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Fish & Shellfish Immunology 34 (2013) 339e347

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Fish & Shellfish Immunology

journal homepage: www.elsevier .com/locate / fs i

In vivo effects of Escherichia coli lipopolysaccharide on regulation of immuneresponse and protein expression in striped catfish (Pangasianodon hypophthalmus)

Bui Thi Bich Hang a,b,*, Sylvain Milla a,c, Virginie Gillardin a, Nguyen Thanh Phuong b, Patrick Kestemont a

aResearch Unit in Environmental and Evolutionary Biology, NARILIS, University of Namur, rue de Bruxelles 61, B-5000 Namur, BelgiumbCollege of Aquaculture and Fisheries, Cantho University, Campus II, Cantho, VietnamcURAFPA, University of Lorraine, France

a r t i c l e i n f o

Article history:Received 19 September 2012Received in revised form21 November 2012Accepted 21 November 2012Available online 1 December 2012

Keywords:Escherichia coliImmunityLipopolysaccharidePangasianodon hypophthalmusProteomic

* Corresponding author. Research Unit in EnvirBiology, NARILIS, University of Namur, rue de BruxelleTel.: þ32 81724363; fax: þ32 81724362.

E-mail addresses: btbhang@ctu.edu.vn (B.T. Bichlorraine.fr (S. Milla), virginie.gillardin@fundp.ac.bectu.edu.vn (N.T. Phuong), patrick.kestemont@fundp.ac

1050-4648/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.fsi.2012.11.025

a b s t r a c t

Lipolysaccharide (LPS), a component of outer membrane protein of gram-negative bacteria, reportedlystimulates fish immune system. However, mechanisms driving this immunomodulatory effect are yetunknown. To determine effects of Escherichia coli lipopolysaccharide on regulation of immune responseand protein expression of striped catfish (Pangasianodon hypophthalmus), juvenile fish (20e25 g) wereinjected with 3, 15 or 45 mg E.coli LPS/kg and challenged with Edwardsiella ictaluri. Plasma cortisol andglucose were rather low and did not differ (p< 0.05) among treatments. All LPS treatments differedregarding blood cell count and immune variables such as plasma and spleen lysozyme, complementactivity and antibody titer, 3 mg LPS/kg yielding best results; red blood cell count was not affected by LPStreatment. Accumulated mortalities after bacterial challenge were 23.4, 32.8, 37.7 and 52.5% fortreatment 3, 15, 45 mg LPS/kg fish and control respectively. Proteomic analysis of peripheral bloodmononuclear cells (PBMC) confirmed that LPS induced differentially over-expressed immune proteinssuch as complement component C3 and lysozyme C2 precursor. Regulation of other proteins such asWap65, alpha-2 macroglobulin-3 and transferrin precursor was also demonstrated. Striped catfishinjected with E.coli LPS enhanced innate immune responses.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Non-specific immune system of fish, including humoral andcellular components such as lysozyme, complement factors andleucocytes, plays a major role at all stages of infection [1].Non-specific immune response of fish can be stimulated by variousimmunostimulants such as beta glucan [2,3], vitamin C [4] andlipopolysaccharide (LPS) [5,6]. But their modes of actions remainunclear.

Lipopolysaccharide (LPS) is a component of the cell envelope ofgram-negative bacteria, consisting of lipid A, core polysaccharideand O-specific chain. The lipid A portion of LPS is known as anendotoxin and is responsible for most of the immunomodulatoryeffects of this component [7]. Some information suggests that LPScan enhance non-specific immune response of fish. Paulsen [8]

onmental and Evolutionarys 61, B-5000 Namur, Belgium.

Hang), sylvain.milla@univ-(V. Gillardin), ntphuong@.be (P. Kestemont).

All rights reserved.

reported that LPS stimulated plasma lysozyme activity andthe gene that encoded lysozyme was over-transcribed andaccumulated in response to LPS in head kidney, spleen, liver andintestine. LPS also affected the quantity, proportion and functionof all kinds of blood cells. For instance, LPS induced the productionof antibodies, lysozyme, cytokines like interleukin-2 and -6,pro-inflammatory cytokines like IL-1b, tumor-necrosis factor a andseveral other factors from macrophages [9e13]. At low doses, LPSmay induce beneficial effects to macrophage of treated organisms[11,14] and may enhance protection against disease [6,15,16]. LPScan stimulate the non-specific and specific immune responses infish [17e21]. However, fish were shown to be low sensitive toendotoxins (LPS) from Escherichia coli [22]. In contrast, LPSextraction from Aeromonas hydrophila can effectively stimulateimmune responses in carp (Cyprinus carpio) and protect fish fromthis bacteria by intraperitoneal injection and bathing [6]. Conse-quently, even if there is a general consensus to consider LPS asa potentially efficient immunostimulant, the origin of LPS mayexplain these discrepancies.

Striped catfish (Pangasianodon hypophthalmus), an importantcommercialfish species in South-EastAsia, is usuallyassociatedwithdifferent gram-negative bacteria causing serious diseases. Although

B.T. Bich Hang et al. / Fish & Shellfish Immunology 34 (2013) 339e347340

LPS was reported as an effective immunostimulant on many fishspecies, no published information about the effects of LPS on theimmune system of striped catfish (P. hypophthalmus) is available sofar. Therefore, the present study was carried out to assess the effectsof LPS from E. coli on immunoregulation and to identify newpotential mechanisms of action of LPS in striped catfish. To answerthese questions, selected immune parameters and proteome wereanalyzed in blood and some organs, and peripheral blood mono-nuclear cells (PBMC), respectively, to find out new immunemarkersregulated by LPS.

