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Fish & Shellsh Immunology 34 (2013) 339e347

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Fish & Shellsh Immunology

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In vivo effects of Escherichia coli lipopolysaccharide on regulation of immune response and protein expression in striped catsh (Pangasianodon hypophthalmus)
Bui Thi Bich Hang a, b, *, Sylvain Milla a, c, Virginie Gillardin a, Nguyen Thanh Phuong b, Patrick Kestemont a

Research Unit in Environmental and Evolutionary Biology, NARILIS, University of Namur, rue de Bruxelles 61, B-5000 Namur, Belgium College of Aquaculture and Fisheries, Cantho University, Campus II, Cantho, Vietnam c URAFPA, University of Lorraine, France

a r t i c l e i n f o
Article history: Received 19 September 2012 Received in revised form 21 November 2012 Accepted 21 November 2012 Available online 1 December 2012 Keywords: Escherichia coli Immunity Lipopolysaccharide Pangasianodon hypophthalmus Proteomic

a b s t r a c t
Lipolysaccharide (LPS), a component of outer membrane protein of gram-negative bacteria, reportedly stimulates sh immune system. However, mechanisms driving this immunomodulatory effect are yet unknown. To determine effects of Escherichia coli lipopolysaccharide on regulation of immune response and protein expression of striped catsh (Pangasianodon hypophthalmus), juvenile sh (20e25 g) were injected with 3, 15 or 45 mg E.coli LPS/kg and challenged with Edwardsiella ictaluri. Plasma cortisol and glucose were rather low and did not differ (p < 0.05) among treatments. All LPS treatments differed regarding blood cell count and immune variables such as plasma and spleen lysozyme, complement activity and antibody titer, 3 mg LPS/kg yielding best results; red blood cell count was not affected by LPS treatment. Accumulated mortalities after bacterial challenge were 23.4, 32.8, 37.7 and 52.5% for treatment 3, 15, 45 mg LPS/kg sh and control respectively. Proteomic analysis of peripheral blood mononuclear cells (PBMC) conrmed that LPS induced differentially over-expressed immune proteins such as complement component C3 and lysozyme C2 precursor. Regulation of other proteins such as Wap65, alpha-2 macroglobulin-3 and transferrin precursor was also demonstrated. Striped catsh injected with E.coli LPS enhanced innate immune responses. 2012 Elsevier Ltd. All rights reserved.

1. Introduction Non-specic immune system of sh, including humoral and cellular components such as lysozyme, complement factors and leucocytes, plays a major role at all stages of infection [1]. Non-specic immune response of sh can be stimulated by various immunostimulants such as beta glucan [2,3], vitamin C [4] and lipopolysaccharide (LPS) [5,6]. But their modes of actions remain unclear. Lipopolysaccharide (LPS) is a component of the cell envelope of gram-negative bacteria, consisting of lipid A, core polysaccharide and O-specic chain. The lipid A portion of LPS is known as an endotoxin and is responsible for most of the immunomodulatory effects of this component [7]. Some information suggests that LPS can enhance non-specic immune response of sh. Paulsen [8]

* Corresponding author. Research Unit in Environmental and Evolutionary Biology, NARILIS, University of Namur, rue de Bruxelles 61, B-5000 Namur, Belgium. Tel.: 32 81724363; fax: 32 81724362. E-mail addresses: (B.T. Bich Hang), (S. Milla), (V. Gillardin), ntphuong@ (N.T. Phuong), (P. Kestemont). 1050-4648/$ e see front matter 2012 Elsevier Ltd. All rights reserved.

reported that LPS stimulated plasma lysozyme activity and the gene that encoded lysozyme was over-transcribed and accumulated in response to LPS in head kidney, spleen, liver and intestine. LPS also affected the quantity, proportion and function of all kinds of blood cells. For instance, LPS induced the production of antibodies, lysozyme, cytokines like interleukin-2 and -6, pro-inammatory cytokines like IL-1b, tumor-necrosis factor a and several other factors from macrophages [9e13]. At low doses, LPS may induce benecial effects to macrophage of treated organisms [11,14] and may enhance protection against disease [6,15,16]. LPS can stimulate the non-specic and specic immune responses in sh [17e21]. However, sh were shown to be low sensitive to endotoxins (LPS) from Escherichia coli [22]. In contrast, LPS extraction from Aeromonas hydrophila can effectively stimulate immune responses in carp (Cyprinus carpio) and protect sh from this bacteria by intraperitoneal injection and bathing [6]. Consequently, even if there is a general consensus to consider LPS as a potentially efcient immunostimulant, the origin of LPS may explain these discrepancies. Striped catsh (Pangasianodon hypophthalmus), an important commercial sh species in South-East Asia, is usually associated with different gram-negative bacteria causing serious diseases. Although


