| ObjectiveAtherosclerosis is the most common diseases of heart cerebrovascular system and the leading cause of mortablity in advanced and developing countries. Atherosclerosis is a slowly progressing chronic inflammatory disease occurring in large and medium-sized arteries and is characterized by an accumulation of lipids in the artery wall, together with infiltration of immunocytes, such as macrophages, T cells and mast cells, and the proliferation of vascular smooth muscle cells. Accumulate research showed that innate immune respons (macrophages) and adaptive immune response (CD4+T cells) participated in the development of atherosclerosis. Recently, the effect of CD4+T cells in this disease received widespread concerns. CD4+T cells contain different subsets and compose of conventional Th1and Th2cells and regulatory T cells (Treg) and new subsets Th17cells. Th1cells promoted the development of atherosclerosis. And the effect of Th2cells on the diseases remains controversial. Regulatory T cells play a protective role in atherogenesis and could inhibit the formation of atherosclerotic plaque. However, the function of Th17cells in the development of atherosclerosis remains undefined.Th17cell is characterized by secreting IL-17A. Naive CD4+T cells stimulated by antigens and differentiated into Th17cells with the regulation of transcription factors RORγt and RORoα and in the presence of IL-6and TGF-β.It has been shown that Th17cells and effector IL-17A play a part in many kinds of chronic inflammation and organ-specific autoimmune disease in animals such as experimental autoimmune encephalomyelitis (EAE), rheumatoid arthritis (RA) and colitis. Atherosclerosis is a chronic inflammatory disease occurring in artery vascular walls, previous studies have shown that Th17cells and its major effector cytokine IL-17A are increased in the peripheral blood of patients with coronary atherosclerosis. And T cells infiltrated in the atherosclerotic plaque express IL-17A in human or in mouse models. These suggest that Th17cells and IL-17A probably participate in the development of atherosclerosis. However, whether Th17cells and IL-17A increased with the development of atherosclerosis? What roles Th17cells and effector IL-17A play in the formation and development of atherosclerosis remains debate. Whether could IL-17A as one of potential therapeutic targets for atherosclerosis? What is the mechanism of IL-17A in this disease? All these problems remain to be investigated.To confirm the effect of Th17cells and IL-17A on the development of atherosclerosis, illustrate the mechanism, and provide theoretical basis and experimental data for clinical applications, this paper intends to study from the following two parts:1. To confirm the aggravated role of Th17cells and effector IL-17A in the development of atherosclerosis.1) The establishment of standard atherosclerotic mice model and rapid carotid lesions mice model:ApoE-/-mice fed with high cholesterol diet to establish standard atherosclerotic mice model. And high cholesterol diet combined with placed perivascular constrictive silica collars on the left common carotid artery to establish rapid carotid lesions mice model.2) The expression of Th17cells and IL-17A in atherosclerotic plaque and peripheral immune organ:Results for RT-PCR and immunohistochemistry showed that Th17cells and IL-17A could be detected in atherosclerotic lesions. And flow cytometry analysis found that the percentage of Th17cells and IL-17A in spleen increased by the formation and development of atherosclerosis. Furthermore, the proportion of Th17cells was positive correlated with the area of the plaque and the level of serum lipids (TCH).3) The effect of Thl7cells and IL-17A on the formation of atherosclerotic plaque:We treated ApoE-/-mice with anti-IL-17A neutralizing antibody or exogenous recombinant mouse IL-17A, and from both the positive and negative aspects, we confirmed that IL-17A could promote the formation of atherosclerotic plaque. Furthermore, IL-17A reduces the content of smooth muscle cells in lesions, suggesting increased instability of the plaques.2. To explore the mechanism of IL-17A promote the development of atherosclerosis from macrophage ER stress and cell apoptosis1) The effect and mechanism of IL-17A on macrophage apoptosis:IL-17A could