation assay (x¯ W s)
Table 3
Cytokine levels in the medium secreted by regulatory T cells (pg/ml) (x¯ W s, n U 5)
Table 2
Groups/cytokines High fat diet Control
IL-10 85.5 13.8 158 29.8
TGF-b
108.8 28.9 295.6 34.8
Treg/Te Groups
IFN-g
IFN, interferon; IL, interleukin; TGF, transforming growth factor.
198.9 42.5 55.9 23.1
P < 0.01.
High fat diet Control
1:1 3.8 1.1 8.8 1.6
1:2
3.9 1.0 8.9 1.4
1:4
0:1
3.3 1.2 4.3 1.2
3.2 1.3 4.2 1.0
P < 0.05.
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Effect of hyperlipidemia on Foxp3 expression Wang et al. 277
Fig. 3
(a)
(b)
100– 250– 500– 1000– 2000–
–176 –384
FOXP3 mRNA transcnpl level
9 8 7
Control High fat diet
P < 0.05
6 5 4 3 P < 0.05
2
P < 0.05
1
P < 0.05
0 Thymus
bp
1
2
3
4
5
Spleen
Blood
Plaque
bp
(a) Agarose gel electrophoresis of Foxp3 reverse transcription-PCR (RT-PCR) product 1: DNA Ladder marker; 2–3: b-actin; 4–5: Foxp3 RT-PCR product. (b) RT-PCR showed Foxp3 transcriptional levels in the different tissues in mice.
T lymphocytes, predominantly CD4þ cells, secrete proinflammatory mediators such as IFN-g, tumor-necrosis factor-a, and IL-1 and IL-6, promoting extracellular matrix collagen production and activating macrophages.1 Tregs are an anti-inflammatory subset of CD4þ T cells that can suppress effector CD4þ and CD8þ cells and induce tolerance, thereby modulating adaptive immune responses.11 Several studies have suggested negative roles for Treg cells in atherosclerosis.12–14 However, the specific mechanisms for the negative regulation by Tregs are still unknown. The role of the transcription factor Foxp3, identified as a molecular marker for Tregs, in atherosclerosis of ApoE/ mice has not yet been elucidated. Many studies have supported hyperlipidemia as a risk factor for atherosclerosis.15 The potential connection among hyperlipidemia, Foxp3 and atherosclerosis was explored by determining whether hyperlipidemia affects the expression of Foxp3. This study may provide a novel mechanism by which hyperlipidemia causes atherosclerosis, thereby providing a basis for novel therapeutic target.
Table 4
Primers of Foxp3 and b-actin Sequence
Foxp3
b-Actin
Sense-primer 50 -CAGGAGAAAGCGGATACCAAATG-30 Antisense-primer 50 -ATCTGTGAGGACTACCGAGCC-30 Sense-primer 50 -TGGAATCCTGTGGCATCCATGAAAC-30 Antisense-primer 50 -TAAAACGCAGCTCAGTAACAGTCCG-30
Amplification size (bp) 176
384
Antigen-specific Treg cells secrete cytokines such as IL-10 and TGF-b. Naturally occurring Tregs act through cell-to-cell contact but predominantly through secretion of TGF-b.16 Despite the lack of a universally accepted membrane antigen that characterizes Tregs, the most likely molecule to characterize Tregs on CD4þ cells is CD25. We, therefore, assayed the number of CD4þ CD25þ T cells in the spleen. Indeed, we found that the high fat diet-fed mice had a significantly reduced number of splenic Tregs. Therefore, to further confirm the identity of these Tregs, comparative analysis of CD25þ Foxp3þ cells was made. We also found a reduction in the number of CD25þFoxp3þ cells in the high fat-fed group, which is consistent with the differences in CD4þCD25þ cells. The transcription factor Foxp3, a key regulatory gene of Tregs, plays an essential role in the development and function of Tregs. The lack of Tregs in Foxp3-mutant scurfy (sf) mice and in Foxp3/ mice results in autoimmune disease. An increased percentage of Tregs has been observed in Foxp3 transgenic mice, and in-vitro activation of Tregs increased Foxp3 expression, supporting the hypothesis that Foxp3 expression affects Treg function.17 Ait-Oufella et al.18 found that Tregs have an antiatherogenic role by blocking CD25þ cells using monoclonal antibodies in the ApoE/ mouse model. Our results provided complementary data that further support the potential role of Foxp3 in Tregs during the pathogenesis of atherosclerosis in the mouse. Hyperlipidemia results in a significant increase in atherosclerotic lesion formation. This was validated by separately measuring plaque size in the aortic sinus and
