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Unique phagocytic properties of hemocytes of Pacific oyster Crassostrea gigas against yeast and yeast cell-wall derivatives
Accepted Manuscript Unique phagocytic properties of hemocytes of Pacific oyster Crassostrea gigas against yeast and yeast cell-wall derivatives Keisuke G. Takahashi, Nakako Izumi-Nakajima, Katsuyoshi Mori PII:
S1050-4648(17)30546-6
DOI:
10.1016/j.fsi.2017.09.027
Reference:
YFSIM 4817
To appear in:
Fish and Shellfish Immunology
Received Date: 17 February 2017 Revised Date:
5 September 2017
Accepted Date: 9 September 2017
Please cite this article as: Takahashi KG, Izumi-Nakajima N, Mori K, Unique phagocytic properties of hemocytes of Pacific oyster Crassostrea gigas against yeast and yeast cell-wall derivatives, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.09.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Second revised manuscript: FSIM-D-17-00156
Title. Unique phagocytic properties of hemocytes of Pacific oyster Crassostrea gigas against
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yeast and yeast cell-wall derivatives
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Journal. Fish and Shellfish Immunology
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Authors. Keisuke G Takahashi1,2*, Nakako Izumi-Nakajima3, and Katsuyoshi Mori2
Laboratory of Aquacultural Biology, Graduate School of Agricultural Sciences, Tohoku
University, Aoba-ku, Sendai 980-0845, JAPAN
Oyster Research Institute, Aoba-ku, Sendai 989-3204, JAPAN
3
National Institute of Radiological Sciences, QST, Inage-ku, Chiba-shi 263-8555, JAPAN
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*Corresponding author:
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Keisuke G Takahashi
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Address: Laboratory of Aquacultural Biology, Graduate School of Agricultural Sciences, Tohoku University, Aoba-ku, Sendai 980-0845, JAPAN Phone: +81 22 757 4134; Fax: +81 22 757 4132 E-mail address: [email protected]
Abbreviations: AGs, agranulocytes; Gs, granulocytes; HK yeast, heat-killed yeast; PI, phagocytic index; PR, 1
ACCEPTED MANUSCRIPT phagocytic rate
Conflict of interest:
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None of the authors has a financial relationship with a commercial entity that has an interest
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in the subject of this manuscript.
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ACCEPTED MANUSCRIPT Abstract
For a marine bivalve mollusk such as Pacific oyster Crassostrea gigas, the elimination of
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foreign particles via hemocyte phagocytosis plays an important role in host defense mechanisms. The hemocytes of C. gigas have a high phagocytic ability for baker’s yeast (Saccharomyces cerevisiae) and its cell-wall product zymosan. C. gigas hemocytes might
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phagocytose yeast cells after binding to polysaccharides on the cell-wall surface, but it is unknown how and what kinds of polysaccharide molecules are recognized. We conducted
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experiments to determine differences in the phagocytic ability of C. gigas hemocytes against heat-killed yeast (HK yeast), zymosan and zymocel, which are similarly sized and shaped but differ in the polysaccharide composition of their particle surface. We found that both the agranulocytes and granulocytes exerted strong phagocytic ability on all tested particles. The
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phagocytic index (PI) of granulocytes for zymosan was 9.4 ± 1.7, which significantly differed with that for HK yeast and zymocel (P < 0.05). To evaluate the PI for the three types of
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particles, and especially to understand the outcome of the much higher PI for zymosan, PI was gauged in increments of 5 (1–5, 6–10, 11–15, and ≥16), and the phagocytic frequencies
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were compared according to these increments. The results show that a markedly high PI of ≥16 was exhibited by 18.1% of granulocytes for zymosan, significantly higher than 1.7% and 3.9% shown for HK yeast and zymocel, respectively (P < 0.05). These findings indicate that the relatively high PI for zymosan could not be attributed to a situation wherein all phagocytic hemocytes shared a high mean PI, but rather to the ability of some hemocytes to phagocytose a larger portion of zymosan. To determine whether the phagocytosis of these respective particles depended on the recognition of specific polysaccharide receptors on the 3
ACCEPTED MANUSCRIPT hemocyte surface, C. gigas hemocytes were pretreated with soluble α-mannan or β-laminarin and then allowed to phagocytose the three types of the particles. The percentage of phagocytic cells of β-laminarin-treated granulocytes decreased significantly for zymosan and
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zymocel, but not for yeast. These results suggest that C. gigas might possess at least two types of hemocytes, and that one type of the hemocytes (granulocytes) is more active for phagocytosis. The granulocytes were found to have multiple subtypes with different
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phagocytic abilities and multiple phagocytic receptors. Some of the granulocyte subtypes revealed a much stronger phagocytic ability, depending on the presence of β-glucan receptors
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for phagocytosis.
Highlights
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1. Two sub-populations, AGs and Gs, could be separated from the hemocytes of C. gigas. 2. Both AGs and Gs exhibited phagocytic ability, especially Gs functioned much active
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phagocytes.
3. A part of Gs showed markedly high PI for zymosan.
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4. Soluble laminarin partly inhibited ingestion of zymosan and zymocel by Gs. 5. C. gigas has multiple types of hemocytes with phagocytic ability, some of which depend on β-glucan receptors for phagocytosis.
Keywords: Crassostrea gigas; Oyster; Hemocytes; Granulocytes; Agranulocytes; Phagocytosis; Yeast; Zymosan; β-glucan receptor
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ACCEPTED MANUSCRIPT 1. Introduction
The host defense system of bivalve mollusks is understood to depend mainly on
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circulating hemocytes. These cells are present in the hemolymph and possess a strong migration ability in response to invading microorganisms, including potential pathogens, and will actively phagocytose these invaders [1], [2], [3]. The hemocytes of bivalve mollusks
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morphologically resemble mammalian phagocytic leukocytes, such as macrophages and neutrophils [4], and like leukocytes they have the ability to recognize, engulf, and internally
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degrade biological particles, including microbial pathogens. Therefore, it is believed that the hemocytes of bivalve mollusks might possess a specialized system that is mediated by a hemocyte membrane receptor in a non-self-recognition of foreign particles. In eastern oyster Crassostrea virginica, cell membrane-associated galectin of the
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hemocyte recognizes and functions to phagocytose Perkinsus marinus trophozoites—the etiologic agent in Dermo disease, which is responsible for episodes of mass mortality among
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C. virginica [5], [6]. Receptors of the Nimrod family are found on the hemocytes of C. gigas [7]; these receptors recognize lipopolysaccharides (LPS) and enhance the phagocytosis of bacteria.
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gram-negative
Furthermore,
in
recent
years,
cDNA
sequences
of
pattern-recognition-receptor-related genes have been cloned and analyzed for mRNA expression in bivalve mollusks, and these include LPS and β-1,3-glucan recognition proteins [8], [9], and peptidoglycan-recognition protein [10], [11]. In addition, hemocytes of C. gigas have the function analogous to that human toll-like receptors (TLRs) as the representative pattern recognition receptors (PRRs)[12]. These receptors play an important role in host defense of C. gigas against pathogen infection. 5
ACCEPTED MANUSCRIPT In molluscs, laminarin (soluble β-1,3-glucan) markedly inhibited the phagocytosis of both opsonized and unopsonized, autoclave-killed yeast cells by hemocytes of the snail Biomphalaria glabrata [13]. Notably, β-1,3-glucan specific receptor(s) may occur on the
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hemocyte surface. However, details related to the participation of β-1,3-glucan-specific receptors in phagocytosis by the hemocytes of bivalve mollusks have yet to be demonstrated unequivocally.
