− mice

Cytokine 49 (2010) 185–193

Contents lists available at ScienceDirect

Cytokine journal homepage: www.elsevier.com/locate/issn/10434666

The Th17/Treg functional imbalance during atherogenesis in ApoE/ mice Jiang-jiao Xie a,1, Jun Wang a,1, Ting-ting Tang a, Jian Chen a, Xing-li Gao a, Jing Yuan a, Zi-hua Zhou a, Meng-yang Liao a, Rui Yao a, Xian Yu a, Dan Wang a, Yan Cheng b, Yu-hua Liao a, Xiang Cheng a,* a b

Laboratory of Cardiovascular Immunology, Institute of Cardiology, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430022, China Institute for the Translational Medicine and Therapeutics (ITMAT), University of Pennsylvania School of Medicine, 816 BRB II/III, 421 Curie Boulevard, Philadelphia, PA 19104, USA

a r t i c l e

i n f o

Article history: Received 25 May 2009 Received in revised form 20 August 2009 Accepted 11 September 2009

Keywords: Atherosclerosis Th17 Treg Inflammation

a b s t r a c t Objective: Atherosclerosis is a chronic inflammatory disease regulated by T lymphocyte subsets. Recently, CD4+ CD25+ Foxp3+ regulatory T (Treg) cells and Th17 cells have been described as two distinct subsets and have the opposite effects on autoimmunity. Clinical observation has revealed that the Th17/Treg imbalance exists in patients with acute coronary syndrome. We investigated whether the Th17/Treg functional imbalance existed during atherogenesis in ApoE/ mice. Methods and results: Th17/Treg functions at different levels including cell frequencies, related cytokine secretion and key transcription factors were investigated comparatively between ApoE/ mice and their age-matched C57BL/6J mice. The results demonstrated that ApoE/ mice revealed significantly increased secretion of Th17 related cytokines (IL-17 and IL-6) and expression of transcription factor (RORct) levels and obviously decreased number in Treg cells, secretion of Treg related cytokines (TGF-b1) and expression of transcription factor (Foxp3) levels as compared with age-matched C57BL/6J mice. Th17 related mediators reached their maximum expression values at the early stage (8–16 weeks of age) in ApoE/ mice, and then followed by continuous depression of their expression levels. Meanwhile, the expression of Treg related mediators was much lower in ApoE/ mice than in their age-matched wild-type littermates. Conclusions: Th17/Treg functional imbalance exists during atherogenesis in ApoE/ mice, suggesting a potential role of Th17/ Treg imbalance in the formation and progression of atherosclerosis. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Atherosclerosis is a chronic inflammatory disease involving T lymphocytes, particularly CD4+ T cells [1–3]. The up-regulation of T helper cell 1 (Th1) response has been found in the local atherosclerotic lesions and circulating lymphocytes in atherosclerotic animal models and patients with acute coronary syndrome [ACS, including unstable angina (UA) and acute myocardial infarction (AMI)], suggesting that Th1/Th2 imbalance plays an important role in the development of atherosclerosis and plaque rupture [4–7]. CD4+ CD25+ regulatory T (Treg) cells and Th17 cells have recently been described as two distinct subsets which are different from Th1 and Th2 cells. Treg cells expressing the forkhead/winged helix transcription factor (Foxp3) have anti-inflammatory properties and maintain tolerance against self components by contactdependent suppression or releasing anti-inflammatory cytokines [such as interleukin (IL)-10 and transforming growth factor (TGF)-b1] [8], while Th17 cells expressing retinoic acid-related or* Corresponding author. Tel.: +86 27 85726376; fax: +86 27 85727340. E-mail address: [email protected] (X. Cheng). 1 These authors contributed equally to this work. 1043-4666/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2009.09.007

