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Statins reduce the expressions of Tim-3 on NK cells and NKT cells in atherosclerosis
European Journal of Pharmacology 821 (2018) 49–56
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
Statins reduce the expressions of Tim-3 on NK cells and NKT cells in atherosclerosis
T
Na Zhanga, Min Zhanga, Ru-Tao Liua, Peng Zhanga, Chun-Lin Yanga, Long-Tao Yueb, Heng Lia, ⁎ Yong-Kang Lic, Rui-Sheng Duana, a
Department of Neurology, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014, PR China Central laboratory, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014, PR China c Department of Cardiology, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014, PR China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Statin Atherosclerosis Natural killer cell Natural killer T cell Tim-3
3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors (statins) have an immuno-regulatory effect in addition to lowing-lipids. Accumulated evidence showed that the expressions of T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) on natural killer (NK) cells increased in atherosclerotic patients and animal models. In this study, 14 patients treated with rosuvastatin and 12 patients with atorvastatin for more than 3 months were included and 20 patients without statins treatment as control. Both statins treatment reduced the expressions of Tim-3 on NK cells and their subtypes, natural killer T (NKT) cells and CD3+ T cells, and increased the proportions of NKT cells among peripheral blood mononuclear cells, accompanied by the decreased levels of total cholesterol, low density lipoprotein, and increased ratios of high density lipoprotein to cholesterol. These may contribute to the functions of statins in the treatment of atherosclerosis.
1. Introduction Cardiovascular and cerebrovascular diseases are the main causes of death around the world. Their major underlying cause is atherosclerosis. Atherosclerosis is characterized as a chronic inflammatory disease of the arterial wall, which involves both the innate and adaptive immune responses (Chavez-Sanchez et al., 2014). Natural killer (NK) cells are important components of innate immune system. NK cells in human can be divided into two subtypes: CD56dim NK cells which display potent cytolytic activity and CD56bright NK cells which can produce cytokines (Bonaccorsi et al., 2015). The proportion and cytotoxicity of NK cells decreased in peripheral blood of atherosclerotic patients, which is the same as that in autoimmune diseases (Backteman et al., 2012; Hou et al., 2012; Jonasson et al., 2005). However, the effects of NK cells in atherosclerosis are controversial (Bonaccorsi et al., 2015; Schiller et al., 2002; Selathurai et al., 2014). The natural killer T (NKT) cells own some properties in common with both conventional T cells and NK cells, and they can produce both proand anti-inflammatory cytokines upon activation. NKT cells were detected in atherosclerotic plaques and the number of NKT cells decreased in the peripheral blood of patients with cardiovascular diseases (Bondarenko et al., 2014; Rombouts et al., 2016). Once activated, NK cells and NKT cells can produce IFN-γ and many other cytokines
⁎
(VanderLaan et al., 2007). IFN-γ is regarded as an atherogenic cytokine. T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) is a member of Tim proteins which are type 1 transmembrane proteins, and is expressed on many immune cells (Foks et al., 2013). The expressions of Tim-3 were up-regulated on peripheral NKT cells in chronic hepatitis B patients (Rong et al., 2014). In many diseases, the expressions of Tim-3 on NK cells were found increased accompanied by the decreased cytotoxicity and cytokines production of NK cells (da Silva et al., 2014; Hou et al., 2014, 2012; Wang et al., 2015; Xu et al., 2015, 2014). The expressions of Tim-3 on NK cells were up-regulated in atherosclerotic patients and LDLr−/− mice (Foks et al., 2013; Hou et al., 2012). So, Tim-3 on NK cells and NKT cells may participate in inflammatory diseases as well as atherosclerosis. Statins are the inhibitors of HMG-CoA reductase. They can reduce the levels of total cholesterol and low density lipoprotein (LDL) (Park et al., 2016). Statins alleviate atherosclerosis by not only the lipidlowering activity but also its immunomodulatory effect. Statins can increase the frequency of NK cells, while their effects on the cytotoxicity of NK cells are controversial (Hillyard et al., 2004, 2007; Jonasson et al., 2005; Raemer et al., 2009). Both the negative and positive effects of statins on IFN-γ were reported (Coward et al., 2006; Jameel et al., 2013). Whether statins can influence the expressions of Tim-3 on NK cells and NKT cells remains unclear.
Correspondence to: Department of Neurology, Shandong Provincial Qianfoshan Hospital, Shandong University, No. 16766, Jingshi Road, Jinan, Shandong 250014, PR China. E-mail address: [email protected] (R.-S. Duan).
https://doi.org/10.1016/j.ejphar.2017.12.050 Received 1 May 2017; Received in revised form 20 December 2017; Accepted 21 December 2017 Available online 27 December 2017 0014-2999/ © 2017 Elsevier B.V. All rights reserved.
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into the clinic. In the study, 14 atherosclerotic patients with rosuvastatin treatment (10 mg for 2 patients and 20 mg for the rest patients per day for more than 3 months) (Astrazeneca, England), 12 patients with atorvastatin treatment (5 mg for 1 patient and 10 mg for the rest patients per day for more than 3 months) (Pfizer pharmaceutical company, United States) were included, and 20 atherosclerotic patients without any statin treatment were included as control. The overnight fasting venous blood samples of patients were collected with EDTA-anticoagulant tubes (BD, Franklin Lakes, NJ, USA). Table 1 shows the demographic characteristics of patients included in the study. The sample sizes were determined according to the formula
Table 1 Demographic characteristics of patients included in the study.
Male, n (%) Age, y Diabetes, n (%)
Control (n = 20)
Rosuvastatin (n = 14)
Atorvastatin (n = 12)
P Value
15 (75) 64.0 ± 1.9 4 (20)
11 (78) 63.3 ± 2.4 3 (21)
8 (67) 67.1 ± 2.3 4 (33)
0.78 0.48 0.67
Mean value was expressed as mean ± S.E.M. For continuous variables comparison, ANOVA was performed among three groups; χ2 tests were performed for categorical variables.
n = ψ2
In the present study, we explored the effects of statins on the proportions of NK cells and their subtypes, NKT cells and CD3+ T cells, the expressions of Tim-3 and productions of IFN-γ, and analyzed the levels of total cholesterol, LDL, high density lipoprotein (HDL), as well as the ratios of HDL to cholesterol.
∑ik Si2 / k ∑ik (Xi − X )2 / (k − 1)
(n is on behalf of the sample size. Xi and Si rek
present the mean and standard deviation of sample i . X = ∑i Xi / k . k is the number of groups. ψ is got from the corresponding table. α = 0.05, β = 0.10 . v1 = k − 1, v2 = ∞. Get the corresponding ψ from the table to calculate n1 using the formula. Then get new ψ according to v1 = k −1, v2 = k (n (1) −1) , and calculate n2 using the formula. Repeat the calculation until the result is stable. In this study, k equals 3. According to the calculation, n equals 8). The study was approved by the Ethics Committee of Shandong Provincial Qianfoshan Hospital and each participant was informed about the study.
2. Materials and methods 2.1. Patients included in the trials The atherosclerotic patients demonstrated with image or ultrasound, 35–79 years old, were included from Shandong Provincial Qianfoshan Hospital between January and December of 2016. Patients were excluded for any of the following reasons at the time of screening: history of myocardial infarction or cerebral infarction within 3 months, congestive heart failure, renal failure and dialysis treatment, infections (e.g., acute and chronic viral infection, respiratory infection, intestinal infection), dental problems, connective tissue diseases, autoimmune diseases, any anti-inflammatory treatment, malignancy, any types of surgery in the past 3 months, definite hypersensitivity or contraindication to statins, pregnancy, drug or alcohol abuse, inability to walk
2.2. The study protocol 1 ml anticoagulant venous blood was mixed with 1 ml phosphate buffered saline (PBS) and the peripheral blood mononuclear cells (PBMCs) of the diluted blood were extracted using Ficoll density gradient centrifugation (Dakewe, Shenzhen, China) according to the instruction. The PBMCs were separated into two parts. One part of PBMCs was incubated with FITC-conjugated anti-human CD3 antibody (eBioscience, San Diego, CA, USA), PerCP-conjugated Fig. 1. Representative dot plots of peripheral blood show the gating strategy of different lymphocyte populations. The CD3-CD56+ cells were gated as NK cells and the CD3+CD56+ cells were gated as NKT cells. NK cells were divided into CD56dim NK cells and CD56bright NK cells according to the levels of CD56 expressed on them.
