Early local and systemic innate immune responses in ...

Fish & Shellfish Immunology 22 (2007) 242e251 www.elsevier.com/locate/fsi

Early local and systemic innate immune responses in the teleost gilthead seabream after intraperitoneal injection of whole yeast cells Alberto Cuesta, Alejandro Rodrı´guez, Irene Salinas, Jose´ Meseguer, ´ ngeles Esteban* M. A Fish Innate Immune System Group, Department of Cell Biology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain Received 29 March 2006; revised 17 May 2006; accepted 22 May 2006 Available online 27 May 2006

Abstract The early cellular innate immune responses of the teleost gilthead seabream (Sparus aurata L.) against whole yeast cells were studied. Fish received a single intraperitoneal (i.p.) injection of Saccharomyces cerevisiae and leukocyte mobilization, degranulation, peroxidase content, respiratory burst, phagocytic and cytotoxic activities were assayed in both head-kidney leukocytes (HKLs) and peritoneal exudate leukocytes (PELs). The total number of PELs significantly increased from 4 h post-injection until the end of the experiment (3 days). Interestingly, flow cytometric analysis revealed variations in the proportion of cell-types in the PE. Thus, PE acidophilic granulocytes increased to a significant extent 4 h post-injection and were restored thereafter. Moreover, PE monocyte-macrophages started to increase from 24 h, the enhancement being statistically significant after 48 and 72 h. Degranulation was greater in PELs throughout the assay. The peroxidase content of the leukocytes was affected differently in HKLs and PELs. The respiratory burst activity was not affected in HKLs but significantly increased in PELs from 4 to 48 h post-injection with yeast cells. On the other hand, HKL phagocytosis had decreased 72 h post-injection with yeast cells while it increased after 4 and 24 h postinjection in the PELs. Conversely, the cytotoxic activity was significantly enhanced in HKLs from 24 to 72 h post-injection but slightly decreased in PELs. Finally, our data demonstrate that seabream injected with the yeast Saccharomyces cerevisiae show leukocyte mobilization and cellular innate immune response activation at the site of invasion and also in the head-kidney. The implications of the leukocyte-types and the immune responses observed, as well as analogies with other particulated antigens, will be discussed as possible models for investigating the effect of potential pathogens. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Innate immunity; Yeast cells; Leukocytes; Cytotoxicity; Granulocytes; Mobilization; Fish

1. Introduction Much information exists concerning the effects of yeast components (b-glucans, chitin, RNA, etc.) on the mammalian immune response, while studies involving whole yeast cells are more scarce. The same applies to teleost * Corresponding author. Tel.: þ34 968367665; fax: þ34 968363963. ´ ngeles Esteban). E-mail address: [email protected] (M. A 1050-4648/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2006.05.005

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fish, such studies mainly focusing on the fish immune response after in vitro or in vivo treatment with purified yeast components and to a lesser extent with whole yeasts. In the few studies carried out in this respect, in vitro leukocyte incubation with whole yeast cells led to the phagocytosis of yeasts and leukocyte degranulation [1e4]. Moreover, the phagocytosis of whole yeasts was partially inhibited by certain sugars, demonstrating the involvement of b-glucanand mannose-receptors in the process carried out by phagocytic leukocytes [5e7]. The dietary intake of whole yeast cells has also demonstrated their immunostimulant properties enhancing leukocyte phagocytosis, cytotoxicity and respiratory burst [8e11]. However, all these reports have studied the systemic effects on the humoral (serum) and cellular (head-kidney leukocytes) immune responses but not at the site of administration. The fish immune response, either at local and/or systemic sites, after bacterial injection has been widely reported and leukocyte mobilization, phagocytosis, respiratory burst, non-specific cytotoxic cells (NCC) activity and expression of immune-relevant genes such as IL-1b or TNFa have been described [12e18]. However, little information exists regarding other particulated antigens. Added to this, we have shown some differences in the seabream innate immune responses after intraperitoneal (i.p.) injection of bacteria [15,16] or tumor cells [19]. Strikingly, no study has explored these aspects after injection of whole yeast cells while the use of isolated yeast components, such as b-glucans or chitin, has been undertaken to evaluate the effects on immune responses [20e22]. However, the use of purified/single yeast components or pathogen-associated molecular patterns (PAMPs) may have disadvantages due to the lack of multi-antigenic interactions and the use of whole pathogens is therefore advisable. More recently, the regulation of gene expression after contact with PAMPs or pathogens is providing valuable information about the molecules potentially involved in fish defense mechanisms [15,17,19,23e27]. The aim of the present work was to study the early events in the cellular innate immune system in the response against whole yeast cells. For this, we injected Saccharomyces cerevisiae yeast cells into the peritoneal cavity of gilthead seabream (Sparus aurata L.) and studied leukocyte redistribution, as well as degranulation, the peroxidase content and respiratory burst, phagocytic and cytotoxic activities in local and systemic immune tissues.