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The aim of this study is evaluation of the involvement of β-1,3-glucan and β-1,3-glucan receptors on the hemocyte surface in phagocytic processes of C. gigas
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hemocytes. We examined differences in the phagocytic abilities (phagocytic rate and phagocytic index) of C. gigas hemocytes for three different types of unopsonized foreign particles, namely Saccharomyces cerevisiae, zymosan and zymocel. The three types of particles used in this study have similar sizes and shapes, but differ in the composition of
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polysaccharides on their particle surface. The cell walls of S. cerevisiae consist almost entirely of mannans or mannoproteins (α-linked polymers of mannose; about 40% of dry
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weight of the cell walls) and β-glucans (β-1,3-linked and β-1,6-linked polymers of glucose with many branched chains; about 55% of dry weight of the cell walls), as well as a small
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amount of chitin (about 2% of the dry weight) [14]. The cell-wall outer layers consist of glycosylated mannoproteins emanating from the cell surface [15]. The inner core is composed primarily of β-1,3-glucans linked to β-1,6-glucans and chitin [15]. Chitin is mostly concentrated at the septal region, around the bud scars of the mother cells [16]. There are large amounts of mannoproteins occur on the cell surface in S. cerevisiae; a trace amount of chitin is additionally present on the cell surface. Zymosan is a boiled, trypsin-treated cell-wall derivative of S. cerevisiae [17]. Many mannoproteins are digested by these treatments, and 6
ACCEPTED MANUSCRIPT β-1,3-glucans and β-1,6-glucans are exposed on the surface of zymosan particles. Therefore, β-glucans, mannoproteins, and chitin all occur on the zymosan surface. Zymocel particles comprise β-glucans that are highly purified (>97% β-glucans; <0.05% mannoproteins) from
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the cell walls of S. cerevisiae. The two study objectives were: (1) to examine differences in the phagocytic ability of each subpopulation of C. gigas hemocytes toward the three types of the particles; and, (2)
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to examine the inhibitory effects of soluble α-mannan and β-glucan on phagocytosis of each of the three particle types by C. gigas hemocytes. Finally, we considered whether the
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phagocytic processes were accomplished through the action of the hemocyte-specific receptors that also recognize the polysaccharides of yeast cell walls.
2.1. Oysters
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2. Materials and Methods
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Pacific oyster C. gigas, with an average shell height of 13.7 ± 2.2 cm, were obtained from hanging beds at Matsushima Bay, Miyagi Prefecture, Japan from October to December, 2014.
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The specimens were transferred to a laboratory and held in 150-l aquaria with re-circulating, filtered artificial seawater (Marine Art BR; Senju Pharmaceutical Co. Ltd., Osaka, Japan) for 2–5 days until their use in an experiment. The water temperature was maintained at 15 ± 1°C. A total of 200 oysters was used in this study.
2.2. Collection of hemolymph and the isolation of hemocytes from C. gigas Hemolymph was withdrawn from the blood sinus of the adductor muscle of the oyster using a 7
ACCEPTED MANUSCRIPT tuberculin syringe with a 23-gauge, 1.5-inch needle. Each oyster was bled only once. Hemolymph from five individuals was pooled. The pooled hemolymph was transferred into a 15-ml centrifuge tube and centrifuged at 300 × g for 20 min at 4°C to separate the hemocytes.
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The resulting hemocyte pellet was washed three times with a balanced salt solution (BSS) for C. gigas hemocytes (oyster BSS: 446.6 mM NaCl, 14.5 mM KCl, 14.2 mM MgSO4, 10.6 mM MgCl2, 8.6 mM CaCl2, 3.0 mM NaHCO3, 0.08 mM NaH2PO4, and 5.6 mM glucose; pH
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7.8 [18]). The washed hemocyte pellet was then resuspended to achieve 5 × 106 cells/ml in oyster BSS. More than 95% of the hemocytes were viable based on a dye exclusion test with
maintained on ice until use.
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0.05% trypan blue in oyster BSS. To minimize cell clumping, the hemocyte suspension was
2.3. Separation of subpopulations of C. gigas hemocytes
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To separate subpopulations of hemocytes, density-gradient centrifugation with Percoll (GE Healthcare Life Sciences, Tokyo, Japan) were performed as outlined by Takahashi et al. [19],
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with slight modifications. Briefly, a discontinuous gradient was prepared by overlaying 5 ml of Percoll solution at 1.050 g/ml density (33%), and 5 ml of Percoll at 1.077 g/ml density
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(50%), from top to bottom. Five milliliters of hemocyte suspension (approximately 8 × 106 hemocytes) was applied onto the Percoll gradient and then centrifuged at 450 × g for 15 min at 4°C. After centrifugation, two different subpopulations were clearly visible and each were fractionated. More than 90% of the hemocytes were viable based on a dye exclusion test with 0.05% trypan blue in oyster BSS. The manipulation for separation of the two subpopulations was repeated eight times, using 6 × 107 hemocytes in total. The hemocyte types and purity of the subpopulations in each layer were determined by microscopic observation after staining 8
ACCEPTED MANUSCRIPT with May-Grünwald/Giemsa.
2.4. Preparation of the S. cerevisiae, zymosan, and zymocel
as foreign particles for phagocytosis assay.
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In the present study, S. cerevisiae and its cell-wall products zymosan and zymocel were used
A strain (IAM 4178) of baker’s yeast S. cerevisiae was supplied by the Institute of
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Molecular and Cellular Biosciences, University of Tokyo, Japan. Stock cultures of the S. cerevisiae strain were maintained at 4°C on yeast-mold (YM) agar plates (Difco Laboratories,
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Detroit, MI, USA). Fresh S. cerevisiae were prepared by seeding into 50 ml of YM broth (Difco Laboratories) in conical flasks and then incubating along with gentle shaking for 24 h at 25°C. After incubation, S. cerevisiae cells were harvested by centrifugation at 6500 × g for 30 min at 4°C, and then washed twice with 0.01 M phosphate-buffered 0.15 M saline (PBS,
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pH 7.4). The washed cells were suspended in 0.15 M NaCl solution and killed by heating at 90°C for 60 min, allowed to cool to room temperature, and then washed three times with PBS
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by centrifugation. The heat-killed S. cerevisiae cells (HK yeast) were resuspended in 0.15 M NaCl solution, and their concentration was adjusted to 108 cells/ml.
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Zymosan A (S. cerevisiae) BioParticles were purchased from Thermo Fisher
Scientific K.K. (Tokyo, Japan). Zymocel particles were obtained from Alpha-Beta Technologies (Smithfield, RI, USA). For preparation as foreign particles for use in this study, these particles were resuspended in PBS at a concentration of 10 mg/ml. The suspensions were sonicated for 15 min to dissipate particle clumps. Next, they were boiled at 90°C for 60 min, and then allowed to cool to room temperature. Lastly, they were washed three times with PBS by centrifugation. The concentration of each particle suspension was adjusted to 9
ACCEPTED MANUSCRIPT 200 µg/ml.
2.5. Fluorescent-probe labeling of the HK yeast, zymosan and zymocel
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The HK yeast and zymosan particles were labeled with fluorescein isothiocyanate (FITC; Sigma-Aldrich Japan K.K., Tokyo, Japan). Zymocel particles were conjugated with 5-(4,6-dichlorotriazinyl) aminofluorescein (DTAF) (Molecular Probes Inc.), because zymocel
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is not well labeled with FITC. The HK yeast and zymosan particles were suspended in 0.1 M NaHCO3/Na2CO3 buffer, pH 9.5, containing 0.1 mg/ml FITC, and incubated for 2 h with
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shaking at 20°C in the dark. At the end of the incubation period, the labeled particles were washed three times with PBS to remove free FITC. Finally, the yeast and zymosan were resuspended in oyster BSS to a concentration of 1 × 108 particles/ml. Meanwhile, the zymocel particles were suspended in 0.1 M borate buffer (pH 9.0) containing 0.1 mg/ml
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DTAF; the mixture was allowed to react for 24 h at 20°C, with gentle stirring. The DTAF-coupled zymocel particles were washed three times with PBS and separated from free
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DTAF by centrifugation. Finally, the zymocel particles were resuspended in oyster BSS to a
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concentration of 1 × 108 particles/ml.