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Fig. 2. Statins treatment decreased the percentages of Tim-3+ cells among NK cells and their subtypes. The histograms show means ± S.D. (D and E). Box and whisker plots (F) show median and interquartile range (25th and 75th percentiles) in the box, and the whiskers represent the minimum and maximum values. (** P < 0.01)
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Fig. 3. Statins treatment decreased the MFI of Tim-3 on NK cells and their subtypes. Boxes and whisker plots (D-F) show median and interquartile range (25th and 75th percentiles) in the box, and the whiskers represent the minimum and maximum values. (* P < 0.05 and ** P < 0.01)
One-way ANOVA analysis was used to analyze the continuous variable data that were conformed to normality and homogeneity of variance. The rest continuous variable data were analyzed with non-parametric test (Kruskal-Wallis one-way ANOVA). The categorical variable data were analyzed with chi-square test. P value was considered significant at P < 0.05.
anti-human CD56 antibody (BioLegend, San Diego, CA, USA), and APCconjugated anti-human Tim-3 antibody (BioLegend) for 30 min at 4 °C in a dark room. Then the cells were washed and detected using the flow cytometer (FACSAria II cell sorter of BD). The other part was incubated with 1 μl of cell stimulation cocktail (plus protein transport inhibitors) (500×) (eBioscience) for 2 h in 500 μl complete medium (RPMI 1640 (HyClone, Beijing, China) medium containing 1% (v/v) penicillin-streptomycin (containing 10,000 IU/ml penicillin and 10,000 μg/ml streptomycin; Hyclone, Logan, UT, USA) and 10% fetal bovine serum (FBS; Gibco, Grand Land, NY, USA)) and then collected. The cells were incubated with FITCconjugated anti-human CD3 antibody (eBioscience), PerCP-conjugated anti-human CD56 antibody (BioLegend) for 30 min at 4 °C in a dark room. Then cells were washed and fixed with 2% paraformaldehyde, permeablized with the intracellular staining perm wash buffer (10×) (BioLegend) and incubated with PE-conjugated anti-human IFN-γ antibody (eBioscience) for 30 min at 4 ℃ in a dark room. Finally, the cells were washed and detected using the flow cytometer. The patients included in this study were detected the levels of total cholesterol, LDL, HDL, as well as the ratios of HDL to cholesterol in the peripheral venous blood by the clinical laboratory (the data of two patients after atorvastatin treatment were not available). So, in this section, 10 patients with atorvastatin treatment (10 mg per day for more than 3 months) were included, and the numbers of patients in the other two groups were the same as above.
3. Results 3.1. Statins administration reduced the percentages of Tim-3+ cells among NK cells and their subtypes We gated the various lymphocyte populations as showed in Fig. 1. Compared with control, both rosuvastatin and atorvastatin treatment reduced the percentages of Tim-3+ cells among NK cells (P ＜ 0.01 for both comparisons, Fig. 2 A and D), CD56dim NK cells (P ＜ 0.01 for both comparisons, Fig. 2 B and E) and CD56bright NK cells (P ＜ 0.01 for both comparisons, Fig. 2 C and F). Meanwhile, both rosuvastatin and atorvastatin reduced the mean fluorescence intensity (MFI) of Tim-3 on the surface of NK cells (P ＜ 0.05 for both comparisons, Fig. 3 A and D), CD56dim NK cells (P ＜ 0.05 for both comparisons, Fig. 3 B and E) and CD56bright NK cells (P ＜ 0.01 for both comparisons, Fig. 3 C and F). However, there was no difference in the expressions of Tim-3 on NK cells and their subtypes between rosuvastatin and atorvastatin treatment (Fig. 2D–F and Fig. 3D–F). In addition, both statins treatment did not change the percentages of NK cells and their subtypes (data not show).
2.3. Statistical analysis All data were analyzed using SPSS 20 (SPSS Inc., Chicago, IL, USA). 52
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Fig. 4. Statins treatment increased the numbers of NKT cells and decreased the percentages of Tim-3+ cells among NKT cells. (C) The comparison of the percentages of NKT cells among PBMCs in three groups. Box and whisker plots show median and interquartile range (25th and 75th percentiles) in the box, and the whiskers represent the minimum and maximum values. (D) The comparison of the percentages of Tim-3+ cells among NKT cells in three groups (mean + S.D.). (* P < 0.05 and *** P < 0.001)
3.2. Statins administration reduced the percentages of Tim-3+ cells among NKT cells and CD3+ T cells, and increased the percentages of NKT cells among PBMCs
3.3. Statins administration did not change the productions of IFN-γ by NK, NKT and CD3+ T cells We detected the productions of IFN-γ by NK, NKT and CD3+ T cells using flow cytometry. There was no difference in the productions of IFN-γ among the three groups (data not show).
We detected the percentages of NKT cells among PBMCs (Fig. 4 A) and expressions of Tim-3 on NKT cells and CD3+ T cells (Fig. 4 B and Fig. 5 A). The percentages of NKT cells among PBMCs increased in patients with atorvastatin treatment compared those in the control (P ＜ 0.05, Fig. 4 C). Rosuvastatin treatment increased the NKT cells but without statistical significance (0.05 ＜ P ＜ 0.1). Both statins administration reduced the percentages of Tim-3+ cells among NKT cells (P ＜ 0.001 for both comparisons, Fig. 4 D) and CD3+ T cells compared to control (P ＜ 0.001 for rosuvastatin and P ＜ 0.01 for atorvastatin, Fig. 5 B).
3.4. Statins administration reduced the levels of total cholesterol and LDL, increased the ratios of HDL to cholesterol We analyzed the levels of total cholesterol, LDL, HDL, the ratios of HDL to cholesterol. Both rosuvastatin and atorvastatin reduced the levels of total cholesterol (P ＜ 0.001 for rosuvastatin and P ＜ 0.05 for atorvastatin, Fig. 6 A) and levels of LDL (P ＜ 0.001 for rosuvastatin and 53
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Fig. 5. Statins treatment decreased the percentages of Tim-3+ cells among CD3+ T cells. (B) The comparisons of the percentages of Tim-3+ cells among CD3+ T cells in three groups. Box and whisker plots show median and interquartile range (25th and 75th percentiles) in the box, and the whiskers represent the minimum and maximum values. (** P < 0.01 and *** P < 0.001) Fig. 6. Statins treatment decreased the levels of total cholesterol and LDL, increased the ratios of HDL to cholesterol. Boxes and whisker plots (A-C) show median and interquartile range (25th and 75th percentiles) in the box, and the whiskers represent the minimum and maximum values. (* P < 0.05, ** P < 0.01 and *** P < 0.001)
P ＜ 0.05 for atorvastatin, Fig. 6 B). Both rosuvastatin and atorvastatin increased the ratios of HDL to cholesterol (P ＜ 0.001 for rosuvastatin and P ＜ 0.01 for atorvastatin, Fig. 6 C). There was no difference in the levels of HDL among the three groups (data not show).