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

LPS was reported as an effective immunostimulant on many sh species, no published information about the effects of LPS on the immune system of striped catsh (P. hypophthalmus) is available so far. Therefore, the present study was carried out to assess the effects of LPS from E. coli on immunoregulation and to identify new potential mechanisms of action of LPS in striped catsh. To answer these questions, selected immune parameters and proteome were analyzed in blood and some organs, and peripheral blood mononuclear cells (PBMC), respectively, to nd out new immune markers regulated by LPS. 2. Materials and methods 2.1. Fish and experimental conditions 2.1.1. Fish Farm raised striped catsh juveniles (20e25 g) were acclimatized to laboratory conditions for 15 days and then stocked into 16 composite tanks (500 L) in a ow through freshwater supply system, fed twice a day a commercial feed (30% crude proteins, 2.5 mm, Proconco). Water temperature (30 2  C), dissolved oxygen (5.57 0.01 mg/L) and pH (7.7 0.02) ranged on acceptable values throughout the experimental period. 2.1.2. Experimental design The experimental design included 4 treatments in quadruplicate (70 sh/tank). After acclimation for 15 days, sh were i.p. injected with 0.1 mL of E. coli LPS (Sigma) at 0, 3, 15 and 45 mg LPS/kg on days 1 and 10. Control group received 0.1 mL of phosphate buffer saline (PBS) on the same schedule. Six sh were sampled for blood, plasma, spleen and peripheral blood mononuclear cell (PBMC) on day 14. All samples were kept at 80  C until analysis: blood were used for hematology, plasma was analyzed for cortisol, glucose, lysozyme, complement and total Ig analysis, spleen was used for lysozyme and complement analysis and PBMC were used for proteomics. 2.1.3. Challenge test Bacteria were cultured on Tryptic Soy Agar plate (TSA, Merck) for 48 h at 28  C. The pure colonies of bacteria were checked throughout the shape of colonies and Gram staining under light microscope (Olympus). Then, one colony was collected and put into a 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 and washed 3 times with 0.85% of NaCl solution. The mean colony count used the method of optical density (OD) and OD was adjusted to 0.1 value by spectrophotometer (Thermo spectronic, USA) at 590 nm. Then, this suspension was diluted 1000 times with NaCl solution and injected to the sh. Each of the 4 LPS treatments was divided into two groups (120 sh/group), one group as control injected with 0.1 mL of 0.85% NaCl solution and the second one challenged with 0.1 mL LD50 of E. ictaluri by intra-muscular injection on day 17th. Mortality was recorded daily during 14 days after the challenge test. The head kidney was collected from agonizing sh for bacteria conrmation. 2.2. Bacteria detection 2.2.1. DNA extraction Head kidneys of sh were mixed and grinded with 600 mL lysis buffer (0.5 M NaCl, 0.1 M TriseHCl at pH 8.0, 1% sodium dodecyl sulfate, and 0.1 mM EDTA) and 2.5 mL of proteinase-K solution (40 mg/mL). All mixtures were mixed well and incubated for 15 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 Choloroform:Isoamylalcohol (24:1), the mixtures were centrifuged at 13,000 rpm for 15 min at 4  C. The upper supernatant was collected into new tube. Following addition 600 mL Phenol:Choloroform:Isoamylalcohol (25:24:1), the mixtures were mixed well and centrifuged at 13,000 rpm for 10 min at 4  C. The supernatant was collected and mixed quickly with 500 mL cold isopropanol, and centrifuged at 13,000 rpm for 10 min. The DNA pellets were washed once with 70% ethanol and dried. Before PCR, DNA was dissolved in TE buffer (10 mM TriseHCl and 0.1 mM EDTA at pH 8.0) and stored at e20  C. 2.2.2. PCR amplication PCR reaction was performed to amplify a 407 bp specic DNA fragment of E. ictaluri with forward primer 50 -GTA GCA GGG AGA AAG CTT GC-30 and reverse primer 50 -GAA CGC TAT TAA CGC TCA CAC C-30 [23]. Each 25 mL reaction contained 1.5 mM MgCl2, 0.2 nM dNTPs, 0.4 mM for each primer, 2.5 U of Taq polymerase (Promega) and 100 ng of DNA extracted from sh head kidney. PCR amplication was performed using a thermocycler (Applied Biosystem). The cycling parameters consisted of an initial denaturation at 95  C for 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, and a nal extension at 72  C for 10 min. PCR amplicons were resolved by agarose gel electrophoresis in 1% agarose in 40 mM Triseacetate, 1 mM EDTA, and stained with 1 mg/mL ethidium bromide. 2.3. Hematology and stress parameters 2.3.1. Red blood cell (RBC) counting Total RBC was counted on Neubauer hemocytometer using NattHerrick solution as a diluent stain [24]. First, 10 mL of each blood sample were diluted into 1990 mL of Natt and Herricks solution and mixed gently for at least 3 min. The cell suspension was put into the chamber and allowed to settle for 2e3 min before initiating the count under the light microscope. The RBC was counted in 5 of the 25 small areas. 2.3.2. White blood cell (WBC) counting A small drop of whole blood was smeared on a microscope slide by using a smearing slide (Cover glasses 24 50, Germany). The slide smear was dried quickly, xed in methanol (95%, M1775, Sigma) for 1e2 min and stained with Wrights and Giemsa [25]. Classifying of blood cell types was determined following Supranee [26]. Results of each blood cell were calculated according to Hrubec [27]. 2.3.3. Lysozyme assay The lysozyme assay protocol was adapted from Ellis [28] and Milla [29]. Using sterile potter homogenizers, the spleen lysate was obtained by homogenizing for 30 s, 1 g of spleen in 2.33 mL of buffer containing protease inhibitor cocktail (sodium phosphate buffer 0.067 M and Triton X-100 0.1%/ethanol 95% and acetic acid 1%, v/v, pH 6.2) and then centrifuged at 1000g for 5 min. In microplates 96 wells, the lysozyme activity assay was initiated by mixing 2 mL of spleen lysate or 10 mL of plasma with 130 mL of lyophilized Micrococcus lysodeikticus (Sigma) suspension in phosphate buffer, pH 6.2 (0.6 mg/mL). The difference in absorbance at 450 nm was monitored between 0 and 30 min for plasma (0 and 3 h for the spleen) and used to calculate lysozyme activity in units. One unit represents the amount of lysozyme that caused a 0.001 decrease in absorbance. 2.3.4. Complement assay The alternative complement pathway was assayed using rabbit red blood cells (RRBC, Biomerieux, Craponne, France) as targets

