Role of C3 Diversity in Innate Immune Reactions of Teleost Fish

补体系统由30多种可溶蛋白质和受体组成，在先天免疫和获得性免疫中发挥重要作用。补体系统的关键成分是C3蛋白。一旦激活，C3分子被裂解成C3b和C3a片段，使C3b能够共价结合到外来粒子上，如病毒、细菌、真菌和原生动物。目标颗粒可以被表面带有补体受体的吞噬细胞识别和破坏，也可以进一步裂解。除了硬骨鱼，到目前为止，每一种被研究的动物物种都只含有一种具有功能活性的C3，它是单一基因的产物。相比之下，硬骨鱼表现出独特的功能活性C3蛋白的多样性，这些蛋白是不同基因的产物。这种C3的多样性可能会对涉及C3分子的广泛的免疫机制产生重要的生物学影响。据推测，这种C3多样性背后的功能相关性可能反映在C3功能的多样化和/或微调上，这反过来可能扩大和加强鱼类的先天免疫反应。因此，在本项目中，将分析C3多样性在硬骨鱼先天免疫中的功能相关性。C3蛋白参与了两种主要的先天免疫机制：吞噬和呼吸爆发。这些功能是通过两个主要的C3激活片段C3b和iC3b来发挥的。该项目的具体目标是：目标1：从鲑鱼的三种C3亚型中提纯C3b和iC3b片段，并对其进行生化表征，同时产生相应的IgM/C3b/iC3b包被颗粒。尽管这些成分对鲑鱼没有商业用途，但从以前对鲑鱼补体的工作中开发的方案使Synyer博士能够纯化各种C3亚型，以及产生C3b和iC3b所需的其他蛋白质。目的：分析各种C3b和iC3b片段与鲑鱼吞噬细胞上可能存在的C3受体的相互作用。C3b和iC3b的作用与这些C3衍生物所结合的受体密切相关。桑耶博士计划测试两种可供选择的假说：C3分子的多样化也导致了C3受体及其生物学作用的多样化，或者各种C3蛋白共享相同的受体，它们可能以相似或不同的亲和力结合到这些受体上。为了解决这些假设，将分析各种C3b和iC3b分子与鲑鱼吞噬细胞的结合和内化。此外，研究人员还将评估C3b/iC3b片段与其受体相互作用所产生的可能的受体后事件(细胞内钙浓度的变化和使用膜片钳记录技术分析细胞内信号事件)。这种对C3b和iC3b分子与它们可能的受体的相互作用的分析将有助于解释目标3中获得的关于鲑鱼C3亚型在天然免疫中的功能的功能数据。目的：研究不同C3亚型在吞噬和呼吸爆发两个主要的先天免疫过程中的作用。据推测，硬骨鱼C3分子中产生的多样性也可能导致C3介导的一些功能的多样化和/或微调和专门化。因此，这里需要评估的最关键的问题是，不同C3亚型的各种C3b和iC3b片段是否对吞噬和呼吸爆发有不同的影响，以及这种差异是否与C3b和iC3b分子与鲑鱼白细胞相互作用时观察到的结合、内化和受体后事件有关(目标2)。在这里，将使用双色流式细胞术(FACS)同时测量吞噬作用和呼吸爆发。在这些研究中，虹鱼将被用作硬骨鱼模型，因为研究人员已经产生了这些研究所需的许多特定试剂，包括纯化的鲑鱼补体分子和识别所有这些分子的抗体。这些研究应该会为C3令人着迷的多样性的生物学意义提供新的线索，正是这种多样性使硬骨鱼的补体系统成为一种如此独特的免疫机制。这些研究还有望为补体系统在硬骨鱼先天免疫反应中的作用提供新的见解。其中许多研究既涉及使用同源成分(纯化的鲑鱼分子)，也涉及在比较免疫学领域中不常用的创新技术。因此，这些研究将为鱼类免疫系统的研究建立新的方向，并有望扩大对免疫反应进化的理解。

The complement system consists of a complex group of more than 30 soluble proteins and receptors that play an important role in innate and adaptive immunity. The key component of the complement system is the protein C3. Upon activation, the C3 molecule is cleaved into C3b and C3a fragments, enabling C3b to covalently bind to foreign particles such as viruses, bacteria, fungi, and protozoans. The targeted particles can be recognized and destroyed by phagocytic cells bearing complement receptors on their surfaces, or they can be further lysed. With the exception of teleost fish, each of the animal species studied to date contains only a single form of functionally active C3 that is the product of a single gene. In contrast, teleost fish demonstrate a unique diversity of functionally active C3 proteins, which are the products of different genes. This C3 diversity may have important biological consequences for the wide array of immune mechanisms involving the C3 molecule. It is hypothesized that the functional relevance behind this C3 diversity may be reflected in a diversification and/or fine tuning of the C3 functions, which in turn may both broaden and strengthen the innate immune response of fish. Therefore, in this project the functional relevance of C3 diversity in innate immunity in teleost fish will be analyzed. The C3 protein is involved in two major innate immune mechanisms: phagocytosis and respiratory burst. These functions are exerted through two main C3 activation fragments, C3b and iC3b. The specific Aims of this project are: AIM 1: To purify and biochemically characterize the C3b and iC3b fragments from each of the three trout C3 isoforms, along with generating the corresponding IgM/C3b/iC3b-coated particles.Although these components are not