4. Discussion In the present study, we found that statins reduced the proportions of Tim-3+ cells among peripheral NK cells and their subtypes, NKT cells 54
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and CD3+ T cells in atherosclerotic patients, increased the proportions of NKT cells among PBMCs, but had no effect on the IFN-γ productions of NK, NKT and CD3+ T cells. Statins reduced the levels of total cholesterol and LDL, increased the ratios of HDL to cholesterol. Atherosclerosis is characterized as a chronic inflammatory disease with the involvement of various immune cells and cytokines (ChavezSanchez et al., 2014). To a certain degree, atherosclerosis is considered as an autoimmune disease (Govea-Alonso et al., 2016). The decreased frequencies of NK cells and NKT cells were found in some autoimmune diseases, accompanied with the decreased cytotoxicity of NK cells, especially in the active periods of diseases (Chalan et al., 2016; Chien et al., 2011; Gross et al., 2016; Popko and Gorska, 2015; Suzuki et al., 2005). NK cells were considered to play an immunomodulatory role in autoimmune diseases, because different effects of NK cells were found in different studies. Therefore, NK cells were regarded as a two edged weapon and their effects depended on the different NK cell subtypes, different inflammation microenvironments and different stages of diseases (Zhang and Tian, 2017). Similarly, the frequencies of NK cells, NKT cells and the cytotoxicity of NK cells were found to be decreased in the peripheral blood of atherosclerotic patients (Backteman et al., 2012; Hou et al., 2012; Jonasson et al., 2005). However, the effects of NK cells in animal experiments are inconsistent. Atherosclerosis was alleviated in high-fat diet fed ApoE-/- mice after the depletion of NK cells with anti-Asialo-GM1 antibodies, and NK cells were found to aggravate atherosclerosis by cytotoxicity after the transfer of IFN-γ-deficient NK cells, perforin and granzyme B-deficient NK cells respectively (Bonaccorsi et al., 2015; Selathurai et al., 2014). On the contrary, NK cells were also reported to be anti-atherogenic, which might depend on the cytokines, but not the cytotoxicity of NK cells (Bonaccorsi et al., 2015; Schiller et al., 2002). These different results were probably due to the different roles played by NK cells and their subtypes in the different stages of atherosclerosis, which is the same as that in other autoimmune diseases. Accumulated evidence indicated that IFN-γ is a pivotal cytokine secreted by NK cells and plays an important role in the development of atherosclerosis. IFN-γ could destabilize plaques and exacerbate atherosclerosis by inducing the apoptosis of smooth muscle cells and/or secretion of matrix metalloproteinase (MMP) (Bonaccorsi et al., 2015). IFN-γ deficiency reduced the development of atherosclerotic lesion significantly and restoration of IFN-γ induced lesion development in ApoE-/- mice (Fogg et al., 2006). So we chose IFN-γ as the cytokine to be analyzed in this study. NKT cells can recognize glycolipid antigens presented by CD1d on antigen presenting cells. They can produce IFN-γ, IL-10 and other cytokines after activation. CD4+ NKT cells promoted atherosclerosis with perforin and granzyme B in animals (Li et al., 2015). Using CD1d-dependent lipid antagonist of NKT cells (DPPEPEG350) in ApoE(-/-) mice could reduce atherosclerosis development and delay progression of established atherosclerosis, and using α-galactosylceramide (α-GalCer), an activator of NKT cells, enlarged lesions of atherosclerosis (Braun et al., 2010; Li et al., 2016). However, CD1d dependent NKT cells were also reported to only play a role in the initiation of atherosclerosis and their effect was transient (Aslanian et al., 2005). Furthermore, in the LDL-/- mice treated with α-GalCer, the plaque size was decreased, the proportions of CD3+IL-10+ T cells in spleen and lymph node, and the production of IL-10 were increased, while no changes were found in CD3+IFN-γ+ T cells and IFN-γ production (van Puijvelde et al., 2009). NKT cells were also found to be pro-atherogenic and anti-atherogenic before and after the formation of lesions. So, the effects of NKT cells on atherosclerosis are controversial. Statins can inhibit the cytotoxicity of NK cells by inhibiting isoprenylation, interfering with lymphocyte function associated antigen (LFA)-1-mediated conjugate formation and depleting membrane raft (Hillyard et al., 2004, 2007; Raemer et al., 2009). But statins were also found to enhance the cytotoxicity of NK cells (Jonasson et al., 2005). Tim-3 is constitutively expressed on NK cells. In tumors and infectious diseases, the expressions of Tim-3 on NK cells and NKT cells were upregulated, accompanied by the decreased cytokines production and
cytotoxicity of NK cells (da Silva et al., 2014; Hou et al., 2014; Rong et al., 2014; Wang et al., 2015; Xu et al., 2015, 2014). The expressions of Tim-3 on NK cells were increased obviously in peripheral blood of atherosclerotic patients, which implied the involvement of Tim-3 in atherosclerosis. Our study revealed that statins decreased the expressions of Tim-3 on NK cells and their subtypes, NKT cells and CD3+ T cells. Statins also decreased expressions of Tim-3 on T cells in the HIVinfected individuals (Overton et al., 2014). So we think the effects of statins on the expressions of Tim-3 on NK cells, NKT cells and T cells are non-specific. In our study, we found that statins increased the proportions of NKT cells, but without any effect on the proportions of NK cells and their subtypes as well as the productions of IFN-γ by NK, NKT and CD3+ T cells. Our analysis also verified that statins reduced the levels of total cholesterol and LDL. These demonstrated that statins treatment reduced Tim-3 expressions and increased the proportions of NKT cells in atherosclerotic patients in addition to modulating the lipids. In summary, statins can reduce the expressions of Tim-3 on NK cells and their subtypes, NKT cells and CD3+ T cells, increase the proportions of NKT cells, without affecting the productions of IFN-γ, which probably contribute to the anti-atherogenic effect of statins in atherosclerosis treatment. However, the exact mechanisms behind the phenomena need to be further studied. Acknowledgment This work was supported by Taishan Scholars Construction Engineering of Shandong Province (ts20130914) and partially supported by grants from the National Natural Science Foundation of China (81471222). Competing interests All authors declare no competing interests. References Aslanian, A.M., Chapman, H.A., Charo, I.F., 2005. Transient role for CD1d-restricted natural killer T cells in the formation of atherosclerotic lesions. Arterioscler. Thromb. Vasc. Biol. 25, 628–632. Backteman, K., Andersson, C., Dahlin, L.G., Ernerudh, J., Jonasson, L., 2012. Lymphocyte subpopulations in lymph nodes and peripheral blood: a comparison between patients with stable angina and acute coronary syndrome. PLoS One 7, e32691. Bonaccorsi, I., De Pasquale, C., Campana, S., Barberi, C., Cavaliere, R., Benedetto, F., Ferlazzo, G., 2015. Natural killer cells in the innate immunity network of atherosclerosis. Immunol. Lett. 168, 51–57. Bondarenko, S., Catapano, A.L., Norata, G.D., 2014. The CD1d-natural killer T cell axis in atherosclerosis. J. Innate Immun. 6, 3–12. Braun, N.A., Covarrubias, R., Major, A.S., 2010. Natural killer T cells and atherosclerosis: form and function meet pathogenesis. J. Innate Immun. 2, 316–324. Chalan, P., Bijzet, J., Kroesen, B.J., Boots, A.M., Brouwer, E., 2016. Altered natural killer cell subsets in seropositive arthralgia and early rheumatoid arthritis are associated with autoantibody status. J. Rheumatol. 43, 1008–1016. Chavez-Sanchez, L., Espinosa-Luna, J.E., Chavez-Rueda, K., Legorreta-Haquet, M.V., Montoya-Diaz, E., Blanco-Favela, F., 2014. Innate immune system cells in atherosclerosis. Arch. Med. Res. 45, 1–14. Chien, P.J., Yeh, J.H., Chiu, H.C., Hsueh, Y.M., Chen, C.T., Chen, M.C., Shih, C.M., 2011. Inhibition of peripheral blood natural killer cell cytotoxicity in patients with myasthenia gravis treated with plasmapheresis. Eur. J. Neurol. 18, 1350–1357. Coward, W.R., Marei, A., Yang, A., Vasa-Nicotera, M.M., Chow, S.C., 2006. Statin-induced proinflammatory response in mitogen-activated peripheral blood mononuclear cells through the activation of caspase-1 and IL-18 secretion in monocytes. J. Immunol. 176, 5284–5292. da Silva, I.P., Gallois, A., Jimenez-Baranda, S., Khan, S., Anderson, A.C., Kuchroo, V.K., Osman, I., Bhardwaj, N., 2014. Reversal of NK-cell exhaustion in advanced melanoma by Tim-3 blockade. Cancer Immunol. Res. 2, 410–422. Fogg, D.K., Sibon, C., Miled, C., Jung, S., Aucouturier, P., Littman, D.R., Cumano, A., Geissmann, F., 2006. A clonogenic bone marrow progenitor specific for macrophages and dendritic cells. Science 311, 83–87. Foks, A.C., Ran, I.A., Wasserman, L., Frodermann, V., Ter Borg, M.N., de Jager, S.C., van Santbrink, P.J., Yagita, H., Akiba, H., Bot, I., Kuiper, J., van Puijvelde, G.H., 2013. Tcell immunoglobulin and mucin domain 3 acts as a negative regulator of atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 33, 2558–2565. Govea-Alonso, D.O., Beltran-Lopez, J., Salazar-Gonzalez, J.A., Vargas-Morales, J., Rosales-Mendoza, S., 2016. Progress and future opportunities in the development of vaccines against atherosclerosis. Expert Rev. Vaccin. 1–14.