2. Materials and methods

2.1. Fish and experimental conditions

2.1.1. FishFarm raised striped catfish juveniles (20e25 g) were acclima-

tized to laboratory conditions for 15 days and then stocked into 16composite tanks (500 L) in a flow through freshwater supplysystem, fed twice a day a commercial feed (30% crude proteins,2.5 mm, Proconco). Water temperature (30� 2 �C), dissolvedoxygen (5.57� 0.01 mg/L) and pH (7.7� 0.02) ranged on acceptablevalues throughout the experimental period.

2.1.2. Experimental designThe experimental design included 4 treatments in quadruplicate

(70 fish/tank). After acclimation for 15 days, fish were i.p. injectedwith 0.1 mL of E. coli LPS (Sigma) at 0, 3, 15 and 45 mg LPS/kg ondays 1 and 10. Control group received 0.1 mL of phosphate buffersaline (PBS) on the same schedule. Six fish were sampled for blood,plasma, spleen and peripheral blood mononuclear cell (PBMC) onday 14. All samples were kept at �80 �C until analysis: blood wereused for hematology, plasma was analyzed for cortisol, glucose,lysozyme, complement and total Ig analysis, spleen was used forlysozyme and complement analysis and PBMC were used forproteomics.

2.1.3. Challenge testBacteria were cultured on Tryptic Soy Agar plate (TSA, Merck)

for 48 h at 28 �C. The pure colonies of bacteria were checkedthroughout the shape of colonies and Gram staining under lightmicroscope (Olympus). Then, one colony was collected and put intoa centrifuge tube (50 mL) containing 20 mL of Tryptic Soy Broth(TSB, Merck). This tube was shaken overnight, 180 rpm at 28 �C.Then, bacteria were centrifuged at 5000 rpm at 4 �C for 5 min andwashed 3 times with 0.85% of NaCl solution. Themean colony countused the method of optical density (OD) and ODwas adjusted to 0.1value by spectrophotometer (Thermo spectronic, USA) at 590 nm.Then, this suspension was diluted 1000 times with NaCl solutionand injected to the fish.

Each of the 4 LPS treatments was divided into two groups (120fish/group), one group as control injected with 0.1 mL of 0.85% NaClsolution and the second one challenged with 0.1 mL LD50 ofE. ictaluri by intra-muscular injection on day 17th. Mortality wasrecorded daily during 14 days after the challenge test. The headkidney was collected from agonizing fish for bacteria confirmation.

2.2. Bacteria detection

2.2.1. DNA extractionHead kidneys of fish were mixed and grinded with 600 mL lysis

buffer (0.5 M NaCl, 0.1 M TriseHCl at pH 8.0, 1% sodium dodecylsulfate, and 0.1 mM EDTA) and 2.5 mL of proteinase-K solution(40 mg/mL). All mixtures were mixed well and incubated for15 min at 37 �C and then added 2.5 mL of RNase (2 mg/mL), mixed

and incubated for 30 min at 37 �C. Upon addition of 600 mL Chol-oroform:Isoamylalcohol (24:1), the mixtures were centrifuged at13,000 rpm for 15 min at 4 �C. The upper supernatant was collectedinto new tube. Following addition 600 mL Phenol:Cholor-oform:Isoamylalcohol (25:24:1), the mixtures were mixed well andcentrifuged at 13,000 rpm for 10 min at 4 �C. The supernatant wascollected and mixed quickly with 500 mL cold isopropanol, andcentrifuged at 13,000 rpm for 10 min. The DNA pellets werewashed once with 70% ethanol and dried. Before PCR, DNA wasdissolved in TE buffer (10 mMTriseHCl and 0.1 mM EDTA at pH 8.0)and stored at e20 �C.

2.2.2. PCR amplificationPCR reaction was performed to amplify a 407 bp specific DNA

fragment of E. ictaluri with forward primer 50-GTA GCA GGG AGAAAG CTT GC-30and reverse primer 50-GAA CGC TAT TAA CGC TCACAC C-30 [23]. Each 25 mL reaction contained 1.5 mMMgCl2, 0.2 nMdNTPs, 0.4 mM for each primer, 2.5 U of Taq polymerase (Promega)and 100 ng of DNA extracted from fish head kidney. PCR amplifi-cation was performed using a thermocycler (Applied Biosystem).The cycling parameters consisted of an initial denaturation at 95 �Cfor 4 min, followed by 30 cycles of denaturation at 95 �C for 30 s,annealing at 57 �C for 45 s and extension at 72 �C for 30 s, anda final extension at 72 �C for 10 min. PCR amplicons were resolvedby agarose gel electrophoresis in 1% agarose in 40 mMTriseacetate,1 mM EDTA, and stained with 1 mg/mL ethidium bromide.

2.3. Hematology and stress parameters

2.3.1. Red blood cell (RBC) countingTotal RBCwas counted on Neubauer hemocytometer using Natt-

Herrick solution as a diluent stain [24]. First, 10 mL of each bloodsample were diluted into 1990 mL of Natt and Herrick’s solution andmixed gently for at least 3 min. The cell suspensionwas put into thechamber and allowed to settle for 2e3 min before initiating thecount under the light microscope. The RBC was counted in 5 of the25 small areas.