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278 Journal of Cardiovascular Medicine 2014, Vol 15 No 4
Fig. 4
(a) High fat diet
Control
Plaque
Thymus
β-actin
β-actin
High fat diet
Control
Spleen
High fat diet
Control
High fat diet
Control
Blood
β-actin
β-actin
(b)
Intensities of phosphor-Foxp 3 bands
100
80
Control High fat diet
P < 0.05
60 P < 0.05 40
P < 0.05
20 P < 0.05 0 Thymus
Spleen
Blood
Plaque
(a) Western blot analyzing the expression of Foxp3 protein in the spleen, thymus, plaque and circulating blood in each group. (b) Foxp3 protein level was analyzed by Western blotting and protein quantification densitometric analysis.
the entire longitudinally stripped aortas. The expression level of Foxp3 in different tissues, especially in thymus, was significantly lower in the high fat-fed group. This suggests hyperlipidemia, an important risk factor of atherosclerosis, may inhibit the function and expression of Foxp3. The expression level of Foxp3 was the highest in the thymus, shown by both RT-PCR and Western blot. Naturally occurring murine Tregs differentiate in the thymus. Under normal conditions, Tregs are found primarily in the thymus, peripheral blood, lymph nodes and spleen.19 So from our results, we speculate that the function of Foxp3 is compromised by hyperlipidemia, which affects the maturation of natural Tregs in the thymus. In spite of signaling pathways initiated by T-cell receptor (TCR), IL-2, TGF-b and CD28 have been shown to affect Foxp3 expression.20,21 Whether hyperlipidemia affects the expression
and function of Foxp3 by immuno-inflammatory reaction, or whether there are other factors involved in initiating Foxp3 expression and promoting its immune regulatory characteristics remain to be elucidated. In summary, the role of Foxp3 in autoimmune conditions has been established. Foxp3 expression plays a critical role in the regulation and development of Tregs. Hyperlipidemia can promote the progression of atherosclerosis by inhibiting the expression of Foxp3, especially in the thymus, and consequently affecting the number and function of Tregs. These data provide potential novel therapeutic targets for the management and prevention of atherosclerosis as well as other inflammatory and autoimmune conditions. However, our study is limited by the relatively small number of mice included, and by the matching system used to identify a control
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Effect of hyperlipidemia on Foxp3 expression Wang et al. 279
group which does not overcome unknown biasing factors. We did not assess extracoronary atherosclerotic burden, which theoretically could also have influenced the panel of cytokines and Tregs that we studied.
9
10 11
Acknowledgements
12
This work was supported by a grant from the Educational Foundation of Hubei province to Zhixiao Wang (No.B20102108). The authors have no conflict of interest.
13 14
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6
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8
Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med 2005; 352:1685–1695. Libby P. Inflammation in atherosclerosis. Nature 2002; 420:868– 874. Binder CJ, Chang MK, Shaw PX, et al. Innate and acquired immunity in atherogenesis. Nat Med 2002; 8:1218–1226. Ait-Oufella H, Salomon BL, Potteaux S, et al. Natural regulatory T cells control the development of atherosclerosis in mice. Nat Med 2006; 12:178–180. Mor A, Planer D, Luboshits G, et al. Role of naturally occurring CD4þCD25þ regulatory T cells in experimental atherosclerosis. Arterioscler Thromb Vasc Biol 2007; 27:893–900. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4þCD25þ regulatory T cells. Nat Immunol 2003; 4:330–336. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science 2003; 299:1057– 1061. Paigen B, Morrow A, Holmes P, et al. Quantitative assessment of atherosclerotic lesions in mice. Atherosclerosis 1987; 68:231– 240.