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


following Sunyer and Tort [30] and adapted by Milla et al. [29]. Briey, 10 mL of RRBC suspension (3%) diluted in veronal buffer (Biomerieux) were mixed with serial dilutions of plasma or spleen lysate (60 mL of total volume). After incubation for 100 min at 28  C, the samples were centrifuged at 2000g for 10 min at room temperature. The spontaneous hemolysis was obtained by adding 60 mL of veronal buffer to 10 mL of RRBC. The total lysis was obtained by adding 60 mL of distilled water to RRBC. The absorbance was measured at 405 nm. Appropriate calculations served to estimate complement activity. 2.3.5. Total Ig assay The total immunoglobulin concentration of sample was measured by the method of Siwicki and Anderson [31], modied by Milla et al. [29]. Briey, immunoglobulins were precipitated with 10,000 kDa polyethylene glycol (PEG, Sigma). Serums were mixed with 12% PEG solution (v:v) for 2 h at room temperature under constant shaking. After centrifugation at 1000g for 10 min, the supernatant was collected and assayed for its protein concentration. The total immunoglobulin concentration was calculated by subtracting this value from the total protein concentration in the plasma before precipitation with PEG. 2.3.6. Cortisol assay Plasma cortisol was assayed in duplicate using a cortisol ELISA kit (DRG Instruments GmbH, Germany) and following manufacturers instructions. 2.3.7. Glucose assay Plasma glycemia was measured as follows: plasma (50 mL) was deproteinized by adding 100 mL of perchloride acid 0.33 M, and centrifuged at 3000g (for 10 min at 4  C). Glucose concentration was determined in the supernatant according to the glucose oxidase peroxidase method of Hugget [32]. 2.4. Proteomics 2.4.1. PBMC isolation The PBMC isolation followed the method of Boyum [33] and Pierrard [34]. Briey, dilution of 2.5 mL of heparinized blood and 4 mL of phosphate-buffered saline (PBS) was quickly carried out, 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). The white cells at the interface were collected and washed twice with 1 mL cold PBS by low speed centrifugation (1000g, 7 min, 4  C). An osmotic shock with distilled water was applied to remove residual red blood cells. The suspension was centrifuged as previously indicated and then the cells were suspended in 30 mL DLA buffer (Urea 7 M, thiourea 2 M, TriseHCl 30 mM pH 8.5 and CHAPS 4%) and stored at 80  C for proteomic analysis. 2.4.2. Protein extraction and CyDye labeling The PBMCs in DLA were sonicated 3 times, 10 s on ice and centrifuged for 10 min at 12,000g. The pH of the supernatant was adjusted to 8.5 by addition of the appropriate volume of 50 mM NaOH. Protein concentration was measured using Bio-Rad protein assay. Samples containing 25 mg of solubilized proteins were minimally labeled with 200 pmol of Cyanine dyes following the manufacturers protocols (GE Healthcare). Protein samples from control and LPS conditions were labeled with Cy3 and Cy5, a mixed of equal amounts of protein from control and LPS treatment was labeled with Cy2 and used as internal standard. Labeling was performed on ice for 30 min in the dark and samples were quenched with 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 room temperature. 