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Deficiency of programmed cell death 4 affects the balance of T cell subsets in hyperlipidemic mice
Molecular Immunology 112 (2019) 387–393
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Deficiency of programmed cell death 4 affects the balance of T cell subsets in hyperlipidemic mice
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Yang Jianga,1, Qi Gaoa,2, Li-Yang Wanga, Tian Maa, Fa-Liang Zhua, Qun Wanga, Fei Gaob, ⁎ Chun Guoa, Li-Ning Zhanga, a b
Department of Immunology, School of Medicine, Shandong University, Jinan 250012, Shandong, China The Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012, Shandong, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Pdcd4 Atherosclerosis CD8+T cells Tregs Co-stimulatory molecules
Programmed cell death 4 (Pdcd4) was found to be related to apoptosis upon first discovery. It was later found to play the role of tumor suppressor gene in a variety of tumors by inhibiting transcription and translation. Recently, it has been proposed that it may play an important role in some inflammatory diseases and in the immune response. In our previous study, deficiency of Pdcd4 was found to attenuate the formation of atherosclerotic plaques. This might be because deficiency of Pdcd4 may increase IL-10 expression and lipoautophagy by macrophages and attenuate the formation of foam cells. However, the effect of Pdcd4 on the subsets of T cells in hyperlipidemic mice still remained unclear. In the present study, results showed that Pdcd4 deficiency decreased the percentage of CD8+ T cells and increased that of regulatory T cells (Tregs) under hyperlipidemic conditions both in vitro and in vivo, which may be due to the reduced expression of co-stimulatory molecules CD28 and CD137, and the enhancive expression of co-inhibitory molecules CTLA-4. These results indicated that endogenous Pdcd4 promotes immune response mediated by T cells through regulation of the co-stimulatory molecules expression, which may contribute to the development of advanced atherosclerotic plaques. The current work provides new data to understand the role of Pdcd4 in different T cell subsets under hyperlipidemic microenvironment.
1. Introduction Atherosclerosis, which is a chronic, multifactorial, progressive process characterized by an inflammatory response of the arterial wall, is a substantial cause of morbidity and mortality (Steinberg and Witztum, 2010). The process is initialized by hyperlipidemia and intimal deposition of lipid in the arteries and then progression into atherosclerotic plaques (Libby et al., 2011). The immune-inflammatory cells involved in atherosclerosis include monocytes and macrophages, which mediate the innate immune response by handling lipoproteins, release inflammatory cytokines, and work as antigen-presenting cells (APC) to stimulate T cells, and antigen-specific T cells that mediate the adaptive immune response by releasing inflammatory cytokines (Libby, 2012). Accumulating evidence indicates that both CD4+ and CD8+ T cells are involved in atherosclerosis (Hansson and Hermansson, 2011).
CD4+ T cells are the most abundant T cells in atherosclerotic plaques and play a pathogenic role in the progression of atherosclerosis (Zhou et al., 2000). It is well established that Th1 cells promote an inflammatory response and the development of atherosclerosis by producing pro-inflammatory cytokine interferon (IFN)-γ (Moss and Ramji, 2015). Tregs (CD4+FoxP3+) are crucial to mediating immune homeostasis and may have a protective role in the formation of atherogenic plaques (Meng et al., 2012). The contradictory effects of Th2 (Davenport and Tipping, 2003; Engelbertsen et al., 2014; K. Meng et al., 2015), Th17 (Danzaki et al., 2012; Jeon et al., 2015) and CD8+ T cells in atherosclerosis have been also reported, which includes the proatherogenic, anti-atherogenic, or even no influence on the formation of plaques (Kolbus et al., 2012; Kyaw et al., 2013; van Duijn et al., 2018). Programmed cell death 4 (Pdcd4) is a well-known tumor suppressor that inhibits neoplastic transformation, tumor progression, gene
Abbreviations: Pdcd4, Programmed cell death 4; Tregs, Regulatory T cells; APC, Antigen-presenting cells; IFN, Interferon; eIF4A, Translation-initiation-factor-4A; EAE, Encephalomyelitis; CTLA-4, Cytotoxic T-lymphocyte antigen-4; MAPK, Mitogen-activated protein kinase; TCR, T cell receptor ⁎ Corresponding author at: Department of Immunology, School of Medicine, Shandong University, Jinan 250012, Shandong, China. E-mail address: [email protected] (L.-N. Zhang). 1 Present address: Department of Hematology, The Second Hospital of Shandong University, Jinan 250033, Shandong, China. 2 Present address: Department of Clinical Laboratory, Provincial Hospital affiliated with Shandong University, Jinan 250021, Shandong, China. https://doi.org/10.1016/j.molimm.2019.06.020 Received 12 February 2019; Received in revised form 10 June 2019; Accepted 28 June 2019 Available online 06 July 2019 0161-5890/ © 2019 Elsevier Ltd. All rights reserved.
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was calculated.
transcription, and translation (Lankat-Buttgereit and Goke, 2009). Pdcd4 inhibits the translation of mRNA through two channels. One of these channels acts in a translation-initiation-factor-4A (eIF4A)-dependent manner. By binding competitively to eIF4A, Pdcd4 inhibits the combination of RNA helicase eIF4A and scaffold protein eIF4G which initiates the translation of mRNA (Waters et al., 2007). The other channel is eIF4A-independent. Pdcd4 binds directly to mRNA to inhibit its translation (Biyanee et al., 2015). Recent studies have shown that Pdcd4 might also be involved in certain inflammatory diseases. For example, Pdcd4-deficient mice are resistant to autoimmune encephalomyelitis (EAE) (Hilliard et al., 2006), the LPS-induced killing effect (Sheedy et al., 2010), type 1 diabetes (Ruan et al., 2011), dietinduced obesity and adipose tissue inflammation (Wang et al., 2013a), and allergic pulmonary inflammation (Zhong et al., 2014). Our and other groups showed that Pdcd4 deficiency regulated the function of macrophages and involved in the formation of atherosclerotic plaques in Apoe−/− mice (Jiang et al., 2016; Liang et al., 2016; Ye et al., 2016). In addition, Pdcd4 was also found to be involved in macrophage- and Tcell-mediated immune responses. Other studies have indicated that Pdcd4 can inhibit the production of IL-10 by macrophages through Twist2/c-Maf pathway (van den Bosch et al., 2014). There are changes of T cell-related cytokines, such as IL-2, IL-4, and IL-17, in Pdcd4 deficient lymphoma and diabetic mice models, but the mechanism underlying these changes is not clear (Hilliard et al., 2006; Ruan et al., 2011). This study showed that knocking out Pdcd4 in hyperlipidemic mice decreased the percentage of CD8+ T cells and increased the percentage of Tregs both in vitro and in vivo. It is possible that Pdcd4 deficiency upregulate the expression of co-stimulatory molecules CD28 and CD137 and downregulate the expression of the co-inhibitory molecule cytotoxic T-lymphocyte antigen (CTLA)-4.