2.6. Phagocytosis assay
To determine differences in the phagocytic capacity of C. gigas hemocytes for HK yeast, zymosan and zymocel, we examined in vitro phagocytosis by C. gigas hemocytes. Phagocytic capacity was evaluated as the percentage of phagocytic cells (phagocytic rate, PR) and the phagocytic index (PI). The PR denotes the percentage of phagocytosis-positive cells; the PI is expressed as the mean number of foreign particles ingested by one phagocytic 10
ACCEPTED MANUSCRIPT hemocyte. Phagocytosis assay was performed according to the methods of Takahashi and Mori [20], with slight modifications. Briefly, 100-µl droplets of hemocyte suspension (5 × 106
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cells/ml) were mounted onto glass coverslips and incubated at 20°C for 30 min. After incubation, the hemocyte monolayers were washed three times with oyster BSS. The monolayers were overlaid with 100 µl containing either 1 × 107 FITC-labeled HK yeast cells,
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FITC-labeled zymosan or DTAF-labeled zymocel, and incubated at 20°C for another 60 min, at a foreign particle to hemocyte ratio of 20:1. At the end of the incubation period, the excess
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or non-internalized particles were removed by thorough washing of the monolayers with oyster BSS, followed by the addition of 200 µl of trypan blue (5 mg/ml in 25 mM citrate-phosphate buffer containing 450 mM NaCl, pH 4.4) for 15 min to extinguish the FITC or DTAF fluorescence of the extracellularly bound particles. After washing twice with oyster
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BSS, the monolayers were again fluorescent-stained with 10 µl of acridine orange (10 µg/ml in oyster BSS) for classification of the agranulocytes and granulocytes in hemocytes of C.
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gigas, as described by Ling et al. [21]. The hemocyte-particle monolayers were mounted (ProLong Antifade Reagent; Invitrogen Corp., Carlsbad, CA, USA) to avoid reduction of the
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fluorescent signals by photobleaching. The extent of phagocytosis was determined by randomly counting a combined total of at least 600 phagocytic and non-phagocytic hemocytes from each monolayer via visual enumeration at 1000× magnification, and using the appropriate fluorescence filters, under a fluorescence microscope (Eclipse E600 System; Nikon Corp., Tokyo, Japan). Six coverslips of the hemocyte-particle monolayers were made for each specimen, and 26 oysters were used for this phagocytosis assay. After observation of the hemocyte monolayers, the PR and the PI of the C. gigas hemocytes were calculated. 11
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2.7. Pretreatment of C. gigas hemocytes using soluble polysaccharides We examined the impairing effects of polysaccharides on the phagocytosis of each of the
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particles (HK yeast, zymosan and zymocel) by C. gigas hemocytes. We performed the phagocytosis assay as described above in the presence of α-mannan and β-1,3-glucan to analyze competitive inhibition of phagocytosis by the soluble polysaccharides. Mannan
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(α-helix form derived from S. cerevisiae; Sigma-Aldrich Corp.) and laminarin (soluble form of β-1,3-glucan prepared from brown macroalga Laminaria digitata; Sigma-Aldrich Corp.)
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were dissolved in 5 mM sodium citrate, 0.15 M NaCl, pH 5.2, at a concentration of 1 mg/ml, and diluted to 100 µg/ml with oyster BSS. Hemocyte monolayers were pre-incubated with 100 µl of each polysaccharide solution, at 20°C for 60 min, before adding the particles to the hemocyte monolayers.
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To determine the influence of laminarin on phagocytosis of zymosan by granulocytes, the PR was gauged by adding different concentrations of the laminarin (i.e., 0,
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12.5, 25, 50, 100 and 200 µg/ml). Phagocytosis assay was performed as described above, and 15 oysters were used for this phagocytosis assay. Hemocytes pre-treated with oyster BSS (0
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µg/ml) were used as a control.
2.8. Statistical analyses
The experiments were conducted in triplicates of six specimens each. Quantitative data are expressed as mean ± standard deviation (SD) of the replicated experiments. All percentage data were arcsine transformed prior to analysis. These data for the analyses were assessed using one-way analysis of variance (ANOVA). Tukey’s multiple comparison test was 12
ACCEPTED MANUSCRIPT conducted to compare the PR and PI associated with the three different particles as a post-hoc analysis if significant differences were found. Dunnett’s method for multiple comparisons was also used to analyze the inhibitory effects of the soluble polysaccharides on the
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phagocytosis of the three types of particles as compared to that of oyster BSS as the control. Williams’ multiple comparison test was also conducted to analyze the influence of the laminarin on the phagocytosis of zymosan by C. gigas granulocytes at different
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concentrations. Statistical analyses were performed using EZR (Saitama Medical Center, Jichi University, Japan), which is a graphical user interface for R (The R Foundation for
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Statistical Computing, Austria). All tests were considered significant at P < 0.05.
3. Results
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3.1. Classification and separation of subpopulations of C. gigas hemocytes In the present study, agranulocytes (AGs) and granulocytes (Gs) were classifiable as the two
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subpopulations of C. gigas hemocytes, using light microscopy with May-Grünwald/Giemsa staining. The Gs were easily distinguishable from AGs because they contained many granules
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that stained red and/or purple (blue). The cytoplasm of AGs was stained faint blue; no or few granules were present in the cytoplasm. The ratio of AGs to Gs varied in the oyster hemocytes over the course of the year; however, the number of AGs was always greater [20]. The mean proportion of AGs to Gs over the experimental period of this study was 82.8 ± 3.6% : 17.2 ± 4.8% (mean ± SD, n = 41). C. gigas hemocytes were separated into two layers by Percoll density-gradient centrifugation. More than 90% of the hemocytes were viable as revealed by trypan blue 13
ACCEPTED MANUSCRIPT staining. Each layer contained one dominant subpopulation and one minor subpopulation (Table 1): AGs dominated (90.9 ± 2.8 purity) in the upper layer, while Gs (88.9 ± 5.1 purity) were mainly in the bottom layer. The Gs were characterized as cells with a relatively large
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size and high granularity.
3.2. Phagocytic ability of the different subpopulations for HK yeast, zymosan and zymocel
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Next, we examined the phagocytic ability of C. gigas hemocytes against the three types of particles, namely HK yeast and its cell-walls derivatives, zymosan and zymocel using 5 × 105
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hemocytes (100 µl of 5 × 106 hemocytes/ml). Both the AGs and Gs showed phagocytic ability towards all the particles tested, but the Gs exhibited much higher phagocytic ability; this reflected the large number of actively phagocytic hemocytes in the Gs fraction (Fig. 1a). The phagocytic rate (PR) of AGs did not significantly differ among the three types of
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particles, and ranged from 10.1 ± 3.3% for zymosan (n = 26) to 11.7 ± 1.7% for HK yeast (n = 26). The Gs were the more active phagocytes for all the particles tested, and their PR
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ranged from 65.8 ± 4.1% (HK yeast) to 89.1 ± 6.9% (zymosan). The PR of Gs for zymosan and zymocel were significantly greater than that for HK yeast (P < 0.05). The estimated
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phagocytic index (PI) of both the AGs and Gs varied for the three particles (Fig. 1b). The PI of AGs for zymosan was significantly higher, at 7.9 ± 1.2, as compared to that for HK yeast and zymocel (5.0 ± 0.5 and 5.9 ± 0.9, respectively) (P < 0.05). Similarly, the PI of Gs significantly differed among the three foreign particles (P < 0.05): the PI of Gs for zymosan was highest, at 9.4 ± 1.7, and that for HK yeast and zymocel were 3.9 ± 1.1 and 5.6 ± 1.9, respectively. To analyze the characteristics of the PI for the three types of particles, especially to 14
ACCEPTED MANUSCRIPT understand the cause of the much higher value of the PI of Gs for zymosan, the PI was divided into increments of 5 (1–5, 6–10, 11–15, and ≥16) and the phagocytic frequencies of the increments were compared (Fig. 2). This experiment used 5 × 105 hemocytes. The results
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showed a markedly high PI of ≥16 exhibited by 18.1% of Gs for zymosan, significantly higher than the PI of 2.7% and 3.9% exhibited for HK yeast and zymocel, respectively (Fig. 2a). There was a greater ratio of AGs (6.6%) possessing a high PI (≥16) for zymosan as
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and Gs, the maximum PI value was 32 for zymosan.
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compared that for HK yeast and zymocel (0.8% and 2.2%, respectively, Fig. 2b). In both AGs
3.3. Effects of polysaccharides on phagocytosis by granulocytes and agranulocytes Laminarin (β-1,3-glucan) inhibited ingestion of zymosan and zymocel by Gs, although α-mannan did not (Fig. 3a). This experiment used 5 × 105 hemocytes. The PR for both
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zymosan and zymocel was respectively reduced by 47.6% (i.e., from 85.5 ± 4.1% to 44.8 ± 5.7%) and 45.4% (i.e., from 82.0 ± 6.4% to 44.8 ± 6.4%). In addition, the PR of
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laminarin-treated Gs was also affected in HK yeast. In contrast, the PI of Gs did not decrease in any of the particle types after treatment with both α-mannan and laminarin (Fig. 3b). In
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addition, the phagocytic ability (i.e., both the PR and PI) of AGs for these particles was almost unaffected by pretreatment with α-mannan and laminarin. Laminarin partly inhibited the phagocytosis of zymosan by Gs, in a dose-dependent
manner (Fig. 4): the PR of Gs significantly decreased at 100 µg/ml (44.8 ± 8.7%) and 200 µg/ml (43.6 ± 7.4%) compared to that of 0 µg/ml (84.3 ± 6.9%). The lowest PR of Gs was 43.6 ± 7.4% by treatment with 200 µg/ml of laminarin.