phan receptor ct (RORct) play critical roles in the development of autoimmunity and allergic reactions by producing IL-17 and, to a lesser extent, IL-6 [9]. Therefore, the balance between Th17 and Treg cells is important in the development/prevention of inflammatory and autoimmune diseases [10]. In the previous study we reported that the Th17/Treg imbalance existed in patients with ACS, suggesting a potential role of Th17/Treg imbalance in plaque destabilization [11]. Recently, de Boer et al. reported that the frequency of naturally occurring Treg cells decreased in all developmental stages of human atherosclerotic lesions [12]. As apolipoprotein E (ApoE)-deficient mice exhibit atherosclerotic lesion formation similar to what was observed in humans, they have often been used to investigate the pathogenesis of atherosclerosis [13]. In another report, Mor et al. showed Treg numbers were reduced in atherosclerotic ApoE/ mice compared with age-matched C57BL/6J littermates [14]. Little is known about whether the Th17/Treg imbalance existed during atherogenesis in ApoE/ mice. In the present study, we investigated Th17/Treg functions at different levels including cell frequencies, related cytokine secretion and key transcription factors in ApoE/ mice and their agematched C57BL/6J mice. The results demonstrated that ApoE/

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mice revealed significantly increased secretion of Th17 related cytokines (IL-17 and IL-6) and expression of transcription factor (RORct) levels and obviously decreased number in Treg cells, secretion of Treg related cytokines (TGF-b1) and expression of transcription factor (Foxp3) levels as compared with age-matched C57BL/6J mice. Results indicate that Th17/Treg functional imbalance exists during atherogenesis in ApoE/ mice, suggesting a potential role of Th17/Treg imbalance in the formation and progression of atherosclerosis. 2. Materials and methods 2.1. Animal models As a model of in vivo atherosclerosis, ApoE/ mice on a C57BL/ 6J background and their wild-type littermates, purchased from Jackson Laboratory (Bar Harbor, Maine, USA), were bred and maintained in the Animal Center of Beijing University. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). All animal studies were reviewed and approved by the Animal Study Committee of Tongji University. Eight-week-old homozygous ApoE/ male mice and their agematched C57BL/6J littermates with a body weight ranging from 20 to 22 g were used in the present study. The animals received a regular diet (mouse food, Oriental Yeast) were kept in a room with 12 h cycle light/dark, and the temperature was kept at 25 °C. Mice were fed for 0, 8, 16 and 24 weeks (n = 12 per group), euthanized, and analyzed. 2.2. Tissue preparation and atherosclerosis lesions evaluation The mice were euthanized at the age of 8, 16, 24 and 32 weeks, blood was collected by cardiac puncture, spleen, heart and aorta were removed rapidly after perfusion with PBS. Thoracic and abdominal aortas were quick-frozen in liquid nitrogen for later extraction of protein and RNA. Heart with the aortic root, was snap-frozen in OCT compound for cryostat section [15]. Atherosclerotic lesions were measured by lipid deposition stained with oil red O. Approximately 100 serial cross sections (10 lm thick) of aortic root were prepared according to the method described by Paigen et al. [16], with a slight modification. In brief, atherosclerotic lesions in aortic sinus region were examined at five locations, each separated by 200 lm, with the most proximal site starting where the first aortic valve appeared. The quantification of lesion areas were performed by a single observer blinded to the experimental protocol. All images were captured and analyzed by computer image analysis software. The average value for the five locations for each animal was used for analysis [17].