2.4.3. Separation of proteins by 2D-DIGE Prior to electrofocusing, IPG strips (24 cm, pH 4e7; GE Healthcare) were passively rehydrated overnight with 450 mL of a standard rehydration solution (7 M urea, 2 M thiourea, 2% CHAPS, 0.5% IPG 4e7 buffer, 2% DTT). Sample sets containing the labeled mixtures were then cup-loaded onto the IPG strips and isoelectric focusing was performed with an Ettan IPGphor II isoelectric focusing unit (GE Healthcare). The electrophoresis conditions were as follows: 20  C for a total of 68,000 V-h. Immobilized pH gradient strips were reduced (1% DTT) and then alkalized (2.5% iodoacetamide) 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 mM glycine, 0.1% SDS) and run in an ETTAN TM electrophoresis unit (GE Healthcare) at constant 2 W/gel at 15  C until the blue dye front had run off the bottom of the gels. 2.4.4. Image analysis and statistics Labeled CyDye gels were visualized using a Typhoon 9400 scanner (GE Healthcare) at wavelengths specic to CyDyes and resolution was 100 mm. Images analysis was carried out with DeCyder BVA 5.0 software (GE Healthcare). At rst, the differential in-gel analysis module co-detected and quantied the protein spots in each image using the internal standard sample as a reference to normalize the data. In next step, biological variation analysis was used to calculate ratios between samples and internal standard abundances by performing a gel to gel matching of the internal standard spot maps from each gel. Results were analyzed by oneway ANOVA (p < 0.05). 2.4.5. Mass spectrometry and protein identication For peptide sequencing and protein identication, preparative gels loaded with 200 mg of proteins of mixed samples were run following the protocol described above except they were poststained with 10% krypton overnight after twice 30 min of xation in 40% ethanol and 10% acetic acid. Peptides were analyzed by using nano-LC-ESI-MS/MS maXis UHR-TOF coupled with a 2D-LC Dionex UltiMate 3000 (Bruker, Bremen, Germany). Spots were excised from preparative gels using the Ettan Spot Picker (GE Healthcare), and proteins were digested with trypsin by in-gel digestion. The gel pieces were shrunk with 100% acetonitrile. The proteolytic digestion was performed by the addition of 3 mL of modied trypsin (Promega, Leiden, Netherlands) suspended in 100 mM NH4HCO cold buffer. Proteolysis was performed overnight at 37  C. The supernatants were collected and kept at 20  C prior to analysis. The digests were separated by reverse-phase liquid chromatography using a 75 mm 150 mm reverse-phase Dionex column (Acclaim PepMap 100 C18) in an Ultimate 3000 liquid chromatography system. Mobile phase A was 95% of 0.1% formic acid in water and 5% acetonitrile. Mobile phase B was 0.1% formic acid in acetonitrile. The digest (1 mL) was injected, and the organic content of the mobile phase was increased linearly from 5% B to 40% in 40 min and from 40% B to 100% B in 5 min. The column efuent was connected to an ESI nano Sprayer (Bruker). In survey scan, MS spectra were acquired for 0.5 s in the m/z range between 50 and 2200. The 3 most intense peptides ions 2 or 3 were sequenced. The collisioninduced dissociation (CID) energy was automatically set according to mass to charge (m/z) ratio and charge state of the precursor ion. MaXis and Dionex systems were piloted by Compass HyStar


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

Table 1 Effects of LPS injection on various blood parameters of striped catsh. Parameters White blood cells (103/mm3) Lymphocytes (103cell/mm3) Monocytes (103cell/mm3) Neutrophils (103cell/mm3) Control (0 mg/kg sh) 170.7 25.1a 95.25 14.1a 4.29 1.44a 8.38 3.94b LPS (3 mg/kg sh) 200.3 36.8b 106.6 27.1a 10.70 3.37c 9.40 3.39bc LPS (15 mg/kg sh) 148.7 29.9a 91.30 37.3a 7.26 2.70b 13.88 8.20c LPS (45 mg/kg sh) 167.7 20.9a 94.04 23.1a 2.49 1.15a 2.92 1.60a