2.4. Flow cytometry Splenocytes from 16- or 24-week-old mice were separated and stained directly with PECy5-conjugated anti-CD4 Ab (H129.19; BD Pharmingen), FITC-conjugated anti-CD8 Ab (53-6.7; BD Pharmingen); and PE-conjugated anti-CD28 Ab (37.51; BD Pharmingen); or PE-conjugated anti-CD137 Ab (17B5; eBioscience, San Diego, CA, U.S.); or PEconjugated anti-CTLA-4 Ab (UC10-4F10-11; BD Pharmingen). The antibodies used for cytokine staining in T intracellular cells included FITCconjugated anti-IFN-γ Ab (XMG1.2; BD Pharmingen), PE-conjugated anti-IL-17 (eBio17B7; eBioscience); and PE-conjugated anti-IL-4 Ab (11B11; eBioscience); and stained intranuclear with PE-conjugated anti-Foxp3 Ab (FJK-16 s; eBioscience). At least 30000 gated cells were acquired and analyzed with a Cytomics FC500 (Beckman Coulter, Brea, CA). 2.5. T cell proliferation Splenocytes from 16-week-old Pdcd4+/+Apoe−/− and Pdcd4−/ Apoe−/− mice fed with high-fat diets were separated and stimulated with functional grade purified anti-mouse CD3e Ab (145-2C11; eBioscience) for 3 days. Since the spleen cells contain many APCs (dendritic cells, macrophages and B cells), which can provide a co-stimulatory signal for T cells activation. So it does not need additional costimulatory stimulation (Kasagi et al., 2019). The proliferation ability of splenocytes was examined using a CCK-8 kit (Dojindo Laboratories, Japan). −
2.6. Statistical analysis
2. Material and methods
All statistical analyses were performed using SPSS 20.0 software. The unpaired Student t tests were used to compare the differences between the two groups. Data are shown as mean ± SEM. P value < 0.05 was considered to be statistically significant.
2.1. Animals Apoe−/− mice and wild-type C57BL/6 mice were purchased from Beijing Vital River Experimental Animal Technology Co. Ltd. (Beijing, China). Pdcd4−/− mice on C57BL/6 background were collected as described previously and hybridized with Apoe−/− mice to produce Pdcd4−/−Apoe−/− mice (Hilliard et al., 2006). All animal experimental procedures were approved by the Animal Care and Utilization Committee of Shandong University.
3. Results 3.1. Pdcd4 deficiency decreased the number of systemic CD8+ T cells and increased that of Tregs in hyperlipidemic mice In order to explore the role of Pdcd4 deficiency on the balance of T cell subsets in hyperlipidemic mice, the relative populations of different types of T cells, Th1, Th2, Th17, and Treg, were examined in the spleens of mice using flow cytometry. The results showed that the percentages of CD8+ and CD4+ T cells in the splenocytes underwent no significant changes after Pdcd4 deficiency as assessed when the mice were 8 weeks old. These mice had not been given a high-fat diet (Fig. 1A, n = 5). However, the percentage and total number of CD8+ T cells in spleen from Pdcd4−/−Apoe−/− mice were significantly lower than that of Pdcd4+/+Apoe−/− mice at both 16 and 24 weeks old (Fig. 1B-C). These mice had been fed a high-fat diet since the age of 8 weeks. In addition, the percentage of CD4+ T cells and their total number (Fig. 1B-C), Th1 (CD4+IFN-γ+), Th2 (CD4+IL-4+) and Th17 (CD4+IL17+) subsets (Fig. S1) displayed no significant differences between the two genotypes of mice at either 16 or 24 weeks, whereas the percentage of Treg (CD4+Foxp3+) cells in CD4+ T cells was significantly higher in Pdcd4−/−Apoe−/− mice than in Pdcd4+/+Apoe−/− mice (16 weeks: P < 0.001, Pdcd4+/+Apoe−/−: n = 5, Pdcd4−/−Apoe−/−: n = 7; 24 weeks: P < 0.05, Pdcd4+/+Apoe−/−: n = 5, Pdcd4−/−Apoe−/−: n = 6; Fig. 1B-C). These data indicate that Pdcd4 deficiency decreased the number of systemic CD8+ T cells and increased that of Tregs in hyperlipidemic mice.
2.2. Induction of atherosclerosis Starting at 8 weeks of age, sex-matched Pdcd4+/+Apoe−/− and Pdcd4−/−Apoe−/− mice were given a high-fat diet (0.25% cholesterol and 15% cocoa butter) to 16 weeks or 24 weeks to induce atherosclerotic plaques. 2.3. Immunohistochemistry and immunofluorescence Mice were sacrificed at 16 or 24 weeks of age, and their hearts and attached aortic roots were extracted and embedded in OCT compound under −20 °C. Serial frozen sections (6 μm thick) were cut along the aortic root with atherosclerotic plaques. Corresponding sections on separate slides were stained with immunohistochemistry for CD8+ T cells using rat anti-mouse CD8 Ab (53-6.7, BD Pharmingen). The expression of Foxp3 was indicated with rabbit anti-mouse Foxp3 Ab (ab54501; Abcam, Cambridge, U.K.) and FITC-conjugated goat antirabbit IgG, and with DAPI for cell nucleus, using the method of immunofluorescence. The different histological stains were observed using an Olympus microscope (IX71; Olympus Corporation, Tokyo, Japan). The positively stained regions and areas of plaques were measured using Image Proplus 6.0 software and the percentage of positive regions 388
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Fig. 1. Relative populations of CD4+ and CD8+ T cells in the spleens of 8-week-old mice and hyperlipidemic mice. (A) Splenocytes from 8-week-old Pdcd4+/ Apoe−/− and Pdcd4−/−Apoe−/− mice (n = 5) were separated, prepared as single cells, stained with PECy5-conjugated CD4 antibody and FITC-conjugated CD8 antibody, and then assayed for assessment of the percentages of CD4+ and CD8+ T cells using flow cytometry. Splenocytes were separated from Pdcd4+/+Apoe−/− and Pdcd4−/−Apoe−/− mice fed with high-fat diets from the age of 8 weeks to (B) 16 (Pdcd4+/+Apoe−/−: n = 5, Pdcd4−/−Apoe−/−: n = 7) or (C) 24 weeks (Pdcd4+/+Apoe−/−: n = 5, Pdcd4-/- Apoe−/−: n = 6). They were counted and stained with PECy5-conjugated CD4, FITC-conjugated CD8, and PE-conjugated Foxp3. The relative numbers of CD4+, CD8+ T cells and Tregs were detected using flow cytometry. Pooled data ± SEM are shown. *P < 0.05; **P < 0.01; ***P < 0.001. +
formation of atherosclerotic plaques.
