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Specificity of anti-Vibrio immune response through p38 MAPK and PKC activation in the hemocytes of the mussel Mytilus galloprovincialis
Journal of Invertebrate Pathology 105 (2010) 49–55
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Specificity of anti-Vibrio immune response through p38 MAPK and PKC activation in the hemocytes of the mussel Mytilus galloprovincialis Caterina Ciacci a, Michele Betti a, Barbara Canonico a, Barbara Citterio b, Philippe Roch c, Laura Canesi d,* a
DISUAN, Dipartimento di Scienze dell’Uomo, dell’Ambiente e della Natura, Italy Dipartimento di Scienze Biomolecolari, Università ‘‘Carlo Bo” di Urbino, Italy c JRU Ecosystèmes Lagunaires, CNRS-Université de Montpellier 2, France d Dipartimento di Biologia, Università di Genova, Italy b
a r t i c l e
i n f o
Article history: Received 8 February 2010 Accepted 13 May 2010 Available online 20 May 2010 Keywords: Bivalves V. splendidus LGP32 V. anguillarum Hemocytes Immune responses Cell signaling
a b s t r a c t In mussel (Mytilus sp.) hemocytes, differential functional responses to injection with different types of live and heat-killed Vibrio species have been recently demonstrated. In this work, responses of Mytilus hemocytes to heat-killed Vibrio splendidus LGP32 and the mechanisms involved were investigated in vitro and the results were compared with those obtained with Vibrio anguillarum (ATCC 19264). Adhesion of hemocytes after incubation with bacteria was evaluated by flow cytometry: both total hemocyte counts (THC) and percentage of hemocyte sub-populations were determined in non-adherent cells. Functional parameters such as lysosomal membrane stability, lysozyme release, extracellular ROS production and NO production were evaluated, as well as the phosphorylation state of the stress-activated p38 MAPK and PKC. Neither Vibrio affected total hemocyte adhesion, while both induced similar lysosomal destabilization and NO production. However, V. splendidus decreased adhesion of large granulocytes, induced rapid and persistent lysozyme release and stimulated extracellular ROS production: these effects were associated with persistent activation of p38 MAPK and PKC. In contrast, V. anguillarum decreased adhesion of large semigranular hemocytes and increased that of hyalinocytes, had no effect on the extracellular ROS production, and induced significantly lower lysozyme release and phosphorylation of p-38 MAPK and PKC than V. splendidus. These data reinforced the existence of specific interactions between mussel hemocytes and V. splendidus LGP32 and suggest that this Vibrio strain affects bivalve hemocytes through disregulation of immune signaling. The results support the hypothesis that responses of bivalve hemocytes to different bacterial stimuli may depend not only on the nature of the stimulus, but also on the cell subtype, thus leading to differential activation of signaling components. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Bivalves (such a mussels, clams, oysters. . .) can accumulate large numbers of bacteria as a consequence of their filter-feeding habit. In particular, Vibrio species are very abundant in coastal waters and are commonly isolated from edible bivalve tissues where they are accumulated (Wright et al., 1996) and where they can persist even after depuration processes (Pruzzo et al., 2005). Such a resistance to elimination is particularly evident for Vibrio spp. (such as Vibrio cholerae O1, Vibrio parahaemolyticus, Vibrio anguillarum) (Pruzzo et al., 2005). Persistence of different bacteria in bivalve tissues largely depends on their sensitivity to the bactericidal activity of circulating hemocytes and hemolymph soluble factors, resulting from complex interactions between bacteria, * Corresponding author. Address: Dipartimento di Biologia, Università di Genova, Corso Europa 26, 16132-Genova, Italy. Fax: +39 0103538267. E-mail address: [email protected] (L. Canesi). 0022-2011/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2010.05.010
bacterial components and host cells (Mitta et al., 2000; Canesi et al., 2002a). Host–pathogen interactions have been increasingly investigated in different bivalves, with the aim of understanding the pathogenesis of diseases in cultured and wild populations of species susceptible to infection by certain Vibrio spp. and strains (Choquet et al., 2003; Lambert et al., 2003; Gay et al., 2004; Allam et al., 2006; Labreuche et al., 2006; Mateo et al., 2009). Some strains of Vibrio splendidus have been associated with the ‘summer mortalities’ syndrome of juvenile oysters Crassostrea gigas in France (Lacoste et al., 2001; Gay et al., 2004). On the other hand, the mussel Mytilus is considered to be particularly resistant to Vibrio infection, due to the presence of potent immune defence mechanisms. In Mytilus, studies involving different bacterial species and strains, heterologous cytokines and hormones, led to the identification of the role of conserved components of kinase-mediated transduction pathways, including cytosolic kinases (such as MAPKs-Mitogen Activated Protein Ki-
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nases, PKC-Protein Kinase C, PI-3K phosphatidyl-inositol 3 kinase) and kinase-activated transcription factors (such as STATs, CREB, NF-kB), in the immune response (reviewed in Canesi et al., 2006a). From these data, a general scenario emerged suggesting close similarities in the signaling pathways involved in cell mediated immunity between bivalve and mammalian immunocytes. In particular, both the extent and duration of activation of kinase-mediated cascade components are crucial in determining the hemocyte response to extracellular stimuli (Canesi et al., 2005a). Recent studies have shown that in vivo mussels show differential functional responses to injection of different types of live and heat killed Gram (+) and Gram () bacteria, including Vibrios (Allam et al., 2006; Cellura et al., 2006, 2007; Li et al., 2008; Parisi et al., 2008; Ciacci et al., 2009; Costa et al., 2009). Differential responses in terms of hemocyte gene expression, bacterial clearance, total and differential hemocyte counts, support the existence of specific interactions between hemolymph components and different Vibrio species (Cellura et al., 2006, 2007; Li et al., 2008; Parisi et al., 2008). In particular, differences in hemocyte lysosomal membrane stability and serum lysozyme release, as well as in total hemocyte counts and bactericidal activity of whole hemolymph samples, were observed in response to in vivo injection with heat-killed V. splendidus LGP32 or V. anguillarum (Ciacci et al., 2009). In general, the in vivo effects due to V. splendidus were stronger and/or more persistent than those due to V. anguillarum. In this work, the short-term responses of Mytilus hemocytes to heat-killed V. splendidus LGP32 were further investigated in vitro in comparison to heat-killed V. anguillarum (ATCC 19264). Adhesion of hemocytes in non-adherent cells after incubation with bacteria was evaluated by flow cytometry as total hemocyte counts (THC) and percentage of hemocyte sub-populations. Lysosomal membrane stability, lysozyme release, extracellular ROS production and NO production were also evaluated, as well as the phosphorylation (activation) state of the stress-activated p38 MAPK as PKC, that play a key role in the response to bacterial challenge (Canesi et al., 2002b, 2005a). 2. Materials and methods 2.1. Mussels and bacteria Adult mussels (Mytilus galloprovincialis) 4–5 cm long, sampled from an unpolluted area at Cattolica (RN-Italy) were obtained from SEA (Gabicce Mare, PU) and kept for 1–3 days in static tanks containing aerated artificial sea water (ASW) (1 l/mussel) at 16 °C. Sea water was changed daily. V. splendidus LGP32 is a Gram () marine bacterium isolated from juvenile oysters, Crassostrea gigas, during summer 2001 mortalities (Gay et al., 2004). V. anguillarum was from Institut Pasteur, France (ATCC 19264). Both Vibrios were grown at 20 °C in marine broth (Becton Dickinson) for 24 h, adjusted at 108/ml with phosphate-buffered saline (PBS–NaCl; 0.1 M KH2PO4, 0.1 M K2HPO4, 0.15 M NaCl, pH 7.2–7.4) and boiled for 10 min before storage at 4 °C for a maximum of 6 days. We ensured that no bacteria grew on the agar plates over a period of 8 days at 20 °C. 2.2. Hemolymph collection, preparation of hemocyte monolayers and incubation with bacteria Hemolymph was extracted from the posterior adductor muscle using a sterile 1 ml syringe with a 18 G1/200 needle. With the needle removed, the hemolymph was filtered through sterile gauze and pooled in 50 ml Falcon tubes at 4 °C. Each experiment involved four pools of hemolymph obtained from at least 5 mussels each.