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ACCEPTED MANUSCRIPT 4. Discussion The experiments in the present study separated two subpopulations of hemocytes from C. gigas: agranulocytes (AGs) and granulocytes (Gs). In our previous study, we separated the
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three subpopulations of C. gigas hemocytes using discontinuous Percoll gradient method [19]. We attempted to separate the subpopulations using the same Percoll gradient used previously, but we obtained fractions with low purity. Therefore, tried to separate the C. gigas hemocytes
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using different Percoll densities. For unknown reasons, we could not separate two subpopulations with high purity.
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In our previous study, we carried out flow cytometry by fluorescence-activated cell sorting to classify and separate subpopulations of C. gigas hemocytes [22]. According to their size, granularity, and complexity using flow cytometry and May-Grünwald/Giemsa staining, the C. gigas hemocytes were divided into three subpopulations: granulocytes, hyalinocytes
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and agranulocytes. The agranulocytes and hyalinocytes in the study of Terahara et al. [22] have been referred to other studies as small hyalinocytes and large hyalinocytes, respectively
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[23], [24], and agranulocytes and semi-granulocytes, respectively [25]. Similar subtypes of hemocytes have been referred to and reported in other species of Crassostrea, for example:
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agranulocytes, semi-granulocytes, and granulocytes in C. virginica [26]; blast-like cells, hyalinocytes, and granulocytes in C. ariakensis [27]; and blast-like cells, agranulocytes, and granulocytes in C. rhizophorae [28]. In the present study, we were not able to separate three subpopulations by Percoll density-gradient centrifugation. We concluded that hyalinocytes were not able to be separated from the other subpopulations and were contained within the agranulocytes (AGs) fraction based on microscopic observations in this study. Some previous studies have also reported differences in phagocytic ability between 16
ACCEPTED MANUSCRIPT agranulocytes and granulocytes in oyster species. For example, agranulocytes might had a lower phagocytic ability than granulocytes in C. gigas [25] and C. virginica
[29], [30].
Similarly, agranulocytes and semi-granulocytes showed either no or only weak phagocytosis,
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while granulocytes showed strong phagocytic ability in C. gigas hemocytes [7]. In a previous study, we found that both agranulocytes and granulocytes showed phagocytic ability against all the particles tested, but granulocytes especially were more active than agranulocytes [20].
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Hine [31] summarized the characteristics of phagocytosis by both agranulocytes and granulocytes in other bivalve species as follows: granulocytes exhibit a high phagocytic
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ability against various foreign particles [4]; agranulocytes might have a non-phagocytic ability in Mediterranean mussel Mytilus galloprovincialis [32], boring clam Tridacna crocea [33], and three species of deep-sea symbiotic Bathymodiolus mussels [34]; and agranulocytes
mercenaria [35].
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might have a lower phagocytic ability than granulocytes in the hard clam Mercenaria
In this study, we obtained comparable results about the phagocytic ability of both
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AGs and Gs for HK yeast, zymosan and zymocel. The phagocytic rate (PR) and phagocytic index (PI) of Gs were much greater than those of AGs. Furthermore, a new finding was that,
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in both the AGs and Gs, the PI for zymosan was much higher than the PI for other particles. We gauged the values of PI in increments of 5 (i.e., 1–5, 6–10, 11–15, and ≥16), and then compared the phagocytic frequencies of these PI increments to better understand the outcome of a much higher mean PI value for zymosan. A markedly high PI of ≥16 was shown by 18.1% of Gs for zymosan, which was significantly higher than the 2.7% and 3.9% for HK yeast and zymocel, respectively. AGs also revealed greater cell ratios possessing higher PI (≥16) for zymosan than those for HK yeast and zymocel. These results indicate that the high 17
ACCEPTED MANUSCRIPT PI for zymosan was not attributable to all phagocytic hemocytes having a high mean PI, but to some portion of the AGs and Gs being able to phagocytize many zymosan particles using different phagocytic mechanisms.
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In our previous study (described above) we examined the effects of various treatments for the same foreign particles on the respective phagocytic ability of Gs [20]. Escherichia coli cells treated with formaldehyde and then autoclaved were strongly resistant
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to phagocytosis by Gs, although living cells or heat-killed cells were avidly phagocytized. Those results suggested that a chemical or physical alteration of the cell surfaces of E. coli
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affects their ability to be engulfed by Gs, but not by the AGs. Tripp [35] reported similar results in relation to phagocytosis by M. mercenaria hemocytes. His results also suggest that granulocytes possess a specialized system that is mediated by membrane receptors in a
mammals.
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non-self-recognition and binding toward foreign particles, similar to that of macrophages in
Recognition and phagocytosis of foreign particles in mollusks might involve lectins
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or lectin-like receptors with specific carbohydrate-binding properties [5], [13], [36]. The involvement of humoral lectins that function as opsonins has been demonstrated for the
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phagocytosis of yeast by hemocytes of common mussel Mytilus edulis [37] and freshwater snail Biomphalaria glabrata [13]. A galectin (CvGal) located on the surface of C. virginica hemocytes was found to recognize and phagocytize a variety of microbe pathogens [6]. Recently, a new type of phagocytic receptor, CgNimC, was reported as strongly binding to LPS and able to promote phagocytosis of Vibrio species by C. gigas hemocytes [7]. In addition, peptidoglycan recognition protein (PGRP) and β-1,3-glucan-binding protein (LGBP) gene was found in the scallop Chlamys farreri, and these proteins were inferred to be 18
ACCEPTED MANUSCRIPT a humoral recognition molecule or a phagocytic receptor [8], [9]. S. cerevisiae (HK yeast), zymosan and zymocel have similar sizes and shapes but differ in the composition of polysaccharides on their surface: S. cerevisiae has a large amount
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of mannan and only a trace amount of chitin; zymosan contains a large amount of β-glucan and low amounts of mannan and chitin; and zymocel is almost entirely composed of β-glucan. Therefore, we proposed that if differences occurred among the three particles in terms of their
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respective phagocytosis by agranulocytes and granulocytes in C. gigas hemocytes, this would be a result of differential recognition of the polysaccharides on the surface.
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Laminarin, a soluble algal product, is structurally and chemically similar to yeast glucan [38] and it is well used as an inhibitor of β-1,3-glucan receptors [39]. In the present study, laminarin partly inhibited ingestion of zymosan and zymocel by Gs, but did not inhibit that of HK yeast. In addition, soluble mannan did not block ingestion of any of the tested
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particle types. These results indicate that the Gs of C. gigas hemocytes might possess β-glucan receptors for phagocytosis on their cell surface. We concluded that β-glucan
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receptors are important for phagocytosis of foreign particles by oyster hemocytes. In fact, there are microorganisms, fungi [40,41] and oomycetes [41], that possess β-glucan on their surface.
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However, the residual phagocytosis, of about 50%, that was not blocked by laminarin suggests that β-1,3-glucan-independent modes of phagocytosis by Gs exist. Laminarin strongly inhibited phagocytosis of unopsonized, autoclave-killed yeast by B. glabrata hemocytes [13]. In this study, however, phagocytosis of HK yeast by both AGs and Gs was unaffected by pretreatment with either α-mannan or laminarin, which contrasts to the situation in mammals and in B. glabrata. The findings of the present study suggest that C. gigas has multiple types of hemocytes with phagocytic ability, some of which depend on 19
ACCEPTED MANUSCRIPT β-glucan receptors for phagocytosis. In conclusion, the results show that both AGs and Gs, as the two subpopulations of C. gigas hemocytes, exerted strong phagocytic ability for baker’s yeast and its cell-walls
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derivatives, zymosan and zymocel. Especially, zymosan was greatly phagocytized by a portion of the hemocytes. Furthermore, the present findings suggest that C. gigas has multiple types of hemocytes with phagocytic ability, some of which depend on β-glucan
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receptors for phagocytosis. Although we did not obtain evidence for this, we speculate that Gs and AGs contain subpopulations that possess different receptors for phagocytosis. The
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β-glucan receptors might mediate the phagocytosis of these particles by Gs, if not all the particles. Apart from these exceptional Gs, however, other types of hemocytes with similarly
Acknowledgements
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high phagocytic ability might phagocytize foreign particles through different mechanisms.