commercially available for trout, protocols developed from previous work with trout complement has enabled Dr. Synyer to purify the various C3 isoforms, along with the other proteins required for the generation of C3b and iC3b. AIM 2: To analyze the interaction of the various C3b and iC3b fragments with putative C3 receptors present on trout phagocytes. The action exerted by C3b and iC3b is intimately related to the receptors to which these C3 derivatives bind. Dr. Sunyer plans to test two alternative hypotheses; that the diversification of the C3 molecules has also resulted in a diversification of the C3-receptors and their biological roles, or the various C3 proteins share the same receptors to which they may bind with similar or different affinities. To address these hypotheses, the binding and internalization of the various C3b and iC3b molecules to trout phagocytes will be analyzed. In addition, the investigator will also assess possible post-receptor events (changes in intracellular calcium concentration and analysis of intracellular signalling events using the patch-clamp recording technique) resulting from the interaction of the C3b/iC3b fragments with their receptors. This analysis of the interaction of the C3b and iC3b molecules with their putative receptors will be useful in interpreting the functional data obtained in Aim 3 concerning the function of the various trout C3 isoforms in innate immunity. AIM 3: To examine the role of the various C3 isoforms in two major innate immune processes, phagocytosis and respiratory burst. It is hypothesized that the diversity generated in the C3 molecule of teleost fish may have also resulted in a diversification and/or fine tuning and specialization of some of the C3-mediated functions. Thus, the most critical question to assess here is whether the various C3b and iC3b fragments from the various C3 isoforms have different effects on phagocytosis and respiratory burst and whether such differences relate to the binding, internalization and post-receptor events observed upon interaction of the C3b and iC3b molecules with the trout leukocytes (Aim 2). Here, a two-color flow cytometric method (FACS) will be used for simultaneous measurement of phagocytosis and respiratory burst.In these studies, the rainbow trout will be used as the teleost fish model, since the investigator has already generated many of the specific reagents that are required for these studies, including purified trout complement molecules and antibodies recognizing all of these molecules. These studies should shed new light on the biological significance of the fascinating diversity in C3 that makes the teleost fish complement system such a unique immune mechanism. These studies are also expected to provide new insights into the role of the complement system in the innate immune reactions of teleost fish. Many of these studies involve both the use of homologous components (purified trout molecules) and innovative techniques that are not commonly used in the field of comparative immunology. Therefore these studies will establish new directions for the study of the fish immune system and are expected to expand understanding of the evolution of immune reactions.