Inflammatory responses in primary muscle cell cultures in Atlantic salmon (Salmo salar)

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1 Pooley et l. BMC Genomics 213, 14:747 ESEACH ATICLE Open Access Inflmmtory responses in primry muscle cell cultures in Atlntic slmon (Slmo slr) Nichols J Pooley 1, Luc Tcchi 1,2, Christopher J Secomes 1 nd Smuel AM Mrtin 1 Astrct Bckground: The reltionship etween fish helth nd muscle growth is criticl for continued expnsion of the quculture industry. The effect of immune stimultion on the expression of genes relted to the energy lnce of fish is poorly understood. In mmmls immune stimultion results in mjor trnscriptionl chnges in muscle, potentilly to llow relloction of mino cids for use in the immune response nd energy homeostsis. The im of this study ws to investigte the effects of immune stimultion on fish muscle gene expression. esults: Atlntic slmon (Slmo slr) primry muscle cell cultures were stimulted with recominnt (r)il-1β, mjor proinflmmtory cytokine, for 24 h in order to simulte n cute immune response. The trnscriptomic response ws determined y NA hyridiztion to 4 44 K Agilent Atlntic slmon microrry pltform. The ril-1β stimultion induced the expression of genes relted to oth the innte nd dptive immune systems. In ddition there were highly significnt chnges in the expression of genes relted to regultion of the cell cycle, growth/structurl proteins, proteolysis nd lipid metolism. Of interest were numer of IGF inding proteins tht were differentilly expressed, which my demonstrte cross tlk etween the growth nd immune systems. Conclusion: We show ril-1β modultes the expression of not only immune relted genes, ut lso tht of genes involved in processes relted to growth nd metolism. Co-stimultion of muscle cells with oth rigf-i nd ril-1β demonstrtes cross tlk etween these pthwys providing potentil venues for further reserch. This study highlights the potentil negtive effects of inflmmtion on muscle protein deposition nd growth in fish nd extends our understnding of energy lloction in ectothermic nimls. Keywords: Trnscriptomics, Atlntic slmon (Slmo slr), Muscle cell culture, Inflmmtion, Ctolism, Cell cycle, IGF inding proteins Bckground Muscle growth involves tightly controlled lnce etween protein synthesis nd degrdtion [1]. Protein synthesis is driven y the growth hormone (GH)/Insulin like growth fctor (IGF)/mmmlin trget of rpmycin (mto) pthwy [2-5], wheres protein degrdtion occurs vi numer of pthwys including uiquitin protesome [6-8], lysosoml [9], poptotic [1] nd the clcium dependnt clpins [11]. These processes nd the pthwys underlying their regultion hve een exmined in Atlntic slmon (Slmo slr) [12], rinow trout (Oncorhynchus mykiss) [13-16] nd other fish [17,18]. ThenoliceffectsoftheGH/IGFsystemhvelso Correspondence: sm.mrtin@dn.c.uk 1 Institute of Biologicl nd Environmentl Sciences, University of Aerdeen, Tillydrone Avenue, Aerdeen AB24 2TZ, UK Full list of uthor informtion is ville t the end of the rticle een studied in ectothermic nimls including Atlntic slmon [12,19,2], rinow trout [21,22] nd other teleosts [23]. The GH/IGF system hs een seen to ctivte the mto pthwy thus directing protein synthesis, nd is highly conserved in teleosts [2-4]. In mmmls the key signls involved in stimulting nolic ctivity re free mino cids, GH nd IGF [24], wheres ctolic signls include nutrient depletion, hormones such s cortisol nd trnscription fctors such s forkhed ox O (FOXOs) [25]. The ctions of mny of these key signls hve een seen to e conserved in slmonid fish [2,12,22]. Despite eing initited y different signls, ctolism nd nolism shre mny spects of downstrem signlling mchinery, providing the possiility of intrcellulr cross tlk etween these two processes [26]. In mmmls undergoing cute inflmmtory responses, muscle tissue goes into immedite ctolic stte [27,28] 213 Pooley et l.; licensee BioMed Centrl Ltd. This is n open ccess rticle distriuted under the terms of the Cretive Commons Attriution License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited.

2 Pooley et l. BMC Genomics 213, 14:747 Pge 2 of 2 where muscle fires re roken down relesing free mino cids, likely to e used for liver protein synthesis of cute phse serum proteins. As skeletl muscle is the principl ody store of proteins, this tissue is the min trget for ctolism nd relese of free mino cids [29]. In mmmls the inflmmtory response leds to loss of skeletl muscle mss in oth cute nd chronic inflmmtory situtions [3]. The current consensus in higher vertertes is tht this increse in muscle trophy cn e medited y proinflmmtory cytokines such s interleukin-1β (IL-1β) [26,31], IL-6 [27,32,33] nd tumor necrosis fctor-α (TNF-α) [26,27,32]. Severl different processes hve een identified y which proinflmmtory cytokines cn negtively ffect muscle mss. IL-1β nd TNFα receptors, on the surfce of the cells, signl vi conserved signl trnsduction pthwys nd lter gene expression, which in muscle tissue normlly induces genes involved in protein degrdtion resulting in the relese of free mino cids [28,31,34-36]. In prllel this cytokine signlling competes with nd decreses the effects of IGF-I signlling, specificlly during downstrem signl trnsduction, hence reducing the nolic hormone effect. Such intrcellulr receptor crosstlk etween cytokines nd nolic hormones cn led to stte of endocrine resistnce wherey no increse in the mount of lignd present will increse the hormonl effects [26,37,38]. This cytokine induced hormone resistnce cn result in condition known s cchexi, one spect of which is chronic increse in proinflmmtory cytokines such s TNFα nd IL-1β [39,4]. The effects of cchexi re loss of ody mss, especilly skeletl muscle protein, nd it is thought tht the ility of cytokines to cuse hormone resistnce is one of the primry meditors of cchexi. This condition differs from simple weight loss since the loss of ody mss will continue despite feeding [26,39]. Trnscriptionl responses to vrious triggers of protein ctolism hve een exmined in slmonid fish, including strvtion [41], strvtion nd refeeding [42], or following extensive norexic