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Whitman SC, Ravisankar P, Daugherty A. Interleukin-18 enhances atherosclerosis in apolipoprotein E(/) mice through release of interferon-gamma. Circ Res 2002; 90:E34–E38. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med 1999; 340:115–126. George J. Mechanisms of disease: the evolving role of regulatory T cells in atherosclerosis. Nat Clin Pract 2008; 5:531–540. Harats D, Yacov N, Gilburd B, et al. Oral tolerance with heat shock protein 65 attenuates Mycobacterium tuberculosis-induced and high-fat-dietdriven atherosclerotic lesions. J Am Coll Cardiol 2002; 40:1333–1338. Yang K, Li D, Luo M, Hu Y. Generation of HSP60-specific regulatory T cells and effect on atherosclerosis. Cell Immunol 2006; 2:90–95. Hu Z, Li D, Hu Y, Yang K. Changes of CD4þCD25þ regulatory T cells in patients with acute coronary syndrome and the effect of atorvastatin. J Huazhong Univ Sci Technolog Med Sci 2007; 27:524–527. Feig JE, Parathath S, Rong JX, et al. Reversal of hyperlipidemia with a genetic switch favorably affects the content and inflammatory state of macrophages in atherosclerotic plaques. Circulation 2011; 123:989– 998. O’Garra A, Vieira P. Regulatory T cells and mechanisms of immune system control. Nat Med 2004; 10:801–805. Yagi H, Nomura T, Nakamura K, et al. Crucial role of FOXP3 in the development and function of human CD25þCD4þ regulatory T cells. Int Immunol 2004; 16:1643–1656. Ait-Oufella H, Taleb S, Mallat Z, Tedgui A. Cytokine network and T cell immunity in atherosclerosis. Semin Immunopathol 2009; 31:23–33. Wei S, Kryczek I, Zou W. Regulatory T-cell compartmentalization and trafficking. Blood 2006; 108:426–431. Scholzen A, Mittag D, Rogerson SJ, et al. Plasmodium falciparummediated induction of human CD25Foxp3 CD4 T cells is independent of direct TCR stimulation and requires IL-2, IL-10 and TGFbeta. PLoS Pathog 2009; 5:e1000543. Liu Y, Amarnath S, Chen W. Requirement of CD28 signaling in homeostasis/survival of TGF-beta converted CD4þCD25þ Tregs from thymic CD4þCD25- single positive T cells. Transplantation 2006; 82:953–964.
Copyright © Italian Federation of Cardiology. Unauthorized reproduction of this article is prohibited.
Effect of hyperlipidemia on Foxp3 expression in apolipoprotein E-knockout mice Zhixiao Wang, Shan Mao, Zhongqun Zhan, Kefei Yu, Chaorong He and Chongquan Wang Background The transcription factor forkhead box P3 (Foxp3) plays an essential role in the development and function of regulatory T cells. Aims To examine the effect of hyperlipidemia on the expression of Foxp3 in mice. Methods Twenty-four 8-week-old male apolipoprotein E (ApoE)S/S mice on a C57BL/6 background were randomly divided into control group and high fat diet group, 12 mice per group. The blood-lipid levels, the number of Foxp3RCD4R CD25R T cells, and the size of the atherosclerotic lesions in every group were measured. The expression levels of Foxp3 in different tissues were detected. Results Compared with the control group, the level of plasma lipids was significantly higher in the high fat-fed group, but the number and function of Foxp3RCD4R CD25R T cells, the levels of Foxp3 protein expression, and Foxp3
Introduction Atherosclerosis is a multifactorial process that involves interactions among endothelial cells, macrophages, smooth muscle cells and lymphocytes.1–3 A subtype of T cells, called regulatory T cells (Tregs), play a critical role in eliminating autoimmune processes and inflammatory responses. Previous studies show Tregs play a role in negatively regulating the development and progression of atherosclerosis. A recent study showed the number of Tregs was decreased and the function of the cells diminished in atherosclerotic patients,4,5 with no exact mechanisms elucidated yet. The transcription factor forkhead box P3 (Foxp3), a key regulatory gene of Tregs, plays an essential role in the development and function of Tregs. Further studies showed an antiatherogenic role of Foxp3.6,7 However, the mechanisms for Foxp3 in the pathogenesis of atherosclerosis remain unclear, and the expression of Foxp3 in vivo has not been previously examined. In this study, we examined the effect of an important risk factor of atherosclerosis, hyperlipidemia, on the expression of Foxp3, in order to further explore the potential mechanism for hyperlipidemia causing atherosclerosis.