2. Materials and methods 2.1. Animals Fifty gilthead seabream (Sparus aurata L.) (70e90 g body wt) were obtained from Culmarex S.A. (Murcia, Spain). Animals were kept in 450e500 l running seawater (28& salinity) aquaria at 20 2 C and a 12 h light:12 h dark photoperiod. They were fed daily with 2 g of a commercial pellet diet (Trowvit, Spain) per fish. Animals were acclimated for 15 days prior to the experiments. The Bioethical Committee of the University of Murcia approved the studies carried out herein. 2.2. Yeast cells Heat-killed and lyophilized Saccharomyces cerevisiae (strain S288C) yeast cells were washed twice in PBS, counted and adjusted to 107 cells ml1. The yeast cells used in phagocytosis assays were previously labelled with 5 mg ml1 fluorescein isothiocyanate (FITC, Sigma) for 15 min, washed and adjusted to 5 107 cells ml1 of sRPMI (RPMI-1640 culture medium (Gibco) with 0.35% sodium chloride, 100 IU ml1 penicillin, 100 mg ml1 streptomycin and 5% fetal bovine serum). 2.3. Experimental design and sampling Fish were randomly divided into two tanks and each fish received a single intraperitoneal injection (i.p.) of 1 ml of sterile PBS alone (control group) or containing 107 yeast cells. Fish were sampled at 4, 24, 48 and 72 h post-injection (6 fish per group and sampling point) and leukocytes from the head-kidney (HKLs) and peritoneal exudate (PELs) were isolated, as described elsewhere [28]. Leukocytes were washed, counted with a Z2 Coulter Particle Counter (Beckman Coulter) and adjusted to 107 cells ml1 of sRPMI.

