Fmr

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Molecular Immunology 62 (2014) 186–198

Contents lists available at ScienceDirect

Molecular Immunology

j ourna l ho me pa g e : www.elsev ier .com/ locate /mol imm

our CISH paralogues are present in rainbow trout Oncorhynchusykiss: Differential expression and modulation during immune

esponses and development

anja Maehra, Jose L. González Vecinob, Simon Wadsworthb, Tiehui Wanga,∗,hristopher J. Secombesa,∗

Scottish Fish Immunology Research Centre, Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen AB24 2TZ, UKEWOS Innovation AS, 4335 Dirdal, Norway

r t i c l e i n f o

rticle history:eceived 17 March 2014eceived in revised form 10 June 2014ccepted 12 June 2014

eywords:ainbow troutISHOCS familyene expressionytokine stimulationacterial infection

a b s t r a c t

Suppressor of cytokine signalling (SOCS) family members are crucial in the control and attenuation ofcytokine induced responses via activation of the JAK/STAT, TLR and NF-kB signalling pathways. SOCSproteins orchestrate the termination of many types of immune responses and are often the targets ofmicrobial pathogens exploiting SOCS mechanisms to evade the host’s immune response. Through wholeand lineage specific genome duplication events, the teleost cytokine/SOCS network is complex. Not onlyare the orthologues of all mammalian SOCS members present, namely cytokine inducible Src homology2 (SH2)-containing protein (CISH) and SOCS-1 to -7, but multiple gene copies exist that may potentiallybecome functionally divergent. In this paper we focus on the CISH genes in rainbow trout (Oncorhynchusmykiss), and have cloned two further paralogues, CISHa2 and CISHb2, additional to the known CISHa1 andCISHb1 genes. We present for the first time a comparative expression analysis of these four paralogues,to establish whether subfunctionalisation is apparent. In vivo examination of gene expression revealeda higher constitutive expression level of CISHa paralogues compared to CISHb expression in adult trouttissues. All CISHs were relatively highly abundant in immune tissues but CISHa2 and CISHb2 had highestexpression in the heart and muscle. An inverse picture of CISH abundance during trout ontogeny wasseen, and further hints at differential roles of the four genes in immune regulation and development.Stimulation of head kidney (HK) leukocytes with trout recombinant interleukin (rIL)-15 and rIL-21 hada major effect on CISHa2 and to a lesser extent CISHa1 expression. In HK macrophages rIL-1�, phyto-

hemagglutinin, and phorbol 12-myristate 13-acetate also had a strong impact on CISHa2 expression.Yersinia ruckeri infection caused a temporally and spatially differential onset of CISH expression that maybe viewed in the context of pathogen evasion strategies. These data, against the backdrop of fish specificwhole genome duplication events and functional divergence, provide the first evidence for differentialroles of the four trout CISH genes in immune control and development.

© 2014 Elsevier Ltd. All rights reserved.

. Introduction

Cytokines are fundamental constituents of many biologicalrocesses, including the regulation of embryonic development,

aematopoiesis, and responses to pathogenic infection by the

mmune system. The biological activity of most cytokines isxerted through Janus kinases (JAKs) that associate with their

∗ Corresponding authors. Tel.: +0044 1224 272872; fax: +0044 1224 272396.E-mail addresses: t.h.wang@abdn.ac.uk (T. Wang),

.secombes@abdn.ac.uk (C.J. Secombes).

ttp://dx.doi.org/10.1016/j.molimm.2014.06.021161-5890/© 2014 Elsevier Ltd. All rights reserved.

cognate receptors to transduce signals influencing survival, pro-liferation, differentiation and functional activity of immune cellsfurther downstream. Once activated, receptor associated JAKsphosphorylate specific receptor tyrosine residues, thus creatingdocking sites for Src homology 2 (SH2)-containing signalling pro-teins such as the signal transducers and activators of transcription(STATs) promoting cytokine induced gene expression throughnuclear translocation and binding of specific enhancer elements

(Schindler et al., 2007). It is not surprising that the duration andmagnitude of a cytokine response can also be regulated by theintegration of attenuators at these SH2 domain docking sites(Piessevaux et al., 2008). Therefore, mechanisms contributing to

Immun

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T. Maehr et al. / Molecular

he negative regulatory network involve JAK dephosphorylationy SH2-containing protein tyrosine phosphatases (SHPs) (Elcheblyt al., 1999; Lund et al., 2005) as well as sumoylation via pro-ein inhibitors of activated STATs (PIAS) (Shuai and Liu, 2005).owever, one of the major mechanisms for JAK/STAT pathwayttenuation and the prevention of detrimental, overshootingytokine responses is mediated by members of the suppressorsf cytokine signalling (SOCS) protein family (Yoshimura et al.,007). Upon a cytokine induced immune response, activatedTAT proteins not only initiate the transcription of a range ofytokine responsive target genes but also SOCS mRNAs. A classicalegative-feedback loop is completed as these proteins then shutff or limit the signalling pathway by which they were induced.

The founding member of the SOCS family, cytokine inducibleH2-containing protein (CISH), and the seven other membersresent in mammals, SOCS-1 to -7, share a common structure. AllOCS proteins contain a central SH2 domain with an N-terminalxtended SH2 subdomain (ESS) and a C-terminal 40 amino acidaa) motif, the SOCS box. The length of their N-terminal, however,aries (Piessevaux et al., 2008). Based on the latter, SOCS proteinsan be divided into two groups. CISH and SOCS-1 to -3 have ahort N-terminal region of less than 50 aa (type I subfamily) whilehe N-termini of SOCS-4 to -7 are composed of more than 200 aatype II subfamily) (Wormald and Hilton, 2004). In addition to thisommon three-domain structure, SOCS-1 and SOCS-3 possess an-terminal kinase inhibitory region (KIR). Thus, whilst it is known

hat all members of the SOCS family can bind phosphotyrosineesidues via their SH2 domain, the different isoforms can modulateytokine signalling in different complementary ways. First, the KIRound in SOCS-1 and SOCS-3 serves as a pseudo-substrate for JAKs,locking JAK function (Sasaki et al., 1999). Second, they may inhibitignalling by direct competition with STAT-SH2 for docking sites atpecific receptor phosphotyrosine residues (CISH, SOCS-2) (Cohneyt al., 1999; Nicholson et al., 1999). Third, an E3 ubiquitin–ligaseomplex bound to the SOCS box motif may provide an additionalevel of regulation by ubiquitinating associated proteins that targethem for protein degradation (Kamizono et al., 2001; Zhang et al.,999).

Type I SOCS have been widely studied in mammals, especiallyn the context of immune regulation. CISH serves as a negativeegulator of erythropoietin (EPO), interleukin (IL)-2, IL-3, prolactinnd growth hormone (GH) action by inhibiting STAT-5 (Yoshimurat al., 1995). SOCS-1 and SOCS-3 expression is disease associated, aseen in allergy, autoimmune diseases, diabetes, obesity, metabolicyndrome and cancer. SOCS-1 interferes with STAT-1 phosphory-ation and thereby attenuates signalling of interferon-� (IFN-�),L-2, IL-4, IL-6, IL-12, IL-15 and tumor necrosis factor-� (TNF-�)Davey et al., 2006; Murray, 2007). SOCS-3 is induced by IL-6-typeytokines and is essential for the suppression of IL-6/glycoprotein30 (gp130) signalling (Sommer et al., 2005), and can interact withhe receptors for leptin, EPO and granulocyte colony-stimulatingactor (G-CSF) (Bjorbaek et al., 1998; Marine et al., 1999). BothOCS-1 and -3 can inhibit IFN-�-induced expression of antiviralroteins (Vlotides et al., 2004) and SOCS gene expression inducedy chemokines and some toll-like receptor (TLR) agonists (Dalpket al., 2001; Hu et al., 2009). SOCS-2 is implicated in the negativeegulation of GH-STAT-5 and insulin-like growth factor-1 (IGF-1),ith SOCS-2 gene deletion in mice resulting in dramatic gigantism

Greenhalgh et al., 2005).The role of SOCS proteins in teleost fish has begun to be unrav-

lled in recent years (Costa et al., 2011; Grayfer et al., 2011; Jint al., 2007a,b; Skjesol et al., 2014; Wang et al., 2010a,b; Wang

nd Secombes, 2008; Xiao et al., 2010; Zhang et al., 2011). Allight mammalian orthologues have been identified and several ofhem have multiple gene copies (Jin et al., 2007a,b; Wang et al.,010a; Wang and Secombes, 2008), suggesting the SOCS system

ology 62 (2014) 186–198 187

is potentially quite complex in fish. Indeed, we recently con-firmed by synteny and phylogenetic analysis that two proposedadditional fish SOCS family members, SOCS-8 and SOCS-9 (Jin et al.,2008), are likely paralogues of CISH and SOCS-5 (Wang et al.,2011b). The discovery of multiple piscine SOCS gene copies is par-alleled by the existence of duplicated cytokines and their receptors(Hong et al., 2013; Husain et al., 2012), and is thought to be, in alarge part, a consequence of a whole genome duplication (WGD)event that occurred in teleost fish before their radiation and afurther WGD in some lineages, as in salmonids (Kassahn et al.,2009).