RPMI-1640 (Gibco, USA), the cell viability detected by trypan blue staining was >95%. For analysis of Th17, the cell suspension was transferred to each well of 24-well plates. Splenocytes were stimulated for 5 h with 50 ng/ml PMA, 1 lM ionomycin and 500 ng/ml monesin (all from Alexis Biochemicals, San Diego, CA). The incubator was set at 37 °C under a 5% CO2 environment. For analysis for Treg cells, splenocytes were aliquoted into tubes for further staining. In addition, naïve CD4+ T cells were separated from spleens of the C57BL/6J mice and Th17 cells were induced in the presence of TGF-b 1, which, together with IL-6 and other pro-inflammatory cytokines according to the protocol described previously [19]. These Th17 cells induced by TGF-b 1 and IL-6 in vitro were used as the positive control for the flow cytometric analysis of Th17 cells. 2.4.2. Flow cytometric analysis of Th17 and Treg cells Cells were aliquoted into tubes and washed once in PBS, and then resuspended to a density of 106 cells/ml. For Th17 analysis, cells were incubated with phycoerythrin-Cy5 (PE/Cy5) antimouse-CD4 at 4 °C for 20 min for cell surface staining. For Treg cells analysis, cells were incubated with PE/Cy5 anti-mouse-CD4 and Fluorescein isothiocyanate (FITC) anti-mouse-CD25. After surface staining, fixation and permeabilization, cells were stained with FITC anti-mouse-IL-17A for Th17 detection or phycoerythrin (PE) anti-mouse-Foxp3 for Treg cells detection according to the manufacturer’s instructions (all of the Abs were from eBioscience, San Diego, CA). Isotype controls were given to enable correct compensation and confirm antibody specificity. Stained cells were analyzed by flow cytometric analysis using a FACScan cytometer equipped with CellQuest software (BD Bioscience Pharminggen). 2.5. Real-time quantitative polymerase chain reaction Total RNA was isolated from spleen and aorta (from beginning of the aortic arch to the iliac bifurcation) using the Trizol reagent (Invitrogen, Carlsbad, Calif) according to the manufacturer’s instructions. After reverse transcription reaction, used by the first strand cDNA synthesis kit (Toyobo Co., Ltd., Life Science Department, Osaka Japan), real-time quantitative polymerase chain reaction (ABI Prism 7000 Sequence Detection System; Applied Biosystems, Foster City, Calif) was performed to determine the mRNA levels of Th17 and Treg cells related inflammatory mediators with the SYBR Green realtime PCR master mix kit (Toyobo Co., Ltd., Life Science Department, Osaka Japan). Each sample was analyzed in triplicate, normalized to GAPDH. The fold changes in mRNA expression of the inflammatory mediators at each time point relative to the 8-week-old C57BL/6J littermates (n = 5 mice per group) were analyzed by the comparative computed tomography method. PCR primers are as follows:

2.3. Plasma lipid and lipoprotein profile

RORct:

Total plasma cholesterol (TC), triglyceride (TG), and high-density lipoprotein cholesterol (HDL-C) levels were detected by autoanalyzer (Hitachi 917).

IL-17 receptor A:

2.4. Cell preparation and flow cytometry 2.4.1. Cell preparation Splenocytes were separated according to the protocol described previously [18]. Briefly, the fresh spleens removed from mice were gently squeezed with sterile needles in the cold PBS and passed through a stainless steel mesh screen, respectively, and the single-cell suspension was prepared. The splenic lymphocytes were isolated by Ficoll-Hypaque and resuspended in complete media:

IL-17A: IL-6: Foxp3: TGF-b1: IL-10: GAPDH:

forward, 50 -TGCAAGACTCATCGACAAGG-30 reverse, 50 -AGGGGATTCAACATCAGTGC-30 forward, 50 -CCGACCTCTTCAACATCACCTC-30 reverse, 50 -CCACTCCTGGAACCTAAGCACA-30 forward, 50 -TCTCTGATGCTGTTGCTGCT-30 reverse, 50 -CGTGGAACGGTTGAGGTAGT-30 forward, 50 -GAGGATACCACTCCCAACAGACC-30 reverse, 50 -AAGTGCATCATCGTTGTTCATACA-30 forward, 50 -TGGAACCACGGGCACTATCACA-30 reverse, 50 -GAGGCTGCGTATGATCAGTTATGC-30 forward, 50 -TGACGTCACTGGAGTTGTACGG-30 reverse, 50 -GGTTCATGTCATGGATGGTGC-30 forward, 50 -GACAACATACTGCTAACCGACTC-30 reverse, 50 -TCACTCTTCACCTGCTCCACTG-30 forward, 50 -AGCAATGCCTCCTGCACCACCAAC-30 reverse, 50 -CCGGAGGGGCCATCCACAGTCT-30

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size increased significantly compared with those in the former time period (p < 0.05).

