| ObjectAtherosclerosis is a chronic arterial wall inflammatory result from abnormal metabolism of fattiness; it is pathologic base of angina, acute myocardial infarction and sudden death. Atherosclerosis has been the common disease in older men of our country, which largely affect patients’life quality, but the mechanism of Atherosclerosis is not clear. More and more evidence proofed that the inflammation mediated by CD4+T cell is very important in atherosclerosis engendering and developing. CD4+T cell including Thl cell, Th2 cell, a new Th17 subset and regulatory T(Treg) cell, multitudinal research attested that Thl cell promote atherosclerosis, whereas the role of Treg cell and Th2 cell is inhibition. Recently Thl7 cells have been shown to be involved in the pathogenesis of atherosclerosis. We and others have previously demonstrated that a critical pro-atherogenic effect of Th17 cell and/or IL-17 on the development of the atherosclerotic lesions and the plaque vulnerability in ApoE-/-mice. However, the mechanisms by which Th17 and IL-17 function in atherosclerosis development remain largely unknown.We investigated the IL-17/Th17 level in peripheral blood of patient with acute coronary syndrome (ACS); found that both of Th17 cell and IL-17 were increased significant. IL-17 is major effect molecule of Th17 cell, and arterial endothelial cell lesion and adhesion increasing are early incident of atherosclerosis. So we investigated IL-17 effect to endothelial cell (Human umbilical vein endothelial cell, HUVEC), found that IL-17 could enhance the expression of VWF (a marker of endothelial cell damnification), and IL-17 promoting apoptosis of HUVEC may be one of mechanisms. Abnormal function of endothelial cell include characteristic of adhesion increasing, so we investigated that IL-17 affect on adhesion between HUVEC and THP-1cell line(a monocyte cell line of human). So our studies include three parts, fist part is "The Investigation about correlation of Th17 to atherosclerosis and acute coronary syndromes", second is "The mechanism of IL-17 effective molecule of Th17 cells promote lesion of endothelium", third is ’The mechanism of IL-17 major effective molecule of Th17 cells enhance adhesion of endothelial cells to monocyte"MethodsI. The correlation of Th17 to human atherosclerosis1. SubjectsTotal of 40 patients who were underwent diagnostic catheterization at Qilu and Shengli Hospital, were classified as acute coronary syndrome (ACS) group (n=24) and stable angina (SA) group (n=16). Healthy individuals (18 persons) without cardiovascular disease.2. Flow cytometryPeripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of patients and healthy individuals using Ficoll density gradient, PBMCs (1×106/ml) were stimulated with 25 ng/ml of PMA, Flow cytometry detected CD4+IL-17+T cell, CD4+IFN-g+T cell and CD4+CD25highCD127low/-T (Treg) cell, and analyzed change of Th17, Thl and Treg in peripheral blood of patients.3. ELISA assayPlasma from human subjects was analyzed for the cytokine levels of IL-17A, IL-6 and TGF-βusing commercial ELISA kits4 Statistical analyses.All analysis was performed by SPSS 11.0 (SPSS Inc, Chicago, USA), Including correlation between Th17 and associated cells, levels of circulation Il-17, TGF- and IL-6, and correlation between IL-17 and other inflammatory factors. Ⅱ. The impact of IL-17 on lesion of human vascular endothelium and underling mechanism1. Analysis of the expression of IL-17A receptor (IL-17RA) on different donor human umbilical vein endothelial cells (HUVECs) by RT-PCR and flow cytometry2. Effect of IL-17 on the expression of VWF in HUVECsHUVECs were cultured in 6-well plate for 24 h and then treated with different concentration of IL-17A for 24h. At the end of experiments, the cells were collected for the analysis of VWF mRNA levels, the supernatant was used to detect the levels of VWF by ELISA.3. Effect of IL-17 on apoptosis of HUVECsHUVECs were treated with different concentration of IL-17A for 48h. At the end of experiments, the cells were collected for the analysis of apoptosis using Annexin V-FITC apoptosis kit.4. Mechanism of IL-17 promoting apoptosis of HUVECsTo determine whether IL-17 could induce apoptosis of