Role of C3 Diversity in Innate Immune Reactions of Teleost Fish

University Of Pennsylvania, Philadelphia PA

Investigators

Abstract

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.