Phylogeny and expression modulation of interleukin 1 receptors in grass carp (Ctenopharyngodon idella)
Introduction
Interleukin (IL) −1 family plays a central role in regulating many immunological processes including inflammatory response and immune homeostasis (Dinarello, 2018; Zou and Secombes, 2016). In humans, it consists of 11 members which are classified into 3 subfamilies, including the IL-1, IL-18 and IL-36 subfamily, based on the conserved signature sequences and binding receptors (Dinarello, 2018). The IL-1 subfamily contains IL-1α, IL-1β, IL-33 and IL-1 receptor antagonist (IL-1Ra). The IL-18 subfamily has two members, IL-18 and IL-37, and the IL-36 subfamily comprises IL-36, IL-36Ra and IL-38. All the IL-1 molecules except IL-1Ra are synthesised as precursor proteins lacking a signal peptide and require enzymatic cleavage to generate bioactive mature peptide. Caspase 1 and several proteases are known to be involved in the processing of IL-1 family precursors. Functionally, IL-1α, IL-1β, IL-18 and IL-36 are pro-inflammatory whilst IL-1Ra, IL-36Ra and IL-37 inhibit inflammatory response.
The receptors activated by the IL-1 family belong to the Toll/IL-1 receptor (TIR) subfamily and contain characteristic features, sharing 1–3 conserved Ig domains in the extracellular region. The Ig domains are critical for ligand binding whilst the intracellular TIR domain is essential for initiating cellular responses. Ten IL-1R members have been found in humans, and recently, a unified nomenclature is proposed to name them as IL-1R1-10 (Boraschi et al., 2018). In general, functional IL-1 receptor complex consists of two IL-1R chains of a ligand-binding receptor and an accessory receptor (Dinarello, 2018). Five ligand binding receptors including IL-1R1 (for IL-1α, IL-1β and IL-1Ra), IL-1R2 (for IL-1β), IL-1R4/ST2 (for IL-33), IL-1R5/IL-18Rα (for IL-18 and IL-37) and IL-1R6/IL-36R (for IL-36α, IL-36β, IL-36γ and IL-36Ra) have been characterized to bind distinct ligands whilst IL-1R3/IL-1RAcP (for IL-1α, IL-1β, IL-36α, IL-36β, IL-36γ and IL-36Ra) and IL-1R7/IL-18Rβ (for IL-18) serve as the accessory receptors. IL-1R8/SIGIRR contains a single Ig domain in the extracellular region and is a unique anti-inflammatory receptor that also possesses a TIR domain in the cytoplasmic region. The ligand of IL-1R8/SIGIRR has not been identified. Both IL-1R9/IL-1RAcPL1 and IL-1R10/IL-1RAcPL2 contain 3 Ig domains and a TIR domain and are also not functionally characterized. Like IL-1R8/SIGIRR, they are orphan receptors.
To date, two of the three IL-1 subfamilies including the IL-1β and IL-18 subfamily have been reported in fish. Fish IL-1β subfamily consists of 3 members, IL-1β, IL-1 family member 1 (IL-1Fm1, also termed novel IL-1 family member or IL-1Fma) and IL-1Fm2 whilst orthologues of IL-1α, IL-1Ra and IL-33 have not been found (Ogryzko et al., 2014). Fish IL-1β was first discovered in rainbow trout (Oncorhynchus mykiss) using a homology cloning approach in 1999 and was the most studied member over the years (Zou et al., 1999; Zou and Secombes, 2016). Fish IL-1βs share the predicted ternary protein structure of 12 β sheets with their mammalian counterparts and are functionally conserved. They are induced in most cells in response to a wide range of pro-inflammatory stimuli and bacterial and viral infections, promoting inflammation to activate defence by enhancing proliferation and migration of phagocytes to the infection sites. Unlike mammalian IL-1βs, fish IL-1βs lack the canonical caspase 1 cleavage site (Zou and Secombes, 2016). It is still under debate whether fish IL-1βs are processed by caspase 1. A recent study reveals that release of seabream IL-1β from bacteria-infected macrophages was not affected by a caspase-1 inhibitor, suggesting that caspase-1 is not involved in secretion of (Lopez-Castejon et al., 2008). Conversely, evidence from seabass and zebrafish work demonstrates that caspase 1 is required for the release of mature IL-1β (Reis et al., 2012; Vojtech et al., 2012). The IL-1Fm1, first identified in rainbow trout, are found to serve as a negative regulator of inflammation (Wang et al., 2009; Yao et al., 2015), akin the IL-1Ra to antagonise the pro-inflammatory activities of IL-1β. However, the functions of IL-1Fm2 is not fully understood. It has been shown that the recombinant IL-1Fm2 can induce expression of pro- and anti-inflammatory genes in gilthead seabream (Angosto et al., 2014). Interestingly, the IL-1Fm2 precursor is processed into the active mature form in a caspase independent manner.