HUVECs through mitochondrial pathway, the expression of apoptosis-associated proteins was investigated by Western-blotting, and the mitochondrial transmembrane potential was examined using the MitoCaptureTM Apoptosis DetectionⅢ. The impact of IL-17 on adhesion of human monocyte to endothelial cells1. Effect of IL-17 on early adhesion between vascular endothelial cell and monocyteWe detected the contact between HUVEC and monocyte (THP-1) real-timely using Live Cell Station, found that IL-17 promoted adhesion between HUVEC and monocyte (THP-1) very quickly. To investigated adhesion quantificationally, we treated HUVEC with different concentration of IL-17A for various periods of time(0.5 or 3 h), then co-culture with THP-1 stained by calcein AM。Quantitative analysis on effect of IL-17 on adhesion between HUVECs and THP-1 was performed by Multilabel Counter 2. Detection of effect of IL-17 on adhesion molecules expression by real-time PCRHUVECs were treated with IL-17A (10ng/ml) for various periods of time (0.5, 1 and 3h), Total RNA was isolated from HUVECs using Trizol, E-selectin, P-selectin, ICAM-1 and VCAM-1 were detect by real-time PCR3. IL-17 promoted HUVEC polarizationCell polarization is early affair of adhesion between cells, is necessary for adhesion. Whether IL-17 could promote cell polarization, there has not been reported. So, we stimulated HUVEC with IL-17 in different concentration for 30 minutes, we stained cell with FITC-anti-human-VCAM-1 and PE-anti-human-ICAM-1 antibody, and observed with fluorescence microscope.4. Effect of Ca2+ on early adhesion of IL-17HUVECs were treated with different concentration of Ca2+ for 30min,then co-stimulated with IL-17A (10ng/ml) for 30min or 3 hours, and finally co-culture with THP-1, stained by calcein AM for 30min, Quantitative analysis was performed by Multilabel Counter.5. Effect of IL-17A on intracellular Ca2+ concentration of HUVECHUVECs were treated with IL-17A (10ng/ml) for various periods of time(5,15 and 30min),then free-Ca2+ in HUVECs were marked with fluo-3A and detected by flow cytometry. Phosphorylation of PLC 1 and PLC 3 was analyzed using Western blotting. HUVECs were treated with IL-17A (1 Ong/ml) for various periods of time(2,5 and 15min),cells were lysed in PLB,stored in-80. PLC 1 and PLCb3 and phosphorylation of PLC 1 and PLC 3 were detected using Western blotting5. Cell polarization mediated by Ca2+ and pro-adhesion effect of IL-17THP-1 was stained with CFSE and cultured for 24h, and HUVEC was stimulated with IL-17 for 30min, then these two cells were co-cultured for 15 and 30min.Finally HUVEC was stained, and observed with fluorescence microscope. ResultsI. The correlation of Th17 to human atherosclerosis1. Increase in circulating Th17 cells and IL-17 in patients with ACSTo investigate the role of different subset of CD4+T cell in the development of ACS, we first analyzed the frequency of CD4+IL-17+T cell, CD4+IFN-g+T cell and CD4+CD25highCD127low/-T (Treg) cell using flow cytometry in peripheral blood of patients with ACS and health control, showed that circulating Thl 7 and Thl cells significantly increased in the patients with ACS compared with healthy individuals and patients with stable angina pectoris, and Treg cell was decreased. There was no difference between patients with stable angina pectoris and healthy subjects. Consistent with the upregulation of Th17 cells, the levels of the circulating IL-17 in the patients with ACS also increased. These data suggested that IL-17 is involved in the pathogenesis of ACS.2. An increased IL-6 and decreased TGF-βin the peripheral blood of patients with ACS-Since both IL-6 and TGF-P have been reported to be required to the differentiation of Th17, we next studied the levels of these two cytokines in the blood of the ACS with patients and performed a correlation assay between plasma IL-17 and IL-6 or TGF-β, respectively. The patients with ACS showed significantly higher levels of IL-6 in the blood compared with that in normal individuals or with patients with stable angina pectoris, whereas the levels of TGF-βin patients with ACS were significantly lower than that in normal individuals or in patients with stable angina pectoris. Notably, the