2.6. Western blot analysis Thoracic and abdominal aortas were used. Total proteins were prepared from pooled arteries (six arteries for each group). The concentrations of proteins were determined by BCA protein assay (Pierce). Each sample containing 30–50 lg protein was separated on a 7.5%, 9% and 15% SDS–PAGE and electrotransferred onto nitrocellulose membrane. The membrane was sequentially blocked in TBS containing 5% skim milk and then incubated with primary antibodies [anti-IL-17A, anti-IL-17RA, anti-TGF-b1 (1:200, Santa Cruz Biotechnology), anti-Foxp3, anti-RORct (1:500, Biolegend, San Diego), 4 °C, overnight], washed, and further incubated with horseradish peroxidase-conjugated secondary antibody (1:10,000, 37 °C, 1 h). After several washes, immunopositive bands were detected by electrochemiluminescence (Pierce), and the bands were semi-quantitatively analyzed using densitometric methods. 2.7. Cytokine detection by ELISA The concentrations of IL-17, IL-6, TGF-b1 and IL-10 in serum were measured by enzyme-linked immunosorbent assay (ELISA) following the manufacture’s instructions (ELISA kits for IL-17, IL6 and IL-10, Bender Medsysterms, Vienna, Austria, Europe; ELISA kits for TGF-b1, R&D system, Minneapolis, MN, USA). The minimal detectable concentrations were 1.6 pg/ml for IL-17, 7.92 pg/ml for IL-6, 5 pg/ml for IL-10 and 4.61 pg/ml for TGF-b1. Intra-assay and inter-assay coefficients of variation for all ELISA were <5% and <10%, respectively. All assays were performed in triplicate. 2.8. Statistical analyses Values are expressed as means ± SD. Differences between the groups were considered significant at p < 0.05 using the 2-tailed Student t test or the Mann–Whitney U Wilcoxon sum test. 3. Results 3.1. Basic physiological parameters We have shown the body weight and plasma cholesterol gain of ApoE/ mice and the wild-type littermates C57BL/6J mice over a 32-week period (Table 1). 3.2. Atherosclerosis lesion area As shown in Fig. 1, ApoE/ mice had prominent atherosclerotic lesions with progressive development whereas C57BL/6J mice nearly did not have visible atherosclerotic lesions stained with oil red O. We fed eight-week-old ApoE/ mice with a regular diet for 4 time periods: 0, 8, 16 and 24 weeks. Atherosclerotic lesion

3.3. Th17/Treg functional imbalance in spleen 3.3.1. Expression of Th17/Treg transcription factors in spleen As shown in Fig. 2A, the levels of RORct expression were significantly higher in ApoE/ mice than those in their wild-type littermates at all time points (p < 0.05), moreover, it achieved the maximal value at early age (8- to 16-week-old), which were at the onset of the lesion formation. The levels of Foxp3 expression were markedly lower in ApoE/ mice at all time points (p < 0.05), and reached the minimal value at later age (32-weekold, Fig. 2B). Consistent with RORct, the IL-17A mRNA expression in the spleen of ApoE/ mice were markedly higher compared with age-matched C57BL/6J mice and achieved the maximal value at the early age (8- to 16-week-old, Fig. 2C). The level of TGF-b1 expression in ApoE/ mice obviously decreased compared with age-matched C57BL/6J mice, although there was no statistical significance between the two groups at the early age (8-week-old, p > 0.05, Fig. 2D). 3.3.2. Th17 and Treg frequencies in spleen Although we had detected the high expression of RORct and IL17A mRNA in spleen of ApoE/ mice, we had not obtained effective evidence to conform it through flow cytometric analysis. Neither ApoE/ mice nor C57BL/6J mice displayed visible Th17 frequencies in Fig. 2E. The Th17 cells induced by TGF-b1 and IL-6 in vitro were used as the positive control for the flow cytometric analysis of Th17 cells, and the proportion of these Th17 cells (ratio of CD4+ IL-17+ in total CD4+ T cells) was 23.8%. It indicated that Th17 cells without induction by TGF-b1 and IL-6 in spleen were not easy to be detected by FACS at physiological stage. By FACS analysis of spleen cells from 6 mice per group at each time point, we found the number of Treg cells (ratio of CD4+ CD25+ Foxp3+ in total CD4+ T cells) was significantly reduced in ApoE/ mice compared with age-matched C57BL/6J mice, and reached the minimal value at later age (32-week-old, Fig. 2F). The proportion of CD4+ CD25+ T cells (ratio of CD4+ CD25+ in total CD4+ T cells) maintained a similar level in ApoE/ mice and C57BL/6J mice over time. The percentage of Treg cells (CD4+ CD25+ Foxp3+) in CD4+ CD25+ T cells significantly decreased in ApoE/ mice while it remained a stable level in C57BL/6J mice over time (Table 2). 3.4. Th17/Treg functional imbalance in serum IL-17 concentration in ApoE/ mice was significantly higher compared with the age-matched C57BL/6J mice and achieved the maximal value at early age (8- to 16-week-old, Fig. 3A). The change