WGD mechanisms have been a significant force drivinggenetic diversity and radiation in the evolutionary history ofearly vertebrates and have also been associated with impor-tant immunobiological developments such as the genesis ofthe adaptive immune system. Following such mechanisms, theresulting duplicated genomes eventually retain only a small pro-portion of duplicated genes, while seemingly redundant copiesare inactivated by gene fractionation (Berthelot et al., 2014). Thus,duplication of the entire set of genes paves the way for neo-and subfunctionalisation as well as reciprocal gene loss (Semonand Wolfe, 2007). Having experienced tetraploidisation in theirancestor lineage and with some parts of their genome not hav-ing fully reverted to diploidy, salmonid fish provide an excellentmodel to study both the origins and consequences of polyploidyin relation to the immune system. This could not only reveal newevolutionary mechanisms unheard of in the study of immunol-ogy but the characterisation of paralogous genes may also helpto resolve their potentially different functions with regard topathways that are crucial to fish health and growth in aquacul-ture.

Hence, in order to elucidate the mechanisms of control of thefish cytokine network, it is of particular importance to identify allparalogues of SOCS genes and establish possible regulatory andfunctional divergence. In this study, we report the sequencing oftwo further rainbow trout (Oncorhynchus mykiss) CISH molecules,CISHa2 and CISHb2, adding further components to the fish nega-tive regulatory control of cytokine signalling. The expression profileof the new CISH paralogues is presented and compared with thealready known trout CISHa and CISHb (now termed CISHa1 andCISHb1) (Wang et al., 2010a,b) in healthy adult fish and duringtheir ontogeny. In addition, the immune modulation of expressionof the four trout CISH genes has been examined in vivo after bac-terial infection of fish and in vitro after stimulation of leukocyteswith pathogen associated molecular patterns (PAMPs), recombi-nant trout cytokines and other immune stimulants. Our findingsgive the first detailed insight into the subfunctionalisation of fourCISH paralogues present in salmonids, during immune stimulationand ontogeny.

2. Materials and methods

2.1. Fish

Rainbow trout used for examination of CISH expression intissues, in vitro experiments with primary leukocyte culturesand for bacterial challenge were purchased from a local fishfarm at Almondbank, Perthshire. These fish were maintainedin 1 m-diameter fibreglass tanks supplied with a continuousflow of freshwater at 15 ± 1 ◦C within the aquarium facility ofthe Scottish Fish Immunology Research Centre. Fish were fedtwice daily on standard commercial pellets (EWOS), and were

given at least a 2-week acclimatisation period prior to treat-ment. The Institut National de la Recherche Agronomique (INRA,France) provided samples from trout ontogenic stages as describedbelow.

188 T. Maehr et al. / Molecular Immunology 62 (2014) 186–198

Table 1Primers used for cloning and real-time PCR.

Amplicon Primer name Sequence (5′to 3′) Application

EF-1� EF-1�-F CAAGGATATCCGTCGTGGCA Real-time PCREF-1�-R ACAGCGAAACGACCAAGAG Real-time PCR

CISHa1 CISHa1-F CATTCTACCTTGATACCTCAGGCTGGT Real-time PCRCISHa1-R CCTGCTGCACCTTCCTCCC Real-time PCR

CISHa2 CISHa2-F1 GAATGAGTTGATTCATTTTCGGATG Cloning (3′-RACE)CISHa2-F2 CGTCTCTATTCACTGAACATTTGCG Cloning (3′-RACE)CISHa2-F TCTTCTACCTTGATACCTCAGGCTGGT Real-time PCRCISHa2-R CCTTGCCCTTCTGTACCTTCCTTGT Real-time PCR

CISHb1 CISHb1-F GAATGATGGTGAGAGAGGAGATTTGTGT Real-time PCRCISHb1-R GACCCCCAGTACCAACCTGAGTT Real-time PCR

CISHb2 CISHb2-F1 GACAGTTGTTCTTGGCGAGTGG Cloning (3′-RACE)TCACT ′

GATCAGACCC

2i

qd1ntmpaaawC

2o

atts(stpessa(cames

sotmCmCCT

CISHb2-F2

CISHb2-F

CISHb2-R

.2. Identification and cloning of a second CISHa and CISHb genen rainbow trout

Rainbow trout CISHa and CISHb sequences were used asueries for a TBLASTN search for CISH paralogues in the NCBIatabase (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al.,997). This returned two trout expressed sequence tags (EST) (acc.o. CA345090 and EZ854609) which when translated encoded pep-ides with homology to the N-termini of trout CISHa and CISHb

olecules, respectively. To obtain the full-length sequence of theseutative CISH paralogues, primers (F1/F2; Table 1) were designedgainst the 5′-untranslated region (UTR) and used for 3′-rapidmplification of cDNA ends (RACE) as described previously (Wangnd Secombes, 2003). Two 3′-RACE products, of 1.28 kb and 1.32 kb,ere obtained from HK and liver SMART cDNA, for CISHa2 andISHb2, respectively.

.2.1. Sequence analysis of the two novel CISH paralogues andrganisation of all four CISH genes present in rainbow trout

The DNA sequences produced by cloning were assembled andnalysed with the AlignIR programme (LI-COR Inc.). Analysis ofhe translated protein sequences was carried out by applyinghe ExPaSy tool package (http://www.expasy.org/tools). Domaintructures were predicted by employing the SMART 6 programmehttp://smart.embl-heidelberg.de) (Letunic et al., 2009). Proteinequences used for analysis of similarity were retrieved fromhe ExPaSy or NCBI databases and aa homology comparison waserformed using the MatGat programme (v2.02) (Campanellat al., 2003), choosing the scoring matrix BLOSUM62. Compari-on between more than two sequences was achieved by multipleequence alignments created by ClustalW2 (Chenna et al., 2003)nd conserved residues were shaded using the BOXSHADE serverv3.21; http://www.ch.embnet.org/software/BOX form.html). Theonstruction of a phylogenetic tree was based on the ClustalW2lignments and was generated by the Neighbour–Joining (N–J)ethod within the MEGA package (v5.1; Tamura et al., 2011). To

valuate the strength of support for nodes, the tree was boot-trapped 10,000 times.

The gene organisation of all four members of the trout CISHubfamily was inferred taking advantage of the recent releasef rainbow trout whole genome shotgun (WGS) sequence con-igs that were from a single homozygous doubled haploid YY

ale (Berthelot et al., 2014). Three contigs, CCAF010023507,CAF010055202 and CCAF010044231, were identified and

atched the cDNA sequences for trout CISHa1, CISHa2 and

ISHb1, respectively. In addition, two overlapping contigs,CAF010010398 and CCAF010010399 matched the CISHb2 cDNA.he gene organisation was determined by comparing the CISH

GCGTTGGACCTAGCCTATC Cloning (3 -RACE)TGGTGAGAGAGGAGGGTCA Real-time PCRCCAGTACCAACCTGAGTT Real-time PCR

cDNAs against their genomic sequences using the online Spideyprogramme (http://www.ncbi.nlm.nih.gov/spidey) at NCBI.