3.2. Pdcd4 deficiency also decreased the number of CD8+ T cells and increased that of Tregs in atherosclerotic plaques
3.3. Pdcd4 deficiency decreased the proliferation of CD8+ T cells in vitro
To determine whether the changes in T cell subsets attributable to Pdcd4 deficiency may also occur in the aortic plaques, corresponding sections of the aortic root were prepared on separate slides to detect CD8 and Foxp3 expression using immunohistochemistry and immunofluorescence. Results showed that CD8 expression was markedly lower (P < 0.05, n=5; Fig. 2A) in the aortic plaques of Pdcd4−/−Apoe−/− mice than in those of Pdcd4+/+Apoe−/− mice. Meanwhile, the relative number of Foxp3+ T cells in the aortic plaques was increased in response to Pdcd4 deficiency (P < 0.05, n=5; Fig. 2B). These results indicate that local changes in T subsets in atherosclerotic plaque attributable to Pdcd4 deficiency were consistent with the changes in systemic T subsets, which may have contributed to inhibition of the
To explore the direct effect of Pdcd4 deficiency on the function of T cells, splenocytes were isolated from mice that had been fed a high-fat diet (16 weeks old) and then stimulated with an anti-CD3 monoclonal antibody for 3 days. The proliferation and subsets of T cells were examined using a CCK8 kit and flow cytometry, respectively. Results showed that the ability of T cells from Pdcd4−/−Apoe−/− mice to proliferate was significantly weaker than that of Pdcd4+/+Apoe−/− mice (n = 4, P < 0.001; Fig. 3A). Results also showed that Pdcd4 deficiency could attenuate the proliferation of CD8+ T cells but exerted no effects on CD4+ T cells (P < 0.05, Pdcd4+/+Apoe−/−: n = 7, Pdcd4−/−Apoe−/−: n = 6; Fig. 3B). 389
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Fig. 2. Relative populations of CD8+ T cells and Tregs in the plaques of Pdcd4 deficient hyperlipidemic mice. Pdcd4+/+Apoe−/− and Pdcd4−/−Apoe−/− mice were fed with a high-fat diet from the age of 8 weeks to 24 weeks. (A) Serial frozen sections were stained for CD8 by the method of immunohistochemistry (original magnification ×200) (n = 5). (B) Serial frozen sections were stained for Foxp3 with primary antibody and FITC-conjugated second antibody, and for cell nucleus with DAPI, respectively, using immunofluorescence (original magnification ×200). The white arrow and magnified image (original magnification ×400) indicate the positive cells. n = 5; *P < 0.05.
inhibitory CTLA-4 may contribute to the reduced proliferation of T cells under Pdcd4-deficient conditions.
3.4. Pdcd4 deficiency regulated the expression of co-stimulatory and coinhibitory molecules in proliferated T cells in vitro To determine why Pdcd4 deficiency attenuated T cell proliferation, co-stimulatory molecules CD28 and CD137, and co-inhibitory molecule CTLA-4 were examined using flow cytometry, which provided the second signal for T cell activation or inhibition. As shown in Fig. 4, the expressions of both CD28 and CD137 on CD4 + T cells were low and had no differences between the two genotype mice, but, their expressions on CD8+ T cells were high and lower in Pdcd4−/−Apoe−/− mice than in Pdcd4+/+Apoe−/− mice (CD8+CD28+: P < 0.05; CD8+CD137+: P < 0.05). However, the expression of CTLA-4 was higher on both CD4+ and CD8+ T cells (CD4+CTLA-4+: P < 0.01; CD8+CTLA-4+: P < 0.05; Pdcd4+/+Apoe−/−: n = 5, Pdcd4−/ − Apoe−/−: n = 4). These results suggest that down-regulation of costimulatory molecules CD28 and CD137 and up-regulation of co-
4. Discussion The present study demonstrated that deficiency in Pdcd4 can change the relative populations of different types of T-cells in hyperlipidemic mice by regulating the expression of co-stimulatory or co-inhibitory molecules. This may be one of the mechanisms by which Pdcd4 deficiency attenuates the formation of atherosclerotic plaques. Pdcd4 has been proven to be a well-documented tumor suppressor gene that can inhibit neoplastic transformation, progression, and the development of the malignant phenotype (Allgayer, 2010; LankatButtgereit and Goke, 2009; Zhang et al., 2010). Recent studies have shown Pdcd4 to be involved in inflammation and autoimmune diseases, such as EAE, LPS-induced killing effect, type 1 diabetes, diet-induced
Fig. 3. The ability of T cells lacking Pdcd4 to proliferate in vitro. Splenocytes from Pdcd4+/+Apoe−/− and Pdcd4−/−Apoe−/− mice fed with high-fat diets from the age of 8 weeks to 16 weeks were stimulated with anti-CD3 monoclonal antibody (5 μg/mL) in vitro for 3 days. (A) Cell proliferation was examined using a CCK-8 kit (n = 4). (B) The percentages of CD4+ and CD8+ in proliferated T cells were examined using flow cytometry. Pdcd4+/+Apoe−/−: n = 7, Pdcd4−/−Apoe−/−: n = 6; *P < 0.05; ***P < 0.001. 390
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Fig. 4. The expression of co-stimulatory and co-inhibitory molecules in T-cells lacking Pdcd4. Splenocytes from Pdcd4+/+Apoe−/− and Pdcd4−/−Apoe−/− mice fed with high-fat diets from the age of 8 weeks to 16 weeks were stimulated with anti-CD3 monoclonal antibody (5 μg/mL) in vitro for 3 days. The relative populations of CD4+/CD8+CD28+ (left panel), CD4+/CD8+CD137+ (middle panel) and CD4+/CD8+CTLA-4+ (right panel) were detected using flow cytometry after proliferation. Pdcd4+/+Apoe−/−: n = 5, Pdcd4−/−Apoe−/−: n = 4; *P < 0.05; **P < 0.01.