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2.4. Leukocyte mobilization studied by flow cytometry To examine possible leukocyte mobilization or tissue-redistributions, HKL or PEL suspensions were analyzed in a flow cytometer (Becton Dickinson) with an argon-ion laser of 488 nm. Analyses were performed on 30,000 cells, which were acquired at a rate of 300 cells/s. Side scatter (complexity, SSC), forward scatter (size, FSC), green (FL1) and red fluorescence (FL2) parameters were represented in dot-plots or histograms. Gilthead seabream leukocyte populations have previously been characterized by means of flow cytometry, microscopy and functional techniques [28e32]. 2.5. Leukocyte innate immune responses 2.5.1. Degranulation and leukocyte peroxidase The peroxidase content of the HKLs and PELs and in the PELs medium (because of degranulation) was measured as an indicator of leukocyte activation [4]. Thus, degranulation of PELs (PEL-med; peroxidase released by PELs to the medium injected into the peritoneal cavity) and the peroxidase content of HKLs and PELs were determined by a colorimetric method. Briefly, aliquots of PEL-med or lysed leukocytes were dispensed in a 96-well plate containing 10 mM 3,30 ,5,50 -tetramethylbenzidine hydrochloride (TMB) and 5 mM H2O2. The color-change reaction was stopped after 2 min by adding 50 ml of 2 M sulfuric acid and the optical density was read at 450 nm in a plate reader. Standard samples without PEL-med or leukocytes were used as blanks. 2.5.2. Respiratory burst activity The respiratory burst activity of gilthead seabream HKLs and PELs was studied by a chemiluminescence method [33]. Briefly, samples of 106 leukocytes in sRPMI were placed in the wells of a flat-bottomed 96-well microtiter plate, to which was added 100 ml of HBSS containing 1 mg ml1 phorbol myristate acetate (PMA, Sigma) and 104 M luminol (Sigma). The plate was shaken and immediately read in a plate reader for 1 h at 2 min intervals. The kinetic of the reactions was analyzed and the maximum slope of each curve calculated. Backgrounds of luminescence were calculated using reactant solutions containing luminol but not PMA. 2.5.3. Phagocytic activity The phagocytosis of Saccharomyces cerevisiae (strain S288C) by gilthead seabream HKLs and PELs was studied by flow cytometry [4]. Phagocytosis samples consisted of labelled-yeast cells and leukocytes (6.25 yeast cells:leukocyte). Samples were mixed, centrifuged (400 g, 5 min, 22 C), resuspended in sRPMI and incubated at 22 C for 30 min. At the end of the incubation time, the samples were placed on ice and 400 ml ice-cold PBS was added to each sample to stop phagocytosis. The fluorescence of the extracellular yeasts was quenched by adding 40 ml ice-cold trypan blue (0.4% in PBS). Standard samples of FITC-labelled S. cerevisiae or leukocytes were included in each phagocytosis assay. All samples were analyzed in a flow cytometer set to analyze the phagocytic cells. Phagocytic ability was defined as the percentage of cells with ingested yeast cells (green-FITC fluorescent cells, FL1þ) within the phagocyte cell population. The relative number of ingested yeasts per cell (phagocytic capacity) was assessed in arbitrary units from the mean fluorescence intensity of the phagocytic cells. 2.5.4. Cytotoxic activity The natural cytotoxic or tumoricidal activity of gilthead seabream HKLs and PELs was evaluated using a flow cytometry technique based on double-fluorescent labelling [34]. Briefly, tumor target cells from the L-1210 line (mouse lymphoma, ATCC CCL-219) in exponential growth were labelled with 10 mg ml1 of 3,30 -dioctadecyloxacarbocyanine perchlorate (DiO, Sigma) for 1 h in darkness. After labelling, free DiO was removed by washing three times in PBS and cell-staining uniformity was examined by flow cytometry. Leukocytes in sRPMI (effectors) were mixed with DiO-labelled L-1210 cells (targets) (effector:target ratio of 50:1). The samples were centrifuged (400 g, 1 min, 22 C) and incubated at 22 C for 2 h. Cytotoxic samples incubated for 0 h (control) were used to determine initial target viability. After incubation, 30 ml of propidium iodide (400 mg/ml, Sigma) were added and all the samples were analyzed in a flow cytometer set to accept the positive FL1 region, which corresponds to DiO-labelled target cells (FL1þFL2). The percentage of dead or non-viable target cells showing green and red fluorescence (FL1þFL2þ)


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was related to the cytotoxic activity of gilthead seabream leukocytes. Cytotoxic activity, a parameter describing the percentage of non-viable target cells, was calculated by the formula: Cytotoxic activityð%Þ ¼ 100 ð%sample %controlÞ=ð100 %controlÞ:

