Fish & Shellfish Immunology 36 (2014) 113e119
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Probioticsepathogen interactions elicit differential regulation of cutaneous immune responses in epidermal cells of Atlantic cod Gadus morhua Carlo C. Lazado a, Christopher Marlowe A. Caipang b, * a b
Aquaculture Genomics Research Unit, Faculty of Biosciences and Aquaculture, University of Nordland, Bodø 8049, Norway Institute of Marine Research, Bergen 5817, Norway
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 August 2013 Received in revised form 19 October 2013 Accepted 20 October 2013 Available online 28 October 2013
Little is known on the cutaneous immune responses during probioticsepathogen interactions in fish. Thus, this study employed Atlantic cod primary epidermal (EP) cell cultures as a model to understand this interaction. The probioticsepathogen interactions in the EP cell cultures were elucidated using Vibrio anguillarum 2133 (VA) as the pathogen and two host-derived bacteria (GP21 and GP12) as the probiotics. There was a regional size difference on the EP cells; i.e., EP cells from the dorsal region were significantly larger than the EP cells at the ventral side. VA significantly decreased viability of EP cells. In the presence of probiotics, this inhibition was mitigated. The probiotics reduced VA-induced cellular apoptosis and the probioticsepathogen interactions influenced cellular myeloperoxidase activity during the latter stage of co-incubation. The probioticsepathogen interactions triggered differential regulation of immune-related genes and the effects of the interaction were dependent on the region where the cells were isolated and the length of the co-incubation period. In most cases, the presence of probiotics alone showed no significant change on the mRNA level of immune genes in the EP cells but triggered immunostimulatory activity when incubated with VA. This study showed that the virulence of VA in EP cells could be modulated by host-derived probiotics and the immunomodulatory characteristics of the two candidate probionts advanced their immune-related probiotic potential. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Probiotics Pathogen Mucosal Skin Epidermal cell
1. Introduction The skin of the fish provides a physical barrier between the internal milieu and the external environment [1]. This immunologically and metabolically active tissue functions as a primary line of defense against invading pathogens and plays a significant role on nutrient and solute transport [2,3]. In addition, the diversity and uniqueness of the skin in terms of histological features [4] are crucial on the adaptation of the organism to the physical, chemical and biological properties of the aquatic environment [1]. The epidermis of the skin contains cells with significant role in maintaining the cutaneous immunological barrier especially when the host is exposed to harmful foreign bodies such as bacterial and viral pathogens. The cell populations in the epidermal layer are made up of epithelial cells, mucus cells, club cells and numerous other cell types, and each of them plays important functions on the adaptation of the organism to its environment [1,5,6]. Other than
* Corresponding author. Tel.: þ47 55 23 85 00. E-mail address: [email protected] (C.M.A. Caipang). 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.10.017
these cells, there is also an infiltration of neutrophils and macrophages during injury or parasitic infection [6]. These cells are vital in the production of immunoactive substances such as proteases, lysozymes, mucins, antimicrobial peptides, lectins and immunoglobulins. The different epidermal cell populations in fish had been characterized by immunohistochemistry [1,5e8] and these reports substantiated the uniqueness and diversity of the teleostean integument. However, only few papers [9,10] have discussed the isolation and culture of these cells and their use to understand specific responses in the presence of biological and physical stimuli. The intimate contact of the fish with the environment makes the fish skin an ideal tissue to study mucosal immune response. Relative to systemic immunity, studies on mucosal immunity in fish particularly on how pathogens and natural microbiota interact are limited in scope [1]. Atlantic cod is an economically important aquaculture species especially in the Northern hemisphere. The occurrence of numerous diseases such as vibriosis and furunculosis posed serious threats in the sustainability of the aquaculture industry. The industry is heading to a more sustainable way of combating bacterial diseases by limiting the use of antibiotics and investing more on the
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application of natural antimicrobials such as probiotics. Several trials on the application of terrestrial-derived probiotics have already been conducted in Atlantic cod [11,12]. However, there is a strong demand on using probiotic bacteria isolated from the host environment or from the host itself as these microorganisms are expected to perform at their optimum in their natural environment [13]. There were several host-derived bacteria that have been identified and characterized in cod [14e16] but the mechanisms on how they influence the host were fragmentary and least explored. In developing probiotics for aquaculture, it is necessary that the mechanisms on how the candidate bacteria influence the target species be explored holistically. We have previously isolated and characterized two candidate host-derived probiotics in cod namely GP21 (Pseudomonas sp.) and GP12 (Psychrobacter sp.) [17,18]. These bacteria produced beneficial enzymes and influenced digestive physiology [17,19], stimulated immune responses in the head kidney and intestine [17,20,21] and interfered with the adhesion of bacterial pathogens [20]. Previously it has been shown that the skin of naïve Atlantic cod is immunologically active with significant differences on the expression of immune and stress genes depending on the origin of the skin tissue [22]. In addition, unraveling mucus proteome revealed a significant number of immune competent molecules [23]. Up to this date, there are no reports discussing how the interactions of pathogens and probiotics influence cutaneous immunity, particularly the responses of epidermal cells in Atlantic cod. GP21 and GP12 are two candidate bacteria with immune-related beneficial features as shown by their immunomodulatory capabilities in the head kidney and gut of cod [18,20,21]. However, their influence on the skin is yet to be explored. Thus, this study was conducted to characterize the immunomodulatory features of these bacteria in the skin of cod using primary epidermal (EP) cell cultures as a model. Further, the study determined the responses of EP cells in the presence of a pathogen and explored how these hostderived bacteria influenced cutaneous immune responses during probioticsepathogen interactions. 2. Materials and methods 2.1. Bacterial identity, origin and culture conditions The two probiotics used in this study were isolated from the intestinal tract of Atlantic cod and were identified as Pseudomonas sp. (GP21) and Psychrobacter sp. (GP12) [17]. Both bacteria were previously shown to be potential probiotics for Atlantic cod [17e 21]. The pathogenic strain of Vibrio anguillarum 2133 (VA) [24] was obtained from the Institute of Marine Research, Bergen, Norway and further maintained in the culture collection of the Aquatic Animal Health Research Group Laboratory. Unless specified, the bacteria were cultured in Tryptic soy broth (TSB; pH 7.5 0.2, Fluka Biochemika, Germany) supplemented with 1.5 NaCl (w/v) and incubated at 15 C for 24e48 h. Glycerol stocks of the bacteria were maintained at 80 C. 2.2. Isolation and culture of Atlantic cod primary epidermal cells The Atlantic cod that were used in this study were obtained from Codfarmers AS, Bodø, Norway and were maintained at the Mørkvedbukta Research Station of the University of Nordland, Norway. Only apparently healthy fish weighing 300e400 g and free from different pathogens (bacteria, viruses and parasites) were used in this experiment. The experimental procedures were in accordance to the guidelines set by the National Animal Research Authority (FDU) in Norway.