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

3.2 (Bruker). Peak lists were created using DataAnalysis 4.0 (Bruker) and saved as XML le for use with ProteinScape 2.0 (Bruker) with Mascot 2.2 as search engine (Matrix Science). Enzyme specicity was set to trypsin, and the maximum number of missed cleavages per peptide was set at one. Carbamidomethylation was allowed 219 as xed modication and oxidation of methionine as variable modication. Mass tolerance for monoisotopic peptide window was 10 ppm and MS/MS tolerance window was set to 0.05 Da. The peak lists were searched against the full National Center for Biotechnology Information non-redundant (NCBInr) database (11,759,209 sequences downloaded on January the 24th 2011). Scaffold (version Scaffold2_06_01, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identications. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.2) and X!Tandem (The GPM,; version 2007.01.01.). Peptide identications were accepted if they could be established at greater than 95% probability as specied by the Peptide Prophet alogarithm [35]. Protein identications were accepted if they could be established at greater than 99 % probability and contained at least 1 identied peptide. Protein probabilities were assigned by the Protein Prophet algorithm [36]. 2.4.6. Statistical analysis The statistical package for social science (SPSS) software (version 13.0) was used to analyze the data. One-way analysis of variance (ANOVA) was performed to compare the stress, hematological and immune parameters between the different LPS treatments (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 signicant difference between control and LPS treated groups. Similarly, the plasma glucose remained low (53.5e 61.6 mg/100 mL) and there were no signicant differences among treatments (p > 0.05). 3.2. Blood parameters Abundance of red blood cells did not show any signicant difference in all treatments (Table 1). In contrast, total white blood cell number was signicantly higher in treatment 3 mg LPS/kg while those of treatments 15 and 45 mg LPS/kg did not differ from the control. Lymphocytes in treatment 3 mg LPS/kg (107 103 cell/mm3) were more abundant, without signicance, than in the other treatments (p > 0.05). The quantity of monocytes also reached higher values in treatment 3 mg LPS/kg, whereas neutrophils were signicantly more abundant in treatment 15 mg/kg when compared with the other LPS treatments (p < 0.05). Both monocyte and neutrophil numbers were strongly reduced by 42% and 62% in treatment 45 mg LPS/kg, compared with the control group, respectively.

3.3. Immune responses 3.3.1. Plasma and spleen lysozyme In control groups, lysozyme activity was low in both plasma and spleen while it increased signicantly (p < 0.05) in sh injected with 3 and 15 mg LPS/kg (Fig. 1). Fish injected with 3 mg LPS/kg displayed signicantly higher values of lysozyme activity compared with the other treatments while the groups treated with 45 mg LPS/kg did not differ signicantly from the control. 3.3.2. Plasma and spleen complement In control sh, complement activity in both plasma and spleen was signicantly lower (p < 0.05) than in sh injected with 3 and 15 mg LPS/kg (Fig. 2). Fish in treatment (3 mg LPS/kg) displayed signicantly higher values of complement activity compared with the other treatments. Fish treated with 45 mg LPS/kg did not differ from the control. 3.3.3. Total immunoglobulins (Ig) The total amount of immunoglobulins in sh 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 of immunoglobulin than the other groups (p < 0.05) while sh injected with 45 mg LPS/kg did not show signicant difference from the control.
140 120 100 80 60 40 20 0 0 3 15 45 [LPS] (mg/kg fish)
Lysozyme in spleen (U/mg protein)

Lysozyme in plasma (U/ml)

b ab a

12 10 8 a 6 4 2 0 0 3

a a



[LPS] (mg/kg fish)

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

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


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

3.4. Proteome analysis A 2D-DIGE analysis was conducted on PBMC 14 days after the injection with LPS. The mean number of spots detected in 6 gels was 3064 212 and the mean number of spots matched across the gels was 1127 156 (Fig. 4). The one-way ANOVA test among all experimental groups revealed that 31 protein spots displayed signicant differences between control and LPS treatment. A total of 10 differentially expressed proteins were identied using nanoLC-ESI-MS/MS and searches in NCBInr database (Table 2). The identied proteins were separated into 3 groups that belong to distinct functional classes. The rst one corresponds to immune response, among which complement component C3, represented by three different spots (352, 1502 and 1705), was signicantly over-expressed in both LPS treatments (3 mg and 15 mg/kg) except the spot 352. Lysozyme C 2 precursor was signicantly overexpressed in spot 1684. The warm temperature protein relate 65 kDa 1 (Wap 65-1) was also over-expressed in spot 337 and the series of Wap 65-2 was represented by 3 different spots (302, 1038 and 1136) showing a contrasted regulation according to the targeted spot. The alpha-2 macroglobulin-3 was signicantly overexpressed in many spots (120, 140, 971 and 1038) and most of them were over-expressed in treatment 3 mg LPS. In contrast, the immunoglobulin heavy chain variable region, presented by spot 357, was under-expressed. The second protein class was related to transport protein, in which transferrin precursor (spots 576 and 1136) was signicantly over-expressed after LPS administration. In mammals, transferin does not only function as a transport protein but is also related to immune response. Finally, the last identied group of proteins was involved in various biological processes including zgc:112265, zgc:56119 and lamin A (spot 140, 913 and
Fig. 4. Representative 2D gel showing the protein expression proles in striped catsh. Identied protein spots were indicated by Decyder software with signicant changes (ANOVA, p < 0.05).