atherosclerotic plaque lesions in both Apoe−/− mice and Ldlr-/atherosclerosis mouse models, which was attributed to the downregulation of several pro-inflammatory cytokines (Jeon et al., 2010). The activation of CD137 signaling also could promote angiogenesis in atherosclerosis both in vivo and in vitro by modulating the Smad1/ 5‐NFATc1 pathway (Weng et al., 2017). In the plaques of Apoe−/− mice, both T cells and macrophages expressed CD137, which could stimulate T cells to produce IFN-γ and activated MAPK signaling in macrophages to upregulate MMP-9 expression (Jung et al., 2014). Combined with the above data and our research, we hypothesized that Pdcd4 might upregulate the expression of CD137 to adjust the immune responses mediated by T cells and macrophages in atherosclerotic mice. In this study, results showed that deficiency of Pdcd4 could up-regulate co-inhibitory molecule CTLA-4 and down-regulate co-stimulatory molecules CD28 and CD137 on CD8+ T cells. The binding of CD28 to CD80/CD86 promotes T cell activation and accelerates inflammation, and interaction of CTLA4 to CD80 or CD86 inhibits T cells activation and attenuates inflammation (Afek et al., 2004; Gotsman et al., 2008). It also has been reported that the increased numbers of Tregs expressing immune checkpoints (such as CTLA-4) could inhibit the proliferation and activation of cytotoxic CD8+ T cells (Saleh and Elkord, 2019). Tregs have also been proven to have an anti-atherogenic effect (AitOufella et al., 2006; van Es et al., 2010). It has been reported that CD28−/−Ldlr−/− mice developed more serious atherosclerosis, most likely due to the lack of Tregs (Ait-Oufella et al., 2006). CTLA4 has been identified as a crucial negative regulator of T-cell activation by inhibiting the CD28 signal. The high levels of CTLA-4 on Tregs are required for their inhibitory effect on effector cells (Wing et al., 2008). Pretreatment with CTLA4-IgG, to destroy CD28-dependent T-cell costimulatory signaling, could ameliorate hyperhomocysteinaemia-accelerated atherosclerosis in Apoe-/- mice (Ma et al., 2013). And CTLA-4 overexpression in Apoe−/− mice significantly reduced the formation of atherosclerotic lesion and accumulation of macrophages and CD4+ T cells in the plaques (Matsumoto et al., 2016). These results indicate that up-regulation of CTLA-4 but down-regulation of CD28 and CD137 on CD8+ T cells after Pdcd4 deficiency may lead to a weak proliferation of CD8+ T cells. While the decrease of CD8+ T cells, there is no difference in the proportion of CD4+ T cells between two groups, so the proportion of CD4-CD8- cells were increased. The function and characteristics of this cell subset need to be further studied. Taken together, this work indicated that Pdcd4 might affect T cell
obesity and adipose tissue inflammation, allergic pulmonary inflammation, LPS/D-galactosamine-induced acute liver injury, and atherosclerosis (Hilliard et al., 2006; Jiang et al., 2016; Ruan et al., 2011; Sheedy et al., 2010; Wang et al., 2013a, b; Zhong et al., 2014). Pdcd4 has been shown to promote or inhibit inflammation in different models. Previous studies have indicated that Pdcd4 is also involved in macrophage-mediated immune response. Pdcd4 was found to inhibit macrophage apoptosis and the production of IL-10 through Twist2/cMaf or ERK/p38 pathway on the level of transcription (Jiang et al., 2016; Shang et al., 2015; van den Bosch et al., 2014). However, the role of Pdcd4 in T-cell-mediated immune response is not precisely understood. Changes in T-cell-secreted cytokines, such as IL-2, IL-4, and IL17, have been observed in mouse models of Pdcd4-deficient lymphoma, diabetes, and atherosclerosis (Hilliard et al., 2006; Jiang et al., 2016; Ruan et al., 2011), but the role of Pdcd4 on the relative numbers of T cell subsets and their mechanism has not been reported previously. Recently, studies have demonstrated that both miR-16 and miR-155 targeting Pdcd4 could suppress the expression of pro-inflammatory factors IL-6 and enhance the expression of anti-inflammatory factor IL10 secreted by macrophages through mitogen-activated protein kinase (MAPK) and NF-κB, and SOCS1-STAT3 signaling, respectively, in atherosclerosis (Liang et al., 2016; Ye et al., 2016). And our previous studies also have shown that Pdcd4 deficiency can attenuate atherosclerosis in Apoe−/− mice, in which process macrophages produced more IL-10 through activating ERK1/2 and p38 pathway on the level of transcription, and T cells produced more IL-17 but had no effects on the formation of atherosclerotic plaques (Jiang et al., 2016). Results also showed that attenuated atherosclerosis in Apoe−/− mice attributable to Pdcd4 deficiency may be due to enhanced lipoautophagy of macrophages and attenuated foam cell formation (Wang et al., 2016). Both these studies above focused on the function of macrophages, not on the balance of T cell subsets, in the process of Pdcd4-deficiency- attenuated atherosclerotic plaques. In this study, we demonstrated that T cells lacking Pdcd4 had fewer effector cells (CD8+) and more Tregs under high blood lipid conditions in vivo and that the absence of Pdcd4 attenuated the ability of T cell receptor (TCR) signal stimulation to proliferate of CD8+ T cells in vitro. Previous studies have shown that the increased inflammation and the higher proportion of CD8+ T cells attributable to injection of a CD137 agonist could accelerate atherosclerosis in Apoe−/− mice (Soderstrom et al., 2017). CD137 deficiency had been found to induce a reduction in 391
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subsets by regulating the expression of co-stimulatory molecules under hyperlipidemic conditions, which might affect the formation of atherosclerotic plaques. Such our result provides new data to understand the role of Pdcd4 in regulating different T cell subsets under hyperlipidemic microenvironment.
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Conflict of interest The authors declared that they have no conflict of interest. Authors’ contribution Y. J.: performed the experiments, manuscript writing and data analysis; Q.G., L.Y.W., T.M.: assist in the experiments; C.G., F.L.Z., Q.W.: supervised the study; F.G.: contributed materials and analysis tools; L.N.Z.: concept and design, manuscript writing and final approval of the manuscript. All authors read and approved the manuscript. Funding This work was supported by the National Natural Science Foundation of China (81600176) and the Natural Science Foundation of Shandong Province (grant no. ZR2016HB71). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.molimm.2019.06.020. References Afek, A., Harats, D., Roth, A., Keren, G., George, J., 2004. Evidence for the involvement of T cell costimulation through the B-7/CD28 pathway in atherosclerotic plaques from apolipoprotein E knockout mice. Exp. Mol. Pathol. 76, 219–223. https://doi.org/10. 1016/j.yexmp.2003.12.001. Ait-Oufella, H., Salomon, B.L., Potteaux, S., Robertson, A.K., Gourdy, P., Zoll, J., Merval, R., Esposito, B., Cohen, J.L., Fisson, S., Flavell, R.A., Hansson, G.K., Klatzmann, D., Tedgui, A., Mallat, Z., 2006. Natural regulatory T cells control the development of atherosclerosis in mice. Nat. Med. 12, 178–180. https://doi.org/10.1038/nm1343. Allgayer, H., 2010. Pdcd4, a colon cancer prognostic that is regulated by a microRNA. Crit. Rev. Oncol. Hematol. 