- Email: [email protected]
Treg balance in patients with acute coronary syndrome
Biochemical and Biophysical Research Communications 394 (2010) 836–842
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
The role of oxidized low-density lipoprotein in breaking peripheral Th17/Treg balance in patients with acute coronary syndrome Qing Li a,b,c,d,1, Yi Wang a,b,c,1, Ke Chen e, Qing Zhou a,b,c, Wei Wei a,c, Yiping Wang f, Yuan Wang a,b,c,* a
Institute of Clinical Pharmacology, Anhui Medical University, Hefei, Anhui 230032, PR China Laboratory of Molecular Biology and Department of Biochemistry, Anhui Medical University, Hefei, Anhui 230032, PR China c Key Laboratory of Gene Resource Utilization for Severe Disease and Anti-inflammatory and Immunopharmacology (Anhui Medical University), Ministry of Education and Anhui Province, Hefei, Anhui 230032, PR China d The Central Laboratory of Medical Research Center, Anhui Provincial Hospital, Hefei, Anhui 230001, PR China e Department of Cardiovascular Disease, Anhui Provincial Hospital, Hefei, Anhui 230001, PR China f The Centre for Transplantation and Renal Research, Western Clinical School, University of Sydney, Westmead, NSW Australia b
a r t i c l e
i n f o
Article history: Received 6 March 2010 Available online 17 March 2010 Keywords: Oxidized low-density lipoprotein Acute coronary syndrome T-helper 17 Regulatory T cells
a b s t r a c t Oxidized low-density lipoprotein (ox-LDL) is an instrumental factor in atherogenesis, however, the effects of ox-LDL on the balance of Th17/Treg in acute coronary syndrome [ACS, including unstable angina (UA) and acute myocardial infarction (AMI)] is still unclear. CD4+CD25+ regulatory T (Treg) cells and Th17 cells, subsets of T-helper cells, play important roles in peripheral immunity and their imbalance leads to the development of tissue inflammation and autoimmune diseases. However, few studies have explored the effect of Th17/Treg balance in plaque destabilization and the onset of ACS. To explore the shift of Th17/Treg balance in ACS patients and the effect of ox-LDL on the balance, we examined the frequencies of Th17 and Treg cells, key transcription factors and relevant cytokines in patients with AMI, UA, stable angina (SA) and controls. We analysed the correlations of serum ox-LDL to Th17/Treg frequency, and the effects of ox-LDL on Th17/Treg cells in vitro. Our study demonstrated that ACS patients have shown a significant increase of Th17 frequency, RORct expression and serum Interleukin 17 (IL-17), and a obvious decline of Treg frequency, Foxp3 expression, suppressive function, and serum IL-10. Serum ox-LDL positively correlated with the frequency of Th17 cells and negatively correlated with the frequency of Treg cells. In vitro incubation of peripheral blood mononuclear cells from controls with ox-LDL resulted in a significant reduction of Treg cells and a significant elevation of Th17 cells in a dose- and time-dependant manner. Treg and Th17 cells from ACS patients were significantly more susceptible to ox-LDL-mediated alterations. Th17/Treg numerical and functional imbalance exists in ACS patients, and ox-LDL has a direct effect on Th17/Treg imbalance which may contribute to the occurrence of ACS. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Atherosclerosis (AS) is a chronic inflammatory disease in which immune mechanisms appear to make great effect, and atherogenesis involves various immune cells, particularly T lymphocytes, such as CD4+ T-helper cells [1,2]. Acute coronary syndrome (ACS) occurs as a consequence of coronary plaque erosion or rupture and T lymphocytes play an important role in these coronary events [3]. CD4+CD25+ regulatory T (Treg) cells and T-helper 17 (Th17) cells are 2 original subsets distinguished from Th1 and Th2 cells. Treg cells expressing the forkhead/winged helix transcription factor (Foxp3), a fraction of inflammatory-regulated negative cells, have important effects * Corresponding author at: Laboratory of Molecular Biology, Anhui Medical University, Hefei 230032, Anhui Province, PR China. E-mail address: [email protected] (Y. Wang). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.03.090
on the maintenance of immune tolerance and immune homeostasis by contact-dependent suppression or by the release of anti-inflammatory cytokines, such as interleukin (IL)-10 and transforming growth factor (TGF)-b [4]. Th17 cells expressing retinoic acid-related orphan receptor ct (RORct) exert an important effect on the pathogenesis of many experimental autoimmune diseases and human inflammatory conditions by producing IL-17, tumor necrosis factor (TNF)-a and IL-6 [5]. The Th17/Treg imbalance could be critical in the development of tissue inflammation and autoimmune diseases [6]. However, Th17/Treg balance in AS, especially ACS patients has not been fully investigated. Oxidized low-density lipoprotein (ox-LDL) is a key factor in the initiation and progression of atherosclerosis and contributes to endothelial dysfunction and plaque destabilization through multiple mechanisms [7,8]. It is unclear whether ox-LDL also affects AS and ACS patients by disrupting the balance of the peripheral Tregs and Th17 cells.
Q. Li et al. / Biochemical and Biophysical Research Communications 394 (2010) 836–842
837
In this study, we have examined the balance of Th17/Treg cells in ACS patients, and the effects of ox-LDL on these cells.
as a percentage of CD4+ T cells by sequential gating on lymphocytes and CD4+ T cells.
2. Materials and methods
2.4. Flow sorting
2.1. Patients
CD4+CD25+CD127low and CD4+CD25 cells were sorted using the gates on a Beckman Counter flow cytometry cell-sorter (ALTRA HyPerSort System). We obtained a consistent purity of >90% for both CD4+CD25+CD127low and CD4+CD25 cell fractions.
All patients gave written informed consent prior enrolment into the study. We examined patients at Anhui provincial hospital who underwent diagnostic catheterisation between July 2007 and June 2009. Patients were classified into 4 groups: acute myocardial infarction (AMI) patients, unstable angina (UA) patients, stable angina (SA) patients and control subjects with normal coronary arteries (NCA), Patients with ACS and SA had a similar extent of coronary atherosclerosis. There were no evident differences between the 4 groups with regard to age. No patient was treated with anti-inflammatory drugs and/or immunosuppressive agents. None had thromboembolism, disseminated intravascular coagulation, advanced liver disease, renal failure, malignant disease, other inflammatory disease, chronic-immunemediated disorders, valvular heart disease, atrial fibrillation or pacemaker. 2.2. Blood samples We collected 5–10 ml of peripheral blood (PB) from all the patients, in a fasting state, on the morning following admission. The time interval between symptom onset and blood sampling was less than 24 h in all cases. PB mononuclear cells (PBMCs) were prepared by Ficoll density gradient for analysis of flow cytometry (FCM) and reverse transcription-polymerase chain reaction (RT-PCR). Serum was obtained after centrifugation and stored at 80 °C until further use. 2.3. Cell separation and flow cytometry 2.3.1. Cell preparation For the analysis of Th17 cells, PBMCs were suspended at a density of 2.0 106 cells/ml in complete culture medium (Gibco BRL, USA). The cells were stimulated with phorbol myristate acetate (PMA, 25 ng/ml) plus ionomycin (1 lg/ml) for 4 h, in the presence of monensin (1.7 lg/ml, all from Alexis Biochemicals, San Diego, CA, USA). Cells were incubated at 37 °C under a 5% CO2 environment. For the analysis of Treg, 100 ll of PBMCs (106) was added to tubes for further staining. 