Characterisation and expression analysis of the Atlantic halibut (Hippoglossus hippoglossus L.) cytokines: IL-1β, IL-6, IL-11, IL-12β and IFNγ

Abstract

Genes encoding the five Atlantic halibut (Hippoglossus hippoglossus L.) cytokines; interleukin (IL)-1β, IL-6, IL-11b, IL-12βc, and interferon (IFN) γ, were cloned and characterised at a molecular level. The genomic organisation of the halibut cytokine genes was similar to that seen in mammals and/or other fish species. Several mRNA instability motifs were found within the 3′-untranslated region (UTR) of all cytokine cDNA sequences. The putative cytokine protein sequences showed a low sequence identity with the corresponding homologues in mammals, avian and other fish species. Nevertheless, important structural features were presumably conserved such as the presence, or absence in the case of IL-1β, of a signal peptide, secondary structure and family signature motifs. The relative expression pattern of the cytokine genes was analyzed in several halibut organs, revealing a constitutive expression in both lymphoid and non-lymphoid organs. Interestingly, the gills showed a relatively high expression of IL-1β, IL-12βc and IFNγ. The real time RT-PCR data also showed that the mRNA level of IL-1β, IL-6, IL-12βc and IFNγ was high in the thymus, while IL-11b was relatively highly expressed in the posterior kidney and posterior gut. Moreover, the halibut brain showed a relatively high level of IL-6 transcripts. Anterior kidney leucocytes in vitro stimulated with imiquimod showed a significant increase in mRNA level of the five halibut cytokine genes. The sequence and characterisation data presented here will be useful for further investigation of both innate and adaptive immune responses in halibut, and be helpful in the design of vaccines for the control of various infectious diseases.Electronic supplementary materialThe online version of this article (doi:10.1007/s11033-011-0969-x) contains supplementary material, which is available to authorized users.

The cytokine networks of adaptive immunity in fish

“…IL-6 genes haves been cloned in a few teleost fish species including fugu [119], rainbow trout [120], spotted green pufferfish (Bird and Secombes, 2006;unpublished), Japanese flounder [121] sea bream [122], orange-spotted grouper [123] and Atlantic halibut Hippoglossus hippoglossus [124]. In addition both receptor subunits, IL-6Ra and gp130 have been describes in rainbow trout [125] and fugu [126].…”

Characterization, promoter analysis and expression of the interleukin-6 gene in blunt snout bream, Megalobrama amblycephala

“…As all other known vertebrate IL-6, MamIL-6 possessed a signal peptide, 4 conserved a-helices and a IL-6/G-CSF/MGF family consensus pattern (C-x9-Cx6-G-L-x2-[FY]-x3-L), which are well conserved in vertebrates (Bird et al 2005;Castellana et al 2008;Iliev et al 2007;Nam et al 2007;Øvergård et al 2012;Schneider et al 2001;Simpson et al 1997;Yan et al 2013). The predicted a-helixes in MamIL-6 according well to the a-helixes in the human IL-6 (Bazan 1990;Sprang and Bazan 1993) infer that MamIL-6 belongs to the long-chain 4-a-helical bundle protein superfamily (Nicola 1994).…”

“…Despite the vast information on il-6 in mammals, much less is known in fish species, mainly because the sequence identity is low between fish and mammalian il-6s and the gene is transcribed temporarily at low levels under normal circumstance (Castellana et al 2008). Since the first teleosts' il-6 sequence was identified in Japanese pufferfish (Takifugu rubripes) in 2005 (Bird et al 2005), il-6 has been characterized from several other fish species including Japanese flounder (Paralichthys olivaceus), rainbow trout (Oncorhynchus mykiss), gilthead seabream (Sparus aurata), Atlantic halibut (Hippoglossus hippoglossus L.) and grass carp (Ctenopharyngodon idellus) (Nam et al 2007;Iliev et al 2007;Castellana et al 2008;Øvergård et al 2012;Yan et al 2013).…”

Characterization of Novel Precursor miRNAs Using Next Generation Sequencing and Prediction of miRNA Targets in Atlantic Halibut

“…Previously, the expression of cytokines under normal physiological conditions has been demonstrated in Atlantic halibut. For instance, Il-1β is expressed in various organs; Il-12β is highly expressed in immune related organs; and Il-6 exhibits more limited expression pattern and is primarily detected in thymus and brain [40]. Vibrio harveyi challenge experiment in Asian seabass ( Lates calcarifer ) has shown rapid transient elevated expression of both Il-1β and miR-21 in three immune related organs, indicating role of miRNAs in acute inflammatory immune responses [28].…”