2.3. Real-time PCR analysis of gene expression

RNA preparation, cDNA synthesis and real-time PCR analysisusing a LightCycler® 480 System (Roche Applied Science, UK) wereas described elsewhere (Wang et al., 2011a). Gene-specific primerpairs were designed so that at least one of the primers would spanan intron to prevent the amplification of genomic DNA and prod-uct sequence verified (Table 1). To quantify the expression of troutCISH paralogues as well as that of the house keeping gene elonga-tion factor-1� (EF-1�), a standard was constructed by preparing amixture of equal mole amounts of purified PCR products amplifiedfrom cDNA for each gene to be studied. As a reference for quantifica-tion, a serial dilution of these standards was run in the same 96-wellPCR plate as the cDNA samples. The expression level of each genewas calculated as arbitrary units (AU) normalised to the expressionof EF-1�. The fold changes were calculated as the average expres-sion of the treatment groups divided by that of the relevant controlgroup.

2.3.1. Tissue distributionSix healthy fish with an average weight of 120 g were killed and

15 tissues (tail fin, adipose fin, scales, skin, muscle, brain, gills, thy-mus, spleen, HK, liver, heart, intestine, gonad and blood) collectedfor RNA extraction. Samples were homogenised and kept in TRIReagent (Sigma) until processed for cDNA synthesis and real-timePCR examination of CISH gene expression. The comparative expres-sion level of each gene was calculated as AU where one unit isequal to the lowest average expression level seen among all tissuesinvestigated, which was found for CISHb1 in liver.

2.3.2. Expression of the four rainbow trout CISH genes in early lifestages

Whether there is a differential expression of the four CISH trans-cripts during the development of rainbow trout was examinedusing samples from different ontogenetic stages of rainbow trout.Whole animal samples from juvenile stages of rainbow trout thathad been raised at 10 ◦C in recirculated water at the INRA exper-imental fish facility in Jouy-en-Josas, were as described by Wanget al. (2010b), and included: eyed eggs (egg; 17/12/09, ∼280 degreedays; DD), immediate post-hatch fry (hatch; 16/01/09, ∼370 DD),pre-first feeding fry at the stage of full disappearance of the yolk sac(PFF; 4/2/09, 560 DD), and fry 3 weeks following first feeding (fry;

25/2/09, 770 DD). Total RNA preparation was as described previ-ously (Wang et al., 2010b). Each sample contained two eyed eggsor two larvae at hatching, but enough RNA could be obtained froma single PFF or fry. Six samples for each developmental stage were

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T. Maehr et al. / Molecular

repared and real-time PCR quantification of gene expression wass described above. The comparative expression level of each geneas expressed as AU where one unit is equal to the average expres-

ion level of CISHb1 in eyed eggs, where the lowest expressionmong all samples studied was found.

.3.3. Cytokine induced expression of trout CISH paralogues in HKells

HK cells prepared as described previously (Wang et al., 2011a)ere adjusted to 1–1.5 × 106 leukocytes/ml in L-15 medium

upplemented with 10% foetal calf serum (FCS) and penicillin100 U/ml)/streptomycin (100 �g/ml; P/S; all medium componentsrom Gibco), and inoculated into 25-cm2 tissue culture flasks at 5 mler vessel. Freshly prepared HK cells from four fish were left alones control or stimulated with recombinant (r) IFN-� (20 ng/ml)Wang et al., 2011c), rIL-15 (200 ng/ml) (Wang et al., 2007) and rIL-1 (50 ng/ml) (Wang et al., 2011a). The stimulants were diluted in-15 medium just before addition to the cells and 0.1 ml of dilutedtimulants were added to each flask. The cells were then incubatedor 4 h, 8 h and 24 h, then processed for RNA extraction as outlinedbove. Real-time PCR analysis of gene expression was as describedbove and the expression level of each sample was first normalisedo that of EF-1� and then divided by the average expression levelf the time-matched controls.

.3.4. Modulated expression of rainbow trout CISH paralogues inK macrophages

Preparation of primary macrophages was as described pre-iously (Costa et al., 2011). Four-day old primary macrophageultures from four trout were left alone as control or were incu-ated with known stimulatory concentrations of LPS (25 �g/ml;rom Escherichia coli strain 055:B5), polyinosinic:polycytidylic acidpoly(I:C); 50 �g/ml; Sigma), rIFN-� (20 ng/ml), rIL-1� (20 ng/ml)Hong et al., 2001), phytohemagglutinin (PHA; from red kidneyean Phaesolus vulgaris; 10 �g/ml; Sigma), phorbol 12-myristate3-acetate (PMA; 0.1 �g/ml) and calcium ionophore (CI; 500 ng/ml)Wang et al., 2008). All chemicals were from Sigma. The stimulationas terminated after 4 h, 8 h and 24 h and expression quantification

f all four CISH paralogues was determined as above.

.3.5. Expression of the trout CISH paralogues after bacterialnfection

The preparation of a pathogenic strain of Yersinia ruckeriMT3072), the challenge procedure and tissue sampling wass described previously (Harun et al., 2011). Briefly, bacteria0.5 ml/fish, 1 × 106 cfu/ml in phosphate buffered saline; PBS) orBS alone (0.5 ml/fish) were injected intraperitoneally (i.p.) to tworoups of trout (∼200 g/fish). At 6 h, 24 h, 48 h and 72 h post-njection (p.i.), five fish from each group were killed and the HKnd gills sampled aseptically and immediately homogenised inNA-Stat 60TM (AMS Biotechnology). These tissues were chosen asepresentative systemic and mucosal immune sites, with i.p. injec-ion of live bacteria known to distribute the bacteria widely andlicit host responses at both of these sites. RNA isolation, cDNAynthesis, and real-time PCR analysis of gene expression were asbove. The expression level of each sample was expressed as AUfter normalisation to the expression level of EF-1� and the foldhange calculated as the average expression level of the bacteri-lly challenged samples divided by that of the PBS injected controlamples at the same time point.

.3.6. Statistical analysisReal-time quantitative PCR measurements were analysed using

he SPSS package 20.0 (SPSS Inc. Chicago, Illinois). The expressionata from adult trout tissues and juvenile trout stages consisted of

ology 62 (2014) 186–198 189

samples from six individual fish or six independent pools, respec-tively. Since the data sets had been log2 transformed to improvenormal distribution as described by Wang et al. (2011a), statis-tical evaluation of CISH paralogue expression within and acrosstissues/stages was done by comparing the means of the trans-formed data by a Paired-Sample-T-test. However, it was noted thatthe basal CISH expression levels varied greatly within differentfish/juvenile individuals. Therefore, in Figs. 4 and 5 the medianinstead of the mean of the non-log2 transformed raw data are pre-sented to better reflect the distribution of the CISH paralogues.Log2 transformation was also used for statistical analysis of theexpression data from HK cells, primary macrophages and Y. ruckeriinfected fish. A Paired-Sample-T-test was used to find significantdifferences between cell cultures and a one-way analysis of vari-ance (ANOVA) and the least significant difference (LSD) post hoctest were used for comparison of data from bacterially infectedand uninfected fish. Since the basal levels of expression were rel-atively consistent in these data sets, the means of the non-log2transformed raw data are shown in Figs. 6–8.

3. Results

3.1. Classification of trout CISH genes and sequence analysis

Two additional trout CISH cDNA sequences, CISHa2 and CISHb2,have been identified that consist of 1286 bp and 1324 bp, respec-tively (Supplementary Figs. 1 and 2). CISHa2 has a 5′-UTR of 135 bp,an open reading frame (ORF) of 678 bp encoding for 225 aa anda 3′-UTR of 473 bp. There are two potential polyadenylation sites(ATTAAA) located 16 bp and 5 bp upstream of the poly(A) tail anda further one located 18 bp downstream of the stop codon (Sup-plementary Fig. 1). CISHb2 contains a 5′-UTR of 306 bp with fiveupstream ATGs (uATG), all with in-frame stop codons before themain ORF, hinting at translational regulation. The sequence ofthe coding region is 612 bp encoding a protein of 203 aa, 22 aashorter than trout CISHb1. The 3′-UTR is 406 bp, with two poten-tial polyadenylation sites (AATAAA) 42 and 14 bp upstream of thepoly(A) tail. The CISHb2 3′-UTR also possesses a sinlge mRNA insta-bility motif (ATTTA).

