Molecular evidence for the involvement of RORα and RORγ in immune response in teleost

https://doi.org/10.1016/j.fsi.2012.05.033Get rights and content

Abstract

In mammals, retinoid-related orphan receptors (ROR) consist of three members as RORα, RORβ and RORγ. It is well known that RORα plays a critical role in cerebellum development while RORγt directs T helper 17 (Th17) cell differentiation. So far, the knowledge on fish ROR family is limited as only zebrafish ROR family members have been characterized, showing that they play roles in embryonic and cerebellar development. In this study, we have cloned two paralogues for RORα (RORα1 and RORα2) and RORγ (RORγ1 and RORγ2) from grass carp (Ctenopharyngodon idellus). Phylogenetic analysis showed that grass carp RORα and RORγ were grouped in the clade of zebrafish RORα and RORγ, respectively. Real-time RT-PCR assay revealed that these four ROR transcripts exhibited similar expression patterns, in particular the high levels in pituitary, brain and some immune-related tissues, suggesting that all of them may play a role in endocrine and immune system of teleost. To explore the immune roles of grass carp RORα and RORγ, their expression was detected in periphery blood lymphocytes (PBLs) challenged by immune stimuli. Results showed that both RORα and RORγ mRNA levels were up-regulated by PHA but not LPS in PBLs, suggesting that their expression may be subject to different immune processes. In the same cell model, poly I:C stimulation induced RORγ1/2 but not RORα1/2 expression, pointing to the different roles of grass carp RORα and RORγ in immune response. Consistently, bacterial challenge significantly up-regulated the expression of these four ROR genes in spleen, headkidney and thymus. These results not only contribute to elucidate the roles of ROR in fish immunity but also facilitate to further clarify the existence of Th17-like cells in fish.

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).

References (32)

  • S.W. Xu et al.

    Molecular cloning and expression of orange-spotted grouper (Epinephelus coioides) CD8alpha and CD8beta genes

    Fish Shellfish Immunol

    (2011)
  • M. Yang et al.

    Characterization of grass carp (Ctenopharyngodon idellus) Foxp1a/1b/2: evidence for their involvement in the activation of peripheral blood lymphocyte subpopulations

    Fish Shellfish Immunol

    (2010)
  • X.N. Huang et al.

    Characterization of Toll-like receptor 3 gene in large yellow croaker, Pseudosciaena crocea

    Fish Shellfish Immunol

    (2011)
  • J.M. Dijkstra et al.

    Identification and characterization of a second CD4-like gene in teleost fish

    Mol Immunol

    (2006)
  • R.M. Evans

    The steroid and thyroid hormone receptor superfamily

    Science

    (1988)
  • V. Giguere

    Orphan nuclear receptors: from gene to function

    Endocr Rev

    (1999)
  • Cited by (19)

    • Teleost CD4<sup>+</sup> helper T cells: Molecular characteristics and functions and comparison with mammalian counterparts

      2021, Veterinary Immunology and Immunopathology
      Citation Excerpt :

      They also restrain aberrant or excessive immune responses to nonself antigens at sites of inflammation (Sakaguchi et al., 2010) by producing two powerful anti-inflammatory cytokines, namely, TGF-β and IL-10 (Loo et al., 2018). Previous studies on gene expression in a large number of bony fish have demonstrated the important immune functions of cytokines and master transcription factors, including T-bet (Mitra et al., 2010; Wang et al., 2012; Kumari et al., 2015), IFN-γ (Milev-Milovanovic et al., 2006; Chen et al., 2010, 2015), GATA3 (Chi et al., 2012; Wang et al., 2012), IL-4/13 (a molecule related to mammalian IL-4 and IL-13) (Wang and Secombes, 2015; Wang et al., 2016; Yang et al., 2016; Stocchi et al., 2017), ROR-γ (Flores et al., 2007; Du et al., 2012), IL-17A/F (teleost homolog to mammalian IL-17A and IL-17 F) (Kono et al., 2011; Costa et al., 2012; Du et al., 2015; Ding et al., 2016), FOXP3 (Wen et al., 2011; Yang et al., 2012; Wei et al., 2013), IL-10 (Savan et al., 2003; Piazzon et al., 2016), and TGF-β (Maehr et al., 2012; Zhan et al., 2015), associated with CD4+ helper T cells. Notably, the functions of these cytokines and master transcription factors in teleost are similar to those of their mammal counterparts (Fig. 2).

    • Identification of two Stat3 variants lacking a transactivation domain in grass carp: New insights into alternative splicing in the modification of teleost Stat3 signaling

      2018, Fish and Shellfish Immunology
      Citation Excerpt :

      Cells were resuspended in Opti-MEM (Invitrogen) for further use. The cDNA preparation and qRT-PCR analysis with the gene-specific primers (Supplementary Table 1) were performed as described previously [17,22]. The expression plasmids of grass carp Stat3β1/2 were generated by cloning their coding sequences into pcDNA3.1/myc-His (−) (Thermo Scientific) or pEGFP-N1 (Clontech Laboratories, Palo Alto, CA).

    • Identification and functional evaluation of two STAT3 variants in grass carp: Implication for the existence of specific alternative splicing of STAT3 gene in teleost

      2017, Developmental and Comparative Immunology
      Citation Excerpt :

      Head kidney was collected from six fish each in the infection and control group at day 1, 3 and 7 post-infection. The mRNA levels of gcSTAT3α1/2 and β-actin in the head kidneys were determined by qRT-PCR with gene-specific primers (Supplementary Table 1) following our previous experimental procedures (Du et al., 2012). qRT-PCR was performed on a Bio-Rad CFX96™ Real-time detection system (Bio-Rad, Hercules, CA, USA).

    View all citing articles on Scopus
    View full text