migrtions [18] nd vitellogenesis [14,43]. However to dte only limited numer of investigtions hve ddressed the effects of infection or immune stimultion on muscle growth in fish [44,45]. Previously cchexi model in rinow trout ws developed y chronic stimultion with lipopolyscchrides (LPS) [44], mimicking sepsis nd chronic ckground infection. In these fish, muscle protein content ws decresed, ut levels of MyoD nd myosin were unffected indicting tht while muscle ccretion ws ltered, the mechnisms my e different to those known in mmmls. In generl the response ws much less drmtic thn is oserved in mmmls, proly reflecting the different control of mino cid relloction in ectothermic fish. Proinflmmtory cytokines, which include IL-1β, re the primry meditors of the innte immune system [46] nd show rpid response t the trnscriptionl level following recognition of pthogens including cteril nd virl products [47]. IL-1β is secreted s the mture form following clevge of the precursor molecule y interleukin 1 converting enzyme (ICE). The mture solule protein inds to the IL-1 receptor 1 (IL-11) receptor which then recruits the IL-1 receptor ccessory protein (IL-1AcP) nd initites the signl cscde [47,48]. The signlling cscde ctivtes pthwys tht positively regulte the ctivity of trnscription fctor nucler fctor-κβ (NFκB) nd the mitogen ctivted protein kinses p38 (MAPK p38) nd c-jun N-terminl kinses (JNK) [26,48,49]. It is through the ctivtion of these pthwys tht IL-1β is thought to negtively ffect nolism while stimulting ctolism [26,34,37]. Whilst there is some controversy s to how IL-1β is processed in fish [5-52], nevertheless functionl mture peptide hs een produced in severl species [53] nd the receptor genes hve een cloned [54,55]. This pper investigtes the effects of cute proinflmmtory stimultion on the trnscriptome of Atlntic slmon primry myocyte cells. We hypothesise tht the inflmmtory stimuli will cuse significnt chnges in the expression of genes relted to immune function, protein metolism nd other cellulr processes. Further to this, we hypothesize tht co-incution of cell cultures with IGF-I s well s ril-1β will led to n ttenution of the metolic ctions of inflmmtion. esults Cell culture nd stimultion Primry muscle cell cultures were ssessed for differentition nd purity y light microscopy t 4 nd 1 mgnifiction (Dt not shown). Nine grms of white skeletl muscle pooled from six fish provided sufficient cells to rech confluence when evenly split etween two 6 well pltes. Prior to performing the microrry nlysis, confirmtion tht the cells responded to ril-1β ws crried out y rel time PC using IL-1β itself s mrker gene since it is known to increse in expression in response to ril-1β stimultion. IL-1β expression ws significntly incresed (541 fold) in the stimulted smples compred to the control smples. Microrry nlysis Following filtering nd qulity control proes were retined for sttisticl nlysis. Of these 7649 were significntly ltered in expression t P <.5 following correction for multiple tests. We further filtered this set of genes y retining those with fold chnge of >2 leving differentilly regulted set of 254 genes for nlysis (Tle 1, full list of genes Additionl file 1: Tle S1). Within the gene set 129 fetures were incresed nd 1295 fetures decresed in expression.the gene with the highest

3 Pooley et l. BMC Genomics 213, 14:747 Pge 3 of 2 Tle 1 Microrry nlysis showing the numers of trnscripts found to e differentilly expressed following stimultion of primry muscle cells y ril-1β compred to unstimulted cells with vrious P vlue (corrected y Benjmini Hocherg FD) nd Fold chnge (FC) cutoffs P ll P <.5 P <.2 P <.1 P <.5 P <.1 FC ll FC > FC > FC > FC > Expected y chnce up-regultion is the cytokine TNFα2 with 216 fold increse, whilst quporin 1 ws the most decresed in expression with 125 fold reduction in expression. Confirmtion of microrry expression ws conducted using seven key genes nlysed with reltime PC (Additionl file 2: Figure S1) where highly significnt correltion (r 2 =.8763 & P <.1) etween qpc nd microrry dt ws found. In order to etter understnd the chnges in whole cell trnscriptomic output, gene ontology nlysis ws used to indicte the iologicl processes tht were modulted y the IL-1β stimultion. From the 254 fetures retined for nlysis, 2196 (88%) were nnotted to functionl protein nd 1945 (78%) were ssigned t lest one gene ontology (GO) identifier for iologicl process, enling further ssessment of iologicl function. These proportions reflect the nnottion of ll fetures on the microrry slide. Sttisticl nlysis for enrichment for iologicl processes resulted in 1195 iologicl process GO terms eing identified. The nture of GO nlysis mens tht mny of these re overlpping nd only the non-redundnt mjor groupings re presented (Figure 1). Oservtion of oth the GO nlysis nd mnul ssignment identifiction of functions ws used to ssign genes to functionl groups. The differentilly expressed genes could e defined s elonging to numer of distinct functionl clsses, especilly immune response, proteolysis, growth regultion nd structurl proteins, cell cycle nd lipid metolism. The directionl expression chnges indicted how these processes were eing ffected, with generl increse in the expression of immune relted nd protein metolism genes, wheres growth, structurl proteins nd cell cycle showed negtive trend, with mjority of genes eing down regulted in expression. A complex response ws found for genes encoding lipid metolism proteins, indicting mjor trnscriptionl chnges relting to lipid moilistion. Immune response genes There ws cler increse in genes relted to immune function (Tle 2) most notly in the high increse of expression of mnas encoding proinflmmtory cytokines such s IL-1β nd TNFα (1/2)swellschemokinessuch s IL-8. Trnscription fctors involved in IL-1β signlling were lso incresed in expression with suunits of NFκB nd its inhiitor, MAP kinse-intercting serine/threonine kinse 2, MAPK ctivted jun-b nd CCAAT/enhncer inding protein ll eing up regulted. Components of the IL-1β receptor mchinery were lso incresed including IL-1 receptor ccessory protein, IL-1 