Our gene organisation analysis confirmed that all four troutCISH genes can be found in a single trout since their exon/intronstructure was predicted from WGS sequences derived from ahomozygous doubled haploid individual (Berthelot et al., 2014).Each of the genes has a three exon/two intron organisation (Fig. 1),as seen in other vertebrate CISH genes (Wang et al., 2011b), with thetwo introns typically interrupting the coding sequence although inthe case of some CISHb genes intron 1 can also be within the 5′-UTR.

A multiple sequence alignment of the trout CISH sequenceswas generated with selected fish CISH molecules (Fig. 2). Thisrevealed areas of good conservation within the SH2 domain ofthe two trout CISHa aa sequences (96.6% identity) and the twoCISHb sequences (88.8%) while CISHa and CISHb isoforms werebetween 65.2 and 69.7% identical. Although the percent identityof the CISHa1 and CISHa2 SOCS box was relatively high (88.9%),remarkably, this domain was more homologous when comparingCISHa and CISHb aa sequences (83.3 to 86.1%) than when comparingthe CISHb isoforms (80.6%). The N-terminal that contains the ESS isquite divergent between CISH molecules from mammals, birds andamphibians (Wang et al., 2011b) and also appeared to be more vari-able across the four trout CISH proteins ranging from 28.8% identitybetween CISHa2 and CISHb2 to 81.4% between CISHa1 and CISHa2.

In terms of full protein homology, the trout CISHa1 and CISHa2aa sequences shared 89.8% identity and the two CISHb moleculeswere 70.8% identical (Table 2). Identities between the trout CISHaand the trout CISHb sequences were between 55.1 to 56.7%. Prior

190 T. Maehr et al. / Molecular Immunology 62 (2014) 186–198

Fig. 1. Gene organisation of the four trout CISH paralogues and comparison to human CISH and other teleost CISHa and CISHb genes. The trout CISH cDNA sequences used forprediction with the Spidey programme are given in Fig. 3. Trout genomic sequences are from WGS contigs CCAF010023507 (CISHa1), CCAF010055202 (CISHa2), CCAF01004423(CISHb1) and CCAF010010398 and CCAF010010399 (both CISHb2). Gene organisation for comparison with, human, zebrafish and tetraodon was from Wang et al. (2011b).The black and white boxes represent amino acid coding regions and untranslated regions within exons, respectively, and the black bars represent introns. The sizes (bp) ofexons are numbered in the boxes and the intron phases are indicated under the bar.

Trout-a1 1 MILC VQ----- SPRP LLSGTP -TEGH LGM RTGSVSSPHCIHSTSPQWDPTKDL RTITNNTFYLDTSGWYWGAITAGQAHETrout-a2 1 MILCVQ-----GPRPLLSGSP-TEGPVGMRTGSISSPHCLHSTPPQWDPTNDLRTIAKNFFYLDTSGWYWGAITASQAHA Salmon-a1 1 MILC VQ---- -GPRPLLSGSP -TEGPVGMRTGSISSPHCLHSTP PQWDPTNDLRTIAKNFFYLDTSGWYWGAITAGQAHA Salmon-a2 1 MILCVQ-----GPRPLLSGTP-TEGHLGMRTGSVSSPHCIHSTSPQWDPTKDL RTIANNTFYL DTSGWYWGAITAGQAHA Stickleback-a 1 MILCVP-----GPRASLPGAL STESPRGMRAGAAPSTPCLQSTP PPWDP TKDLRAIASNFCYLENSGWYWGAVTAAQA HA Fugu-a 1 MILCVP-----GPRA-LPAAPA PEAPRGMRAGTGTPTPCLQSTHPLWDPTKDLRAIASNFCYLENSGWYWGAITAAQAHA Medaka-a 1 MILCVP---- -GPTA LLPAGP SSEAR WGM RTGTAPPTPCLQST PAKWDP TKDL RAIASNFCYL ENSGWYWGAITAAQA HA Trout-b1 1 MIYCVQRALLRGPPSDMIARMMSSMQQ--NDGERGDLCCQNPTAPPCDPTEDLCCITTTFQNLQNSGWYW GSISAREARDTrout-b2 1 ----------------MIVRT MSSMQQ--DHGERGG-SCQNPAAPLYDSTEDLCSITNTFQYLQNSGWYW GSISASEARDSockeye-b 1 MIYCVQRALLRGPPSDMIARM MSSMQQ--NDGERG DSCCQN PTAPLCDP TEDLCCIT TTFQNLQNSGWYWGSIS AREARDStickleback-b 1 ----------------MVARALTIFHH----NEQGASCRPHPFPPPNDQA EDL RCIT TTFQYLQTSGWYWHSIS ASEAREMedaka-b 1 ----------------MVARTGTILCH----KGQGGSCCPH PSP PPWDPAEDLRCITTTFQYLQASGWYWGSISA GEAKEFugu-b 1 ----------------MVARA MSLFRQ----EKDKGSCCPPA PPAAWGPSEDYRSITATFRYL ETSGWYW GPTSVS DAQEconsensus 1 ..... .. . . .. ... . . .. .... .... *.. *.... .*. *****.......*..

Trout-a1 75 AL QAASEGAFLIRDSSHPLYMLTLSVRTARGPTSIRIQYSGARFLLDSSSPAR PSLLSFPDVPSMV QYYV GPGRKVQ--- Trout-a2 75 AL QAASEGAFLIRDSSHPLYMLTLSVRTARGPTSIRIQYSGAR FLLDSSSPARPSLLSFPDVPSMVQYYV GPTRKVQ--- Salmon-a1 75 AL QAASEGAFLIRDSSHPLYMLTLSVRTARGPTSIRIQYSGAR FLLDSSSPAR PSLLSFPDVPSMV QYYV GPTRKVQ--- Salmon-a2 75 AL QTASEGAFLIRDSSHPLYMLTLSVRTARGPTSIRIQYSGARFLLDSSSPARPSLLSFPDVPSMVQYYV GPGRKVQ--- Stickleback-a 76 ALQEASEGAFLVRDSSHPLYMLTLSVRTARGPTSIRIQYSGAMFLLDSSSPARPTLSSFPNVPSLVQHYMGPERQAE--- Fugu-a 75 AL QEASEGAFLVRDSSHPLYMLTLSVRTARGPTSIRIQYSSAQFLLDSSSPARPNLSSF PNVPSLVQYYM GPKNKAD--- Medaka-a 76 AL QDASEGAFLVRDSSHPLYMLTLSVRTARGPTSIRIQYSG AQFQLDSSTQAR PSLSSF PSVPSLVQHY MRAERTAE--- Trout-b1 79 ALLK VSVGTFLVRDSSHPLYMLTLSVKT ACGP TNVR IEYSGGR FRLDSSSP GPPHLLSFPD VCSLVQHYV GSGQT QQGKR Trout-b2 62 ALLKMSAGTFLVRDSSHPLYMLTLSVKTAFDPTNVR IEYIGGR FRLDSSSP GPPHLLSFPDVCSLVQHY V---------- Sockeye-b 79 ALLK VSVGTFLVRDSSHPLYMLTLSVK TVCGP TNVRIEYSGGRFRLDSSSPGPPHLLSFPDVCSLVQHYVGSGQTQQGKR Stickleback-b 61 ALFQQSEGTFLVRDSSHPQYMLALSVKTRCGPTSIRINYSRGSFWLDSISPSLPRLQSFPDVVSLIQHYKASGHSPR--- Medaka-b 61 ALLTKPEGTFLLRDSSHPQFM LALSVKTRCGPTSVR IDYNRGCFWLD SISRGAPHLKTFSDVLSLIQH YTV SGQTSQ--- Fugu-b 61 ALLRKSEGTFLMRDSNHPKHMLTLSVK TCCGPTSVRIEYSRGSFWLD SIA AGQLRTQTFPD VVSLVQHYMTLGHVPQ--- consensus 81 **. ...*.**.***.**..**.***.*...**..**.*....* ***...... ...*..*.*..*.*.. .. .