Materials and methods All procedures in the present study conformed to the principles outlined in the Guide for the Care and Use of 1558-2027 ß 2014 Italian Federation of Cardiology
gene transcript in selected tissues were lower in the high fat-fed group. Conclusion Hyperlipidemia inhibits the expression and function of Foxp3 in various immune organs, which may be one of the mechanisms by which hyperlipidemia aggravates the formation of atherosclerosis. J Cardiovasc Med 2014, 15:273–279 Keywords: atherosclerosis, Foxp3, Foxp3RCD4R CD25R T cells, hyperlipidemia Department of Cardiology, Taihe Hospital, Hubei University of Medicine, Shiyan, China Correspondence to Zhixiao Wang, Department of Cardiology, Taihe Hospital, Hubei University of Medicine, No. 32 South People’s Road, Shiyan, China, 442000 E-mail: [email protected] Received 12 January 2012 Revised 1 June 2013 Accepted 4 June 2013
Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). Male ApoE/ mice on the background of C57BL/ 6 were purchased from Peking University and kept under standard laboratory conditions in Tongji Medical College, Huazhong University of Science and Technology, Wuhan. The C57BL/6 mouse is one of the most commonly used strains of mice for biomedical research. It is an inbred line of mice, which have the characteristics of high precision, good comparability and uniform stress response. At 8 weeks of age, 12 male ApoE/ mice were fed with a high fat diet. And the other half of the mice were used as a control group, which were fed with a normal chow diet with the following ingredients per kg: 140 g casein, 715 g corn flour, 50 g solkafloc, 40 g corn oil, 50 g vitamin and mineral mixture, 2 g choline bitartrate, 2 g L-cystine, and 1 g DL-methionine. In the high fat diet, 180 g/kg corn flour in the basal diet was replaced with 157.5 g/kg of fat and 12.5 g/kg of cholesterol for 12 weeks. Plasma lipid analysis
Plasma from the high fat-fed ApoE/ mice was diluted with buffer containing 150 mmol/l NaCl, 1 mmol/l EDTA (pH 7.4) to adjust the OD measurement into the range of the standard curve. Total cholesterol (TC) and triacylglycerol were assayed enzymatically using commercial DOI:10.2459/JCM.0b013e3283641b9c
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274 Journal of Cardiovascular Medicine 2014, Vol 15 No 4
kits (Merck, Darmstadt, Germany). High-density lipoprotein-cholesterol (HDL-C) and low-density lipoprotein-cholesterol (LDL-C) were determined by precipitation with phosphotungstic acid/magnesium chloride or with heparin/sodium citrate, respectively, using reagents supplied by Merck. Very low-density lipoprotein (VLDL) and intermediate-density lipoproteincholesterol (IDL-C) (VLDLþIDL-C) were determined by subtracting HDL-C and LDL-C from TC. Quantification of atherosclerotic lesions in the aortic root
Atherosclerotic lesion size in the ascending aorta was determined based on eight hematoxylin–eosin-stained serial sections, 8 mm thick, starting from the region where the aortic sinus becomes the ascending aorta, as described previously.8,9 Morphometric analysis of plaques was carried out by computerized morphometry (Tonx-432, JVC, Japan). Flow cytometry
Splenocytes were costained with monoclonal antibodies fluorescein isothiocyanate-labeled anti-CD4 (7D4; Miltenyi Biotec, Cologne, Germany) and phycoerythrinlabeled anti-CD25 (GK1.5; Miltenyi Biotec). For fluorescence-activated cell sorting (FACS) analysis of intracellular Foxp3 expression, fluorescein isothiocyanate antimouse Foxp3 staining set was used (clone FJK-16s, e-Bioscience, Shanghai, China). Stained cells were analyzed on a FACS with Cell Quest software (BectonDickinson, New York, USA).