receptor kinse nd n IL-1 receptor ntgonist protein mna (Tle 2). Other innte immune relted genes were lso incresed including complement components, C-type lectins nd the ntimicroil proteins hepcidin nd ferritin. Both these ltter two genes hve roles in iron inding. Severl negtive regultors of inflmmtion were lso found to e incresed including two suppressors of cytokine signlling (SOCS) genes, SOCS 1 nd 3, the nti-inflmmtory cytokine IL-1, nd s mentioned erlier n IL-1 ntgonist (nil-1 F). Proteolysis Genes relted to protein metolism were modulted y the IL-1β stimultion including those involved in oth synthesis nd degrdtion (Tle 3). The lrgest group of protein metolism genes found to e incresed in expression were those relted to proteolysis, specificlly the uiquitin protesome pthwy (UBP). Severl E3 uiquitin ligses, uiquitin like proteins nd four 2S protesome suunits ll incresed in expression. Other genes encoding proteolytic proteins found to e incresed in expression included collgense 3 nd cytosolic dipeptidse. A numer of proteses were decresed in expression including suunit of clpin 1, serine protese htr1 nd 35, cysttin B nd uiquitin-conjugting enzyme E2 T. Growth regultion nd structurl proteins An interesting group of genes tht cn e regrded s controllers of nolic signlling were lso modulted. Most notle were the IGF inding proteins (IGFBPs), where IGFBP-6 ws found incresed in expression following the inflmmtory stimulus wheres IGFBPs -4, 5 nd rp1 decresed in expression (Tle 4). Genes controlling muscle cell differentition were lso chnged

4 Pooley et l. BMC Genomics 213, 14:747 Pge 4 of 2 immune system process immune response DNA metolic process cell cycle ctolic process proteolysis regultion of poptotic process iologicl dhesion cellulr locliztion lipid metolic process cell prolifertion growth nion trnsport cell division regultion of growth Enrichment Figure 1 Br chrt showing the 15 Gene ontologies found to e most highly sttisticlly enriched in response to ril-1β stimultion of muscle cells in vitro. Gene ontology enrichment crried out using GOEAST, GOslimming of the susequent list performed with EVIGO. in expression including the trnscriptionl repressor yin-yng 1 (YY1) which showed n up regultion nd myogenic regultory fctor 5 (MyF5) which ws down regulted (Tle 4). Structurl protein encoding mnas showed mrked tendency to e down regulted, s seen with the collgens nd the myosins, β-ctin, nd troponin (Tle 4). Cell cycle nd DNA metolism The expression of genes regulting the cell cycle ws clerly ltered, with the mjority of them eing reduced in expression (Tle 5). Five cyclins (A2, B1/2, E1/2), two cyclin dependent kinses, nd severl cell division cycle proteins were ll reduced in expression. However, two cyclins (D1 nd G2) were incresed in expression. DNA metolism genes were lso generlly decresed in expression, including severl minichromosome mintennce complex components, DNA repliction complex nd DNA repliction licensing fctor mcm2. Lipid nd sterol metolism Finlly, stimultion with ril-1β cused chnges in the expression of genes involved in lipid metolism (Tle 6). These included the increse in expression of severl cholesterol trnsport proteins such s polipoprotein (Apo) L3 nd lipoprotein lipse. However there ws lso down regultion in other similr genes such s Apo A1 inding protein nd Apo B nd down regultion of proteins involved in sterol synthesis. Temporl response nd interction of IGF nd IL-1β To ssess the effect of time of ril-1β stimultion on primry myocytes on gene expression, ril-1β stimultion ws performed t 6, 24 nd 48 h nd four key mrker genes from the microrry nlysis (IL-1β, TNFα, MyF5 nd IGFBP-6) were exmined y rel time PC (Figure 2). IL-1β ws highly incresed in expression t ll-time points ut it ws t 48 h tht the highest increse in expression ws found. TNFα lso showed the gretest fold increse t 48 h however this ws more due to reduction in the control expression seen t 48 h, thn n increse in the stimulted cells. MyF5 ws consistently down regulted t ll time points with no increse in effect seen fter 6 h. Finlly IGFBP-6 ws incresed t ll 3 times, ut with mximum fold increse t 24 h nd 48 h. To ssess the interction etween ril-1β nd rigf-i primry myocyte cultures were stimulted with ril-1β (25 ng/ml), rigf-i (1nM), ril-1β (25 ng/ml) + rigf-i (1nM) or mintined s control. These stimultions were crried out for oth 6 h nd 24 h to determine if ril-1β interfered with erly effects tht IGF-I my hve on the cell cultures. The genes nlysed were chosen to represent the immune response (IL-1β, TNFα nd hepcidin) nd protein metolism/growth (trogin-1, MyF5, IGFBPs-4, 5 & 6). At 6 h co-stimultion of cells there ws n up regultion of IL-1β nd TNFα expression in response to ril-1β stimultion, nd this ws not significntly ltered y co-incution with ril-1 β + rigf-i (Figure 3). Hepcidin ws lso found to e up regulted in response to ril-1β (5.8 fold), with co-incution with ril-1β +rigf-i reducing the mgnitude of this increse ~3% (Figure 3). egrding the expression of the IGFBPs, there ws no effect of ny tretment on the expression of IGFBP-5. IGFBP-6 ws up regulted in response to ril-1β (13. fold) nd this effect ws not ltered y co-incution with