2.3.2. Detection of Treg and Th17 cells For the analysis of Treg cells, cell surface staining was performed by the use of fluorescein isothiocyanate(FITC)-conjugated antiCD4(13B8.2 clone; Beckman Coulter-Immunotech, Marseille, France), phycoerythrin(PE)-conjugated anti-CD25(B1.49.9 clone, Beckman Coulter-Immunotech), PE-cy7-conjugated anti-CD127 (ebioRDR5 clone, eBioscience, San Diego, CA, USA) and appropriate isotype controls for 20 min at room temperature in the dark, followed by washing in phosphate buffered solution (PBS). Cells were fixed and permeabilized with the Fix/Perm reagent (Beckman Coulter-Immunotech), incubated with PE-cy5-conjugated antiFoxp3(PCH101 clone, eBioscience) and its isotype control antibody, washed with PBS and analysed by FCM. For Th17 analysis, the cells were incubated with FITC-conjugated anti-CD4 at 4 °C for 15 min and then stained with PE-conjugated anti-IL-17A (ebio64DEC17 clone, eBioscience) after fixation and permeabilization according to the manufacturer’s instructions. Stained cells were assessed by FCM using the COULTER EPICS ALTRA HyPerSort flow cytometer with EXPO 32 MULTICOMP Software (Beckman Coulter, Miami, FL, USA). The frequency of Treg (CD4+CD25high, CD4+CD25+CD127low and CD4+CD25+Foxp3+) and Th17(CD4+IL17+) cells was expressed
2.5. Functional assay of Treg cells Freshly purified CD4+CD25+CD127low T cells from NCA controls and from patients with AMI, UA and SA (n = 5 in each group) were assayed for their suppressive activity in the allogeneic mixed lymphocyte response (MLR) assay. Suppression was expressed as a percentage of the positive control. 2.6. RORct and Foxp3 expression levels Total RNA was extracted with TRIzol (Invitrogen, USA) according to the manufacturer’s instructions. cDNA was synthesized by using random hexamer primers and RNase H-reverse transcriptase (Invitrogen). TaqMan primers and probes for human Foxp3 and RORct were purchased from Biosune Biotechnology. The following primer pairs were used: Foxp3: F: 50 -CACGCATGTTTGCCTTCTTCAGA-30 , R: 50 -GTAGGGTTGGAACACCTG CTGGG-30 (181 bp), and RORct: F: 50 GCAATGGAAGTGGTGCTGGTT-30 , R: 50 -AGGATGCTTTGGCGATGAGT C-30 (320 bp). The quality of cDNA subjected to the RT-PCR was controlled by amplification of transcripts of b-actin. b-Actin was analysed using the following primers: F: 50 -ATCTGGCACCACACCT TC-30 , R: 50 -AGCCAGGTCCAGACGCA-30 (296 bp). PCR products were subjected to electrophoresis on 1.5% agarose gels stained with ethidium bromide. Semi-quantitative analyses presented the comparison of FoxP3 and RORct PCR products normalized to the co-amplified b-actin-PCR product using Kodak Digital Science ID software. 2.7. Cytokines and ox-LDL in Serum The levels of Interleukin 10 (IL-10), IL-17 and ox-LDL in serum were examined by the enzyme-linked immunosorbent assay (ELISA) and measured at 450 nm on Biocell HT1 ELISA microplate reader. (IL-17 and IL-10 ELISA kits, both from Bender MedSystems, Vienna, Austria; ox-LDL ELISA kits from Uscnlife, Missouri, USA.) The minimal detectable concentrations were 0.5 pg/ml for IL-17, 1.0 pg/ml for IL-10, and 4.5 lg/L for ox-LDL. Intra- and inter-assay coefficients of variation for all ELISA were <5%. All samples were measured in duplicate. 2.8. Effects of ox-LDL on Treg and Th17 cells 2.8.1. Preparation of LDL and ox-LDL Blood for lipoprotein isolation was collected from NCA subjects after 12 h of fasting. LDL was isolated by the ultracentrifugation of serum as previously described [9]. The LDL oxidation assay was performed as previously described [10], and the extent of LDL oxidation was measured by the thiobarbituric acid reactive substances (TBARS) assay, using malondialdehyde (MDA) for the standard curve [11]. 2.8.2. ox-LDL induction experiments PBMCs (1.0 106 cells/ml) from NCA subjects (n = 5) were incubated with culture medium (RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum), including various concentrations of ox-LDL (0, 0.1, 1, 5, 10, 50, and 100 lg/ml) for 24 and
838
Q. Li et al. / Biochemical and Biophysical Research Communications 394 (2010) 836–842
Fig. 1. Expression of RORct and Foxp3 in PBMCs from NCA, SA, UA, and AMI patients was determined by reverse transcription-polymerase chain reaction (PCR). (A) Representative PCR images: 1: NCA, 2: SA, 3: UA, 4: AMI. (B) Ratios of Foxp3/b-actin and RORct/b-actin for the gray-scale value were compared in 4 groups. *P < 0.05 vs. SA and NCA.
48 h in vitro. After incubation, the frequency of Treg and Th17 cells was measured, and analysis of Th17 was carried out after stimulation just as before. To compare the effects of ox-LDL on Treg and Th17 cells from AMI, UA, SA, and NCA subjects, PBMCs (n = 5 from each group) were incubated with 1 lg/ml ox-LDL for 48 h in vitro, and the frequency of Treg and Th17 cells was measured using FCM. The suppressive functions of Tregs were assessed in the presence or absence of ox-LDL (1 lg/ml). 2.9. Statistical analysis Values were expressed as the mean ± standard deviation (SD). Data were analysed by using statistical software (SPSS 11.0; LEAD Technologies, Inc., Chicago, IL, USA). Statistical significance for the
differences in the groups was assessed by one-way analysis of variance (ANOVA). Spearman’s correlation was used as a test of correlation between two continuous variables. P < 0.05 was considered to be statistically significant. 3. Results 3.1. Characteristics of patients Table 1 shows the characteristics of patients. There were no significant differences in age, gender, CAD extent, hypertension, diabetes mellitus, smoking rate, obesity, high-density lipoprotein-cholesterol (HDL-C), and very low-density lipoprotein-cholesterol (VLDL-C) concentrations among patients with AMI, UA, and SA.
Table 1 Patient characteristics. Item
NCA (n = 40)
SA (n = 31)
UA (n = 32)
AMI (n = 30)
Age Gender (male/female) CAD extent (n vessels) Hypertension, n (%) Diabetes mellitus, n (%) Smoking rate, n (%) Obesity, n (%) TC (mmol/L) TG (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) VLDL-C (mmol/L)
56.8 ± 16.4 28/12 0 16 (40) 4 (10) 9 (22.5) 7 (17.5) 4.32 ± 0.61 1.12 ± 0. 41 1.26 ± 0.12 2.41 ± 0.36 0.46 ± 0.13
61.7 ± 10.3 20/11 2.0 ± 0.7 15 (48.3) 8 (25.8) 8 (25.8) 5 (16.1) 4.58 ± 0.86 1.23 ± 0. 53 1.22 ± 0.26 2.60 ± 0.38 0.43 ± 0.12
64.8 ± 11.2 19/13 1.8 ± 0.6 16 (50) 9 (28.1) 7 (21.9) 6 (18.8) 5.08 ± 0.76**,*** 1.66 ± 0. 57*,*** 1.04 ± 0.24 2.92 ± 0.52* 0.55 ± 0.21
56.9 ± 17.8 18/12 2.0 ± 0.8 14 (46.7) 8 (26.7) 9 (30) 5 (16.7) 5.23 ± 0. 87**,**** 1.87 ± 0. 49**,*** 1.09 ± 0.25 3.15 ± 0.71**,*** 0.52 ± 0.18
Values are expressed as mean ± SD or number. AMI: acute myocardial infarction; UA: unstable angina; SA: stable angina; NCA: subjects with normal coronary arteries; CAD: coronary artery disease; BFS: blood-fasting sugar; TC: total cholesterol; TG: total triglyceride; HDL-C: high-density lipoprotein-cholesterol; LDL-C: low-density lipoproteincholesterol; VLDL-C: very low-density lipoprotein-cholesterol. * P < 0.05 vs. NCA. ** P < 0.01 vs. NCA. *** P < 0.05 vs. SA. **** P < 0.01 vs. SA.
Q. Li et al. / Biochemical and Biophysical Research Communications 394 (2010) 836–842 Table 2 Treg cells in patients with ACS. NCA (n = 40) CD4+CD25high/CD4+ T cells (%) CD4+CD25+CD127lo/ CD4+ T cells (%) CD4+CD25+Foxp3+/ CD4+ T cells (%) * ** ***
SA (n = 31)
UA (n = 32)
AMI (n = 30)
839
3.2.4. Expression of RORct in PBMCs The expression of RORct in PBMCs was markedly higher in the AMI and UA patients than in the SA and NCA groups (P < 0.05, Fig. 1).