promote macrophage apoptosis in vitro. Further mechanism study found that IL-17A induced ER stress in murine and human-derived macrophages in vitro. In addition, IL-17A could also induce ER stress in macrophage-dense areas of the atherosclerotic lesions in ApoE-/-mice. However, in vivo, blocking of ER stress by chemical chaperone (PBA) alleviated IL-17A-induced ER stress and reduced the numbers of apoptotic macrophages in lesions, suggesting IL-17A could promote macrophage apoptosis through inducing ER stress.2) The mechanism of IL-17A aggravated the development of atherosclerosis:We treated ApoE-/-mice with IL-17A or IL-17A combined with PBA, and confirmed that IL-17A could promote macrophages apoptosis by activating ER stress response and accelerate the formation of atherosclerotic plaque.3) The mechanism of IL-17A induced ER stress in macrophage:In vitro, IL-17A could up-regulate the expression of aP2and activate NF-κB and MAPK signal pathway (ERK, p38and JNK). Furthermore, we found that IL-17A up-regulated aP2through activating NF-κB and p38,ERK pathway. Finally, we confirmed that aP2mediated IL-17A-induced ER stress in macrophages.Methods1. To confirm the aggravated role of Th17cells and effector IL-17A in the development of atherosclerosis.1) The establishment of standard atherosclerotic mice model and rapid carotid lesions mice model:i) Standard mice model:8weeks old male ApoE-/-mice fed with high cholesterol diet to16weeks or24weeks old. And same strain C57BL/6mice also give high cholesterol diet as control.ii) Rapid carotid lesions mice model:8weeks old male ApoE-/-mice fed with high cholesterol diet for2weeks, and then carotid atherosclerotic lesions were induced by perivascular constrictive silica collars placed on the left common carotid artery. Mice were going on high cholesterol diet to16weeks or24weeks old. And same strain C57BL/6mice as control.2) The expression of Th17cells and IL-17A in atherosclerotic plaque and peripheral immune organ:i) Separate the left common carotid arteries and the aortic root vessels and prepare serial cryosections, HE staining and Oil red O staining detected the formation of atherosclerotic lesions and the content of lipids. And immunohistochemistry detected the expression of IL-17A and IFN-y in plaque, and confirmed the resource of IL-17A with immunofluorescence double staining.ii) Separate the aortic arch to extract RNA and detect the expression of IL-17A, RORyt and IFN-γ, T-bet in atherosclerotic plaque with RT-PCR.iii) Splenocytes were prepared from different age mice and flow cytometry detected the percentage of Thl and Th17cells.iv) To analyze the correlation of the proportion of Thl and Th17cells with the size of atherosclerotic plaque and the level of serum lipids.3) The effect of Th17cells and IL-17A on the formation of atherosclerotic plaque:i) Anti-IL-17A neutralizing antibody treatment:a) Twenty8weeks old male ApoE-/-mice fed with high cholesterol diet for2weeks, and then carotid atherosclerotic lesions were induced by perivascular constrictive silica collars placed on the left common carotid artery. Five days later, mice were randomly divided into two groups and treated with anti-IL-17A neutralization monoclonal antibody (50μg/mouse/time) or isotype antibody (rat IgG2A), once a week for4weeks.b) During this interventional period, plaque sizes were evaluated by Micro-ultrasound imaging, and then plaque morphology was detected by histopathology after mice were sacrificed at one week following the last challenge. Oil red O staining detected the size of lesions.c) Immunohistochemistry detected the expression of macrophage and smooth muscle cells in lesions.ii) Application of exogenous IL-17A:a) Ten8weeks old male ApoE-/-mice were randomly divided into two groups and fed with high cholesterol diet, and11weeks old mice were given intraperitoneal injection with recombinant mice IL-17A (2μg/mice/time) once a week for5weeks and normal saline as control.b) At one week following the last challenge, separate the aortic root vessels and prepare serial cryosections. Oil red O staining observed the size of plaque.c) Immunohistochemistry detected the expression of macrophage and smooth muscle cells in lesions.2. To explore the mechanism of IL-17A promote the development of atherosclerosis from macrophage ER stress and