Details

Autor(en) / Beteiligte

• van der Aa, Lieke M

Titel

Trim Proteins and Cxs Chemokines : Evolutionary Dynamics and Functional Characterization of Two Large Protein Families in Teleost Fish

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

2012

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• Pathogens are a major driving force for the immune system to diversify. The battle between pathogens and their hosts is often referred as an evolutionary race. Pathogens on one hand continuously evolve new strategies to evade host immune responses, while the immune system on the other hand constantly evolves new mechanisms to combat invading pathogens. Diversification of the host immune system offers an advantage at two levels. First, the immune system is in the possession of a large and diverse set of receptors that allows recognition of a wide range of pathogens. Pathogen recognition is a key step for the immune system to initiate the immune response. Secondly, diversification of intracellular and extracellular signaling molecules has generated specialized molecules to finely regulate immune responses. Tight regulation of the immune response is essential, as uncontrolled immune responses are detrimental to the host.Two protein families of the immune system that typically co-evolved with pathogens are TRIM proteins and chemokines. These families highly expanded in gene numbers and are implicated in multiple biological processes. TRIM proteins are named after a conserved tri-partite motif at the N-terminal side of the protein: a RING zinc finger domain, one or two B-boxes and coiled coil domain. TRIM proteins are intracellular proteins that restrict viruses. Chemokines are secreted cytokines that are specialized in orchestrating cell migration. Leukocytes recruitment to the site of infection is essential during the immune response, as leukocytes are specialized in eliminating infectious agents and clearing of infected cells. The recruitment of leukocytes is preceded by vasodilation and increased vascular permeability; these processes together are named inflammation. The diversification of the chemokine family enables the immune system to direct the sequential recruitment of distinct leukocyte subsets, required during different phases of the inflammatory response. A higher degree of diversification and specialization of the immune system is observed with increasing complexity of multicellular organisms. Teleost fish emerged early during vertebrate evolution and already have a fully functional immune system. Its characterization improved our insight in the evolution of this complex system. In this thesis, the evolutionary dynamics of TRIM proteins and chemokines were studied in fish and a characterization of their function was made.In part one of this thesis, a full description was made of the TRIM gene family in zebrafish and tetraodon. These are two fish species for which the whole genome sequence is available. It was first found that numerous TRIM genes are conserved between fish and mammals. Further, a high number of TRIM genes are not well conserved between fish and mammals contain an additional domain at the C-terminus that is named B30.2. Interestingly, it was discovered that among TRIM genes containing a B30.2 domain, certain genes have undergone a dramatic expansion that generated three multigene TRIM families, a feature unique to fish. Orthologous genes were found in mammals for two multigene families and named TRIM35 and TRIM39/bloodthirsty. No orthologs could be found for the third multigene family, which was therefore named finTRIM, for fish novel TRIM. The finTRIM gene family showed a high degree of diversification: diversity in length and number of protein domains for which they encode, diversification in sequence and in gene numbers among different fish species. The finding of sites that evolve under positive selection in the B30.2 domain of all three multigene families, indicates that they evolve under pathogen diversifying pressure (Chapters 2 and 3).In order to find a function for fish TRIM proteins in antiviral immunity, finTRIMs were further investigated in rainbow trout, a well-established model to study viral infection. Also in trout, a high number of finTRIM transcripts of diverse lengths were identified. The induction in finTRIM gene expression by the fish rhabdovirus VHSV and polyI:C indeed indicated a role of trout finTRIMs in antiviral immunity (Chapter 2). This induction of finTRIM gene