The primary epidermal (EP) cells of Atlantic cod were isolated following the modified procedures of Tsutsui et al. [10] and Kilemade et al. [9]. Six fish were anesthesized using MS222 (80 mg mL1, Sigma Aldrich, Germany) and were killed with a strong cranial blow. The skin from both the dorsal and ventral region was dissected aseptically and was washed with cold 1 Phosphate Buffer Saline (PBS) with PenicillineStreptomycin (PS) antibiotic supplement. The dissected skin were carefully checked free of muscle and fibrous tissue to prevent fibroblastic growth during culture. The skin sections were cut into small pieces (approximately 5 mm 5 mm), placed in 20 ml L-15 medium containing 5% (v/v) trypsin-EDTA solution (Sigma Aldrich) and vortexed at 1800 rpm for 20 min at 4 C. Thereafter, the dissociated cell-skin mixture was centrifuged at 800 g for 10 min at 4 C. The supernatant was decanted and the isolated cells were suspended in L-15 medium with PS and 10% (v/v) Fetal Bovine Serum (Sigma Aldrich) followed by three washings. The viability of the cells was determined by staining with 4% tryphan blue. Two ml cell suspension was seeded onto 12-well polypropylene cell culture plate (BD FalconTM, New Jersey, USA) and the cells were allowed to attach for 24 h at15 C. All EP cultures were washed and the culture medium was changed prior to their applications in all subsequent analyses. A separate experiment was conducted to determine the viability of the EP cells during a 5-day culture period with change of medium every other day. The cells were visualized and photographed by Leica Confocal Microsystems (Wetzlar, Germany). For cell size measurement, 5 microscopic fields were photographed and the photos were converted to jpeg format for further analysis. The converted photos were uploaded to ImageJ Image Visualization Software (http://rsb.info.nih.gov/nih-image/), and the scaling was standardized for all photos. At least 50 cells were measured from each photographed field. Six fish were used in this morphometric experiment. 2.3. Probioticsepathogen interactions Three hours prior to bacterial exposure, the media of the cultured EP cells was removed and replaced with freshly prepared L-15 medium free of antibiotics. There were 5 bacterial exposure treatments: GP21 only, GP12 only, VA only, GP21 þ VA and GP12 þ VA. About 100 ml of the bacterial suspension with an initial concentration of 106 CFU ml1 were seeded into the cultured EP cells. The samples were collected at 3- and 24 h post incubation (hpi). The effects of bacterial exposure on the EP cells were assessed by determining cellular responses and by profiling the expression of immune-related genes. For gene expression studies, about threefourths of the media-bacterial suspension mixture was aspirated and 1 mL of TRIzolÒ (Invitrogen, USA) was added to the remaining cell-suspension mixture. The cells were scraped and stored at 80 C prior to RNA extraction. 2.4. Assays for cellular response Cellular viability was assayed using 3 e (4,5 dimethylthiazol-2yl)-2-5-diphenyl tetrazolium bromide (MTT) with modifications for Atlantic cod [17]. Bacterial stimulation studies were conducted with the cells cultured in 96-well plate. Freshly prepared MTT solution (0.5 mg mL1) was added to the bacteria-exposed EP cells at a rate of 20 ml per well. The plate was incubated for 30 min at 15 C, devoid of light. The crystals formed in the well were dissolved with 125 ml solution containing 100 ml of dimethylsulfoxide and 25 ml of glycine buffer (0.1 M glycine, 0.1 M NaCl [pH 10.5]) and incubated further for 10 min at 15 C. The absorbance was read at 570 nm using a spectrophotometer (Fluostar Optima, BMG Labtech GmbH, Offenburg, Germany).
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The apoptotic activity of cells exposed to both the probiotics and the pathogen were detected by quantifying caspase-3 activity using Caspase-3 Colorimetric Assay Kit (BioVision, CA, USA) which was standardized previously for Atlantic cod [20]. Protein concentration was determined using the fluorometer, QubitÒ following the manufacturer’s protocol (Molecular Probes, Eugene, OR, USA) and the samples were standardized to contain approximately 150 mg ml1 protein for the assay. The cellular myeloperoxidase was determined by lysing the bacteria-exposed cells with 60 ml of 0.2% cetyltrimethylammonium bromide (CTAB, Sigma) and assayed following previously reported protocol [25] with modifications. The lysed EP cells were added with 35 ml of fresh L-15 medium containing 20 mM 3,30 ,5,50 -tetramethyl benzidine hydrochloride (Sigma) and 5 mM H2O2, and were incubated for 2 min. The reaction was stopped by adding 35 ml of 4 M sulphuric acid and the absorbance was determined at 450 nm using Fluostar spectrophotomer.
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of the probiotics to modulate and stimulate immune responses of the EP cells during bacterial interaction was evident in their influences on cellular proliferation and humoral defenses during coinfection. In addition, the molecular responses during bacterial interactions revealed that regional cutaneous response profile existed in cod supporting our initial report on the differences of immunological features between the dorsal and the ventral regions of the skin in cod [22]. 3.1. Epidermal (EP) cells from the dorsal and ventral skin We were able to isolate at least 2.0 107 cells ml1 from both regions of the skin and no significant intra-fish variation was observed. The mixture was made up of epithelial, mucus and club cells similar to what had been observed in previous studies employing the isolation method used in this study [10]. Interestingly, we have observed a substantial number of goblet-like mucus cells from the mixed EP cell cultures. The mucous cell densities in
2.5. Isolation of total RNA and gene expression analyses The infected cells were lysed in TRIzolÒ reagent by repetitive pipetting and the total RNA was extracted following the manufacturer’s protocol. qScriptÔ cDNA SuperMix (Quanta Biosciences, USA) was used for first strand cDNA synthesis with an initial RNA input of 2 mg. The cDNAs were quantified using QubitÒ dsDNA BR Assay Kit (Molecular Probes, Eugene, OR, USA), normalized at a concentration of 10 mg mL1 in 1 TE buffer and stored at 20 C for subsequent PCR reactions. Four groups of immune-related genes namely bacterial defense, cytokines, cell-mediated immunity and oxidative stress were used for expression studies. The primers used in the study were obtained from previous published papers [22,24,26e28]. The PCR amplification was carried out using the C1000Ô Thermal Cyler (BioRad, Norway) following this standardized protocol: initial denaturation at 95 C for 3 min, followed by 35 cycles of denaturation at 95 C for 30 s, annealing at 53 C for 30 s and elongation at 72 C for 1 min and a final elongation step at 72 C for 5 min to complete the amplification reaction. The relative expression level of each gene was quantified by densitometric evaluation using Image J software (http://rsb.info.nih.gov/nih-image/) and normalized relative to the expression of b-actin. 2.6. Statistical analyses and data presentation The data are presented as mean SD of values from 6 individual fish. The statistical analyses were performed using Prism 5 (GraphPad Software, Inc., La Jolla, USA). The difference between cell sizes was analyzed using Student’s t-test for independent samples. For gene expression studies, the assumptions for ANOVA were checked prior to carrying out the analyses. If the data did not follow the Gaussian distribution, transformation was conducted prior to parametric one-way ANOVA; thereafter the differences between the groups were checked by Tukey’s Multiple Comparison Test. KruskaleWallis test followed by Dunn’s Multiple Comparison Test was used for non-parametric data. In all cases, significance was noted at P < 0.05. The heatmap of gene expression was designed using TIGR Multiexperiment Viewer (MeV; Dana-Farber Cancer Institute, USA). 3. Results and discussion This study attempted the isolation and in vitro culture of epidermal (EP) cells in Atlantic cod. The primary EP cell cultures appeared to be a promising model to study cutaneous immune responses during probioticsepathogen interactions. The capability
Fig. 1. Epidermal (EP) cells of Atlantic cod in culture. EP cells from the A) dorsal region were significantly larger than the EP cells from the B) ventral region. The inserted photos (A0 and B0 ) are magnified fields showing the size distinction of cod EP cells. mc ¼ mucus-like cell; epc ¼ epithelial cell. Scale bar ¼ 500 mm.