5 Pooley et l. BMC Genomics 213, 14:747 Pge 5 of 2 Tle 2 Differentil expression of genes relted to the immune response Gene ID 1 Annottion 2 Men fold chnge± SE 3 Identity 4 Ss#S AY ± 58.8 TNFα 2 Omy#gi NM_ ± 79.1 IL-1B Ss#STI83_4 AY ± 17.9 TNFα 1 Omy#gi AJ ± 15.4 IL-8 Ss#S AM ± 5.1 Complement c3 Ss#STI4816 BT ± 13.1 Hepcidin Omy#S EF ± 1.1 Prostglndin G/H synthse 2 Ss#STI8688 TC ± 4.9 Ferritin Ss#S AY ±.2 C type lectin receptor A Ss#STI35259 NM_ ±.9 Complement protein component c7-1 Ss#STI23928 NM_ ± 1.8 Complement fctor H precursor Ss#S _S NM_ ± 2.5 CD4-like protein Ss#STI84_4 DW ±.3 NF-kpp- inhiitor lph Ss#S AJ ±.3 IL-1 receptor ccessory protein Ss#STI14647 TC ±. MAP kinse-intercting serine/threonine kinse 2 Ss#S DW ±.2 Suppressor of cytokine signling 1 Ss#S EG ±.3 NF-kpp-B p1 suunit Ss#S EG ±.2 Complement c1q-like protein 4 Ss#S DY ±.2 IL-1 receptor et chin precursor Omy#gi NM_ ±.2 IL-1 receptor ntgonist Ss#KSS392 NM_ ±.3 IL-1 receptor-ssocited kinse 4 Ss#CB5163 CB ±.3 NF-kpp-B 1 p15 suunit Ss#KSS366 BT ±.3 NF-kpp-B inhiitor epsilon Ss#STI8793 TC ±.1 Suppressor of cytokine signling 3 Ss#STI87_4 DW ±.2 IL-1 Ss#STI12117 TC ±.1 Trnscription fctor jun-b Omy#S BX ±.1 Interleukin-6 receptor suunit lph precursor Ss#DW691 DW ±.2 MHC clss i ntigen Ss#TC1654 TC ±.1 TNF receptor-ssocited fctor 6 Ss#STI8822 TC ±.1 IL-15 Ss#S _S BT ±.2 Complement C4-1 Ss#S EG ±.8 Complement component 6 precursor Ss#S EG ± 8.3 Complement fctor D precursor List of selected mnas ssocited with the immune response found to e incresed or decresed in expression in response to ril-1β stimultion. Genes were ssigned to the tle sed upon oth their GO identifier nd previous knowledge of their functions. Genes with gretest fold differences in expression re presented, the genes tht re lower in expression re denoted y (-) vlue. The genes shown were significnt t p <.5 following t- tests with Benjmini-Hocherg FD nd greter thn 2-fold chnge. 1 Indictes the unique code for the feture on the microrry, 2 Accession numer of the cdna sequence, ccession numers eginning with TC re for oligos from the TIG Atlntic Slmon Gene Index. 3 Fold chnge, in the cse of oligos tht fetured multiple times in the gene list the one with the highest fold chnge is reported. 4 Identity of the proe trget s determined y BLASTX nd BLASTN serches. rigf-i. However, stimultion with just rigf-i led to significnt reduction in the expression of IGFBP-6 (Figure 3). Curiously IGFBP-4 ws found to e significntly down regulted in response to co-incution with ril-1β + rigf-i (-2.4 fold) ut not y either tretment lone. MyF5 ws found to e down regulted in response to ril-1β (-7.8 fold) nd this effect ws not significntly ltered y co-incution (-1.7 fold) (Figure 3). Lstly, trogin-1 ws found to e significntly down regulted in response to stimultion with rigf-i (-4.9 fold) ut unltered y ril-1β tretment (Figure 3). Co-incution with ril-1β + rigf-i however lted the rigf-i effect. At 24 h co-stimultion of cells with ril-1β + rigf-i significntly reduced the expression of IL-1β reltive to cells only stimulted with ril-1β, from 654 fold to 427

6 Pooley et l. BMC Genomics 213, 14:747 Pge 6 of 2 Tle 3 Differentil expression of genes relted to proteolysis Gene ID 1 Annottion 2 Men fold chnge± SE 3 Identity 4 Ss#CL13Contig1 CL13Contig1 1. ± 1.8 Collgense 3 Ss#S DW ±.4 Angiotensinogen Ss#CK CK ±.5 Cytosolic non-specific dipeptidse Ss#S EG ±.1 Uiquitin-like protein 1 Ss#S18895 CB ±.2 E3 uiquitin-protein ligse NF144A-A Ss#DW DW ±.4 Ur5 protein Ss#CL3Ctg1 CL3Contig1 2.4 ±.1 Protesome suunit et type-6 precursor Ss#KSS4965 KSS ±.1 Protesome suunit et type 7 Ss#STI415 BT ±.1 Protesomeet type 8 Ss#S DW ±.1 Protesome suunit lph type-6 Ss#S EG ±. E3 uiquitin-protein ligse CHF Ss#S EG ±.1 Uiquitin-conjugting enzyme E2 T Ss#S EG ±.2 Clpin smll suunit 1 Ss#S EG ±.1 Cysttin-B Ss#S DY ±.2 Uiquitin- contining phd nd ring finger 1 Ss#S NM_ ±.2 Serine protese htr1 Ss#S EG ±.9 Protese, serine, 35 List of selected mnas relted to proteolysis found to e incresed or decresed in expression in response to ril-1β stimultion. Genes were ssigned to the tle sed upon oth their GO identifier nd previous knowledge of their functions. Genes with gretest fold differences in expression re presented, the genes tht re lower in expression re denoted y (-) vlue. The genes shown were significnt t p <.5 following t- tests with Benjmini-Hocherg FD nd greter thn 2-fold chnge. 1 Indictes the unique code for the feture on the microrry, 2 Accession numer of the cdna sequence, ccession numers eginning with TC re for oligos from the TIG Atlntic Slmon Gene Index. 3 Fold chnge, in the cse of oligos tht fetured multiple times in the gene list the one with the highest fold chnge is reported. 4 Identity of the proe trget s determined y BLASTX nd BLASTN serches. fold (Figure 4). No significnt effect of co-incution of ril-1β + rigf-i ws found on the expression of TNFα or hepcidin (Figure 4). Additionlly co-incution did not lter the expression of MyF5 or ny of the IGFBPs. While ril-1β lone significntly incresed the expression of trogin-1 (2.8 fold) this increse ws not found in cells co-incuted with ril-1β + rigf-i (Figure 4). However the co-incuted cells hd significntly incresed expression of trogin-1 compred to cells stimulted with just rigf-i. rigf-i lone lso significntly reduced the expression of hepcidin (-1.9 fold) ut hd no effect on the other genes. All the genes tested tht were lso hyridised with sufficient intensity on the microrry showed the sme direction nd similr mgnitude of response in this cell culture experiment. Discussion egultion of muscle mss is under the control of multitude of regultors relted to oth nolic nd ctolic processes. We hypothesised tht the muscle cells would respond to the inflmmtory stimulus y signlling the induction of inflmmtory responsive genes in ddition to other pthwys relted to protein metolism for relese of free mino cids s occurs during the cute phse response [56], or for gluconeogenesis nd energy relloction. Our pproch of using primry cells to exmine the trnscriptomic responses of muscle cells stimulted with IL-1β voids complex host nd cell type responses oserved during in vivo experiments. The response to the recominnt cytokine resulted in lrge pnel of genes tht were significntly modulted eing oth incresed nd decresed in expression. Using gene ontology enrichment nlysis for iologicl processes five key enriched processes were reveled: immune function, protein ctolism, IGF nd growth regultion, cell cycle nd lipid metolism. Immune response The immune genes up regulted included severl proinflmmtory cytokines such s TNF-α, IL-1β nd IL-8, indicting tht stimulted myocytes re cple of synthesising these cytokines nd re undergoing proinflmmtory response. The response to IL-1β is extremely rpid in other cell types in fish [57,58] nd it is likely tht within 24 h these molecules will hve een secreted into the medium. Severl genes in the inflmmtory signlling cscde were induced including NFκB suunits p1 nd p15, nd the NFκ inhiitor (IκB), s seen during inflmmtion in other cell types [58]. Under norml conditions IκB inds to NFκB to inctivte it ut IκB is phosphorylted y IκB kinse (IKK) nd susequently uiquitinted nd destroyed y the protesome