73, 185–191. https://doi.org/10.1016/j.critrevonc.2009. 09.001. Biyanee, A., Ohnheiser, J., Singh, P., Klempnauer, K.H., 2015. A novel mechanism for the control of translation of specific mRNAs by tumor suppressor protein Pdcd4: inhibition of translation elongation. Oncogene 34, 1384–1392. https://doi.org/10. 1038/onc.2014.83. Danzaki, K., Matsui, Y., Ikesue, M., Ohta, D., Ito, K., Kanayama, M., Kurotaki, D., Morimoto, J., Iwakura, Y., Yagita, H., Tsutsui, H., Uede, T., 2012. Interleukin-17A deficiency accelerates unstable atherosclerotic plaque formation in apolipoprotein Edeficient mice. Arterioscler. Thromb. Vasc. Biol. 32, 273–280. https://doi.org/10. 1161/ATVBAHA.111.229997. Davenport, P., Tipping, P.G., 2003. The role of interleukin-4 and interleukin-12 in the progression of atherosclerosis in apolipoprotein E-deficient mice. Am. J. Pathol. 163, 1117–1125. https://doi.org/10.1016/S0002-9440(10)63471-2. Engelbertsen, D., Rattik, S., Knutsson, A., Bjorkbacka, H., Bengtsson, E., Nilsson, J., 2014. Induction of T helper 2 responses against human apolipoprotein B100 does not affect atherosclerosis in ApoE-/- mice. Cardiovasc. Res. 103, 304–312. https://doi.org/10. 1093/cvr/cvu131. Gotsman, I., Sharpe, A.H., Lichtman, A.H., 2008. T-cell costimulation and coinhibition in atherosclerosis. Circ. Res. 103, 1220–1231. https://doi.org/10.1161/CIRCRESAHA. 108.182428. Hansson, G.K., Hermansson, A., 2011. The immune system in atherosclerosis. Nat. Immunol. 12, 204–212. https://doi.org/10.1038/ni.2001. Hilliard, A., Hilliard, B., Zheng, S.J., Sun, H., Miwa, T., Song, W., Goke, R., Chen, Y.H., 2006. Translational regulation of autoimmune inflammation and lymphoma genesis by programmed cell death 4. J. Immunol. 177, 8095–8102. Jeon, H.J., Choi, J.H., Jung, I.H., Park, J.G., Lee, M.R., Lee, M.N., Kim, B., Yoo, J.Y., Jeong, S.J., Kim, D.Y., Park, J.E., Park, H.Y., Kwack, K., Choi, B.K., Kwon, B.S., Oh, G.T., 2010. CD137 (4-1BB) deficiency reduces atherosclerosis in hyperlipidemic mice. Circulation 121, 1124–1133. https://doi.org/10.1161/CIRCULATIONAHA. 109.882704. Jeon, U.S., Choi, J.P., Kim, Y.S., Ryu, S.H., Kim, Y.K., 2015. The enhanced expression of IL-17-secreting T cells during the early progression of atherosclerosis in ApoE-deficient mice fed on a western-type diet. Exp. Mol. Med. 47, e163. https://doi.org/10. 1038/emm.2015.19. Jiang, Y., Gao, Q., Wang, L., Guo, C., Zhu, F., Wang, B., Wang, Q., Gao, F., Chen, Y.,
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Deficiency of programmed cell death 4 affects the balance of T cell subsets in hyperlipidemic mice
T
Yang Jianga,1, Qi Gaoa,2, Li-Yang Wanga, Tian Maa, Fa-Liang Zhua, Qun Wanga, Fei Gaob, ⁎ Chun Guoa, Li-Ning Zhanga, a b
Department of Immunology, School of Medicine, Shandong University, Jinan 250012, Shandong, China The Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital, Shandong University, Jinan 250012, Shandong, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Pdcd4 Atherosclerosis CD8+T cells Tregs Co-stimulatory molecules
Programmed cell death 4 (Pdcd4) was found to be related to apoptosis upon first discovery. It was later found to play the role of tumor suppressor gene in a variety of tumors by inhibiting transcription and translation. Recently, it has been proposed that it may play an important role in some inflammatory diseases and in the immune response. In our previous study, deficiency of Pdcd4 was found to attenuate the formation of atherosclerotic plaques. This might be because deficiency of Pdcd4 may increase IL-10 expression and lipoautophagy by macrophages and attenuate the formation of foam cells. However, the effect of Pdcd4 on the subsets of T cells in hyperlipidemic mice still remained unclear. In the present study, results showed that Pdcd4 deficiency decreased the percentage of CD8+ T cells and increased that of regulatory T cells (Tregs) under hyperlipidemic conditions both in vitro and in vivo, which may be due to the reduced expression of co-stimulatory molecules CD28 and CD137, and the enhancive expression of co-inhibitory molecules CTLA-4. These results indicated that endogenous Pdcd4 promotes immune response mediated by T cells through regulation of the co-stimulatory molecules expression, which may contribute to the development of advanced atherosclerotic plaques. The current work provides new data to understand the role of Pdcd4 in different T cell subsets under hyperlipidemic microenvironment.
1. Introduction Atherosclerosis, which is a chronic, multifactorial, progressive process characterized by an inflammatory response of the arterial wall, is a substantial cause of morbidity and mortality (Steinberg and Witztum, 2010). The process is initialized by hyperlipidemia and intimal deposition of lipid in the arteries and then progression into atherosclerotic plaques (Libby et al., 2011). The immune-inflammatory cells involved in atherosclerosis include monocytes and macrophages, which mediate the innate immune response by handling lipoproteins, release inflammatory cytokines, and work as antigen-presenting cells (APC) to stimulate T cells, and antigen-specific T cells that mediate the adaptive immune response by releasing inflammatory cytokines (Libby, 2012). Accumulating evidence indicates that both CD4+ and CD8+ T cells are involved in atherosclerosis (Hansson and Hermansson, 2011).
CD4+ T cells are the most abundant T cells in atherosclerotic plaques and play a pathogenic role in the progression of atherosclerosis (Zhou et al., 2000). It is well established that Th1 cells promote an inflammatory response and the development of atherosclerosis by producing pro-inflammatory cytokine interferon (IFN)-γ (Moss and Ramji, 2015). Tregs (CD4+FoxP3+) are crucial to mediating immune homeostasis and may have a protective role in the formation of atherogenic plaques (Meng et al., 2012). The contradictory effects of Th2 (Davenport and Tipping, 2003; Engelbertsen et al., 2014; K. Meng et al., 2015), Th17 (Danzaki et al., 2012; Jeon et al., 2015) and CD8+ T cells in atherosclerosis have been also reported, which includes the proatherogenic, anti-atherogenic, or even no influence on the formation of plaques (Kolbus et al., 2012; Kyaw et al., 2013; van Duijn et al., 2018). Programmed cell death 4 (Pdcd4) is a well-known tumor suppressor that inhibits neoplastic transformation, tumor progression, gene
Abbreviations: Pdcd4, Programmed cell death 4; Tregs, Regulatory T cells; APC, Antigen-presenting cells; IFN, Interferon; eIF4A, Translation-initiation-factor-4A; EAE, Encephalomyelitis; CTLA-4, Cytotoxic T-lymphocyte antigen-4; MAPK, Mitogen-activated protein kinase; TCR, T cell receptor ⁎ Corresponding author at: Department of Immunology, School of Medicine, Shandong University, Jinan 250012, Shandong, China. E-mail address: [email protected] (L.-N. Zhang). 1 Present address: Department of Hematology, The Second Hospital of Shandong University, Jinan 250033, Shandong, China. 2 Present address: Department of Clinical Laboratory, Provincial Hospital affiliated with Shandong University, Jinan 250021, Shandong, China. https://doi.org/10.1016/j.molimm.2019.06.020 Received 12 February 2019; Received in revised form 10 June 2019; Accepted 28 June 2019 Available online 06 July 2019 0161-5890/ © 2019 Elsevier Ltd. All rights reserved.