2.31 ± 0.52 2.05 ± 0.39*** 1.28 ± 0.29** 0.93 ± 0.24**
3.3. Decrease in suppression of Tregs from ACS 5.82 ± 0.56 5.01 ± 0.49*
3.32 ± 0.56** 2.51 ± 0.69**
3.88 ± 0.51 3.16 ± 0.38*
2.17 ± 0.39** 1.65 ± 0.46**
P < 0.05 vs. NCA. P < 0.01 vs. NCA and SA. P > 0.05 vs. NCA.
3.2. Frequency of CD4+CD25+ Treg and Th17 cells 3.2.1. Decrease of Treg cells in ACS patients The frequencies of Treg (CD4+CD25high/CD4+, CD4+CD25+CD127lo/ CD4+, and CD4+CD25+Foxp3+/CD4+ T cells) cells were significantly lower in AMI and UA patients than in the SA and NCA groups (P < 0.01; Table 2). The frequencies of CD4+CD25+CD127lo and CD4+CD25+Foxp3+ cells in the SA patients were also markedly lower than in the NCA group (P < 0.05). 3.2.2. Expression of Foxp3 in PBMCs Foxp3 levels in the AMI and UA groups were significantly lower than in the SA and NCA groups (P < 0.05; Fig. 1). 3.2.3. Increase of Th17 cells in ACS patients The frequencies of Th17 (CD4+IL17+/CD4+ T cells) were markedly higher in AMI and UA patients than in the SA and NCA groups (P < 0.01; Fig. 2). There was also an obvious difference between the SA and NCA groups (P < 0.05).
The function of Treg cells was assessed by inhibition of the proliferation of CD4+CD25 cells in NCA controls and SA, UA, and AMI patients. CD4+CD25+CD127low cells showed a different suppressive rate: 82.3 ± 4.8%, 73.7 ± 4.3%, 49.3 ± 3.4%, and 36.7 ± 2.9%, respectively. Suppressive rates of Treg cells were significantly lower in the UA and AMI patients than in the NCA and SA groups (P < 0.01). Suppressive rates of Treg cells were also significantly lower in the SA patients than in the NCA group (P < 0.05). 3.4. Changes of serum cytokines in patients with ACS The levels of IL-10 were significantly lower in AMI and UA patients than in the SA and NCA groups (P < 0.01). The levels of IL17 were markedly higher in the AMI and UA patients than in the SA and NCA groups. Moreover, a decrease in the levels of IL-10 and an increase in the levels of IL-17 were significant for SA patients than for NCA group (P < 0.05; Table 3). 3.5. Role of ox-LDL in the balance of Th17/Treg cells 3.5.1. Correlation of serum ox-LDL levels to Th17/Treg cells in ACS patients The concentration of ox-LDL increased more significantly in the AMI and UA patients than in the SA and NCA groups (P < 0.01; Table 3). In addition, ox-LDL concentrations in serum were negatively correlated with the frequency of Treg cells (P < 0.01 and r = 0.784,
Fig. 2. The frequencies of Th17 increased in patients with ACS. (A) Representative FACS figures from a single patient in each group. The percentage of positive cells is shown in each panel. (B) Comparison of Th17 expression among 4 groups. *P < 0.01 vs. SA and NCA; #P < 0.05 vs. NCA.
840
Q. Li et al. / Biochemical and Biophysical Research Communications 394 (2010) 836–842
Table 3 Serum levels of cytokines and ox-LDL in the 4 groups. NCA (n = 40)
SA (n = 31)
UA (n = 32)
AMI (n = 30) Ug/ml
11.90 ± 1.49** 6.78 ± 1.74** IL-10 25.30 ± 3.43 20.10 ± 2.17* (pg/ml) IL-17 14.70 ± 3.02 20.60 ± 4.09* 50.40 ± 8.61** 58.70 ± 9.36** (pg/ml) 308.70 ± 27.10 335.20 ± 25.90* 392.10 ± 37.80** 433.10 ± 57.20** ox-LDL (lg/L) * **
P < 0.05 vs. NCA. P < 0.01 vs. NCA and SA.
0.815, and 0.776, respectively), and positively correlated with the frequency of Th17 cells (P < 0.01 and r = 0.793). Furthermore, There was an inverse correlation between the concentrations of ox-LDL and IL-10 in serum (P < 0.01 and r = 0.847). There were also positive correlations between the serum ox-LDL concentration and serum IL-17 concentrations (P < 0.01 and r = 0.818). 3.5.2. Effects of ox-LDL on Treg and Th17 cells in vitro We examined the effects of various concentrations of ox-LDL and at different times points on the numbers of Treg and Th17 cells in NCA subjects. With increased ox-LDL concentrations and prolonged incubation times, the frequency of Treg cells was decreased while the frequency of Th17 cells was elevated (Fig. 3A and B). We investigated the sensitivity of the ox-LDL-mediated decrease of Treg cells, compromise of function and increase of Th17 cells in the ACS, SA, and NCA groups. There were significant changes in the number of Treg and Th17 cells by ox-LDL in AMI
Fig. 3. Effect of ox-LDL on the expression of Treg and Th17 cells from NCA subjects. (A) PBMCs were incubated with different concentrations of ox-LDL for 24 h. (B) PBMCs were incubated with different concentrations of ox-LDL for 48 h.
(approximately 52% reduction of Treg and 34% increase of Th17 cells) and UA (about 48% reduction of Treg and 31% increase of Th17 cells) patients than in the SA (approximately 26% reduction of Treg and 19% increase of Th17 cells) and NCA (approximately 20% reduction of Treg and 17% increase of Th17 cells) groups (Fig. 4A and B). However, there was no difference in the numbers of CD4+ cells in cultured PBMCs derived from NCA, SA, and ACS patients treated with ox-LDL. In addition, the suppressive functions of Treg cells from all groups were compromised after exposure to ox-LDL. Incubation of Treg cells with 1 lg/ml ox-LDL, resulted in a significant attenuation of their ability to suppress the proliferation of CD4+CD25+ cells (P < 0.05 for NCA and P < 0.01 for SA, UA, and AMI). It is noteworthy that attenuation of the suppressive properties of Treg cells in AMI and UA patients was also more remarkably than in the SA and NCA groups (P < 0.01) (Fig. 4C and D). 4. Discussion Our study indicate that the shift of the Th17/Treg cell balance from Treg cells towards Th17 cells exists in ACS patients, and that this ox-LDL-mediated change is associated with the imbalance of Th17/Treg cells. Treg cells may suppress proatherogenic immune responses partly by secretion of anti-inflammatory cytokines, such as IL-10. The normal function of Treg cells may be essential to maintain the homeostasis of different subsets of T cells in the vessel wall [12]. There are reduced numbers of Treg cells in atherosclerotic mice, and the adoptive transfer of Treg cells can greatly reduce plaque size [13]. Experimental models revealed an impaired Treg-regulated immune mechanism in atherosclerosis [14]. In our study, Treg number and Foxp3 expression were significantly compromised in ACS patients. Treg cells from ACS patients also hampered the inhibition of responder CD4+CD25 T-cell proliferation in comparison to those from SA and NCA subjects, which is in line with the results of Mor et al. [15]. We also found that the levels of serum IL-10 were decreased in the ACS group and positively correlated with the levels of Treg cells. These results further support the hypothesis that Treg cells may play a protective role against the onset of ACS. Th17 cells are the newest members of the Th cell family and are characterised by their ability to produce specific cytokines such as IL-17. IL-17 has proinflammatory properties and is a key cytokine for the recruitment, activation and migration of neutrophils [16]. Mice with decreased IL-17 levels develop fewer lesions; increases in the levels of IL-17 may enhance early lesion formation [17], suggesting a potential role for Th17 cells in the promotion of atherogenesis. In human lesions, it has been reported that Th17 may participate in the inflammatory process of plaque destabilisation and ACS development [18]. Th17 cells has pleiotropic effects, stimulating epithelial, endothelial, and fibroblastic cells to produce inflammatory cytokines/ chemokines. In addition, Th17 cells are considered to be involved in the aggregation and activation of macrophages in atherosclerotic lesions, which may contribute to lesion progression by releasing matrix metalloproteases. Accordingly, the onset of ACS may be the result of Th17-mediated mechanisms. We found that ACS patients exhibited a marked increase in Th17 number, RORct expression and IL-17 levels when compared with SA and NCA subjects. Also even SA patients showed a statistically significant increase in Th17 levels as compared with NCA subjects, which was different from the results of Cheng et al. who detected no obvious difference in the number of Th17 cells between SA and NCA subjects [19]. The reason could be related to relatively large number of patients in our study.
