Role of C3 Diversity in Innate Immune Reactions of Teleost Fish

补体系统由30多种可溶性蛋白质和受体组成，在先天性和适应性免疫中发挥重要作用。补体系统的关键成分是蛋白质C3。在活化时，C3分子被切割成C3 b和C3 a片段，使C3 b能够共价结合到外来颗粒，如病毒、细菌、真菌和原生动物。靶向颗粒可以被其表面上携带补体受体的吞噬细胞识别和破坏，或者它们可以被进一步裂解。除了硬骨鱼之外，迄今为止研究的每一种动物物种都只含有单一形式的功能活性C3，这是单一基因的产物。与此相反，硬骨鱼表现出独特的多样性的功能活性C3蛋白，这是不同基因的产物。 这种C3多样性可能对涉及C3分子的广泛的免疫机制具有重要的生物学后果。据推测，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/或微调中，这反过来又可能扩大和加强鱼类的先天免疫反应。因此，在本项目中，将分析硬骨鱼先天免疫中C3多样性的功能相关性。C3蛋白参与两种主要的先天免疫机制：吞噬作用和呼吸爆发。这些功能通过两个主要的C3激活片段C3 b和iC 3b发挥作用。该项目的具体目标是：目标1：纯化并生化表征来自三种鳟鱼C3同种型中的每一种的C3 b和iC 3 b片段，沿着产生相应的IgM/C3 b/iC 3 b包被颗粒。尽管这些组分在市场上不能用于鳟鱼，但从先前使用鳟鱼补体的工作中开发的方案使Synyer博士能够纯化各种C3同种型，沿着产生C3 b和iC 3 b所需的其他蛋白质。 目的2：分析各种C3 b和iC 3b片段与鳟鱼吞噬细胞上存在的假定C3受体的相互作用。C3 b和iC 3b发挥的作用与这些C3衍生物结合的受体密切相关。 Sunyer博士计划测试两种替代假设; 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。为了解决这些假设，结合和内化的各种C3 b和iC 3b分子鳟鱼吞噬细胞将进行分析。 此外，研究者还将评估C3 b/iC 3b片段与其受体相互作用可能导致的受体后事件（细胞内钙浓度变化和使用膜片钳记录技术分析细胞内信号传导事件）。C3 b和iC 3b分子与其假定受体的相互作用的分析将有助于解释目的3中获得的关于各种鳟鱼C3同种型在先天免疫中的功能的功能数据。 目的3：研究各种C3亚型在两个主要的先天免疫过程中的作用，吞噬作用和呼吸爆发。 据推测，硬骨鱼的C3分子中产生的多样性也可能导致了一些C3介导的功能的多样化和/或微调和专业化。因此，这里要评估的最关键的问题是来自各种C3同种型的各种C3 b和iC 3b片段是否对吞噬作用和呼吸爆发具有不同的作用，以及这种差异是否与C3 b和iC 3b分子与鳟鱼白细胞相互作用后观察到的结合、内化和受体后事件有关（目的2）。 在此，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，虹鳟鱼将被用作硬骨鱼模型，因为研究者已经制备了这些研究所需的许多特定试剂，包括纯化的鳟鱼补体分子和识别所有这些分子的抗体。这些研究应该为C3中令人着迷的多样性的生物学意义提供新的线索，这种多样性使硬骨鱼补体系统成为一种独特的免疫机制。这些研究也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。这些研究中的许多都涉及同源成分（纯化的鳟鱼分子）和创新技术的使用，这些技术在比较免疫学领域并不常用。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.