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was calculated.
transcription, and translation (Lankat-Buttgereit and Goke, 2009). Pdcd4 inhibits the translation of mRNA through two channels. One of these channels acts in a translation-initiation-factor-4A (eIF4A)-dependent manner. By binding competitively to eIF4A, Pdcd4 inhibits the combination of RNA helicase eIF4A and scaffold protein eIF4G which initiates the translation of mRNA (Waters et al., 2007). The other channel is eIF4A-independent. Pdcd4 binds directly to mRNA to inhibit its translation (Biyanee et al., 2015). Recent studies have shown that Pdcd4 might also be involved in certain inflammatory diseases. For example, Pdcd4-deficient mice are resistant to autoimmune encephalomyelitis (EAE) (Hilliard et al., 2006), the LPS-induced killing effect (Sheedy et al., 2010), type 1 diabetes (Ruan et al., 2011), dietinduced obesity and adipose tissue inflammation (Wang et al., 2013a), and allergic pulmonary inflammation (Zhong et al., 2014). Our and other groups showed that Pdcd4 deficiency regulated the function of macrophages and involved in the formation of atherosclerotic plaques in Apoe−/− mice (Jiang et al., 2016; Liang et al., 2016; Ye et al., 2016). In addition, Pdcd4 was also found to be involved in macrophage- and Tcell-mediated immune responses. Other studies have indicated that Pdcd4 can inhibit the production of IL-10 by macrophages through Twist2/c-Maf pathway (van den Bosch et al., 2014). There are changes of T cell-related cytokines, such as IL-2, IL-4, and IL-17, in Pdcd4 deficient lymphoma and diabetic mice models, but the mechanism underlying these changes is not clear (Hilliard et al., 2006; Ruan et al., 2011). This study showed that knocking out Pdcd4 in hyperlipidemic mice decreased the percentage of CD8+ T cells and increased the percentage of Tregs both in vitro and in vivo. It is possible that Pdcd4 deficiency upregulate the expression of co-stimulatory molecules CD28 and CD137 and downregulate the expression of the co-inhibitory molecule cytotoxic T-lymphocyte antigen (CTLA)-4.
2.4. Flow cytometry Splenocytes from 16- or 24-week-old mice were separated and stained directly with PECy5-conjugated anti-CD4 Ab (H129.19; BD Pharmingen), FITC-conjugated anti-CD8 Ab (53-6.7; BD Pharmingen); and PE-conjugated anti-CD28 Ab (37.51; BD Pharmingen); or PE-conjugated anti-CD137 Ab (17B5; eBioscience, San Diego, CA, U.S.); or PEconjugated anti-CTLA-4 Ab (UC10-4F10-11; BD Pharmingen). The antibodies used for cytokine staining in T intracellular cells included FITCconjugated anti-IFN-γ Ab (XMG1.2; BD Pharmingen), PE-conjugated anti-IL-17 (eBio17B7; eBioscience); and PE-conjugated anti-IL-4 Ab (11B11; eBioscience); and stained intranuclear with PE-conjugated anti-Foxp3 Ab (FJK-16 s; eBioscience). At least 30000 gated cells were acquired and analyzed with a Cytomics FC500 (Beckman Coulter, Brea, CA). 2.5. T cell proliferation Splenocytes from 16-week-old Pdcd4+/+Apoe−/− and Pdcd4−/ Apoe−/− mice fed with high-fat diets were separated and stimulated with functional grade purified anti-mouse CD3e Ab (145-2C11; eBioscience) for 3 days. Since the spleen cells contain many APCs (dendritic cells, macrophages and B cells), which can provide a co-stimulatory signal for T cells activation. So it does not need additional costimulatory stimulation (Kasagi et al., 2019). The proliferation ability of splenocytes was examined using a CCK-8 kit (Dojindo Laboratories, Japan). −
2.6. Statistical analysis
2. Material and methods
All statistical analyses were performed using SPSS 20.0 software. The unpaired Student t tests were used to compare the differences between the two groups. Data are shown as mean ± SEM. P value < 0.05 was considered to be statistically significant.
2.1. Animals Apoe−/− mice and wild-type C57BL/6 mice were purchased from Beijing Vital River Experimental Animal Technology Co. Ltd. (Beijing, China). Pdcd4−/− mice on C57BL/6 background were collected as described previously and hybridized with Apoe−/− mice to produce Pdcd4−/−Apoe−/− mice (Hilliard et al., 2006). All animal experimental procedures were approved by the Animal Care and Utilization Committee of Shandong University.
3. Results 3.1. Pdcd4 deficiency decreased the number of systemic CD8+ T cells and increased that of Tregs in hyperlipidemic mice In order to explore the role of Pdcd4 deficiency on the balance of T cell subsets in hyperlipidemic mice, the relative populations of different types of T cells, Th1, Th2, Th17, and Treg, were examined in the spleens of mice using flow cytometry. The results showed that the percentages of CD8+ and CD4+ T cells in the splenocytes underwent no significant changes after Pdcd4 deficiency as assessed when the mice were 8 weeks old. These mice had not been given a high-fat diet (Fig. 1A, n = 5). However, the percentage and total number of CD8+ T cells in spleen from Pdcd4−/−Apoe−/− mice were significantly lower than that of Pdcd4+/+Apoe−/− mice at both 16 and 24 weeks old (Fig. 1B-C). These mice had been fed a high-fat diet since the age of 8 weeks. In addition, the percentage of CD4+ T cells and their total number (Fig. 1B-C), Th1 (CD4+IFN-γ+), Th2 (CD4+IL-4+) and Th17 (CD4+IL17+) subsets (Fig. S1) displayed no significant differences between the two genotypes of mice at either 16 or 24 weeks, whereas the percentage of Treg (CD4+Foxp3+) cells in CD4+ T cells was significantly higher in Pdcd4−/−Apoe−/− mice than in Pdcd4+/+Apoe−/− mice (16 weeks: P < 0.001, Pdcd4+/+Apoe−/−: n = 5, Pdcd4−/−Apoe−/−: n = 7; 24 weeks: P < 0.05, Pdcd4+/+Apoe−/−: n = 5, Pdcd4−/−Apoe−/−: n = 6; Fig. 1B-C). These data indicate that Pdcd4 deficiency decreased the number of systemic CD8+ T cells and increased that of Tregs in hyperlipidemic mice.