cell apoptosis1) The effect and mechanism of IL-17A on macrophage apoptosis and the formation of atherosclerotic plaque:i) Experiment in vitroa) Isolating of mice peritoneal macrophages and stimulated with IL-17A, Hoechst staining observed cell apoptosis from cytomorphology and flow cytometry (Annexin V/PI) detected the numbers of apoptotic cells.b) Mice macrophage cell line RAW264.7and peritoneal macrophages stimulated by IL-17A, then Western Blotting detected the expression of ER stress markers p-PERK, p-eIF2a and XBP1s.c) Human monocyte cell line Thp-1and peripheral blood mononuclear cells were induced into macrophages and stimulated with human IL-17A, then Western Blotting detected the expression of ER stress markers p-PERK, p-eIF2α and XBP1s.d) Mice peritoneal macrophages stimulated by IL-17A, then Western Blotting detected the expression of CHOP, caspase12and caspase3cleavage.ii) Experiment in vivoa) Thirty8weeks old male ApoE-/-mice fed with high cholesterol diet, and11weeks old mice were randomly divided into3group:Control (i.p. normal saline), IL-17A-treated group (i.p. mice IL-17A,2μg/mice/time, once a week), IL-17A combined with PBA treated group (i.p. IL-17A,2μg/mice/time, once a week and PBA,100mg/kg, twice a week).b) At one week following the last challenge, separate the aortic root vessels and prepare serial cryosections. Immunohistochemistry detected the expression of Moma-2(for macrophage), p-PERK, p-eIF2α and CHOP, caspase12in atherosclerotic lesions. And Tunnel staining for apoptotic macrophages in lesions. Separate the aortic arch to extract proteins, Western Blotting detected the expression of ER stress markers (p-PERK, p-eIF2α and XBP1s) and CHOP, caspase12, and caspase3cleavage.c) Oil red O observed the size of atherosclerotic plaque.2) The mechanism of IL-17A induced ER stress in macrophage:i) Mice peritoneal macrophages stimulated by IL-17A, RT-PCR and Western Blotting detected the expression of aP2.ii) Mice peritoneal macrophages with aP2inhibitor (BMS309403)(50uM) pre-treated three hours and then stimulated by IL-17A, Western Blotting detected the expression of p-eIF2α and XBPls.iii) Mice peritoneal macrophages stimulated by IL-17A, Western Blotting detected the expression of p-IκB, p-ERK, p-p38and p-JNK. Furthermore, mice peritoneal macrophages with NF-κB (PDTC), ERK (PD98059), JNK (SP600125) and p38(SB203580) inhibitor pre-treated one hour and then stimulated by IL-17A, Western Blotting detected the expression of aP2.Results1. We confirmed that Th17cells and effector IL-17A aggravated the development of atherosclerosis with experiment in vivo and in vitro1) The expression of Th17cells and IL-17A in atherosclerotic plaque and peripheral immune organi) The expression of IL-17A and IFN-y in atherosclerotic plaqueTo study the effect of Th17cells and IL-17A on atherogenesis, we established rapid carotid lesions mice model and standard atherosclerotic model with ApoE-/-mice. And we separated the left common carotid arteries and the aortic root vessels and prepare serial cryosections, HE staining, Oil red O staining and immunohisto-chemistry detected the formation of atherosclerotic lesions and the expression of IL-17A and IFN-y. Results showed that8week-old ApoE-/-mice had no significant plaque formation in carotids and vascular intima only appeared slightly thickening. At this time, no IL-17A and IFN-y was detected in lesions. When mice fed with high cholesterol diet for additional eight weeks,16weeks old ApoE-/-mice showed that vascular intima significantly thicken and proliferation of smooth muscle cells. Atherosclerotic plaque characterized by thicker fibrous cap and much of nucleated cells infiltrating and was defined as the early stage of atherosclerosis. At16weeks old ApoE-/-mice, substantial expression of IL-17A and IFN-y was detected. The plaque was more severe when ApoE-/-mice were continuously fed with high cholesterol diet for16weeks,24weeks old ApoE-/-mice showed that the size increased obviously, fibrous cap became thinner, the number of smooth muscle cells reduced and many vacuoles present in lesions, that was the late stage of atherosclerosis. This stage, we could detect IL-17A in lesions and almost no IFN-y was found. In contrast, the age-matched wild-type C57BL/6mice (fed with the same regimen as ApoE-/-mice) had no