7 Pooley et l. BMC Genomics 213, 14:747 Pge 7 of 2 Tle 4 Differentil expression of genes for growth regultion & structurl proteins Gene ID 1 Annottion 2 Men fold chnge± SE 3 Identity 4 Ss#S DW ± 2.5 IGF inding protein 6 Ss#S AY ±.2 Growth hormone receptor isoform 1 precursor Ss#S EG ±.2 CCAAT/enhncer inding protein delt Ss#DW DW ±.4 Signl trnsducer nd ctivtor of trnscription 2 Ss#STI16259 TC ±.1 YY1 trnscription fctor Ss#STI319 BT ±. egultor of g-protein signling 18 SsHomCont3_8 SsHomContl3 2.2 ±.3 Bet-ctin Ss#S DW ±.1 Collgen lph 2 type VI Omy#TC TC ±.2 Collgen lph 2 type V preproprotein Ss#S CB ±.4 Type I collgen lph 2 chin Ss#S DQ ±.2 Growth hormone receptor isoform 2 precursor Ss#S EG ±. Collgen lph 1 type II isoform 1 precursor Ss#STI176 TC ±.2 Growth rrest-specific 1 Ss#CA4182 CA ±.2 Trnsforming growth fctor, et receptor III Ss#S EG ±.2 Vsculr endothelil growth fctor D Ss#CA37592 CA ±.2 Myosin IB Ss#S DY ±.2 Lminin, et 1 Ss#S EG ±.3 Collgen lph 1 type V Ss#S DY ±.1 Myosin phosphtse-ho intercting protein isoform 1 Ss#STI2556 TC ±.1 Type i kertin s8 Ss#S EU ±.1 IGF inding protein 5 Ss#S DY ±.5 Corticotropin relesing fctor precursor Ss#STI5529 BT ±.2 Collgen triple helix repet contining 1 Ss#TC91867 TC ±.3 Collgen lph 1 type XI isoform A preproprotein Ss#S EG ±.5 Troponin I, slow skeletl muscle Ss#STI115_3 BT ±.6 Tropomyosin-1 lph chin Ss#STI119 TC ±.4 Myosin ic Ss#S EF ±.7 IGF inding 7 precursor Ss#S EF ±.4 IGF inding protein 4 Ss#S EG ± 5.3 Collgen lph 1 type X precursor Ss#STI3922 TC ± 2.7 Myf5 protein List of selected mnas relted to growth regultion & structurl proteins found to e incresed or decresed in expression in response to ril-1β stimultion. Genes were ssigned to the tle sed upon oth their GO identifier nd previous knowledge of their functions. Genes with gretest fold differences in expression re presented, the genes tht re lower in expression re denoted y (-) vlue. The genes shown were significnt t p <.5 following t- tests with Benjmini-Hocherg FD nd greter thn 2-fold chnge. 1 Indictes the unique code for the feture on the microrry, 2 Accession numer of the cdna sequence, ccession numers eginning with TC re for oligos from the TIG Atlntic Slmon Gene Index. 3 Fold chnge, in the cse of oligos tht fetured multiple times in the gene list the one with the highest fold chnge is reported. 4 Identity of the proe trget s determined y BLASTX nd BLASTN serches. [59,6]. A relted key signlling molecule up regulted ws MAP kinse-intercting serine/threonine kinse 2, centrl to the MAPK pthwys involved in IL-1β signlling [61], nd with dditionl roles in the regultion of IGF signlling [26]. Another importnt trnscription fctor up regulted ws the MAPK ctivted jun-b which increses trnscription of IL-1β responsive genes generlly t AP-1 responsive sites [62]. Interestingly, lthough jun-bmyessocitedwith inflmmtory signlling, it lso hs role in mintining muscle mss nd its over expression in mmmls cn induce hypertrophy [63], indicting complex regultion of trnscriptionl mchinery. In prllel to this, severl genes encoding proteins tht hve roles s nti-inflmmtory fctors were ctivted; these include two suppressors of cytokine signlling (SOCS 1 nd 3), IL-1 nd n IL-1 receptor chin. SOCS proteins re often co-regulted during inflmmtion to prevent cellulr dmge nd re negtive regultors of cytokine signlling nd function tht interferes with signl trnsduction from cytokine receptors. The SOCS genes hve een chrcterised in slmonid fish

8 Pooley et l. BMC Genomics 213, 14:747 Pge 8 of 2 Tle 5 Differentil expression of genes relted to the cell cycle & DNA repliction Gene ID 1 Annottion 2 Men fold chnge± SE 3 Identity 4 Ss#S DW ±.3 Cyclin D1 Ss#S DY ±.2 Cell division cycle ssocited 4 isoform 14 Ss#S EG ±.1 Cyclin G2 Ss#S32917 DW ±.1 Cyclin-dependent kinse 2 isoform 1 Ss#STI1834 TC ±.1 Cyclin B1 Omy#S C ±.2 Cyclin B2 Ss#S EG ±.2 Cell cycle progression 1 isoform 2 Ss#S EG ±.1 Meditor of NA polymerse II trnscription suunit 22 Ss#TC1912 TC ±.2 Cyclin E1 isoform 1 Ss#KSS3754 NM_ ±.2 Minichromosome mintennce complex component 4 Ss#STI128 TC ±.1 Cell division control protein 2 Ss#TC13697_S TC ±.1 DNA repliction licensing fctor mcm2 Ss#S32962 DW ±.1 Cyclin-dependent kinse 4 Ss#S EG ±.1 Cyclin A2 Ss#S CB ±.3 Minichromosome mintennce complex component 2 Ss#S EG ±.1 Minichromosome mintennce complex component 3 Ss#S EG ±.4 DNA repliction complex GINS protein PSF1 Ss#S DW ±.6 Spindle pole ody component 24 homolog Ss#STI15543 TC ±.5 Minichromosome mintennce complex component 7 Ss#S CB ±.6 Minichromosome mintennce complex component 5 Ss#S DW ±.4 Cell division cycle ssocited 7 isoform 1 Omy#S C ± 8.7 Cyclin E2 Omy#S CU ± 2.8 Cell division cycle 6 protein List of selected mnas relted to the cell cycle & DNA repliction found to e incresed or decresed in expression in response to ril-1β stimultion. Genes were ssigned to the tle sed upon oth their GO identifier nd previous knowledge of their functions. Genes with gretest fold differences in expression re presented, the genes tht re lower in expression re denoted y (-) vlue. The genes shown were significnt t p <.5 following t- tests with Benjmini-Hocherg FD nd greter thn 2-fold chnge. 1 Indictes the unique code for the feture on the microrry, 2 Accession numer of the cdna sequence, ccession numers eginning with TC re for oligos from the TIG Atlntic Slmon Gene Index. 3 Fold chnge, in the cse of oligos tht fetured multiple times in the gene list the one with the highest fold chnge is reported. 