Cloning and expression analysis of two ROR-γ homologues (ROR-γa1 and ROR-γa2) in rainbow trout Oncorhynchus mykiss

Abstract

This paper describes the cloning and characterisation of two retinoid-related orphan receptor (ROR)-γ homologues (ROR-γa1 and -γa2) in rainbow trout (Oncorhynchus mykiss). The coding region predicted for both homologues consists of 1410 base pairs (bp), which translate into two 469 amino acid (aa) proteins. The trout ROR-γs revealed a high conservation of both DNA- and ligand-binding domains (functional regions of the nuclear receptor family), and shared a high homology to mammalian ROR-γt. A phylogenetic tree containing ROR family members confirmed that both trout homologues clustered within the ROR-γ group. Both results suggested that these molecules are likely to be ROR-γ homologues, more similar to the mammalian splice variant ROR-γt than the full length ROR-γ. Expression analysis of tissues obtained from healthy fish revealed highest constitutive expression of trout ROR-γ in muscle, followed by the brain, heart and skin. This suggests that these genes may play an important role in such tissues. In vitro studies, using trout cell lines, demonstrated that ROR-γ is induced significantly by LPS and down-regulated by the presence of PolyI:C and recombinant interferon (IFN)-γ. Moreover, analysis of this gene in head kidney macrophages and mixed primary leucocyte cultures indicated that differences were apparent between the different cell types/sources used, indicating that its expression may be cell-type dependent. Additional studies to investigate the regulation of this gene in vivo demonstrated that its expression was significantly higher in vaccinated vs unvaccinated fish following bacterial (Yersinia ruckeri) challenge but it was down-regulated after a viral (VHSV) infection. This suggests a potential role of trout ROR-γ, a putative T(H)17 transcription factor, in protection against extracellular bacteria.

An Overview of the Immunological Defenses in Fish Skin

“…In vitro studies using trout cell lines demonstrated that ROR-g is induced significantly by LPS and downregulated by the presence of Poly I:C and recombinant interferon (IFN)-g. In vivo studies demonstrated that its expression was significantly higher in vaccinated versus unvaccinated fishes following bacterial (Yersinia ruckeri) challenge, but it was downregulated after a viral (VHSV) infection. All the data suggest a potential role of trout ROR-g, a putative TH17 transcription factor, in protection against extracellular bacteria [353].…”

The cytokine networks of adaptive immunity in fish

“…In fish, ROR-g orthologues have been identified in zebrafish [143], grass carp [144] and rainbow trout [145], with two paralogues in each species. Phylogenetic tree analysis revealed that the two cyprinid RORg molecules from the same species form two groups, whilst the two trout RORgs group within a single clade, suggesting that the two cyprinid RORgs arose from the teleost-wide WGD event but that the trout paralogues arose from an additional WGD in salmonids.…”

“…Mammalian RORgts are produced using an alternative promoter in the second intron of RORg genes, and are 21 amino acids shorter at the N-terminal. Multiple alignment of protein sequences and gene organization analysis suggests that the fish RORg is more similar to mammalian RORgt than to RORg [145].…”

“…Trout RORg is widely expressed but with low levels of expression in immune relevant tissues [145]. Trout RORg expression is inhibited by PMA, calcium ionophore and PHA, factors that induce IL-17A/F2 expression in the same system [113].…”

“…In vivo bacterial infection (Y. ruckeri) upregulates the expression of both trout RORg and IL-17A/F2 in spleen. However, VHSV infection down-regulates trout RORg expression but upregulates IL-17A/F2 expression [113,145]. The dissociation of expression of trout RORg and IL-17A/F2 poses a question as to whether RORg can control IL-17A/F expression in fish.…”