Hemocytes were incubated at 16 °C with different bacteria suspension to yield a final bacteria/hemocyte ratio of 50:1 for different periods of time, as indicated in each experiment. Untreated and control vehicle hemocyte samples were run in parallel. 2.3. Adhesion assay The capacity of hemocytes to adhere to culture dishes during incubation with bacteria was estimated according to a method adapted from Choquet et al. (2003). Briefly, aliquots of 1 ml of hemolymph were incubated with either V. splendidus or V. anguillarum resuspended in 1 ml of sterile ASW to obtain a final ratio of 50 bacteria per hemocyte. Control samples were incubated with 1 ml of sterile ASW. After incubation (3 h at 16 °C), 2 ml of 6% formalin in ASW was added for fixation and flow-cytometry analysis. Supernatants (containing non-adherent cells) were transferred into 5-ml polystyrene tubes (FalconÒ) and analyzed using a FACSCalibur flow cytometer (BD Biosciences). Data acquisition and analysis were performed with BD CellQuest software using the parameters of relative size (FSC) and granularity (SSC). Total hemocyte count (THC) was performed adding Counting beads (Dako Cytocount™) in 50 ll to each tube. Six gates were set up to identify four cell sub-populations, as well as spermatozoa, cell debris, and aggregates, that were not considered for further analysis. Hemocyte sub-populations were characterized according to García-García et al. (2008): large granular (R1), large semigranular (R2), small semigranular (R3) and small agranular (hyaline) (R4) hemocytes. Results were expressed as percentage of non-adherent hemocytes in samples incubated with bacteria with respect to controls. 2.4. Lysosomal membrane stability Lysosomal membrane stability was evaluated by the NRR assay as previously described (Canesi et al., 2003) following the procedure of Lowe et al. (1995). Hemocyte monolayers on glass slides were pre-incubated for 30 min with different bacteria in hemolymph serum. Control hemocytes were run in parallel. Hemocyte monolayers were washed out and incubated with 30 ll of a neutral red (NR) solution (final concentration 40 lg ml1 from a stock solution of NR 20 mg ml1 DMSO). After 15 min, excess dye was washed out, 30 ll of artificial sea water was added, and slides were sealed with a coverslip. Every 15 min, slides were examined under optical microscope and the percentage of cells showing loss of dye from lysosomes in each field was evaluated. For each time point, 10 fields were randomly observed, each containing 8–10 cells. End point of the assay was defined as the time at which 50% of the cells showed sign of lysosomal leaking, i.e. the cytosol becoming red and the cells rounded. Triplicate preparations were performed for each sample. All incubations were carried out at 16 °C. 2.5. Lysozyme release Lysozyme activity in the extracellular medium was evaluated as the ability to lyse a standard suspension of M. lysodeikticus (15 mg/ 100 ml in 66 mM phosphate buffer, pH 6.4) and measured as decrease in absorbance at 450 nm, as previously reported (Canesi et al., 2003). Briefly, hemolymph serum was obtained by centrifugation of whole hemolymph at 200 g for 10 min, and the supernatant was sterilized through a 0.22 lm-pore filter. Lysozyme activity was determined in aliquots of serum incubated with or without bacteria for different periods of time (from 15 to 120 min). Hen egg-white (HEW) lysozyme was used as a concentration reference and lysozyme activity was expressed as HEW lysozyme equivalents/mg protein/ml.
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2.6. Detection of extracellular ROS production Extracellular generation of superoxide by mussel hemocytes was measured using the reduction of cytochrome c according to Pipe et al. (1995) with slight modifications. Hemolymph was extracted into an equal volume of TBS (0.05 M Tris–HCl buffer, pH 7.6, containing 2% NaCl) and protein content was evaluated as described by Harthree (1972) using serum albumin as a standard. Aliquots (500 ll) of hemocyte suspension in triplicate were incubated with 500 ll of cytochrome c solution (75 lM ferricytochrome c in TBS), with or without bacterial suspension. Cytochrome c in TBS was used as blank. Experiments were carried out also in the presence of added extracellular superoxide dismutase (SOD) (300 mU/ml). After 0 and 30 min samples were centrifuged at 1000 rpm for 10 min and the absorption of supernatants was measured at 550 nm. The results were expressed as changes in OD per mg protein. 2.7. Nitric oxide production Nitric oxide (NO) production by mussel hemocytes was evaluated as described previously (Canesi et al., 2006b) using the Griess reaction, which quantifies the nitrite (NO 2 ) content of supernatants. Aliquots of hemocyte suspensions (1.5 ml) were incubated at 16 °C with bacterial suspension (hemocyte/bacteria ratio 50:1) for 0–3 h. Every 60 min, samples were frozen and stored at 80 °C until analysis. Before analysis, samples were thawed and centrifuged (12,000g for 30 min at 4 °C), and supernatants were analyzed for NO 2 content. Aliquots (300 ll) in triplicate were incubated for 10 min in the dark with 300 ll of 1% (wt/v) sulphanilamide in 5% H3PO4 and 300 ll of 0.1% (wt/v) N-(1-naphthy)ethylenediamine dihydrochloride. Sample absorptions were measured at 540 nm, and the molar concentration of NO 2 in the sample was calculated from standard curves generated using known concentrations of sodium nitrite. 2.8. Electrophoresis and Western blotting Hemolymph serum was obtained by centrifugation of whole hemolymph at 200 g for 10 min and the supernatant was sterilized through a 0.22 lm-pore filter. Hemocyte monolayers were prepared as previously described (Canesi et al., 2002b). Each bacterial suspension was diluted in hemolymph serum and 1.5 ml were added to each hemocyte monolayer to yield a final bacteria/hemocyte ratio of 50:1. Control hemocytes received only 1.5 ml of hemolymph serum. Incubations were carried out at 16 °C for different periods of time. Levels of phosphorylated MAPKs and PKC in protein extracts from hemocyte monolayers were determined by SDS–PAGE and Western blotting using phosphospecific antibodies as previously described (Canesi et al., 2002b, 2005a). Supernatants from each culture dish were discarded and the hemocytes were lysed with 0.1 vol. of ice-cold lysis buffer and sonicated for 45 s at 50 W. Samples were boiled for 4 min and then centrifuged for 10 min at 14,000g to remove insoluble debris. Supernatants were mixed 1:1 (v:v) with sample buffer (0.5 M Tris–HCl pH 6.8, 2% SDS, 10% glycerol, 4% 2-mercaptoethanol, 0.05% Bromophenol blue) and samples (normalized to 30 lg of protein before loading) were resolved by 12% (for MAPK) or by 10% (for PKC) SDS–polyacrylamide gel electrophoresis (Laemmli, 1970). Pre-stained molecular mass markers (Bio-Rad) were run on adjacent lanes. The gels were electro-blotted and stained with Coomassie blue according to Towbin et al. (1979). Blots were probed with human recombinant-specific, anti-phospho-p38 MAPK (Thr180/Tyr182, New England Biolabs Inc) and anti-phospho-PKC(pan) (ser660, New England Biolabs Inc.) diluted 1:1000 as primary antibodies, and horseradish perox-
51
idase-conjugated goat anti-rabbit IgG (Sigma) diluted 1:3000 as secondary antibody. Nitrocellulose membranes were stripped and re-probed with rabbit polyclonal anti-actin antibody (Sigma) diluted 1:1000 as loading controls (Canesi et al., 2002b). Immune complexes were visualized using an enhanced chemiluminescence Western blotting analysis system (Amersham–Pharmacia) following the manufacturer’s specifications. Western blot films were digitized (Chemidoc-Biorad) and band optical densities were quantified using a computerized imaging system (QuantityOne). Relative optical densities (arbitrary units) at each time point were normalized against those of each control group. 2.9. Statistical analyses Data are the mean ± SD (triplicates) of at least four independent experiments. Statistical analysis was performed using ANOVA followed by Bonferroni post hoc test. Data from Western blot analyses were analyzed by the Mann–Whitney U test with significance at p < 0.05. 3. Results 3.1. Effects on hemocyte adhesion Hemocyte adhesion was assessed by flow-cytometry analysis of total hemocyte count (THC) in the supernatants of hemocyte monolayers incubated for 3 h with either V. splendidus or V. anguillarum. As shown in Fig. 1A, both Vibrios induced a non-significant increase in THC in non-adherent hemocytes. On the other hand, analysis of the non-adherent hemocyte sub-populations revealed significant differences in samples incubated with Vibrios (Fig. 1B). V. splendidus induced a significant increase in the percentage of large granulocytes (R1, +34%, p < 0.05). Others sub-populations did not increase or decrease significantly. With V. anguillarum, large granulocytes (R1) were not significantly increased, whereas an increase in large semigranular hemocytes (R2, +27%, p < 0.05) and a decrease in small hyaline hemocytes (R4, 25%, p < 0.05) were observed. 3.2. Effects on lysosomal membrane stability (LMS) and lysozyme activity LMS was evaluated by the NR retention time assay. As shown in Fig. 2A, incubation with V. splendidus for 30 min induced a significant decrease in hemocyte LMS with respect to controls (61%, p < 0.01). A similar effect was observed with V. anguillarum (55%, p < 0.01). V. splendidus induced a rapid (since 15 min after contact) and significant lysosomal enzyme release, evaluated as lysozyme activity in the extracellular medium (Fig. 2B). The effect gradually increased until at least 120 min (about +100% with respect to controls, p < 0.05). Increased lysozyme activity was also induced by V. anguillarum peaking at 30 min, followed by a decrease at longer times of incubation. At 60 and 120 min, extracellular lysozyme activity was significantly higher with V. splendidus than with V. anguillarum (+44 and +55% respectively, p < 0.05). 3.3. Effects on ROS and nitrite production The effect of Vibrios on extracellular ROS production was evaluated by the cyt c reduction assay after 30 min of incubation (Fig. 3). V. splendidus induced a significant increase in ROS production (+97%, p < 0.05). ROS production was not significantly inhibited by addition of superoxide dismutase (SOD), indicating that superoxide (O 2 ) was not the main oxygen radical form involved in the
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Fig. 1. Effects of heat-killed V. splendidus LGP32 (V.s.) and V. anguillarum (V.a.) on hemocyte adhesion. C = control. (A) Changes in total hemocyte counts (THC); (B) changes in hemocyte sub-populations. R1 = large granular; R2 = large semigranular; R3 = small semigranular; R4 = small agranular (hyaline) hemocytes. Data (arithmetic mean ± SD) were collected from two aliquots of four replicates for each time point, and expressed as percentages with respect to control, analyzed by ANOVA followed by Bonferroni post hoc test. = p < 0.05.
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Fig. 2. Effects of heat-killed V. splendidus LGP32 (V.s.) and V. anguillarum (V.a.) on hemocyte lysosomal membrane stability (LMS) (A) and extracellular lysozyme activity (B). Data, representing the mean ± SD of four experiments in triplicate, were analyzed by ANOVA followed by Bonferroni post hoc test. For A: a = treatments vs. control (C), p < 0.01; and for B: a = treatments vs. control; b = V. anguillarum vs. V. splendidus, p < 0.05.