2.3.2. White blood cell (WBC) countingA small drop of whole blood was smeared on a microscope slide

byusinga smearing slide (Cover glasses 24� 50,Germany). The slidesmear was dried quickly, fixed in methanol (95%, M1775, Sigma) for1e2 min and stained with Wright’s and Giemsa [25]. Classifying ofblood cell types was determined following Supranee [26]. Results ofeach blood cell were calculated according to Hrubec [27].

2.3.3. Lysozyme assayThe lysozyme assay protocol was adapted from Ellis [28] and

Milla [29]. Using sterile potter homogenizers, the spleen lysate wasobtained by homogenizing for 30 s, 1 g of spleen in 2.33 mL ofbuffer containing protease inhibitor cocktail (sodium phosphatebuffer 0.067 M and Triton X-100 0.1%/ethanol 95% and acetic acid1%, v/v, pH 6.2) and then centrifuged at 1000g for 5 min. Inmicroplates 96 wells, the lysozyme activity assay was initiated bymixing 2 mL of spleen lysate or 10 mL of plasma with 130 mL oflyophilized Micrococcus lysodeikticus (Sigma) suspension in phos-phate buffer, pH 6.2 (0.6 mg/mL). The difference in absorbance at450 nmwasmonitored between 0 and 30 min for plasma (0 and 3 hfor the spleen) and used to calculate lysozyme activity in units. Oneunit represents the amount of lysozyme that caused a 0.001decrease in absorbance.

2.3.4. Complement assayThe alternative complement pathway was assayed using rabbit

red blood cells (RRBC, Biomerieux, Craponne, France) as targets

B.T. Bich Hang et al. / Fish & Shellfish Immunology 34 (2013) 339e347 341

following Sunyer and Tort [30] and adapted by Milla et al. [29].Briefly, 10 mL of RRBC suspension (3%) diluted in veronal buffer(Biomerieux) were mixed with serial dilutions of plasma or spleenlysate (60 mL of total volume). After incubation for 100 min at 28 �C,the samples were centrifuged at 2000g for 10 min at roomtemperature. The spontaneous hemolysis was obtained by adding60 mL of veronal buffer to 10 mL of RRBC. The total lysis was obtainedby adding 60 mL of distilled water to RRBC. The absorbance wasmeasured at 405 nm. Appropriate calculations served to estimatecomplement activity.

2.3.5. Total Ig assayThe total immunoglobulin concentration of sample was

measured by the method of Siwicki and Anderson [31], modified byMilla et al. [29]. Briefly, immunoglobulins were precipitated with10,000 kDa polyethylene glycol (PEG, Sigma). Serums were mixedwith 12% PEG solution (v:v) for 2 h at room temperature underconstant shaking. After centrifugation at 1000g for 10 min, thesupernatant was collected and assayed for its protein concentra-tion. The total immunoglobulin concentration was calculated bysubtracting this value from the total protein concentration in theplasma before precipitation with PEG.

2.3.6. Cortisol assayPlasma cortisol was assayed in duplicate using a cortisol ELISA

kit (DRG Instruments GmbH, Germany) and following manufac-turer’s instructions.

2.3.7. Glucose assayPlasma glycemia was measured as follows: plasma (50 mL) was

deproteinized by adding 100 mL of perchloride acid 0.33 M, andcentrifuged at 3000g (for 10 min at 4 �C). Glucose concentrationwas determined in the supernatant according to the glucoseoxidase peroxidase method of Hugget [32].

2.4. Proteomics

2.4.1. PBMC isolationThe PBMC isolation followed the method of Boyum [33] and

Pierrard [34]. Briefly, dilution of 2.5 mL of heparinized blood and4 mL of phosphate-buffered saline (PBS) was quickly carriedout, the mix was poured over a layer of 6 mL Ficoll Paque Plus(1.077 g/mL, GE Healthcare, Uppsala, Sweden) and centrifuged(800g, 20 min, 28 �C). Thewhite cells at the interfacewere collectedand washed twice with 1 mL cold PBS by low speed centrifugation(1000g, 7 min, 4 �C). An osmotic shock with distilled water wasapplied to remove residual red blood cells. The suspension wascentrifuged as previously indicated and then the cells weresuspended in 30 mL DLA buffer (Urea 7 M, thiourea 2 M, TriseHCl30 mM pH 8.5 and CHAPS 4%) and stored at �80 �C for proteomicanalysis.

2.4.2. Protein extraction and CyDye labelingThe PBMCs in DLA were sonicated 3 times, 10 s on ice and

centrifuged for 10 min at 12,000g. The pH of the supernatant wasadjusted to 8.5 by addition of the appropriate volume of 50 mMNaOH. Protein concentration was measured using Bio-Rad proteinassay. Samples containing 25 mg of solubilized proteins wereminimally labeled with 200 pmol of Cyanine dyes following themanufacturer’s protocols (GE Healthcare). Protein samples fromcontrol and LPS conditions were labeled with Cy3 and Cy5, a mixedof equal amounts of protein from control and LPS treatment waslabeled with Cy2 and used as internal standard. Labeling was per-formed on ice for 30 min in the dark and samples were quenchedwith 1 mM lysine for 10 min on ice in the dark. Then, an equal

volume of reduction buffer (7 M urea, 2 M thiourea, 2% DTT, 2%CHAPS, 2% IPG 4e7 buffer) was added for 15 min at roomtemperature.