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Fig. 2. Responses elicited by the EP cells during probioticsepathogen interactions were assessed by A) cellular viability, B) apoptotic activity and C) myeloperoxidase activity. Column bars with same letters indicate no significant difference (P > 0.05) Legend: CON ¼ Control; GP21 ¼ Pseudomonas sp.; GP12 Psychrobacter sp.; VA ¼ Vibrio anguillarum 2133; GP21-VA ¼ Vibrio anguillarum 2133 and Pseudomonas sp.; and GP12-VA ¼ Vibrio anguillarum 2133 and Psychrobacter sp. N ¼ 6.
skin act as a sensitive first line of immune defense parameter in fish [29]. They are responsible for the production of mucosal layer [30] and could also be involved in mucus production during transfer of material from the secondary circulatory system [31]. The substantial presence of these cells in the culture could be related to the initial findings of the immune-rich potential of Atlantic cod skin and its mucus [22,23]. The previous report on the skin structure of cod focused on skin pigmentation [7] and failed to mention the different cell populations comprising the epidermal layer. It would be interesting to account in the future the different cell populations
of the epidermal layer of the cod skin. The percentage of surviving EP cells exceeded 80% after 24 hpi and the viability could be maintained for 5 days with the change of medium every other day. Though dead cells and tissue debris were observed, they were removed gradually by daily washing and changing of the culture media. Remarkably, there was a significant regional difference on the size of the isolated EP cells in cod. To our knowledge, this regional difference on EP cell sizes has not been previously accounted in other teleost species. The EP cells from the dorsal region (average cell size: w268 mm) of the skin were significantly
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larger than the cells from the ventral region (average cell size: w205 mm) (Fig. 1). Since we did not have a specific count of each cell population, the regional size difference observed was a general account regardless of cell type as the population of both regions was observed and assumed to be homogenous. This regional difference could be a strong point to further explore and characterize the cell types and populations of the epidermal skin of Atlantic cod. In the naïve cod, immune features of the dorsal and ventral regions varied significantly with the latter region showing significantly higher expression of immune genes even at the basal physiological conditions [22]. The regional difference on cell size noted in this study could substantiate the differences that were previously observed because cell size could influence different physiological responses of the cell.
cells (Fig. 3C). There were instances that the presence of bacteria decreased myeloperoxidase activity and this was typified by GP21 at 3 hpi, regardless of cutaneous region. There was a significant increase on myeloperoxidase activity in VA-exposed EP cells except in the dorsal region at 3 hpi. This could be a protective reaction of the cells as the bacteria were recognized as virulent. Longer exposure of the EP cells to both probiotics and pathogen significantly elevated myeloperoxidase activity. The presence of probiotics could have an additive influence on the myeloperoxidasemediated reaction of the cells by stimulating enzyme production for a more effective immune response against a pathogen.
3.2. Stimulated activities of the EP cells during probioticsepathogen interactions
The modulated expression profile during probioticsepathogen interactions corroborated with the differences observed between cutaneous regions on the expression of immune- and stress-related genes in the skin of Atlantic cod [22]. Bactericidal/permeabilityincreasing protein (BPI) and lipopolysaccharide-binding protein (LBP) is an important member of the innate immune system both in mammals and in Atlantic cod [35,36]. In most cases, the expression of bpi/lbp in both cutaneous regions was not significantly affected by probiotics exposure except at 24 hpi in the dorsal EP cells (Fig. 3A). bpi/lbp was significantly up-regulated in the presence of VA at the dorsal EP cells regardless of exposure time. Interestingly, the expression of bpi/lbp remained unchanged in the ventral EP cells during bacterial interactions in contrast to the significant upregulation observed in the dorsal EP cells. Lysozyme has been shown to be highly expressed in the skin of a naïve Atlantic cod suggesting that it is an essential component of the skin as an immunologically active tissue [22]. The exposure of dorsal EP cells to probiotics did not elicit significant up-regulation of lyz expression. On the other hand, VA elicited significant elevation of lyz expression and the expression was significantly promoted during
The effect of bacterial exposure on cellular viability was assessed by determining the activity of the EP cells through time and the bacteria-induced changes were observed to be temporal and regional-dependent. The host-derived probiotics did not elicit significant change on the viability of EP cells from both regions at both time points (Fig. 2A). The exposure of EP cells with VA significantly reduced the viability of EP cells on both cutaneous regions except that the effect on ventral EP cells was observed only at 24 hpi. The simultaneous exposure of EP cells with pathogens and probiotics significantly mitigated the inhibitory feature of the former on cellular viability except at 3 hpi in the dorsal region. The presence of the probiotics could have elicited a protective effect on the EP cells by counteracting the inhibition of cellular proliferation induced by VA. Apoptosis has duality in its feature as it can be both a damaging pathway induced by a pathogen to enter the host or as a protective mechanism against the pathogen [32]. The caspase-3 activity of the cells showed an inverse profile relative to cellular proliferation and this was typified when EP cells were exposed to VA (Fig. 2B). This implies that the inhibition observed in the proliferation of the cells could be due to the induction of apoptosis in the presence of the pathogen. VA was previously found to have the capability to induce cellular apoptosis in the intestinal epithelial cells of cod [20] and the same virulence mechanism was observed in the present study with the EP cultures. It was also found that the pathogenicity of VA in the head kidney leukocytes of sea bass was due to the downregulation of caspases [33]. The differences on how cellular apoptosis in fish are influenced by VA could be dependent on the species, types of cell as well as the pathogenic strains of the bacteria. There were no significant changes on caspase-3 activity when the EP cells were exposed to probiotics alone indicating that the EP cells recognized the bacteria as non-pathogenic and natural residents of the host thus the reaction was at the basal level. Interestingly, there were two evident apoptosis-related reactions during bacterial interactions. The probiotics influenced VA-induced apoptosis by diminishing the effect thereby protecting the cells. This was observed in the dorsal EP cells at 24 hpi and in the ventral EP cells at 3 hpi. On the other hand, the significant increase in the caspase-3 activity observed in the dorsal EP cells at 3 hpi and in the ventral EP cells at 24 hpi could be explained in two possibilities: 1) the VA-mediated apoptosis was strong enough for the probiotics to act upon or 2) the protective mechanism of the probiotics was through the activation of apoptosis. The production of peroxidase enzyme during bacterial interaction was measured by myeloperoxidase activity which is also related to neutrophil antimicrobial activity [34]. In most cases, the presence of bacteria elevated the myeloperoxidase activity of the EP
3.3. Differential regulation of immune genes during probioticse pathogen interactions
Fig. 3. Heatmap of the expression of A) bacterial defense (bpi/lbp and lyz) and, B) cytokine (il1b) genes during probioticsepathogen interactions. Legend: CON ¼ Control; GP21 ¼ Pseudomonas sp.; GP12 Psychrobacter sp.; VA ¼ Vibrio anguillarum 2133; GP21VA ¼ Vibrio anguillarum 2133 and Pseudomonas sp.; GP12-VA ¼ Vibrio anguillarum 2133 and Psychrobacter sp.; D3 ¼ dorsal 3 hpi; D24 ¼ dorsal 24 hpi; V3 ¼ ventral 3 hpi; and V24 ¼ ventral 24 hpi. Expression values of target genes were expressed relative to bactin expression. Asterisk (*) indicates that the expression is significantly different from the control group. N ¼ 6.