818), that were signicantly under-expressed except zgc:112265 in treatment 3 mg LPS/kg sh. 3.5. Fish mortality after challenged by E. ictaluri The mortality in control groups was 52.46% (Fig. 5). The injected LPS groups displayed a lower mortality throughout the challenge test 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 of bacteria identication showed that E. ictaluri were detected in all bacterial infection samples even if the level of detection was variable among sh (Fig. 6). 4. Discussion In this study, impact of E. coli LPS on innate immune system of striped catsh was assessed. We clearly showed that the innate immunity of striped catsh was responsive to all LPS treatments and the strongest stimulation was reached at the low dose of 3 mg LPS/kg. Plasma cortisol and glucose were measured to evaluate the stress status of sh and they were not affected by LPS treatments. Plasma cortisol is an indicator of the primary stress response and is relevant to evaluate sh welfare [37]. Furthermore, cortisol level is closely linked to many of the adverse consequences of stress including effects on growth [38], reproduction [39], and the immune system [40]. According to Wendelaar Bonga [41] blood glucose levels also increased in sh following exposure to stressful conditions. In pallid sturgeon (Scaphirhynchus albus), the plasma concentration of cortisol after injection with LPS did not differ from that of control sh [42]. In contrast, in yellow perch (Perca avescens) LPS increased the plasma cortisol after 6 h post-injection when compared with sh injected with saline [43]. All together, we hypothesize that the period between LPS administration and plasma collection was too long in our study to observe a putative rise of cortisolemia. In the present study, LPS enhanced total white blood cells, the proportion and quantity of some leucocyte populations but did not alter the abundance of red blood cells. The increase of total leucocytes may be considered as a good indicator of activatory response of sh cellular immunity to LPS stimulation. Among white blood cells, the quantity of monocytes and neutrophils was increased in LPS injected sh and monocytes reached the highest values at a concentration of 3 mg LPS/kg. However, the enhancement in

45 40 c

Total of Ig (mg/ml)

35 30 25 20 15 10 5 0 0 3 15 45 b a ab

LPS (mg/kg fish)

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


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

Table 2 List of identied proteins differentially expressed in PBMC of striped catsh after LPS injection. Spot No Accession No. Protein name Species Matching peptides Theorical pI/Mw (kDa) Fold change 3 vs. 0 mg LPS/kg 1.1 2.4 1.1 2.5 1.5 1.1 1.5 1.23 2.6 1.9 1.3 1.5 1.03 15 vs. 0 mg LPS/kg 1.3 1.2 1.3 1.1 1.1 1.3 1.2 1.6 1.2 1.2 1.01 1.2 1.6

Immune response proteins 352 F5HT30 1502 F5HT30 1705 F5HT30 1684 B5XA65 337 B1NQM9 302 B1NQN2 1038 B1NQN2 1136 B1NQN2 120 Q9PVU3 140 Q9PVU3 971 Q9PVU3 1038 Q9PVU3 357 ACD38374 Transport protein 576 C9W3Q0 1136 C9W3Q0 Other function proteins 140 Q6WQW2 913 Q5RH28 818 Q1WCE8

Complement component C3 Complement component C3 Complement component C3 Lysozyme C 2 precursor Wap 65-1 Wap 65-2 Wap 65-2 Wap 65-2 Alpha-2 macroglobulin-3 Alpha-2 macroglobulin-3 Alpha-2 macroglobulin-3 Alpha-2 macroglobulin-3 Immunoglobulin heavy chain variable region Transferrin precursor Transferrin precursor Zgc:112265 Zgc:56119 Filamin A

Clarias macrocephalus C. macrocephalus C. macrocephalus Salmo salar Ictalurus punctatus Ictalurus punctatus I. punctatus I. punctatus Common carp Common carp Common carp Common carp I. punctatus

2 2 2 2 2 2 2 2 2 2 2 2 1

6.65/147 6.65/147 6.65/147 6.12/16 5.52/54 6.19/51 6.19/51 6.19/51 6.12/87 6.12/87 6.12/87 6.12/87 5.4/16