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Fig. 3. Effects of heat-killed V. splendidus LGP32 (V.s.) and V. anguillarum (V.a.) on extracellular ROS production, as measured by cyt c reduction. C: Control. SOD: superoxide dismutase. Data presented and statistic calculations as in Fig. 1. = p < 0.05.
response. On the other hand, V. anguillarum did not significantly affect extracellular ROS production. NO production by hemocytes was evaluated as nitrite accumulation. Hemocyte incubation with both V. splendidus and V. anguil-
Fig. 4. Effects of heat-killed V. splendidus LGP32 (V.s.) and V. anguillarum (V.a.) on NO production by mussel hemocytes. Data presented and statistic calculations as in Fig. 1. = p < 0.05.
larum induced a significant and time dependent increase in NO production after 1 h and particularly at 3 h of incubation (Fig. 4). 3.4. Effect on p-38 MAPK and PKC phosphorylation Hemocytes were incubated for different periods of time (from 5 to 60 min) with heat-killed V. splendidus or V. anguillarum and the
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phosphorylation state of p38 MAPK and PKC was evaluated in hemocyte protein extracts by electrophoresis and Western blotting with anti-phospho-MAPK or anti-phospho-PKC(pan) antibodies. Fig. 5A shows representative blots of p-p38 and p-PKC obtained after incubation with V. splendidus or V. anguillarum. Densitometry analysis of phosphorylated protein bands are reported in Fig. 5B for p-p38 and in Fig. 5C and D for p-PKC. V. splendidus induced a time dependent increase in phosphorylation of p38 MAPK (Fig. 5B). The effect was significant since 5 min (1.6-fold the control, p < 0.05), and the level of p-p38 steadily increased up to 2.6-fold the control after 60 min incubation (p < 0.05). A slower and smaller effect was observed with V. anguillarum (1.6 and 1.8-fold the control, respectively, after 15 and 60 min, p < 0.05). In particular, after 60 min, p38 MAPK phosphorylation induced by V. splendidus was significantly higher than that induced by V. anguillarum (+44%, p < 0.05). PKC phosphorylation was evaluated utilizing anti-pan-phospho-PKC antibodies, that recognize two phosphorylated protein bands of 70 and 75 kDa, respectively, in mussel hemocytes (Fig. 5A) as in mammalian cells (Canesi et al., 2006b). V. splendidus induced a large and persistent phosphorylation of both protein bands that was significant from 15 to 60 min (Fig. 5C and D). The level of 75 kDa p-PKC labeled band was maximal at 15 min incubation (2.1-fold the control, p < 0.05). Lower but sustained phosphorylation was observed at longer incubation times (1.6–1.5-fold the controls at 30 and 60 min, p < 0.05) (Fig. 5C). On the other hand, V. anguillarum did not significantly affect phosphorylation of the 75 kDa PKC at any time of incubation. V. splendidus induced a large and time dependent increase in phosphorylation of the 70 kDa PKC isoform (from 2.4 to 3.1-folds the controls at 15 and 60 min, respectively p < 0.05) (Fig. 5D). A smaller effect was induced by V. anguillarum (1.4–1.6-fold the controls at 30 and 60 min, respectively, p < 0.05). The effect of V. splendidus was higher than that of V. anguillarum (from +71% to +93%, at 30 and 60 min, respectively, p < 0.05). No time-dependent changes in band intensities of p-p38 and p-PKC were observed in control samples incubated with hemolymph serum for different periods of time (data not shown).
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A number of studies demonstrated that different alive Vibrio species can elicit distinct responses in bivalve hemocytes both in vitro and in vivo (Choquet et al., 2003; Lambert et al., 2003; Gay et al., 2004; Allam et al., 2006; Labreuche et al., 2006; Cellura et al., 2006, 2007; Li et al., 2008; Parisi et al., 2008; Ciacci et al., 2009; Costa et al., 2009; Mateo et al., 2009). In the present study, the short-term responses of M. galloprovincialis hemocytes to in vitro challenge with heat-killed V. splendidus LGP32 were investigated, and the results compared with those obtained with V. anguillarum. The results demonstrate that V. splendidus LGP32 rapidly induced significant changes in hemocyte adhesion, lysosomal membrane stability, lysosomal enzyme release, extracellular ROS production and NO production. These responses were associated with rapid and persistent activation of immune signaling components (p38 MAPK and PKC). These are the first data on the multiple short-term responses elicited by V. splendidus in M. galloprovincialis hemocytes and on the mechanisms involved. Also V. anguillarum induced lysosomal destabilization and NO production; however, V. anguillarum did not stimulate extracellular ROS production, and showed significantly less intense effects on lysozyme activity and phosphorylation of p-38 MAPK and PKC with respect to V. splendidus. Incubation with either V. splendidus or V. anguillarum did not affect THC in non-adherent cells, indicating that, in our assays, neither Vibrio caused major cell damage. However, significant differences could be appreciated when analyzing the percentage of different non-adherent hemocyte sub-populations. A loss in hemocyte adhesion induced by V. splendidus was mainly ascribed to large granular hemocytes (R1). On the other hand, an increase in non-adherent large semigranular hemocytes (R2) was observed with V. anguillarum, in parallel with a decrease in small hyaline hemocytes (R4). Lysosomal membrane stability is a sensitive indicator of bivalve cellular stress (Lowe et al., 1995), including bacterial challenge (Hauton et al., 2001; Canesi et al., 2005a). Decreases in LMS have been shown to be mediated by activation of different signaling
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Fig. 5. Changes in phosphorylation of immunoreactive p-38 MAPK- and PKC-like proteins in mussel hemocytes incubated with heat-killed V. splendidus LGP32 (V.s.) and V. anguillarum (V.a.) in the presence of hemolymph serum for different periods of time. (A) Representative blots of three independent experiments. Anti-actin blots are shown as loading controls. (B–D) densitometric measurements of blots from four independent experiments presented as mean ± SD. B: p-p38 MAPK; C: 75 kDa p-PKC; D: 70 kDa p-PKC. a = treatments vs. control; b = V. anguillarum vs. V. splendidus; p < 0.05 Mann–Whitney U test.
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components, including p38 MAPK, PKC, PI-3 kinase (Canesi et al., 2004, 2006b). Incubation of hemocyte monolayers with both Vibrios for 30 min induced a significant lysosomal membrane destabilization, as previously observed with living bacteria, including V. cholerae (Canesi et al., 2005a). The lack of differential effects on hemocyte LMS observed between the two Vibrios can partly be explained when considering that the NRR assay was performed on adherent granular hemocytes, and that the assay does not differentiate among granulocyte sub-populations. Both Vibrios induced rapid lysozyme release from the hemocytes. Although the extent of the response was similar to that previously observed with different strains of living V. cholerae and E. coli (Pruzzo et al., 2005), a different time course was observed. The increase in lysozyme release induced by heat-killed V. splendidus and V. anguillarum was persistent up to 120 min, whereas the effect of living bacteria was generally transient (Pruzzo et al., 2005). Such a difference may be partly related to the persistence of bacterial components in the extracellular medium, whereas living bacteria may be more rapidly engulfed by the hemocytes. Moreover, at longer times of incubation, the effect of V. splendidus on lysozyme release was significantly higher than that of V. anguillarum. Since lysozyme release is due to degranulation processes, these results further support the hypothesis that at longer times of incubation V. splendidus could mainly affect mature, large granulocytes. Only granular hemocyte sub-populations were capable of significant ROS production in M. galloprovincialis (García-García et al., 2008). Incubation for 30 min with V. splendidus induced a twofold increase in extracellular oxygen radical production, evaluated by cyt c reduction, whereas no significant effects were observed with V. anguillarum. ROS production was not significantly reduced by adding extracellular SOD, indicating that ROS species other than O 2 were involved. The lack of effect observed with V. anguillarum confirmed data obtained in vitro at short times of incubation in M. galloprovincialis hemocytes with living V. anguillarum (Costa et al., 2009). In M. galloprovincialis all hemocyte sub-populations, including hyalinocytes, were capable of producing NO in response to zymosan (García-García et al., 2008). Our data showed that both Vibrios induced a similar time dependent increase in NO production, evaluated by the Griess reaction. The effect of heat-killed V. anguillarum was lower than that previously observed with living V. anguillarum (Costa et al., 2009). In M. galloprovincialis hemocytes different functional responses can be modulated by multiple kinase-mediated pathways, each signaling component showing a different time course and extent of activation/inactivation depending on the stimulus (Betti et al., 2006; Canesi et al., 2006a,b). MAPKs, in particular the stress-activated MAPKs p38 and JNK (c-Jun terminal kinases), and PKC have been shown to play a crucial role in mediating the responses to live Gram () bacteria (Canesi et al., 2006a). The extent and time course of activation (phosphorylation) of these signaling components are crucial in determining the overall response to bacterial challenge: in particular, a rapid and transient phosphorylation of both p38 and JNK MAPKs is associated with efficient bacterial killing (Canesi et al., 2005a), whereas persistent phosphorylation leads to cell damage and immunotoxic effects (Canesi et al., 2003, 2005b; Betti et al., 2006). Our data showed that heat-killed Vibrio species are able to induce both p38 MAPK and PKC activation in mussel hemocytes. However, V. splendidus induced a stronger phosphorylation of both p38 MAPK and PKC than V. anguillarum, indicating that distinct bacterial components may be responsible for activation of these different kinases. The effects of different pathogenassociated molecular patterns (PAMPs) on functional responses and gene expressions in Mytilus hemocytes have been recently investigated both in vitro and in vivo (Costa et al., 2009). When