2.4.3. Separation of proteins by 2D-DIGEPrior to electrofocusing, IPG strips (24 cm, pH 4e7; GE Health-

care) were passively rehydrated overnight with 450 mL of a stan-dard rehydration solution (7 M urea, 2 M thiourea, 2% CHAPS, 0.5%IPG 4e7 buffer, 2% DTT). Sample sets containing the labeledmixtures were then cup-loaded onto the IPG strips and isoelectricfocusing was performed with an Ettan IPGphor II isoelectricfocusing unit (GE Healthcare). The electrophoresis conditions wereas follows: 20 �C for a total of 68,000 V-h. Immobilized pH gradientstrips were reduced (1% DTT) and then alkalized (2.5% iodoaceta-mide) in equilibration buffer (50 mM Tris, 6 M urea, 30% glycerol,2% SDS, pH 8.8). The second dimension was run on a 11%, 24 cm,1 mm thick acrylamide gel. The strips were overlaid with 1%agarose in SDS running buffer DALTsix (25 mM Tris, 192 mMglycine, 0.1% SDS) and run in an ETTAN TM electrophoresis unit (GEHealthcare) at constant 2 W/gel at 15 �C until the blue dye front hadrun off the bottom of the gels.

2.4.4. Image analysis and statisticsLabeled CyDye gels were visualized using a Typhoon 9400

scanner (GE Healthcare) at wavelengths specific to CyDyes andresolution was 100 mm. Images analysis was carried out withDeCyder BVA 5.0 software (GE Healthcare). At first, the differentialin-gel analysis module co-detected and quantified the protein spotsin each image using the internal standard sample as a reference tonormalize the data. In next step, biological variation analysis wasused to calculate ratios between samples and internal standardabundances by performing a gel to gel matching of the internalstandard spot maps from each gel. Results were analyzed by one-way ANOVA (p< 0.05).

2.4.5. Mass spectrometry and protein identificationFor peptide sequencing and protein identification, preparative

gels loaded with 200 mg of proteins of mixed samples were runfollowing the protocol described above except they were post-stained with 10% krypton overnight after twice 30 min of fixa-tion in 40% ethanol and 10% acetic acid. Peptides were analyzedby using nano-LC-ESI-MS/MS maXis UHR-TOF coupled witha 2D-LC Dionex UltiMate 3000 (Bruker, Bremen, Germany). Spotswere excised from preparative gels using the Ettan Spot Picker(GE Healthcare), and proteins were digested with trypsin byin-gel digestion. The gel pieces were shrunk with 100% acetoni-trile. The proteolytic digestion was performed by the addition of3 mL of modified trypsin (Promega, Leiden, Netherlands)suspended in 100 mM NH4HCO cold buffer. Proteolysis wasperformed overnight at 37 �C. The supernatants were collectedand kept at �20 �C prior to analysis. The digests were separatedby reverse-phase liquid chromatography using a 75 mm� 150 mmreverse-phase Dionex column (Acclaim PepMap 100 C18) in anUltimate 3000 liquid chromatography system. Mobile phase Awas 95% of 0.1% formic acid in water and 5% acetonitrile. Mobilephase B was 0.1% formic acid in acetonitrile. The digest (1 mL) wasinjected, and the organic content of the mobile phase wasincreased linearly from 5% B to 40% in 40 min and from 40% B to100% B in 5 min. The column effluent was connected to an ESInano Sprayer (Bruker). In survey scan, MS spectra were acquiredfor 0.5 s in the m/z range between 50 and 2200. The 3 mostintense peptides ions 2þ or 3þ were sequenced. The collision-induced dissociation (CID) energy was automatically set accord-ing to mass to charge (m/z) ratio and charge state of the precursorion. MaXis and Dionex systems were piloted by Compass HyStar

Table 1Effects of LPS injection on various blood parameters of striped catfish.

Parameters Control (0 mg/kg fish) LPS (3 mg/kg fish) LPS (15 mg/kg fish) LPS (45 mg/kg fish)

White blood cells (�103/mm3) 170.7� 25.1a 200.3� 36.8b 148.7� 29.9a 167.7� 20.9a

Lymphocytes (�103cell/mm3) 95.25� 14.1a 106.6� 27.1a 91.30� 37.3a 94.04� 23.1a

Monocytes (�103cell/mm3) 4.29� 1.44a 10.70� 3.37c 7.26� 2.70b 2.49� 1.15a

Neutrophils (�103cell/mm3) 8.38� 3.94b 9.40� 3.39bc 13.88� 8.20c 2.92� 1.60a

Different letters in row indicate significant differences (p< 0.05) in mean (�SD) values.

a

c

b

ab

0

20

40

60

80

100

120

140

0 3 15 45

Lyso

zym

e in

p

lasm

a (U

/m

l)

[LPS] (mg/kg fish)

a

b

aa

0

2

4

6

8

10

12

14

0 3 15 45

Ly

so

zy

me

in

sp

le

en

(U

/m

g p

ro

te

in

)

[LPS] (mg/kg fish)

Fig. 1. Effects of LPS on plasma lysozyme in striped catfish.