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bacterial interactions. The ventral EP cells showed a clear profile of lyz up-regulation during bacterial exposure and interactions where elevated transcript levels were observed at both exposure times. The antibacterial responses of the EP cells through the upregulation of bpi/lbp and lyz supported the initial observation that these molecules are important in the bacterial defense system of Atlantic cod skin [22]. These molecules are believed to be contributors of the antibacterial activity observed in the cutaneous extract of cod [37]. Further, the capability of EP cells to respond against the invading pathogen was enhanced in the presence of probiotics through the modulation of the bacterial defense genes. These host-derived bacteria have been previously observed to have stimulatory effect on the antibacterial activity of head kidney leukocytes of cod [21]. The present study supported this immunostimulatory feature and the beneficial characteristic is not limited to only one immune-related tissue. Inflammation is one of the many immune responses of an organism to exogenous stimuli, specifically those of biological threats with il-1b as one of the genes involved in the pro-inflammatory cascade. The up-regulation of il-1b has been demonstrated as a pro-inflammatory response of the fish skin to a pathogen [38,39]. Even though there was a significant up-regulation of il-1b expression in the EP cells exposed to probiotics, the expression was merely instantaneous as no significant expression was observed at 24 hpi in both cutaneous regions (Fig. 3C). This expression profile was similar to what have been observed with these probiotics in head kidney leukocytes [21]. This corroborates with the previous reports that this gene is responsible for the early pro-inflammatory response in fish [40]. Regardless of cutaneous region, exposure of EP cells with VA triggered significant up-regulation of il-1b. This significant up-regulation was also observed during probioticse pathogen interactions in the dorsal region at both time points. Interestingly during probioticsepathogen interactions in the ventral EP cells, the mRNA level of il-1b was brought to the basal level implying the capability of probiotics to reduce inflammatory reactions induced by the presence of VA. The expression of two cell-mediated immunity genes, nccrp and gzma, is shown in Fig. 4A. The expression of nccrp was significantly up-regulated in the presence of probiotics, pathogen and during their interactions and these changes were only observed at the latter stage of exposure. NCCRP-1 is an important molecule in target cell recognition and activation of NCC in cod [41] and it was not surprising to observe the significant up-regulation during pathogen exposure. The significant up-regulation in the dorsal EP cells with the probiotics at 24 hpi could be explained that the probiotics played as an additive factor in eliciting and priming a better immune response during probioticsepathogen interactions. This influence was regional-dependent because changes were only observed in the dorsal EP cells. We have not observed any significant and remarkable changes on gzma expression during bacterial exposure. Traditionally, gzma are potent members of serine proteases family that are induced mostly during virus infection [42]. It is interesting to explore the possible functions of gzma in cod as it was previously been observed to be up-regulated in the spleen in the presence of a bacterial antigen [26]. It might have anti-viral and antibacterial properties and could be tissue-dependent on its immunological significance. The presence of VA elicited significant up-regulation of cat and sod in the EP cells (Fig. 4B). In particular, there was a significant expression of cat in both cutaneous regions and this effect was observed until 24 hpi. Bacterial antigen could influence the expression of cat as previously observed in the up-regulation of this gene in the spleen of vaccinated cod [26]. This suggests that cat is an important gene on the oxidative stress response of cod during VA exposure and eventual infection. The same pattern of
Fig. 4. Heatmap of the expression of A) cell-mediated immunity (nccrp and gzma) and, B) oxidative stress (cat and sod) genes during probioticsepathogen interactions. Legend: CON ¼ Control; GP21 ¼ Pseudomonas sp.; GP12 Psychrobacter sp.; VA ¼ Vibrio anguillarum 2133; GP21-VA ¼ Vibrio anguillarum 2133 and Pseudomonas sp.; GP12VA ¼ Vibrio anguillarum 2133 and Psychrobacter sp.; D3 ¼ dorsal 3 hpi; D24 ¼ dorsal 24 hpi; V3 ¼ ventral 3 hpi; and V24 ¼ ventral 24 hpi. Expression values of target genes were expressed relative to b-actin expression. Asterisk (*) indicates that the expression is significantly different from the control group. N ¼ 6.
expression was observed with sod transcription during bacterial exposure. It was interesting to observe that the induction of oxidative stress gene expression during VA infection could be reduced in the presence of the probiotics. This was evidently observed during bacterial interactions wherein the expression of cat and sod showed no significant difference with the control group. This suggests a logical possibility that these bacteria have the capability to reduce oxidative stress induced by VA infection. Besides their immune-related and nutritional importance, probiotics could also protect the fish from chemical, physical and biological stress [43]. GP21 and GP12 exhibit these probiotic features and need to be explored in future studies. 4. Conclusions This study supported and provided additional knowledge on the immunological properties of Atlantic cod skin and its components. Further, the immunostimulatory activities of two candidate probionts, GP21 and GP12 and their capacity to modulate immune responses of EP cells against VA infection expanded their probiotic potentials. These results substantiate the broad range of probiotic properties of these host-derived bacteria from cod. The primary culture of Atlantic cod EP cells can be a good model in studying cutaneous immune responses to bacteria and can be applied to other pathogens including viruses and parasites. However, additional studies should be conducted focusing on the identification and characterization of the different cell populations in the skin. By doing so, there will be sufficient data as to which particular cell types in the skin that are directly interacting during probioticse pathogen exposure of the fish.
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Acknowledgment This study was financed by the project “Mucosal immune system of Atlantic cod e creating a knowledge base” (Project Number 184703 funded by the Research Council of Norway). Hilde Ribe is also acknowledged for her technical help.
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[21] Lazado CC, Caipang CMA, Gallage S, Brinchmann MF, Kiron V. Expression profiles of genes associated with immune response and oxidative stress in Atlantic cod, Gadus morhua head kidney leukocytes modulated by live and heat-inactivated intestinal bacteria. Comp Biochem Physiol e B Biochem Mol Biol 2010;155:249e55. [22] Caipang CMA, Lazado CC, Brinchmann MF, Rombout JHWM, Kiron V. Differential expression of immune and stress genes in the skin of Atlantic cod (Gadus morhua). Comp Biochem Physiol e Part D: Genomics Proteomics 2011;6:158e62. [23] Rajan B, Fernandes JMO, Caipang CMA, Kiron V, Rombout JHWM, Brinchmann MF. Proteome reference map of the skin mucus of Atlantic cod (Gadus morhua) revealing immune competent molecules. Fish Shellfish Immunol 2011;31:224e31. [24] Caipang CMA, Hynes N, Puangkaew J, Brinchmann MF, Kiron V. Intraperitoneal vaccination of Atlantic cod, Gadus morhua with heat-killed Listonella anguillarum enhances serum antibacterial activity and expression of immune response genes. Fish Shellfish Immunol 2008;24:314e22. [25] Quade MJ, Roth JA. A rapid, direct assay to measure degranulation of bovine neutrophil primary granules. Vet Immunol Immunopathol 1997;58:239e48. [26] Caipang CMA, Brinchmann MF, Kiron V. Profiling gene expression in the spleen of Atlantic cod, Gadus morhua upon vaccination with Vibrio anguillarum antigen. Comp Biochem Physiol e B Biochem Mol Biol 2009;153:261e7. [27] Caipang CMA, Lazado CC, Brinchmann MF, Kiron V. Infection-induced changes in expression of antibacterial and cytokine genes in the gill epithelial cells of Atlantic cod, Gadus morhua during incubation with bacterial pathogens. Comp Biochem Physiol e B Biochem Mol Biol 2010;156:319e25. [28] Caipang CMA, Lazado CC, Brinchmann MF, Kiron V. Transcription of selected immune-related genes in spleen cells of cod, Gadus morhua following incubation with alginic acid and b-glucan. J Exp Mar Biol Ecol 2012;416e417: 202e7. [29] Olafsdottir SH, Buchmann K. Dexamethasone treatment affects skin mucous cell density in Gyrodactylus derjavini infected Salmo salar. J Helminthol 2004;78:87e90. [30] Harris JE, Hunt S. The fine structure of the epidermis of two species of salmonid fish, the Atlantic salmon (Salmo salar L.) and the brown trout (Salmo trutta L.). Cell Tissue Res 1975;163:535e43. [31] Easy RH, Ross NW. Changes in Atlantic salmon (Salmo salar) epidermal mucus protein composition profiles following infection with sea lice (Lepeophtheirus salmonis). Comp Biochem Physiol Part D: Genomics Proteomics 2009;4:159e67. [32] Grassmé H, Jendrossek V, Gulbins E. Molecular mechanisms of bacteria induced apoptosis. Apoptosis 2001;6:441e5. [33] Sepulcre MP, Sarropoulou E, Kotoulas G, Meseguer J, Mulero V. Vibrio anguillarum evades the immune response of the bony fish sea bass (Dicentrarchus labrax L.) through the inhibition of leukocyte respiratory burst and down-regulation of apoptotic caspases. Mol Immunol 2007;44:3751e7. [34] Swain P, Dash S, Sahoo PK, Routray P, Sahoo SK, Gupta SD, et al. Non-specific immune parameters of brood Indian major carp Labeo rohita and their seasonal variations. Fish Shellfish Immunol 2007;22:38e43. [35] Levy O. Antimicrobial proteins