the in vitro responses to different stimuli were analyzed in different hemocyte sub-populations, they were shown to depend not only on the nature of the stimulus, but also on the cell subtype (García-García et al., 2008). Different hemocyte subtypes, or hemocyte maturation stages, could differentially express still unidentified receptors whose interactions with distinct bacterial components may lead to activation/inactivation of different signaling pathways. Actually, our data indicate that the differences observed in responses to different Vibrios may be explained in terms of differential activation of cell signaling components. In M. galloprovincialis hemocytes, PKC plays a key role in mediating the activation of the oxidative burst by different stimuli (Canesi et al., 2005b, 2006b). Large granular and semigranular hemocytes have been shown to present the strongest respiratory burst (García-García et al., 2008). The increase in PKC phosphorylation induced by V. splendidus may be responsible for the observed induction of extracellular ROS production that was not observed with V. anguillarum, thus suggesting specific interactions between V. splendidus components and large granulocytes. PKC phosphorylation was evaluated utilizing anti-pan-phospho-PKC antibodies that detect a-, bI-, bII-, d-, e-, and g-isoforms of mammalian PKC only when phosphorylated at a COOH-terminal residue homologous to Ser660 of human PKC-bII, and that recognize two phosphorylated protein bands of 75 and 70 kDa in mussel hemocytes (Canesi et al., 2006b). In these cells, the 75-kDa PKC isoform was previously shown to correspond to PKC-a and -b isoforms, as evaluated with a specific anti-phospho-PKC-a/bII antibody directed towards phosphorylated Thr638/641 residues (Canesi et al., 2006b). Both bands were phosphorylated in response to V. splendidus, as previously observed with living V. cholerae (Canesi et al., 2005a). On the other hand, V. anguillarum did not affect phosphorylation of the 75-kDa PKC and showed a much smaller effect on phosphorylation of the 70-kDa isoform compared with V. splendidus. Meanwhile, the exact role of the different PKC isoforms in mediating the response to different Vibrios requires further investigation. In M. galloprovincialis, the utilization of different kinase inhibitors showed that PI-3K, and possibly ERK MAPK, but not PKC, were involved in mediating NO production (García-García et al., 2008). Moreover, PKA was suggested as the major activating agent of the constitutive NOS (NO synthase) (Novas et al., 2004). Our data indicated that both Vibrios induced similar NO production, this supporting the hypothesis of the involvement of multiple kinases in mediating the response. On the other hand, at longer times of incubation, the sustained phosphorylation of both p38 MAPK and PKC induced by V. splendidus might lead to subsequent impairment of immune functions. Previous data obtained in vivo with heat-killed V. splendidus and V. anguillarum indicated that some effects of V. splendidus were stronger than those elicited by V. anguillarum at longer time post-injection (Ciacci et al., 2009). In particular at 48 h p.i., mussels injected with V. splendidus showed persistent hemocyte lysosomal destabilization and decrease in percentage of small and large granulocytes. In addition, only the mussels injected with V. anguillarum showed recovery of both parameters and increased in vitro bactericidal activity of whole hemolymph samples towards E. coli and V. anguillarum. The present results supported the hypothesis that the distinct effects of the two Vibrios may reflect distinct interactions of each bacterium with different hemocyte sub-populations as previously suggested (Parisi et al., 2008). Overall, the results demonstrate that heat-killed Vibrio species can rapidly induce significant functional responses in Mytilus hemocytes through activation of kinase-mediated signaling components. These data also support the hypothesis that differential responses can be elicited by distinct bacterial components of the
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Vibrio genus. In particular, the results obtained with V. splendidus LGP32, a pathogen associated with oyster larvae and juvenile stage mortalities (Gay et al., 2004), suggest that this Vibrio strain might affect bivalve host cells through disregulation of their signaling pathways. V. splendidus toxicity was shown to be related to thermo labile extracellular metalloproteases (Gomez-Leon et al., 2005). In V. splendidus LGP32, expression of a vms gene coding for a metalloprotease has been recently identified as an important virulence factor (Binesse et al., 2008). Our results, obtained using heat-killed V. splendidus LGP32, indicated that interactions between bacteria and hemolymph components other than secretion of metalloproteases may be also involved in determining the distinct responses observed to different bacterial species. Our results confirmed that the Mediterranean mussel M. galloprovincialis is endowed with a potent innate immune system able to respond to Vibrio infestation and can be usefully used to investigate the mechanisms of Vibrio-host cell interactions in invertebrates. Acknowledgment This work was supported by the EU program IMAQUANIM (FOOD-CT-2005-007103) subcontract CNRS 009252. References Allam, B., Paillard, C., Auffret, M., Ford, S.E., 2006. Effects of the pathogenic Vibrio tapetis on defence factors of susceptible and non-susceptible bivalve species: II. Cellular and biochemical changes following in vivo challenge. Fish Shellfish Immunol. 20, 384–397. Betti, M., Ciacci, C., Lorusso, L.C., Canonico, B., Falcioni, T., Gallo, G., Canesi, L., 2006. Effects of tumor necrosis factor alpha (TNFalpha) on Mytilus hemocytes: role of stress-activated MAP kinases. Biol. Cell. 98, 233–244. Binesse, J., Delsert, C., Saulnier, D., Champomier-Vergès, M.C., Zagorec, M., MunierLehmann, H., Mazel, D., Le Roux, F., 2008. Metalloprotease vsm is the major determinant of toxicity for extracellular products of Vibrio splendidus. Appl. Environ. Microbiol. 74, 7108–7117. Canesi, L., Gavioli, M., Pruzzo, C., Gallo, G., 2002a. Bacteria–hemocyte interactions and phagocytosis in marine bivalves. Microsc. Res. Tech. 57, 469–476. Canesi, L., Betti, M., Ciacci, C., Scarpato, A., Citterio, B., Pruzzo, C., Gallo, G., 2002b. Signalling pathways involved in the physiological response of mussel hemocytes to bacterial challenge: the role of p38 MAP kinase. Dev. Comp. Immunol. 26, 325–334. Canesi, L., Ciacci, C., Betti, M., Scarpato, A., Citterio, B., Pruzzo, C., Gallo, G., 2003. Effects of PCB congeners on the immune function of Mytilus hemocytes: alterations of tyrosine kinase-mediated cell signaling. Aquat. Toxicol. 63, 293– 306. Canesi, L., Ciacci, C., Betti, M., Lorusso, L.C., Marchi, B., Burattini, S., Falcieri, E., Gallo, G., 2004. Rapid effect of 17beta-estradiol on cell signaling and function of Mytilus hemocytes. Gen. Comp. Endocrinol. 136, 58–71. Canesi, L., Betti, M., Ciacci, C., Lorusso, L.C., Gallo, G., Pruzzo, C., 2005a. Interactions between Mytilus hemocytes and different strains of Escherichia coli and Vibrio cholerae O1 El Tor: role of kinase-mediated signalling. Cell. Microbiol. 7, 667– 674. Canesi, L., Lorusso, L.C., Ciacci, C., Betti, M., Gallo, G., 2005b. Effects of the brominated flame retardant tetrabromobisphenol-A (TBBPA) on cell signaling and function of Mytilus hemocytes: involvement of MAP kinases and protein kinase C. Aquat. Toxicol. 75, 277–287. Canesi, L., Betti, M., Ciacci, C., Lorusso, L.C., Pruzzo, C., Gallo, G., 2006a. Cell signalling in the immune response of mussel hemocytes. Inv. Surv. J. 3, 40–49. Canesi, L., Ciacci, C., Lorusso, L.C., Betti, M., Guarnieri, T., Tavolari, S., Gallo, G., 2006b. Immunomodulation by 17b-estradiol in bivalve hemocytes. Am. J. Physiol. Regul. Integr. Comp. Physiol. 291, R664–673. Cellura, C., Toubiana, M., Parrinello, N., Roch, P., 2006. HSP70 gene expression in Mytilus galloprovincialis hemocytes is triggered by moderate heat shock and Vibrio anguillarum, but not by V. Splendidus or Micrococcus lysodeikticus. Dev. Comp. Immunol. 30, 984–997. Cellura, C., Toubiana, M., Parrinello, N., Roch, P., 2007. Specific expression of antimicrobial peptide and HSP70 genes in response to heat-shock and
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several bacterial challenges in mussels. Fish Shellfish Immunol. 22, 340– 350. Choquet, G., Soudant, P., Lambert, C., Nicolas, J.L., Paillard, C., 2003. Reduction of adhesion properties of Ruditapes philippinarum hemocytes exposed to Vibrio tapetis. Dis. Aquat. Organ. 57, 109–116. Ciacci, C., Citterio, B., Betti, M., Canonico, B., Roch, P., Canesi, L., 2009. Functional differential immune responses of Mytilus galloprovincialis to bacterial challenge. Comp. Biochem. Physiol. B, Biochem. Mol. Biol. 153, 365–371. Costa, M.M., Prado-Alvarez, M., Gestal, C., Li, H., Roch, P., Novoa, B., Figueras, A., 2009. Functional and molecular immune response of mediterranean mussel (Mytilus galloprovincialis) haemocytes against pathogen-associated molecular patterns and bacteria. Fish Shellfish Immunol. 26, 515–523. García-García, E., Prado-Alvarez, M., Novoa, B., Figueras, A., Rosales, C., 2008. Immune responses of mussel hemocyte subpopulations are differentially regulated by enzymes of the PI 3-K, PKC, and ERK kinase families. Dev. Comp. Immunol. 32, 637–653. Gay, M., Berthe, F.C.J., Le Roux, F., 2004. Screening of Vibrio isolates to develop an experimental infection model in the Pacific oyster, Crassostrea gigas. Dis. Aquat. Org. 59, 49–56. Gomez-Leon, J., Villamil, L., Lemos, M.L., Novoa, B., Figueras, A., 2005. Isolation of Vibrio alginolyticus and Vibrio splendidus from aquacultured carpet shell clam (Ruditapes decussatus) larvae associated with mass mortalities. Appl. Environ. Microbiol. 71, 98–104. Harthree, E.F., 1972. Determination of protein. A modification of the Lowry method that gives a linear photometric response. Anal. Biochem. 48, 422–427. Hauton, C., Hawkins, L.E., Hutchinson, S., 2001. Response of hemocyte lysosomes to bacterial inoculation in the oysters Ostrea edulis L. and Crassostrea gigas (Thunberg) and the scallop Pecten maximus (L.). Fish Shellfish Immunol. 