B.T. Bich Hang et al. / Fish & Shellfish Immunology 34 (2013) 339e347342

3.2 (Bruker). Peak lists were created using DataAnalysis 4.0(Bruker) and saved as XML file for use with ProteinScape 2.0(Bruker) with Mascot 2.2 as search engine (Matrix Science).Enzyme specificity was set to trypsin, and the maximum numberof missed cleavages per peptide was set at one. Carbamidome-thylation was allowed 219 as fixed modification and oxidation ofmethionine as variable modification. Mass tolerance for mono-isotopic peptide window was 10 ppm and MS/MS tolerancewindow was set to 0.05 Da. The peak lists were searched againstthe full National Center for Biotechnology Informationnon-redundant (NCBInr) database (11,759,209 sequences down-loaded on January the 24th 2011). Scaffold (version Scaffold-2_06_01, Proteome Software Inc., Portland, OR) was used tovalidate MS/MS based peptide and protein identifications. AllMS/MS samples were analyzed using Mascot (Matrix Science,London, UK; version 2.2) and X!Tandem (The GPM, thegpm.org;version 2007.01.01.). Peptide identifications were accepted ifthey could be established at greater than 95% probabilityas specified by the Peptide Prophet alogarithm [35]. Proteinidentifications were accepted if they could be established atgreater than 99 % probability and contained at least 1 identifiedpeptide. Protein probabilities were assigned by the ProteinProphet algorithm [36].

2.4.6. Statistical analysisThe statistical package for social science (SPSS) software

(version 13.0) was used to analyze the data. One-way analysis ofvariance (ANOVA) was performed to compare the stress, hemato-logical and immune parameters between the different LPS treat-ments (p< 0.05).

3. Results

3.1. Plasma cortisol and glucose levels

The plasma cortisol levels ranged between 35.07 and 50.29 ng/mL, without any significant difference between control and LPStreated groups. Similarly, the plasma glucose remained low (53.5e61.6 mg/100 mL) and there were no significant differences amongtreatments (p> 0.05).

3.2. Blood parameters

Abundance of red blood cells did not show any significantdifference in all treatments (Table 1). In contrast, total white bloodcell number was significantly higher in treatment 3 mg LPS/kgwhile those of treatments 15 and 45 mg LPS/kg did not differfrom the control. Lymphocytes in treatment 3 mg LPS/kg(107�103 cell/mm3) were more abundant, without significance,than in the other treatments (p> 0.05). The quantity of monocytesalso reached higher values in treatment 3 mg LPS/kg, whereasneutrophils were significantly more abundant in treatment15 mg/kg when compared with the other LPS treatments (p< 0.05).Both monocyte and neutrophil numbers were strongly reduced by42% and 62% in treatment 45 mg LPS/kg, compared with the controlgroup, respectively.

3.3. Immune responses

3.3.1. Plasma and spleen lysozymeIn control groups, lysozyme activity was low in both plasma and

spleen while it increased significantly (p< 0.05) in fish injectedwith 3 and 15 mg LPS/kg (Fig. 1). Fish injected with 3 mg LPS/kgdisplayed significantly higher values of lysozyme activity comparedwith the other treatments while the groups treated with 45 mgLPS/kg did not differ significantly from the control.

3.3.2. Plasma and spleen complementIn control fish, complement activity in both plasma and spleen

was significantly lower (p< 0.05) than in fish injected with 3 and15 mg LPS/kg (Fig. 2). Fish in treatment (3 mg LPS/kg) displayedsignificantly higher values of complement activity compared withthe other treatments. Fish treated with 45 mg LPS/kg did not differfrom the control.

3.3.3. Total immunoglobulins (Ig)The total amount of immunoglobulins in fish injected with

LPS was increased when compared with control group (Fig. 3,p< 0.05). Fish injected with 3 mg LPS/kg displayed higher values ofimmunoglobulin than the other groups (p< 0.05) while fishinjected with 45 mg LPS/kg did not show significant differencefrom the control.

Fig. 2. Effect of LPS on complement activity in plasma and spleen of striped catfish.

Fig. 4. Representative 2D gel showing the protein expression profiles in striped catfish.Identified protein spots were indicated by Decyder software with significant changes(ANOVA, p< 0.05).

B.T. Bich Hang et al. / Fish & Shellfish Immunology 34 (2013) 339e347 343

3.4. Proteome analysis

A 2D-DIGE analysis was conducted on PBMC 14 days after theinjection with LPS. The mean number of spots detected in 6 gelswas 3064� 212 and the mean number of spots matched across thegels was 1127�156 (Fig. 4). The one-way ANOVA test among allexperimental groups revealed that 31 protein spots displayedsignificant differences between control and LPS treatment. A totalof 10 differentially expressed proteins were identified using nano-LC-ESI-MS/MS and searches in NCBInr database (Table 2). Theidentified proteins were separated into 3 groups that belong todistinct functional classes. The first one corresponds to immuneresponse, among which complement component C3, representedby three different spots (352, 1502 and 1705), was significantlyover-expressed in both LPS treatments (3 mg and 15 mg/kg) exceptthe spot 352. Lysozyme C 2 precursor was significantly over-expressed in spot 1684. The warm temperature protein relate65 kDa 1 (Wap 65-1) was also over-expressed in spot 337 and theseries of Wap 65-2 was represented by 3 different spots (302, 1038and 1136) showing a contrasted regulation according to the tar-geted spot. The alpha-2 macroglobulin-3 was significantly over-expressed in many spots (120, 140, 971 and 1038) and most ofthem were over-expressed in treatment 3 mg LPS. In contrast, theimmunoglobulin heavy chain variable region, presented by spot357, was under-expressed. The second protein class was related totransport protein, in which transferrin precursor (spots 576 and1136) was significantly over-expressed after LPS administration. Inmammals, transferin does not only function as a transport proteinbut is also related to immune response. Finally, the last identifiedgroup of proteins was involved in various biological processesincluding zgc:112265, zgc:56119 and filamin A (spot 140, 913 and

a

c

b ab

0

5

10

15

20

25

30

35

40

45

0 3 15 45

To

tal o

f Ig

(m

g/m

l)