2.6. Statistical analysis The data from the flow cytometric assays were analyzed using the statistical option of the Lysis Software Package (Becton Dickinson). The data are represented as means þ SE and analyzed by one-way analysis of variance (ANOVA) and Tukey’s comparison of means when applicable. 3. Results 3.1. Leukocyte mobilization and redistribution Firstly, we evaluated the changes in the number of PELs resulting from the injection of yeast cells. While the total number of isolated PELs in the control fish ranged from 35 to 45 million (Fig. 1), it reached values of 80e340 million in yeast cell-injected fish being significantly increased during the trial. We also analyzed by flow cytometry possible variations in the leukocyte subpopulations due to the injected yeasts, based on the different FSC vs. SSC parameters of the cells (Fig. 2). In HKLs, little and no significant differences were observed in the two populations present. The PELs were distributed in four subpopulations and, 4 h post-injection of yeast cells, the percentage of the R1 population had increased (1.5-fold) while the other leukocyte populations had slightly decreased. Moreover, R4 PELs gradually increased from 24 to 72 h (up to 3.2-fold). On the other hand, PE R2 and R3 leukocytes remained at similar levels to those found in control fish. 3.2. Systemic (HK) and local (PE) cellular innate immune responses We studied the early systemic immune responses in the HKL because of its importance as a primary and secondary lymphoid organ in teleost fish, as well as in the site of injection, the PE. The HKL peroxidase (Fig. 3) content decreased significantly 24 h post-injection but increased in PELs at all the assayed times, although the increments were statistically significant only at 4 h post-injection. On the other hand, PEL degranulation, measured as the content 250

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of peroxidase in the isolation medium of PELs, was greatly enhanced (up to 3.4-fold) (Fig. 3) at all the sampling times, the highest value being observed 4 h post-injection. Concomitantly, respiratory burst activity was unaffected (Fig. 4) in HKLs but significantly enhanced in PELs from 4 to 48 h post-injection of S. cerevisiae. Furthermore, 72 h postinjection, the yeast cells produced an inhibition of the HKL phagocytic response (Fig. 5) in the percentage of phagocytic cells (phagocytic ability) and in the number of ingested particles (phagocytic capacity). However, the phagocytic ability had significantly increased in PELs 4 h and 24 h post-injection but the phagocytic capacity had significantly decreased 4 h after injection (Fig. 5). Finally, HKL cytotoxic activity (Fig. 6) was significantly enhanced from 24 (2.3-fold) to 72 h post-injection but was slightly inhibited in PELs, although the inhibition was only statistically significant at 72 h post-injection. 4. Discussion While activation of the fish immune response after intraperitoneal injection of bacteria has been reported [13e18] little information exists concerning other different particulated antigens [19,35,36]. Looking at this gap, in this paper we have evaluated the early innate immune responses in local and systemic tissues of gilthead seabream specimens after injection with Saccharomyces cerevisiae yeast cells. In infected fish, both naturally and experimentally induced, mobilization and redistribution of leukocytes from hematopoietic tissues to the inflammation site has been demonstrated. This is regarded as the first step whereby fish recruit leukocytes to the site where they are needed to play their role in the fish defense. Evidence for this

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phenomenon is shown by the great increase in leukocyte numbers present in the peritoneal cavity after the injection of yeast cells, as also occurs in seabream specimens injected with bacteria and tumor cells [16,19]. Moreover, thanks to morpho-functional studies [28e32] we are able to differentiate some of the leukocyte populations and their variations by means of flow cytometry. Thus, the HKL R1 population consists of acidophilic granulocytes, while R2 is formed of a mix of thrombocytes, lymphocytes and monocyte-macrophages. PEL populations, on the other hand, correspond to acidophilic granulocytes (R1), neutrophils (R2), lymphocytes (R3) and monocyte-macrophages (R4). Thus, with the help of flow cytometry, we could detect differences in the leukocyte types present in the peritoneum after the injection of yeast. Acidophils increased after 4 h while monocyte-macrophages did so gradually from 24 to 72 h after the i.p. injection of whole yeast cells. When yeast cell components, such as b-glucans, were injected leukocyte redistributions in carp were also observed, namely, the total number of blood leukocytes and, more specifically, the percentage of neutrophils and monocytes increased [36]. Similar to our findings, the presence of particulated antigens, such us