2.2. Induction of atherosclerosis Starting at 8 weeks of age, sex-matched Pdcd4+/+Apoe−/− and Pdcd4−/−Apoe−/− mice were given a high-fat diet (0.25% cholesterol and 15% cocoa butter) to 16 weeks or 24 weeks to induce atherosclerotic plaques. 2.3. Immunohistochemistry and immunofluorescence Mice were sacrificed at 16 or 24 weeks of age, and their hearts and attached aortic roots were extracted and embedded in OCT compound under −20 °C. Serial frozen sections (6 μm thick) were cut along the aortic root with atherosclerotic plaques. Corresponding sections on separate slides were stained with immunohistochemistry for CD8+ T cells using rat anti-mouse CD8 Ab (53-6.7, BD Pharmingen). The expression of Foxp3 was indicated with rabbit anti-mouse Foxp3 Ab (ab54501; Abcam, Cambridge, U.K.) and FITC-conjugated goat antirabbit IgG, and with DAPI for cell nucleus, using the method of immunofluorescence. The different histological stains were observed using an Olympus microscope (IX71; Olympus Corporation, Tokyo, Japan). The positively stained regions and areas of plaques were measured using Image Proplus 6.0 software and the percentage of positive regions 388
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Fig. 1. Relative populations of CD4+ and CD8+ T cells in the spleens of 8-week-old mice and hyperlipidemic mice. (A) Splenocytes from 8-week-old Pdcd4+/ Apoe−/− and Pdcd4−/−Apoe−/− mice (n = 5) were separated, prepared as single cells, stained with PECy5-conjugated CD4 antibody and FITC-conjugated CD8 antibody, and then assayed for assessment of the percentages of CD4+ and CD8+ T cells using flow cytometry. Splenocytes were separated from Pdcd4+/+Apoe−/− and Pdcd4−/−Apoe−/− mice fed with high-fat diets from the age of 8 weeks to (B) 16 (Pdcd4+/+Apoe−/−: n = 5, Pdcd4−/−Apoe−/−: n = 7) or (C) 24 weeks (Pdcd4+/+Apoe−/−: n = 5, Pdcd4-/- Apoe−/−: n = 6). They were counted and stained with PECy5-conjugated CD4, FITC-conjugated CD8, and PE-conjugated Foxp3. The relative numbers of CD4+, CD8+ T cells and Tregs were detected using flow cytometry. Pooled data ± SEM are shown. *P < 0.05; **P < 0.01; ***P < 0.001. +
formation of atherosclerotic plaques.
3.2. Pdcd4 deficiency also decreased the number of CD8+ T cells and increased that of Tregs in atherosclerotic plaques
3.3. Pdcd4 deficiency decreased the proliferation of CD8+ T cells in vitro
To determine whether the changes in T cell subsets attributable to Pdcd4 deficiency may also occur in the aortic plaques, corresponding sections of the aortic root were prepared on separate slides to detect CD8 and Foxp3 expression using immunohistochemistry and immunofluorescence. Results showed that CD8 expression was markedly lower (P < 0.05, n=5; Fig. 2A) in the aortic plaques of Pdcd4−/−Apoe−/− mice than in those of Pdcd4+/+Apoe−/− mice. Meanwhile, the relative number of Foxp3+ T cells in the aortic plaques was increased in response to Pdcd4 deficiency (P < 0.05, n=5; Fig. 2B). These results indicate that local changes in T subsets in atherosclerotic plaque attributable to Pdcd4 deficiency were consistent with the changes in systemic T subsets, which may have contributed to inhibition of the
To explore the direct effect of Pdcd4 deficiency on the function of T cells, splenocytes were isolated from mice that had been fed a high-fat diet (16 weeks old) and then stimulated with an anti-CD3 monoclonal antibody for 3 days. The proliferation and subsets of T cells were examined using a CCK8 kit and flow cytometry, respectively. Results showed that the ability of T cells from Pdcd4−/−Apoe−/− mice to proliferate was significantly weaker than that of Pdcd4+/+Apoe−/− mice (n = 4, P < 0.001; Fig. 3A). Results also showed that Pdcd4 deficiency could attenuate the proliferation of CD8+ T cells but exerted no effects on CD4+ T cells (P < 0.05, Pdcd4+/+Apoe−/−: n = 7, Pdcd4−/−Apoe−/−: n = 6; Fig. 3B). 389
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Fig. 2. Relative populations of CD8+ T cells and Tregs in the plaques of Pdcd4 deficient hyperlipidemic mice. Pdcd4+/+Apoe−/− and Pdcd4−/−Apoe−/− mice were fed with a high-fat diet from the age of 8 weeks to 24 weeks. (A) Serial frozen sections were stained for CD8 by the method of immunohistochemistry (original magnification ×200) (n = 5). (B) Serial frozen sections were stained for Foxp3 with primary antibody and FITC-conjugated second antibody, and for cell nucleus with DAPI, respectively, using immunofluorescence (original magnification ×200). The white arrow and magnified image (original magnification ×400) indicate the positive cells. n = 5; *P < 0.05.
inhibitory CTLA-4 may contribute to the reduced proliferation of T cells under Pdcd4-deficient conditions.
3.4. Pdcd4 deficiency regulated the expression of co-stimulatory and coinhibitory molecules in proliferated T cells in vitro To determine why Pdcd4 deficiency attenuated T cell proliferation, co-stimulatory molecules CD28 and CD137, and co-inhibitory molecule CTLA-4 were examined using flow cytometry, which provided the second signal for T cell activation or inhibition. As shown in Fig. 4, the expressions of both CD28 and CD137 on CD4 + T cells were low and had no differences between the two genotype mice, but, their expressions on CD8+ T cells were high and lower in Pdcd4−/−Apoe−/− mice than in Pdcd4+/+Apoe−/− mice (CD8+CD28+: P < 0.05; CD8+CD137+: P < 0.05). However, the expression of CTLA-4 was higher on both CD4+ and CD8+ T cells (CD4+CTLA-4+: P < 0.01; CD8+CTLA-4+: P < 0.05; Pdcd4+/+Apoe−/−: n = 5, Pdcd4−/ − Apoe−/−: n = 4). These results suggest that down-regulation of costimulatory molecules CD28 and CD137 and up-regulation of co-
4. Discussion The present study demonstrated that deficiency in Pdcd4 can change the relative populations of different types of T-cells in hyperlipidemic mice by regulating the expression of co-stimulatory or co-inhibitory molecules. This may be one of the mechanisms by which Pdcd4 deficiency attenuates the formation of atherosclerotic plaques. Pdcd4 has been proven to be a well-documented tumor suppressor gene that can inhibit neoplastic transformation, progression, and the development of the malignant phenotype (Allgayer, 2010; LankatButtgereit and Goke, 2009; Zhang et al., 2010). Recent studies have shown Pdcd4 to be involved in inflammation and autoimmune diseases, such as EAE, LPS-induced killing effect, type 1 diabetes, diet-induced
Fig. 3. The ability of T cells lacking Pdcd4 to proliferate in vitro. Splenocytes from Pdcd4+/+Apoe−/− and Pdcd4−/−Apoe−/− mice fed with high-fat diets from the age of 8 weeks to 16 weeks were stimulated with anti-CD3 monoclonal antibody (5 μg/mL) in vitro for 3 days. (A) Cell proliferation was examined using a CCK-8 kit (n = 4). (B) The percentages of CD4+ and CD8+ in proliferated T cells were examined using flow cytometry. Pdcd4+/+Apoe−/−: n = 7, Pdcd4−/−Apoe−/−: n = 6; *P < 0.05; ***P < 0.001. 390
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Fig. 4. The expression of co-stimulatory and co-inhibitory molecules in T-cells lacking Pdcd4. Splenocytes from Pdcd4+/+Apoe−/− and Pdcd4−/−Apoe−/− mice fed with high-fat diets from the age of 8 weeks to 16 weeks were stimulated with anti-CD3 monoclonal antibody (5 μg/mL) in vitro for 3 days. The relative populations of CD4+/CD8+CD28+ (left panel), CD4+/CD8+CD137+ (middle panel) and CD4+/CD8+CTLA-4+ (right panel) were detected using flow cytometry after proliferation. Pdcd4+/+Apoe−/−: n = 5, Pdcd4−/−Apoe−/−: n = 4; *P < 0.05; **P < 0.01.