expression was attributed to the whole set of finTRIM genes, demonstrated by a high-throughput sequencing approach. Two finTRIM proteins were found to localize in the cytosol, which allows interaction with other cytosolic factors (Chapter 4). In addition to their role as direct viral restriction factors, an E3 ubiquitin ligase activity is demonstrated for multiple mammalian TRIM proteins. E3 ubiquitin ligases catalyze the conjugation of ubiquitin to a substrate protein and recent data indicate an important role for E3 ubiquitin ligases in regulating signaling pathways (discussed in Chapter 8). For two trout finTRIMs it was found that they display E3 ubiquitin ligase activity, implying that they can control the activity of other proteins, possibly those involved in signaling pathways associated with antiviral immunity (Chapter 4).In part two of this thesis, inflammatory CXC chemokines were investigated. Zebrafish and common carp, which both belong to the cyprinid lineage, were used as a model. Two CXC chemokines were earlier identified in carp, of which their sequence most resembles mammalian CXCL8 and CXCL9/10/11 chemokines, but true orthology could not be demonstrated by phylogeny studies. These two chemokines were alternatively named CXCa and CXCb, for the CXCL8-like and CXCL9/10/11-like chemokines, respectively. It was found that chemokines belonging to both the CXCa and CXCb subset diversified in cyprinid fish (Chapters 5 and 7). Two CXCL8-like lineages were identified, and named CXCa_L1 and CXCL8_L2. CXCa_L1 has orthologous genes in other fish lineages, while for CXCL8_L2 no orthologs were identified in fish outside the cyprinid lineage. For CXCb, a similar diversification was observed. A second CXCb gene was cloned in carp that was named CXCb2. In zebrafish, a cluster of seven CXCb-related genes was identified on chromosome five.In vitro studies demonstrated that chemokines within the CXCL8-like subset, or the CXCb subset, respond differently towards microbial stimuli (Chapters 5 and 7). Further, it was observed that the promoter regions of the individual zebrafish CXCb genes contain different transcription factor binding sites, indicating that these genes are regulated by different stimuli. Moreover, in two carp in vivo inflammation models, a more potent induction was observed in gene expression for CXCL8_L2 over CXCa_L1. Based on these results, it is speculated that the multiple lineages belonging to either the CXCL8-like subset, or the CXCb subset, have diverged functions. The fish CXC chemokines behave as “true” chemokines, as chemotactic activity of carp leukocytes towards recombinant proteins prepared for CXCa_L1, CXCL8_L2 and CXCb was demonstrated in vitro and in vivo (Chapters 5 and 6). Further, intracellular calcium mobilization was observed upon stimulation of granulocytes with CXCa_L1, indicating signaling via typical chemokine G-protein coupled receptors (Chapter 6). Based on the kinetics of CXCa_L1, CXCL8_L2 and CXCb1 gene expression in a carp zymosan-induced peritonitis model, it was hypothesized that these chemokines are functional homologs of their mammalian counterparts (Chapter 5). By an in vivo study in which the recombinant proteins were administrated intraperitoneally, it was observed that both CXCa_L1 and CXCL8_L2 strongly recruit leukocytes, predominantly of the neutrophilic granulocyte population (Chapter 6). Together with a stimulatory effect on carp phagocytes in vitro, a pro-inflammatory function was attributed to both CXCL8-like lineages. CXCb was not potent in leukocyte recruitment in vivo. It was speculated that CXCb recruits monocytes and enables macrophage polarization towards the M2 subset, which acts in tissue repair and remodeling. In addition, the two carp CXCb chemokines both resemble the mammalian IFN-γ inducible CXCL9-11 chemokines, as CXCb1 and CXCb2 genes were both inducible by IFN-γ2, moreover, CXCb1 was also chemotactic for lymphocytes (Chapters 6 and 7).In conclusion, the results obtained in this thesis indicate that specialized functions of TRIM proteins and CXC chemokines in immunity were already present in early vertebrate evolution. Based on the features whereby TRIM genes evolved in fish, it is hypothesized that fish multigene TRIM proteins are implicated in pathogen sensing and possibly represent a novel class of pattern-recognition receptors. Future work on chemokines should aim at the identification of receptors, to further delineate their role during inflammation.