1 : 4, and 0 : 1), in a 96-well microplate at 378C with 5%CO2 air. The final volume was 200 ml per well. On the 2nd, 3rd, 4th and 5th days, 25 ml diluted 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added into each well and incubated for 4 h, followed by addition of 100 ml dimethyl sulphoxide into each well. The microplates were incubated at 378C overnight to dissolve MTT crystals before optical density values were determined by ELISA reader at a wavelength of 490 nm. Interferon-g (IFN-g), interleukin-10 (IL-10) and transforming growth factor-b (TGF-b) in the supernatants were determined by ELISA according to the manufacturer’s protocol (Jingmei Biotech Co. Ltd, Shenzhen, China). Real-time quantitative reverse transcription-PCR for the expression of Foxp3 mRNA in various tissues and blood cells
Quantitative reverse transcription-PCR (RT-PCR) was used to assess the transcriptional level of Foxp3 mRNA in spleen, thymus gland, atherosclerosis plaque and circulating blood of the mice. Western blot analysis of Foxp3 protein
Western blotting was performed with mouse serum antifoxp3 (monoclonal antibody; Abcam, Cambridge, UK) at a dilution of 1 : 100 and a secondary antibody, peroxidase-conjugated AffiniPure goat antimouse IgG (Abcam). b-Actin served as a control protein.
CD4RCD25R T-cell purification
Spleen and lymph node cell suspensions were concentrated with monoclonal antibodies fluorescein isothiocyanate-labeled anti-CD4 (7D4; Miltenyi Biotec) and phycoerythrin-labeled anti-CD25 (GK1.5; Miltenyi Biotec). CD25þ T cells were isolated from total splenocytes by a first step of negative sorting of CD4 with a cocktail of hapten-conjugated CD8, CD11b, CD16, CD19, CD36 and CD56 antibodies, and microbeads coupled with an antihapten monoclonal antibody (CD4 T-cell isolation kit; Miltenyi Biotec). This was followed by a selection of CD25-positive cells by microbead separation (CD25 microbeads; Miltenyi Biotech), providing more than 90% purity assessed by FACS analysis. Function of CD4RCD25R regulatory T cells and cytokine assay
To verify the dysfunction of Tregs, an in-vitro assay was performed. For this assay, different ratios of CD4þ CD25þ T cells (suppressor) and freshly purified CD4þCD25 T cells (responder; 1 105 cells per well), with the addition of splenocytes devoid of red blood corpuscles as antigen-presenting cells (2 104 cells per well) and ox-LDL (0.5 mg) were cocultured (1 : 1, 1 : 2,
Statistics
Statistical analyses were performed with SPSS 17.0 software (SPSS Inc., Chicago, Illinois, USA). Data are presented as mean SD. Statistical differences in the amounts of continuous variables were compared by t-test. All tests were two-sided and the differences were considered statistically significant if P was less than 0.05.
Experimental results Body weight and plasma lipid levels in apolipoprotein ES/S mice
The body weight of the 12 mice was 20 1 g in each group prior to the study (8 weeks of age). At the end of the study at 20 weeks of age, the body weight was 28 2 g for control group and 29 3 g for the high fat diet-fed group. There was no statistical difference in body weight between the two groups. High fat diet resulted in a significant increase in plasma triacylglycerol, TC, LDL and VLDLþIDL-C. In contrast, there was no statistical difference in plasma HDL-C between control mice and high fat diet-fed mice (Table 1).