atherosclerotic plaque lesions in both Apoe−/− mice and Ldlr-/atherosclerosis mouse models, which was attributed to the downregulation of several pro-inflammatory cytokines (Jeon et al., 2010). The activation of CD137 signaling also could promote angiogenesis in atherosclerosis both in vivo and in vitro by modulating the Smad1/ 5‐NFATc1 pathway (Weng et al., 2017). In the plaques of Apoe−/− mice, both T cells and macrophages expressed CD137, which could stimulate T cells to produce IFN-γ and activated MAPK signaling in macrophages to upregulate MMP-9 expression (Jung et al., 2014). Combined with the above data and our research, we hypothesized that Pdcd4 might upregulate the expression of CD137 to adjust the immune responses mediated by T cells and macrophages in atherosclerotic mice. In this study, results showed that deficiency of Pdcd4 could up-regulate co-inhibitory molecule CTLA-4 and down-regulate co-stimulatory molecules CD28 and CD137 on CD8+ T cells. The binding of CD28 to CD80/CD86 promotes T cell activation and accelerates inflammation, and interaction of CTLA4 to CD80 or CD86 inhibits T cells activation and attenuates inflammation (Afek et al., 2004; Gotsman et al., 2008). It also has been reported that the increased numbers of Tregs expressing immune checkpoints (such as CTLA-4) could inhibit the proliferation and activation of cytotoxic CD8+ T cells (Saleh and Elkord, 2019). Tregs have also been proven to have an anti-atherogenic effect (AitOufella et al., 2006; van Es et al., 2010). It has been reported that CD28−/−Ldlr−/− mice developed more serious atherosclerosis, most likely due to the lack of Tregs (Ait-Oufella et al., 2006). CTLA4 has been identified as a crucial negative regulator of T-cell activation by inhibiting the CD28 signal. The high levels of CTLA-4 on Tregs are required for their inhibitory effect on effector cells (Wing et al., 2008). Pretreatment with CTLA4-IgG, to destroy CD28-dependent T-cell costimulatory signaling, could ameliorate hyperhomocysteinaemia-accelerated atherosclerosis in Apoe-/- mice (Ma et al., 2013). And CTLA-4 overexpression in Apoe−/− mice significantly reduced the formation of atherosclerotic lesion and accumulation of macrophages and CD4+ T cells in the plaques (Matsumoto et al., 2016). These results indicate that up-regulation of CTLA-4 but down-regulation of CD28 and CD137 on CD8+ T cells after Pdcd4 deficiency may lead to a weak proliferation of CD8+ T cells. While the decrease of CD8+ T cells, there is no difference in the proportion of CD4+ T cells between two groups, so the proportion of CD4-CD8- cells were increased. The function and characteristics of this cell subset need to be further studied. Taken together, this work indicated that Pdcd4 might affect T cell
obesity and adipose tissue inflammation, allergic pulmonary inflammation, LPS/D-galactosamine-induced acute liver injury, and atherosclerosis (Hilliard et al., 2006; Jiang et al., 2016; Ruan et al., 2011; Sheedy et al., 2010; Wang et al., 2013a, b; Zhong et al., 2014). Pdcd4 has been shown to promote or inhibit inflammation in different models. Previous studies have indicated that Pdcd4 is also involved in macrophage-mediated immune response. Pdcd4 was found to inhibit macrophage apoptosis and the production of IL-10 through Twist2/cMaf or ERK/p38 pathway on the level of transcription (Jiang et al., 2016; Shang et al., 2015; van den Bosch et al., 2014). However, the role of Pdcd4 in T-cell-mediated immune response is not precisely understood. Changes in T-cell-secreted cytokines, such as IL-2, IL-4, and IL17, have been observed in mouse models of Pdcd4-deficient lymphoma, diabetes, and atherosclerosis (Hilliard et al., 2006; Jiang et al., 2016; Ruan et al., 2011), but the role of Pdcd4 on the relative numbers of T cell subsets and their mechanism has not been reported previously. Recently, studies have demonstrated that both miR-16 and miR-155 targeting Pdcd4 could suppress the expression of pro-inflammatory factors IL-6 and enhance the expression of anti-inflammatory factor IL10 secreted by macrophages through mitogen-activated protein kinase (MAPK) and NF-κB, and SOCS1-STAT3 signaling, respectively, in atherosclerosis (Liang et al., 2016; Ye et al., 2016). And our previous studies also have shown that Pdcd4 deficiency can attenuate atherosclerosis in Apoe−/− mice, in which process macrophages produced more IL-10 through activating ERK1/2 and p38 pathway on the level of transcription, and T cells produced more IL-17 but had no effects on the formation of atherosclerotic plaques (Jiang et al., 2016). Results also showed that attenuated atherosclerosis in Apoe−/− mice attributable to Pdcd4 deficiency may be due to enhanced lipoautophagy of macrophages and attenuated foam cell formation (Wang et al., 2016). Both these studies above focused on the function of macrophages, not on the balance of T cell subsets, in the process of Pdcd4-deficiency- attenuated atherosclerotic plaques. In this study, we demonstrated that T cells lacking Pdcd4 had fewer effector cells (CD8+) and more Tregs under high blood lipid conditions in vivo and that the absence of Pdcd4 attenuated the ability of T cell receptor (TCR) signal stimulation to proliferate of CD8+ T cells in vitro. Previous studies have shown that the increased inflammation and the higher proportion of CD8+ T cells attributable to injection of a CD137 agonist could accelerate atherosclerosis in Apoe−/− mice (Soderstrom et al., 2017). CD137 deficiency had been found to induce a reduction in 391
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subsets by regulating the expression of co-stimulatory molecules under hyperlipidemic conditions, which might affect the formation of atherosclerotic plaques. Such our result provides new data to understand the role of Pdcd4 in regulating different T cell subsets under hyperlipidemic microenvironment.
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