Q. Li et al. / Biochemical and Biophysical Research Communications 394 (2010) 836–842
841
Fig. 4. ox-LDL induces a more evident alteration of Treg and Th17 cells from ACS patients. (A) PBMCs from the NCA, SA, UA and AMI groups were incubated with 1.0 lg/ml oxLDL for 48 h. (B) Average change of Treg and Th17 numbers in the 4 groups after a 48 h co-culture with 1.0 lg/ml ox-LDL. (C) Treg cells from the NCA, SA, UA and AMI groups are compromised in their suppressive properties after a 48 h co-culture with 1.0 lg/ml ox-LDL. (D) Average attenuation of the suppressive function of Treg cells in the 4 groups when exposed to ox-LDL.
Both Treg cells and Th17 cells have shown to be involved in pathogenesis of atherosclerosis and ACS. These roles of Treg and Th17 cells suggest a balance between them. Our study demonstrated that the balance of Th17/Treg cells was impaired seriously in ACS patients.
It has reported that ox-LDL plays an important role in the promotion of atherosclerosis initiation, progression, and plaque destabilisation [1]. Circulating ox-LDL levels reflect the burden of oxidative-induced endothelial damage and foam cell formation [20].
842
Q. Li et al. / Biochemical and Biophysical Research Communications 394 (2010) 836–842
In our study, ox-LDL serum concentrations were negatively correlated with the frequency of Treg cells and IL-10 levels, and positively correlated with the frequency of Th17 cells and IL-17 levels. Moreover, ox-LDL not only compromised the expression of Treg cells and their suppressive functions, but also enhanced the expression of Th17 cells in vitro. Our findings suggest, for the first time, that ox-LDL could influence Th17 expression and lead to the imbalance of Th17/Treg cells. Furthermore, Treg and Th17 cells from AMI and UA patients were more susceptible to the influence of ox-LDL when compared to those in NCA and UA subjects. Accordingly, it could be considered that differential sensitivity to ox-LDL results in the aggravation of the Th17/Treg imbalance, which promotes plaque inflammation and destabilisation in ACS. The mechanisms in which ox-LDL directly affect the Th17/Treg balance have not been investigated. We hypothesised that ox-LDL may affect the balance by 2 pathways. Firstly, ox-LDL might have a potential role in the differentiation of Th17 and Treg cells. Secondly, ox-LDL-mediated cell apoptosis and proliferation could have been partially responsible for the change in the balance of Th17/Treg cells. However, exact mechanisms are on the way to be examined. These different pathways may alter the fragile balance between Treg and Th17 cells, which destabilizes the fibrous cap and increases the risk of plaque rupture and ACS. Ox-LDL is thus an important factor in determining stabilization of the plaques by effect the Th17/Treg balance. Control ox-LDL level would promote the stability of vulnerable plaques and prevent the onset of ACS. In conclusion, our findings show that Th17/Treg numerical and functional imbalance exists in patients with ACS. Ox-LDL has a direct effect on Th17/Treg imbalance which may contribute to plaque destabilization and pathogenesis of ACS. Acknowledgments This work was supported by National Natural Science Foundation of China (No. 30971226) and Natural Science Foundation of Anhui province of China (No. 090413091). References [1] G.K. Hansson, Inflammation, atherosclerosis, and coronary artery disease, N. Engl. J. Med. 352 (2005) 1685–1695.
[2] C.J. Binder, M.K. Chang, P.X. Shaw, Y.I. Miller, K. Hartvigsen, A. Dewan, J.L. Witztum, Innate and acquired immunity in atherogenesis, Nat. Med. 8 (2002) 1218–1226. [3] G.K. Hansson, P. Libby, U. Schonbeck, Z.Q. Yan, Innate and adaptive immunity in the pathogenesis of atherosclerosis, Circ. Res. 91 (2002) 281–291. [4] S. Sakaguchi, M. Ono, R. Setoguchi, H. Yagi, S. Hori, Z. Fehervari, J. Shimizu, T. Takahashi, T. Nomura, Foxp3+CD25+CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease, Immunol. Rev. 212 (2006) 8–27. [5] E. Bettelli, M. Oukka, V.K. Kuchroo, T(H)-17 cells in the circle of immunity and autoimmunity, Nat. Immunol. 8 (2007) 345–350. [6] B. Homey, After TH1/TH2 now comes Treg/TH17: significance of T helper cells in immune response organization, Hautarzt 57 (2006) 730–732. [7] U. Landmesser, D.G. Harrison, Oxidant stress as a marker for cardiovascular events: ox marks the spot, Circulation 104 (2001) 2638–2640. [8] J. Davignon, P. Ganz, Role of endothelial dysfunction in atherosclerosis, Circulation 109 (2004) 27–32. [9] M. Aviram, M. Rosenblat, S. Billecke, J. Erogul, R. Sorenson, C.L. Bisgaier, R.S. Newton, B. La Du, Human serum paraoxonase (PON 1) is inactivated by oxidized low density lipoprotein and preserved by antioxidants, Free Radic. Biol. Med. 26 (1999) 892–904. [10] G. Sobal, J.E. Menzel, H. Sinzinger, Influence of acetylsalicylic acid on oxidation of native and glycated low-density lipoprotein, Life Sci. 66 (2000) 1987–1998. [11] J.A. Buege, S.D. Aust, Microsomal lipid peroxidation, Methods Enzymol. 52 (1978) 302–310. [12] J. Feng, Z. Zhang, W. Kong, B. Liu, Q. Xu, X. Wang, Regulatory T cells ameliorate hyperhomocysteinaemia-accelerated atherosclerosis in apoE/ mice, Cardiovas Res. 84 (2009) 155–163. [13] A. Mor, D. Planer, G. Luboshits, A. Afek, S. Metzger, T. Chajek-Shaul, G. Keren, J. George, Role of naturally occurring CD4+CD25+ regulatory T cells in experimental atherosclerosis, Arterioscler. Thromb. Vasc. Biol. 27 (2007) 893–900. [14] I. Gotsman, N. Grabie, R. Gupta, R. Dacosta, M. MacConmara, J. Lederer, G. Sukhova, J.L. Witztum, A.H. Sharpe, A.H. Lichtman, Impaired regulatory T-cell response and enhanced atherosclerosis in the absence of inducible costimulatory molecule, Circulation 114 (2006) 2047–2055. [15] A. Mor, G. Luboshits, D. Planer, G. Keren, J. George, Altered status of CD4+CD25+ regulatory T cells in patients with acute coronary syndromes, Eur. Heart J. 27 (2006) 2530–2537. [16] T. Korn, E. Bettelli, M. Oukka, V.K. Kuchroo, IL-17 and Th17 Cells, Annu. Rev. Immunol. 27 (2009) 485–517. [17] L. Song, C. Schindler, IL-6 and the acute phase response in murine atherosclerosis, Atherosclerosis 177 (2004) 43–51. [18] S. Hashmi, Q.T. Zeng, Role of interleukin-17 and interleukin-17-induced cytokines interleukin-6 and interleukin-8 in unstable coronary artery disease, Coron. Artery Dis. 17 (2006) 699–706. [19] X. Cheng, X. Yu, Y.J. Ding, Q.Q. Fu, J.J. Xie, T.T. Tang, R. Yao, Y. Chen, Y.H. Liao, The Th17/Treg imbalance in patients with acute coronary syndrome, Clin. Immunol. 127 (2008) 89–97. [20] K. Shimada, H. Mokuno, E. Matsunaga, T. Miyazaki, K. Sumiyoshi, K. Miyauchi, H. Daida, Circulating oxidized low-density lipoprotein is an independent predictor for cardiac event in patients with coronary artery disease, Atherosclerosis 174 (2004) 343–347.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
The role of oxidized low-density lipoprotein in breaking peripheral Th17/Treg balance in patients with acute coronary syndrome Qing Li a,b,c,d,1, Yi Wang a,b,c,1, Ke Chen e, Qing Zhou a,b,c, Wei Wei a,c, Yiping Wang f, Yuan Wang a,b,c,* a