55
European Journal of Pharmacology 821 (2018) 49–56
N. Zhang et al.
blind, randomized study. J. Am. Coll. Cardiol. 67, 1772–1783. Popko, K., Gorska, E., 2015. The role of natural killer cells in pathogenesis of autoimmune diseases. Cent.-Eur. J. Immunol. 40, 470–476. Raemer, P.C., Kohl, K., Watzl, C., 2009. Statins inhibit NK-cell cytotoxicity by interfering with LFA-1-mediated conjugate formation. Eur. J. Immunol. 39, 1456–1465. Rombouts, M., Ammi, R., Van Brussel, I., Roth, L., De Winter, B.Y., Vercauteren, S.R., Hendriks, J.M., Lauwers, P., Van Schil, P.E., De Meyer, G.R., Fransen, E., Cools, N., Schrijvers, D.M., 2016. Linking CD11b (+) dendritic cells and natural killer T cells to plaque inflammation in atherosclerosis. Mediat. Inflamm. 2016, 6467375. Rong, Y.H., Wan, Z.H., Song, H., Li, Y.L., Zhu, B., Zang, H., Zhao, Y., Liu, H.L., Zhang, A.M., Xiao, L., Xin, S.J., You, S.L., 2014. Tim-3 expression on peripheral monocytes and CD3+CD16/CD56+natural killer-like T cells in patients with chronic hepatitis B. Tissue Antigens 83, 76–81. Schiller, N.K., Boisvert, W.A., Curtiss, L.K., 2002. Inflammation in atherosclerosis: lesion formation in LDL receptor-deficient mice with perforin and Lyst(beige) mutations. Arterioscler. Thromb. Vasc. Biol. 22, 1341–1346. Selathurai, A., Deswaerte, V., Kanellakis, P., Tipping, P., Toh, B.H., Bobik, A., Kyaw, T., 2014. Natural killer (NK) cells augment atherosclerosis by cytotoxic-dependent mechanisms. Cardiovasc. Res. 102, 128–137. Suzuki, Y., Onodera, H., Tago, H., Saito, R., Ohuchi, M., Shimizu, M., Itoyama, Y., 2005. Altered populations of natural killer cell and natural killer T cell subclasses in myasthenia gravis. J. Neuroimmunol. 167, 186–189. van Puijvelde, G.H., van Wanrooij, E.J., Hauer, A.D., de Vos, P., van Berkel, T.J., Kuiper, J., 2009. Effect of natural killer T cell activation on the initiation of atherosclerosis. Thromb. Haemost. 102, 223–230. VanderLaan, P.A., Reardon, C.A., Sagiv, Y., Blachowicz, L., Lukens, J., Nissenbaum, M., Wang, C.R., Getz, G.S., 2007. Characterization of the natural killer T-cell response in an adoptive transfer model of atherosclerosis. Am. J. Pathol. 170, 1100–1107. Wang, F., Hou, H., Wu, S., Tang, Q., Huang, M., Yin, B., Huang, J., Liu, W., Mao, L., Lu, Y., Sun, Z., 2015. Tim-3 pathway affects NK cell impairment in patients with active tuberculosis. Cytokine 76, 270–279. Xu, L., Huang, Y., Tan, L., Yu, W., Chen, D., Lu, C., He, J., Wu, G., Liu, X., Zhang, Y., 2015. Increased Tim-3 expression in peripheral NK cells predicts a poorer prognosis and Tim-3 blockade improves NK cell-mediated cytotoxicity in human lung adenocarcinoma. Int. Immunopharmacol. 29, 635–641. Xu, L.Y., Chen, D.D., He, J.Y., Lu, C.C., Liu, X.G., Le, H.B., Wang, C.Y., Zhang, Y.K., 2014. Tim-3 expression by peripheral natural killer cells and natural killer T cells increases in patients with lung cancer–reduction after surgical resection. Asian Pac. J. Cancer Prev.: APJCP 15, 9945–9948. Zhang, C., Tian, Z., 2017. NK cell subsets in autoimmune diseases. J. Autoimmun.
Gross, C.C., Schulte-Mecklenbeck, A., Runzi, A., Kuhlmann, T., Posevitz-Fejfar, A., Schwab, N., Schneider-Hohendorf, T., Herich, S., Held, K., Konjevic, M., Hartwig, M., Dornmair, K., Hohlfeld, R., Ziemssen, T., Klotz, L., Meuth, S.G., Wiendl, H., 2016. Impaired NK-mediated regulation of T-cell activity in multiple sclerosis is reconstituted by IL-2 receptor modulation. Proc. Natl. Acad. Sci. USA 113, E2973–E2982. Hillyard, D.Z., Cameron, A.J., McDonald, K.J., Thomson, J., MacIntyre, A., Shiels, P.G., Panarelli, M., Jardine, A.G., 2004. Simvastatin inhibits lymphocyte function in normal subjects and patients with cardiovascular disease. Atherosclerosis 175, 305–313. Hillyard, D.Z., Nutt, C.D., Thomson, J., McDonald, K.J., Wan, R.K., Cameron, A.J., Mark, P.B., Jardine, A.G., 2007. Statins inhibit NK cell cytotoxicity by membrane raft depletion rather than inhibition of isoprenylation. Atherosclerosis 191, 319–325. Hou, H., Liu, W., Wu, S., Lu, Y., Peng, J., Zhu, Y., Lu, Y., Wang, F., Sun, Z., 2014. Tim-3 negatively mediates natural killer cell function in LPS-induced endotoxic shock. PLoS One 9, e110585. Hou, N., Zhao, D., Liu, Y., Gao, L., Liang, X., Liu, X., Gai, X., Zhang, X., Zhu, F., Ni, M., Zhang, Y., Sun, W., Ma, C., 2012. Increased expression of T cell immunoglobulin- and mucin domain-containing molecule-3 on natural killer cells in atherogenesis. Atherosclerosis 222, 67–73. Jameel, A., Ooi, K.G., Jeffs, N.R., Galatowicz, G., Lightman, S.L., Calder, V.L., 2013. Statin Modulation of human T-cell proliferation, IL-1beta and IL-17 production, and IFNgamma T cell expression: synergy with conventional immunosuppressive agents. Int. J. Inflamm. 2013, 434586. Jonasson, L., Backteman, K., Ernerudh, J., 2005. Loss of natural killer cell activity in patients with coronary artery disease. Atherosclerosis 183, 316–321. Li, Y., Kanellakis, P., Hosseini, H., Cao, A., Deswaerte, V., Tipping, P., Toh, B.H., Bobik, A., Kyaw, T., 2016. A CD1d-dependent lipid antagonist to NKT cells ameliorates atherosclerosis in ApoE-/- mice by reducing lesion necrosis and inflammation. Cardiovasc. Res. 109, 305–317. Li, Y., To, K., Kanellakis, P., Hosseini, H., Deswaerte, V., Tipping, P., Smyth, M.J., Toh, B.H., Bobik, A., Kyaw, T., 2015. CD4+ natural killer T cells potently augment aortic root atherosclerosis by perforin- and granzyme B-dependent cytotoxicity. Circ. Res. 116, 245–254. Overton, E.T., Sterrett, S., Westfall, A.O., Kahan, S.M., Burkholder, G., Zajac, A.J., Goepfert, P.A., Bansal, A., 2014. Effects of atorvastatin and pravastatin on immune activation and T-cell function in antiretroviral therapy-suppressed HIV-1-infected patients. Aids 28, 2627–2631. Park, S.J., Kang, S.J., Ahn, J.M., Chang, M., Yun, S.C., Roh, J.H., Lee, P.H., Park, H.W., Yoon, S.H., Park, D.W., Lee, S.W., Kim, Y.H., Lee, C.W., Mintz, G.S., Han, K.H., Park, S.W., 2016. Effect of statin treatment on modifying plaque composition: a double-
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Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
Statins reduce the expressions of Tim-3 on NK cells and NKT cells in atherosclerosis
T
Na Zhanga, Min Zhanga, Ru-Tao Liua, Peng Zhanga, Chun-Lin Yanga, Long-Tao Yueb, Heng Lia, ⁎ Yong-Kang Lic, Rui-Sheng Duana, a
Department of Neurology, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014, PR China Central laboratory, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014, PR China c Department of Cardiology, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014, PR China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Statin Atherosclerosis Natural killer cell Natural killer T cell Tim-3
3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors (statins) have an immuno-regulatory effect in addition to lowing-lipids. Accumulated evidence showed that the expressions of T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) on natural killer (NK) cells increased in atherosclerotic patients and animal models. In this study, 14 patients treated with rosuvastatin and 12 patients with atorvastatin for more than 3 months were included and 20 patients without statins treatment as control. Both statins treatment reduced the expressions of Tim-3 on NK cells and their subtypes, natural killer T (NKT) cells and CD3+ T cells, and increased the proportions of NKT cells among peripheral blood mononuclear cells, accompanied by the decreased levels of total cholesterol, low density lipoprotein, and increased ratios of high density lipoprotein to cholesterol. These may contribute to the functions of statins in the treatment of atherosclerosis.