Table 1 Body weight and plasma lipid levels. Age (weeks)

Mice

Weight (g)

TC (mmol/L)

TG (mmol/L)

HDL-C(mmol/L)

8

C57BL/6J ApoE/ C57BL/6J ApoE/ C57BL/6J ApoE/ C57BL/6J ApoE/

20.26 ± 0.52 21.52 ± 0.74 22.28 ± 0.75 24.13 ± 1.51 25.73 ± 2.07 26.05 ± 4.9 26.83 ± 2.06 27.89 ± 3.2

2.87 ± 1.18 11.13 ± 4.3* 2.53 ± 0.63 11.34 ± 2.09* 1.87 ± 0.29 14.4 ± 3.82* 1.93 ± 0.35 16.87 ± 2.77*

0.91 ± 0.32 1.55 ± 0.85 1.21 ± 0.28 1.71 ± 0.53 0.96 ± 0.17 1.74 ± 1.38 1.11 ± 0.29 1.81 ± 0.96

1.51 ± 0.09 0.94 ± 0.1* 1.53 ± 0.43 0.72 ± 0.17* 1.39 ± 0.22 0.87 ± 0.18* 1.3 ± 0.33 0.59 ± 0.14*

16 24 32

Values are expressed as means ± SD. TC, total cholesterol; TG, triglycerides; HDL-C, high-density lipoprotein cholesterol. * p < 0.05 vs. the age-matched C57BL/6J littermates.

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A

tical significance exited among the different time point in ApoE/ mice (Fig. 3C, 3D). Moreover, the increased amplitude between ApoE/ mice and their age-matched C57BL/6J mice were lower in IL-10 [ApoE//C57BL/6J (fold): 8–32w:1.8; 2.4; 1.9; 1.7] than that in IL-17 [ApoE//C57BL/6J (fold): 8–32w: 6.6; 6.2; 5.0; 2.9] at each time point.

ApoE-/-

C57BL/6J

Age (weeks)

8w

3.5. Th17/Treg functional imbalance in plaques

a

e

b

f

c

g

By real-time quantitative polymerase chain reaction (PCR) and Western blot analysis, we detected the mRNA and protein levels of Th17/Treg related cytokines and their transcription factors in the atherosclerotic plaques (Figs. 4 and 5). The expressions of Th17 related cytokine (IL-17) in ApoE/ mice significantly increased compared with the age-matched C57BL/6J mice and the level of IL-17 expression achieved the maximal value at the early age (8- to 16-week-old). Moreover, there was a similar tendency in the change of RORct and IL-17 receptor (IL-17R) expression. We also detected Treg cells related cytokines (TGF-b1) and transcription factor (Foxp3) expressions within the plaques in mRNA and protein levels. The level of TGF-b1 expression in ApoE/ mice decreased obviously compared with the age-matched C57BL/6J mice, there was no statistical significance between the two groups at the early age (8-week-old, p > 0.05). In contrast, Foxp3 expression in ApoE/ mice was higher compared with their age-matched C57BL/6J mice, but it achieved similar values in the two groups at the later age (32-week-old, p > 0.05). Meanwhile the increased amplitude of Foxp3 between ApoE/ mice and their wild-type littermates was lower than that of RORct at each time point. [mRNA: Foxp3: ApoE//C57BL/6J (fold): 8–32w: 2.1; 1.6; 2.0; 1.1; RORct: ApoE//C57BL/6J (fold): 8–32w: 6.2; 6.8; 3.7; 2.9; protein: Foxp3: ApoE//C57BL/6J (fold): 8–32w:1.8; 1.7; 1.7; 1.1; RORct: ApoE// C57BL/6J (fold): 8–32w: 4.1; 4.9; 3.9; 3.1].