4 Identity of the proe trget s determined y BLASTX nd BLASTN serches. [64] nd re incresed in expression following stimultion with severl different cytokines including IL-1β, TNFα nd IL-6. Other immune relted genes such s hepcidin, ferritin, C type lectin nd the complement system were lso significntly incresed in expression. Both hepcidin nd ferritin control iron vilility nd hve ntimicroil ctions with ferritin sequestering iron to reduce vilility to microes [65], wheres hepcidin lso hs direct ntimicroil properties nd is often descried s n ntimicroil peptide [66-68]. C-type lectins recognise crohydrte moieties nd re often induced y proinflmmtory signls [58,69], to regulte vriety of immune processes including the complement system [7-72]. There ws lso ctivtion of some genes tht re components of the dptive immune system, such s mjor histocomptiility complex (MHC) clss I nd CD4-like protein, ut t the time point we exmined the predominnt immune gene response ws y molecules of the innte defences. Protein ctolic processes A mjor proteolytic pthwy in muscle is the uiquitin protesome pthwy, which in mmmls is elieved to e responsile for the mjority of muscle protein degrdtion initited y numer of different stimuli including inflmmtion in mmmls [3]. This pthwy hs lso een seen to e ctivted in slmonid fish during muscle trophy induced y food deprivtion [45,73,74], hormonl chnges [75], with some evidence of severl components eing modulted during immune responses [45,76]. The end product of proteolysis is the relese of free mino cids for de novo protein synthesis or for the oxidtion of the mino cids nd gluconeogenesis. Following the inflmmtory stimulus, severl components of the UBP were incresed in expression in myocytes. Severl uiquitin E3 ligses, which initite the trgeting of proteins for degrdtion nd numer of protesomesuunitsfromthectlyticcoreoftheprotesome

9 Pooley et l. BMC Genomics 213, 14:747 Pge 9 of 2 Tle 6 Differentil expression of genes relted to lipid nd sterol metolism Gene ID 1 Annottion 2 Men fold chnge± SE 3 Identity 4 Ss#S CB ± 2.2 Cretine kinse, uiquitous mitochondril precursor Ss#STI12_4 AY ± 1.1 Prostglndin-endoperoxide synthse 2 Ss#S DW ±.3 Sphingomyelin synthse 1 Ss#STI22551 TC ±.2 Lipoprotein lipse Ss#TC15353 TC ± 1. Mecr protein Ss#STI1271 TC ±.1 etinol dehydrogense 3 Ss#STI31819 TC ±.3 Glucose-6-phosphte-1-dehydrogense Ss#S NM_ ±.7 Myotuulrin Ss#S DY ±.1 Cytochrome c oxidse suunit 5B, mitochondril Ss#S DY ±.1 PPA-lph intercting complex protein 285 isoform 1 Ss#KSS1976 KSS ±.2 78 kd glucose-regulted protein Ss#S EG ±.2 Apolipoprotein-L3 Ss#S DW ±.2 Glycolipid trnsfer protein Ss#S DY ±.1 StA-relted lipid trnsfer domin contining 3 Ss#CL5Contig2 CL5Contig2 2.3 ±.1 Fructose-isphosphte ldolse A Ss#S DY ±.2 Adipose differentition-relted protein Ss#STI13627 TC ±.2 Cox18 cytochrome c oxidse ssemly homolog Ss#CA43659 CA ±.2 Apolipoprotein B precursor Ss#DW DW ±.1 Mitochondril uncoupling protein 2 Ss#STI21893 TC ±.1 Cretine kinse -type Ss#S EG ±.1 Lipid phosphte phosphohydrolse 1 Ss#STI2245 TC ±.1 Lipse Ss#S DW ±.1 Lipid phosphte phosphohydrolse 2 Ss#S32465 DW ±.3 Glycerldehyde-3-phosphte dehydrogense-2 Ss#TC1874 TC ±.2 Lipoclin precursor Ss#STI21285 TC ±.3 Glutmine synthetse Ss#S EG ±.5 Cholesteryl ester trnsfer protein, plsm Ss#STI2265 TC ± 1.4 Apolipoprotein -i inding protein List of selected mnas relted to the lipid & sterol metolism found to e incresed or decresed in expression in response to ril-1β stimultion. Genes were ssigned to the tle sed upon oth their GO identifier nd previous knowledge of their functions. Genes with gretest fold differences in expression re presented, the genes tht re lower in expression re denoted y (-) vlue. The genes shown were significnt t p <.5 following t- tests with Benjmini-Hocherg FD nd greter thn 2-fold chnge. 1 Indictes the unique code for the feture on the microrry, 2 Accession numer of the cdna sequence, ccession numers eginning with TC re for oligos from the TIG Atlntic Slmon Gene Index. 3 Fold chnge, in the cse of oligos tht fetured multiple times in the gene list the one with the highest fold chnge is reported. 4 Identity of the proe trget s determined y BLASTX nd BLASTN serches. (β suunits 6, 7 nd 8 nd α suunit 6), were incresed in expression. We hypothesise tht these chnges would result in incresed protein degrdtion nd reduced muscle growth relesing free mino cids, which in vivo would e rellocted to other orgns, such s the liver s occurs in mmmls [77,78]. Although the predominnt proteolytic genes modulted were relted to the UBP system, cysttin B, n inhiitor of the cidic lysosoml cthepsins ws down regulted, possily indicting n increse in cthepsin iovilility nd ctivity [79]. In ddition the clcium dependnt protese clpin suunit 1 ws down regulted following the IL-1β stimultion. This protese hs roles in positive regultion of myofusion inhiiting the differentition of myocyte cells [8,81] nd this my indicte reduction of muscle cell differentition. Other proteses oserved to e incresed included collgense 3, tht is incresed in expression in NFkB medited inflmmtion in mmmls [82-84] nd during vitellogenesis induced muscle trophy in slmonids [43]. Angiotensinogen, the precursor of oth ngiotensin I & II, ws lso incresed in expression, nd is known to interfere with the ctions nd production of IGF-I, which in mmmls is medited y the NFκB pthwy in collortion with protein kinse C [85,86]. In generl there ws cler effect of ril-1β on the expression of genes relted to ctolism s evidenced

10 Pooley et l. BMC Genomics 213, 14:747 Pge 1 of 2 Figure 2 Grph showing the fold effects of ril-1β stimultion compred to control on the expression of genes involved in the immune response nd growth fter 6, 24 nd 48 h stimultion. Sttistics were crried out using one wy ANOVA. Time points tht do not shre letter re sttisticlly different from ech other. All mnas exmined here were significntly ltered in expression t ll time points reltive to the unstimulted control. y trnscriptomic shift towrds muscle ctolism y the increse in mnas relted to protein degrdtion nd the down regultion of protein degrdtion relted genes tht hve positive effects on growth. IGFBPs The IGF system is instrumentl in the control of protein synthesis nd growth in oth mmmls nd fish [87]. The ctivity of IGF is under tight control, often y fmily of IGF inding proteins (IGFBPs), which hve recently een chrcterised in slmonid fish [88]. They function y either stilising the IGF or y competitively