Comparative studies of complement responses to ICs

补体对 IC 反应的比较研究

• 批准号: 10645672
• 负责人: Gowthami M Arepally
• 金额: $ 24.15万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10645672
• 关键词: Address; Animal Model; Animals; Antibodies; Antigen-Antibody Complex; Autoimmune; Binding; Biological; Cavia; Classical Complement Pathway; Clinical; Cold Hemagglutinin Disease; Comparative Study; Complement; Complement 3 Convertase; Complement 3b; Complement 3c; Complement Activation; Complement Inactivators; Complex; Data; Development; Dinitrophenols; Disease; Erythrocytes; Feedback; Funding; Future; Generations; Genetic; Genetic Variation; Genomics; Goals; Graft Rejection; Hemolysis; Hemolytic-Uremic Syndrome; Heparin; Human; Immune; Immunological Models; Impairment; Keyhole Limpet Hemocyanin; Laboratory Animals; Malignant Neoplasms; Mediating; Medical; Molecular; Monoclonal Antibodies; Mus; Mutation; Oryctolagus cuniculus; PF4 Gene; Pathway interactions; Physiological; Plasma; Proteins; Proteome; Proteomics; Publishing; Rattus; Serum; Study models; System; Testing; Therapeutic; Variant; Whole Blood; complement pathway; complement system; differential expression; hereditary angioneurotic edema; inhibitor; mouse model; paroxysmal nocturnal hemoglobinuria; research and development; response; species difference; success; therapeutic evaluation

The complement systems of humans and laboratory animals (mice and rats) show striking species differences in function, particularly with regards to the classical pathway of complement activation. Indeed, studies since the 1950’s have documented markedly reduced classical pathway function in mice as compared to rats and humans. In preliminary data, we confirm and extend these findings using model immune complexes (ICs) formed by monoclonal antibodies to platelet factor 4 (PF4) and heparin (KKO ICs) or polyclonal rabbit anti-dinitrophenol (DNP) antibodies to DNP conjugated with keyhole limpet hemocyanin (DNP ICs). Specifically, we show: 1) robust C3 activation by KKO and DNP ICs in whole blood and plasma from mice, rats and humans 2) classical pathway requirements for complement activation by KKO ICs in rats and human, but alternative pathway requirements in mice 3) incorporation of alternative pathway proteins into KKO ICs in mice, but not rats or humans 4) comparable requirements for the alternative pathway among four murine strains and 5) differential expression of alternative pathway proteins by proteomics in mice as compared to rats and humans. Based on these preliminary data and published observations, we will test the overall hypothesis that impaired classical pathway activity in mice is counterbalanced by the alternative pathway C3b feedback cycle. To test this hypothesis, we propose the following two aims: 1) Comparative studies of human, mouse, and rat complement systems. In this aim, we will test that hypothesis that genetic variation in murine classical pathway proteins contributes to impaired classical pathway function. We will apply molecular approaches to compare genetic sequences and perform structural homologies of classical pathway proteins in mice v rats and humans. In other studies, we will investigate the plasma proteome of mice, rats, and humans to examine protein/immune complex interactions, co-expression of complement and complement-associated proteins and correlate complement protein levels with functional responses to KKO and DNP ICs. 2) Functional studies of murine and human complement systems. In this aim, we will test the hypothesis that mice utilize the C3b feedback cycle of the alternative pathway as a compensatory response to low classical pathway activity. To test this hypothesis, we will use purified human and mouse complement proteins to demonstrate reduced impaired classical C3 convertase activity and normal/heightened activity of the C3b feedback cycle in mice, examine the effects of interchanging human and mouse complement proteins, and identify potential murine inhibitors of classical pathway activation from mouse serum. Together, these studies are expected to delineate the molecular and functional basis of divergent complement pathway responses in mice and humans. We expect these studies to generate preliminary data for an expanded application to elucidate the molecular and structural divergence of murine complement responses with the long term goal of developing translationally relevant animal models.

人类和实验室动物（小鼠和大鼠）的补体系统在功能上显示出惊人的物种差异，特别是关于补体活化的经典途径。事实上，自20世纪50年代以来的研究已经证明，与大鼠和人类相比，小鼠中的经典途径功能显著降低。在初步的数据，我们确认和扩展这些发现使用模型免疫复合物（IC）形成的单克隆抗体血小板因子4（PF 4）和肝素（KKO IC）或多克隆兔抗二硝基苯酚（DNP）抗体DNP与钥孔血蓝蛋白（DNP IC）共轭。具体而言，我们显示：1）小鼠、大鼠和人的全血和血浆中KKO和DNP IC的稳健C3活化2）大鼠和人中KKO IC的补体活化的经典途径要求，但小鼠中的旁路途径要求3）小鼠中旁路途径蛋白质掺入KKO IC中，但不包括大鼠或人类4）四种鼠品系之间对替代途径的要求相当，以及5）通过蛋白质组学在小鼠中与大鼠和人相比的旁路途径蛋白质的差异表达。基于这些初步数据和已发表的观察结果，我们将测试小鼠中受损的经典途径活性被替代途径C3 b反馈循环抵消的总体假设。为了验证这一假设，我们提出了以下两个目标：1）人，小鼠和大鼠补体系统的比较研究。在这个目标中，我们将测试的假设，在小鼠经典途径蛋白质的遗传变异，导致经典途径功能受损。我们将应用分子生物学的方法来比较基因序列，并在小鼠、大鼠和人类中进行经典途径蛋白质的结构同源性。在其他研究中，我们将研究小鼠、大鼠和人的血浆蛋白质组，以检查蛋白质/免疫复合物相互作用、补体和补体相关蛋白的共表达，并将补体蛋白水平与对KKO和DNP IC的功能反应相关联。2)鼠和人补体系统的功能研究。在这个目标中，我们将测试的假设，小鼠利用C3 b反馈循环的替代途径作为一种补偿性反应，以低经典途径的活动。为了检验这一假设，我们将使用纯化的人和小鼠补体蛋白来证明小鼠中受损的经典C3转化酶活性降低和C3 b反馈循环的正常/升高的活性，检查互换人和小鼠补体蛋白的影响，并从小鼠血清中鉴定经典途径活化的潜在鼠抑制剂。总之，这些研究有望描绘小鼠和人类不同补体途径反应的分子和功能基础。我们希望这些研究能够产生初步数据，用于扩展应用，以阐明小鼠补体应答的分子和结构差异，长期目标是开发与免疫相关的动物模型。