16 w

24 w

4. Discussion

32 w

B

atherosclerotic lesion area (% of total area)

d

h 35 C57BL/6J ApoE-/-

30

*#

25 *

20 *

15 10 5 0

8w

16w 24w Age (weeks)

32w

Fig. 1. Atherosclerotic lesion area. (A) Atherosclerotic lesion in ApoE/ mice and age-matched C57BL/6J mice, shown as cryostat section of aortic root stained with oil red O (Magnification 40). a ? d, C57BL/6J mice; e ? h, ApoE/ mice. (B) The percentage of atherosclerotic lesion area/lumina in aortic root. *p < 0.05 vs. the agematched C57BL/6J mice. #p < 0.05 between 8 weeks and 32 weeks. : ApoE/ mice; s: C57BL/6J mice.

of TGF-b1 showed a striking contrast: its concentration in ApoE/ mice was obviously lower than the age-matched C57BL/6J mice and achieved the minimal value at the later age (32-week-old, Fig. 3B). Both IL-6 and IL-10 concentration in ApoE/ mice were higher than their age-matched C57BL/6J mice over time, no statis-

The Th17/Treg balance controls inflammation and may be important in atherosclerosis. To investigate whether the Th17/Treg functional imbalance existed during atherogenesis in mice, we detected Th17/Treg functions comparatively between ApoE/ mice and age-matched C57BL/6J littermates. The results demonstrated that ApoE/ mice revealed significant increase in Th17 related cytokines (IL-17 and IL-6) and transcription factor (RORct) levels and obvious decrease in Treg number, Treg related cytokines (TGF-b1) and transcription factor (Foxp3) levels as compared with age-matched C57BL/6J mice. Our results indicated that Th17/Treg functional imbalance existed during atherogenesis in ApoE/ mice, suggesting a potential role of Th17/Treg imbalance in the formation and progression of atherosclerosis. Atherosclerosis is a chronic inflammatory disease involving T lymphocytes [1]. Numerous studies have demonstrated that Th1/ Th2 imbalance exits in atherosclerotic animal models and patients with ACS [4]. Therefore the Th1 response, which has a proatherogenic potential, plays an important role in the development of atherosclerosis [18]. The Th17 cell expressing RORct is a distinct subset different from Th1 and Th2 cells which plays a critical role in the development of autoimmunity and allergic reactions by producing IL-17 and, to a lesser extent, IL-6 [9]. Recently, as the development of immunology, the concept that autoimmune diseases were exclusively mediated by Th1 cells has been challenged, and the idea that Th17 cells contribute to the induction and development of the autoimmune reaction has emerged [9]. The most classic model is experimental autoimmune encephalomyelitis (EAE). Mice which are deficient for the Th1 cytokine IFN-c developed en-

189

A

ROR γ t mRNA expression in spleen (fold change)

J.-j. Xie et al. / Cytokine 49 (2010) 185–193

16

E

E (1)

*

14 12

*

10 8 *

6

*#

4 2 0

1.2 1 0.8

* *

0.6

* *

0.4 0.2

10 9 8 7 6 5 4 3 2 1 0

8w

16w 24w Age (weeks)

CD4+IL17+/CD4+ T cells (%)

E (2)

1.4

C57BL/6J ApoE-/-

0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 8w

32w

16w

24w

32w

Age (weeks)

F

* * *

a1

7.1%

b1

7.8%

c1

5.5%

a2

86.9%

b2

89.3%

c2

69.7%

d2

34.2%

*

8w

16w 24w Age (weeks)

32w

FOXP3 PE

IL-17A mRNA expression in spleen (fold change) TGF- β 1 mRNA expression in spleen (fold change)

D

32w

1.6

0

C

16w 24w Age (weeks)

1.8

CD25 FITC

B

Foxp3 mRNA expression in spleen (fold change)

8w

1.6 1.4 1.2 1 0.8

* *

0.6

*

0.4 0.2 0

8w

16w 24w Age (weeks)

32w

d1

6.2%

CD4 PE-Cy5 Fig. 2. Expression of transcription factors, cytokines and Th17/Treg frequencies in spleen. (A and B) The levels of RORct (transcription factor for Th17 cells) mRNA and Foxp3 (transcription factor for Treg cells) mRNA expression in ApoE/ mice and their wild-type littermates at all time points. (C and D) The levels of IL-17A (the important cytokine of Th17 cells) mRNA and TGF-b1 (the important cytokine of Treg cells) expression in spleen. (E) Th17 frequencies detected by flow cytometric analysis. (E (1)) Representative FACS pictures from a single mice in each group. (E (2)) Collective analyses of result at all time points. F. the percentage of CD4+ CD25+ T subsets (a1 ? d1) and representative Foxp3 expression in CD4+ CD25+ T subsets (a2 ? d2) from a single mice in each group. The percentage of positive cells was shown in each panel. a1 and a2: C57BL/6J 8w; b1 and b2: C57BL/6J 32w; c1 and c2: ApoE/ 8w; d1 and d2: ApoE/ 32w. *p < 0.05 vs. the age-matched C57BL/6J mice. #p < 0.05 between 8 weeks and 32 weeks. : ApoE/ mice; s: C57BL/6J mice.

