Immunization against Atherosclerosis

预防动脉粥样硬化的免疫

• 批准号: 8029358
• 负责人: AOSHUANG CHEN
• 金额: $ 23.55万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-15 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8029358
• 关键词: Accounting; Adjuvant; Adoptive Transfer; Animals; Antiatherogenic; Antigen Targeting; Antigens; Apolipoprotein E; Arteries; Atherosclerosis; Autoimmune Diabetes; B-Lymphocytes; Blood; Cell physiology; Cells; Cessation of life; Cholesterol; Chronic; Collagen; Cryoultramicrotomy; Data; Delayed Hypersensitivity; Developed Countries; Development; Dexamethasone; Disease; Equilibrium; Eragrostis; Flow Cytometry; Glucocorticoids; Grant; Health; Heart Diseases; IL2RA gene; Immune; Immunity; Immunization; Immunologic Deficiency Syndromes; Immunosuppressive Agents; Inflammatory; Inflammatory Response; Insulin-Dependent Diabetes Mellitus; Intervention; Lead; Lesion; Microbe; Mus; Ovalbumin; Pathologic; Pathway interactions; Peptides; Play; Process; Regimen; Regulatory T-Lymphocyte; Role; Site; Smooth Muscle Myocytes; Specificity; Spleen; Stroke; Structure of brachiocephalic artery; T cell response; T-Lymphocyte; T-Lymphocyte Subsets; Testing; Time; Toxic effect; aortic arch; atherogenesis; desensitization; design; enzyme linked immunospot assay; in vivo; insight; macrophage; male; mouse model; novel; peripheral blood; peripheral tolerance; protective efficacy; response; tumor

DESCRIPTION (provided by applicant): Atherosclerosis is driven partially by pathogenic CD4+ effector T cells (Teff) reactive to antigens upregulated in the lesion ("athero-antigens"). Importantly, CD4+CD25+Foxp3+ regulatory T cells (Treg), a subset of T cells that function to suppress the response of Teff, have been shown to play a role in controlling atherosclerosis. Within the context of atherosclerosis an imbalance may exist between pathogenic Teff and suppressive Treg in response to athero-antigens, leading to the promotion of atherogenesis. These notions prompted us to design an anti-atherosclerosis strategy aimed at resetting the balance between Treg and Teff via increasing athero-specific Treg in vivo. Previously, mirroring the use of the conventional adjuvants for enhancing immunity, we explored the concept of "tolerogenic adjuvant" and designed a novel immunization strategy. In mouse models of delayed-type hypersensitivity (DTH) and spontaneous type I diabetes, we showed that immunization with peptide antigen, when performed under the influence of the immunosuppressant dexamethasone (DEX) serving as tolerogenic adjuvant (a strategy we termed "suppressed immunization"), caused long-term desensitization of DTH and blockade of autoimmune diabetes, respectively. We showed that such protective efficacy was associated with selective expansion of antigen-specific Treg. Encouraged by these results, we here will test our hypothesis that suppressed immunization (SI) using DEX and athero-antigens may selectively expand athero-specific Treg and reduce atherosclerosis. Under Specific Aim #1, we will determine whether SI increases the ratio of Treg to Teff at the lesion site and in peripheral blood in treated mice. To that end, male ApoE-/-/Foxp3-GFP+/+ mice (we have recently generated) will be treated with SI combining DEX with HSP60-derived peptide HP1. As controls, mice will be treated with PBS or HP1 alone. Innominate arteries from treated mice will be cryosectioned, and immunohistochemical analysis will be performed to quantify Teff (CD4+Foxp3-) and Treg (CD4+Foxp3-GFP+). Antigen specificity of Treg will be analyzed via ELISPOT with single cells prepared from the aortic arch. In addition, Teff and Treg in peripheral blood will be quantified via immunostaining and flow cytometry, and antigen specificity of the Treg will be assessed by their proliferation in response to the immunizing peptide HP1. Under Specific Aim #2, we will assess anti-atherogenic efficacy of SI in treated animals. To that end, male ApoE-/- mice with established atherosclerosis will be divided into 3 groups (n e 6). One group will be dissected before treatment to establish the baseline for lesion extent. Each of the 2 treatment groups will be treated with HP1 alone (control) or SI. Subsequently, lesions in the innominate artery will be quantified by histochemical/morphometric analysis. In addition, sections of the innominate artery will be analyzed for contents of macrophage, smooth muscle cell (SMC), and collagen. Statistical analyses will be performed on obtained data. Under Specific Aim #3, we will exploit adoptive transfer to determine whether SI-induced Treg contribute to the anti-atherogenic efficacy of SI. To that end, ApoE-/-/Foxp3-GFP+ mice will be treated with SI; 4 weeks later, Treg (Foxp3-GFP+) will be isolated from the spleen and used as donor Treg (test Treg). Treg isolated from non-treated mice will be used as control Treg. CD4+/Foxp3- cells isolated from ApoE-/- mice that have developed advanced atherosclerosis will be used as pathogenic Teff. Subsequently, pathogenic Teff will be adoptively transferred alone (control), or in combination (in various ratios) with test Treg or control Treg, into recipient ApoE-/-/Rag-2-/- mice (which are T cell- and B cell-deficient). Six weeks later, atherosclerosis lesion extent in recipients will be analyzed. Completion of these specific aims is likely to generate a safe, effective, and practical anti-atherosclerosis therapy. In addition, this study will further define the role of athero-specific Treg in controlling atherosclerosis and provide useful insights pertaining to targeting these Treg optimally.