and peptides: anti-infective molecules of mammalian leukocytes. J Leukoc Biol 2004;76:909e25. [36] Solstad T, Stenvik J, Jørgensen TØ. mRNA expression patterns of the BPI/LBP molecule in the Atlantic cod (Gadus morhua L. Fish Shellfish Immunol 2007;23:260e71. [37] Ruangsri J, Fernandes JMO, Brinchmann M, Kiron V. Antimicrobial activity in the tissues of Atlantic cod (Gadus morhua L.). Fish Shellfish Immunol 2010;28: 879e86. [38] Gonzalez SF, Buchmann K, Nielsen ME. Real-time gene expression analysis in carp (Cyprinus carpio L.) skin: inflammatory responses caused by the ectoparasite Ichthyophthirius multifiliis. Fish Shellfish Immunol 2007;22:641e50. [39] Gonzalez SF, Huising MO, Stakauskas R, Forlenza M, Lidy Verburg-van Kemenade BM, Buchmann K, et al. Real-time gene expression analysis in carp (Cyprinus carpio L.) skin: inflammatory responses to injury mimicking infection with ectoparasites. Develop Comp Immunol 2007;31:244e54. [40] Secombes CJ, Wang T, Hong S, Peddie S, Crampe M, Laing KJ, et al. Cytokines and innate immunity of fish. Develop Comp Immunol 2001;25:713e23. [41] Seppola M, Robertsen B, Jensen I. The gene structure and expression of the non-specific cytotoxic cell receptor protein (NCCRP-1) in Atlantic cod (Gadus morhua L.). Comp Biochem Physiol e B Biochem Mol Biol 2007;147:199e208. [42] Wernersson S, Reimer JM, Poorafshar M, Karlson U, Wermenstam N, Bengtén E, et al. Granzyme-like sequences in bony fish shed light on the emergence of hematopoietic serine proteases during vertebrate evolution. Develop Comp Immunol 2006;30:901e18. [43] Mohapatra S, Chakraborty T, Kumar V, Deboeck G, Mohanta KN. Aquaculture and stress management: a review of probiotic intervention. J Anim Physiol Anim Nutr 2013;97:405e30.
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Full length article
Probioticsepathogen interactions elicit differential regulation of cutaneous immune responses in epidermal cells of Atlantic cod Gadus morhua Carlo C. Lazado a, Christopher Marlowe A. Caipang b, * a b
Aquaculture Genomics Research Unit, Faculty of Biosciences and Aquaculture, University of Nordland, Bodø 8049, Norway Institute of Marine Research, Bergen 5817, Norway
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 August 2013 Received in revised form 19 October 2013 Accepted 20 October 2013 Available online 28 October 2013
Little is known on the cutaneous immune responses during probioticsepathogen interactions in fish. Thus, this study employed Atlantic cod primary epidermal (EP) cell cultures as a model to understand this interaction. The probioticsepathogen interactions in the EP cell cultures were elucidated using Vibrio anguillarum 2133 (VA) as the pathogen and two host-derived bacteria (GP21 and GP12) as the probiotics. There was a regional size difference on the EP cells; i.e., EP cells from the dorsal region were significantly larger than the EP cells at the ventral side. VA significantly decreased viability of EP cells. In the presence of probiotics, this inhibition was mitigated. The probiotics reduced VA-induced cellular apoptosis and the probioticsepathogen interactions influenced cellular myeloperoxidase activity during the latter stage of co-incubation. The probioticsepathogen interactions triggered differential regulation of immune-related genes and the effects of the interaction were dependent on the region where the cells were isolated and the length of the co-incubation period. In most cases, the presence of probiotics alone showed no significant change on the mRNA level of immune genes in the EP cells but triggered immunostimulatory activity when incubated with VA. This study showed that the virulence of VA in EP cells could be modulated by host-derived probiotics and the immunomodulatory characteristics of the two candidate probionts advanced their immune-related probiotic potential. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Probiotics Pathogen Mucosal Skin Epidermal cell
1. Introduction The skin of the fish provides a physical barrier between the internal milieu and the external environment [1]. This immunologically and metabolically active tissue functions as a primary line of defense against invading pathogens and plays a significant role on nutrient and solute transport [2,3]. In addition, the diversity and uniqueness of the skin in terms of histological features [4] are crucial on the adaptation of the organism to the physical, chemical and biological properties of the aquatic environment [1]. The epidermis of the skin contains cells with significant role in maintaining the cutaneous immunological barrier especially when the host is exposed to harmful foreign bodies such as bacterial and viral pathogens. The cell populations in the epidermal layer are made up of epithelial cells, mucus cells, club cells and numerous other cell types, and each of them plays important functions on the adaptation of the organism to its environment [1,5,6]. Other than
* Corresponding author. Tel.: þ47 55 23 85 00. E-mail address: [email protected] (C.M.A. Caipang). 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.10.017
these cells, there is also an infiltration of neutrophils and macrophages during injury or parasitic infection [6]. These cells are vital in the production of immunoactive substances such as proteases, lysozymes, mucins, antimicrobial peptides, lectins and immunoglobulins. The different epidermal cell populations in fish had been characterized by immunohistochemistry [1,5e8] and these reports substantiated the uniqueness and diversity of the teleostean integument. However, only few papers [9,10] have discussed the isolation and culture of these cells and their use to understand specific responses in the presence of biological and physical stimuli. The intimate contact of the fish with the environment makes the fish skin an ideal tissue to study mucosal immune response. Relative to systemic immunity, studies on mucosal immunity in fish particularly on how pathogens and natural microbiota interact are limited in scope [1]. Atlantic cod is an economically important aquaculture species especially in the Northern hemisphere. The occurrence of numerous diseases such as vibriosis and furunculosis posed serious threats in the sustainability of the aquaculture industry. The industry is heading to a more sustainable way of combating bacterial diseases by limiting the use of antibiotics and investing more on the
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application of natural antimicrobials such as probiotics. Several trials on the application of terrestrial-derived probiotics have already been conducted in Atlantic cod [11,12]. However, there is a strong demand on using probiotic bacteria isolated from the host environment or from the host itself as these microorganisms are expected to perform at their optimum in their natural environment [13]. There were several host-derived bacteria that have been identified and characterized in cod [14e16] but the mechanisms on how they influence the host were fragmentary and least explored. In developing probiotics for aquaculture, it is necessary that the mechanisms on how the candidate bacteria influence the target species be explored holistically. We have previously isolated and characterized two candidate host-derived probiotics in cod namely GP21 (Pseudomonas sp.) and GP12 (Psychrobacter sp.) [17,18]. These bacteria produced beneficial enzymes and influenced digestive physiology [17,19], stimulated immune responses in the head kidney and intestine [17,20,21] and interfered with the adhesion of bacterial pathogens [20]. Previously it has been shown that the skin of naïve Atlantic cod is immunologically active with significant differences on the expression of immune and stress genes depending on the origin of the skin tissue [22]. In addition, unraveling mucus proteome revealed a significant number of immune competent molecules [23]. Up to this date, there are no reports discussing how the interactions of pathogens and probiotics influence cutaneous immunity, particularly the responses of epidermal cells in Atlantic cod. GP21 and GP12 are two candidate bacteria with immune-related beneficial features as shown by their immunomodulatory capabilities in the head kidney and gut of cod [18,20,21]. However, their influence on the skin is yet to be explored. Thus, this study was conducted to characterize the immunomodulatory features of these bacteria in the skin of cod using primary epidermal (EP) cell cultures as a model. Further, the study determined the responses of EP cells in the presence of a pathogen and explored how these hostderived bacteria influenced cutaneous immune responses during probioticsepathogen interactions. 2. Materials and methods 2.1. Bacterial identity, origin and culture conditions The two probiotics used in this study were isolated from the intestinal tract of Atlantic cod and were identified as Pseudomonas sp. (GP21) and Psychrobacter sp. (GP12) [17]. Both bacteria were previously shown to be potential probiotics for Atlantic cod [17e 21]. The pathogenic strain of Vibrio anguillarum 2133 (VA) [24] was obtained from the Institute of Marine Research, Bergen, Norway and further maintained in the culture collection of the Aquatic Animal Health Research Group Laboratory. Unless specified, the bacteria were cultured in Tryptic soy broth (TSB; pH 7.5 0.2, Fluka Biochemika, Germany) supplemented with 1.5 NaCl (w/v) and incubated at 15 C for 24e48 h. Glycerol stocks of the bacteria were maintained at 80 C. 2.2. Isolation and culture of Atlantic cod primary epidermal cells The Atlantic cod that were used in this study were obtained from Codfarmers AS, Bodø, Norway and were maintained at the Mørkvedbukta Research Station of the University of Nordland, Norway. Only apparently healthy fish weighing 300e400 g and free from different pathogens (bacteria, viruses and parasites) were used in this experiment. The experimental procedures were in accordance to the guidelines set by the National Animal Research Authority (FDU) in Norway.