1. Introduction Cardiovascular and cerebrovascular diseases are the main causes of death around the world. Their major underlying cause is atherosclerosis. Atherosclerosis is characterized as a chronic inflammatory disease of the arterial wall, which involves both the innate and adaptive immune responses (Chavez-Sanchez et al., 2014). Natural killer (NK) cells are important components of innate immune system. NK cells in human can be divided into two subtypes: CD56dim NK cells which display potent cytolytic activity and CD56bright NK cells which can produce cytokines (Bonaccorsi et al., 2015). The proportion and cytotoxicity of NK cells decreased in peripheral blood of atherosclerotic patients, which is the same as that in autoimmune diseases (Backteman et al., 2012; Hou et al., 2012; Jonasson et al., 2005). However, the effects of NK cells in atherosclerosis are controversial (Bonaccorsi et al., 2015; Schiller et al., 2002; Selathurai et al., 2014). The natural killer T (NKT) cells own some properties in common with both conventional T cells and NK cells, and they can produce both proand anti-inflammatory cytokines upon activation. NKT cells were detected in atherosclerotic plaques and the number of NKT cells decreased in the peripheral blood of patients with cardiovascular diseases (Bondarenko et al., 2014; Rombouts et al., 2016). Once activated, NK cells and NKT cells can produce IFN-γ and many other cytokines
⁎
(VanderLaan et al., 2007). IFN-γ is regarded as an atherogenic cytokine. T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) is a member of Tim proteins which are type 1 transmembrane proteins, and is expressed on many immune cells (Foks et al., 2013). The expressions of Tim-3 were up-regulated on peripheral NKT cells in chronic hepatitis B patients (Rong et al., 2014). In many diseases, the expressions of Tim-3 on NK cells were found increased accompanied by the decreased cytotoxicity and cytokines production of NK cells (da Silva et al., 2014; Hou et al., 2014, 2012; Wang et al., 2015; Xu et al., 2015, 2014). The expressions of Tim-3 on NK cells were up-regulated in atherosclerotic patients and LDLr−/− mice (Foks et al., 2013; Hou et al., 2012). So, Tim-3 on NK cells and NKT cells may participate in inflammatory diseases as well as atherosclerosis. Statins are the inhibitors of HMG-CoA reductase. They can reduce the levels of total cholesterol and low density lipoprotein (LDL) (Park et al., 2016). Statins alleviate atherosclerosis by not only the lipidlowering activity but also its immunomodulatory effect. Statins can increase the frequency of NK cells, while their effects on the cytotoxicity of NK cells are controversial (Hillyard et al., 2004, 2007; Jonasson et al., 2005; Raemer et al., 2009). Both the negative and positive effects of statins on IFN-γ were reported (Coward et al., 2006; Jameel et al., 2013). Whether statins can influence the expressions of Tim-3 on NK cells and NKT cells remains unclear.
Correspondence to: Department of Neurology, Shandong Provincial Qianfoshan Hospital, Shandong University, No. 16766, Jingshi Road, Jinan, Shandong 250014, PR China. E-mail address: [email protected] (R.-S. Duan).
https://doi.org/10.1016/j.ejphar.2017.12.050 Received 1 May 2017; Received in revised form 20 December 2017; Accepted 21 December 2017 Available online 27 December 2017 0014-2999/ © 2017 Elsevier B.V. All rights reserved.
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into the clinic. In the study, 14 atherosclerotic patients with rosuvastatin treatment (10 mg for 2 patients and 20 mg for the rest patients per day for more than 3 months) (Astrazeneca, England), 12 patients with atorvastatin treatment (5 mg for 1 patient and 10 mg for the rest patients per day for more than 3 months) (Pfizer pharmaceutical company, United States) were included, and 20 atherosclerotic patients without any statin treatment were included as control. The overnight fasting venous blood samples of patients were collected with EDTA-anticoagulant tubes (BD, Franklin Lakes, NJ, USA). Table 1 shows the demographic characteristics of patients included in the study. The sample sizes were determined according to the formula
Table 1 Demographic characteristics of patients included in the study.
Male, n (%) Age, y Diabetes, n (%)
Control (n = 20)
Rosuvastatin (n = 14)
Atorvastatin (n = 12)
P Value
15 (75) 64.0 ± 1.9 4 (20)
11 (78) 63.3 ± 2.4 3 (21)
8 (67) 67.1 ± 2.3 4 (33)
0.78 0.48 0.67
Mean value was expressed as mean ± S.E.M. For continuous variables comparison, ANOVA was performed among three groups; χ2 tests were performed for categorical variables.
n = ψ2
In the present study, we explored the effects of statins on the proportions of NK cells and their subtypes, NKT cells and CD3+ T cells, the expressions of Tim-3 and productions of IFN-γ, and analyzed the levels of total cholesterol, LDL, high density lipoprotein (HDL), as well as the ratios of HDL to cholesterol.
∑ik Si2 / k ∑ik (Xi − X )2 / (k − 1)
(n is on behalf of the sample size. Xi and Si rek
present the mean and standard deviation of sample i . X = ∑i Xi / k . k is the number of groups. ψ is got from the corresponding table. α = 0.05, β = 0.10 . v1 = k − 1, v2 = ∞. Get the corresponding ψ from the table to calculate n1 using the formula. Then get new ψ according to v1 = k −1, v2 = k (n (1) −1) , and calculate n2 using the formula. Repeat the calculation until the result is stable. In this study, k equals 3. According to the calculation, n equals 8). The study was approved by the Ethics Committee of Shandong Provincial Qianfoshan Hospital and each participant was informed about the study.
2. Materials and methods 2.1. Patients included in the trials The atherosclerotic patients demonstrated with image or ultrasound, 35–79 years old, were included from Shandong Provincial Qianfoshan Hospital between January and December of 2016. Patients were excluded for any of the following reasons at the time of screening: history of myocardial infarction or cerebral infarction within 3 months, congestive heart failure, renal failure and dialysis treatment, infections (e.g., acute and chronic viral infection, respiratory infection, intestinal infection), dental problems, connective tissue diseases, autoimmune diseases, any anti-inflammatory treatment, malignancy, any types of surgery in the past 3 months, definite hypersensitivity or contraindication to statins, pregnancy, drug or alcohol abuse, inability to walk
2.2. The study protocol 1 ml anticoagulant venous blood was mixed with 1 ml phosphate buffered saline (PBS) and the peripheral blood mononuclear cells (PBMCs) of the diluted blood were extracted using Ficoll density gradient centrifugation (Dakewe, Shenzhen, China) according to the instruction. The PBMCs were separated into two parts. One part of PBMCs was incubated with FITC-conjugated anti-human CD3 antibody (eBioscience, San Diego, CA, USA), PerCP-conjugated Fig. 1. Representative dot plots of peripheral blood show the gating strategy of different lymphocyte populations. The CD3-CD56+ cells were gated as NK cells and the CD3+CD56+ cells were gated as NKT cells. NK cells were divided into CD56dim NK cells and CD56bright NK cells according to the levels of CD56 expressed on them.