11, 143–153. Labreuche, Y., Lambert, C., Soudant, P., Boulo, V., Huvet, A., Nicolas, J., 2006. Cellular and molecular hemocyte responses of the Pacific oyster, Crassostrea gigas, following bacterial infection with Vibrio aestuarianus strain 01/32. Microb. Infect. 8, 2715–2724. Lacoste, A., Jalabert, F., Malham, S., Cueff, A., Gelebart, F., Cordevant, C., Lange, M., Poulet, S.A., 2001. A Vibrio splendidus strain is associated with summer mortality of juvenile oysters Crassostrea gigas in the Bay of Morlaix (North Brittany, France). Dis. Aquat. Org. 46, 139–145. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lambert, C., Soudant, P., Choquet, G., Paillard, C., 2003. Measurement of Crassostrea gigas hemocyte oxidative metabolism by flow cytometry and the inhibiting capacity of pathogenic vibrios. Fish Shellfish Immunol. 15, 225–240. Li, H., Parisi, M.G., Toubiana, M., Cammarata, M., Roch, P., 2008. Lysozyme gene expression and hemocyte behaviour in the mediterranean mussel, Mytilus galloprovincialis, after injection of various bacteria or temperature stresses. Fish Shellfish Immunol. 25, 143–152. Lowe, D.M., Fossato, V.U., Depledge, M.H., 1995. Contaminant-induced lysosomal membrane damage in blood cells of mussels Mytilus galloprovincialis from the Venice Lagoon: an in vitro study. Mar. Ecol. Prog. Ser. 129, 89–196. Mateo, D.R., Siah, A., Araya, M.T., Berthe, F.C.J., Johnson, G.R., Greenwood, S.J., 2009. Differential in vivo response of soft-shell clam hemocytes against two strains of Vibrio splendidus: changes in cell structure, numbers and adherence. J. Invert. Pathol. 102, 50–56. Mitta, G., Vandenbulcke, F., Roch, P., 2000. Original involvement of antimicrobial peptides in mussel innate immunity. FEBS Lett. 486, 185–190. Novas, A., Cao, A., Barcia, R., Ramos-Martınez, J.I., 2004. 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Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Specificity of anti-Vibrio immune response through p38 MAPK and PKC activation in the hemocytes of the mussel Mytilus galloprovincialis Caterina Ciacci a, Michele Betti a, Barbara Canonico a, Barbara Citterio b, Philippe Roch c, Laura Canesi d,* a
DISUAN, Dipartimento di Scienze dell’Uomo, dell’Ambiente e della Natura, Italy Dipartimento di Scienze Biomolecolari, Università ‘‘Carlo Bo” di Urbino, Italy c JRU Ecosystèmes Lagunaires, CNRS-Université de Montpellier 2, France d Dipartimento di Biologia, Università di Genova, Italy b
a r t i c l e
i n f o
Article history: Received 8 February 2010 Accepted 13 May 2010 Available online 20 May 2010 Keywords: Bivalves V. splendidus LGP32 V. anguillarum Hemocytes Immune responses Cell signaling
a b s t r a c t In mussel (Mytilus sp.) hemocytes, differential functional responses to injection with different types of live and heat-killed Vibrio species have been recently demonstrated. In this work, responses of Mytilus hemocytes to heat-killed Vibrio splendidus LGP32 and the mechanisms involved were investigated in vitro and the results were compared with those obtained with Vibrio anguillarum (ATCC 19264). Adhesion of hemocytes after incubation with bacteria was evaluated by flow cytometry: both total hemocyte counts (THC) and percentage of hemocyte sub-populations were determined in non-adherent cells. Functional parameters such as lysosomal membrane stability, lysozyme release, extracellular ROS production and NO production were evaluated, as well as the phosphorylation state of the stress-activated p38 MAPK and PKC. Neither Vibrio affected total hemocyte adhesion, while both induced similar lysosomal destabilization and NO production. However, V. splendidus decreased adhesion of large granulocytes, induced rapid and persistent lysozyme release and stimulated extracellular ROS production: these effects were associated with persistent activation of p38 MAPK and PKC. In contrast, V. anguillarum decreased adhesion of large semigranular hemocytes and increased that of hyalinocytes, had no effect on the extracellular ROS production, and induced significantly lower lysozyme release and phosphorylation of p-38 MAPK and PKC than V. splendidus. These data reinforced the existence of specific interactions between mussel hemocytes and V. splendidus LGP32 and suggest that this Vibrio strain affects bivalve hemocytes through disregulation of immune signaling. The results support the hypothesis that responses of bivalve hemocytes to different bacterial stimuli may depend not only on the nature of the stimulus, but also on the cell subtype, thus leading to differential activation of signaling components. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Bivalves (such a mussels, clams, oysters. . .) can accumulate large numbers of bacteria as a consequence of their filter-feeding habit. In particular, Vibrio species are very abundant in coastal waters and are commonly isolated from edible bivalve tissues where they are accumulated (Wright et al., 1996) and where they can persist even after depuration processes (Pruzzo et al., 2005). Such a resistance to elimination is particularly evident for Vibrio spp. (such as Vibrio cholerae O1, Vibrio parahaemolyticus, Vibrio anguillarum) (Pruzzo et al., 2005). Persistence of different bacteria in bivalve tissues largely depends on their sensitivity to the bactericidal activity of circulating hemocytes and hemolymph soluble factors, resulting from complex interactions between bacteria, * Corresponding author. Address: Dipartimento di Biologia, Università di Genova, Corso Europa 26, 16132-Genova, Italy. Fax: +39 0103538267. E-mail address: [email protected] (L. Canesi). 0022-2011/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2010.05.010
bacterial components and host cells (Mitta et al., 2000; Canesi et al., 2002a). Host–pathogen interactions have been increasingly investigated in different bivalves, with the aim of understanding the pathogenesis of diseases in cultured and wild populations of species susceptible to infection by certain Vibrio spp. and strains (Choquet et al., 2003; Lambert et al., 2003; Gay et al., 2004; Allam et al., 2006; Labreuche et al., 2006; Mateo et al., 2009). Some strains of Vibrio splendidus have been associated with the ‘summer mortalities’ syndrome of juvenile oysters Crassostrea gigas in France (Lacoste et al., 2001; Gay et al., 2004). On the other hand, the mussel Mytilus is considered to be particularly resistant to Vibrio infection, due to the presence of potent immune defence mechanisms. In Mytilus, studies involving different bacterial species and strains, heterologous cytokines and hormones, led to the identification of the role of conserved components of kinase-mediated transduction pathways, including cytosolic kinases (such as MAPKs-Mitogen Activated Protein Ki-
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nases, PKC-Protein Kinase C, PI-3K phosphatidyl-inositol 3 kinase) and kinase-activated transcription factors (such as STATs, CREB, NF-kB), in the immune response (reviewed in Canesi et al., 2006a). From these data, a general scenario emerged suggesting close similarities in the signaling pathways involved in cell mediated immunity between bivalve and mammalian immunocytes. In particular, both the extent and duration of activation of kinase-mediated cascade components are crucial in determining the hemocyte response to extracellular stimuli (Canesi et al., 2005a). Recent studies have shown that in vivo mussels show differential functional responses to injection of different types of live and heat killed Gram (+) and Gram () bacteria, including Vibrios (Allam et al., 2006; Cellura et al., 2006, 2007; Li et al., 2008; Parisi et al., 2008; Ciacci et al., 2009; Costa et al., 2009). Differential responses in terms of hemocyte gene expression, bacterial clearance, total and differential hemocyte counts, support the existence of specific interactions between hemolymph components and different Vibrio species (Cellura et al., 2006, 2007; Li et al., 2008; Parisi et al., 2008). In particular, differences in hemocyte lysosomal membrane stability and serum lysozyme release, as well as in total hemocyte counts and bactericidal activity of whole hemolymph samples, were observed in response to in vivo injection with heat-killed V. splendidus LGP32 or V. anguillarum (Ciacci et al., 2009). In general, the in vivo effects due to V. splendidus were stronger and/or more persistent than those due to V. anguillarum. In this work, the short-term responses of Mytilus hemocytes to heat-killed V. splendidus LGP32 were further investigated in vitro in comparison to heat-killed V. anguillarum (ATCC 19264). Adhesion of hemocytes in non-adherent cells after incubation with bacteria was evaluated by flow cytometry as total hemocyte counts (THC) and percentage of hemocyte sub-populations. Lysosomal membrane stability, lysozyme release, extracellular ROS production and NO production were also evaluated, as well as the phosphorylation (activation) state of the stress-activated p38 MAPK as PKC, that play a key role in the response to bacterial challenge (Canesi et al., 2002b, 2005a). 2. Materials and methods 2.1. Mussels and bacteria Adult mussels (Mytilus galloprovincialis) 4–5 cm long, sampled from an unpolluted area at Cattolica (RN-Italy) were obtained from SEA (Gabicce Mare, PU) and kept for 1–3 days in static tanks containing aerated artificial sea water (ASW) (1 l/mussel) at 16 °C. Sea water was changed daily. V. splendidus LGP32 is a Gram () marine bacterium isolated from juvenile oysters, Crassostrea gigas, during summer 2001 mortalities (Gay et al., 2004). V. anguillarum was from Institut Pasteur, France (ATCC 19264). Both Vibrios were grown at 20 °C in marine broth (Becton Dickinson) for 24 h, adjusted at 108/ml with phosphate-buffered saline (PBS–NaCl; 0.1 M KH2PO4, 0.1 M K2HPO4, 0.15 M NaCl, pH 7.2–7.4) and boiled for 10 min before storage at 4 °C for a maximum of 6 days. We ensured that no bacteria grew on the agar plates over a period of 8 days at 20 °C. 2.2. Hemolymph collection, preparation of hemocyte monolayers and incubation with bacteria Hemolymph was extracted from the posterior adductor muscle using a sterile 1 ml syringe with a 18 G1/200 needle. With the needle removed, the hemolymph was filtered through sterile gauze and pooled in 50 ml Falcon tubes at 4 °C. Each experiment involved four pools of hemolymph obtained from at least 5 mussels each.