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Effect of hyperlipidemia on Foxp3 expression Wang et al. 275
Table 1 Plasma lipid profiles in apolipoprotein E-knockout mice fed control or high fat diets
a
TG (mmol/l) TCb (mmol/l) HDL-Cb (mmol/l) LDL-Cb (mmol/l) VLDL þ IDL-Cb (mmol/l)
Control
High fat diet
1.0 0.13 10.6 0.12 1.0 0.1 2.2 0.5 8.0 1.6
2.8 0.19 28.2 1.2 1.1 0.3 5.8 0.2 20 0.2
Values are mean SD (n ¼ 12 for each group). TC, total cholesterol; TG, triacylglycerol; HDL, high-density lipoprotein; IDL-C, intermediate-density lipoprotein-cholesterol; LDL, low-density lipoprotein; VLDL, very low-density P < 0.01. P < 0.001. a For TG, 1 mmol/l equals 88.0 g/dl. lipoprotein. b For cholesterol (C), 1 mmol/l is 25.91 g/dl.
Morphometric analysis of plaques
Aortic roots of mice were stained by hematoxylin–eosin (Fig. 1) and the size of plaques was analyzed using computerized morphometry. The plaque area of mice in the high fat diet group (25 044.32 4920.42 mm2) was significantly larger than the control group (15 292.78 3309.10 mm2, P 0.01; and at a 0 : 1 ratio, P > 0.01, respectively; P values indicate the average of five separate experiments; Table 3). No significant differences were found in responder cell proliferation in the control mice when the assay was conducted in the presence of activated protein C (APC) from the high fat-fed mice or the control mice. When CD4þCD25þ cells from the high fat diet mice were used as responders in the described suppressive assay, the ability to inhibit the proliferation of Tregs in the high fat-fed mice was reduced, compared with the cells from the control mice (the mean value of reduction was 32.%, P < 0.01). Assessment of Foxp3 transcript levels
Foxp3 mRNA expression (Fig. 3a) was quantified using a SYBR Green I and then analyzed using real-time quantitative RT-PCR with the listed primers (Table 4). The Foxp3 mRNA expression level in various tissues was significantly lower in high fat diet group than the control group (P < 0.05) (Fig. 3b).
Fig. 1
(a)
(b)
100 µm
100 µm
Representative photomicrographs from aortic sinus sections of the control group (a) and the high fat diet group (b), stained with hematoxylin–eosin (magnification is 40).
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276 Journal of Cardiovascular Medicine 2014, Vol 15 No 4
Fig. 2
3 2
10
CD25-PE
3
10 10
10
2
CD25-PE
10
10
4
4
10
10
5
5
(a)
2
3
10
4
10
5
10
10
10
(b)
2
10
3
10
4
10
5
(c) CD4-FITC
CD4-FITC 10
22 20 P < 0.05
P < 0.05
8
16 %Foxp3/CD25I
%CD4/CD25/CD4
18 14 12 10 8
6
4
6 2
2 4
0
0 Control
High fat diet
Control
High fat diet
Comparative analysis of the number and function of regulatory T cells (Tregs) between the two groups of mice. (b) Spleen cells were obtained from 3-month-old mice and stained with antibodies to CD4 and CD25 as outlined in Methods. Data are presented from eight mice in each group. (c) Staining of splenocytes from the two groups of mice was performed for detection of CD25þFoxp3þ cells. Representative fluorescence-activated cell sorting (FACS) sheets are provided in (a).
Discussion
Quantitative assessment of the expression of Foxp3 protein
Foxp3 expression was quantified by the specific binding recorded on X-ray film. NIH Image Version 1.61 was used to quantify the intensities of the bands (Fig. 4a). Foxp3 expression (as assessed by the mean intensity of bands on the X-ray film) in the spleen, thymus, circulating blood and plaque of high fat diet group was significantly lower than that in the control group (P < 0.01) (Fig. 4b).