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JP16K07866.
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This work was supported in part by JSPS KAKENHI Grant Numbers 17380112 and
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Takahashi et al., Fig. 2
27
zymosan
HK yeast
zymocel
(a)
100 90 a
70 60
b
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b
50 40 30 20
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a
10
b
0
6~10
1~5
11~15
b
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100 90 a
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(b)
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HK yeast
70
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Phagocytic index
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40 30 20
a, b b a
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Phagocytic index
Takahashi et al., Fig. 3 28
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Takahashi et al., Fig. 4 29
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Takahashi et al., Table 30
ACCEPTED MANUSCRIPT Table 1 Purity of hemocyte sub-populations in layers separated by Percoll density
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gradient
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ACCEPTED MANUSCRIPT Figure captions
Fig. 1 Phagocytosis of HK yeast, zymosan, and zymocel by agranulocytes and granulocytes
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of C. gigas: (a) phagocytic rate (i.e., percentage of phagocytosis-positive cells); (b) phagocytic index (i.e., mean number of foreign particles ingested by one phagocytic hemocyte). The data are expressed as mean ± SD in columns having different alphabetical
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designations to show significant differences using Tukey’s multiple comparison test (P <
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0.05; n = 26).
Fig. 2 Frequency of the phagocytic index values (in increments of 5: 1–5, 6–10, 11–15, and ≥16) for the granulocytes (a) and agranulocytes (b) in response to HK yeast, zymosan and zymocel. The data are expressed as mean ± SD in columns having different alphabetical
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0.05; n = 26).
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designations to show significant differences using Tukey’s multiple comparison test (P <
Fig. 3 Effects of soluble α-mannan or laminarin on the phagocytosis of HK yeast, zymosan
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and zymocel by granulocytes: (a) phagocytic rate; (b) phagocytic index. *Significantly lower than values for treatment with oyster balanced salt solution (BSS) as a control, as determined by Dunnett’s multiple comparison test (P < 0.05; n = 15).
Fig. 4 Dose dependency of the inhibitory effects of laminarin on the phagocytosis of zymosan by granulocytes. The data are expressed as mean ± SD. *Significantly lower than the values for treatment with oyster balanced salt solution (0 µg/ml of laminarin) as a control, as 32
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determined by Williams’ multiple comparison test (P < 0.05; n = 6).
33
ACCEPTED MANUSCRIPT Highlights
1. Two sub-populations, AGs and Gs, could be separated from the hemocytes of C. gigas.
phagocytes. 3. A part of Gs showed markedly high PI for zymosan.
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2. Both AGs and Gs exhibited phagocytic ability, especially Gs functioned much active
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4. Soluble laminarin partly inhibited ingestion of zymosan and zymocel by Gs.
5. C. gigas has multiple types of hemocytes with phagocytic ability, some of which depend
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on β-glucan receptors for phagocytosis.
S1050-4648(17)30546-6
DOI:
10.1016/j.fsi.2017.09.027
Reference:
YFSIM 4817
To appear in:
Fish and Shellfish Immunology
Received Date: 17 February 2017 Revised Date:
5 September 2017
Accepted Date: 9 September 2017
Please cite this article as: Takahashi KG, Izumi-Nakajima N, Mori K, Unique phagocytic properties of hemocytes of Pacific oyster Crassostrea gigas against yeast and yeast cell-wall derivatives, Fish and Shellfish Immunology (2017), doi: 10.1016/j.fsi.2017.09.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Second revised manuscript: FSIM-D-17-00156
Title. Unique phagocytic properties of hemocytes of Pacific oyster Crassostrea gigas against
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yeast and yeast cell-wall derivatives
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Journal. Fish and Shellfish Immunology
1
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Authors. Keisuke G Takahashi1,2*, Nakako Izumi-Nakajima3, and Katsuyoshi Mori2
Laboratory of Aquacultural Biology, Graduate School of Agricultural Sciences, Tohoku
University, Aoba-ku, Sendai 980-0845, JAPAN
Oyster Research Institute, Aoba-ku, Sendai 989-3204, JAPAN
3
National Institute of Radiological Sciences, QST, Inage-ku, Chiba-shi 263-8555, JAPAN
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*Corresponding author:
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2
Keisuke G Takahashi
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Address: Laboratory of Aquacultural Biology, Graduate School of Agricultural Sciences, Tohoku University, Aoba-ku, Sendai 980-0845, JAPAN Phone: +81 22 757 4134; Fax: +81 22 757 4132 E-mail address: [email protected]
Abbreviations: AGs, agranulocytes; Gs, granulocytes; HK yeast, heat-killed yeast; PI, phagocytic index; PR, 1
ACCEPTED MANUSCRIPT phagocytic rate
Conflict of interest:
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None of the authors has a financial relationship with a commercial entity that has an interest
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in the subject of this manuscript.
2
ACCEPTED MANUSCRIPT Abstract
For a marine bivalve mollusk such as Pacific oyster Crassostrea gigas, the elimination of
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foreign particles via hemocyte phagocytosis plays an important role in host defense mechanisms. The hemocytes of C. gigas have a high phagocytic ability for baker’s yeast (Saccharomyces cerevisiae) and its cell-wall product zymosan. C. gigas hemocytes might
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phagocytose yeast cells after binding to polysaccharides on the cell-wall surface, but it is unknown how and what kinds of polysaccharide molecules are recognized. We conducted
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experiments to determine differences in the phagocytic ability of C. gigas hemocytes against heat-killed yeast (HK yeast), zymosan and zymocel, which are similarly sized and shaped but differ in the polysaccharide composition of their particle surface. We found that both the agranulocytes and granulocytes exerted strong phagocytic ability on all tested particles. The
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phagocytic index (PI) of granulocytes for zymosan was 9.4 ± 1.7, which significantly differed with that for HK yeast and zymocel (P < 0.05). To evaluate the PI for the three types of
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particles, and especially to understand the outcome of the much higher PI for zymosan, PI was gauged in increments of 5 (1–5, 6–10, 11–15, and ≥16), and the phagocytic frequencies
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were compared according to these increments. The results show that a markedly high PI of ≥16 was exhibited by 18.1% of granulocytes for zymosan, significantly higher than 1.7% and 3.9% shown for HK yeast and zymocel, respectively (P < 0.05). These findings indicate that the relatively high PI for zymosan could not be attributed to a situation wherein all phagocytic hemocytes shared a high mean PI, but rather to the ability of some hemocytes to phagocytose a larger portion of zymosan. To determine whether the phagocytosis of these respective particles depended on the recognition of specific polysaccharide receptors on the 3
ACCEPTED MANUSCRIPT hemocyte surface, C. gigas hemocytes were pretreated with soluble α-mannan or β-laminarin and then allowed to phagocytose the three types of the particles. The percentage of phagocytic cells of β-laminarin-treated granulocytes decreased significantly for zymosan and
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zymocel, but not for yeast. These results suggest that C. gigas might possess at least two types of hemocytes, and that one type of the hemocytes (granulocytes) is more active for phagocytosis. The granulocytes were found to have multiple subtypes with different
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phagocytic abilities and multiple phagocytic receptors. Some of the granulocyte subtypes revealed a much stronger phagocytic ability, depending on the presence of β-glucan receptors
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for phagocytosis.
Highlights
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1. Two sub-populations, AGs and Gs, could be separated from the hemocytes of C. gigas. 2. Both AGs and Gs exhibited phagocytic ability, especially Gs functioned much active
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phagocytes.
3. A part of Gs showed markedly high PI for zymosan.
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4. Soluble laminarin partly inhibited ingestion of zymosan and zymocel by Gs. 5. C. gigas has multiple types of hemocytes with phagocytic ability, some of which depend on β-glucan receptors for phagocytosis.
