TRIM proteins and CXC chemokines Evolutionary dynamics and functional characterization of two large protein families in teleost fish Lieke M. van der Aa TRIM proteins and CXC chemokines Evolutionary dynamics and functional characterization of two large protein families in teleost fish Lieke M. van der Aa Thesis committee Thesis supervisor Prof. dr. ir. H.F.J. Savelkoul Professor of Cell Biology and Immunology Wageningen University, the Netherlands Thesis co-supervisors Dr. B.M.L. van Kemenade Assistant professor, Cell Biology and Immunology group Wageningen University, the Netherlands Dr. P. Boudinot Directeur de Recherches, Virologie et Immunologie Moléculaires Institut National de la Recherche Agronomique, Jouy-en-Josas, France Other members Prof. Dr. Just Vlak Wageningen University, the Netherlands Prof Dr. Chris J. Secombes University of Aberdeen, Scotland Dr. Annemarie H. Meijer Leiden University, the Netherlands Dr. Sebastian Nisole Pasteur Institute, Paris, France This research was conducted under the auspices of the Graduate School of the Wageningen Institute of Animal Sciences, WIAS TRIM proteins and CXC chemokines Evolutionary dynamics and functional characterization of two large protein families in teleost fish Lieke M. van der Aa Thesis at Wageningen University submitted in fulfillment of the requirements for the degree of doctor Prof. Dr. M.J. Kropff, by the authorityin the presence of the Rector of the Magnificus Thesis Committee appointed by the Academic Board to be defended in public on Friday 20 January 2012 at 4 p.m. in the Aula. Lieke M. van der Aa TRIM proteins and CXC chemokines, Evolutionary dynamics and functional PhDcharacterization thesis, Wageningen of two large University, protein the families Netherlands, in teleost 2012 fish With references, with summaries in English and Dutch ISBN: 978-94-6173-120-3 Table of Contents Chapter 1 General Introduction 6 Part I: TRIM proteins Chapter 2 22 A large new subset of TRIM genes highly diversified Chapter 3 Origin and evolution of TRIM proteins: new insights 58 by duplication and positive selection in teleost fish from the complete TRIM repertoire of zebrafish and Chapter 4 88 pufferfish ubiquitin ligase activity FinTRIMs, fish virus-inducible proteins with E3 Part II: CXC chemokines Chapter 5 108 CXCL8 chemokines in teleost fish: two lineages with distinct expression profiles during early phases of Chapter 6 132 inflammation CXCb chemokines Pro-inflammatory functions for carp CXCL8-like and Chapter 7 152 Diversification of IFNγ-inducible CXCb chemokines in cyprinid fish Chapter 8 General Discussion 176 Summaries and Acknowledgements 202 Summary Samenvatting Acknowledgements About the author 218 Curriculum Vitae List of Publications Overview of completed training activities Un problème sans solution est un problème mal posé. - Albert Einstein - Chapter 1 General Introduction Chapter 1 Pathogens are a major driving force for the immune system to evolve. Pathogens drive the immune system to constantly re-adapt and to create a well-balanced response to combat microbes that invade and inhabit them. An interesting class of the animal successful animal group that represents more than half of all living vertebrate species kingdom to study the evolution of the immune system are teleost fish, an extremely developed innate immune system, and a functional adaptive immune system, they can provide[1]. Teleosts interesting originated clues 450 about million the early years evolved ago and defense because system teleost against fish pathogens. have a well- In this thesis, two large protein families were studied: the TRIM protein family and the chemokine family. TRIM proteins are intracellular proteins that control immunity from within the cell, while chemokines are secreted cytokines that orchestrate immunity at systemic levels. The evolution of both TRIM and chemokine genes is characterized processes generated two protein families that both play a crucial role in the immune system.by species-specific The interest duplication in TRIM proteins events, in followed mammals by has functional grown enormously diversification. in the These last few years when their importance in the antiviral immune response was discovered. moonshine),At the start ofwhich this both thesis function project, in hematopoiesisresearch on TRIM [2, 3], proteins a TRIM proteinin fish wasin Japanese limited eel to the description of only two TRIM proteins in zebrafish (named bloodthirsty and that is expressed in branchial epithelial cells [4] and TRIM genes identified by a global matterscreening of debateof the genome whether of there tetraodon is true and orthology. zebrafish Due [5]. to Within the low the sequence chemokine similarity family, certain genes have clear orthologs in fish and