Hemocytes were incubated at 16 °C with different bacteria suspension to yield a final bacteria/hemocyte ratio of 50:1 for different periods of time, as indicated in each experiment. Untreated and control vehicle hemocyte samples were run in parallel. 2.3. Adhesion assay The capacity of hemocytes to adhere to culture dishes during incubation with bacteria was estimated according to a method adapted from Choquet et al. (2003). Briefly, aliquots of 1 ml of hemolymph were incubated with either V. splendidus or V. anguillarum resuspended in 1 ml of sterile ASW to obtain a final ratio of 50 bacteria per hemocyte. Control samples were incubated with 1 ml of sterile ASW. After incubation (3 h at 16 °C), 2 ml of 6% formalin in ASW was added for fixation and flow-cytometry analysis. Supernatants (containing non-adherent cells) were transferred into 5-ml polystyrene tubes (FalconÒ) and analyzed using a FACSCalibur flow cytometer (BD Biosciences). Data acquisition and analysis were performed with BD CellQuest software using the parameters of relative size (FSC) and granularity (SSC). Total hemocyte count (THC) was performed adding Counting beads (Dako Cytocount™) in 50 ll to each tube. Six gates were set up to identify four cell sub-populations, as well as spermatozoa, cell debris, and aggregates, that were not considered for further analysis. Hemocyte sub-populations were characterized according to García-García et al. (2008): large granular (R1), large semigranular (R2), small semigranular (R3) and small agranular (hyaline) (R4) hemocytes. Results were expressed as percentage of non-adherent hemocytes in samples incubated with bacteria with respect to controls. 2.4. Lysosomal membrane stability Lysosomal membrane stability was evaluated by the NRR assay as previously described (Canesi et al., 2003) following the procedure of Lowe et al. (1995). Hemocyte monolayers on glass slides were pre-incubated for 30 min with different bacteria in hemolymph serum. Control hemocytes were run in parallel. Hemocyte monolayers were washed out and incubated with 30 ll of a neutral red (NR) solution (final concentration 40 lg ml1 from a stock solution of NR 20 mg ml1 DMSO). After 15 min, excess dye was washed out, 30 ll of artificial sea water was added, and slides were sealed with a coverslip. Every 15 min, slides were examined under optical microscope and the percentage of cells showing loss of dye from lysosomes in each field was evaluated. For each time point, 10 fields were randomly observed, each containing 8–10 cells. End point of the assay was defined as the time at which 50% of the cells showed sign of lysosomal leaking, i.e. the cytosol becoming red and the cells rounded. Triplicate preparations were performed for each sample. All incubations were carried out at 16 °C. 2.5. Lysozyme release Lysozyme activity in the extracellular medium was evaluated as the ability to lyse a standard suspension of M. lysodeikticus (15 mg/ 100 ml in 66 mM phosphate buffer, pH 6.4) and measured as decrease in absorbance at 450 nm, as previously reported (Canesi et al., 2003). Briefly, hemolymph serum was obtained by centrifugation of whole hemolymph at 200 g for 10 min, and the supernatant was sterilized through a 0.22 lm-pore filter. Lysozyme activity was determined in aliquots of serum incubated with or without bacteria for different periods of time (from 15 to 120 min). Hen egg-white (HEW) lysozyme was used as a concentration reference and lysozyme activity was expressed as HEW lysozyme equivalents/mg protein/ml.
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2.6. Detection of extracellular ROS production Extracellular generation of superoxide by mussel hemocytes was measured using the reduction of cytochrome c according to Pipe et al. (1995) with slight modifications. Hemolymph was extracted into an equal volume of TBS (0.05 M Tris–HCl buffer, pH 7.6, containing 2% NaCl) and protein content was evaluated as described by Harthree (1972) using serum albumin as a standard. Aliquots (500 ll) of hemocyte suspension in triplicate were incubated with 500 ll of cytochrome c solution (75 lM ferricytochrome c in TBS), with or without bacterial suspension. Cytochrome c in TBS was used as blank. Experiments were carried out also in the presence of added extracellular superoxide dismutase (SOD) (300 mU/ml). After 0 and 30 min samples were centrifuged at 1000 rpm for 10 min and the absorption of supernatants was measured at 550 nm. The results were expressed as changes in OD per mg protein. 2.7. Nitric oxide production Nitric oxide (NO) production by mussel hemocytes was evaluated as described previously (Canesi et al., 2006b) using the Griess reaction, which quantifies the nitrite (NO 2 ) content of supernatants. Aliquots of hemocyte suspensions (1.5 ml) were incubated at 16 °C with bacterial suspension (hemocyte/bacteria ratio 50:1) for 0–3 h. Every 60 min, samples were frozen and stored at 80 °C until analysis. Before analysis, samples were thawed and centrifuged (12,000g for 30 min at 4 °C), and supernatants were analyzed for NO 2 content. Aliquots (300 ll) in triplicate were incubated for 10 min in the dark with 300 ll of 1% (wt/v) sulphanilamide in 5% H3PO4 and 300 ll of 0.1% (wt/v) N-(1-naphthy)ethylenediamine dihydrochloride. Sample absorptions were measured at 540 nm, and the molar concentration of NO 2 in the sample was calculated from standard curves generated using known concentrations of sodium nitrite. 2.8. Electrophoresis and Western blotting Hemolymph serum was obtained by centrifugation of whole hemolymph at 200 g for 10 min and the supernatant was sterilized through a 0.22 lm-pore filter. Hemocyte monolayers were prepared as previously described (Canesi et al., 2002b). Each bacterial suspension was diluted in hemolymph serum and 1.5 ml were added to each hemocyte monolayer to yield a final bacteria/hemocyte ratio of 50:1. Control hemocytes received only 1.5 ml of hemolymph serum. Incubations were carried out at 16 °C for different periods of time. Levels of phosphorylated MAPKs and PKC in protein extracts from hemocyte monolayers were determined by SDS–PAGE and Western blotting using phosphospecific antibodies as previously described (Canesi et al., 2002b, 2005a). Supernatants from each culture dish were discarded and the hemocytes were lysed with 0.1 vol. of ice-cold lysis buffer and sonicated for 45 s at 50 W. Samples were boiled for 4 min and then centrifuged for 10 min at 14,000g to remove insoluble debris. Supernatants were mixed 1:1 (v:v) with sample buffer (0.5 M Tris–HCl pH 6.8, 2% SDS, 10% glycerol, 4% 2-mercaptoethanol, 0.05% Bromophenol blue) and samples (normalized to 30 lg of protein before loading) were resolved by 12% (for MAPK) or by 10% (for PKC) SDS–polyacrylamide gel electrophoresis (Laemmli, 1970). Pre-stained molecular mass markers (Bio-Rad) were run on adjacent lanes. The gels were electro-blotted and stained with Coomassie blue according to Towbin et al. (1979). Blots were probed with human recombinant-specific, anti-phospho-p38 MAPK (Thr180/Tyr182, New England Biolabs Inc) and anti-phospho-PKC(pan) (ser660, New England Biolabs Inc.) diluted 1:1000 as primary antibodies, and horseradish perox-
51
idase-conjugated goat anti-rabbit IgG (Sigma) diluted 1:3000 as secondary antibody. Nitrocellulose membranes were stripped and re-probed with rabbit polyclonal anti-actin antibody (Sigma) diluted 1:1000 as loading controls (Canesi et al., 2002b). Immune complexes were visualized using an enhanced chemiluminescence Western blotting analysis system (Amersham–Pharmacia) following the manufacturer’s specifications. Western blot films were digitized (Chemidoc-Biorad) and band optical densities were quantified using a computerized imaging system (QuantityOne). Relative optical densities (arbitrary units) at each time point were normalized against those of each control group. 2.9. Statistical analyses Data are the mean ± SD (triplicates) of at least four independent experiments. Statistical analysis was performed using ANOVA followed by Bonferroni post hoc test. Data from Western blot analyses were analyzed by the Mann–Whitney U test with significance at p < 0.05. 3. Results 3.1. Effects on hemocyte adhesion Hemocyte adhesion was assessed by flow-cytometry analysis of total hemocyte count (THC) in the supernatants of hemocyte monolayers incubated for 3 h with either V. splendidus or V. anguillarum. As shown in Fig. 1A, both Vibrios induced a non-significant increase in THC in non-adherent hemocytes. On the other hand, analysis of the non-adherent hemocyte sub-populations revealed significant differences in samples incubated with Vibrios (Fig. 1B). V. splendidus induced a significant increase in the percentage of large granulocytes (R1, +34%, p < 0.05). Others sub-populations did not increase or decrease significantly. With V. anguillarum, large granulocytes (R1) were not significantly increased, whereas an increase in large semigranular hemocytes (R2, +27%, p < 0.05) and a decrease in small hyaline hemocytes (R4, 25%, p < 0.05) were observed. 3.2. Effects on lysosomal membrane stability (LMS) and lysozyme activity LMS was evaluated by the NR retention time assay. As shown in Fig. 2A, incubation with V. splendidus for 30 min induced a significant decrease in hemocyte LMS with respect to controls (61%, p < 0.01). A similar effect was observed with V. anguillarum (55%, p < 0.01). V. splendidus induced a rapid (since 15 min after contact) and significant lysosomal enzyme release, evaluated as lysozyme activity in the extracellular medium (Fig. 2B). The effect gradually increased until at least 120 min (about +100% with respect to controls, p < 0.05). Increased lysozyme activity was also induced by V. anguillarum peaking at 30 min, followed by a decrease at longer times of incubation. At 60 and 120 min, extracellular lysozyme activity was significantly higher with V. splendidus than with V. anguillarum (+44 and +55% respectively, p < 0.05). 3.3. Effects on ROS and nitrite production The effect of Vibrios on extracellular ROS production was evaluated by the cyt c reduction assay after 30 min of incubation (Fig. 3). V. splendidus induced a significant increase in ROS production (+97%, p < 0.05). ROS production was not significantly inhibited by addition of superoxide dismutase (SOD), indicating that superoxide (O 2 ) was not the main oxygen radical form involved in the
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B
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Fig. 1. Effects of heat-killed V. splendidus LGP32 (V.s.) and V. anguillarum (V.a.) on hemocyte adhesion. C = control. (A) Changes in total hemocyte counts (THC); (B) changes in hemocyte sub-populations. R1 = large granular; R2 = large semigranular; R3 = small semigranular; R4 = small agranular (hyaline) hemocytes. Data (arithmetic mean ± SD) were collected from two aliquots of four replicates for each time point, and expressed as percentages with respect to control, analyzed by ANOVA followed by Bonferroni post hoc test. = p < 0.05.