Several receptors have been described for the IL-1R family members in fish. They include IL-1R1 from Atlantic salmon (Subramaniam et al., 2002), common carp (Metz et al., 2006), Dolly Varden char (Meng et al., 2018), large yellow croaker (Ao et al., 2016), orange-spotted grouper (Lu et al., 2013) and rainbow trout (Scapigliati et al., 2004); IL-1R2 from Atlantic salmon (Morrison et al., 2012), flounder (Fan et al., 2010), gilthead seabream (Lopez-Castejon et al., 2007), grass carp (Yang et al., 2013), rainbow trout (Sangrador-Vegas et al., 2000); IL-1R3/IL-1RAcP from Atlantic salmon (Stansberg et al., 2005); IL-1R8/SIGIRR from zebrafish (Feng et al., 2016); a homologue of IL-1R8 containing 2 Ig domains (DIGIRR) from Fugu, tetraodon and three spine sticklebacks (Gu et al., 2011); IL-1R9/IL-1RAcPL1 from zebrafish (Yoshida and Mishina, 2008). Like mammals, all the fish IL-1Rs possess the characteristic Ig domains in the extracellular region and a cytoplasmic TIR domain, with the exception of IL-1R2 which lacks the TIR domain for signalling. DIGIRR is a homologue of SIGIRR with two duplicated Ig domains and found only in limited teleost species such as Fugu, tetraodon and three spine sticklebacks. Fish IL-1Rs are constitutively expressed in most tissues but can be induced in response to inflammatory stimuli such as LPS and pathogen infection (Subramaniam et al., 2002). A soluble form of IL-1R1 has also been found in orange-spotted grouper and is inducible in response to excessive IL-1β, suggesting that it may act as antagonist to inhibit IL-1β actions and play a role in maintaining immune homeostasis (Lu et al., 2013). On the other hand, pathogens such as Neoparamoeba perurans can manipulate the effects of IL-1Rs at the infection sites to suppress the host immune response to establish infection (Morrison et al., 2012). The functions of most IL-Rs have yet been experimentally characterized but recent studies demonstrate that the anti-inflammatory properties of several inhibitory receptors seem to be conserved during evolution (Feng et al., 2016; Gu et al., 2011). For instance, zebrafish IL-1R8/SIGIRR is able to inhibit TRIF-mediated inflammatory response activated by TLR3/TLR22, whilst tetraodon DIGIRR, when over-expressed in zebrafish embryos, down-regulates IL-1β-mediated signalling and inflammatory response. To allow in-depth functional analysis of IL-1R family members, the present study identified five IL-1Rs including IL-1R3/IL-1RAcP, IL-1R8/SIGIRR, IL-1R9a/IL-1RAcPL1a, IL-1R9b/IL-1RAcPL1b and IL-1R10/IL-1RAcPL2 in grass carp and analysed their phylogeny and expression modulation during bacterial and viral infection. The results add further knowledge to understand the evolution of the IL-1R family and their roles in regulating immune response in teleost fish.
Section snippets
Experimental fish
Healthy grass carp (Ctenopharyngodon idella) (120 ± 10 g) were purchased from a local farm in Shanghai, China, and maintained under laboratory conditions at 25 ± 3 °C) for at least 2 weeks prior to the experimental procedures. Fish were anesthetized with 0.05% MS222 (Sigma-Aldrich, USA) before injection and sampling. All experiments were conducted under the national regulations on use of laboratory animals and local guidelines of use of animals for research.
Cloning and identification of CiIL-1Rs
Total RNA was extracted from grass
Identification of CiIL-1Rs
The coding sequences of CiIL-1Rs were obtained by local BLAST search and verified by sequencing of PCR products. The full length cDNA of CiIL-1R3/IL-1RAcP was 2371 bp with an open reading frame (ORF) of 1791 bp encoding a membrane protein of 596 aa (Supplement Fig. 1). The compiled CiIL-1R9a/IL-1RAcPL1a cDNA was 2635 bp in length and translated into a protein of 701 aa with no transmembrane region predicted (Supplement Fig. 2). The cDNA of CiIL-1R9b/IL-1RAcPL1b was 3006 bp with an ORF of 2106
Discussion
The IL-1 cytokines regulate inflammation via activation of a panel of receptors containing extracellular immunoglobulin domains and a cytoplasmic TIR domain family. The IL-1R family consists of 10 members in humans and form heterodimeric receptor complex for ligand binding to promote or inhibit inflammation. To date, six IL-1 family receptors (IL-1R1-6) have been characterized to bind to known IL-1 ligands whilst four are orphan receptors (IL-1R7-10) with ligands yet to be determined. In the
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgement
This work is supported by the National Key R&D Program of China (2018YFD0900304) and the National Natural Science Foundation of China (Grant numbers U1605211 and 31272666).
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