detectable plaque in carotids throughout the whole experimental periods.In addition, we detected the expression of IL-17A, RORyt and IFN-γ, T-bet with RT-PCR. Consistent with the results from immunohistochemistry or immune-fluorescence staining, the expression of IL-17A in ApoE-/-mice with the early and late stage of plaque was markedly higher than that of wild-type control mice. However, IFN-y only was detected in early stage of plaque and could not be detected in late stage of plaque. Notably, the expression of RORyt and T-bet, the transcription factors controlling Th17and Thl cell differentiation respectively, was significantly higher in the vascular wall of ApoE-/-mice than in that of control mice at as early as8weeks. The levels of RORyt and T-bet continued to increase and were maintained at higher levels in ApoE-/-mice at the early and late stages of atherosclerotic plaque (16-24weeks). To confirm the cellular sources of IL-17A, we co-stained IL-17A with CD4+T cells and macrophages with immunofluorescence staining. IL-17A was mainly expressed in both CD4+T cells and macrophages infiltrated into the atherosclerotic plaque. The data suggest that both Th17and Thl cells were involved in the development of the atherosclerotic inflammation, and Th17play more important roles in late stage of atherosclerosis.ii) Increased Th17and Thl cells in the spleen of ApoE-/-mice with atherosclerotic plaqueTo observe the change of Th17and Thl cells in peripheral immune organ, we detected the number of Th17(CD4+IL-17A+) and Thl (CD4+IFN-γ+) in spleen with flow cytometry. Results showed that the pre-symptomatic ApoE-/-mice (8weeks-old) had minimal numbers of Th17cells with no statistical difference compared with control mice. Although the number of Thl cells was higher than Th17, there was no statistical difference between ApoE-/-and control mice. However, the frequencies of Th17and Thl cells in16week-old ApoE-/-mice with plaque was significantly higher than that in control mice, which were further increased in the ApoE-/-mice with advanced atherosclerotic plaque (24weeks-old). In addition, we observed a population of CD4+T cells expressing both IL-17+and IFN-γ+. The frequency of CD4+IL-17+IFN-γ+T cells significantly increased in the late stage atherosclerotic ApoE-/-mice compared with age-matched control mice.Furthermore, simple linear regression analysis, we observed that the area of the plaque and the level of TCH was not only correlated with the proportion of Thl cells, but also closely associated with Th17cell subset and CD4+IL17+IFN-γ+cells. These results suggested that Th17and Thl cells both probably participated in atherogenesis.2) The influence of Th17cells and IL-17A on the development of atherosclerosisTo confirm the role of Th17cells and IL-17A on the development of atherosclerosis, we treated ApoE-/-mice with anti-IL-17A neutralizing antibody and exogenous IL-17A. Results from micro-ultrasound imaging and Oil red O staining showed that neutralizing IL-17A antibody significantly inhibited the formation of atherosclerotic plaque, however, exogenous IL-17A obviously accelerated atherogenesis. Immunohistochemistry show that IL-17A treatment had no significant effect on the number of macrophage and it decreased the numbers of smooth muscle cells in plaques. These results confirmed from positive and negative aspects that IL-17A could promote the development of atherosclerosis and increased instability of the plaques.2. IL-17A-induced ER stress in macrophage promote cell apoptosis and aggravate atherogenesis by up-regulating NF-κB and p38, ERK pathway mediated the expression of aP21) IL-17A promote macrophage apoptosis by inducing ER stressi) IL-17A promote macrophage apoptosisMacrophage apoptosis is an important cause for the development of atherosclerosis. To explore the mechanism of IL-17A accelerate the formation of atherosclerotic plaque, we stimulated macrophage with IL-17A in vitro. Results from Hoechst staining showed that macrophages treated by IL-17A revealed increase of apoptotic bodies. Next, results from flow cytometry showed that IL-17A promote the number of late apoptotic macrophages increasing markedly. ii) IL-17A induced endoplasmic reticulum stress in murine and human-derivedmacrophage in vitroSince increasing evidence supports that ER stress is one