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Fig. 2. Effects of heat-killed V. splendidus LGP32 (V.s.) and V. anguillarum (V.a.) on hemocyte lysosomal membrane stability (LMS) (A) and extracellular lysozyme activity (B). Data, representing the mean ± SD of four experiments in triplicate, were analyzed by ANOVA followed by Bonferroni post hoc test. For A: a = treatments vs. control (C), p < 0.01; and for B: a = treatments vs. control; b = V. anguillarum vs. V. splendidus, p < 0.05.
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Fig. 3. Effects of heat-killed V. splendidus LGP32 (V.s.) and V. anguillarum (V.a.) on extracellular ROS production, as measured by cyt c reduction. C: Control. SOD: superoxide dismutase. Data presented and statistic calculations as in Fig. 1. = p < 0.05.
response. On the other hand, V. anguillarum did not significantly affect extracellular ROS production. NO production by hemocytes was evaluated as nitrite accumulation. Hemocyte incubation with both V. splendidus and V. anguil-
Fig. 4. Effects of heat-killed V. splendidus LGP32 (V.s.) and V. anguillarum (V.a.) on NO production by mussel hemocytes. Data presented and statistic calculations as in Fig. 1. = p < 0.05.
larum induced a significant and time dependent increase in NO production after 1 h and particularly at 3 h of incubation (Fig. 4). 3.4. Effect on p-38 MAPK and PKC phosphorylation Hemocytes were incubated for different periods of time (from 5 to 60 min) with heat-killed V. splendidus or V. anguillarum and the
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phosphorylation state of p38 MAPK and PKC was evaluated in hemocyte protein extracts by electrophoresis and Western blotting with anti-phospho-MAPK or anti-phospho-PKC(pan) antibodies. Fig. 5A shows representative blots of p-p38 and p-PKC obtained after incubation with V. splendidus or V. anguillarum. Densitometry analysis of phosphorylated protein bands are reported in Fig. 5B for p-p38 and in Fig. 5C and D for p-PKC. V. splendidus induced a time dependent increase in phosphorylation of p38 MAPK (Fig. 5B). The effect was significant since 5 min (1.6-fold the control, p < 0.05), and the level of p-p38 steadily increased up to 2.6-fold the control after 60 min incubation (p < 0.05). A slower and smaller effect was observed with V. anguillarum (1.6 and 1.8-fold the control, respectively, after 15 and 60 min, p < 0.05). In particular, after 60 min, p38 MAPK phosphorylation induced by V. splendidus was significantly higher than that induced by V. anguillarum (+44%, p < 0.05). PKC phosphorylation was evaluated utilizing anti-pan-phospho-PKC antibodies, that recognize two phosphorylated protein bands of 70 and 75 kDa, respectively, in mussel hemocytes (Fig. 5A) as in mammalian cells (Canesi et al., 2006b). V. splendidus induced a large and persistent phosphorylation of both protein bands that was significant from 15 to 60 min (Fig. 5C and D). The level of 75 kDa p-PKC labeled band was maximal at 15 min incubation (2.1-fold the control, p < 0.05). Lower but sustained phosphorylation was observed at longer incubation times (1.6–1.5-fold the controls at 30 and 60 min, p < 0.05) (Fig. 5C). On the other hand, V. anguillarum did not significantly affect phosphorylation of the 75 kDa PKC at any time of incubation. V. splendidus induced a large and time dependent increase in phosphorylation of the 70 kDa PKC isoform (from 2.4 to 3.1-folds the controls at 15 and 60 min, respectively p < 0.05) (Fig. 5D). A smaller effect was induced by V. anguillarum (1.4–1.6-fold the controls at 30 and 60 min, respectively, p < 0.05). The effect of V. splendidus was higher than that of V. anguillarum (from +71% to +93%, at 30 and 60 min, respectively, p < 0.05). No time-dependent changes in band intensities of p-p38 and p-PKC were observed in control samples incubated with hemolymph serum for different periods of time (data not shown).
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5
A number of studies demonstrated that different alive Vibrio species can elicit distinct responses in bivalve hemocytes both in vitro and in vivo (Choquet et al., 2003; Lambert et al., 2003; Gay et al., 2004; Allam et al., 2006; Labreuche et al., 2006; Cellura et al., 2006, 2007; Li et al., 2008; Parisi et al., 2008; Ciacci et al., 2009; Costa et al., 2009; Mateo et al., 2009). In the present study, the short-term responses of M. galloprovincialis hemocytes to in vitro challenge with heat-killed V. splendidus LGP32 were investigated, and the results compared with those obtained with V. anguillarum. The results demonstrate that V. splendidus LGP32 rapidly induced significant changes in hemocyte adhesion, lysosomal membrane stability, lysosomal enzyme release, extracellular ROS production and NO production. These responses were associated with rapid and persistent activation of immune signaling components (p38 MAPK and PKC). These are the first data on the multiple short-term responses elicited by V. splendidus in M. galloprovincialis hemocytes and on the mechanisms involved. Also V. anguillarum induced lysosomal destabilization and NO production; however, V. anguillarum did not stimulate extracellular ROS production, and showed significantly less intense effects on lysozyme activity and phosphorylation of p-38 MAPK and PKC with respect to V. splendidus. Incubation with either V. splendidus or V. anguillarum did not affect THC in non-adherent cells, indicating that, in our assays, neither Vibrio caused major cell damage. However, significant differences could be appreciated when analyzing the percentage of different non-adherent hemocyte sub-populations. A loss in hemocyte adhesion induced by V. splendidus was mainly ascribed to large granular hemocytes (R1). On the other hand, an increase in non-adherent large semigranular hemocytes (R2) was observed with V. anguillarum, in parallel with a decrease in small hyaline hemocytes (R4). Lysosomal membrane stability is a sensitive indicator of bivalve cellular stress (Lowe et al., 1995), including bacterial challenge (Hauton et al., 2001; Canesi et al., 2005a). Decreases in LMS have been shown to be mediated by activation of different signaling
p-PKC 70Kd (fold change vs control)
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4. Discussion
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Fig. 5. Changes in phosphorylation of immunoreactive p-38 MAPK- and PKC-like proteins in mussel hemocytes incubated with heat-killed V. splendidus LGP32 (V.s.) and V. anguillarum (V.a.) in the presence of hemolymph serum for different periods of time. (A) Representative blots of three independent experiments. Anti-actin blots are shown as loading controls. (B–D) densitometric measurements of blots from four independent experiments presented as mean ± SD. B: p-p38 MAPK; C: 75 kDa p-PKC; D: 70 kDa p-PKC. a = treatments vs. control; b = V. anguillarum vs. V. splendidus; p < 0.05 Mann–Whitney U test.