levels of IL-17 had a closely positive correlation with IL-6, but a negative correlation with TGF-β. The high levels of IL-6 and moderate levels of TGF-βin the patients with ACS likely favor the Th17 differentiation from naive CD4+T cell; on the other hand, IL-17 may also trigger more IL-6 production from macrophages and endothelial cells, forming a positive feedback loop.4. The relation between IL-17 and other danger factor Next we analyzed whether the plasma IL-17 was associated with the relevant cardiovascular risk factors in patients with ACS, including blood lipid or glucose metabolic parameters cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), and glucose. We showed that the levels of circulating IL-17 had no significant correlation with the levels of plasma HDL, LDL, TG, cholesterol, and glucose, suggesting a lack of a direct influence of IL-17 to the lipid and glucose metabolism during the pathogenesis of ACS.Ⅱ. The impact of IL-17 on lesion of human vascular endothelium and underling mechanism1. HUVECs constitutively express low-level IL-17AR.Despite the increase in IL-17 amounts in the onset of ACS, a direct causative role of IL-17 to the pathogenesis of ACS remained unclear. Considering the crucial role of endothelial dysfunction and injury in the development of atherosclerosis and plaque instability, we studied the direct effects of IL-17 on human vascular endothelial cells. We first analyzed the expression of IL-17A receptor (IL-17RA) on human umbilical vein endothelial cells (HUVECs) by RT-PCR and flow cytometry. HUVECs constitutively expressed low-level IL-17AR mRNA and proteins, suggesting vascular endothelial cells as a potential target for IL-172. IL-17 damages endothelium via inducing VWF secretion by endothelial cellsVWF is a large and circulating glycoprotein that plays an essential role in haemostasis. It mediates platelet adhesion to the vascular wall, platelet aggregation and serves as a plasma carrier for factorⅧ, stabilizing the factorⅧin the circulation. Plasma VWF has been proposed as a marker of endothelial dysfunction since it is almost exclusively produced by endothelial cells and its release is increased when endothelial cells are damaged. We showed that IL-17 markedly enhanced amounts of VWF mRNA expression and protein production by HUVECs in a dose-dependent manner, indicating that IL-17A induced the endothelial injury or dysfunction.3. IL-17 induces apoptosis of endothelial cells To understand the molecular mechanisms responsible for the damaging effects of IL-17 on endothelial cells, we studied the roles of IL-17 in endothelial apoptosis. IL-17 induced the early apoptosis of HUVECs in a dose-dependent manner in vitro. Low concentration of IL-17 (1 to 5 ng/ml) had no obvious effects on the endothelial apoptosis (PI-, Annexin V+), but high concentration of IL-17(10 ng/ml) significantly increased the apoptosis of HUVECs; the pro-apoptotic effect of IL-17 peaked at the concentration of 50 ng/ml, and maintained thereafter. These data suggest that IL-17-mediated endothelial damage is caused by apoptosis of endothelial cells.4. IL-17 induces apoptosis of endothelial cells through mitochondrial pathwayTo determine the pathway by which IL-17A induced the apoptosis of HUVECs, we examined the expression of apoptosis-associated proteins including cleaved caspase-3,8 and 9, Bcl-2 and Bax. Cleaved caspase-3 and 9 significantly increased in HUVECs treated with IL-17 (10ng/ml and 50ng/ml) for 48h compared with that in untreated HUVECs, on the contrary, no significant difference was observed on the expression of cleaved caspase-8. Importantly, Bax, a critical pro-apoptotic protein involved in mitochondrial apoptosis pathway, markedly increased in IL-17-treated cells, which led to the ratio of Bax to Bcl-2 increased significantly.As the disruption of the mitochondrial transmembrane potential is one of the earliest intracellular events that occur following induction of apoptosis, we then analyzed the mitochondrial transmembrane potential using the MitoCaptureTM Apoptosis Detection, result shown that IL-17 enhanced mitochondrial membrane damage, and led to apoptosis of endothelial cells.III. The impact of IL-17 on adhesion of