Cloning of a novel interleukin (IL)-20-like gene in rainbow trout Oncorhynchus mykiss gives an insight into the evolution of the IL-10 family §,§§ Tiehui Wang, Patricia Dı ´az-Rosales, Sam A.M. Martin, Christopher J. Secombes * Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, UK 1. Introduction Interleukin (IL)-10, initially described as a cytokine synthesis inhibitory factor (CSIF) [1], is an anti-inflammatory and immune regulatory cytokine [2]. The IL-10 family of cytokines was determined recently after the discovery of several IL-10 related cytokines, IL-19 [3], IL-20 [4], IL-22/IL-TIF [5], IL-24/Mda-4 [6], and IL-26/AK155 [7]. A number of viruses, including Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV), also encode viral members of the IL-10 family [8]. The human IL-10 family of cytokines resembles each other in having 20–30% identity in their primary sequences, and conserved amino acid residues important for their characteristic a-helical secondary structure [9]. IL-10 is an intercalated dimer made of two identical polypeptides, packed in two compact, six-helix bundle domains [10]. IL-26 also forms homodimers similar to IL-10 [11]. IL-19, IL-20, IL-22, and IL-24 were suggested to form a unique IL-19 subfamily characterized by their structural features and aggregation state as monomers [12]. Crystal structures of IL-19 and IL-22 showed that these cytokines consist of seven amphipathic helices of different lengths, covering an extensive hydrophobic core [13]. The IL-10 cytokine family is clustered on two chromosomes; IL-10, IL-19, IL-20 and IL-24 are clustered on chromosome 1, whilst IL-22 and IL-26 are neighbours on chromosome 12 [2,9]. The IL-10 family of cytokines signals through receptors that are dimers of an R1 type of receptor with a long cytoplasmic domain and an R2 type of receptor with a short cytoplasmic domain [9,14]. For example, IL-10 signals through IL-10R1 and IL-10R2. An important feature of the IL-10 family members is sharing of the receptor subunits. IL-19, IL-20 and IL-24 share a receptor complex of IL-20R1/ IL-20R2. IL-20 and IL-24 also share another receptor containing IL- 22R1 and IL-20R2. The IL-10R2 subunit is shared by IL-10, IL-22 and Developmental and Comparative Immunology 34 (2010) 158–167 ARTICLE INFO Article history: Received 20 July 2009 Received in revised form 7 September 2009 Accepted 8 September 2009 Available online 23 September 2009 Keywords: Rainbow trout IL-20L IL-10 family Gene organisation Bacterial infection Interferon-gamma IL-1beta Expression Modulation ABSTRACT A novel IL-20-like (IL-20L) gene has been analyzed in rainbow trout. It has a six exon/five intron gene organisation and is translated into 181 amino acids (aa) with a signal peptide of 25 aa and a mature peptide of 156 aa. The trout IL-20L translation shares highest identities to other IL-20L molecules from fish and to IL-20 in mammals. Phylogenetic analysis showed that the fish IL-20L molecules form an independent clade but cluster with the group containing the IL-19, IL-20 and IL-24 molecules from higher vertebrates with a high bootstrap value (89%). The fish IL-20L genes are syntenically in the same location as mammalian IL-19/IL-20/IL-24. Gene organisation and multiple alignment also showed a close relationship of fish IL-20L genes to the mammalian IL-19/IL-20/IL-24 subfamily. These data suggest that the fish IL-20L molecules may have arisen from an ancestral gene that gave rise to IL-19, IL-20 and IL-24 in higher vertebrates. A high level of IL-20L expression in immune related tissues and in the brain, suggests an important role of the fish IL-20L molecule in both the fish immune system and nervous system. Although the exact cell types expressing IL-20L have yet to be defined, macrophages express IL-20L. Moreover, the IL-20L expression in the macrophage cell line RTS-11 was modulated by pro-inflammatory cytokines, signalling pathway activators, microbial mimics and the immuno-suppressor dexamethasone. These data suggest that trout IL-20L is an important player in the cytokine network. The increased expression of IL-20L was only detected at late stages (4–24 h) of LPS stimulation in RTS-11 cells and in spleen 24–72 h after infection with Yersinia ruckeri, and suggests that the increased expression of IL-20L by LPS and infection may be via the rapid increase of pro-inflammatory cytokines (e.g., IL-1b) and other factors that is known to occur. ß 2009 Elsevier Ltd. All rights reserved. § This work was supported financially by the European Commission, Contract No. 007103 (IMAQUANIM—Improved Immunity of Aquacultured Animals). §§ The nucleotide sequence data will appear in the EMBL/DDBJ/GenBank nucleotide sequence database under the following accession numbers: FN386779 (IL-20L cDNA) and FN386780 (IL-20L genomic DNA). * Corresponding author. Tel.: +44 1224 272872; fax: +44 1224 272396. E-mail address: c.secombes@abdn.ac.uk (C.J. Secombes). Contents lists available at ScienceDirect Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci 0145-305X/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2009.09.003