I. punctatus I. punctatus Danio rerio D. rerio I. punctatus

1 2 3 2 2

6.19/74 6.19/74 6.02/103 7.18/106 5.73/10

2.2 1.2 1.9 2.9 1.04

1.01 1.6 1.2 1.1 1.4

leucocyte population was not due to the increase of lymphocyte number. According to Kozinska and Guz [11], total leucocyte count increased in sh injected with 50 and 1250 mg LPS/sh when compared with control group. Moreover, phagocytic activity was also increased in sh treated with 1250 mg LPS/sh on day 30 after injection. A previous study reported that sh phagocyte activity was enhanced when using antigens from sh pathogenic gramnegative bacteria [44]. Kolman et al. [45] demonstrated an increase in phagocytic index and phagocytosis capacity in bester (Huso huso L.Acipenser ruthenus L.) immunized with Aeromonas salmonicida outer membrane antigens. The granulocytes and monocytes/macrophages are important cells of the non-specic immune pathways in sh, therefore their level tends to increase when using LPS administration [1]. In accordance with the literature, our study supports the statement that the increased number of monocytes in sh treated with LPS is linked to the stimulation of non-specic phagocytosis activity. In sh, LPS often stimulates immune system and protects sh against several sh pathogens [5,6,16,46,]. LPS is also reported to stimulate non-specic immune parameters of animals by enhancing lysozyme gene expression and activity [8]. In this experiment, the increased lysozyme activity in plasma and spleen also demonstrated the efciency of LPS treatment in striped catsh. Similarly to lysozyme, the plasma and spleen complement activity were induced after LPS treatments. Swain [7] reported that complement system can be activated either by classical or
70 60 50 40 30 20 10 0 0

[Mortality (%)

c b ab a

3 15 [LPS] (mg/kg bw)


Fig. 5. Effects of LPS injected in striped catsh on the mortality induced after challenge test with E. ictaluri.

alternative pathways depending upon the smooth or rough type of LPS. The complement system plays key roles in innate and adaptive immunity through mediating phagocytosis, respiratory burst, chemotaxis and cell lysis [47]. Our result thus supports the immunostimulatory effect of LPS in striped catsh. In this study, total immunoglobulins in plasma were increased in low dose of LPS treatments (3 and 15 mg/kg). The O polysaccharide chain in LPS can act as antigenic determinant, therefore, sh respond to LPS sometimes by producing antibody against O side chains [7]. This statement comes from many studies which reported that LPS can enhance humoral immune response in treated sh. Injection of LPS into carp, brown trout, channel catsh, turbot, eels and rainbow trout produced higher antibody titer against Aeromonas bestiarum, Salmonella typhimurium, E. ictaluri, cytophaga-like bacteria, Edwardsiella tarta and Flavobacterium psychrophilum [11,17e20,48,49]. All together, we suggest that LPS is a potent activator of innate and probably acquired immunity in striped catsh. In the current study, proteomic analysis showed that many immune proteins were regulated in PBMC following LPS treatments. Complement component C3 is one of the major protein over-expressed in two LPS treatments. Similarly, changes in the expression level of C3-1, C3-3 and C3-4 mRNAs in rainbow trout liver, spleen and head kidney were reported after 48h of stimulation with LPS [50]. In embryo and larvae of zebrash (Danio rerio) many genes linked to the complement pathway (C3, C1r/s, C4, C6, Bf, MBL and MASP) were differentially expressed [51]. Following the LPS injection to striped sh, over-expression of C3 complement component was related to an increase of complement activity in plasma and spleen. With the past literature, our results thus show that complement pathway is induced at the transcriptional, translational and post-translational levels. The third component of complement (C3) is central to all activation pathways of the complement system [52], which explain the reported relationship between induction of C3 and the whole complement activity in the blood. It is also thought to play a role in the inammation process, leucocyte chemotaxis, B lymphocyte activity, opsonisation of exogen particles and phagocytosis [47,52,53], which translates a positive role of LPS in the immunoregulation.