human monocyte to endothelial cells1. IL-17 promote the adhesion between endothelial cell and monocyteAdhesion between endothelial cell and monocyte played an important role in the development of atherosclerosis, however, the report of which IL-17 is very little. So we investigated effect of IL-17 on adhesion between endothelial cells and monocyte real-timely or quantificationally. The data demonstrated that pro-adhesion effect of IL-17 was dose-dependent, and peak at 50ng/ml. whereas no obvious effect was observed in low dose. Interestingly, the pro-adhesion effect of IL-17 was obvious when HUVEC was treated with IL-17 for just 30min.2. IL-17 induce expression of adhesion molecule on HUVECsThe adhesion molecules expressed on endothelial mainly include ICAM-1, VCAM-1 and E-selectin. Real-time PCR demonstrated that IL-17 could not upregulate the expression of adhesion molecules mRNA in HUVEC after stimulating with IL-17(10ng/ml) for 30 minutes and 1 hour, and result of flow cytometry was sameness. According to the results of adhesion assay, the pro-adhesion effect of IL-17 was independent on the upregulation of adhesion molecules.3. Ca2+ involved in HUVEC polarization induced by IL-17In the progress of cell adhesion, cross-linking cells may induce cell polarization. We found that IL-17 promote polarization distribution of adhesion molecules on HUVEC. Importantly, the pro-polarization effect of IL-17 reduced when HUVEC were treated with Ca2+ inhibitor.4. The pro-adhesion effect of IL-17 is extracellular Ca2+ dependentCa2+ participated in the adhesion progression between endothelial cells and monocyte, however, whether Ca2+ was involved in the pro-adhesion of IL-17 remained unclear. To investigate the role of Ca2+ in this progress, we inhibit the effect of extracellular Ca2+ using Ca2+ chelate (EGTA).Suppression of extracellular Ca2+ significantly inhibit pro-adhesion effect of IL-17, and this inhibition was obvious even in a low-dose EGTA.Ca2+ played an important role in cell adhesion. Influxing of Ca2+ promote adhesion between endothelial and monocytes. Next we investigated whether IL-17 (10ng/ml) could promote influx of Ca2+, and found that IL-17 can promote Ca2+ influx in a time dependent manner, peaking in 5min and recovering to normal level in 15min.These results indicate that IL-17 could transiently upregulate the function of intracellular Ca2+ in HUVEC, suggesting IL-17 promote adhesion between endothelial cells and monocyte by contributing to Ca2+ influx.5. IL-17 enhanced influx by activating PL 1There are two pathways mediating Ca2+ influx. One is G-protein coupled receptor pathway which induce Ca2+ influx by activating PLC 3. Another is protein tyrosine kinase receptor pathway which promotes PLC 1 activation. So we detect PLC 3 and pPLC 3、PLC 1 and pPLC 1 by Western blotting. We demonstrated that IL-17 can phosphorylate PLC 1, whereas have no effect on PLC 3.We identify for the first time that IL-17 could activate PLC 1, promoting Ca2+ influx, which mediate the pro-adhesion effect of IL-17.Conclusion1. Increase in circulating Th17 cells and IL-17 in patients with ACS, on the contrary, Treg cell was decrease. And there was no different between stable angina and health control.2. The levels of circulating IL-17 in patients with ACS had a closely positive correlation with IL-6, but a negative correlation with TGF-(3. And the levels of circulating IL-17 had no significant correlation with the levels of plasma HDL, LDL, TG, cholesterol, and glucose, suggesting a lack of a direct influence of IL-17 to the lipid and glucose metabolism during the pathogenesis of ACS.3. Our findings have revealed a previously unrecognized function of IL-17: IL-17 promoted endothelial damage via induced apoptosis of endothelial cells, contributing to the development of atherosclerosis and formation of plaque. IL-17 induced the apoptosis of vascular endothelial cells via a mitochondrial pathway.4. IL-17 promote the pro-adhesion between endothelial cells and monocytes, and the pro-adhesion effect of IL-17 was independent on the upregulation of adhesion molecules on HUVECs.5. The pro-adhesion effect of IL-17 was extracellular Ca2+-dependent, IL-17 can upregulate concentration