Keywords: Crassostrea gigas; Oyster; Hemocytes; Granulocytes; Agranulocytes; Phagocytosis; Yeast; Zymosan; β-glucan receptor
4
ACCEPTED MANUSCRIPT 1. Introduction
The host defense system of bivalve mollusks is understood to depend mainly on
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circulating hemocytes. These cells are present in the hemolymph and possess a strong migration ability in response to invading microorganisms, including potential pathogens, and will actively phagocytose these invaders [1], [2], [3]. The hemocytes of bivalve mollusks
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morphologically resemble mammalian phagocytic leukocytes, such as macrophages and neutrophils [4], and like leukocytes they have the ability to recognize, engulf, and internally
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degrade biological particles, including microbial pathogens. Therefore, it is believed that the hemocytes of bivalve mollusks might possess a specialized system that is mediated by a hemocyte membrane receptor in a non-self-recognition of foreign particles. In eastern oyster Crassostrea virginica, cell membrane-associated galectin of the
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hemocyte recognizes and functions to phagocytose Perkinsus marinus trophozoites—the etiologic agent in Dermo disease, which is responsible for episodes of mass mortality among
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C. virginica [5], [6]. Receptors of the Nimrod family are found on the hemocytes of C. gigas [7]; these receptors recognize lipopolysaccharides (LPS) and enhance the phagocytosis of bacteria.
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gram-negative
Furthermore,
in
recent
years,
cDNA
sequences
of
pattern-recognition-receptor-related genes have been cloned and analyzed for mRNA expression in bivalve mollusks, and these include LPS and β-1,3-glucan recognition proteins [8], [9], and peptidoglycan-recognition protein [10], [11]. In addition, hemocytes of C. gigas have the function analogous to that human toll-like receptors (TLRs) as the representative pattern recognition receptors (PRRs)[12]. These receptors play an important role in host defense of C. gigas against pathogen infection. 5
ACCEPTED MANUSCRIPT In molluscs, laminarin (soluble β-1,3-glucan) markedly inhibited the phagocytosis of both opsonized and unopsonized, autoclave-killed yeast cells by hemocytes of the snail Biomphalaria glabrata [13]. Notably, β-1,3-glucan specific receptor(s) may occur on the
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hemocyte surface. However, details related to the participation of β-1,3-glucan-specific receptors in phagocytosis by the hemocytes of bivalve mollusks have yet to be demonstrated unequivocally.
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The aim of this study is evaluation of the involvement of β-1,3-glucan and β-1,3-glucan receptors on the hemocyte surface in phagocytic processes of C. gigas
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hemocytes. We examined differences in the phagocytic abilities (phagocytic rate and phagocytic index) of C. gigas hemocytes for three different types of unopsonized foreign particles, namely Saccharomyces cerevisiae, zymosan and zymocel. The three types of particles used in this study have similar sizes and shapes, but differ in the composition of
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polysaccharides on their particle surface. The cell walls of S. cerevisiae consist almost entirely of mannans or mannoproteins (α-linked polymers of mannose; about 40% of dry
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weight of the cell walls) and β-glucans (β-1,3-linked and β-1,6-linked polymers of glucose with many branched chains; about 55% of dry weight of the cell walls), as well as a small
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amount of chitin (about 2% of the dry weight) [14]. The cell-wall outer layers consist of glycosylated mannoproteins emanating from the cell surface [15]. The inner core is composed primarily of β-1,3-glucans linked to β-1,6-glucans and chitin [15]. Chitin is mostly concentrated at the septal region, around the bud scars of the mother cells [16]. There are large amounts of mannoproteins occur on the cell surface in S. cerevisiae; a trace amount of chitin is additionally present on the cell surface. Zymosan is a boiled, trypsin-treated cell-wall derivative of S. cerevisiae [17]. Many mannoproteins are digested by these treatments, and 6
ACCEPTED MANUSCRIPT β-1,3-glucans and β-1,6-glucans are exposed on the surface of zymosan particles. Therefore, β-glucans, mannoproteins, and chitin all occur on the zymosan surface. Zymocel particles comprise β-glucans that are highly purified (>97% β-glucans; <0.05% mannoproteins) from
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the cell walls of S. cerevisiae. The two study objectives were: (1) to examine differences in the phagocytic ability of each subpopulation of C. gigas hemocytes toward the three types of the particles; and, (2)
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to examine the inhibitory effects of soluble α-mannan and β-glucan on phagocytosis of each of the three particle types by C. gigas hemocytes. Finally, we considered whether the
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phagocytic processes were accomplished through the action of the hemocyte-specific receptors that also recognize the polysaccharides of yeast cell walls.
2.1. Oysters
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2. Materials and Methods
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Pacific oyster C. gigas, with an average shell height of 13.7 ± 2.2 cm, were obtained from hanging beds at Matsushima Bay, Miyagi Prefecture, Japan from October to December, 2014.
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The specimens were transferred to a laboratory and held in 150-l aquaria with re-circulating, filtered artificial seawater (Marine Art BR; Senju Pharmaceutical Co. Ltd., Osaka, Japan) for 2–5 days until their use in an experiment. The water temperature was maintained at 15 ± 1°C. A total of 200 oysters was used in this study.
2.2. Collection of hemolymph and the isolation of hemocytes from C. gigas Hemolymph was withdrawn from the blood sinus of the adductor muscle of the oyster using a 7
ACCEPTED MANUSCRIPT tuberculin syringe with a 23-gauge, 1.5-inch needle. Each oyster was bled only once. Hemolymph from five individuals was pooled. The pooled hemolymph was transferred into a 15-ml centrifuge tube and centrifuged at 300 × g for 20 min at 4°C to separate the hemocytes.
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The resulting hemocyte pellet was washed three times with a balanced salt solution (BSS) for C. gigas hemocytes (oyster BSS: 446.6 mM NaCl, 14.5 mM KCl, 14.2 mM MgSO4, 10.6 mM MgCl2, 8.6 mM CaCl2, 3.0 mM NaHCO3, 0.08 mM NaH2PO4, and 5.6 mM glucose; pH
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7.8 [18]). The washed hemocyte pellet was then resuspended to achieve 5 × 106 cells/ml in oyster BSS. More than 95% of the hemocytes were viable based on a dye exclusion test with
maintained on ice until use.
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0.05% trypan blue in oyster BSS. To minimize cell clumping, the hemocyte suspension was
2.3. Separation of subpopulations of C. gigas hemocytes
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To separate subpopulations of hemocytes, density-gradient centrifugation with Percoll (GE Healthcare Life Sciences, Tokyo, Japan) were performed as outlined by Takahashi et al. [19],
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with slight modifications. Briefly, a discontinuous gradient was prepared by overlaying 5 ml of Percoll solution at 1.050 g/ml density (33%), and 5 ml of Percoll at 1.077 g/ml density
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(50%), from top to bottom. Five milliliters of hemocyte suspension (approximately 8 × 106 hemocytes) was applied onto the Percoll gradient and then centrifuged at 450 × g for 15 min at 4°C. After centrifugation, two different subpopulations were clearly visible and each were fractionated. More than 90% of the hemocytes were viable based on a dye exclusion test with 0.05% trypan blue in oyster BSS. The manipulation for separation of the two subpopulations was repeated eight times, using 6 × 107 hemocytes in total. The hemocyte types and purity of the subpopulations in each layer were determined by microscopic observation after staining 8
ACCEPTED MANUSCRIPT with May-Grünwald/Giemsa.
2.4. Preparation of the S. cerevisiae, zymosan, and zymocel
as foreign particles for phagocytosis assay.
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In the present study, S. cerevisiae and its cell-wall products zymosan and zymocel were used
A strain (IAM 4178) of baker’s yeast S. cerevisiae was supplied by the Institute of
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Molecular and Cellular Biosciences, University of Tokyo, Japan. Stock cultures of the S. cerevisiae strain were maintained at 4°C on yeast-mold (YM) agar plates (Difco Laboratories,
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Detroit, MI, USA). Fresh S. cerevisiae were prepared by seeding into 50 ml of YM broth (Difco Laboratories) in conical flasks and then incubating along with gentle shaking for 24 h at 25°C. After incubation, S. cerevisiae cells were harvested by centrifugation at 6500 × g for 30 min at 4°C, and then washed twice with 0.01 M phosphate-buffered 0.15 M saline (PBS,
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pH 7.4). The washed cells were suspended in 0.15 M NaCl solution and killed by heating at 90°C for 60 min, allowed to cool to room temperature, and then washed three times with PBS
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by centrifugation. The heat-killed S. cerevisiae cells (HK yeast) were resuspended in 0.15 M NaCl solution, and their concentration was adjusted to 108 cells/ml.