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bacteria, parasites or tumor cells, primes fish leukocytes to mobilize and redistribute to the tissues where the antigen appears [12,14,16,19,37e40]. Systemic and local innate immune responses were determined in fish injected with yeast cells. Firstly, we measured the presence of a lysosome enzyme (peroxidase) and free oxygen radicals. When a fish encounters a pathogen, leukocytes produce toxic radicals (O 2 , NO, etc.) to kill the pathogens. Moreover, at the end of this respiratory burst cascade, myeloperoxidase and eosinophil peroxidase (MPO and EPO, respectively) uses H2O2 and halide ions to form chlorides and chloramines to help in the fight. Both enzymes are contained in granules of phagocytic cells and can be released by degranulation when properly activated [4,41,42]. Therefore, peroxidases (released by degranulation or inside the cells) and the production of reactive oxygen intermediaries (ROIs) have been shown to be good indicators of leukocyte activation. Consequently, PEL degranulation was greatly enhanced following injection of yeast cells throughout the experiment. Similarly, seabream HKLs and mammalian leukocytes were activated to degranulate (measured by MPO and/or EPO release) after contact with whole yeast cells or zymosan [4,43e45]. Moreover, the peroxidase content of PELs after 4 h was also increased. Concomitantly, we found increased ROI production in PELs from 4 to 48 h post-injection while it remained unaffected in HKLs. Although we do not know the in vitro effect of whole yeast cells on ROI production, isolated components like b-glucans, chitin or zymosan can be used to trigger their production in fish leukocytes [17,46e48]. Moreover, the dietary intake of yeast cells increases the leukocyte

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content and ROI production of HK leukocytes [8e10] but, unfortunately, nothing is known about the effect on the gut-associated lymphoid tissue. Similar results have been obtained when using other particulated antigens such us bacteria, parasites and tumor cells [16,19,36,39]. The other two main cellular innate immune responses, phagocytosis and cytotoxicity, were also determined. The phagocytosis of yeast cells decreased in HKLs 72 h post-injection but was primed in PELs 4 and 24 h after injection. Strikingly, 4 h post-injection, the PEL phagocytic capacity had decreased. These same yeast cells are rapidly ingested by seabream leukocytes in vitro, a process that may be blocked by sugars [4,6] and demonstrates the involvement of b-glucan and mannose-receptors in the phagocytic process though others such as TLRs may also be considered. Finally, the cytotoxic activity was greatly enhanced in HKLs from 24 h to the end of the assay but not in the PELs. Both immune responses also increased after the dietary intake of whole yeast cells [8e10] as occurs after injection. For example, the presence of particulate antigens such us bacteria, tumor cells, parasites and, now, yeast cells led to a great increase in the NCC activity, at least, in HK leukocytes [13,19,27,38,49]. Further characterization of the phagocytes and NCCs at the cellular and molecular level will help to understand their function against pathogens. Unfortunately, we were unable to make a simple correlation between the leukocyte types present at each sampling point and the immune response elicited. Firstly, apart from the activation status, the great increase in the number of PELs in yeast-injected fish could be responsible for the massive degranulation observed at the same sampling times. Moreover, the increased percentage of highly active acidophils in PELs 4 h post-injection could explain the increased leukocyte peroxidase content, ROI production and phagocytosis. However, the increased PELs ROI production and phagocytosis observed in PELs at other sampling points do not support this hypothesis. Moreover, while the proportions of HK leukocyte types remained apparently unchanged, the leukocyte peroxidase content and phagocytosis were punctually decreased while the NCC activity was increased at 24 h and afterwards. These findings suggest that the immune response is the result of a sum of factors where the number of leukocytes, maturation status and activation elicited are operating in the immune response to the antigen. To conclude, we have shown for the first time that in fish injected intraperitoneally with whole yeast cells, Saccharomyces cerevisiae, leukocytes are rapidly recruited to the injection site. Leukocyte degranulation as well as peroxidase, respiratory burst, phagocytic and NCC activities increased in yeast cell-injected fish.

Acknowledgements This work has been partially funded by the European Commission (QLRT-2001-00722). A. Cuesta and I. Salinas have fellowships from Fundacioń Caja Murcia and Fundacioń Seńeca, respectively.

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