Molecular evidence for the involvement of RORα and RORγ in immune response in teleost
Highlights
► We have cloned two paralogues for RORα and RORγ from grass carp. ► Four ROR transcripts exhibited similar expression patterns in the tissues examined. ► PHA but not LPS stimulated RORα and RORγ expression in periphery blood lymphocytes. ► Poly I:C induced RORγ but not RORα expression in periphery blood lymphocytes.
Introduction
In mammals, retinoid-related orphan receptors (ROR), consisting of RORα, RORβ and RORγ (also referred to as RORA-C or NR1F1-3), belong to the nuclear hormone receptor family [1]. Each of them contains a highly conserved DNA-binding domain (DBD) and a C-terminal ligand-binding domain (LDB) which are characteristics for ROR family [2], [3], [4]. Of particular note is that most ROR genes generate several isoforms through alternative exon splicing or different promoter usage. Four isoforms (RORα 1–4) have been identified in human, while only two RORα isoforms have been found in mouse. In addition, both human and mouse RORγ genes generate two isoforms as γ1 and γ2 [1]. However, the mouse RORβ gene generates two isoforms while human RORβ gene appears to express unique isoform. Interestingly, these isoforms for RORα, RORβ and RORγ differ from each other only in their N-terminus [1]. For example, RORγ2, most commonly referred to as RORγt, differs from the RORγ1 with a truncation of N-terminus [5].
These ROR isoforms are expressed in a variety of tissues and involved in different physiological processes. In contrast to RORα and RORγ, RORβ expression is largely restricted in central nervous system and retina [6], [7]. Actually, RORα and RORγ have been implicated in immune functions. RORα has been reported to express with particularly high levels in lymphoid cells [8]. In this context, RORα may play a role in thymopoiesis and lymphocyte development through regulating cytokine production and the numbers of both T and B lymphocytes [8]. Among ROR family, RORγ has attracted the most interest in view of its important role in immune system [5], showing that RORγ1 is essential for differentiation and development of secondary lymphoid organs [5], [9]. In particular, RORγt directs Th17 lineage differentiation through the interaction of RORγt with Runx1 to promote IL-17 expression [10], [11]. Further study demonstrates that both RORα and RORγt are essential for Th17 differentiation in which RORα appears to regulate the expression of IL-17, IFN-γ and T-bet [12]. These findings provided the basis and impulse for us to identify the immune functions of RORα and RORγ in lower vertebrates.
In teleost, only zebrafish ROR genes, including RORα, RORβ and RORγ, have been identified [13] and their expression patterns in embryonic development have also been investigated [13], [14]. However, the roles of RORs in fish immunity and their regulation in fish are still unknown. With respect to the potential roles of RORα and RORγ in immunity, we isolated RORα and RORγ cDNA from grass carp (Ctenopharyngodon idellus). Unlike those in mammals, both grass carp RORα and RORγ have two paralogues and their isoforms were not detected. Meanwhile, these four ROR genes were widely expressed in a variety of tissues. Furthermore, using a static incubation approach, the effects of immune stimulants (PHA, LPS and poly I:C) on the mRNA expression of grass carp RORα and RORγ were examined in periphery blood lymphocytes (PBLs), thereby providing the evidence for their involvement in immune response. In support of this notion, we also detected their expression in major lymphoid organs after intraperitoneal injection with bacteria. This is the first study that elucidates the roles of ROR in fish immunity and our results facilitate to further clarify the existence of Th17-like cells in fish.
Section snippets
Animals
One-year-old grass carp, weighing 0.75–1.0 kg, were purchased from Chengdu Tongwei Aquatic Science and Technology Company (China) and were maintained in laboratory with room temperature and natural photoperiod for 2 weeks before experimental processing. The tissues for expression analysis and periphery blood were obtained from the freshly killed fish according to the Regulation of Animal Use in Sichuan province, China.
Cloning of grass carp RORα and RORγ
Total RNA was isolated from grass carp intestine, gill and thymus using
Molecular cloning of grass carp RORα and RORγ
We cloned the full-length cDNA sequences of RORα1 (GenBank ID: JN882279), RORα2 (GenBank ID: JN882280), RORγ1 (GenBank ID: JN882281) and RORγ2 (GenBank ID: JN882282) from grass carp. The cDNA of grass carp RORα1 contained a 90 bp 5′UTR, an ORF of 1425 bp encoding a 474-amino acid (aa) polypeptide, and a 3′UTR of 240 bp (Supplementary Fig. S1). RORα2 sequence consisted of an 84 bp 5′UTR, an 822 bp 3′UTR and a 1407 bp CDS encoding a 468-aa protein (Supplementary Fig. S2). The ORF of grass carp
Discussion
In the present study, we cloned two paralogues for RORα (RORα1, RORα2) and RORγ, (RORγ1 and RORγ2) from grass carp. However, no isoform for these four genes were detected although RORα and RORγ gene generate four and two isoforms in human, respectively [19]. In agreement with two RORα paralogues in grass carp, two RORα genes (also named RORα1 and RORα2) have been identified from zebrafish genome [14]. Although only one zebrafish RORγ has been reported [13], another zebrafish RORγ sequence can
Acknowledgments
This work was supported by the grants from the National Natural Science Foundation of China (30972280) and the Science and Technology Committee of Sichuan Province (2011FZ0007).
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