Functional characterization of interleukin (IL)-22 and its inhibitor, IL-22 binding protein (IL-22BP) in Mandarin fish, Siniperca chuatsi

https://doi.org/10.1016/j.dci.2019.03.007Get rights and content

Highlights

  • IL-22 and IL-22BP genes were cloned from mandarin fish S. chuatsi.

  • IL-22 induced the expression of AMP genes, such as hepcidin and LEAP-2 in intestinal cells.

  • IL-22BP inhibited completely the IL-22 induced downstream gene expression.

  • IL-22 and IL-22BP interacted as revealed through yeast two-hybrid assay.

  • Conserved residues were found in IL-22BP in mandarin fish, as reported in mammals.

Abstract

As an important immune regulatory molecule, interleukin (IL)-22 has been reported in several species of fish, but its soluble receptor, IL-22 binding protein (IL-22BP), discovered as a natural antagonist of IL-22 in mammals, has not been functionally characterized in fish to date. In the present study, IL-22 and IL-22BP genes were cloned in mandarin fish Siniperca chuatsi. They all exhibited a high basal expression level in mucosa-enriched tissues, implying their possible roles in mucosal immunity. The IL-22 was found to show a potent response to LPS stimulation, acting as an inducer of antimicrobial peptide (AMP) genes, such as hepcidin and Liver-expressed antimicrobial peptide-2 (LEAP-2) in intestinal cells. IL-22BP, via co-incubation with IL-22, inhibited completely the induction of downstream genes by IL-22. Through a yeast two-hybrid assay, the interaction between IL-22BP and IL-22 was confirmed, which may account for the inhibitory effect of IL-22BP. Moreover, two hot spot residues for IL-22 binding, as reported in mammalian IL-22BP, were found to be conserved both in sequence location and function in mandarin fish IL-22BP, indicating that the interaction mode between IL-22 and IL-22BP may be also conserved in fish and mammals. In conclusion, the mandarin fish IL-22 and IL-22BP are conserved in their interaction and function with their mammalian orthologues, and these findings provide basis for future research on IL-22-IL-22BP axis in fish immunity.

Introduction

Interleukin (IL)-22 was first cloned in IL-9 stimulated T cells in mouse and was named initially as IL-10-related T cell-derived inducible factor (IL-TIF) (Dumoutier et al., 2000). It shares sequence homology with IL-10 and belongs to the IL-10 family of cytokines, which includes IL-10, the IL-20 subfamily of cytokines (IL-19, IL-20, IL-22, IL-24, IL-26) and the distantly related type Ⅲ interferons (IFNs) (Ouyang et al., 2011). IL-22 is generated primarily in immune cells, and is considered primarily to be a Th1-associated cytokine (Gurney, 2004; Wolk et al., 2002; Wolk and Sabat, 2006). Later, other two subsets of T helper cells, Th17 and Th22 cells, are found to secret IL-22 (Liang et al., 2006; Trifari et al., 2009). Some other cell lineages can also generate IL-22, like NKp46+ cells, lymphoid tissue inducer-like cells, γδT cells and NKT cells (Colonna, 2009; Goto et al., 2009; Mabuchi et al., 2011; Satoh-Takayama et al., 2008).

IL-22 is multifunctional in immunity. It can promote host immune defense against bacterial pathogens via enhancing the expression of antimicrobial peptides (AMPs) (Aujla et al., 2008; Wolk et al., 2004; Zheng et al., 2008), and can also contribute to tissue regeneration and wound healing, mainly by promoting proliferation, survival and repair of tissue epithelial cells (Dudakov et al., 2012; Radaeva et al., 2004). It can also promote inflammatory responses, by inducing the expression of proinflammatory molecules, such as IL-6, IL-8, LPS-binding protein, and serum amyloid A (Andoh et al., 2005; Liang et al., 2010; Wolk et al., 2007). It seems possible that the functional outcomes of IL-22 expression can be complicated. For example, IL-22 promoted epithelial cell proliferation, which is protective in tissue damage repair, but its proinflammatory behaviour can induce pathological inflammation when dysregulated, leading to tissue destruction. The different functional outcomes may be related with the context where IL-22 is expressed (Dudakov et al., 2015; Sonnenberg et al., 2011).