In the current study, we investigated the effect of hyperlipidemia on the expression and function of Foxp3 in experimental atherosclerosis. Atherosclerosis is a chronic disease of the arterial wall,1 a multifactorial process that involves interactions among endothelial cells, macrophages, smooth muscle cells and lymphocytes. Inflammation, oxidative stress and endothelial dysfunction are thought to play an active role in the initiation and progression of atherosclerosis.10 Activated Comparison of value A in every group in lymphocyte proliferation assay (x¯ W s)
Table 3
Cytokine levels in the medium secreted by regulatory T cells (pg/ml) (x¯ W s, n U 5)
Table 2
Groups/cytokines High fat diet Control
IL-10 85.5 13.8 158 29.8
TGF-b
108.8 28.9 295.6 34.8
Treg/Te Groups
IFN-g
IFN, interferon; IL, interleukin; TGF, transforming growth factor.
198.9 42.5 55.9 23.1
P < 0.01.
High fat diet Control
1:1 3.8 1.1 8.8 1.6
1:2
3.9 1.0 8.9 1.4
1:4
0:1
3.3 1.2 4.3 1.2
3.2 1.3 4.2 1.0
P < 0.05.
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Effect of hyperlipidemia on Foxp3 expression Wang et al. 277
Fig. 3
(a)
(b)
100– 250– 500– 1000– 2000–
–176 –384
FOXP3 mRNA transcnpl level
9 8 7
Control High fat diet
P < 0.05
6 5 4 3 P < 0.05
2
P < 0.05
1
P < 0.05
0 Thymus
bp
1
2
3
4
5
Spleen
Blood
Plaque
bp
(a) Agarose gel electrophoresis of Foxp3 reverse transcription-PCR (RT-PCR) product 1: DNA Ladder marker; 2–3: b-actin; 4–5: Foxp3 RT-PCR product. (b) RT-PCR showed Foxp3 transcriptional levels in the different tissues in mice.
T lymphocytes, predominantly CD4þ cells, secrete proinflammatory mediators such as IFN-g, tumor-necrosis factor-a, and IL-1 and IL-6, promoting extracellular matrix collagen production and activating macrophages.1 Tregs are an anti-inflammatory subset of CD4þ T cells that can suppress effector CD4þ and CD8þ cells and induce tolerance, thereby modulating adaptive immune responses.11 Several studies have suggested negative roles for Treg cells in atherosclerosis.12–14 However, the specific mechanisms for the negative regulation by Tregs are still unknown. The role of the transcription factor Foxp3, identified as a molecular marker for Tregs, in atherosclerosis of ApoE/ mice has not yet been elucidated. Many studies have supported hyperlipidemia as a risk factor for atherosclerosis.15 The potential connection among hyperlipidemia, Foxp3 and atherosclerosis was explored by determining whether hyperlipidemia affects the expression of Foxp3. This study may provide a novel mechanism by which hyperlipidemia causes atherosclerosis, thereby providing a basis for novel therapeutic target.