inding the IGF to prevent ttchment to the IGF receptor [87] nd thus reducing the nolic effects of IGF on the cells. We found severl IGFBP encoding mnas were modulted y the proinflmmtory stimulus. IGFBP-6 is thought to hve inding preference for IGF-II ut lso inds IGF-I [89]. These direct effects on the ctivity of oth IGFs might drive the cells wy from high levels of protein synthesis nd nolism towrds stte of ctolism [9,91]. Previous studies indicte IGFBP-6 expression is ssocited with the inhiition of cell prolifertion in oth fish [12] nd mmmls [89,92]. Additionlly IGFBP-6 expression is reduced during resumption of growth following strvtion [2,93]. These findings tend to indicte tht IGFBP-6 expression hs negtive reltionship with growth due to the ility of IGFBP-6 to ct s negtive regultor of IGF-I & II ctivity, thus mking n increse in the expression of IGFBP-6 potentil mrker of inflmmtion induced ctolism in slmon muscle. Other IGFBPs 4, 5 nd rp1 were ll decresed in expression following the inflmmtory stimulus. In slmonids IGFBP-4 expression in muscle is incresed y nolic stimuli such s refeeding fter strvtion [2,93] nd is positively relted to the expression of the promyogenic trnscription fctors MyoD nd MyF5 in vitro [12]. IGFBP-5 cn potentite the effects of IGF-I especilly with regrd to one [94] nd muscle differentition [9]. In rinow trout IGFBP-5 incresed in expression in muscle during refeeding fter strvtion [93] nd, in Atlntic slmon primry myocytes, the expression of IGFBP-5 decresed during cell prolifertion suggesting this protein is ssocited with entry to cell cycle [12]. Together these results suggest the IGFBPs re responding in coordinted fshion to reduce IGF signlling nd ltering the lnce etween nolic nd ctolic pthwys. Growth regultion nd structurl proteins Mny trnscription fctors involved in growth regultion were ltered. CCAAT/enhncer inding protein delt

11 Pooley et l. BMC Genomics 213, 14:747 Pge 11 of 2 IL-1B TNF Hepcidin Atrogin c c MyF IGFBP-4-1 IGFBP IGFBP Figure 3 (See legend on next pge.)

12 Pooley et l. BMC Genomics 213, 14:747 Pge 12 of 2 (See figure on previous pge.) Figure 3 Fold chnge of genes involved in oth the immune response nd growth in response to 6 h stimultion with either ril-1β (25 ng/ml), ril-1β (25 ng/ml) + rigf(1nm), or rigf(1nm). represents significnt difference from control, rs which shre letter re not significntly different. All fold chnges were clculted compred to unstimulted control smples. Comprtive gene expression ws mesured with qpc. ws incresed, nd is trnscription fctor with multiple functions, tht is positively relted to myosttin expression in mmmls [95]. In rinow trout muscle it is incresed during energy relloction cused y vitellogenesis [43] indicting locking of muscle growth. A second key trnscription fctor, NFκB, is often ssocited solely with immune function ut lso negtively regultes myogenesis vi the trnscriptionl repressor YY1 [34,96]. Both of these molecules were incresed in this experiment y IL-1β. YY1 is likely to e meditor of NFκB induced muscle growth inhiition, chieving this y silencing myofirillr promoters in myolsts [34,96]. MyF5, muscle specific trnscription fctor, regultes muscle cell differentition [3,97] nd reduction in its expression level in this experiment fits with our nticipted reduction of muscle growth mrkers in response to ril-1β stimultion.additionllywefoundgenerldecreseinexpression of mnas coding for muscle structurl proteins such s collgens, myosins, ctin nd kertin, consistent with the hypothesis tht the muscle cells re undergoing reduction in growth in response to immune stimultion, s previously showninmmmls[98]. Cell cycle The cell cycle is lrgely medited through the ctions of cyclin/cyclin dependent kinse complexes [99]. In the slmon myocytes multiple cyclins were modulted y IL-1β stimultion strongly suggesting cell cycle ctivity is eing ltered. For exmple, cyclin D1 expression ws incresed nd functions in comintion with cyclin dependent kinses to initite nd progress through the G1 phse of the cell cycle [99,1]. The increse of cyclin D1 my e relted to NFκB meditedrrestofmuscle growth y preventing myocyte differentition [11]. Cyclin G2 ws lso incresed nd my inhiit entry into the cell cycle [12,13]. The remining cyclins A2, B (1 nd 2) nd E (1 nd 2) were ll decresed in expression. Cyclin A2 is rte limiting fctor during S-phse nd DNA synthesis nd entry to mitosis [14], wheres cyclins E1 nd E2 re responsile for the trnsition from G1 to S phses nd initition of DNA repliction [15]. Cyclins B1 & B2 hve roles during the S-phse nd the M-phse nd re crucil for mintennce of the mitotic stte [16]. Severl other cyclin relted kinses, cell division proteins nd minichromosome mintennce complex components were generlly down regulted indicting mjor reduction in cell cycle ctivity nd DNA metolism in these primry muscle cells under n inflmmtory stimulus. Lipid nd sterol metolism A finl group of genes found to e ltered were those relted to lipid nd sterol metolism, here severl cholesterol trnsport proteins were incresed in expression including Apo L3, glycolipid trnsfer protein nd lipoprotein lipse. Apo-L3 is known to e TNF-α inducile protein nd its expression is known to e involved in the ctivtion of the NFκB signlling pthwy ctivted y cytokines in mmmls [17]. The increse in the lipoprotein lipse could reflect n incresed rekdown oflipoproteinsforimmuneorcellulrprocesses;thisgene is under the control of mny different signls in mmmls including insulin, nutritionl stte nd cytokines [18]. Prostglndin-endoperoxide synthse 2, gene known in mmmls to e inducile y vriety of inflmmtory sustnces [19], ws lso incresed s result of the ril-1β stimultion. Apo A1 inding protein nd Apo B were reduced in expression s well s severl other sterol synthesis proteins. These results indicte tht lipid metolism is eing ctively chnged in these myocytes under the inflmmtory stimulus, resulting in complex chnges in trnscription of their mnas. Mny of these chnges could e medited through intrcellulr crosstlk with the IGF/insulin pthwy(s) [26,11]. Interction etween IGF-I nd IL-1β esults from the microrry clerly indicted genes involved in the IGF regultion were eing modulted y IL-1β, especilly numer IGF inding proteins. We performed dditionl experiments to ddress if trnscripts ltered y inflmmtion would e modulted y IGF-1, hormone which drives cells towrds n nolic sttus. Atrogin -1 key gene involved in protein degrdtion ws reduced in expression y incution of cells with rigf-1 s previously reported, conversely it is incresed following incution with IL-1β s occurs in mmmls [31]. When cells were co-incution with ril-1β nd rigf-i n lmost totl inhiition of the trogin-1 down regultion ws found, suggesting the proinflmmtory signl is locking the nolic effect of IGF-1. IL-1β lone results in n increse in trogin -1 expression t 24 h s found in mmmlin cells stimulted y proinflmmtory cytokines