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Fig. 2. Statins treatment decreased the percentages of Tim-3+ cells among NK cells and their subtypes. The histograms show means ± S.D. (D and E). Box and whisker plots (F) show median and interquartile range (25th and 75th percentiles) in the box, and the whiskers represent the minimum and maximum values. (** P < 0.01)
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Fig. 3. Statins treatment decreased the MFI of Tim-3 on NK cells and their subtypes. Boxes and whisker plots (D-F) show median and interquartile range (25th and 75th percentiles) in the box, and the whiskers represent the minimum and maximum values. (* P < 0.05 and ** P < 0.01)
One-way ANOVA analysis was used to analyze the continuous variable data that were conformed to normality and homogeneity of variance. The rest continuous variable data were analyzed with non-parametric test (Kruskal-Wallis one-way ANOVA). The categorical variable data were analyzed with chi-square test. P value was considered significant at P < 0.05.
anti-human CD56 antibody (BioLegend, San Diego, CA, USA), and APCconjugated anti-human Tim-3 antibody (BioLegend) for 30 min at 4 °C in a dark room. Then the cells were washed and detected using the flow cytometer (FACSAria II cell sorter of BD). The other part was incubated with 1 μl of cell stimulation cocktail (plus protein transport inhibitors) (500×) (eBioscience) for 2 h in 500 μl complete medium (RPMI 1640 (HyClone, Beijing, China) medium containing 1% (v/v) penicillin-streptomycin (containing 10,000 IU/ml penicillin and 10,000 μg/ml streptomycin; Hyclone, Logan, UT, USA) and 10% fetal bovine serum (FBS; Gibco, Grand Land, NY, USA)) and then collected. The cells were incubated with FITCconjugated anti-human CD3 antibody (eBioscience), PerCP-conjugated anti-human CD56 antibody (BioLegend) for 30 min at 4 °C in a dark room. Then cells were washed and fixed with 2% paraformaldehyde, permeablized with the intracellular staining perm wash buffer (10×) (BioLegend) and incubated with PE-conjugated anti-human IFN-γ antibody (eBioscience) for 30 min at 4 ℃ in a dark room. Finally, the cells were washed and detected using the flow cytometer. The patients included in this study were detected the levels of total cholesterol, LDL, HDL, as well as the ratios of HDL to cholesterol in the peripheral venous blood by the clinical laboratory (the data of two patients after atorvastatin treatment were not available). So, in this section, 10 patients with atorvastatin treatment (10 mg per day for more than 3 months) were included, and the numbers of patients in the other two groups were the same as above.
3. Results 3.1. Statins administration reduced the percentages of Tim-3+ cells among NK cells and their subtypes We gated the various lymphocyte populations as showed in Fig. 1. Compared with control, both rosuvastatin and atorvastatin treatment reduced the percentages of Tim-3+ cells among NK cells (P ＜ 0.01 for both comparisons, Fig. 2 A and D), CD56dim NK cells (P ＜ 0.01 for both comparisons, Fig. 2 B and E) and CD56bright NK cells (P ＜ 0.01 for both comparisons, Fig. 2 C and F). Meanwhile, both rosuvastatin and atorvastatin reduced the mean fluorescence intensity (MFI) of Tim-3 on the surface of NK cells (P ＜ 0.05 for both comparisons, Fig. 3 A and D), CD56dim NK cells (P ＜ 0.05 for both comparisons, Fig. 3 B and E) and CD56bright NK cells (P ＜ 0.01 for both comparisons, Fig. 3 C and F). However, there was no difference in the expressions of Tim-3 on NK cells and their subtypes between rosuvastatin and atorvastatin treatment (Fig. 2D–F and Fig. 3D–F). In addition, both statins treatment did not change the percentages of NK cells and their subtypes (data not show).
2.3. Statistical analysis All data were analyzed using SPSS 20 (SPSS Inc., Chicago, IL, USA). 52
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Fig. 4. Statins treatment increased the numbers of NKT cells and decreased the percentages of Tim-3+ cells among NKT cells. (C) The comparison of the percentages of NKT cells among PBMCs in three groups. Box and whisker plots show median and interquartile range (25th and 75th percentiles) in the box, and the whiskers represent the minimum and maximum values. (D) The comparison of the percentages of Tim-3+ cells among NKT cells in three groups (mean + S.D.). (* P < 0.05 and *** P < 0.001)
3.2. Statins administration reduced the percentages of Tim-3+ cells among NKT cells and CD3+ T cells, and increased the percentages of NKT cells among PBMCs
3.3. Statins administration did not change the productions of IFN-γ by NK, NKT and CD3+ T cells We detected the productions of IFN-γ by NK, NKT and CD3+ T cells using flow cytometry. There was no difference in the productions of IFN-γ among the three groups (data not show).
We detected the percentages of NKT cells among PBMCs (Fig. 4 A) and expressions of Tim-3 on NKT cells and CD3+ T cells (Fig. 4 B and Fig. 5 A). The percentages of NKT cells among PBMCs increased in patients with atorvastatin treatment compared those in the control (P ＜ 0.05, Fig. 4 C). Rosuvastatin treatment increased the NKT cells but without statistical significance (0.05 ＜ P ＜ 0.1). Both statins administration reduced the percentages of Tim-3+ cells among NKT cells (P ＜ 0.001 for both comparisons, Fig. 4 D) and CD3+ T cells compared to control (P ＜ 0.001 for rosuvastatin and P ＜ 0.01 for atorvastatin, Fig. 5 B).
3.4. Statins administration reduced the levels of total cholesterol and LDL, increased the ratios of HDL to cholesterol We analyzed the levels of total cholesterol, LDL, HDL, the ratios of HDL to cholesterol. Both rosuvastatin and atorvastatin reduced the levels of total cholesterol (P ＜ 0.001 for rosuvastatin and P ＜ 0.05 for atorvastatin, Fig. 6 A) and levels of LDL (P ＜ 0.001 for rosuvastatin and 53
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Fig. 5. Statins treatment decreased the percentages of Tim-3+ cells among CD3+ T cells. (B) The comparisons of the percentages of Tim-3+ cells among CD3+ T cells in three groups. Box and whisker plots show median and interquartile range (25th and 75th percentiles) in the box, and the whiskers represent the minimum and maximum values. (** P < 0.01 and *** P < 0.001) Fig. 6. Statins treatment decreased the levels of total cholesterol and LDL, increased the ratios of HDL to cholesterol. Boxes and whisker plots (A-C) show median and interquartile range (25th and 75th percentiles) in the box, and the whiskers represent the minimum and maximum values. (* P < 0.05, ** P < 0.01 and *** P < 0.001)
P ＜ 0.05 for atorvastatin, Fig. 6 B). Both rosuvastatin and atorvastatin increased the ratios of HDL to cholesterol (P ＜ 0.001 for rosuvastatin and P ＜ 0.01 for atorvastatin, Fig. 6 C). There was no difference in the levels of HDL among the three groups (data not show).