hanced EAE [20], deficient for IL-12 p35 (specific for IL-12) did not alter the progression of EAE, but deficient for either p40

(shared by IL-12 and IL-23) or p19 (specific for IL-23, which is important in expansion and IL-17 production of Th17 cells

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Table 2 Treg frequencies in spleen. Age (weeks)

Mice

CD4+ CD25+/CD4+ T cells (%)

CD4+ CD25+ Foxp3+/CD4+ CD25+ T cells (%)

CD4+ CD25+ Foxp3+/CD4+ T cells (%)

8

C57BL/6J ApoE/ C57BL/6J ApoE/ C57BL/6J ApoE/ C57BL/6J ApoE/

6.41 ± 0.78 5.97 ± 1.04 6.56 ± 0.76 5.74 ± 0.56 7.01 ± 0.97 5.25 ± 1.53 7.36 ± 0.92 6.38 ± 1.82

87.5 ± 3.73 76.3 ± 5.64 84.2 ± 4.83 70.2 ± 2.32 84.7 ± 6.13 48.8 ± 6.26 87.2 ± 6.23 29.5 ± 3.44#

5.77 ± 0.65 4.55 ± 0.93* 5.72 ± 0.81 4.12 ± 0.49* 6.03 ± 1.16 2.88 ± 0.77* 6.41 ± 1.26 1.89 ± 0.71*,#

16 24 32

Values are expressed as means ± SD. * p < 0.05 vs. the age-matched C57BL/6J littermates. # p < 0.05 vs. 8-week-old ApoE/ mice.

although it is dispensable in commitment of Th17) resulted in a decrease in the number of Th17 cells and a protective function from EAE [21,22], the transfer of myelin-reactive IL-17-producing T cells expanded with IL-23 in vitro induced severe EAE [23], moreover, IL-17 directly induced tissue damage in various autoimmune diseases. In a word, Th17 cells are not only important in autoimmunity on the whole but also participate in the local inflammatory process in tissues. It has been demonstrated that patients with ACS exhibited a significant increase in peripheral Th17 number, related cytokines (IL-17, IL-6 and IL-23) and RORct levels [11,24]. In the present study we detected whether Th17 functional change exited in the atherosclerotic animal model according to these data. We presented the following conclusions: ApoE/ mice revealed significant increase in Th17 related cytokines (IL-17 and IL-6) and transcription factor (RORct) level compared with age-matched C57BL/6J mice; the Th17 related mediators, which elevated slightly over time but did not have noticeably statistical difference between the groups in C57BL/6J mice, increased markedly and reached their maximum expressive values at early stage (8–16 weeks of age) in ApoE/ mice, then followed by continuous depression of their expression levels. On the whole, although we found the high expression of the RORct and IL-17A mRNA in the spleen of ApoE/ mice, we had not obtained effective evidence to conform by flow cytometric analysis, which may be explained by the low sensitivity of flow cytometry. Elevated Th17 related mediators have a considerable link with atherosclerosis. IL-17 is involved in the attraction and activation of macrophages in atherosclerotic lesions through up-regulation of IL-17 receptor in local vascular walls [25], which may contribute to lesion progression by releasing of proteolytic factors such as matrix metalloproteases [26]. Increased vascular IL-17 and TNF-a level may act synergistically to create a pro-inflammatory microenvironment promoting the development of atherosclerotic vascular disease [27]. Interestingly, we have noticed the phenomenon that Th17 related mediators increased markedly and reached their maximum expression values at early stage (8–16 weeks of age) in ApoE/ mice, which may contribute to the initiation of the atherosclerosis. Th17/Treg imbalance plays a critical role in regulating inflammatory and autoimmune diseases, Treg cells help to dampen inflammatory processed and Th17 cells promote various aspects of immune activation [10]. The differentiation of naïve Th cells into Treg cells or Th17 cells depends on a unique combination of stimulating factors, e.g. TGF-b, with or with not IL-6, and subsequent activation of diverse transcription factors, e.g. Foxp3 or RORct. TGF-b is a critical regulator of the signaling pathways that initiate and maintain Foxp3 expression, which is a master control protein for the Treg induction, thus facilitate Treg generation from de novo.