important mechanism for macrophage apoptosis, to explore the mechanism of IL-17A promote macrophage apoptosis. Mice macrophage cell line RAW264.7and mice peritoneal macrophage were stimulated by IL-17A, Western Blotting analysis showed that the expression of ER stress markers p-PERK,p-eIF2α and XBP1s increased significantly. This suggested that IL-17A could induce ER stress in murine-derived macrophages. To observe whether IL-17A play the same roles on human-derived macrophage, we induced Thp-1and PBMC into macrophages and then stimulated with IL-17A, Western Blotting analysis showed that the expression of p-PERK,p-eIF2α and XBP1s increased significantly. These results indicated that IL-17A could induce ER stress in human and murine-derived macrophages in vitro.iii) Exogenous IL-17A induced ER stress in macrophage-dense areas of the atherosclerotic lesions in ApoE-/-miceTo confirm the impact of IL-17A on ER stress in vivo, we treated ApoE-/-mice fed with high cholesterol diet utilizing exogenous IL-17A, Western blotting analysis showed that p-PERK, p-eIF2α and XBP-1s were significantly up-regulated in aortic vessels with atherosclerotic plaque in IL-17A-treated mice compared with control. Consist with results from Western Blotting, immunohistochemistry staining revealed that increased expression of p-eIF2a and p-PERK mainly located in macrophage-dense areas of the atherosclerotic lesions. These indicate that IL-17A is able to induce directly ER stress response in macrophage both in vitro and in vivo.iv) IL-17A promote macrophage apoptosis by inducing ER stressIt has been shown that prolonged activation of ER-associated CHOP and caspase12pathways are involved in the ER stress-mediated apoptosis. To explore whether IL-17A promote macrophage apoptosis by inducing ER stress, we found that IL-17A significantly promote the expression of CHOP and caspase3cleavage, and slightly up-regulate caspasel2in vitro. To confirm the effect in vivo, we divided ApoE-/-mice into3groups:Control, IL-17A-treated group and IL-17A combined with PBA treated group. Western Blotting and immunohistochemistry showed that the expression of CHOP and caspase3cleavage up-regulated markedly in lesions of IL-17A-treated mice, and caspase12had no alteration. However, the expression of p-PERK and p-eIF2α diminished obviously in lesions of IL-17A combined PBA-treated mice, suggesting PBA could alleviate ER stress in lesions. At the same time, CHOP and caspase3cleavage reduced significantly and caspase12had no obvious change. Tunnel staining showed that IL-17A leads to a marked increase in apoptotic cells in atherosclerotic lesions and IL-17A comined with PBA could reduce apoptotic cells. Those indicated that IL-17A could induce apoptosis in macrophages mainly via ER stress-CHOP-caspase3axis.2) IL-17A accelerated the progression of atherosclerosis via ER stress-induced apoptosis in macrophageApoE-/-mice divided into three groups:control, IL-17A-treated group and IL-17A combined with PBA treated group. Oil red O detected the size of the aortic root for lesions, results showed that mice receiving IL-17A and PBA treatment had an obvious reduction in plaque size compared with IL-17A-treated mice. It follows that IL-17A could induce apoptosis in macrophages mainly via ER stress-CHOP-caspase3axis by which it accelerated progression of atherosclerosis.3) IL-17A up-regulated expression of aP2by activating NF-κB and ERK/p38MAPK pathways and further induced ER stress in macrophagei) The up-regulated expression of aP2is required for IL-17A-induced ER stress in macrophagesRecent study has suggested that aP2mediates saturated fatty acid-induced ER stress and apoptosis in macrophage, to study the mechanism of IL-17A induced ER stress in macrophage. Results from RT-PCR and Western Blotting both showed that IL-17A up-regulated directly aP2transcription and translation in macrophages in vitro. Importantly, IL-17A also led to a significant increase in the expression of aP2in atherosclerotic lesions in vivo. To clarify the role of aP2in IL-17A-induced ER stress, we treated macrophages with aP2inhibitor (BMS309403) in presence of IL-17A and found that accompanying inhibition of aP2, ER stress indicators p-eIF2α and the splice of XBP-1in macrophages diminished obviously correspondingly. This data clearly demonstrate that