Trout-a1 152 ---QGKVEETHGGKPAQRTVQESTVLLKLKRALHKPQAFPSLQH LARLTI NCSTDCPDQLPLPRPLVRFLQDYP FQV 22 5Trout-a2 152 ---KGKVEETHGT QPAQRTVQESTVVLKLKRALHKPQAFPSLQHLTRLTINRSTGCPDQLPLPRPLVRYLQDYPFHV 22 5Salmon-a1 152 ---KGKVEETHGAQPAQRTVQESTVVLKLKRALHKPQAFPSLQHLTRLTINRSTGCPDQLPLPRPLVRYLQDYP FHV 225Salmon-a2 152 ---QGKVEETHCGKPAQRTVQESTVLLKLKRALHKPQAFPSLQHLARLTINCSTDCPDQLPLPRPLVRFLQDYPFQV 225Stickleback-a 153 ---EGKVVEDAPS KPSQQTIQETSVVLKLKRAVFKPQGLPSLQHLTR LVINRHSDCPEQLPLPRPLLRYVQDYPFKV 22 6Fugu-a 152 ---GGGLEK EGPSKSCAE PIQSTSVVLKLKRAVYKPHSLPSLQHLTRLVIN RHSDCPEQLPLPKPLLRYVQDYP FKV 22 5Medaka-a 153 ---EGRVEDEAPSKPCH NTFEETSLVLKL RSAVYKPQ GLPSLQHLTR LVIN RHTDCPDQLPLPRPLLRYMQEYP FKV 226Trout-b1 159 VESEGPSKPNAKPSPSQPSAKDNAVLLK LMRPL--PQAFPSLQHLTRLTI NCQ IDCIDQLPLPRPLVHYLQDYP FQV 233Trout-b2 132 ---EDTPQPKAKPRSPQPAVKGNAVLLKLLRPL--PQAFPSLQHLTRLTI NHHTDC PYQLPL PCPLVRYLQDYPFQV 20 3Sockeye-b 159 VESEGPSKPNAKPSPSQPSAKDNAVLLKLMRPL--PQAFPSLQHLTRLTINCQIDCIDQLPLPRPL VHYLQDYPFQV 23 3Stickleback-b 138 DPAS----DGIHPQTKPEAA KDSVVPLKLSHPLHKPEAFPSLQHVTRLTIN RHA HCPDQLPLP KTLLRYLQEYP FTI 210Medaka-b 138 DLAS----FKTKADPSQHAAKDAGVPLRLVRPLHRREGFPSLQHLARLTINKQTNCPDQLPLPKPLLDFLQNYP FII 201Fugu-b 138 SHTHSDMYQ RAKPDPSGDAARGC DVPLK LMF PLHK PGGFPSLRHLTR LAIN RHA SCPDQLPLP KLLLRYLQDYP FHI 214consensus 161 . . . . . . .. .....*.*..........***.*..**.**. ..*..*****..*....*.*** . 237

SH2 domain

SOCS bo x

SH2 domainESS

Fig. 2. Multiple alignment of the deduced aa sequences of trout CISHa and CISHb paralogues with CISH protein sequences from selected fish species. The multiple alignment wasproduced using ClustalW2 and conserved aa highlighted using BOXSHADE (v3.21). The location of the extended Src homology 2 (SH2) subdomain (ESS), SH2 domain andSOCS box are indicated by horizontal arrows above the alignment. The accession numbers for sequences used in this analysis are given in Fig. 3.

T. Maehr et al. / Molecular Immunology 62 (2014) 186–198 191

Table 2Amino acid (aa) identities (top right) and similarities (bottom left) of the CISHa and CISHb molecules from fish and CISH sequences from selected birds, amphibians andmammals. The accession numbers of the protein sequences are given in Fig. 3.

aa 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

1 Trout-a1 225 89.8 97.8 90.2 65.3 69.0 64.6 52.2 56.7 55.1 45.5 48.4 48.9 47.6 41.2 40.0 37.0 38.62 Trout-a2 225 93.3 90.2 99.1 68.4 72.6 67.7 52.9 55.5 55.6 46.8 50.2 45.8 48.9 43.8 42.6 40.1 41.93 Salmon-a1 225 97.8 93.8 90.7 66.7 70.4 65.9 52.2 55.7 54.7 44.7 47.6 48.4 47.6 41.9 40.8 37.7 39.44 Salmon-a2 225 93.8 99.1 94.2 68.4 72.6 67.7 52.4 55.5 55.1 46.4 49.8 46.7 49.3 43.8 42.6 40.1 41.55 Tetraodon-a 225 80.4 82.7 81.8 82.2 84.1 76.5 51.1 50.6 49.8 43.3 47.2 42.2 44.9 40.5 40.6 39.3 40.56 Stickleback-a 226 81.0 84.1 82.3 83.6 92.0 81.4 53.9 54.6 50.4 43.7 45.6 42.9 46.9 41.2 42.2 40.5 42.67 Medaka-a 226 77.9 79.6 79.2 79.2 85.8 89.4 50.4 51.1 47.8 41.1 45.8 42.5 48.7 40.2 41.4 40.1 40.78 Zebrafish-a 212 63.6 65.3 64.0 64.9 67.1 69.0 67.3 49.4 51.9 43.3 47.9 47.0 50.0 38.1 41.8 38.9 39.19 Trout-b1 233 68.7 67.0 67.0 67.0 64.8 67.8 64.4 59.7 70.8 52.5 53.2 54.0 50.6 43.6 46.6 42.7 43.810 Trout-b2 203 65.8 64.0 64.4 63.6 65.8 64.2 61.9 59.4 73.0 49.8 56.6 54.5 55.1 43.2 43.4 40.5 40.911 Tetraodon-b 214 56.7 57.6 55.8 57.1 59.7 58.4 55.8 55.0 63.9 61.0 58.0 54.1 43.3 37.8 36.8 38.0 38.112 Stickleback-b 210 59.1 59.6 58.2 59.1 61.3 59.3 58.8 59.0 62.7 66.7 69.3 67.1 50.0 38.8 40.2 43.0 40.913 Medaka-b 201 62.7 59.1 62.2 60.0 59.1 59.7 60.2 62.3 66.1 66.2 66.2 76.2 46.2 37.8 41.8 38.5 38.814 Zebrafish-b 204 62.2 62.2 62.2 62.7 62.2 60.2 61.1 66.5 60.9 67.2 56.7 62.4 63.8 37.0 37.8 37.0 36.815 Xenopus 257 54.1 56.0 54.5 55.6 53.3 55.3 52.1 49.8 54.9 52.1 51.8 50.2 51.4 47.9 60.4 53.7 55.316 Chicken 249 57.0 56.6 57.4 56.6 57.4 57.4 56.6 52.2 58.6 53.0 52.6 51.4 52.2 51.4 70.8 66.0 66.517 Mouse 257 51.0 53.3 50.2 52.1 54.5 53.7 53.7 50.2 54.1 48.2 51.0 52.9 48.6 47.5 70.0 74.7 90.7

50

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sporTmtCpts3Ts4

nAcitC

3

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ferent developmental stages, with its transcript level being 61 timeshigher in fry compared to eyed eggs (Fig. 5). However, CISHb2 was

18 Human 258 51.9 53.9 52.3 53.5 58.1 55.8 53.5

o the discovery of the additional trout CISH paralogues presentedere, we reported the cloning of trout CISHb1 in a recent reviewn teleost SOCS (Wang et al., 2011b). In this review two Atlanticalmon CISH sequences (which can be found in GenBank underhe acc. nos. B9EPA9 and B5XCB4) were referred to as CISHa1nd CISHa2, respectively. However, when all four trout CISH pro-ein sequences are now compared to these aa sequences it islear that trout CISHa1 has highest identity to the Atlantic salmonequence termed CISHa2 (97.8%) and vice versa, trout CISHa2 hasigh identity (99.1%) with salmon CISHa1. Their homology togetherith phylogenetic tree analysis (described later), suggests thetlantic salmon CISH sequences should be renamed and the salmonequence with the acc. no. B5XCB4 will be referred to as CISHa1 andhe sequence B9EPA9 as CISHa2 in this study.

