Cloning, expression analysis and bioactivity studies of rainbow trout (Oncorhynchus mykiss) interleukin-22 Milena M. Monte a,⇑ , Jun Zou a , Tiehui Wang a , Allison Carrington b , Chris J. Secombes a a Scottish Fish Immunology Research Centre, University of Aberdeen, Zoology Building, Tillydrone Avenue, Aberdeen AB24 2TZ, Scotland, UK b Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland, UK article info Article history: Received 26 October 2010 Received in revised form 15 March 2011 Accepted 15 March 2011 Available online 21 April 2011 Keywords: Oncorhynchus mykiss Interleukin-22 Mucosal expression Recombinant IL-22 Antimicrobial peptides abstract This report describes the cloning and characterisation of rainbow trout (Oncorhynchus mykiss) interleukin (IL)-22, and presents studies of the functional activity of its recombinant protein for the first time in a non-mammalian species. The predicted IL-22 coding region consists of 522 nucleotides which translates into a 173 amino acid protein, that contains an IL-10 family signature which is reasonably well conserved with other vertebrate IL-22 molecules. Expression analysis in tissues from healthy fish revealed a higher constitutive expression of IL-22 in mucosal tissues, suggesting a potentially important role in mucosal immunity. In vitro studies demonstrated that IL-22 expression was induced significantly by PHA and PMA in splenocyte primary cultures 4 h post-stimulation. Expression was also induced in the spleen upon infection of fish with the Gram-negative bacterium Yersinia ruckeri, suggesting a potential role of IL-22 in vivo in defence against bacterial diseases. The Escherichia coli produced recombinant IL-22 enhanced the expression of a number of antimicrobial peptides, promoting host innate immunity against microbes and revealing a biological similarity with its mammalian counterpart. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction IL-22 was first described 10 years ago both in humans and mice, and initially named IL-10-related T cell-derived inducible factor (IL-TIF) [1–3]. It is a novel member of the IL-10 family, which in- cludes IL-10, IL-19, IL-20, IL-24 and IL-26 [4]. Both the human and mouse IL-22 proteins consist of 179 amino acids (aa) contain- ing a signal peptide, and are secreted as a 146 aa polypeptide. They share a similar gene structure to most of the IL-10 family members, defined by the presence of 5 coding exons [1–3,5]. IL-22 is mainly produced by a newly discovered lineage of T helper (T H ) cells that produce a unique profile of effector cytokines and develop via an independent differentiation mechanism, rela- tive to T H 1 and T H 2 cell development. This subset has been named T H 17, as they secrete IL-17A and IL-17F, as well as IL-22 [6–9]. They are known to be involved in innate immunity against extracellular pathogens, microbes that probably are not completely eradicated by the other T H cell subsets, and in the development of autoim- mune disease [10,11]. IL-22 can also be produced, at lower levels, by natural killer (NK) cells and other cell lineages [4,8,9,12]. For example, recent findings revealed that a new subpopulation of mucosal NK-like cells secrete IL-22, particularly in the intestine [13]. In addition, a different subset of NK cells, named NK-22, has been shown to be specialised in IL-22 production in mucosal-asso- ciated lymphoid tissue in mouse and human, providing mucosal protection [14]. It has also been reported recently that a new line- age of CD4 + cells exists with a similar role to T H 17 cells, that pro- duces IL-22 but not IL-17, and as such is distinct from the other lineages already characterised [15]. Functionally, IL-22 has a crucial antimicrobial role against bac- terial pathogens because it enhances the expression of antimicro- bial peptides (such as b-defensins 2 and 3) and pro-inflammatory cytokines (such as IL-6 and IL-8) [4,9,16–19]. It brings about these effects by signalling via a heterodimeric receptor complex com- posed of two chains, IL-10Rb and IL-22R, belonging to the class II cytokine receptor family [3,4,20]. In lower vertebrates, IL-22 homologues were first identified in fugu (Takifugu rubripes) in 2004 [21], followed by zebrafish (Danio rerio) [22] and the amphibian Xenopus tropicalis [23]. Most recently, this molecule was also cloned in two gadoid species (Atlantic cod, Gadus morhua, and haddock, Melanogrammus aeglefinus) [24]. How- ever, to date the activity of the recombinant IL-22 protein remains unstudied in any non-mammalian vertebrate. In this report we describe the sequencing of the IL-22 molecule and its characterisation in a commercially important fish species, the rainbow trout (Oncorhynchus mykiss). Additionally, in order to gain a better insight into the biological function of piscine IL-22, the recombinant protein (rIL-22) was produced in Escherichia coli and its bioactivity studied. 1043-4666/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2011.03.015 ⇑ Corresponding author. Tel.: +44 1224 272870; fax: +44 1224 272396. E-mail address: m.monte@abdn.ac.uk (M.M. Monte). Cytokine 55 (2011) 62–73 Contents lists available at ScienceDirect Cytokine journal homepage: www.elsevier.com/locate/issn/10434666