Institute of Clinical Pharmacology, Anhui Medical University, Hefei, Anhui 230032, PR China Laboratory of Molecular Biology and Department of Biochemistry, Anhui Medical University, Hefei, Anhui 230032, PR China c Key Laboratory of Gene Resource Utilization for Severe Disease and Anti-inflammatory and Immunopharmacology (Anhui Medical University), Ministry of Education and Anhui Province, Hefei, Anhui 230032, PR China d The Central Laboratory of Medical Research Center, Anhui Provincial Hospital, Hefei, Anhui 230001, PR China e Department of Cardiovascular Disease, Anhui Provincial Hospital, Hefei, Anhui 230001, PR China f The Centre for Transplantation and Renal Research, Western Clinical School, University of Sydney, Westmead, NSW Australia b
a r t i c l e
i n f o
Article history: Received 6 March 2010 Available online 17 March 2010 Keywords: Oxidized low-density lipoprotein Acute coronary syndrome T-helper 17 Regulatory T cells
a b s t r a c t Oxidized low-density lipoprotein (ox-LDL) is an instrumental factor in atherogenesis, however, the effects of ox-LDL on the balance of Th17/Treg in acute coronary syndrome [ACS, including unstable angina (UA) and acute myocardial infarction (AMI)] is still unclear. CD4+CD25+ regulatory T (Treg) cells and Th17 cells, subsets of T-helper cells, play important roles in peripheral immunity and their imbalance leads to the development of tissue inflammation and autoimmune diseases. However, few studies have explored the effect of Th17/Treg balance in plaque destabilization and the onset of ACS. To explore the shift of Th17/Treg balance in ACS patients and the effect of ox-LDL on the balance, we examined the frequencies of Th17 and Treg cells, key transcription factors and relevant cytokines in patients with AMI, UA, stable angina (SA) and controls. We analysed the correlations of serum ox-LDL to Th17/Treg frequency, and the effects of ox-LDL on Th17/Treg cells in vitro. Our study demonstrated that ACS patients have shown a significant increase of Th17 frequency, RORct expression and serum Interleukin 17 (IL-17), and a obvious decline of Treg frequency, Foxp3 expression, suppressive function, and serum IL-10. Serum ox-LDL positively correlated with the frequency of Th17 cells and negatively correlated with the frequency of Treg cells. In vitro incubation of peripheral blood mononuclear cells from controls with ox-LDL resulted in a significant reduction of Treg cells and a significant elevation of Th17 cells in a dose- and time-dependant manner. Treg and Th17 cells from ACS patients were significantly more susceptible to ox-LDL-mediated alterations. Th17/Treg numerical and functional imbalance exists in ACS patients, and ox-LDL has a direct effect on Th17/Treg imbalance which may contribute to the occurrence of ACS. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Atherosclerosis (AS) is a chronic inflammatory disease in which immune mechanisms appear to make great effect, and atherogenesis involves various immune cells, particularly T lymphocytes, such as CD4+ T-helper cells [1,2]. Acute coronary syndrome (ACS) occurs as a consequence of coronary plaque erosion or rupture and T lymphocytes play an important role in these coronary events [3]. CD4+CD25+ regulatory T (Treg) cells and T-helper 17 (Th17) cells are 2 original subsets distinguished from Th1 and Th2 cells. Treg cells expressing the forkhead/winged helix transcription factor (Foxp3), a fraction of inflammatory-regulated negative cells, have important effects * Corresponding author at: Laboratory of Molecular Biology, Anhui Medical University, Hefei 230032, Anhui Province, PR China. E-mail address: [email protected] (Y. Wang). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.03.090
on the maintenance of immune tolerance and immune homeostasis by contact-dependent suppression or by the release of anti-inflammatory cytokines, such as interleukin (IL)-10 and transforming growth factor (TGF)-b [4]. Th17 cells expressing retinoic acid-related orphan receptor ct (RORct) exert an important effect on the pathogenesis of many experimental autoimmune diseases and human inflammatory conditions by producing IL-17, tumor necrosis factor (TNF)-a and IL-6 [5]. The Th17/Treg imbalance could be critical in the development of tissue inflammation and autoimmune diseases [6]. However, Th17/Treg balance in AS, especially ACS patients has not been fully investigated. Oxidized low-density lipoprotein (ox-LDL) is a key factor in the initiation and progression of atherosclerosis and contributes to endothelial dysfunction and plaque destabilization through multiple mechanisms [7,8]. It is unclear whether ox-LDL also affects AS and ACS patients by disrupting the balance of the peripheral Tregs and Th17 cells.
Q. Li et al. / Biochemical and Biophysical Research Communications 394 (2010) 836–842
837
In this study, we have examined the balance of Th17/Treg cells in ACS patients, and the effects of ox-LDL on these cells.
as a percentage of CD4+ T cells by sequential gating on lymphocytes and CD4+ T cells.
2. Materials and methods
2.4. Flow sorting
2.1. Patients
CD4+CD25+CD127low and CD4+CD25 cells were sorted using the gates on a Beckman Counter flow cytometry cell-sorter (ALTRA HyPerSort System). We obtained a consistent purity of >90% for both CD4+CD25+CD127low and CD4+CD25 cell fractions.
All patients gave written informed consent prior enrolment into the study. We examined patients at Anhui provincial hospital who underwent diagnostic catheterisation between July 2007 and June 2009. Patients were classified into 4 groups: acute myocardial infarction (AMI) patients, unstable angina (UA) patients, stable angina (SA) patients and control subjects with normal coronary arteries (NCA), Patients with ACS and SA had a similar extent of coronary atherosclerosis. There were no evident differences between the 4 groups with regard to age. No patient was treated with anti-inflammatory drugs and/or immunosuppressive agents. None had thromboembolism, disseminated intravascular coagulation, advanced liver disease, renal failure, malignant disease, other inflammatory disease, chronic-immunemediated disorders, valvular heart disease, atrial fibrillation or pacemaker. 2.2. Blood samples We collected 5–10 ml of peripheral blood (PB) from all the patients, in a fasting state, on the morning following admission. The time interval between symptom onset and blood sampling was less than 24 h in all cases. PB mononuclear cells (PBMCs) were prepared by Ficoll density gradient for analysis of flow cytometry (FCM) and reverse transcription-polymerase chain reaction (RT-PCR). Serum was obtained after centrifugation and stored at 80 °C until further use. 2.3. Cell separation and flow cytometry 2.3.1. Cell preparation For the analysis of Th17 cells, PBMCs were suspended at a density of 2.0 106 cells/ml in complete culture medium (Gibco BRL, USA). The cells were stimulated with phorbol myristate acetate (PMA, 25 ng/ml) plus ionomycin (1 lg/ml) for 4 h, in the presence of monensin (1.7 lg/ml, all from Alexis Biochemicals, San Diego, CA, USA). Cells were incubated at 37 °C under a 5% CO2 environment. For the analysis of Treg, 100 ll of PBMCs (106) was added to tubes for further staining. 2.3.2. Detection of Treg and Th17 cells For the analysis of Treg cells, cell surface staining was performed by the use of fluorescein isothiocyanate(FITC)-conjugated antiCD4(13B8.2 clone; Beckman Coulter-Immunotech, Marseille, France), phycoerythrin(PE)-conjugated anti-CD25(B1.49.9 clone, Beckman Coulter-Immunotech), PE-cy7-conjugated anti-CD127 (ebioRDR5 clone, eBioscience, San Diego, CA, USA) and appropriate isotype controls for 20 min at room temperature in the dark, followed by washing in phosphate buffered solution (PBS). Cells were fixed and permeabilized with the Fix/Perm reagent (Beckman Coulter-Immunotech), incubated with PE-cy5-conjugated antiFoxp3(PCH101 clone, eBioscience) and its isotype control antibody, washed with PBS and analysed by FCM. For Th17 analysis, the cells were incubated with FITC-conjugated anti-CD4 at 4 °C for 15 min and then stained with PE-conjugated anti-IL-17A (ebio64DEC17 clone, eBioscience) after fixation and permeabilization according to the manufacturer’s instructions. Stained cells were assessed by FCM using the COULTER EPICS ALTRA HyPerSort flow cytometer with EXPO 32 MULTICOMP Software (Beckman Coulter, Miami, FL, USA). The frequency of Treg (CD4+CD25high, CD4+CD25+CD127low and CD4+CD25+Foxp3+) and Th17(CD4+IL17+) cells was expressed