LPS (mg/kg fish)

Fig. 3. Effects of LPS on total of immunoglobulins of striped catfish.

818), that were significantly under-expressed except zgc:112265 intreatment 3 mg LPS/kg fish.

3.5. Fish mortality after challenged by E. ictaluri

The mortality in control groups was 52.46% (Fig. 5). The injectedLPS groups displayed a lower mortality throughout the challengetest and reached an accumulated mortality of 23.4, 32.8 and 37.7%in treatments 3, 15, and 45 mg/kg LPS, respectively. The results ofbacteria identification showed that E. ictaluri were detected in allbacterial infection samples even if the level of detection was vari-able among fish (Fig. 6).

4. Discussion

In this study, impact of E. coli LPS on innate immune system ofstriped catfish was assessed. We clearly showed that the innateimmunity of striped catfish was responsive to all LPS treatmentsand the strongest stimulation was reached at the low dose of 3 mgLPS/kg. Plasma cortisol and glucose were measured to evaluate thestress status of fish and they were not affected by LPS treatments.Plasma cortisol is an indicator of the primary stress response and isrelevant to evaluate fish welfare [37]. Furthermore, cortisol level isclosely linked to many of the adverse consequences of stressincluding effects on growth [38], reproduction [39], and theimmune system [40]. According to Wendelaar Bonga [41] bloodglucose levels also increased in fish following exposure to stressfulconditions. In pallid sturgeon (Scaphirhynchus albus), the plasmaconcentration of cortisol after injectionwith LPS did not differ fromthat of control fish [42]. In contrast, in yellow perch (Perca fla-vescens) LPS increased the plasma cortisol after 6 h post-injectionwhen compared with fish injected with saline [43]. All together,we hypothesize that the period between LPS administration andplasma collection was too long in our study to observe a putativerise of cortisolemia.

In the present study, LPS enhanced total white blood cells, theproportion and quantity of some leucocyte populations but did notalter the abundance of red blood cells. The increase of total leuco-cytes may be considered as a good indicator of activatory responseof fish cellular immunity to LPS stimulation. Among white bloodcells, the quantity of monocytes and neutrophils was increased inLPS injected fish and monocytes reached the highest values ata concentration of 3 mg LPS/kg. However, the enhancement in

Table 2List of identified proteins differentially expressed in PBMC of striped catfish after LPS injection.

Spot No Accession No. Protein name Species Matchingpeptides

TheoricalpI/Mw (kDa)

Fold change

3 vs. 0 mgLPS/kg

15 vs. 0 mgLPS/kg

Immune response proteins352 F5HT30 Complement component C3 Clarias macrocephalus 2 6.65/147 �1.1 �1.31502 F5HT30 Complement component C3 C. macrocephalus 2 6.65/147 þ2.4 þ1.21705 F5HT30 Complement component C3 C. macrocephalus 2 6.65/147 þ1.1 �1.31684 B5XA65 Lysozyme C 2 precursor Salmo salar 2 6.12/16 þ2.5 þ1.1337 B1NQM9 Wap 65-1 Ictalurus punctatus 2 5.52/54 þ1.5 þ1.1302 B1NQN2 Wap 65-2 Ictalurus punctatus 2 6.19/51 þ1.1 �1.31038 B1NQN2 Wap 65-2 I. punctatus 2 6.19/51 þ1.5 þ1.21136 B1NQN2 Wap 65-2 I. punctatus 2 6.19/51 þ1.23 þ1.6120 Q9PVU3 Alpha-2 macroglobulin-3 Common carp 2 6.12/87 þ2.6 �1.2140 Q9PVU3 Alpha-2 macroglobulin-3 Common carp 2 6.12/87 þ1.9 �1.2971 Q9PVU3 Alpha-2 macroglobulin-3 Common carp 2 6.12/87 �1.3 þ1.011038 Q9PVU3 Alpha-2 macroglobulin-3 Common carp 2 6.12/87 þ1.5 þ1.2357 ACD38374 Immunoglobulin heavy chain

variable regionI. punctatus 1 5.4/16 �1.03 �1.6

Transport protein576 C9W3Q0 Transferrin precursor I. punctatus 1 6.19/74 þ2.2 þ1.011136 C9W3Q0 Transferrin precursor I. punctatus 2 6.19/74 þ1.2 þ1.6

Other function proteins140 Q6WQW2 Zgc:112265 Danio rerio 3 6.02/103 þ1.9 �1.2913 Q5RH28 Zgc:56119 D. rerio 2 7.18/106 �2.9 þ1.1818 Q1WCE8 Filamin A I. punctatus 2 5.73/10 �1.04 �1.4

