Intensive statin treatment ameliorate the Th17/Treg functional imbalance in patients with non‐ST elevation acute coronary syndrome underwent percutaneous coronary intervention

Abstract Background Inflammation plays important roles in the pathogenesis of acute coronary syndrome (ACS). Statins exert positive effects on the plaque stabilization through anti‐inflammation, however, the detailed mechanism is still under investigation. Hypothesis Studies suggest that the Th17/Treg functional imbalance takes key part in the plaque destabilization and the onset of ACS. We hypothesized that intensive statin therapy could ameliorate the Th17/Treg imbalance in patients with ACS. Methods Sixty‐six patients with non‐ST elevation acute coronary syndrome (NSTE‐ACS) were randomized to conventional group and intensive group. Peripheral blood samples were collected on admission and after atorvastatin treatment. The frequencies of circulating Th17 cells and Treg cells, the levels of cytokines associated with Th17 cells (IL‐17, IL‐6 and IL‐23) and associated with Treg cells (IL‐10 and TGF‐β1) were measured through flow cytometry and ELISA assay respectively. Results One week after therapy, the frequencies of circulating Th17 cells of both the groups decreased and the frequencies of circulating Treg cells increased significantly, compared with the basal levels. Furthermore, the decreased frequencies of circulating Th17 cells and the increased frequencies of circulating Treg cells in the intensive group were significantly higher than those in the conventional group. In consistence, the decreased accumulation of IL‐17, IL‐6 and IL‐23 (cytokines relevant to Th17 cells) and the increased accumulation of IL‐10 and TGF‐β1 in peripheral blood were displayed in both groups. The changes are more significant in the intensive group. Conclusion Intensive statins therapy could ameliorate the Th17 and Treg functional imbalance in patients with ACS.


| INTRODUCTION
Non-ST elevation acute coronary syndrome (NSTE-ACS, including unstable angina pectoris and non-ST elevation acute myocardial infarction) occurs as a consequence of coronary plaque erosion or rupture. It is thought that inflammation and immune mechanisms play an important role in the pathogenesis of ACS. Recently, CANTOS study 1 showed that pure anti-inflammatory treatment could significantly reduce cardiovascular events, which provided definitive evidence-based medical evidence for the atherosclerotic inflammation hypothesis. Th17 and Treg are two subpopulations of CD4+ T lymphocytes and the Th17/Treg functional imbalance may be important in the pathogenesis of plaque destabilization and the onset of ACS. 2,3 Early and intensive statin treatment is an efficient intervention to reduce hard cardiovascular events after ACS. 4 Studies showed that statins have immunomodulatory property and the effect of statins on atherosclerosis depends in part on the immunomodulatory mechanism. 5,6 However, the mechanisms of Th17/Treg imbalance in ACS patients and the effect of statins on it are still unclear. Our study observed the effect of intensive statin therapy on the Th17/Treg functional imbalance in patients with ACS.

| Treatments
All patients were treated with dual antiplatelet therapy consisting of aspirin 100 mg once a day and clopidogrel 75 mg once a day or ticagrelor 90 mg twice a day. β-Blocker, angiotensin-converting enzyme inhibitor/angiotensin receptor blocker, nitrates and low molecular weight heparin were administered by the attending physician when necessary. Patients were randomized to two groups, the conventional group with 20 mg atorvastatin and intensive group with 40 mg atorvastatin(Pfizer Ireland Pharmaceuticals，SFDA: J20120049). All the patients underwent selective PCI during hospital.
On admission and 1 week after statin therapy, blood samples were withdrawn for cytometric analysis of the stained Th17 cells

| Statistical analysis
All statistical analyses were performed using SPSS 20.0 (SPSS Inc., Chicago, Illinois,). P < .05 was considered to indicate statistical significance. Continuous variables are presented as mean ± SD, differences between groups were compared by the method of one-way ANOVA.
Categorical variables are presented as number and percentage, and the distribution of these variables between two groups was compared using Chi-squared test.

| Patient characteristics
Baseline characteristics of the two groups were listed in Table 1.
There were no significant differences between the two groups with regard to age, sex, mean blood pressure, heart rate, medications, number of stents, and doses of contrast. There were no major complications in either group and all survived to hospital discharge. The levels of hsTnT, NT-proBNP, LDL-C, Scr had no significant difference in both of the two groups on admission.

| Circulating Th17 cells frequencies
The circulating Th17 cells frequencies in patients of the control group was 0.63 ± 0.25%. The circulating Th17 cells frequencies in patients with ACS were listed in Table 2 and Figure 1. The frequencies of circulating Th17 cells in the conventional group and intensive group were significantly higher than that in the control group on admission (P < .05). There was no significant difference between the two groups. One week after statins therapy, the frequencies of circulating Th17 cells of both the groups decreased significantly. Furthermore, the decreased frequencies of circulating Th17 cells in the intensive group were significantly higher than that in the conventional group.