It has been demonstrated in mammals that IL-22 has a transmembrane receptor complex consisting of two chains, IL-22RA1 and IL-10R2 (Kotenko et al., 2001a). In contrast to the ubiquitous expression of IL-10R2, the expression of IL-22RA1 has a limited pattern, being expressed mainly on nonhaematopoietic cells, especially epithelial cells, which results in a restricted distribution of functional targets of IL-22. Upon ligand and receptor binding, Janus kinases (Jak)-signal transducers and activators of transcription (STAT) signalling pathway is activated, and the phosphorylation of STAT3 is primarily important in IL-22 signalling (Dudakov et al., 2015).

In addition to the membrane-bound receptors, a secreted receptor, named IL-22 binding protein (IL-22BP, also called IL-22RA2), is identified. IL-22BP contains typical fibronectin type Ⅲ (FNⅢ) domains, and shares protein sequence homology with the extracellular part of IL-22RA1 with higher affinity for IL-22 than the IL-22RA1-IL-10R2 complex. IL-22BP binds to IL-22 tightly, hindering this cytokine from interacting with the cell surface receptor complex, thus neutralizing its activity (Dumoutier et al., 2001; Kotenko et al., 2001b; Xu et al., 2001). IL-22BP can be produced by immature dendritic cells (DCs) and can be induced potently by retinoic acid, but its expression level decreases when DCs undergo maturation (Martin et al., 2014).

To date, the IL-22 orthologues have been cloned in several fish species, including zebrafish (Dario rerio) (Igawa et al., 2006), Atlantic cod (Gadus morhua), haddock (Melanogrammus aeglefinus) (Corripio-Miyar et al., 2009), rainbow trout (Oncorhynchus mykiss) (Monte et al., 2011), turbot (Scophthalmus maximus) (Costa et al., 2012), so-iny mullet (Liza haematocheila) (Qi et al., 2015) and yellow catfish (Pelteobagrus fulvidraco) (Jiang et al., 2018). The function of IL-22 in promoting host defense against bacterial infection has been reported in zebrafish, turbot, and so-iny mullet (Costa et al., 2013; Qi et al., 2015). The piscine IL-22BP gene was initially predicted in fugu (Takifugu rubripes), tetraodon (Tetraodon nigroviridis) and zebrafish via phylogenetic analysis (Stein et al., 2007), with the cDNA sequence cloned in zebrafish (Levraud et al., 2007) and recently in yellow catfish (Jiang et al., 2018). But, functional study of IL-22BP has never been performed in fish. In this study, IL-22 and IL-22BP were identified in mandarin fish, and the bioactivity of IL-22 was tested in intestinal cells, and the inhibitory effect of IL-22BP was also characterized for the first time in fish.

Section snippets

Fish and cell line

The mandarin fish Siniperca chuatsi were purchased from a fish market in Wuhan, Hubei Province, China, and kept in recirculating freshwater system at 28 °C with aeration for at least two weeks before experiments. Animals were treated according to the regulation of the Care and Use of Laboratory Animals of the Chinese Academy of Sciences, with also the approval from the Institute of Hydrobiology.

Chinese hamster ovary cells (CHO-K1) were cultured in F-12K (Kaighn's) Medium (Gibco) supplemented

Sequence characters of IL-22 and IL-22BP in Mandarin fish

The cDNA sequence of IL-22 and IL-22BP (GenBank accession number: MH352457 and MH352458, respectively) was cloned in mandarin fish using RACE PCRs. The ORF of mandarin fish IL-22 contains 573 bp, encoding 190 amino acids (aa). A signal peptide of 35 aa was predicted at the N-terminal region of the protein. Two possible N-glycosylation sites (Asn-Xaa-Thr/Ser) and an IL-10 family signature motif were found at the C-terminal part. Human IL-22 protein had six alpha helices, i.e. helices A, B, C, D,

Discussion

Since IL-22BP was reported as a natural antagonist of IL-22 in mammals, continuous research has been carried out over the last 20 years on its cellular source, upstream regulation and function in mucosal immunity (Dumoutier et al., 2001; Huber et al., 2012; Martin et al., 2014). However, although the IL-22BP orthologues have been identified in several teleost species (Jiang et al., 2018; Levraud et al., 2007), the function of IL-22BP in fish has never been reported. In the present study, IL-22

Acknowledgements

Professor Yong-An Zhang and Dr. Kai-song Peng in the Institute of Hydrobiology, Chinese Academy of Science, Wuhan, China are deeply acknowledged for helps in intestinal cell isolation. The present research was financially supported by National Natural Science Foundation of China (30230075), and by China Agriculture Research System (CARS-46), and also by a special top talent plan “One Thing One Decision (Yishiyiyi) and “First class fishery discipline” programme in Shandong Province, China.

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