B.T. Bich Hang et al. / Fish & Shellfish Immunology 34 (2013) 339e347344

leucocyte population was not due to the increase of lymphocytenumber. According to Kozinska and Guz [11], total leucocyte countincreased in fish injected with 50 and 1250 mg LPS/fish whencompared with control group. Moreover, phagocytic activity wasalso increased in fish treated with 1250 mg LPS/fish on day 30 afterinjection. A previous study reported that fish phagocyte activitywas enhanced when using antigens from fish pathogenic gram-negative bacteria [44]. Kolman et al. [45] demonstrated anincrease in phagocytic index and phagocytosis capacity in bester(Huso huso L.�Acipenser ruthenus L.) immunized with Aeromonassalmonicida outer membrane antigens. The granulocytes andmonocytes/macrophages are important cells of the non-specificimmune pathways in fish, therefore their level tends to increasewhen using LPS administration [1]. In accordance with the litera-ture, our study supports the statement that the increased numberof monocytes in fish treated with LPS is linked to the stimulation ofnon-specific phagocytosis activity.

In fish, LPS often stimulates immune system and protectsfish against several fish pathogens [5,6,16,46,]. LPS is alsoreported to stimulate non-specific immune parameters of animalsby enhancing lysozyme gene expression and activity [8]. In thisexperiment, the increased lysozyme activity in plasma and spleenalso demonstrated the efficiency of LPS treatment in striped catfish.Similarly to lysozyme, the plasma and spleen complement activitywere induced after LPS treatments. Swain [7] reported thatcomplement system can be activated either by classical or

c

aab

b

010203040506070

0 3 15 45

[M

ortality (%

)

[LPS] (mg/kg bw)

Fig. 5. Effects of LPS injected in striped catfish on the mortality induced after challengetest with E. ictaluri.

alternative pathways depending upon the smooth or rough type ofLPS. The complement system plays key roles in innate and adaptiveimmunity through mediating phagocytosis, respiratory burst,chemotaxis and cell lysis [47]. Our result thus supports theimmunostimulatory effect of LPS in striped catfish. In this study,total immunoglobulins in plasmawere increased in low dose of LPStreatments (3 and 15 mg/kg). The ‘O’ polysaccharide chain in LPScan act as antigenic determinant, therefore, fish respond to LPSsometimes by producing antibody against ‘O’ side chains [7]. Thisstatement comes from many studies which reported that LPScan enhance humoral immune response in treated fish. Injectionof LPS into carp, brown trout, channel catfish, turbot, eels andrainbow trout produced higher antibody titer against Aeromonasbestiarum, Salmonella typhimurium, E. ictaluri, cytophaga-likebacteria, Edwardsiella tarta and Flavobacterium psychrophilum[11,17e20,48,49]. All together, we suggest that LPS is a potentactivator of innate and probably acquired immunity in stripedcatfish.

In the current study, proteomic analysis showed that manyimmune proteins were regulated in PBMC following LPS treat-ments. Complement component C3 is one of the major proteinover-expressed in two LPS treatments. Similarly, changes in theexpression level of C3-1, C3-3 and C3-4 mRNAs in rainbow troutliver, spleen and head kidney were reported after 48h of stimula-tion with LPS [50]. In embryo and larvae of zebrafish (Danio rerio)many genes linked to the complement pathway (C3, C1r/s, C4, C6,Bf, MBL and MASP) were differentially expressed [51]. Followingthe LPS injection to striped fish, over-expression of C3 complementcomponent was related to an increase of complement activity inplasma and spleen. With the past literature, our results thus showthat complement pathway is induced at the transcriptional,translational and post-translational levels. The third component ofcomplement (C3) is central to all activation pathways of thecomplement system [52], which explain the reported relationshipbetween induction of C3 and the whole complement activity in theblood. It is also thought to play a role in the inflammation process,leucocyte chemotaxis, B lymphocyte activity, opsonisation of exo-gen particles and phagocytosis [47,52,53], which translates a posi-tive role of LPS in the immunoregulation.

Fig. 6. Bacteria confirmation by PCR (M: marker, (�): negative control, (þ): positive control, 1, 2, 3, 4, 5, 7, 8, 16: fish were injected with NaCl and 6, 9, 10, 11, 12, 13, 14, 15: fish wereinjected with E. ictaluri).

B.T. Bich Hang et al. / Fish & Shellfish Immunology 34 (2013) 339e347 345

An over-expression of lysozyme C 2 precursor was observed inthis study, in accordance with the increase of lysozyme activity inplasma and spleen of LPS treated fish. The lysozyme c-type hasbeen identified in fish and was firstly isolated from rainbow trout[54]. It is considered as one of the most important anti-bacterialmolecules in fish [46,55,56]. Three genes encoding c-type lyso-zymes, C1-, C2-, C3-type, were obtained from blue tilapia (Oreo-chromis aureus) [57]. Lysozyme C2 can be active against a range ofGram-positive and Gram-negative bacteria and it is more effectivethan lysozyme C1 in killing Gram-negative bacteria. In kelp grouper(Epinephelus bruneus), the expression level of lysozyme c-type wasup-regulated mainly in heart, kidney and spleen between 24 and48 h after the fish were challenged with LPS [58]. Moreover, inAtlantic salmon Salmo salar, mRNA levels of the lysozyme c-typewere increased in liver, spleen and intestine 7 days after injectionwith LPS [8]. Like complement, we deduce from our study thatlysozyme is a major target of LPS in striped catfish and bothinductions of these immune factors may explain the higher resis-tance to pathogen infection in LPS treated fish.