描述（由申请人提供）：动脉粥样硬化部分由与病变中上调的抗原（“动脉粥样硬化抗原”）反应的致病性CD 4+效应T细胞（Teff）驱动。重要的是，CD 4 + CD 25 + Foxp 3+调节性T细胞（Treg），一种功能抑制Teff反应的T细胞亚群，已被证明在控制动脉粥样硬化中发挥作用。在动脉粥样硬化的背景下，致病性Teff和抑制性Treg之间可能存在对动脉粥样硬化抗原应答的不平衡，导致动脉粥样硬化形成的促进。这些概念促使我们设计一种抗动脉粥样硬化策略，旨在通过增加体内动脉粥样硬化特异性Treg来重置Treg和Teff之间的平衡。在此之前，我们借鉴传统佐剂的免疫增强作用，探索了“致耐受性佐剂”的概念，并设计了一种新的免疫策略。在迟发型超敏反应（DTH）和自发性I型糖尿病的小鼠模型中，我们发现，当在免疫抑制剂地塞米松（DEX）作为致耐受性佐剂（我们称之为“抑制免疫”的策略）的影响下进行肽抗原免疫时，分别引起DTH的长期脱敏和自身免疫性糖尿病的阻断。我们发现这种保护功效与抗原特异性Treg的选择性扩增相关。受这些结果的鼓舞，我们在这里将测试我们的假设，即使用DEX和动脉粥样硬化抗原的抑制免疫（SI）可能选择性地扩增动脉粥样硬化特异性Treg并减少动脉粥样硬化。在特定目标#1下，我们将确定SI是否增加经治疗小鼠中病变部位处和外周血中Treg与Teff的比率。为此，雄性ApoE-/-/Foxp 3-GFP+/+小鼠（我们最近产生的）将用组合DEX与HSP 60衍生肽HP 1的SI处理。作为对照，小鼠将单独用PBS或HP 1处理。将处理小鼠的无名动脉冷冻切片，并进行免疫组织化学分析以定量Teff（CD 4 + Foxp 3-）和Treg（CD 4 + Foxp 3-GFP+）。Treg的抗原特异性将通过ELISPOT与从主动脉弓制备的单个细胞进行分析。此外，外周血中的Teff和Treg将通过免疫染色和流式细胞术进行定量，并且Treg的抗原特异性将通过它们响应于免疫肽HP 1的增殖来评估。在特定目标#2下，我们将评估SI在治疗动物中的抗动脉粥样硬化功效。为此，将具有确定的动脉粥样硬化的雄性ApoE-/-小鼠分成3组（n = 6）。治疗前将对一组进行解剖，以确定病变范围的基线。2个治疗组中的每一个将用单独的HP 1（对照）或SI治疗。随后，将通过组织化学/形态学分析对无名动脉中的病变进行定量。此外，将分析无名动脉切片中巨噬细胞、平滑肌细胞（SMC）和胶原蛋白的含量。将对获得的数据进行统计分析。在具体目标#3下，我们将利用过继转移来确定SI诱导的Treg是否有助于SI的抗动脉粥样硬化功效。为此，ApoE-/-/Foxp 3-GFP+小鼠将用SI处理; 4周后，Treg（Foxp 3-GFP+）将从脾分离并用作供体Treg（测试Treg）。从未处理的小鼠分离的Treg将用作对照Treg。从已经发展晚期动脉粥样硬化的ApoE-/-小鼠分离的CD 4 +/Foxp 3-细胞将用作致病性Teff。随后，将致病性Teff单独（对照）或与测试Treg或对照Treg组合（以各种比例）过继转移到受体ApoE-/-/Rag-2-/-小鼠（其为T细胞和B细胞缺陷型）中。六周后，将分析受体的动脉粥样硬化病变程度。这些特定目标的完成可能会产生一种安全，有效和实用的抗动脉粥样硬化治疗。此外，本研究将进一步确定动脉粥样硬化特异性Treg在控制动脉粥样硬化中的作用，并提供有关最佳靶向这些Treg的有用见解。