2.5. Functional assay of Treg cells Freshly purified CD4+CD25+CD127low T cells from NCA controls and from patients with AMI, UA and SA (n = 5 in each group) were assayed for their suppressive activity in the allogeneic mixed lymphocyte response (MLR) assay. Suppression was expressed as a percentage of the positive control. 2.6. RORct and Foxp3 expression levels Total RNA was extracted with TRIzol (Invitrogen, USA) according to the manufacturer’s instructions. cDNA was synthesized by using random hexamer primers and RNase H-reverse transcriptase (Invitrogen). TaqMan primers and probes for human Foxp3 and RORct were purchased from Biosune Biotechnology. The following primer pairs were used: Foxp3: F: 50 -CACGCATGTTTGCCTTCTTCAGA-30 , R: 50 -GTAGGGTTGGAACACCTG CTGGG-30 (181 bp), and RORct: F: 50 GCAATGGAAGTGGTGCTGGTT-30 , R: 50 -AGGATGCTTTGGCGATGAGT C-30 (320 bp). The quality of cDNA subjected to the RT-PCR was controlled by amplification of transcripts of b-actin. b-Actin was analysed using the following primers: F: 50 -ATCTGGCACCACACCT TC-30 , R: 50 -AGCCAGGTCCAGACGCA-30 (296 bp). PCR products were subjected to electrophoresis on 1.5% agarose gels stained with ethidium bromide. Semi-quantitative analyses presented the comparison of FoxP3 and RORct PCR products normalized to the co-amplified b-actin-PCR product using Kodak Digital Science ID software. 2.7. Cytokines and ox-LDL in Serum The levels of Interleukin 10 (IL-10), IL-17 and ox-LDL in serum were examined by the enzyme-linked immunosorbent assay (ELISA) and measured at 450 nm on Biocell HT1 ELISA microplate reader. (IL-17 and IL-10 ELISA kits, both from Bender MedSystems, Vienna, Austria; ox-LDL ELISA kits from Uscnlife, Missouri, USA.) The minimal detectable concentrations were 0.5 pg/ml for IL-17, 1.0 pg/ml for IL-10, and 4.5 lg/L for ox-LDL. Intra- and inter-assay coefficients of variation for all ELISA were <5%. All samples were measured in duplicate. 2.8. Effects of ox-LDL on Treg and Th17 cells 2.8.1. Preparation of LDL and ox-LDL Blood for lipoprotein isolation was collected from NCA subjects after 12 h of fasting. LDL was isolated by the ultracentrifugation of serum as previously described [9]. The LDL oxidation assay was performed as previously described [10], and the extent of LDL oxidation was measured by the thiobarbituric acid reactive substances (TBARS) assay, using malondialdehyde (MDA) for the standard curve [11]. 2.8.2. ox-LDL induction experiments PBMCs (1.0 106 cells/ml) from NCA subjects (n = 5) were incubated with culture medium (RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum), including various concentrations of ox-LDL (0, 0.1, 1, 5, 10, 50, and 100 lg/ml) for 24 and
838
Q. Li et al. / Biochemical and Biophysical Research Communications 394 (2010) 836–842
Fig. 1. Expression of RORct and Foxp3 in PBMCs from NCA, SA, UA, and AMI patients was determined by reverse transcription-polymerase chain reaction (PCR). (A) Representative PCR images: 1: NCA, 2: SA, 3: UA, 4: AMI. (B) Ratios of Foxp3/b-actin and RORct/b-actin for the gray-scale value were compared in 4 groups. *P < 0.05 vs. SA and NCA.
48 h in vitro. After incubation, the frequency of Treg and Th17 cells was measured, and analysis of Th17 was carried out after stimulation just as before. To compare the effects of ox-LDL on Treg and Th17 cells from AMI, UA, SA, and NCA subjects, PBMCs (n = 5 from each group) were incubated with 1 lg/ml ox-LDL for 48 h in vitro, and the frequency of Treg and Th17 cells was measured using FCM. The suppressive functions of Tregs were assessed in the presence or absence of ox-LDL (1 lg/ml). 2.9. Statistical analysis Values were expressed as the mean ± standard deviation (SD). Data were analysed by using statistical software (SPSS 11.0; LEAD Technologies, Inc., Chicago, IL, USA). Statistical significance for the
differences in the groups was assessed by one-way analysis of variance (ANOVA). Spearman’s correlation was used as a test of correlation between two continuous variables. P < 0.05 was considered to be statistically significant. 3. Results 3.1. Characteristics of patients Table 1 shows the characteristics of patients. There were no significant differences in age, gender, CAD extent, hypertension, diabetes mellitus, smoking rate, obesity, high-density lipoprotein-cholesterol (HDL-C), and very low-density lipoprotein-cholesterol (VLDL-C) concentrations among patients with AMI, UA, and SA.
Table 1 Patient characteristics. Item
NCA (n = 40)
SA (n = 31)
UA (n = 32)
AMI (n = 30)
Age Gender (male/female) CAD extent (n vessels) Hypertension, n (%) Diabetes mellitus, n (%) Smoking rate, n (%) Obesity, n (%) TC (mmol/L) TG (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) VLDL-C (mmol/L)
56.8 ± 16.4 28/12 0 16 (40) 4 (10) 9 (22.5) 7 (17.5) 4.32 ± 0.61 1.12 ± 0. 41 1.26 ± 0.12 2.41 ± 0.36 0.46 ± 0.13
61.7 ± 10.3 20/11 2.0 ± 0.7 15 (48.3) 8 (25.8) 8 (25.8) 5 (16.1) 4.58 ± 0.86 1.23 ± 0. 53 1.22 ± 0.26 2.60 ± 0.38 0.43 ± 0.12
64.8 ± 11.2 19/13 1.8 ± 0.6 16 (50) 9 (28.1) 7 (21.9) 6 (18.8) 5.08 ± 0.76**,*** 1.66 ± 0. 57*,*** 1.04 ± 0.24 2.92 ± 0.52* 0.55 ± 0.21
56.9 ± 17.8 18/12 2.0 ± 0.8 14 (46.7) 8 (26.7) 9 (30) 5 (16.7) 5.23 ± 0. 87**,**** 1.87 ± 0. 49**,*** 1.09 ± 0.25 3.15 ± 0.71**,*** 0.52 ± 0.18
Values are expressed as mean ± SD or number. AMI: acute myocardial infarction; UA: unstable angina; SA: stable angina; NCA: subjects with normal coronary arteries; CAD: coronary artery disease; BFS: blood-fasting sugar; TC: total cholesterol; TG: total triglyceride; HDL-C: high-density lipoprotein-cholesterol; LDL-C: low-density lipoproteincholesterol; VLDL-C: very low-density lipoprotein-cholesterol. * P < 0.05 vs. NCA. ** P < 0.01 vs. NCA. *** P < 0.05 vs. SA. **** P < 0.01 vs. SA.
Q. Li et al. / Biochemical and Biophysical Research Communications 394 (2010) 836–842 Table 2 Treg cells in patients with ACS. NCA (n = 40) CD4+CD25high/CD4+ T cells (%) CD4+CD25+CD127lo/ CD4+ T cells (%) CD4+CD25+Foxp3+/ CD4+ T cells (%) * ** ***
SA (n = 31)
UA (n = 32)
AMI (n = 30)
839
3.2.4. Expression of RORct in PBMCs The expression of RORct in PBMCs was markedly higher in the AMI and UA patients than in the SA and NCA groups (P < 0.05, Fig. 1).