The percent identity of trout CISHa1 to other fish CISHaequences was 52.2% to 69.0% (Table 2). Approximately the sameercent identities were seen for CISHa2. The aa sequence identitiesf CISHb1 to the other fish CISHb sequences were markedly lower,anging from 50.6% (Zebrafish CISHb) to 54.0% (Medaka CISHb).rout CISHb2 also shared lower identities with other fish CISHbolecules which were in the range of 49.8% (Tetraodon CISHb)

o 56.6% (Stickleback CISHb). The overall identity between teleostISHa and CISHb aa sequences lay between 41.1% and 56.7% whileercent identities between all fish CISH molecules and those ofetrapods ranged from 36.8% to 46.6%. When comparing the latterequences to the trout CISHa1 sequence, identities were between7.0% and 41.2% while trout CISHa2 was 40.1% to 43.8% identical.he trout CISHb1 sequence had slightly more identity to tetrapodequences (42.7% to 46.6%) while the trout CISHb2 molecule shared0.5% to 43.4% sequence identity at the aa level.

Phylogenetic tree analysis of SOCS protein sequences supportedaming the two new trout sequences as CISHa2 and CISHb2 (Fig. 3).s seen in the tree, all the CISH molecules formed an independentlade grouping with tetrapod CISH. The fish CISH clade was dividednto a CISHa and a CISHb sub-clade, in which trout CISHa1 groupedogether with trout CISHa2, and trout CISHb1 grouped with troutISHb2.

.2. Tissue distribution of the four trout CISH genes

Constitutive expression of the four trout CISH transcripts wasound in all fifteen tissues examined by real-time PCR (Fig. 4).owever, CISH abundance in some tissues was very weak, espe-ially for the expression of the CISHb paralogues. Indeed, the lowest

.4 55.4 50.8 50.0 50.0 48.1 47.7 69.4 75.6 92.2

expression level amongst the four CISH genes was found in liverfor CISHb1, which was defined as an AU of 1. All CISH isoformswere relatively highly expressed in at least one immune rele-vant tissue, however, differential expression of the four paralogueswas noted. CISHa1 expression was highest in blood, spleen andHK while CISHa2 had highest transcript levels in heart, spleen,blood and brain (Fig. 4A). The CISHb1 transcript was most abun-dant in HK, blood and heart, whilst CISHb2 was most highlyexpressed in heart and muscle (Fig. 4B). Notably, of the four troutCISH genes, CISHa1 had high constitutive expression levels noless than the other paralogues in all tissues with the exceptionof muscle. The expression level of CISHa1 was significantly higherthan that of CISHa2 in all the tissues examined, except for brain,liver and tail fins. CISHa1 also had significantly higher expres-sion compared to that of CISHb1 (ranging from 2.8 to 127 timeshigher in the skin and liver, respectively) in all tissues investi-gated except muscle. Similarly, the constitutive abundance of theCISHa1 transcript was significantly higher than that of the CISHb2paralogue in most tissues (3.7 to 21.9 times) but with no differ-ence in heart and lower expression in muscle. Lastly, a significantdifference between the CISHb paralogues was observed in thespleen, HK, heart, thymus, brain, liver, muscle and gills where theexpression level of CISHb2 was higher in the spleen, heart, thy-mus, liver, muscle and gills but significantly lower in the HK andbrain.

3.3. Expression of trout CISH paralogues during the ontogeny ofrainbow trout

The in vivo ontogenetic expression of all four CISH copies waslowest at the egg stage and gradually increased after hatching to thePFF and fry stage (Fig. 5), with significant differences seen betweenthese stages (egg vs PFF, egg vs fry) for all paralogues. In addi-tion, CISHa1 and CISHb1 expression levels were significantly higherin PFF fry vs immediate post-hatch fry, but no significant differ-ences were found between PFF fry and (3 weeks post-feeding) fry.CISHb1 showed the largest increase in expression between the dif-

the most prominent isoform across all stages. In eggs it was sig-nificantly higher compared to the other three paralogues and afterhatching it remained significantly higher than CISHa1 and CISHa2(Fig. 5).

192 T. Maehr et al. / Molecular Immunology 62 (2014) 186–198

Fig. 3. Phylogenetic tree showing the relationship of the trout CISHa and CISHb with other known SOCS family members. The aa sequences were aligned using ClustalW2and the tree constructed by the N–J method supported with 10,000 bootstrap replications using MEGA5 software. The evolutionary distances were computed using theJones–Thornton–Taylor (JTT) matrix based method. All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons(Pairwise deletion option). The name of the molecule and the common species name are followed by the accession number. The trout CISH molecules reported in this paperare shaded and without accession numbers.

T. Maehr et al. / Molecular Immunology 62 (2014) 186–198 193

0.1

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Blood Spl een HK He art Gonad Intestine Thymus Skin Sca les Br ain Li ver Mus cle Gill s Tail fins AF

CISHa1CISHa2

A.

B.

CISHb1CISHb2

1

Fig. 4. Relative expression of the four trout CISH paralogues in vivo. The relative expression level of the trout CISHa (A) and CISHb (B) genes from 15 different tissues from 6 fishw � the

l ned, wt an + in

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as determined by real-time PCR. After normalising against the expression of EF-1iver, which was the lowest level among the four CISH genes in all the tissues examihe relative expression of all four paralogues. The results are presented as the medi

.4. Modulation of trout CISH paralogues in cytokine stimulatedK leukocytes

Since the HK had relatively high expression of all four CISHranscripts, HK primary cultures were used to examine whetherheir expression could be modulated with selected recombinantytokines (Fig. 6). After stimulation with rIL-15 and rIL-21, CISHa1nd CISHa2 were the only responsive paralogues. CISHa1 showed a

mall but significant up-regulation by rIL-15 at 4 h, 8 h and 24 h andt 4 h post-stimulation with rIL-21 (Fig. 6A). A more prominent up-egulation of CISHa2 was induced by rIL-15 and rIL-21 at all time

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CISHa 1CISHa 2CISHb1CISHb2

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ig. 5. Expression of trout CISH paralogues during the ontogeny of rainbow trout. Thexpression of the trout CISHa and CISHb paralogues was examined as in Fig. 4 inour early life stages, eyed eggs (egg), immediate post-hatch fry (hatch), pre-firsteeding fry at the stage of full disappearance of the yolk sac (PFF), and fry 3 weeksollowing first feeding (fry). The median expression level of CISHb1 in eggs washe lowest level among the four genes in the developmental stages examined andas defined as 1 (indicated by the black box above the bar) to enable comparison

f the relative expression of all four paralogues. The results are presented as theedian + interquartile range from six samples.

values were expressed as arbitrary units. The median expression level of CISHb1 inas defined as 1 (indicated by the black box above the bar) to enable comparison ofterquartile range.

points (Fig. 6B), with the highest increases seen at 4 h following rIL-15 (14.5-fold) or rIL-21 (17.8-fold) stimulation. Interestingly, whilerIFN-� could not elicit any changes in CISHa1, CISHa2 or CISHb2expression, a significant 3.3-fold increase in CISHb1 was seen after4 h of stimulation (Fig. 6C).

3.5. Modulation of trout CISH paralogues in immune stimulatedHK macrophages

Several studies have shown that SOCS/CISH expression can beinduced in innate immune cells, including macrophages and den-dritic cells (Bode et al., 1999; Crespo et al., 2000). To study thisin trout a variety of immune agents, including pro-inflammatorycytokines (rIL-1�, rIFN-�), PAMPs (LPS and poly(I:C)), stimulatorsof cell signalling pathways (PMA, CI), and a lectin (PHA) wereused to stimulate primary HK macrophages and examine CISH par-alogue expression. The expression of CISHb1 and CISHb2 in HKmacrophages was very weak and was not modulated by any of thestimulants used (data not shown). In contrast, CISHa1 and CISHa2were found to be modulated to some degree (Fig. 7). CISHa1 hada higher constitutive expression level than CISHa2 (AU of 7.6 vs1.5) and was significantly up-regulated 4.5-fold by poly(I:C) and10.4-fold by CI at 24 h. Interestingly, CISHa2 was the most highlymodulated transcript in HK macrophages and was up-regulated byLPS, poly(I:C), rIL-1� and PHA (Fig. 7B). The highest induction wastriggered by poly(I:C), where a 24.8-fold increase in expression was

seen after 24 h. The pro-inflammatory cytokine IL-1� was also astrong inducer of CISHa2 and elevated expression 20.6-fold at 24 h.A small but significant down-regulation of CISHa2 was seen after8 h incubation with PMA (Fig. 7B).