Additionally, the LPS influenced the expression of warmtemperature acclimation protein 65 kDa (Wap65) in spots 302, 337,1038 and 1136. Wap65 are glycoproteins present in plasma andliver of teleost fish. Two different isoforms (Wap65-1 and Wap65-2) have been isolated in several fish species [59,60]. They play anessential role in the acclimation of fish to warm temperatures [61].Besides this major function in thermal acclimation, they are tightlyassociated with other biological pathways, especially the acuteimmune response [62,63]. This is supported by the up-regulatedexpression of Wap65 genes in fish after LPS injection [63] orexperimental infection with pathogenic microorganisms [64,65].Kikuchi [66] also reported that Wap65 mRNA level increased in thehepatopancreas of the goldfish (Carassius auratus) following LPSadministration and Wap65-2 gene was also up-regulated in theliver of channel catfish (Ictalurus punctatus) after bacterial infectionwith E. ictaluri [59]. Contrary to complement and lysozyme whosefunctionality has been well characterized, Wap65 may appear asa new candidate of the immune response in teleost fish.

In the current study, 2D-DIGE analysis revealed that alpha-2macroglobulins were identified in several spots (120, 140, 971 and1038) with over-expression except in spot 140 (treatment 15 mgLPS) and 971 (treatment 3 mg LPS). Alpha-2 macroglobulins arenon-pecific protease inhibitors, which mediate the clearance ofendogenous and exogenous proteases [67]. Activated form ofalpha-2 macroglobulins can also interact with many kinds of cyto-kines, lectin or lipopolysaccharide, and contribute to the regulationof immune reactions [68,69]. Previous studies in common carp

(Cyprinus carpio) suggested a modest increase in gene transcriptionof alpha-2 macroglobulins in the liver of carp infected with Trypa-noplasmaborreli [70]. Inmudcrab, the injectionof LPSwas causedbya striking up-regulation of alpha-2 macroglobulin expression in thehemocytes [71]. It was shown that alpha-2 macroglobulin tran-scription level in amphioxus (Branchiostoma japonicum) increasedafter acute challenge with LPS, hinting at the clue that alpha-2macroglobulin may be an immune-relevant molecule involved inacute phase response [72]. Our study is the first to reveal in stripedfish that alpha-2 macroglobulin protein may be a marker of expo-sure to LPS and putatively implicated in the immune response.

In this study, the immunoglobulin heavy chain variable regionwas under-expressed in 3 mg and 15 mg LPS treatments. In teleostfish, the most prevalent immunoglobulin in plasma is tetramericIgM. Each subunit is composed by two heavy and light chains thatare linked by covalent and noncovalent bonds [73,74]. Severalprotein spots (576, 1136) were identified as fish transferrin, over-expressed after LPS injection in different doses. Transferrins area superfamily of iron-binding proteins in vertebrates [75]. Serumtransferrin is an important iron transporter. Through the bindingand transporting of iron, transferrin participates in immune regu-lation, antimicrobial and antioxidant activity, cytoprotection andelectron transport [76,77]. Furthermore, transferrin-derivedsynthetic peptide induces highly conserved pro-inflammatoryresponse of macrophages [78]. In roughskin sculpin (Trachidermusfasciatus), transferrin mRNA expression was significantly up-regulated in immune organs (skin, blood and spleen) during theLPS challenge and heavy metal exposure experiment [79]. Theseresults suggested that transferrin was involved in the innateimmune response in striped fish after LPS stimulation. The pro-teomic analysis also identified other proteins that are not classicallyincluded in the immune response group. Protein zgc:112265 wasidentified in spot 140 and over-expressed at 3 mg LPS and under-expressed at 15 mg LPS. This protein was novel protein detectionin zebrafish and up to now, its function is unclear. Another proteinzgc:56119 was identified in spot 913 with under expression in 3 mgLPS and over-expression in 15 mg LPS. This protein plays a role ofserine-type endopeptidase inhibitor. The filamin Awas identified inspot 818 and was under-expressed in both LPS treatments. FilaminA is an actin-binding protein with a well-established role in thecytoskeleton, where it determines cell shape and locomotion bycross-linking actin filaments [80]. These proteins may be potentialfactors of the fish immune response and further studies would beneeded to elucidate their functions.

In summary, it can be concluded that LPS administrationby injection in striped catfish stimulates non-specific immune

B.T. Bich Hang et al. / Fish & Shellfish Immunology 34 (2013) 339e347346

response as well as humoral immune system and differentiallyexpressed several important immune proteins. The dose at3 mg LPS/kg fish induced highest significant values and effectivelyprotected fish from bacteria E. ictaluri. Further study is needed toset up the in vitro effects of LPS in PBMC of striped catfish to betterunderstand the mechanism of action of LPS.

Acknowledgments

The authors thank E. Delaive and M. Dieu from URBC, Universityof Namur, for valuable help during proteomic analysis. We arethankful to CUD (Coopération Universitaire pour le Développe-ment) and the General Directorate for Cooperation in Belgium forfinancial support through the bilateral PIC project Deltaquasafebetween the University of Namur and Liège in Belgium and Can ThoUniversity in Vietnam. B.T.B.Hang was supported by a CUD grant.

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