mammals, but for many genes is still a between chemokine genes from fish and mammals and the small size of these proteins, theirit is difficult biological to establish function theiris not evolutionary yet well known. relatedness In this thesis, [6-9]. the Although evolutionary gene expression dynamics ofdata TRIM indicate and CXCthat chemokinecertain fish geneschemokines were investigated play a role inand immunity a characterization and inflammation, of their Danio rerio Cyprinius carpio) and Onchorynchus mykiss) were used hereby as model species. functions was made in fish. Zebrafish ( ), common carp ( 1.1rainbow Pathogen trout ( recognition and activation of the immune system Pathogens present in air, water, food, or carried and transmitted by other organisms, form a continuous challenge to a host. The epithelial lining of the skin, respiratory airways and gastro-intestinal tract form the physical barrier between the outside and inside world of an organism. When a pathogen succeeds in trespassing one of these barriers, the immune system plays a crucial role in eliminating the intruder before it causes irreversible harm to the host. High numbers of innate immune cells are residing in the extravascular tissue, the space between the epithelium and blood vessels. The main task of these immune cells is to quickly detect and eliminate intruding pathogens. Pathogen recognition is a key step to initiate immune responses. Pathogens have characteristic well-conserved molecules that are important for their virulence and survival, and that are usually not expressed by the host. These so called pathogen-associated molecular 10 General Introduction expressed by cells of the host. Based on distinct protein subdomains of which PRRs patterns (PAMPs) are recognized by pathogen recognition receptors (PRRs), which are byconsist, [10-13]). they PRRs are classified are mostly into expressed four major by innateclasses: immune Toll-like cells receptors and are (TLRs), localized NOD-like on the receptors (NLRs), RIG-I-like receptors (RLRs) and the C-type lectins (CLRs) (reviewed Recognition of PAMPs by PRRs facilitates the onset of an immune response tailored to a cell surface (TLRs), in phagosomal vacuoles (TLRs), or in the cytosol (NLRs, RLRs, CLRs). pathways that lead to the activation of transcription factors. These include nuclear factor- specific type of pathogen. Pathogen recognition by PRRs activates intracellular signaling κB (NF-κB), AP-1 and interferon-regulatory factors (IRFs). These transcription factors cellsregulate to killcytokine the pathogen. gene expression, In case likeof viral the pro-inflammatoryinfection, certain cytokinestypes of interferons interleukin-1β are especially(IL-1β) and important tumor necrosis for the inductionfactor-α (TNF-α), of an anti-viral which togetherstate in bothactivate immune local andimmune non- immune cells, by stimulating the production of anti-viral proteins. Proteins belonging to the TRIM protein family control the production of interferons and have also anti- cytokines produced upon PRR activation mediate in the induction of a systemic immuneviral activity responsive. (reviewed Hereby, by [14-17]). immune Incells addition are recruited to evoking from a the local blood immune circulation response, and specialized lymphoid tissues and are activated to mediate in the process of pathogen killing and elimination, to prevent a systemic infection. The process of vascular dilation, increased permeability and edema, together with leukocyte accumulation at the site Chemokines are specialized cytokines that play of infection, is called inflammation. an important role in orchestrating cell recruitment during inflammation. A schematic 1.2overview TRIM of theproteins: first steps E3 of ligasesthe immune functioning response is depictedin antiviral in figure immunity 1. The immune response upon viral infection is becoming well characterized in mammals. Cytokines specifically induced upon detection of viral RNA and DNA by the PRRs TLR3, TLR7-9, RIG-I, MDA-5, PKR and DAI, are type I interferons (IFNs) [12, 18]. IFNs are classified into type I, II and III, based on their binding to distinct receptors. Type I IFNs include IFN-α and IFN-β, which are produced by both immune and non- stimulatesimmune cells macrophages upon virus and detection. plays a Typerole in II theIFN, onset IFN-γ, of isthe induced adaptive upon immune viral andresponse. other pathogen detection and is expressed by specific cell types of the immune system. IFN-γ Type III IFN, IFN-λ, also has anti-viral activity. Secreted IFN-α/β acts on nearby cells STATs)that express and subsequent the receptor expression for IFNα/β of (IFNAR).hundreds Triggering of genes thatof the mediate IFNAR, in which clearance signals of through the Jak/Stat