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Zymosan A (S. cerevisiae) BioParticles were purchased from Thermo Fisher
Scientific K.K. (Tokyo, Japan). Zymocel particles were obtained from Alpha-Beta Technologies (Smithfield, RI, USA). For preparation as foreign particles for use in this study, these particles were resuspended in PBS at a concentration of 10 mg/ml. The suspensions were sonicated for 15 min to dissipate particle clumps. Next, they were boiled at 90°C for 60 min, and then allowed to cool to room temperature. Lastly, they were washed three times with PBS by centrifugation. The concentration of each particle suspension was adjusted to 9
ACCEPTED MANUSCRIPT 200 µg/ml.
2.5. Fluorescent-probe labeling of the HK yeast, zymosan and zymocel
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The HK yeast and zymosan particles were labeled with fluorescein isothiocyanate (FITC; Sigma-Aldrich Japan K.K., Tokyo, Japan). Zymocel particles were conjugated with 5-(4,6-dichlorotriazinyl) aminofluorescein (DTAF) (Molecular Probes Inc.), because zymocel
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is not well labeled with FITC. The HK yeast and zymosan particles were suspended in 0.1 M NaHCO3/Na2CO3 buffer, pH 9.5, containing 0.1 mg/ml FITC, and incubated for 2 h with
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shaking at 20°C in the dark. At the end of the incubation period, the labeled particles were washed three times with PBS to remove free FITC. Finally, the yeast and zymosan were resuspended in oyster BSS to a concentration of 1 × 108 particles/ml. Meanwhile, the zymocel particles were suspended in 0.1 M borate buffer (pH 9.0) containing 0.1 mg/ml
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DTAF; the mixture was allowed to react for 24 h at 20°C, with gentle stirring. The DTAF-coupled zymocel particles were washed three times with PBS and separated from free
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DTAF by centrifugation. Finally, the zymocel particles were resuspended in oyster BSS to a
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concentration of 1 × 108 particles/ml.
2.6. Phagocytosis assay
To determine differences in the phagocytic capacity of C. gigas hemocytes for HK yeast, zymosan and zymocel, we examined in vitro phagocytosis by C. gigas hemocytes. Phagocytic capacity was evaluated as the percentage of phagocytic cells (phagocytic rate, PR) and the phagocytic index (PI). The PR denotes the percentage of phagocytosis-positive cells; the PI is expressed as the mean number of foreign particles ingested by one phagocytic 10
ACCEPTED MANUSCRIPT hemocyte. Phagocytosis assay was performed according to the methods of Takahashi and Mori [20], with slight modifications. Briefly, 100-µl droplets of hemocyte suspension (5 × 106
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cells/ml) were mounted onto glass coverslips and incubated at 20°C for 30 min. After incubation, the hemocyte monolayers were washed three times with oyster BSS. The monolayers were overlaid with 100 µl containing either 1 × 107 FITC-labeled HK yeast cells,
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FITC-labeled zymosan or DTAF-labeled zymocel, and incubated at 20°C for another 60 min, at a foreign particle to hemocyte ratio of 20:1. At the end of the incubation period, the excess
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or non-internalized particles were removed by thorough washing of the monolayers with oyster BSS, followed by the addition of 200 µl of trypan blue (5 mg/ml in 25 mM citrate-phosphate buffer containing 450 mM NaCl, pH 4.4) for 15 min to extinguish the FITC or DTAF fluorescence of the extracellularly bound particles. After washing twice with oyster
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BSS, the monolayers were again fluorescent-stained with 10 µl of acridine orange (10 µg/ml in oyster BSS) for classification of the agranulocytes and granulocytes in hemocytes of C.
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gigas, as described by Ling et al. [21]. The hemocyte-particle monolayers were mounted (ProLong Antifade Reagent; Invitrogen Corp., Carlsbad, CA, USA) to avoid reduction of the
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fluorescent signals by photobleaching. The extent of phagocytosis was determined by randomly counting a combined total of at least 600 phagocytic and non-phagocytic hemocytes from each monolayer via visual enumeration at 1000× magnification, and using the appropriate fluorescence filters, under a fluorescence microscope (Eclipse E600 System; Nikon Corp., Tokyo, Japan). Six coverslips of the hemocyte-particle monolayers were made for each specimen, and 26 oysters were used for this phagocytosis assay. After observation of the hemocyte monolayers, the PR and the PI of the C. gigas hemocytes were calculated. 11
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2.7. Pretreatment of C. gigas hemocytes using soluble polysaccharides We examined the impairing effects of polysaccharides on the phagocytosis of each of the
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particles (HK yeast, zymosan and zymocel) by C. gigas hemocytes. We performed the phagocytosis assay as described above in the presence of α-mannan and β-1,3-glucan to analyze competitive inhibition of phagocytosis by the soluble polysaccharides. Mannan
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(α-helix form derived from S. cerevisiae; Sigma-Aldrich Corp.) and laminarin (soluble form of β-1,3-glucan prepared from brown macroalga Laminaria digitata; Sigma-Aldrich Corp.)
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were dissolved in 5 mM sodium citrate, 0.15 M NaCl, pH 5.2, at a concentration of 1 mg/ml, and diluted to 100 µg/ml with oyster BSS. Hemocyte monolayers were pre-incubated with 100 µl of each polysaccharide solution, at 20°C for 60 min, before adding the particles to the hemocyte monolayers.
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To determine the influence of laminarin on phagocytosis of zymosan by granulocytes, the PR was gauged by adding different concentrations of the laminarin (i.e., 0,
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12.5, 25, 50, 100 and 200 µg/ml). Phagocytosis assay was performed as described above, and 15 oysters were used for this phagocytosis assay. Hemocytes pre-treated with oyster BSS (0
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µg/ml) were used as a control.
2.8. Statistical analyses
The experiments were conducted in triplicates of six specimens each. Quantitative data are expressed as mean ± standard deviation (SD) of the replicated experiments. All percentage data were arcsine transformed prior to analysis. These data for the analyses were assessed using one-way analysis of variance (ANOVA). Tukey’s multiple comparison test was 12
ACCEPTED MANUSCRIPT conducted to compare the PR and PI associated with the three different particles as a post-hoc analysis if significant differences were found. Dunnett’s method for multiple comparisons was also used to analyze the inhibitory effects of the soluble polysaccharides on the
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phagocytosis of the three types of particles as compared to that of oyster BSS as the control. Williams’ multiple comparison test was also conducted to analyze the influence of the laminarin on the phagocytosis of zymosan by C. gigas granulocytes at different
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concentrations. Statistical analyses were performed using EZR (Saitama Medical Center, Jichi University, Japan), which is a graphical user interface for R (The R Foundation for
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Statistical Computing, Austria). All tests were considered significant at P < 0.05.
3. Results
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3.1. Classification and separation of subpopulations of C. gigas hemocytes In the present study, agranulocytes (AGs) and granulocytes (Gs) were classifiable as the two
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subpopulations of C. gigas hemocytes, using light microscopy with May-Grünwald/Giemsa staining. The Gs were easily distinguishable from AGs because they contained many granules
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that stained red and/or purple (blue). The cytoplasm of AGs was stained faint blue; no or few granules were present in the cytoplasm. The ratio of AGs to Gs varied in the oyster hemocytes over the course of the year; however, the number of AGs was always greater [20]. The mean proportion of AGs to Gs over the experimental period of this study was 82.8 ± 3.6% : 17.2 ± 4.8% (mean ± SD, n = 41). C. gigas hemocytes were separated into two layers by Percoll density-gradient centrifugation. More than 90% of the hemocytes were viable as revealed by trypan blue 13
ACCEPTED MANUSCRIPT staining. Each layer contained one dominant subpopulation and one minor subpopulation (Table 1): AGs dominated (90.9 ± 2.8 purity) in the upper layer, while Gs (88.9 ± 5.1 purity) were mainly in the bottom layer. The Gs were characterized as cells with a relatively large
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size and high granularity.