IL-17A for up-regulated aP2expression is required, at least partially, for IL-17A-induced ER stress in macrophages.ii) IL-17A up-regulated expression of aP2by activating NF-κB and ERK/p38MAPK pathways Previous works have shown that IL-17A can induce multiple genes via activating the nuclear factor (NF)-κB pathway and MAPK pathway. To explore the mechanism of IL-17A up-regulated the expression of aP2, macrophages were stimulated by IL-17A. And Western Blotting analysis showed that IL-17A could rapidly up-regulate the expression of p-IκB, p-ERK and p-p38, p-JNK. Subsequently, to clarify causative role of these signal pathways in IL-17A-upregulated aP2, we treated mouse peritoneal macrophages with the inhibitor of NF-κB, ERK, JNK and p38and then detected the aP2expression by Western Blotting. Results revealed that the inhibitors of NF-κB, ERK and p38diminish significantly the expression of aP2. However, JNK inhibitor did not suppress the aP2expression. Collectively, IL-17A up-regulates the expression of aP2through activating NF-κB and ERK/p38MAPK signal pathways in macrophages.Conclusions1. Th17cells and effector IL-17A could promote the development of atherosclerosisThe expression of Th17cells and IL-17A in atherosclerotic plaque increased and the proportion of Th17cells in spleen increased by the development of atherosclerosis and correlated with the size of atherosclerotic plaque and the level of TCH. In addition, in vivo, we confirm that Th17cells and effector IL-17A promote the formation of atherosclerotic plaque.2. IL-17A induced ER stress in macrophage and secondary apoptosis is one of mechanisms that IL-17A accelerates the formation of atherosclerotic plaque IL-17A induced ER stress in macrophage in vitro culture and in lesions. Prolongation of ER stress promotes macrophage apoptosis and accelerates the formation of atherosclerotic plaque through activating ER stress-CHOP-caspase3pathway.3. IL-17A up-regulating the expression of aP2is one of mechanisms of IL-17A induced ER stress in macrophageFurther study verify that IL-17A induces ER stress in macrophage through activating NF-κB and p38,ERK pathway and up-regulating the expression of aP2. And aP2inhibitor could alleviate ER stress that IL-17A induced in macrophage. Originality and SignificanceIn this research, we systematicaly study the effect and mechanism of Th17cells and its effector IL-17A in the development of atherosclerosis with experiment in vivo and in vitro. These have important theoretical and potential application value for revealing immunological mechanism of atherosclerosis and preventing atherosclerotic related cardiovascular disease. The originality and significance as follows:1. We confirm that Th17cells and IL-17A participated in the development of atherosclerosis. And exogenous IL-17A treatment obviously promotes the formation of atherosclerotic plaque. However, neutralizing IL-17A antibody could reduce the size of plaque. These foundings provide strong theoretical and experimental basis for treatment of atherosclerosis with IL-17A as clinical therapy target.2. We demonstrate, for the first time, that IL-17A by oneself can induce ER stress in macrophage. Secondary apoptosis of prolongation ER stress is one of mechanisms that IL-17A promotes the development of atherosclerosis. Furthermore, the inhibiting of ER stress with chemical chaperon PBA effectively control IL-17A-aggravated atherosclerosis.3. For the first time, we demonstrate that IL-17A up-regulate the expression of aP2and then induce ER stress in macrophage. And firstly, we establish a previously unrecognized link between cytokines and lipid metabolism through cytosolic lipid chaperone aP2in macrophage. These provide new ideas for studying the relationship and mechanism between inflammation and lipid metabolism diseases.4. We confirm that that IL-17A antibody and PBA could inhibit atherogenesis, aP2inhibitor could alleviate ER stress in macrophage. All these provide potent theoretical and experimental basis for treatment target as inflammatory cytokines, ER stress and lipid metabolism.Limitations of this study1. The mechanism of IL-17A induced ER stress need to be further investigated.2. The effect of aP2on IL-17A-induced ER stress and promoted atherosclerosis need experiments in vivo to be verified. |
PDF Full Text Request