The primary epidermal (EP) cells of Atlantic cod were isolated following the modified procedures of Tsutsui et al. [10] and Kilemade et al. [9]. Six fish were anesthesized using MS222 (80 mg mL1, Sigma Aldrich, Germany) and were killed with a strong cranial blow. The skin from both the dorsal and ventral region was dissected aseptically and was washed with cold 1 Phosphate Buffer Saline (PBS) with PenicillineStreptomycin (PS) antibiotic supplement. The dissected skin were carefully checked free of muscle and fibrous tissue to prevent fibroblastic growth during culture. The skin sections were cut into small pieces (approximately 5 mm 5 mm), placed in 20 ml L-15 medium containing 5% (v/v) trypsin-EDTA solution (Sigma Aldrich) and vortexed at 1800 rpm for 20 min at 4 C. Thereafter, the dissociated cell-skin mixture was centrifuged at 800 g for 10 min at 4 C. The supernatant was decanted and the isolated cells were suspended in L-15 medium with PS and 10% (v/v) Fetal Bovine Serum (Sigma Aldrich) followed by three washings. The viability of the cells was determined by staining with 4% tryphan blue. Two ml cell suspension was seeded onto 12-well polypropylene cell culture plate (BD FalconTM, New Jersey, USA) and the cells were allowed to attach for 24 h at15 C. All EP cultures were washed and the culture medium was changed prior to their applications in all subsequent analyses. A separate experiment was conducted to determine the viability of the EP cells during a 5-day culture period with change of medium every other day. The cells were visualized and photographed by Leica Confocal Microsystems (Wetzlar, Germany). For cell size measurement, 5 microscopic fields were photographed and the photos were converted to jpeg format for further analysis. The converted photos were uploaded to ImageJ Image Visualization Software (http://rsb.info.nih.gov/nih-image/), and the scaling was standardized for all photos. At least 50 cells were measured from each photographed field. Six fish were used in this morphometric experiment. 2.3. Probioticsepathogen interactions Three hours prior to bacterial exposure, the media of the cultured EP cells was removed and replaced with freshly prepared L-15 medium free of antibiotics. There were 5 bacterial exposure treatments: GP21 only, GP12 only, VA only, GP21 þ VA and GP12 þ VA. About 100 ml of the bacterial suspension with an initial concentration of 106 CFU ml1 were seeded into the cultured EP cells. The samples were collected at 3- and 24 h post incubation (hpi). The effects of bacterial exposure on the EP cells were assessed by determining cellular responses and by profiling the expression of immune-related genes. For gene expression studies, about threefourths of the media-bacterial suspension mixture was aspirated and 1 mL of TRIzolÒ (Invitrogen, USA) was added to the remaining cell-suspension mixture. The cells were scraped and stored at 80 C prior to RNA extraction. 2.4. Assays for cellular response Cellular viability was assayed using 3 e (4,5 dimethylthiazol-2yl)-2-5-diphenyl tetrazolium bromide (MTT) with modifications for Atlantic cod [17]. Bacterial stimulation studies were conducted with the cells cultured in 96-well plate. Freshly prepared MTT solution (0.5 mg mL1) was added to the bacteria-exposed EP cells at a rate of 20 ml per well. The plate was incubated for 30 min at 15 C, devoid of light. The crystals formed in the well were dissolved with 125 ml solution containing 100 ml of dimethylsulfoxide and 25 ml of glycine buffer (0.1 M glycine, 0.1 M NaCl [pH 10.5]) and incubated further for 10 min at 15 C. The absorbance was read at 570 nm using a spectrophotometer (Fluostar Optima, BMG Labtech GmbH, Offenburg, Germany).
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The apoptotic activity of cells exposed to both the probiotics and the pathogen were detected by quantifying caspase-3 activity using Caspase-3 Colorimetric Assay Kit (BioVision, CA, USA) which was standardized previously for Atlantic cod [20]. Protein concentration was determined using the fluorometer, QubitÒ following the manufacturer’s protocol (Molecular Probes, Eugene, OR, USA) and the samples were standardized to contain approximately 150 mg ml1 protein for the assay. The cellular myeloperoxidase was determined by lysing the bacteria-exposed cells with 60 ml of 0.2% cetyltrimethylammonium bromide (CTAB, Sigma) and assayed following previously reported protocol [25] with modifications. The lysed EP cells were added with 35 ml of fresh L-15 medium containing 20 mM 3,30 ,5,50 -tetramethyl benzidine hydrochloride (Sigma) and 5 mM H2O2, and were incubated for 2 min. The reaction was stopped by adding 35 ml of 4 M sulphuric acid and the absorbance was determined at 450 nm using Fluostar spectrophotomer.