of intracellular Ca2+ via activating PLC-1, and Pro-adhesion effect of IL-17 is mediated by Ca2+-promoting cell polarization. In a words, IL-17/Th17 was increased in peripheral blood of patients with ACS, IL-17, a major effect molecules of Th17 cells, could enhance the development of atherosclerosis, and both inducing endothelial lesion and promoting adhesion function of endothelial cells are the potential mechanisms. Part II Inhibitory effects of CD28-binding peptoids on T cell mediated Immune responseObjectsAtherosclerosis is a chronic autoimmune disease, T cells are involved in the development of atherosclerosis and formation of plaque. Cross-linking of CD28 and B7 induced co-stimulatory signal which is the most important co-stimulatory signal in T cell activation. Blockade of CD28 can effectively inhibit T cell activation. Several different strategies for blocking the CD28-B7 interactions have been used, including Anti-CD28 Abs and Polypeptide. However, the extensive applications of the Ab-based antagonism to clinical treatment are limited by apparent disadvantages such as inherent immunogenicity, unwanted Fc signaling, poor tissue penetration and bioinstability. And there are some limitations for peptide drugs including short half-life, rapid metabolism, vulnerable to protease and poor bioavailability. Recent research has been directed toward the creation of non-natural, sequence-specific biomimetic oligomers with bioinspired structures that capture the amino-acid interface of the targeted proteins. One such family of molecules is the poly-N-substituted glycines or peptoids, which have close structural similarity to peptides but are essentially invulnerable to protease degradation. To screen for peptoid that specifically target CD28, we first designed and chemically synthesized 19 candidate peptoids based on molecular modeling and docking. Using the phage-displaying system that expresses the extracellular domain of the CD28 homodimer and contains the core B7-binding motif, a peptoid (No.9), was identified to display the highest binding activity to CD28.I. Material and Methods1. Assay of lymphocyte proliferation of human peripheral blood mononuclear cells (PBMCs) stimulated by the anti-CD28 Ab and inhibited by the CD28-binding peptoidsThe proliferation assay was employed to detect the effects of CD28-binding peptoid on the CD28-dependent lymphocyte proliferation. Mononuclear cells were separated from human peripheral blood by Ficoll density gradient centrifugation, and stimulated by phytohemagglutinin(PHA), PHA+anti-human-CD28 Ab, or PHA (50μg/ml)+anti-human-CD28 Ab+peptoid for 56 h at 37℃in an incubator. For an additional 72 h of incubation,3H-thymidine was added to each well. The cells were then collected and the radioactivity was measured by a liquid scintillation counter.2. Assay of lymphocyte proliferation of mouse splenocyte by stimulation with anti-CD28 Abs and by mixed lymphocyte reactionPrior to assaying of lymphocyte proliferation in mice, splenocyte suspension was prepared. Red blood cells were removed via lysing. Measurements of proliferation stimulated by antihuman CD28 and suppressed by CD28-targeting peptoid were performed using the same method as in the above proliferation assay for human PBMCs. For mixed lymphocyte reaction, lymphocytes suspensions were prepared from C57BL/6 mice for responder cells and from BALB/c mice for stimulator cells, as described above. For making responder cells, mitomycin C was added into splenocyte suspended, and treated at 37℃for 30 min, cells were spun down and washed, and then resuspended 1 ml of RPMI-1640 supplied with 15% of fetal bovine serum, The responders and stimulators were then mixed at a ratio of 1:1 and cultured with or without the human CD28-targeting peptoid, as described above, except that 0.5 mCi/well of thymidine was added 16 h before the radioactivity measurement.3. Mouse bone-marrow transplantation, peptoid treatment and phenotype observationThe mouse model of bone-marrow transplantation was established using C57BL/6 mice as the donors and BALB/c mice as the recipients. The bone-marrow cells and splenocytes of donor were mixed with a ratio of 1:1 as transplantation preparation. Four