4. Discussion In the present study, we found that statins reduced the proportions of Tim-3+ cells among peripheral NK cells and their subtypes, NKT cells 54
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and CD3+ T cells in atherosclerotic patients, increased the proportions of NKT cells among PBMCs, but had no effect on the IFN-γ productions of NK, NKT and CD3+ T cells. Statins reduced the levels of total cholesterol and LDL, increased the ratios of HDL to cholesterol. Atherosclerosis is characterized as a chronic inflammatory disease with the involvement of various immune cells and cytokines (ChavezSanchez et al., 2014). To a certain degree, atherosclerosis is considered as an autoimmune disease (Govea-Alonso et al., 2016). The decreased frequencies of NK cells and NKT cells were found in some autoimmune diseases, accompanied with the decreased cytotoxicity of NK cells, especially in the active periods of diseases (Chalan et al., 2016; Chien et al., 2011; Gross et al., 2016; Popko and Gorska, 2015; Suzuki et al., 2005). NK cells were considered to play an immunomodulatory role in autoimmune diseases, because different effects of NK cells were found in different studies. Therefore, NK cells were regarded as a two edged weapon and their effects depended on the different NK cell subtypes, different inflammation microenvironments and different stages of diseases (Zhang and Tian, 2017). Similarly, the frequencies of NK cells, NKT cells and the cytotoxicity of NK cells were found to be decreased in the peripheral blood of atherosclerotic patients (Backteman et al., 2012; Hou et al., 2012; Jonasson et al., 2005). However, the effects of NK cells in animal experiments are inconsistent. Atherosclerosis was alleviated in high-fat diet fed ApoE-/- mice after the depletion of NK cells with anti-Asialo-GM1 antibodies, and NK cells were found to aggravate atherosclerosis by cytotoxicity after the transfer of IFN-γ-deficient NK cells, perforin and granzyme B-deficient NK cells respectively (Bonaccorsi et al., 2015; Selathurai et al., 2014). On the contrary, NK cells were also reported to be anti-atherogenic, which might depend on the cytokines, but not the cytotoxicity of NK cells (Bonaccorsi et al., 2015; Schiller et al., 2002). These different results were probably due to the different roles played by NK cells and their subtypes in the different stages of atherosclerosis, which is the same as that in other autoimmune diseases. Accumulated evidence indicated that IFN-γ is a pivotal cytokine secreted by NK cells and plays an important role in the development of atherosclerosis. IFN-γ could destabilize plaques and exacerbate atherosclerosis by inducing the apoptosis of smooth muscle cells and/or secretion of matrix metalloproteinase (MMP) (Bonaccorsi et al., 2015). IFN-γ deficiency reduced the development of atherosclerotic lesion significantly and restoration of IFN-γ induced lesion development in ApoE-/- mice (Fogg et al., 2006). So we chose IFN-γ as the cytokine to be analyzed in this study. NKT cells can recognize glycolipid antigens presented by CD1d on antigen presenting cells. They can produce IFN-γ, IL-10 and other cytokines after activation. CD4+ NKT cells promoted atherosclerosis with perforin and granzyme B in animals (Li et al., 2015). Using CD1d-dependent lipid antagonist of NKT cells (DPPEPEG350) in ApoE(-/-) mice could reduce atherosclerosis development and delay progression of established atherosclerosis, and using α-galactosylceramide (α-GalCer), an activator of NKT cells, enlarged lesions of atherosclerosis (Braun et al., 2010; Li et al., 2016). However, CD1d dependent NKT cells were also reported to only play a role in the initiation of atherosclerosis and their effect was transient (Aslanian et al., 2005). Furthermore, in the LDL-/- mice treated with α-GalCer, the plaque size was decreased, the proportions of CD3+IL-10+ T cells in spleen and lymph node, and the production of IL-10 were increased, while no changes were found in CD3+IFN-γ+ T cells and IFN-γ production (van Puijvelde et al., 2009). NKT cells were also found to be pro-atherogenic and anti-atherogenic before and after the formation of lesions. So, the effects of NKT cells on atherosclerosis are controversial. Statins can inhibit the cytotoxicity of NK cells by inhibiting isoprenylation, interfering with lymphocyte function associated antigen (LFA)-1-mediated conjugate formation and depleting membrane raft (Hillyard et al., 2004, 2007; Raemer et al., 2009). But statins were also found to enhance the cytotoxicity of NK cells (Jonasson et al., 2005). Tim-3 is constitutively expressed on NK cells. In tumors and infectious diseases, the expressions of Tim-3 on NK cells and NKT cells were upregulated, accompanied by the decreased cytokines production and
cytotoxicity of NK cells (da Silva et al., 2014; Hou et al., 2014; Rong et al., 2014; Wang et al., 2015; Xu et al., 2015, 2014). The expressions of Tim-3 on NK cells were increased obviously in peripheral blood of atherosclerotic patients, which implied the involvement of Tim-3 in atherosclerosis. Our study revealed that statins decreased the expressions of Tim-3 on NK cells and their subtypes, NKT cells and CD3+ T cells. Statins also decreased expressions of Tim-3 on T cells in the HIVinfected individuals (Overton et al., 2014). So we think the effects of statins on the expressions of Tim-3 on NK cells, NKT cells and T cells are non-specific. In our study, we found that statins increased the proportions of NKT cells, but without any effect on the proportions of NK cells and their subtypes as well as the productions of IFN-γ by NK, NKT and CD3+ T cells. Our analysis also verified that statins reduced the levels of total cholesterol and LDL. These demonstrated that statins treatment reduced Tim-3 expressions and increased the proportions of NKT cells in atherosclerotic patients in addition to modulating the lipids. In summary, statins can reduce the expressions of Tim-3 on NK cells and their subtypes, NKT cells and CD3+ T cells, increase the proportions of NKT cells, without affecting the productions of IFN-γ, which probably contribute to the anti-atherogenic effect of statins in atherosclerosis treatment. However, the exact mechanisms behind the phenomena need to be further studied. Acknowledgment This work was supported by Taishan Scholars Construction Engineering of Shandong Province (ts20130914) and partially supported by grants from the National Natural Science Foundation of China (81471222). Competing interests All authors declare no competing interests. References Aslanian, A.M., Chapman, H.A., Charo, I.F., 2005. Transient role for CD1d-restricted natural killer T cells in the formation of atherosclerotic lesions. Arterioscler. Thromb. Vasc. Biol. 25, 628–632. Backteman, K., Andersson, C., Dahlin, L.G., Ernerudh, J., Jonasson, L., 2012. Lymphocyte subpopulations in lymph nodes and peripheral blood: a comparison between patients with stable angina and acute coronary syndrome. PLoS One 7, e32691. Bonaccorsi, I., De Pasquale, C., Campana, S., Barberi, C., Cavaliere, R., Benedetto, F., Ferlazzo, G., 2015. Natural killer cells in the innate immunity network of atherosclerosis. Immunol. Lett. 168, 51–57. Bondarenko, S., Catapano, A.L., Norata, G.D., 2014. The CD1d-natural killer T cell axis in atherosclerosis. J. Innate Immun. 6, 3–12. Braun, N.A., Covarrubias, R., Major, A.S., 2010. Natural killer T cells and atherosclerosis: form and function meet pathogenesis. J. Innate Immun. 2, 316–324. Chalan, P., Bijzet, J., Kroesen, B.J., Boots, A.M., Brouwer, E., 2016. Altered natural killer cell subsets in seropositive arthralgia and early rheumatoid arthritis are associated with autoantibody status. J. Rheumatol. 43, 1008–1016. Chavez-Sanchez, L., Espinosa-Luna, J.E., Chavez-Rueda, K., Legorreta-Haquet, M.V., Montoya-Diaz, E., Blanco-Favela, F., 2014. Innate immune system cells in atherosclerosis. Arch. Med. Res. 45, 1–14. Chien, P.J., Yeh, J.H., Chiu, H.C., Hsueh, Y.M., Chen, C.T., Chen, M.C., Shih, C.M., 2011. Inhibition of peripheral blood natural killer cell cytotoxicity in patients with myasthenia gravis treated with plasmapheresis. Eur. J. Neurol. 18, 1350–1357. Coward, W.R., Marei, A., Yang, A., Vasa-Nicotera, M.M., Chow, S.C., 2006. Statin-induced proinflammatory response in mitogen-activated peripheral blood mononuclear cells through the activation of caspase-1 and IL-18 secretion in monocytes. J. Immunol. 176, 5284–5292. da Silva, I.P., Gallois, A., Jimenez-Baranda, S., Khan, S., Anderson, A.C., Kuchroo, V.K., Osman, I., Bhardwaj, N., 2014. Reversal of NK-cell exhaustion in advanced melanoma by Tim-3 blockade. Cancer Immunol. Res. 2, 410–422. Fogg, D.K., Sibon, C., Miled, C., Jung, S., Aucouturier, P., Littman, D.R., Cumano, A., Geissmann, F., 2006. A clonogenic bone marrow progenitor specific for macrophages and dendritic cells. Science 311, 83–87. Foks, A.C., Ran, I.A., Wasserman, L., Frodermann, V., Ter Borg, M.N., de Jager, S.C., van Santbrink, P.J., Yagita, H., Akiba, H., Bot, I., Kuiper, J., van Puijvelde, G.H., 2013. Tcell immunoglobulin and mucin domain 3 acts as a negative regulator of atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 33, 2558–2565. Govea-Alonso, D.O., Beltran-Lopez, J., Salazar-Gonzalez, J.A., Vargas-Morales, J., Rosales-Mendoza, S., 2016. Progress and future opportunities in the development of vaccines against atherosclerosis. Expert Rev. Vaccin. 1–14.