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of the probiotics to modulate and stimulate immune responses of the EP cells during bacterial interaction was evident in their influences on cellular proliferation and humoral defenses during coinfection. In addition, the molecular responses during bacterial interactions revealed that regional cutaneous response profile existed in cod supporting our initial report on the differences of immunological features between the dorsal and the ventral regions of the skin in cod [22]. 3.1. Epidermal (EP) cells from the dorsal and ventral skin We were able to isolate at least 2.0 107 cells ml1 from both regions of the skin and no significant intra-fish variation was observed. The mixture was made up of epithelial, mucus and club cells similar to what had been observed in previous studies employing the isolation method used in this study [10]. Interestingly, we have observed a substantial number of goblet-like mucus cells from the mixed EP cell cultures. The mucous cell densities in
2.5. Isolation of total RNA and gene expression analyses The infected cells were lysed in TRIzolÒ reagent by repetitive pipetting and the total RNA was extracted following the manufacturer’s protocol. qScriptÔ cDNA SuperMix (Quanta Biosciences, USA) was used for first strand cDNA synthesis with an initial RNA input of 2 mg. The cDNAs were quantified using QubitÒ dsDNA BR Assay Kit (Molecular Probes, Eugene, OR, USA), normalized at a concentration of 10 mg mL1 in 1 TE buffer and stored at 20 C for subsequent PCR reactions. Four groups of immune-related genes namely bacterial defense, cytokines, cell-mediated immunity and oxidative stress were used for expression studies. The primers used in the study were obtained from previous published papers [22,24,26e28]. The PCR amplification was carried out using the C1000Ô Thermal Cyler (BioRad, Norway) following this standardized protocol: initial denaturation at 95 C for 3 min, followed by 35 cycles of denaturation at 95 C for 30 s, annealing at 53 C for 30 s and elongation at 72 C for 1 min and a final elongation step at 72 C for 5 min to complete the amplification reaction. The relative expression level of each gene was quantified by densitometric evaluation using Image J software (http://rsb.info.nih.gov/nih-image/) and normalized relative to the expression of b-actin. 2.6. Statistical analyses and data presentation The data are presented as mean SD of values from 6 individual fish. The statistical analyses were performed using Prism 5 (GraphPad Software, Inc., La Jolla, USA). The difference between cell sizes was analyzed using Student’s t-test for independent samples. For gene expression studies, the assumptions for ANOVA were checked prior to carrying out the analyses. If the data did not follow the Gaussian distribution, transformation was conducted prior to parametric one-way ANOVA; thereafter the differences between the groups were checked by Tukey’s Multiple Comparison Test. KruskaleWallis test followed by Dunn’s Multiple Comparison Test was used for non-parametric data. In all cases, significance was noted at P < 0.05. The heatmap of gene expression was designed using TIGR Multiexperiment Viewer (MeV; Dana-Farber Cancer Institute, USA). 3. Results and discussion This study attempted the isolation and in vitro culture of epidermal (EP) cells in Atlantic cod. The primary EP cell cultures appeared to be a promising model to study cutaneous immune responses during probioticsepathogen interactions. The capability
Fig. 1. Epidermal (EP) cells of Atlantic cod in culture. EP cells from the A) dorsal region were significantly larger than the EP cells from the B) ventral region. The inserted photos (A0 and B0 ) are magnified fields showing the size distinction of cod EP cells. mc ¼ mucus-like cell; epc ¼ epithelial cell. Scale bar ¼ 500 mm.
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Fig. 2. Responses elicited by the EP cells during probioticsepathogen interactions were assessed by A) cellular viability, B) apoptotic activity and C) myeloperoxidase activity. Column bars with same letters indicate no significant difference (P > 0.05) Legend: CON ¼ Control; GP21 ¼ Pseudomonas sp.; GP12 Psychrobacter sp.; VA ¼ Vibrio anguillarum 2133; GP21-VA ¼ Vibrio anguillarum 2133 and Pseudomonas sp.; and GP12-VA ¼ Vibrio anguillarum 2133 and Psychrobacter sp. N ¼ 6.
skin act as a sensitive first line of immune defense parameter in fish [29]. They are responsible for the production of mucosal layer [30] and could also be involved in mucus production during transfer of material from the secondary circulatory system [31]. The substantial presence of these cells in the culture could be related to the initial findings of the immune-rich potential of Atlantic cod skin and its mucus [22,23]. The previous report on the skin structure of cod focused on skin pigmentation [7] and failed to mention the different cell populations comprising the epidermal layer. It would be interesting to account in the future the different cell populations
of the epidermal layer of the cod skin. The percentage of surviving EP cells exceeded 80% after 24 hpi and the viability could be maintained for 5 days with the change of medium every other day. Though dead cells and tissue debris were observed, they were removed gradually by daily washing and changing of the culture media. Remarkably, there was a significant regional difference on the size of the isolated EP cells in cod. To our knowledge, this regional difference on EP cell sizes has not been previously accounted in other teleost species. The EP cells from the dorsal region (average cell size: w268 mm) of the skin were significantly
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larger than the cells from the ventral region (average cell size: w205 mm) (Fig. 1). Since we did not have a specific count of each cell population, the regional size difference observed was a general account regardless of cell type as the population of both regions was observed and assumed to be homogenous. This regional difference could be a strong point to further explore and characterize the cell types and populations of the epidermal skin of Atlantic cod. In the naïve cod, immune features of the dorsal and ventral regions varied significantly with the latter region showing significantly higher expression of immune genes even at the basal physiological conditions [22]. The regional difference on cell size noted in this study could substantiate the differences that were previously observed because cell size could influence different physiological responses of the cell.
cells (Fig. 3C). There were instances that the presence of bacteria decreased myeloperoxidase activity and this was typified by GP21 at 3 hpi, regardless of cutaneous region. There was a significant increase on myeloperoxidase activity in VA-exposed EP cells except in the dorsal region at 3 hpi. This could be a protective reaction of the cells as the bacteria were recognized as virulent. Longer exposure of the EP cells to both probiotics and pathogen significantly elevated myeloperoxidase activity. The presence of probiotics could have an additive influence on the myeloperoxidasemediated reaction of the cells by stimulating enzyme production for a more effective immune response against a pathogen.
3.2. Stimulated activities of the EP cells during probioticsepathogen interactions
The modulated expression profile during probioticsepathogen interactions corroborated with the differences observed between cutaneous regions on the expression of immune- and stress-related genes in the skin of Atlantic cod [22]. Bactericidal/permeabilityincreasing protein (BPI) and lipopolysaccharide-binding protein (LBP) is an important member of the innate immune system both in mammals and in Atlantic cod [35,36]. In most cases, the expression of bpi/lbp in both cutaneous regions was not significantly affected by probiotics exposure except at 24 hpi in the dorsal EP cells (Fig. 3A). bpi/lbp was significantly up-regulated in the presence of VA at the dorsal EP cells regardless of exposure time. Interestingly, the expression of bpi/lbp remained unchanged in the ventral EP cells during bacterial interactions in contrast to the significant upregulation observed in the dorsal EP cells. Lysozyme has been shown to be highly expressed in the skin of a naïve Atlantic cod suggesting that it is an essential component of the skin as an immunologically active tissue [22]. The exposure of dorsal EP cells to probiotics did not elicit significant up-regulation of lyz expression. On the other hand, VA elicited significant elevation of lyz expression and the expression was significantly promoted during
The effect of bacterial exposure on cellular viability was assessed by determining the activity of the EP cells through time and the bacteria-induced changes were observed to be temporal and regional-dependent. The host-derived probiotics did not elicit significant change on the viability of EP cells from both regions at both time points (Fig. 2A). The exposure of EP cells with VA significantly reduced the viability of EP cells on both cutaneous regions except that the effect on ventral EP cells was observed only at 24 hpi. The simultaneous exposure of EP cells with pathogens and probiotics significantly mitigated the inhibitory feature of the former on cellular viability except at 3 hpi in the dorsal region. The presence of the probiotics could have elicited a protective effect on the EP cells by counteracting the inhibition of cellular proliferation induced by VA. Apoptosis has duality in its feature as it can be both a damaging pathway induced by a pathogen to enter the host or as a protective mechanism against the pathogen [32]. The caspase-3 activity of the cells showed an inverse profile relative to cellular proliferation and this was typified when EP cells were exposed to VA (Fig. 2B). This implies that the inhibition observed in the proliferation of the cells could be due to the induction of apoptosis in the presence of the pathogen. VA was previously found to have the capability to induce cellular apoptosis in the intestinal epithelial cells of cod [20] and the same virulence mechanism was observed in the present study with the EP cultures. It was also found that the pathogenicity of VA in the head kidney leukocytes of sea bass was due to the downregulation of caspases [33]. The differences on how cellular apoptosis in fish are influenced by VA could be dependent on the species, types of cell as well as the pathogenic strains of the bacteria. There were no significant changes on caspase-3 activity when the EP cells were exposed to probiotics alone indicating that the EP cells recognized the bacteria as non-pathogenic and natural residents of the host thus the reaction was at the basal level. Interestingly, there were two evident apoptosis-related reactions during bacterial interactions. The probiotics influenced VA-induced apoptosis by diminishing the effect thereby protecting the cells. This was observed in the dorsal EP cells at 24 hpi and in the ventral EP cells at 3 hpi. On the other hand, the significant increase in the caspase-3 activity observed in the dorsal EP cells at 3 hpi and in the ventral EP cells at 24 hpi could be explained in two possibilities: 1) the VA-mediated apoptosis was strong enough for the probiotics to act upon or 2) the protective mechanism of the probiotics was through the activation of apoptosis. The production of peroxidase enzyme during bacterial interaction was measured by myeloperoxidase activity which is also related to neutrophil antimicrobial activity [34]. In most cases, the presence of bacteria elevated the myeloperoxidase activity of the EP
3.3. Differential regulation of immune genes during probioticse pathogen interactions
Fig. 3. Heatmap of the expression of A) bacterial defense (bpi/lbp and lyz) and, B) cytokine (il1b) genes during probioticsepathogen interactions. Legend: CON ¼ Control; GP21 ¼ Pseudomonas sp.; GP12 Psychrobacter sp.; VA ¼ Vibrio anguillarum 2133; GP21VA ¼ Vibrio anguillarum 2133 and Pseudomonas sp.; GP12-VA ¼ Vibrio anguillarum 2133 and Psychrobacter sp.; D3 ¼ dorsal 3 hpi; D24 ¼ dorsal 24 hpi; V3 ¼ ventral 3 hpi; and V24 ¼ ventral 24 hpi. Expression values of target genes were expressed relative to bactin expression. Asterisk (*) indicates that the expression is significantly different from the control group. N ¼ 6.