2.31 ± 0.52 2.05 ± 0.39*** 1.28 ± 0.29** 0.93 ± 0.24**
3.3. Decrease in suppression of Tregs from ACS 5.82 ± 0.56 5.01 ± 0.49*
3.32 ± 0.56** 2.51 ± 0.69**
3.88 ± 0.51 3.16 ± 0.38*
2.17 ± 0.39** 1.65 ± 0.46**
P < 0.05 vs. NCA. P < 0.01 vs. NCA and SA. P > 0.05 vs. NCA.
3.2. Frequency of CD4+CD25+ Treg and Th17 cells 3.2.1. Decrease of Treg cells in ACS patients The frequencies of Treg (CD4+CD25high/CD4+, CD4+CD25+CD127lo/ CD4+, and CD4+CD25+Foxp3+/CD4+ T cells) cells were significantly lower in AMI and UA patients than in the SA and NCA groups (P < 0.01; Table 2). The frequencies of CD4+CD25+CD127lo and CD4+CD25+Foxp3+ cells in the SA patients were also markedly lower than in the NCA group (P < 0.05). 3.2.2. Expression of Foxp3 in PBMCs Foxp3 levels in the AMI and UA groups were significantly lower than in the SA and NCA groups (P < 0.05; Fig. 1). 3.2.3. Increase of Th17 cells in ACS patients The frequencies of Th17 (CD4+IL17+/CD4+ T cells) were markedly higher in AMI and UA patients than in the SA and NCA groups (P < 0.01; Fig. 2). There was also an obvious difference between the SA and NCA groups (P < 0.05).
The function of Treg cells was assessed by inhibition of the proliferation of CD4+CD25 cells in NCA controls and SA, UA, and AMI patients. CD4+CD25+CD127low cells showed a different suppressive rate: 82.3 ± 4.8%, 73.7 ± 4.3%, 49.3 ± 3.4%, and 36.7 ± 2.9%, respectively. Suppressive rates of Treg cells were significantly lower in the UA and AMI patients than in the NCA and SA groups (P < 0.01). Suppressive rates of Treg cells were also significantly lower in the SA patients than in the NCA group (P < 0.05). 3.4. Changes of serum cytokines in patients with ACS The levels of IL-10 were significantly lower in AMI and UA patients than in the SA and NCA groups (P < 0.01). The levels of IL17 were markedly higher in the AMI and UA patients than in the SA and NCA groups. Moreover, a decrease in the levels of IL-10 and an increase in the levels of IL-17 were significant for SA patients than for NCA group (P < 0.05; Table 3). 3.5. Role of ox-LDL in the balance of Th17/Treg cells 3.5.1. Correlation of serum ox-LDL levels to Th17/Treg cells in ACS patients The concentration of ox-LDL increased more significantly in the AMI and UA patients than in the SA and NCA groups (P < 0.01; Table 3). In addition, ox-LDL concentrations in serum were negatively correlated with the frequency of Treg cells (P < 0.01 and r = 0.784,
Fig. 2. The frequencies of Th17 increased in patients with ACS. (A) Representative FACS figures from a single patient in each group. The percentage of positive cells is shown in each panel. (B) Comparison of Th17 expression among 4 groups. *P < 0.01 vs. SA and NCA; #P < 0.05 vs. NCA.
840
Q. Li et al. / Biochemical and Biophysical Research Communications 394 (2010) 836–842
Table 3 Serum levels of cytokines and ox-LDL in the 4 groups. NCA (n = 40)
SA (n = 31)
UA (n = 32)
AMI (n = 30) Ug/ml
11.90 ± 1.49** 6.78 ± 1.74** IL-10 25.30 ± 3.43 20.10 ± 2.17* (pg/ml) IL-17 14.70 ± 3.02 20.60 ± 4.09* 50.40 ± 8.61** 58.70 ± 9.36** (pg/ml) 308.70 ± 27.10 335.20 ± 25.90* 392.10 ± 37.80** 433.10 ± 57.20** ox-LDL (lg/L) * **
P < 0.05 vs. NCA. P < 0.01 vs. NCA and SA.
0.815, and 0.776, respectively), and positively correlated with the frequency of Th17 cells (P < 0.01 and r = 0.793). Furthermore, There was an inverse correlation between the concentrations of ox-LDL and IL-10 in serum (P < 0.01 and r = 0.847). There were also positive correlations between the serum ox-LDL concentration and serum IL-17 concentrations (P < 0.01 and r = 0.818). 3.5.2. Effects of ox-LDL on Treg and Th17 cells in vitro We examined the effects of various concentrations of ox-LDL and at different times points on the numbers of Treg and Th17 cells in NCA subjects. With increased ox-LDL concentrations and prolonged incubation times, the frequency of Treg cells was decreased while the frequency of Th17 cells was elevated (Fig. 3A and B). We investigated the sensitivity of the ox-LDL-mediated decrease of Treg cells, compromise of function and increase of Th17 cells in the ACS, SA, and NCA groups. There were significant changes in the number of Treg and Th17 cells by ox-LDL in AMI
Fig. 3. Effect of ox-LDL on the expression of Treg and Th17 cells from NCA subjects. (A) PBMCs were incubated with different concentrations of ox-LDL for 24 h. (B) PBMCs were incubated with different concentrations of ox-LDL for 48 h.
(approximately 52% reduction of Treg and 34% increase of Th17 cells) and UA (about 48% reduction of Treg and 31% increase of Th17 cells) patients than in the SA (approximately 26% reduction of Treg and 19% increase of Th17 cells) and NCA (approximately 20% reduction of Treg and 17% increase of Th17 cells) groups (Fig. 4A and B). However, there was no difference in the numbers of CD4+ cells in cultured PBMCs derived from NCA, SA, and ACS patients treated with ox-LDL. In addition, the suppressive functions of Treg cells from all groups were compromised after exposure to ox-LDL. Incubation of Treg cells with 1 lg/ml ox-LDL, resulted in a significant attenuation of their ability to suppress the proliferation of CD4+CD25+ cells (P < 0.05 for NCA and P < 0.01 for SA, UA, and AMI). It is noteworthy that attenuation of the suppressive properties of Treg cells in AMI and UA patients was also more remarkably than in the SA and NCA groups (P < 0.01) (Fig. 4C and D). 4. Discussion Our study indicate that the shift of the Th17/Treg cell balance from Treg cells towards Th17 cells exists in ACS patients, and that this ox-LDL-mediated change is associated with the imbalance of Th17/Treg cells. Treg cells may suppress proatherogenic immune responses partly by secretion of anti-inflammatory cytokines, such as IL-10. The normal function of Treg cells may be essential to maintain the homeostasis of different subsets of T cells in the vessel wall [12]. There are reduced numbers of Treg cells in atherosclerotic mice, and the adoptive transfer of Treg cells can greatly reduce plaque size [13]. Experimental models revealed an impaired Treg-regulated immune mechanism in atherosclerosis [14]. In our study, Treg number and Foxp3 expression were significantly compromised in ACS patients. Treg cells from ACS patients also hampered the inhibition of responder CD4+CD25 T-cell proliferation in comparison to those from SA and NCA subjects, which is in line with the results of Mor et al. [15]. We also found that the levels of serum IL-10 were decreased in the ACS group and positively correlated with the levels of Treg cells. These results further support the hypothesis that Treg cells may play a protective role against the onset of ACS. Th17 cells are the newest members of the Th cell family and are characterised by their ability to produce specific cytokines such as IL-17. IL-17 has proinflammatory properties and is a key cytokine for the recruitment, activation and migration of neutrophils [16]. Mice with decreased IL-17 levels develop fewer lesions; increases in the levels of IL-17 may enhance early lesion formation [17], suggesting a potential role for Th17 cells in the promotion of atherogenesis. In human lesions, it has been reported that Th17 may participate in the inflammatory process of plaque destabilisation and ACS development [18]. Th17 cells has pleiotropic effects, stimulating epithelial, endothelial, and fibroblastic cells to produce inflammatory cytokines/ chemokines. In addition, Th17 cells are considered to be involved in the aggregation and activation of macrophages in atherosclerotic lesions, which may contribute to lesion progression by releasing matrix metalloproteases. Accordingly, the onset of ACS may be the result of Th17-mediated mechanisms. We found that ACS patients exhibited a marked increase in Th17 number, RORct expression and IL-17 levels when compared with SA and NCA subjects. Also even SA patients showed a statistically significant increase in Th17 levels as compared with NCA subjects, which was different from the results of Cheng et al. who detected no obvious difference in the number of Th17 cells between SA and NCA subjects [19]. The reason could be related to relatively large number of patients in our study.