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components, including p38 MAPK, PKC, PI-3 kinase (Canesi et al., 2004, 2006b). Incubation of hemocyte monolayers with both Vibrios for 30 min induced a significant lysosomal membrane destabilization, as previously observed with living bacteria, including V. cholerae (Canesi et al., 2005a). The lack of differential effects on hemocyte LMS observed between the two Vibrios can partly be explained when considering that the NRR assay was performed on adherent granular hemocytes, and that the assay does not differentiate among granulocyte sub-populations. Both Vibrios induced rapid lysozyme release from the hemocytes. Although the extent of the response was similar to that previously observed with different strains of living V. cholerae and E. coli (Pruzzo et al., 2005), a different time course was observed. The increase in lysozyme release induced by heat-killed V. splendidus and V. anguillarum was persistent up to 120 min, whereas the effect of living bacteria was generally transient (Pruzzo et al., 2005). Such a difference may be partly related to the persistence of bacterial components in the extracellular medium, whereas living bacteria may be more rapidly engulfed by the hemocytes. Moreover, at longer times of incubation, the effect of V. splendidus on lysozyme release was significantly higher than that of V. anguillarum. Since lysozyme release is due to degranulation processes, these results further support the hypothesis that at longer times of incubation V. splendidus could mainly affect mature, large granulocytes. Only granular hemocyte sub-populations were capable of significant ROS production in M. galloprovincialis (García-García et al., 2008). Incubation for 30 min with V. splendidus induced a twofold increase in extracellular oxygen radical production, evaluated by cyt c reduction, whereas no significant effects were observed with V. anguillarum. ROS production was not significantly reduced by adding extracellular SOD, indicating that ROS species other than O 2 were involved. The lack of effect observed with V. anguillarum confirmed data obtained in vitro at short times of incubation in M. galloprovincialis hemocytes with living V. anguillarum (Costa et al., 2009). In M. galloprovincialis all hemocyte sub-populations, including hyalinocytes, were capable of producing NO in response to zymosan (García-García et al., 2008). Our data showed that both Vibrios induced a similar time dependent increase in NO production, evaluated by the Griess reaction. The effect of heat-killed V. anguillarum was lower than that previously observed with living V. anguillarum (Costa et al., 2009). In M. galloprovincialis hemocytes different functional responses can be modulated by multiple kinase-mediated pathways, each signaling component showing a different time course and extent of activation/inactivation depending on the stimulus (Betti et al., 2006; Canesi et al., 2006a,b). MAPKs, in particular the stress-activated MAPKs p38 and JNK (c-Jun terminal kinases), and PKC have been shown to play a crucial role in mediating the responses to live Gram () bacteria (Canesi et al., 2006a). The extent and time course of activation (phosphorylation) of these signaling components are crucial in determining the overall response to bacterial challenge: in particular, a rapid and transient phosphorylation of both p38 and JNK MAPKs is associated with efficient bacterial killing (Canesi et al., 2005a), whereas persistent phosphorylation leads to cell damage and immunotoxic effects (Canesi et al., 2003, 2005b; Betti et al., 2006). Our data showed that heat-killed Vibrio species are able to induce both p38 MAPK and PKC activation in mussel hemocytes. However, V. splendidus induced a stronger phosphorylation of both p38 MAPK and PKC than V. anguillarum, indicating that distinct bacterial components may be responsible for activation of these different kinases. The effects of different pathogenassociated molecular patterns (PAMPs) on functional responses and gene expressions in Mytilus hemocytes have been recently investigated both in vitro and in vivo (Costa et al., 2009). When
the in vitro responses to different stimuli were analyzed in different hemocyte sub-populations, they were shown to depend not only on the nature of the stimulus, but also on the cell subtype (García-García et al., 2008). Different hemocyte subtypes, or hemocyte maturation stages, could differentially express still unidentified receptors whose interactions with distinct bacterial components may lead to activation/inactivation of different signaling pathways. Actually, our data indicate that the differences observed in responses to different Vibrios may be explained in terms of differential activation of cell signaling components. In M. galloprovincialis hemocytes, PKC plays a key role in mediating the activation of the oxidative burst by different stimuli (Canesi et al., 2005b, 2006b). Large granular and semigranular hemocytes have been shown to present the strongest respiratory burst (García-García et al., 2008). The increase in PKC phosphorylation induced by V. splendidus may be responsible for the observed induction of extracellular ROS production that was not observed with V. anguillarum, thus suggesting specific interactions between V. splendidus components and large granulocytes. PKC phosphorylation was evaluated utilizing anti-pan-phospho-PKC antibodies that detect a-, bI-, bII-, d-, e-, and g-isoforms of mammalian PKC only when phosphorylated at a COOH-terminal residue homologous to Ser660 of human PKC-bII, and that recognize two phosphorylated protein bands of 75 and 70 kDa in mussel hemocytes (Canesi et al., 2006b). In these cells, the 75-kDa PKC isoform was previously shown to correspond to PKC-a and -b isoforms, as evaluated with a specific anti-phospho-PKC-a/bII antibody directed towards phosphorylated Thr638/641 residues (Canesi et al., 2006b). Both bands were phosphorylated in response to V. splendidus, as previously observed with living V. cholerae (Canesi et al., 2005a). On the other hand, V. anguillarum did not affect phosphorylation of the 75-kDa PKC and showed a much smaller effect on phosphorylation of the 70-kDa isoform compared with V. splendidus. Meanwhile, the exact role of the different PKC isoforms in mediating the response to different Vibrios requires further investigation. In M. galloprovincialis, the utilization of different kinase inhibitors showed that PI-3K, and possibly ERK MAPK, but not PKC, were involved in mediating NO production (García-García et al., 2008). Moreover, PKA was suggested as the major activating agent of the constitutive NOS (NO synthase) (Novas et al., 2004). Our data indicated that both Vibrios induced similar NO production, this supporting the hypothesis of the involvement of multiple kinases in mediating the response. On the other hand, at longer times of incubation, the sustained phosphorylation of both p38 MAPK and PKC induced by V. splendidus might lead to subsequent impairment of immune functions. Previous data obtained in vivo with heat-killed V. splendidus and V. anguillarum indicated that some effects of V. splendidus were stronger than those elicited by V. anguillarum at longer time post-injection (Ciacci et al., 2009). In particular at 48 h p.i., mussels injected with V. splendidus showed persistent hemocyte lysosomal destabilization and decrease in percentage of small and large granulocytes. In addition, only the mussels injected with V. anguillarum showed recovery of both parameters and increased in vitro bactericidal activity of whole hemolymph samples towards E. coli and V. anguillarum. The present results supported the hypothesis that the distinct effects of the two Vibrios may reflect distinct interactions of each bacterium with different hemocyte sub-populations as previously suggested (Parisi et al., 2008). Overall, the results demonstrate that heat-killed Vibrio species can rapidly induce significant functional responses in Mytilus hemocytes through activation of kinase-mediated signaling components. These data also support the hypothesis that differential responses can be elicited by distinct bacterial components of the
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Vibrio genus. In particular, the results obtained with V. splendidus LGP32, a pathogen associated with oyster larvae and juvenile stage mortalities (Gay et al., 2004), suggest that this Vibrio strain might affect bivalve host cells through disregulation of their signaling pathways. V. splendidus toxicity was shown to be related to thermo labile extracellular metalloproteases (Gomez-Leon et al., 2005). In V. splendidus LGP32, expression of a vms gene coding for a metalloprotease has been recently identified as an important virulence factor (Binesse et al., 2008). Our results, obtained using heat-killed V. splendidus LGP32, indicated that interactions between bacteria and hemolymph components other than secretion of metalloproteases may be also involved in determining the distinct responses observed to different bacterial species. Our results confirmed that the Mediterranean mussel M. galloprovincialis is endowed with a potent innate immune system able to respond to Vibrio infestation and can be usefully used to investigate the mechanisms of Vibrio-host cell interactions in invertebrates. Acknowledgment This work was supported by the EU program IMAQUANIM (FOOD-CT-2005-007103) subcontract CNRS 009252. References Allam, B., Paillard, C., Auffret, M., Ford, S.E., 2006. Effects of the pathogenic Vibrio tapetis on defence factors of susceptible and non-susceptible bivalve species: II. Cellular and biochemical changes following in vivo challenge. Fish Shellfish Immunol. 20, 384–397. Betti, M., Ciacci, C., Lorusso, L.C., Canonico, B., Falcioni, T., Gallo, G., Canesi, L., 2006. Effects of tumor necrosis factor alpha (TNFalpha) on Mytilus hemocytes: role of stress-activated MAP kinases. Biol. Cell. 98, 233–244. Binesse, J., Delsert, C., Saulnier, D., Champomier-Vergès, M.C., Zagorec, M., MunierLehmann, H., Mazel, D., Le Roux, F., 2008. Metalloprotease vsm is the major determinant of toxicity for extracellular products of Vibrio splendidus. Appl. Environ. Microbiol. 74, 7108–7117. Canesi, L., Gavioli, M., Pruzzo, C., Gallo, G., 2002a. Bacteria–hemocyte interactions and phagocytosis in marine bivalves. Microsc. Res. Tech. 57, 469–476. Canesi, L., Betti, M., Ciacci, C., Scarpato, A., Citterio, B., Pruzzo, C., Gallo, G., 2002b. Signalling pathways involved in the physiological response of mussel hemocytes to bacterial challenge: the role of p38 MAP kinase. Dev. Comp. Immunol. 26, 325–334. Canesi, L., Ciacci, C., Betti, M., Scarpato, A., Citterio, B., Pruzzo, C., Gallo, G., 2003. Effects of PCB congeners on the immune function of Mytilus hemocytes: alterations of tyrosine kinase-mediated cell signaling. Aquat. Toxicol. 63, 293– 306. Canesi, L., Ciacci, C., Betti, M., Lorusso, L.C., Marchi, B., Burattini, S., Falcieri, E., Gallo, G., 2004. Rapid effect of 17beta-estradiol on cell signaling and function of Mytilus hemocytes. Gen. Comp. Endocrinol. 136, 58–71. Canesi, L., Betti, M., Ciacci, C., Lorusso, L.C., Gallo, G., Pruzzo, C., 2005a. Interactions between Mytilus hemocytes and different strains of Escherichia coli and Vibrio cholerae O1 El Tor: role of kinase-mediated signalling. Cell. Microbiol. 7, 667– 674. Canesi, L., Lorusso, L.C., Ciacci, C., Betti, M., Gallo, G., 2005b. Effects of the brominated flame retardant tetrabromobisphenol-A (TBBPA) on cell signaling and function of Mytilus hemocytes: involvement of MAP kinases and protein kinase C. Aquat. Toxicol. 75, 277–287. Canesi, L., Betti, M., Ciacci, C., Lorusso, L.C., Pruzzo, C., Gallo, G., 2006a. Cell signalling in the immune response of mussel hemocytes. Inv. Surv. J. 3, 40–49. Canesi, L., Ciacci, C., Lorusso, L.C., Betti, M., Guarnieri, T., Tavolari, S., Gallo, G., 2006b. Immunomodulation by 17b-estradiol in bivalve hemocytes. Am. J. Physiol. Regul. Integr. Comp. Physiol. 291, R664–673. Cellura, C., Toubiana, M., Parrinello, N., Roch, P., 2006. HSP70 gene expression in Mytilus galloprovincialis hemocytes is triggered by moderate heat shock and Vibrio anguillarum, but not by V. Splendidus or Micrococcus lysodeikticus. Dev. Comp. Immunol. 30, 984–997. Cellura, C., Toubiana, M., Parrinello, N., Roch, P., 2007. Specific expression of antimicrobial peptide and HSP70 genes in response to heat-shock and
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