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bacterial interactions. The ventral EP cells showed a clear profile of lyz up-regulation during bacterial exposure and interactions where elevated transcript levels were observed at both exposure times. The antibacterial responses of the EP cells through the upregulation of bpi/lbp and lyz supported the initial observation that these molecules are important in the bacterial defense system of Atlantic cod skin [22]. These molecules are believed to be contributors of the antibacterial activity observed in the cutaneous extract of cod [37]. Further, the capability of EP cells to respond against the invading pathogen was enhanced in the presence of probiotics through the modulation of the bacterial defense genes. These host-derived bacteria have been previously observed to have stimulatory effect on the antibacterial activity of head kidney leukocytes of cod [21]. The present study supported this immunostimulatory feature and the beneficial characteristic is not limited to only one immune-related tissue. Inflammation is one of the many immune responses of an organism to exogenous stimuli, specifically those of biological threats with il-1b as one of the genes involved in the pro-inflammatory cascade. The up-regulation of il-1b has been demonstrated as a pro-inflammatory response of the fish skin to a pathogen [38,39]. Even though there was a significant up-regulation of il-1b expression in the EP cells exposed to probiotics, the expression was merely instantaneous as no significant expression was observed at 24 hpi in both cutaneous regions (Fig. 3C). This expression profile was similar to what have been observed with these probiotics in head kidney leukocytes [21]. This corroborates with the previous reports that this gene is responsible for the early pro-inflammatory response in fish [40]. Regardless of cutaneous region, exposure of EP cells with VA triggered significant up-regulation of il-1b. This significant up-regulation was also observed during probioticse pathogen interactions in the dorsal region at both time points. Interestingly during probioticsepathogen interactions in the ventral EP cells, the mRNA level of il-1b was brought to the basal level implying the capability of probiotics to reduce inflammatory reactions induced by the presence of VA. The expression of two cell-mediated immunity genes, nccrp and gzma, is shown in Fig. 4A. The expression of nccrp was significantly up-regulated in the presence of probiotics, pathogen and during their interactions and these changes were only observed at the latter stage of exposure. NCCRP-1 is an important molecule in target cell recognition and activation of NCC in cod [41] and it was not surprising to observe the significant up-regulation during pathogen exposure. The significant up-regulation in the dorsal EP cells with the probiotics at 24 hpi could be explained that the probiotics played as an additive factor in eliciting and priming a better immune response during probioticsepathogen interactions. This influence was regional-dependent because changes were only observed in the dorsal EP cells. We have not observed any significant and remarkable changes on gzma expression during bacterial exposure. Traditionally, gzma are potent members of serine proteases family that are induced mostly during virus infection [42]. It is interesting to explore the possible functions of gzma in cod as it was previously been observed to be up-regulated in the spleen in the presence of a bacterial antigen [26]. It might have anti-viral and antibacterial properties and could be tissue-dependent on its immunological significance. The presence of VA elicited significant up-regulation of cat and sod in the EP cells (Fig. 4B). In particular, there was a significant expression of cat in both cutaneous regions and this effect was observed until 24 hpi. Bacterial antigen could influence the expression of cat as previously observed in the up-regulation of this gene in the spleen of vaccinated cod [26]. This suggests that cat is an important gene on the oxidative stress response of cod during VA exposure and eventual infection. The same pattern of
Fig. 4. Heatmap of the expression of A) cell-mediated immunity (nccrp and gzma) and, B) oxidative stress (cat and sod) genes during probioticsepathogen interactions. Legend: CON ¼ Control; GP21 ¼ Pseudomonas sp.; GP12 Psychrobacter sp.; VA ¼ Vibrio anguillarum 2133; GP21-VA ¼ Vibrio anguillarum 2133 and Pseudomonas sp.; GP12VA ¼ Vibrio anguillarum 2133 and Psychrobacter sp.; D3 ¼ dorsal 3 hpi; D24 ¼ dorsal 24 hpi; V3 ¼ ventral 3 hpi; and V24 ¼ ventral 24 hpi. Expression values of target genes were expressed relative to b-actin expression. Asterisk (*) indicates that the expression is significantly different from the control group. N ¼ 6.
expression was observed with sod transcription during bacterial exposure. It was interesting to observe that the induction of oxidative stress gene expression during VA infection could be reduced in the presence of the probiotics. This was evidently observed during bacterial interactions wherein the expression of cat and sod showed no significant difference with the control group. This suggests a logical possibility that these bacteria have the capability to reduce oxidative stress induced by VA infection. Besides their immune-related and nutritional importance, probiotics could also protect the fish from chemical, physical and biological stress [43]. GP21 and GP12 exhibit these probiotic features and need to be explored in future studies. 4. Conclusions This study supported and provided additional knowledge on the immunological properties of Atlantic cod skin and its components. Further, the immunostimulatory activities of two candidate probionts, GP21 and GP12 and their capacity to modulate immune responses of EP cells against VA infection expanded their probiotic potentials. These results substantiate the broad range of probiotic properties of these host-derived bacteria from cod. The primary culture of Atlantic cod EP cells can be a good model in studying cutaneous immune responses to bacteria and can be applied to other pathogens including viruses and parasites. However, additional studies should be conducted focusing on the identification and characterization of the different cell populations in the skin. By doing so, there will be sufficient data as to which particular cell types in the skin that are directly interacting during probioticse pathogen exposure of the fish.
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Acknowledgment This study was financed by the project “Mucosal immune system of Atlantic cod e creating a knowledge base” (Project Number 184703 funded by the Research Council of Norway). Hilde Ribe is also acknowledged for her technical help.
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