| Circulating Treg cells frequencies in patients with ACS
The circulating Treg cells frequencies in patients of the control was 3.81 ± 1.52%. The circulating Treg cell frequencies in patients with NSTE-ACS were listed in Table 2 and Figure 2. The frequencies of circulating Treg cells in the conventional group and intensive group were significantly lower than that in the control group on admission (P < .05). There was no significant difference between the two groups.
One week after statins therapy, the frequencies of circulating Treg cells of both the groups increased significantly. Furthermore, the increased frequencies of circulating Treg cells in the intensive group were significantly higher than that in the conventional group. 13.53 ± 6.62, and 151.62 ± 58.70 pg/mL. The levels of important cytokines associated with Th17 cells (IL-10 and TGF-β1) were 13.44 ± 6.58 and 808.58 ± 410.21 pg/mL. The levels of IL-17, IL-6, and IL-23 in the conventional group and intensive group were significantly higher than those in the control group on admission (P < 0.05), while the levels of IL-10 and TGF-β1 were significantly lower than those in the control group (P < .05). There were no significant differences between the two groups. One week after statins therapy, the levels of IL-17, IL-6, and IL-23 in both the conventional group and the intensive group decreased, and which decreased much higher in the intensive group than the conventional group. While, the levels of IL-10 and TGF-β1 in both the groups increased and which increased much significantly in the intensive group than the conventional group (Table 3).

| DISCUSSION
The activation of immunity is closely related to atherosclerosis, and the imbalance of regulatory and pathogenic immunity may promote the development of atherosclerosis. 7 Atherogenesis involves various immune cells, particularly CD4+ T-helper cells. 8,9 Th17 and Treg cells, as newly discovered CD4+ T cells, are closely related to inflammation, which might affect the plaque degeneration and the onset of ACS.
Th17 cells are important components of activated T cells, which played a key role in the progression of plaque instability. 10,11 Th17 cells produce the signature cytokines interleukin-17 (IL-17), and to a lesser extent tumor necrosis factor-α (TNF-α), IL-23, and IL-6. IL-17 can also stimulate macrophages to produce inflammatory cytokines, such as IL-6, TNF-a, and IL-1b. 12, 13 Zhu et al 14 demonstrated that F I G U R E 1 The frequencies of circulating Th17 cells in the conventional group and intensive group were significantly higher than that in the control group on admission (P < .05). One week after statins therapy, the frequencies of circulating Th17 cells of both the groups decreased significantly. The decreased frequencies of circulating Th17 cells in the intensive group were significantly higher than that in the conventional group IL-17 not only promotes the production of large amounts of vWF by vascular endothelial cells, but also induces apoptosis of vascular endothelial cells through the mitochondrial pathway. IL-6 is a cytokine that had been implicated in vascular inflammation. 15 IL-6 receptor blocker with tocilizumab could reduce the expression level of C reactive protein and troponin in patients with non-ST segment elevation myocardial infarction. 16 Our study showed that the frequencies of circulating cytokines, such as IL-10 and TGF-β1. 19 Active TGF-β1 is important for Tregs to mediate immunosuppression. 20 IL-10 is an immunoregulatory cytokine and had been shown to have a protective role in both atherosclerotic lesion formation and stability in animal studies. 21 In our study, the frequencies of circulating Treg cells and the levels of TGF-β1 and IL-10 in the conventional group and intensive group were significantly lower than those in the control group on admission.
F I G U R E 2 The frequencies of circulating Treg cells in the conventional group and intensive group were significantly lower than that in the control group on admission (P < .05). One week after statins therapy, the frequencies of circulating Treg cells of both the groups increased significantly. The increased frequencies of circulating Treg cells in the intensive group were significantly higher than that in the conventional group The results suggested that the intensive statins therapy is more conducive to exerting anti-inflammatory effects by inhibiting Th17 cell-mediated pathogenic immune response.

| Limitations
Our study has some limitations. First, guidelines recommended early initiation of statin therapy for patients with ACS. Therefore, based on ethical considerations, we could not set patients who were not treated with statins as a control group. Second, the present cohort consisted of a small number of patients and shorter observation time.
The present results therefore require verification in a larger population and longer follow up.

ACKNOWLEDGMENTS
We thank the respondents for participating in this study, and we are thankful to Guiyue Zhu, Xiaoyan Wu, Kefeng Lu, Bo dong and Bin cui for data collection and experimental aid.

SOURCES OF FUNDING
This work was supported by the National Natural Science Foundation of China (No.81770382).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors received no specific funding for this work.