Meanwhile, TGF-b, which is also essential to allow IL-17 differentiation, acting together with IL-6, another obligatory component in the differentiation of Th17 cells, can also induces the de novo generate pathogenic Th17 cells from naïve precursors [28]. Switch of a Th17/Treg profile plays an important role in controlling immune response and autoimmune diseases. Treg cells efficiently control the autoimmune diseases and their numbers decrease in several autoimmune diseases [29– 32]. It has been reported that Treg frequencies decreased and their functional suppressive properties are compromised in ApoE/ mice compared with the C57BL/6J littermates [14] and in patients with ACS [12,33–34]. Adoptive transferring of Treg cells can attenuate atherosclerosis in animal models [14,35]. However, the changed tendency of Treg cells on atherogenesis in ApoE/ mice over time was still unclear. Here, our results demonstrated that ApoE/ mice revealed obvious decrease in Treg number, Treg related cytokines (TGF-b1) and transcription factor (Foxp3) levels as compared with age-matched C57BL/6J mice over time. It was interesting that although Foxp3 expression in spleen was markedly lower in ApoE/ mice than those in their wild-type littermates, the Foxp3 expression reversed in plaques. We could consider at least two reasonable aspects for this phenomenon: first, its increased amplitude between ApoE/ mice and their age-matched C57BL/6J mice was still lower than that in RORct at each time point, which suggested that the Th17/Treg functional imbalance still existed within the atherosclerotic plaques, especially the Th17 predominated ones; second, the reason for higher Foxp3 expression in ApoE/ mice may be explained by the larger numbers of T cells including Treg cells infiltrated in the atherosclerotic plaques on major arteries such as the aorta when fed with normal diet [36,37], whereas C57BL/6J mice did not have visible atherosclerotic lesions, which meant the Treg cells and foxp3 expression were rare in the normal intima, as de Boer et al. said that T cells were virtually absent in normal vessels without atherosclerotic lesions [12]. The similar phenomena included the increased IL-10 concentrations in serum of ApoE/ mice, and the increased amplitude was still lower than that of IL-17 at each time point. Moreover, in this study, the frequencies of Treg cells in spleen of ApoE/ mice kept on descending over time and reached the minimal value at the late age. This ratio contained two compositions: the proportion of CD4+ CD25+ T cells in total CD4+ T cells, which maintained a similar level in ApoE/ mice over time, and the proportion of CD4+ CD25+ Foxp3+ T cells in CD4+ CD25+ T cells, which decreased significantly in ApoE/ mice over time. CD4+ CD25+ T cells included two subsets: conventional effector T cells (CD4+ CD25+ foxp3-) and Treg cells (CD4+ CD25+ foxp3+) [38]. It suggested that although the percentage of conventional effector T cells (CD4+ CD25+ foxp3) increased during atherogenesis in ApoE/ mice, the proportion of Treg cells (CD4+

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CD25+ foxp3+) decreased on the contrary, which accelerated the atherosclerotic inflammation. The striking decrease of Treg cells and increase of effector T cells in ApoE/ mice at the late stage may contribute to the development of atherosclerosis. In summary, our investigation demonstrated that Th17/Treg functional imbalance exists during atherogenesis in ApoE/ mice, suggesting a potential role of Th17/Treg imbalance in the formation and progression of atherosclerosis. We should prove our con-

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clusion by a larger sample size and make penetrating studies on the mechanism of Th17/Treg functional imbalance in plaque progression and destablilization. Th17/Treg functional imbalance may provide an exciting new target for the treatment of atherosclerosis.

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Disclosures None.

Acknowledgments This work described in this article was supported by National Natural Science Foundation of China (No. 30600234, 30871067) and National Basic Research Program of China (973 Program) 2007CB512000; 2007CB512005.

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