55
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N. Zhang et al.
blind, randomized study. J. Am. Coll. Cardiol. 67, 1772–1783. Popko, K., Gorska, E., 2015. The role of natural killer cells in pathogenesis of autoimmune diseases. Cent.-Eur. J. Immunol. 40, 470–476. Raemer, P.C., Kohl, K., Watzl, C., 2009. Statins inhibit NK-cell cytotoxicity by interfering with LFA-1-mediated conjugate formation. Eur. J. Immunol. 39, 1456–1465. Rombouts, M., Ammi, R., Van Brussel, I., Roth, L., De Winter, B.Y., Vercauteren, S.R., Hendriks, J.M., Lauwers, P., Van Schil, P.E., De Meyer, G.R., Fransen, E., Cools, N., Schrijvers, D.M., 2016. Linking CD11b (+) dendritic cells and natural killer T cells to plaque inflammation in atherosclerosis. Mediat. Inflamm. 2016, 6467375. Rong, Y.H., Wan, Z.H., Song, H., Li, Y.L., Zhu, B., Zang, H., Zhao, Y., Liu, H.L., Zhang, A.M., Xiao, L., Xin, S.J., You, S.L., 2014. Tim-3 expression on peripheral monocytes and CD3+CD16/CD56+natural killer-like T cells in patients with chronic hepatitis B. Tissue Antigens 83, 76–81. Schiller, N.K., Boisvert, W.A., Curtiss, L.K., 2002. Inflammation in atherosclerosis: lesion formation in LDL receptor-deficient mice with perforin and Lyst(beige) mutations. Arterioscler. Thromb. Vasc. Biol. 22, 1341–1346. Selathurai, A., Deswaerte, V., Kanellakis, P., Tipping, P., Toh, B.H., Bobik, A., Kyaw, T., 2014. Natural killer (NK) cells augment atherosclerosis by cytotoxic-dependent mechanisms. Cardiovasc. Res. 102, 128–137. Suzuki, Y., Onodera, H., Tago, H., Saito, R., Ohuchi, M., Shimizu, M., Itoyama, Y., 2005. Altered populations of natural killer cell and natural killer T cell subclasses in myasthenia gravis. J. Neuroimmunol. 167, 186–189. van Puijvelde, G.H., van Wanrooij, E.J., Hauer, A.D., de Vos, P., van Berkel, T.J., Kuiper, J., 2009. Effect of natural killer T cell activation on the initiation of atherosclerosis. Thromb. Haemost. 102, 223–230. VanderLaan, P.A., Reardon, C.A., Sagiv, Y., Blachowicz, L., Lukens, J., Nissenbaum, M., Wang, C.R., Getz, G.S., 2007. Characterization of the natural killer T-cell response in an adoptive transfer model of atherosclerosis. Am. J. Pathol. 170, 1100–1107. Wang, F., Hou, H., Wu, S., Tang, Q., Huang, M., Yin, B., Huang, J., Liu, W., Mao, L., Lu, Y., Sun, Z., 2015. Tim-3 pathway affects NK cell impairment in patients with active tuberculosis. Cytokine 76, 270–279. Xu, L., Huang, Y., Tan, L., Yu, W., Chen, D., Lu, C., He, J., Wu, G., Liu, X., Zhang, Y., 2015. Increased Tim-3 expression in peripheral NK cells predicts a poorer prognosis and Tim-3 blockade improves NK cell-mediated cytotoxicity in human lung adenocarcinoma. Int. Immunopharmacol. 29, 635–641. Xu, L.Y., Chen, D.D., He, J.Y., Lu, C.C., Liu, X.G., Le, H.B., Wang, C.Y., Zhang, Y.K., 2014. Tim-3 expression by peripheral natural killer cells and natural killer T cells increases in patients with lung cancer–reduction after surgical resection. Asian Pac. J. Cancer Prev.: APJCP 15, 9945–9948. Zhang, C., Tian, Z., 2017. NK cell subsets in autoimmune diseases. J. Autoimmun.
Gross, C.C., Schulte-Mecklenbeck, A., Runzi, A., Kuhlmann, T., Posevitz-Fejfar, A., Schwab, N., Schneider-Hohendorf, T., Herich, S., Held, K., Konjevic, M., Hartwig, M., Dornmair, K., Hohlfeld, R., Ziemssen, T., Klotz, L., Meuth, S.G., Wiendl, H., 2016. Impaired NK-mediated regulation of T-cell activity in multiple sclerosis is reconstituted by IL-2 receptor modulation. Proc. Natl. Acad. Sci. USA 113, E2973–E2982. Hillyard, D.Z., Cameron, A.J., McDonald, K.J., Thomson, J., MacIntyre, A., Shiels, P.G., Panarelli, M., Jardine, A.G., 2004. Simvastatin inhibits lymphocyte function in normal subjects and patients with cardiovascular disease. Atherosclerosis 175, 305–313. Hillyard, D.Z., Nutt, C.D., Thomson, J., McDonald, K.J., Wan, R.K., Cameron, A.J., Mark, P.B., Jardine, A.G., 2007. Statins inhibit NK cell cytotoxicity by membrane raft depletion rather than inhibition of isoprenylation. Atherosclerosis 191, 319–325. Hou, H., Liu, W., Wu, S., Lu, Y., Peng, J., Zhu, Y., Lu, Y., Wang, F., Sun, Z., 2014. Tim-3 negatively mediates natural killer cell function in LPS-induced endotoxic shock. PLoS One 9, e110585. Hou, N., Zhao, D., Liu, Y., Gao, L., Liang, X., Liu, X., Gai, X., Zhang, X., Zhu, F., Ni, M., Zhang, Y., Sun, W., Ma, C., 2012. Increased expression of T cell immunoglobulin- and mucin domain-containing molecule-3 on natural killer cells in atherogenesis. Atherosclerosis 222, 67–73. Jameel, A., Ooi, K.G., Jeffs, N.R., Galatowicz, G., Lightman, S.L., Calder, V.L., 2013. Statin Modulation of human T-cell proliferation, IL-1beta and IL-17 production, and IFNgamma T cell expression: synergy with conventional immunosuppressive agents. Int. J. Inflamm. 2013, 434586. Jonasson, L., Backteman, K., Ernerudh, J., 2005. Loss of natural killer cell activity in patients with coronary artery disease. Atherosclerosis 183, 316–321. Li, Y., Kanellakis, P., Hosseini, H., Cao, A., Deswaerte, V., Tipping, P., Toh, B.H., Bobik, A., Kyaw, T., 2016. A CD1d-dependent lipid antagonist to NKT cells ameliorates atherosclerosis in ApoE-/- mice by reducing lesion necrosis and inflammation. Cardiovasc. Res. 109, 305–317. Li, Y., To, K., Kanellakis, P., Hosseini, H., Deswaerte, V., Tipping, P., Smyth, M.J., Toh, B.H., Bobik, A., Kyaw, T., 2015. CD4+ natural killer T cells potently augment aortic root atherosclerosis by perforin- and granzyme B-dependent cytotoxicity. Circ. Res. 116, 245–254. Overton, E.T., Sterrett, S., Westfall, A.O., Kahan, S.M., Burkholder, G., Zajac, A.J., Goepfert, P.A., Bansal, A., 2014. Effects of atorvastatin and pravastatin on immune activation and T-cell function in antiretroviral therapy-suppressed HIV-1-infected patients. Aids 28, 2627–2631. Park, S.J., Kang, S.J., Ahn, J.M., Chang, M., Yun, S.C., Roh, J.H., Lee, P.H., Park, H.W., Yoon, S.H., Park, D.W., Lee, S.W., Kim, Y.H., Lee, C.W., Mintz, G.S., Han, K.H., Park, S.W., 2016. Effect of statin treatment on modifying plaque composition: a double-
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