194 T. Maehr et al. / Molecular Immunology 62 (2014) 186–198

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Fig. 6. Modulation of the expression of trout CISHa1 (A), CISHa2 (B), CISHb1 (C) and CISHb2 (D) in head kidney (HK) leukocytes by cytokine stimulation. Freshly prepared HKl ith rIFF leved nd us

3r

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eukocyte cultures from four different fish were left alone as control or incubated wold changes were calculated by dividing the average of the normalised expressionifferences between the stimulated samples and control at the same time point, fou

.6. Differential modulation of trout CISH paralogues by Y.uckeri infection

To assess whether CISH transcription could be modulated by

acterial infection, rainbow trout were injected i.p. with PBS (con-rol) or Y. ruckeri, the causative agent of yersiniosis or enteric red

outh disease (ERM). CISH gene expression was examined in the

*

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ig. 7. Differential modulation of the expression of CISHa1 (A) and CISHa2 (B) in headidney (HK) macrophages. Four-day old HK macrophage cultures from four differ-nt trout were stimulated with LPS, poly(I:C), rIFN-�, rIL-1�, PHA, PMA and CI for

h, 8 h and 24 h and gene expression analysed as in Fig. 4. Data are presented aseans + SEM. Significant (p < 0.05) differences between the stimulated samples and

ontrol at the same time point, found using a paired-sample T-test, are shown withn asterisk.

N-�, rIL-15 or rIL-21 for 4 h, 8 h and 24 h and gene expression examined as in Fig. 4.ls by that of the controls. Data are presented as means + SEM. Significant (p < 0.05)ing a paired-sample T-test, are shown with an asterisk.

HK and gills 6 h, 24 h, 48 h and 72 h post-infection (p.i.). In HK sam-ples from the infected group the expression level of CISHa1, CISHa2and CISHb2 was significantly up-regulated at 24 h (Fig. 8A), withCISHa2 showing the largest (15-fold) increase. CISHa2 was also sig-nificantly up-regulated (4-fold) at 48 h p.i., whilst CISHa1 showedan additional early elevation (8.5-fold) at 6 h p.i. compared to thePBS control group.

CISH gene expression in gills was markedly different from thatin HK during bacterial infection. No modulation of CISH expressionby Y. ruckeri infection was seen in gills at 6 h and 24 h. However,CISHa2, CISHb1 and CISHb2 were all significantly up-regulated(approximately 3-fold) at 48 h (Fig. 8B). The expression level ofCISHa2 and CISHb2 remained elevated in infected fish at 72 h p.i.but CISHb1 had returned to control levels. Lastly, the expression ofCISHa1 was not modulated in the gills at any time point.

4. Discussion

The complexity of the teleost fish cytokine network is not onlyillustrated by the existence of multiple gene copies of cytokines andtheir receptors but also by multiple paralogues of negative regula-tors of cytokine signalling such as the SOCS genes. We have recentlymade an attempt to clarify the relationships between the differ-ent SOCS subgroups in fish. Our phylogeny and synteny analyseshave confirmed that the proposed additional fish SOCS-8 isoformappears to be a paralogue of teleost CISH that we have termed CISHb(Wang et al., 2011b). In this report, through the identification andcloning of two further CISH gene copies in the rainbow trout, CISHa2and CISHb2, we reveal for the first time that four paralogues of aSOCS subfamily can be present in teleost fish.

The novel trout CISH molecules both contain the SOCS proteinsignature domains, which were well conserved when aligned withother fish CISHa and CISHb sequences (Fig. 2). The novel CISHa2and CISHb2 are 89.8% and 70.8% identical to their trout CISHa1 and

CISHb1 paralogues, respectively (Table 2). A slightly higher homol-ogy between the fish CISHa sequences was seen relative to the fishCISHb molecules and this may reflect the more variable N-terminalaa length noted in the latter. That they are indeed homologues of

T. Maehr et al. / Molecular Immunology 62 (2014) 186–198 195

CISHa1

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Time post-infection

6 h 24 h 48 h 72 h 6 h 24 h 48 h 72 h 6 h 24 h 48 h 72 h6 h 24 h 48 h 72 h

Fig. 8. Modulation of the trout CISH transcripts in head kidney (HK) (A) and gills (B) after Yersinia ruckeri infection. Rainbow trout were injected intraperitoneally with Y. ruckeri( 6 h, 24a d sams

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1 × 106 cfu/0.5 ml/fish) or with PBS (0.5 ml/fish). HK and gill tissues were collected

s means + SEM of five fish. Significant (p < 0.05) differences between the stimulateignificant ANOVA, are shown with an asterisk.

ertebrate CISH molecules was further confirmed by an N–J phy-ogenetic tree, in which CISHa2 and CISHb2 clustered within theespective subgroups that represent piscine CISHa and CISHb, withigh bootstrap support (Fig. 3). These two fish subgroups may haverisen from the teleost 3R WGD event (Kassahn et al., 2009), since

degree of gene synteny is apparent between these two loci inebrafish and medaka vs the mammalian CISH locus (Wang et al.,011b). Each of the trout CISHa molecules clustered together withespective salmon CISHa molecules, while trout CISHb1 clusteredith a sockeye salmon CISHb, suggesting that these additional par-

logues likely arose from the more recent salmonid specific 4RGD. Hence, it appears that besides many cytokine and cytokine

eceptor genes, a formidable number of SOCS genes have beenetained after the teleost 3R and salmonid 4R WGD.

Through divergence in regulatory control or protein activity,owever, duplicated genes may acquire different functions. Thus,hilst all four trout CISH genes consist of three exons and two

ntrons (Wang et al., 2011b) (Fig. 1), similar to other vertebrateomologues, the shortened N-terminals of fish CISHb moleculesay be an indication that functional and regulatory diversification

f the trout CISH isoforms has occurred. In addition, whereas noATGs were found in the 5′-UTR of CISHa2, all the other trout CISHaralogues contain uATGs in their 5′-UTRs and our sequence align-ent and homology analysis clearly revealed some differences in

he primary structure of fish CISHa and CISHb SH2 domains andOCS boxes (Fig. 2). Although aa substitutions in these regionsight have facilitated trout CISH interference with JAK/STAT path-ays in immunity and development, it remains to be elucidated

t the protein level whether or to what extent these changes mayave contributed to the potential subfunctionalisation indicated byur gene expression studies.

Transcription of the four trout CISHs was detectable in all tissuesnvestigated, although their constitutive expression level was rel-tively low (Fig. 4). However, SOCS genes are known to be rapidlynduced by cytokine stimulation and immunological and physio-ogical stresses (Johnston, 2004). While CISHa1 and CISHa2 were

he most prominent paralogues expressed constitutively, all copieshowed relatively high expression levels in spleen, HK, blood andeart (Fig. 4A and B) and may hint at immunological but also phys-

ological roles for the CISH paralogues in rainbow trout.

h, 48 h and 72 h later and gene expression analysed as in Fig. 4. Data are presentedples and control at the same time point, found using an LSD post hoc test after a

Intriguingly, CISH transcription in different developmentalstages of rainbow trout followed an almost reverse picture to theconstitutive levels. CISHb2 contributed most to CISH mRNA abun-dance during ontogeny (Fig. 5) and it is noticeable that, whilst inmost adult tissues the CISHb paralogues were generally less abun-dant, CISHb2 was the most prominent paralogue in muscle. Thus,the data obtained from developmental and adult trout tissues canbe reconciled with the participation of CISH in the regulation of GHsignalling. The development and somatic growth of newly hatchedlarvae into juvenile fish is accomplished by a series of molecular,biochemical and morphological changes with some of these pro-cesses, such as skeletal muscle growth, under the control of GHand IGF-I (Ayson et al., 1994; Duguay et al., 1996; Funkensteinet al., 1997; Gomez-Requeni et al., 2012; Herrero-Turrion et al.,2003; Richardson et al., 1995). CISH and SOCS-1 to -3 are known tohave important functions in the regulation of GH signalling (Crokeret al., 2008), and a recent study by Figueiredo et al. (2012) suggeststhat the same is true in fish, as SOCS-1 and SOCS-3 expression aresignificantly increased in the muscle tissue of GH receptor (GHR)-transgenic zebrafish.