Q. Li et al. / Biochemical and Biophysical Research Communications 394 (2010) 836–842
841
Fig. 4. ox-LDL induces a more evident alteration of Treg and Th17 cells from ACS patients. (A) PBMCs from the NCA, SA, UA and AMI groups were incubated with 1.0 lg/ml oxLDL for 48 h. (B) Average change of Treg and Th17 numbers in the 4 groups after a 48 h co-culture with 1.0 lg/ml ox-LDL. (C) Treg cells from the NCA, SA, UA and AMI groups are compromised in their suppressive properties after a 48 h co-culture with 1.0 lg/ml ox-LDL. (D) Average attenuation of the suppressive function of Treg cells in the 4 groups when exposed to ox-LDL.
Both Treg cells and Th17 cells have shown to be involved in pathogenesis of atherosclerosis and ACS. These roles of Treg and Th17 cells suggest a balance between them. Our study demonstrated that the balance of Th17/Treg cells was impaired seriously in ACS patients.
It has reported that ox-LDL plays an important role in the promotion of atherosclerosis initiation, progression, and plaque destabilisation [1]. Circulating ox-LDL levels reflect the burden of oxidative-induced endothelial damage and foam cell formation [20].
842
Q. Li et al. / Biochemical and Biophysical Research Communications 394 (2010) 836–842
In our study, ox-LDL serum concentrations were negatively correlated with the frequency of Treg cells and IL-10 levels, and positively correlated with the frequency of Th17 cells and IL-17 levels. Moreover, ox-LDL not only compromised the expression of Treg cells and their suppressive functions, but also enhanced the expression of Th17 cells in vitro. Our findings suggest, for the first time, that ox-LDL could influence Th17 expression and lead to the imbalance of Th17/Treg cells. Furthermore, Treg and Th17 cells from AMI and UA patients were more susceptible to the influence of ox-LDL when compared to those in NCA and UA subjects. Accordingly, it could be considered that differential sensitivity to ox-LDL results in the aggravation of the Th17/Treg imbalance, which promotes plaque inflammation and destabilisation in ACS. The mechanisms in which ox-LDL directly affect the Th17/Treg balance have not been investigated. We hypothesised that ox-LDL may affect the balance by 2 pathways. Firstly, ox-LDL might have a potential role in the differentiation of Th17 and Treg cells. Secondly, ox-LDL-mediated cell apoptosis and proliferation could have been partially responsible for the change in the balance of Th17/Treg cells. However, exact mechanisms are on the way to be examined. These different pathways may alter the fragile balance between Treg and Th17 cells, which destabilizes the fibrous cap and increases the risk of plaque rupture and ACS. Ox-LDL is thus an important factor in determining stabilization of the plaques by effect the Th17/Treg balance. Control ox-LDL level would promote the stability of vulnerable plaques and prevent the onset of ACS. In conclusion, our findings show that Th17/Treg numerical and functional imbalance exists in patients with ACS. Ox-LDL has a direct effect on Th17/Treg imbalance which may contribute to plaque destabilization and pathogenesis of ACS. Acknowledgments This work was supported by National Natural Science Foundation of China (No. 30971226) and Natural Science Foundation of Anhui province of China (No. 090413091). References [1] G.K. Hansson, Inflammation, atherosclerosis, and coronary artery disease, N. Engl. J. Med. 352 (2005) 1685–1695.
[2] C.J. Binder, M.K. Chang, P.X. Shaw, Y.I. Miller, K. Hartvigsen, A. Dewan, J.L. Witztum, Innate and acquired immunity in atherogenesis, Nat. Med. 8 (2002) 1218–1226. [3] G.K. Hansson, P. Libby, U. Schonbeck, Z.Q. Yan, Innate and adaptive immunity in the pathogenesis of atherosclerosis, Circ. Res. 91 (2002) 281–291. [4] S. Sakaguchi, M. Ono, R. Setoguchi, H. Yagi, S. Hori, Z. Fehervari, J. Shimizu, T. Takahashi, T. Nomura, Foxp3+CD25+CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease, Immunol. Rev. 212 (2006) 8–27. [5] E. Bettelli, M. Oukka, V.K. Kuchroo, T(H)-17 cells in the circle of immunity and autoimmunity, Nat. Immunol. 8 (2007) 345–350. [6] B. Homey, After TH1/TH2 now comes Treg/TH17: significance of T helper cells in immune response organization, Hautarzt 57 (2006) 730–732. [7] U. Landmesser, D.G. Harrison, Oxidant stress as a marker for cardiovascular events: ox marks the spot, Circulation 104 (2001) 2638–2640. [8] J. Davignon, P. Ganz, Role of endothelial dysfunction in atherosclerosis, Circulation 109 (2004) 27–32. [9] M. Aviram, M. Rosenblat, S. Billecke, J. Erogul, R. Sorenson, C.L. Bisgaier, R.S. Newton, B. La Du, Human serum paraoxonase (PON 1) is inactivated by oxidized low density lipoprotein and preserved by antioxidants, Free Radic. Biol. Med. 26 (1999) 892–904. [10] G. Sobal, J.E. Menzel, H. Sinzinger, Influence of acetylsalicylic acid on oxidation of native and glycated low-density lipoprotein, Life Sci. 66 (2000) 1987–1998. [11] J.A. Buege, S.D. Aust, Microsomal lipid peroxidation, Methods Enzymol. 52 (1978) 302–310. [12] J. Feng, Z. Zhang, W. Kong, B. Liu, Q. Xu, X. Wang, Regulatory T cells ameliorate hyperhomocysteinaemia-accelerated atherosclerosis in apoE/ mice, Cardiovas Res. 84 (2009) 155–163. [13] A. Mor, D. Planer, G. Luboshits, A. Afek, S. Metzger, T. Chajek-Shaul, G. Keren, J. George, Role of naturally occurring CD4+CD25+ regulatory T cells in experimental atherosclerosis, Arterioscler. Thromb. Vasc. Biol. 27 (2007) 893–900. [14] I. Gotsman, N. Grabie, R. Gupta, R. Dacosta, M. MacConmara, J. Lederer, G. Sukhova, J.L. Witztum, A.H. Sharpe, A.H. Lichtman, Impaired regulatory T-cell response and enhanced atherosclerosis in the absence of inducible costimulatory molecule, Circulation 114 (2006) 2047–2055. [15] A. Mor, G. Luboshits, D. Planer, G. Keren, J. George, Altered status of CD4+CD25+ regulatory T cells in patients with acute coronary syndromes, Eur. Heart J. 27 (2006) 2530–2537. [16] T. Korn, E. Bettelli, M. Oukka, V.K. Kuchroo, IL-17 and Th17 Cells, Annu. Rev. Immunol. 27 (2009) 485–517. [17] L. Song, C. Schindler, IL-6 and the acute phase response in murine atherosclerosis, Atherosclerosis 177 (2004) 43–51. [18] S. Hashmi, Q.T. Zeng, Role of interleukin-17 and interleukin-17-induced cytokines interleukin-6 and interleukin-8 in unstable coronary artery disease, Coron. Artery Dis. 17 (2006) 699–706. [19] X. Cheng, X. Yu, Y.J. Ding, Q.Q. Fu, J.J. Xie, T.T. Tang, R. Yao, Y. Chen, Y.H. Liao, The Th17/Treg imbalance in patients with acute coronary syndrome, Clin. Immunol. 127 (2008) 89–97. [20] K. Shimada, H. Mokuno, E. Matsunaga, T. Miyazaki, K. Sumiyoshi, K. Miyauchi, H. Daida, Circulating oxidized low-density lipoprotein is an independent predictor for cardiac event in patients with coronary artery disease, Atherosclerosis 174 (2004) 343–347.