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


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

An over-expression of lysozyme C 2 precursor was observed in this study, in accordance with the increase of lysozyme activity in plasma and spleen of LPS treated sh. The lysozyme c-type has been identied in sh and was rstly isolated from rainbow trout [54]. It is considered as one of the most important anti-bacterial molecules in sh [46,55,56]. Three genes encoding c-type lysozymes, C1-, C2-, C3-type, were obtained from blue tilapia (Oreochromis aureus) [57]. Lysozyme C2 can be active against a range of Gram-positive and Gram-negative bacteria and it is more effective than lysozyme C1 in killing Gram-negative bacteria. In kelp grouper (Epinephelus bruneus), the expression level of lysozyme c-type was up-regulated mainly in heart, kidney and spleen between 24 and 48 h after the sh were challenged with LPS [58]. Moreover, in Atlantic salmon Salmo salar, mRNA levels of the lysozyme c-type were increased in liver, spleen and intestine 7 days after injection with LPS [8]. Like complement, we deduce from our study that lysozyme is a major target of LPS in striped catsh and both inductions of these immune factors may explain the higher resistance to pathogen infection in LPS treated sh. Additionally, the LPS inuenced the expression of warm temperature acclimation protein 65 kDa (Wap65) in spots 302, 337, 1038 and 1136. Wap65 are glycoproteins present in plasma and liver of teleost sh. Two different isoforms (Wap65-1 and Wap652) have been isolated in several sh species [59,60]. They play an essential role in the acclimation of sh to warm temperatures [61]. Besides this major function in thermal acclimation, they are tightly associated with other biological pathways, especially the acute immune response [62,63]. This is supported by the up-regulated expression of Wap65 genes in sh after LPS injection [63] or experimental infection with pathogenic microorganisms [64,65]. Kikuchi [66] also reported that Wap65 mRNA level increased in the hepatopancreas of the goldsh (Carassius auratus) following LPS administration and Wap65-2 gene was also up-regulated in the liver of channel catsh (Ictalurus punctatus) after bacterial infection with E. ictaluri [59]. Contrary to complement and lysozyme whose functionality has been well characterized, Wap65 may appear as a new candidate of the immune response in teleost sh. In the current study, 2D-DIGE analysis revealed that alpha-2 macroglobulins were identied in several spots (120, 140, 971 and 1038) with over-expression except in spot 140 (treatment 15 mg LPS) and 971 (treatment 3 mg LPS). Alpha-2 macroglobulins are non-pecic protease inhibitors, which mediate the clearance of endogenous and exogenous proteases [67]. Activated form of alpha-2 macroglobulins can also interact with many kinds of cytokines, lectin or lipopolysaccharide, and contribute to the regulation of immune reactions [68,69]. Previous studies in common carp

(Cyprinus carpio) suggested a modest increase in gene transcription of alpha-2 macroglobulins in the liver of carp infected with Trypanoplasma borreli [70]. In mud crab, the injection of LPS was caused by a striking up-regulation of alpha-2 macroglobulin expression in the hemocytes [71]. It was shown that alpha-2 macroglobulin transcription level in amphioxus (Branchiostoma japonicum) increased after acute challenge with LPS, hinting at the clue that alpha-2 macroglobulin may be an immune-relevant molecule involved in acute phase response [72]. Our study is the rst to reveal in striped sh that alpha-2 macroglobulin protein may be a marker of exposure to LPS and putatively implicated in the immune response. In this study, the immunoglobulin heavy chain variable region was under-expressed in 3 mg and 15 mg LPS treatments. In teleost sh, the most prevalent immunoglobulin in plasma is tetrameric IgM. Each subunit is composed by two heavy and light chains that are linked by covalent and noncovalent bonds [73,74]. Several protein spots (576, 1136) were identied as sh transferrin, overexpressed after LPS injection in different doses. Transferrins are a superfamily of iron-binding proteins in vertebrates [75]. Serum transferrin is an important iron transporter. Through the binding and transporting of iron, transferrin participates in immune regulation, antimicrobial and antioxidant activity, cytoprotection and electron transport [76,77]. Furthermore, transferrin-derived synthetic peptide induces highly conserved pro-inammatory response of macrophages [78]. In roughskin sculpin (Trachidermus fasciatus), transferrin mRNA expression was signicantly upregulated in immune organs (skin, blood and spleen) during the LPS challenge and heavy metal exposure experiment [79]. These results suggested that transferrin was involved in the innate immune response in striped sh after LPS stimulation. The proteomic analysis also identied other proteins that are not classically included in the immune response group. Protein zgc:112265 was identied in spot 140 and over-expressed at 3 mg LPS and underexpressed at 15 mg LPS. This protein was novel protein detection in zebrash and up to now, its function is unclear. Another protein zgc:56119 was identied in spot 913 with under expression in 3 mg LPS and over-expression in 15 mg LPS. This protein plays a role of serine-type endopeptidase inhibitor. The lamin A was identied in spot 818 and was under-expressed in both LPS treatments. Filamin A is an actin-binding protein with a well-established role in the cytoskeleton, where it determines cell shape and locomotion by cross-linking actin laments [80]. These proteins may be potential factors of the sh immune response and further studies would be needed to elucidate their functions. In summary, it can be concluded that LPS administration by injection in striped catsh stimulates non-specic immune


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