CISH is a specific regulator of cytokines such as IL-2 that induceSTAT-5 activation. In humans, CISH is the gene most consistentlyup-regulated by IL-2 stimulation (Jin et al., 2006) and appears tobe critical for T cell survival and proliferation in response to infec-tions (Kovanen and Leonard, 2004). Mammalian IL-15 and IL-21are related to IL-2 and are pleiotropic cytokines that have a broadspectrum of biological activities on different types of immune cells,and non-immune cells (Leonard et al., 2008; Shurin, 2003). Thus,it was expected that trout rIL-15 and rIL-21 would affect at leastsome cell types in primary HK cultures that contain a mixed popula-tion of leukocytes, with the potential to modulate CISH expression.Indeed, both cytokines were inducers of CISHa1 and strong induc-ers of CISHa2 expression (Fig. 6). The two CISHa paralogous geneswere concomitantly up-regulated by rIL-15 at all time points, albeitin a more modest way regarding CISHa1 expression (Fig. 6) and rIL-21 was a potent inducer of CISHa2 mRNA transcription across all

time points (Fig. 6B). Clearly, these results suggest that the troutCISHa paralogues are involved in the regulation of IL-15 and, partic-ularly CISHa2, in IL-21 signalling. Rainbow trout rIL-21 has alreadybeen demonstrated to induce SOCS-1 and SOCS-3 expression in HK

1 Immu

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96 T. Maehr et al. / Molecular

ells (Wang et al., 2011a) and it is plausible that cross-modulationan occur amongst these family members and between the CISHsoforms. For example, prolactin signalling is attenuated by SOCS-

induction but can be restored by SOCS-2 co-transfection (Pezett al., 1999). In addition, SOCS-2 may compete for Elongin B/C bind-ng with other SOCS proteins, resulting in destabilisation of theatter, as was demonstrated using competition experiments withISH and SOCS-2 (Piessevaux et al., 2008).

Besides the potential for regulatory interference and overlap-ing function of the trout CISH molecules it is also possible that theyave undergone subfunctionalisation. For example, in agreementith findings regarding trout CISHa1 gene modulation in rIFN-�

timulated RTG-2 and RTS-11 cells (Wang et al., 2010a), neitherISHa1 nor CISHa2 were induced by treatment with this pro-

nflammatory cytokine in HK leukocytes in this study (Fig. 6A and). However, the combination of the significant, albeit small, up-egulation of CISHb1 in response to rIFN-� and the refractory naturef the CISHb paralogues to rIL-15 and rIL-21 (Fig. 6C and D), hintst the acquisition of different roles for the four rainbow trout CISHenes. In addition to this, the induction of SOCS gene expressionay not only be cytokine and SOCS member dependent but also

ontingent on the cell type. HK macrophages were chosen to inves-igate CISH modulation further, using a battery of common immunetimulants. While the CISHb genes were very lowly expressed andot modulated in this cell type, HK macrophages strongly expressedISHa2 after 24 h exposure to the bacterial and viral mimics LPSnd poly(I:C), the pro-inflammatory cytokine rIL-1�, and, to aesser extent, by the lectin PHA (Fig. 7A). A slight but significantown-regulation after PMA treatment at 8 h was also observedor CISHa2 (Fig. 7B). Poly(I:C), the strongest inducer of CISHa2 inK cells, was also able to induce CISHa1 in HK macrophages but

o a much lower degree. However, none of the pro-inflammatoryytokines or PHA modulated CISHa1 expression (Fig. 7A), possi-ly because the CISHa1 paralogue may function in response tother physiological processes, as suggested by the ∼10-fold up-egulation after 24 h of treatment with the pathway activator CIhat was not seen for CISHa2. The conclusion that CISHa2 may be the

ore important CISH paralogue for control of immune responses ofK macrophages is further supported by our results showing thatAMPs and PMA did not have an effect on CISHa1 transcription inhe rainbow trout macrophage/monocyte cell line RTS-11 and thebroblastic cell line RTG-2 (Wang et al., 2010a).

The different members of the trout CISH subfamily also followedifferent kinetics during infection with Y. ruckeri, a Gram-negativeacterium that is the aetiological agent of ERM. The bacteriumolonises vascularised tissues such as HK and gills. When compar-ng trout CISH mRNAs in such tissues (Fig. 8), in HK samples fromnfected fish a rapid (8.5-fold) onset of CISHa1 gene expression at

h p.i. was followed by a 15- and a 5-fold induction of CISHa2nd CISHb2, respectively, at 24 h (Fig. 8A) with no modulation ofISHb1 expression in this tissue. In the gills, CISHa1 expression wasot changed and transcription of the other paralogues was induced

ater than in the HK (48 h pi), and in the case of CISHa2 and CISHb2p-regulation was still significant at 72 h p.i. (Fig. 8B).

We have previously demonstrated that a number of pro-nflammatory genes are highly up-regulated in key tissues relevanto this disease after Y. ruckeri infection of naïve fish using the chal-enge model reported here (Harun et al., 2011). For example, in theK, TNF-�1–3 were highly induced at 48 h post-infection (Hongt al., 2013) and in spleen the IL-12 subunit chains p40b and p40cere up-regulated after 24 h and p35 was progressively and highly

xpressed 24 h to 72 h after the onset of infection (Wang et al.,

014). Furthermore, the study by Harun et al. (2011) reported annbalanced inflammatory response with overexpression of pro-

nflammatory cytokines in the spleen of naïve fish compared toaccinated fish, that may contribute to the development of this

nology 62 (2014) 186–198

disease. Such immune responses may be the targets of the inducedCISHs during ERM disease. However, whilst pathogen recognitionvia TLRs can stimulate expression of CISH/SOCS proteins in hostcells (Mansell et al., 2006; Narayana and Balaji, 2008), a strategyof certain viruses, bacteria and parasites is to evade host immuneresponses by manipulating cytokine receptor signalling (Baetzet al., 2007). Thus, some microbial pathogens can utilise the host’sSOCS system to dysregulate cytokine signalling and, speculatively,this may further account for CISH paralogue activation at the heightof Y. ruckeri infection observed in this study (Fig. 8B). Although asuccessful vaccine exists against ERM, the precise mechanisms bywhich it elicits protection are not fully understood. Therefore itmight be worthwhile to investigate in future experiments whetherthe differential modulation of the trout CISH paralogues is alteredas a consequence of vaccination.

In conclusion, the cloning of rainbow trout CISHa2 and CISHb2in addition to CISHa1 and CISHb1 demonstrate that a full comple-ment of this SOCS subfamily exists and has been retained since theautotetraploidisation event in the common ancestor of salmonids.Their differential in vivo gene expression in adult tissues and juve-nile stages of rainbow trout, as well as the differences in in vitromodulation by cytokines, PAMPs and pathway activators, hint atpossible subfunctionalisation and regulatory interference of thedifferent paralogues in immune and developmental control butalso potentially in other biological processes. Differential modu-lation of the trout CISH mRNAs during microbial infection, as seenwith Y. ruckeri, may also foster pathogen evasion strategies. Thisstudy emphasises the importance of paralogue gene discovery inthe complex cytokine system of teleost fish, and highlights thatfish specific gene duplication will hamper studies aimed at deter-mining the function of genes with homology to molecules in othervertebrate groups.

The nucleotide sequence data will appear in theEMBL/DDBJ/GenBank nucleotide sequence database under thefollowing accession numbers: HG003693 (CISHa2) and HG003694(CISHb2).

Acknowledgements

TW received funding from the MASTS pooling initiative (TheMarine Alliance for Science and Technology for Scotland). This workwas supported financially by Contract No. 007103 (IMAQUANIM-Improved immunity of aquacultured animals) and FP7-222719(LIFECYCLE-Building a biological knowledge-base on fish life-cycles for competitive, sustainable European aquaculture) fromthe European Commission. We thank Pierre Boudinot (Virologieet Immunologie Moléculaires, Institut National de la RechercheAgronomique, Jouy-en-Josas, France) and Sam Martin (Scottish FishImmunology Research Centre, University of Aberdeen) for provid-ing samples of rainbow trout developmental stages.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.molimm.2014.06.021.

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