13 Pooley et l. BMC Genomics 213, 14:747 Pge 13 of IL-1B 2 15 TNF IL-1B IL-1B + IGF-I IGF-I c 5 IL-1B IL-1B + IGF-I IGF-I Hepcidin IL-1B IL-1B + IGF-I IGF-I Atrogin-1 IL-1B IL-1B + IGF-I IGF-I c MyF5 IGFBP IL-1B IL-1B + IGF-I IGF-I IL-1B IL-1B + IGF-I IGF-I IGFBP-5.2 IL-1B IL-1B + IGF-I IGF-I Figure 4 (See legend on next pge.) IGFBP-6 IL-1B IL-1B + IGF-I IGF-I

14 Pooley et l. BMC Genomics 213, 14:747 Pge 14 of 2 (See figure on previous pge.) Figure 4 Fold chnge of genes involved in oth the immune response nd growth in response to 24 h stimultion with either ril-1β (25 ng/ml), ril-1β (25 ng/ml) + rigf(1nm), or rigf(1nm). represents significnt difference from control, rs which shre letter re not significntly different. All fold chnges were clculted compred to unstimulted control smples. Comprtive gene expression ws mesured with qpc. [111]. The co-stimultion lso decresed the mgnitude of the response of the ntimicroil peptide hepcidin, highlighting n lterntive lloction of resources depending on the signlling the muscle cells re receiving. Together these results show how nolic signls my ttenute trnscription of immune defence molecules nd tht proinflmmtory signls cn increse ctolic effects in the cells. Conclusions Muscle tissue is complex nd dynmic orgn nd is generlly the only protein storge orgn in the ody; hence it needs to e le to control the synthesis of proteins nd relese of mino cids vi degrdtion under vriety of environmentl nd physiologicl conditions. Muscle does respond to immune insults in fish, [45,76] ut to dte these responses hve not een exmined in n in vitro system removing the mileu of cytokines nd hormones. Here we show direct effect of proinflmmtory cytokine on primry muscle cells tht induces not only immune genes, ut lso lters the wider trnscriptome indicting incresed ctolism, lipid moiliztion nd decresed cell prolifertion with lrge role potentilly for the IGFBPs (Figure 5). Susequent experiments demonstrte tht oth IL-1β nd IGF-1 exert disprte effects on mechnisms tht regulte growth nd other physiologicl responses s highlighted y their interction of expression on trogin-1. These findings will direct future reserch into the control of muscle mss in ectothermic nimls, prticulrly in reltion to helth nd nutrition. Methods Myostellite isoltion nd stimultion Atlntic slmon (men weight of 25 g nd men length of 12 cm) were used for skeletl muscle myostellite cell extrction, s previously descried [ ]. For ech muscle extrction 6 fish were used (~1.5 g tissue from ech fish), this ws to remove ny individul fish effects. No experimentl procedures were crried out on the fish nd fish mintennce ws in line with ntionl ethicl guidelines in n experimentl fcility t University of Aerdeen, UK. Fish were mintined in freshwter nd fed commercil diet t 1.5% ody weight per dy. Fish were killed using schedule one method nd muscle tissue from ove the mid line of the fish ws removed sterilely with sclpel nd forceps. This pooled muscle (pprox 9 g) ws plced into pre-weighed flsk contining 3 ml of Leiovitz L15 medium (Gico) + penicillin/streptomycin 1% (Pen/Strep, Gico, Penicillin 1, units/ml, streptomycin 1, μg/ml) (L15 + P/S). The muscle ws diced into smll locks (2 mm 3 )using sterile procedures nd the diced muscle then centrifuged (3 g, 5 min) nd the superntnt removed. The tissue ws digested in collgense (.2%) t 11 C for 1 h. Following digestion the cell suspension ws centrifuged (3 g, 5 min) nd wshed efore eing centrifuged gin (3 g, 5 min). This pellet ws digested in trypsin (.1%) t 11 C for 3 min. The cell suspension ws gin centrifuged (3 g, 4 sec) nd the remining superntnt ws dded to L15 + P/S plus 1% foetl clf serum (FCS, Sigm) efore eing pssed through 2 μm nylonmesh.the suspension ws centrifuged (3 g, 15 min), the superntnt ws removed nd 12 ml of L15 + P/S + 1% FCS were dded. Finlly the contents of this tue were dded to two 6 well pltes. Prior to plting lminin (mouse lminin, Sigm-Aldrich) ws pplied to the well surfces 24 h efore the cells were plted out, t concentrtion of 1 mg/ml. Cell cultures were then left for 1 h for microstellite cells to ind to the surfce efore the medium ws first chnged nd cells llowed to differentite t 22 C, with the medium eing chnged every 2 dys. Stimultion of cells Cells were cultured for 4 dys to llow cellulr differentition, this ws oserved using light microscopy. Morphology typicl of stellite cells nd time tken to rech confluence in our system ws pproximtely 6 dys. The cells were found to exhiit the typicl growth pttern previously oserved for muscle stellite cells s descried in Bower & Johnston (21) [12]. Initilly cells were mononucleic efore eginning to proliferte nd differentite into spindle shped cells, smll numer of which were eginning to fuse together y dy 4. For the microrry experiment stimultions, the medium ws removed nd 1 ml of new medium (with.5% FCS) contining either 1 μl recominnt trout IL-1β protein (ril-1β) to chieve concentrtion of 25 ng/ml or non-stimulted control with 1 μl PBS. The concentrtion of IL-1β hs previously een determined to e optiml in slmonid cell lines [58]. The cells were then stimulted for 24 h efore NA extrction ws crried out. Susequent experiments were crried out to further investigte the effects of ril-1β t different time points nd