to six hours prior to transplantation, the BALB/c recipient mice were irradiated with a dosage of 6.5 cGy. For transplantation,2×10’cells of the transplantation preparation was injected into the tail veins of the recipient mice. Since the second day of transplantation, recipients were intraperitoneally injected daily with either 100 mg/ml of No.9 peptoid or 0.1 ml of fetal bovine solution. Symptoms such as diarrhea, viability, hairspring and back arching were observed daily and the weight was measured twice a week. Phenotypic severity was assessed and scored as 0,1 and 2, which stood for normal, mild and severe phenotypes, respectively. The survival rate was recorded three times a week.Ⅱ. Results1. The peptoid inhibits proliferation of human and mouse T lymphocytesTo examine whether the peptoid No.9 inhibits the CD28-mediated lymphocyte proliferation in vitro, human PBMCs were stimulated by either PHA or CD28 Ab alone, or along with peptoid No.9 at incremented concentrations. As indicated by 3H-TdR incorporation, the proliferative activity stimulated by anti-CD28 and PHA was significantly inhibited by peptoid No.9 (P=0.01). Interestingly, due to the identical CD28-binding motif of mouse CD28 to its human counterpart, on which the peptoid design was based, this peptoid also inhibited the CD3 Ab- and CD28 Ab-stimulated proliferation of lymphocytes derived from mice (P,0.05).Moreover, in the mixed lymphocyte reaction experiment, the proliferation of responder lymphocytes from C57BL/6 mice stimulated by APCs from BALB/c mice was significantly suppressed by addition of 10-20 mg of peptoid No.9 (P,0.01), indicating an inhibitory effect of the peptoid on the proliferation triggered by alloantigen stimulation.2. The peptoid attenuates the graft-versus-host disease (GVHD) in mice by suppressing alloantigen-induced immunoinflammationThe CD28-dependent costimulatory is crucial for alloantigen-induced T-cell activation and clonal expansion and plays important roles in the pathogenesis of acute and chronic GVHDs. To test whether the CD28-binding peptoid No.9 can interfere with the immunoresponses against alloantigen and alleviate the inflammation in vivo, bone-marrow transplantation was conducted using C57BL/6 mice as the donors and BALB/c mice as the recipients. One to eight days after transplantation, symptoms such as diarrhea, loss of weight, decrease in viability, back bending and developmental retardation occurred in the recipient mice. The mice in the GVHD group without peptoid treatment had the most severe symptoms and their phenotypical severity scores in the rose quickly from day 1 and reached the peak (score 10) at day 5 and maintained for up to 4 weeks. In contrast, the GVHD mice received a daily intraperitoneal injection with 100 mg/ml of peptoid No.9 reduced the severity by nearly two scores from day 5 to day 18.Survival rate observation revealed that the recipient mice without peptoid treatment started to die from day 3. The mortality was 70% on day 14 and went up to 100% on day 28. In contrast, the recipients received peptoid No.9 treatment started to die on day 7 and the mortality on day 14 was declined to 25%, although it also reached 100% on day 28. Histological study on day 19 when symptoms were severe showed that pathological changes were prominent in liver and colon tissues. Changes included bleeding, necrosis, mild edema, etc. The results of H&E staining showed that lymphocyte infiltration was prominent in both liver and colon tissues of the recipient mice without peptoid treatment, whereas the inflammation was significantly suppressed in those pre-treated with 10 mg of the peptoid No.9. Collectively, these results suggested that the peptoid No.9 attenuates the GVHD in mice by suppressing alloantigen-induced immunoinflammation.Conclusion:We first identified a peptoid that binds to CD28 with high affinity and specificity. This peptide can not only effectively inhibit T-cell proliferation in vitro, but also attenuate the GVHD through suppressing the alloantigen-induced immunoresponse in vivo. We synthesized a peptoid which can inhibit T cell activation by suppressing the function of CD28. |
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