Table 4
Primers of Foxp3 and b-actin Sequence
Foxp3
b-Actin
Sense-primer 50 -CAGGAGAAAGCGGATACCAAATG-30 Antisense-primer 50 -ATCTGTGAGGACTACCGAGCC-30 Sense-primer 50 -TGGAATCCTGTGGCATCCATGAAAC-30 Antisense-primer 50 -TAAAACGCAGCTCAGTAACAGTCCG-30
Amplification size (bp) 176
384
Antigen-specific Treg cells secrete cytokines such as IL-10 and TGF-b. Naturally occurring Tregs act through cell-to-cell contact but predominantly through secretion of TGF-b.16 Despite the lack of a universally accepted membrane antigen that characterizes Tregs, the most likely molecule to characterize Tregs on CD4þ cells is CD25. We, therefore, assayed the number of CD4þ CD25þ T cells in the spleen. Indeed, we found that the high fat diet-fed mice had a significantly reduced number of splenic Tregs. Therefore, to further confirm the identity of these Tregs, comparative analysis of CD25þ Foxp3þ cells was made. We also found a reduction in the number of CD25þFoxp3þ cells in the high fat-fed group, which is consistent with the differences in CD4þCD25þ cells. The transcription factor Foxp3, a key regulatory gene of Tregs, plays an essential role in the development and function of Tregs. The lack of Tregs in Foxp3-mutant scurfy (sf) mice and in Foxp3/ mice results in autoimmune disease. An increased percentage of Tregs has been observed in Foxp3 transgenic mice, and in-vitro activation of Tregs increased Foxp3 expression, supporting the hypothesis that Foxp3 expression affects Treg function.17 Ait-Oufella et al.18 found that Tregs have an antiatherogenic role by blocking CD25þ cells using monoclonal antibodies in the ApoE/ mouse model. Our results provided complementary data that further support the potential role of Foxp3 in Tregs during the pathogenesis of atherosclerosis in the mouse. Hyperlipidemia results in a significant increase in atherosclerotic lesion formation. This was validated by separately measuring plaque size in the aortic sinus and
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278 Journal of Cardiovascular Medicine 2014, Vol 15 No 4
Fig. 4
(a) High fat diet
Control
Plaque
Thymus
β-actin
β-actin
High fat diet
Control
Spleen
High fat diet
Control
High fat diet
Control
Blood
β-actin
β-actin
(b)
Intensities of phosphor-Foxp 3 bands
100
80
Control High fat diet
P < 0.05
60 P < 0.05 40
P < 0.05
20 P < 0.05 0 Thymus
Spleen
Blood
Plaque
(a) Western blot analyzing the expression of Foxp3 protein in the spleen, thymus, plaque and circulating blood in each group. (b) Foxp3 protein level was analyzed by Western blotting and protein quantification densitometric analysis.
the entire longitudinally stripped aortas. The expression level of Foxp3 in different tissues, especially in thymus, was significantly lower in the high fat-fed group. This suggests hyperlipidemia, an important risk factor of atherosclerosis, may inhibit the function and expression of Foxp3. The expression level of Foxp3 was the highest in the thymus, shown by both RT-PCR and Western blot. Naturally occurring murine Tregs differentiate in the thymus. Under normal conditions, Tregs are found primarily in the thymus, peripheral blood, lymph nodes and spleen.19 So from our results, we speculate that the function of Foxp3 is compromised by hyperlipidemia, which affects the maturation of natural Tregs in the thymus. In spite of signaling pathways initiated by T-cell receptor (TCR), IL-2, TGF-b and CD28 have been shown to affect Foxp3 expression.20,21 Whether hyperlipidemia affects the expression
and function of Foxp3 by immuno-inflammatory reaction, or whether there are other factors involved in initiating Foxp3 expression and promoting its immune regulatory characteristics remain to be elucidated. In summary, the role of Foxp3 in autoimmune conditions has been established. Foxp3 expression plays a critical role in the regulation and development of Tregs. Hyperlipidemia can promote the progression of atherosclerosis by inhibiting the expression of Foxp3, especially in the thymus, and consequently affecting the number and function of Tregs. These data provide potential novel therapeutic targets for the management and prevention of atherosclerosis as well as other inflammatory and autoimmune conditions. However, our study is limited by the relatively small number of mice included, and by the matching system used to identify a control
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Effect of hyperlipidemia on Foxp3 expression Wang et al. 279
group which does not overcome unknown biasing factors. We did not assess extracoronary atherosclerotic burden, which theoretically could also have influenced the panel of cytokines and Tregs that we studied.
9
10 11
Acknowledgements
12
This work was supported by a grant from the Educational Foundation of Hubei province to Zhixiao Wang (No.B20102108). The authors have no conflict of interest.
13 14
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