3.2. Phagocytic ability of the different subpopulations for HK yeast, zymosan and zymocel
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Next, we examined the phagocytic ability of C. gigas hemocytes against the three types of particles, namely HK yeast and its cell-walls derivatives, zymosan and zymocel using 5 × 105
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hemocytes (100 µl of 5 × 106 hemocytes/ml). Both the AGs and Gs showed phagocytic ability towards all the particles tested, but the Gs exhibited much higher phagocytic ability; this reflected the large number of actively phagocytic hemocytes in the Gs fraction (Fig. 1a). The phagocytic rate (PR) of AGs did not significantly differ among the three types of
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particles, and ranged from 10.1 ± 3.3% for zymosan (n = 26) to 11.7 ± 1.7% for HK yeast (n = 26). The Gs were the more active phagocytes for all the particles tested, and their PR
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ranged from 65.8 ± 4.1% (HK yeast) to 89.1 ± 6.9% (zymosan). The PR of Gs for zymosan and zymocel were significantly greater than that for HK yeast (P < 0.05). The estimated
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phagocytic index (PI) of both the AGs and Gs varied for the three particles (Fig. 1b). The PI of AGs for zymosan was significantly higher, at 7.9 ± 1.2, as compared to that for HK yeast and zymocel (5.0 ± 0.5 and 5.9 ± 0.9, respectively) (P < 0.05). Similarly, the PI of Gs significantly differed among the three foreign particles (P < 0.05): the PI of Gs for zymosan was highest, at 9.4 ± 1.7, and that for HK yeast and zymocel were 3.9 ± 1.1 and 5.6 ± 1.9, respectively. To analyze the characteristics of the PI for the three types of particles, especially to 14
ACCEPTED MANUSCRIPT understand the cause of the much higher value of the PI of Gs for zymosan, the PI was divided into increments of 5 (1–5, 6–10, 11–15, and ≥16) and the phagocytic frequencies of the increments were compared (Fig. 2). This experiment used 5 × 105 hemocytes. The results
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showed a markedly high PI of ≥16 exhibited by 18.1% of Gs for zymosan, significantly higher than the PI of 2.7% and 3.9% exhibited for HK yeast and zymocel, respectively (Fig. 2a). There was a greater ratio of AGs (6.6%) possessing a high PI (≥16) for zymosan as
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and Gs, the maximum PI value was 32 for zymosan.
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compared that for HK yeast and zymocel (0.8% and 2.2%, respectively, Fig. 2b). In both AGs
3.3. Effects of polysaccharides on phagocytosis by granulocytes and agranulocytes Laminarin (β-1,3-glucan) inhibited ingestion of zymosan and zymocel by Gs, although α-mannan did not (Fig. 3a). This experiment used 5 × 105 hemocytes. The PR for both
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zymosan and zymocel was respectively reduced by 47.6% (i.e., from 85.5 ± 4.1% to 44.8 ± 5.7%) and 45.4% (i.e., from 82.0 ± 6.4% to 44.8 ± 6.4%). In addition, the PR of
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laminarin-treated Gs was also affected in HK yeast. In contrast, the PI of Gs did not decrease in any of the particle types after treatment with both α-mannan and laminarin (Fig. 3b). In
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addition, the phagocytic ability (i.e., both the PR and PI) of AGs for these particles was almost unaffected by pretreatment with α-mannan and laminarin. Laminarin partly inhibited the phagocytosis of zymosan by Gs, in a dose-dependent
manner (Fig. 4): the PR of Gs significantly decreased at 100 µg/ml (44.8 ± 8.7%) and 200 µg/ml (43.6 ± 7.4%) compared to that of 0 µg/ml (84.3 ± 6.9%). The lowest PR of Gs was 43.6 ± 7.4% by treatment with 200 µg/ml of laminarin.
15
ACCEPTED MANUSCRIPT 4. Discussion The experiments in the present study separated two subpopulations of hemocytes from C. gigas: agranulocytes (AGs) and granulocytes (Gs). In our previous study, we separated the
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three subpopulations of C. gigas hemocytes using discontinuous Percoll gradient method [19]. We attempted to separate the subpopulations using the same Percoll gradient used previously, but we obtained fractions with low purity. Therefore, tried to separate the C. gigas hemocytes
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using different Percoll densities. For unknown reasons, we could not separate two subpopulations with high purity.
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In our previous study, we carried out flow cytometry by fluorescence-activated cell sorting to classify and separate subpopulations of C. gigas hemocytes [22]. According to their size, granularity, and complexity using flow cytometry and May-Grünwald/Giemsa staining, the C. gigas hemocytes were divided into three subpopulations: granulocytes, hyalinocytes
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and agranulocytes. The agranulocytes and hyalinocytes in the study of Terahara et al. [22] have been referred to other studies as small hyalinocytes and large hyalinocytes, respectively
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[23], [24], and agranulocytes and semi-granulocytes, respectively [25]. Similar subtypes of hemocytes have been referred to and reported in other species of Crassostrea, for example:
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agranulocytes, semi-granulocytes, and granulocytes in C. virginica [26]; blast-like cells, hyalinocytes, and granulocytes in C. ariakensis [27]; and blast-like cells, agranulocytes, and granulocytes in C. rhizophorae [28]. In the present study, we were not able to separate three subpopulations by Percoll density-gradient centrifugation. We concluded that hyalinocytes were not able to be separated from the other subpopulations and were contained within the agranulocytes (AGs) fraction based on microscopic observations in this study. Some previous studies have also reported differences in phagocytic ability between 16
ACCEPTED MANUSCRIPT agranulocytes and granulocytes in oyster species. For example, agranulocytes might had a lower phagocytic ability than granulocytes in C. gigas [25] and C. virginica
[29], [30].
Similarly, agranulocytes and semi-granulocytes showed either no or only weak phagocytosis,
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while granulocytes showed strong phagocytic ability in C. gigas hemocytes [7]. In a previous study, we found that both agranulocytes and granulocytes showed phagocytic ability against all the particles tested, but granulocytes especially were more active than agranulocytes [20].
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Hine [31] summarized the characteristics of phagocytosis by both agranulocytes and granulocytes in other bivalve species as follows: granulocytes exhibit a high phagocytic
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ability against various foreign particles [4]; agranulocytes might have a non-phagocytic ability in Mediterranean mussel Mytilus galloprovincialis [32], boring clam Tridacna crocea [33], and three species of deep-sea symbiotic Bathymodiolus mussels [34]; and agranulocytes
mercenaria [35].
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might have a lower phagocytic ability than granulocytes in the hard clam Mercenaria
In this study, we obtained comparable results about the phagocytic ability of both
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AGs and Gs for HK yeast, zymosan and zymocel. The phagocytic rate (PR) and phagocytic index (PI) of Gs were much greater than those of AGs. Furthermore, a new finding was that,
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in both the AGs and Gs, the PI for zymosan was much higher than the PI for other particles. We gauged the values of PI in increments of 5 (i.e., 1–5, 6–10, 11–15, and ≥16), and then compared the phagocytic frequencies of these PI increments to better understand the outcome of a much higher mean PI value for zymosan. A markedly high PI of ≥16 was shown by 18.1% of Gs for zymosan, which was significantly higher than the 2.7% and 3.9% for HK yeast and zymocel, respectively. AGs also revealed greater cell ratios possessing higher PI (≥16) for zymosan than those for HK yeast and zymocel. These results indicate that the high 17
ACCEPTED MANUSCRIPT PI for zymosan was not attributable to all phagocytic hemocytes having a high mean PI, but to some portion of the AGs and Gs being able to phagocytize many zymosan particles using different phagocytic mechanisms.
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In our previous study (described above) we examined the effects of various treatments for the same foreign particles on the respective phagocytic ability of Gs [20]. Escherichia coli cells treated with formaldehyde and then autoclaved were strongly resistant
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to phagocytosis by Gs, although living cells or heat-killed cells were avidly phagocytized. Those results suggested that a chemical or physical alteration of the cell surfaces of E. coli
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affects their ability to be engulfed by Gs, but not by the AGs. Tripp [35] reported similar results in relation to phagocytosis by M. mercenaria hemocytes. His results also suggest that granulocytes possess a specialized system that is mediated by membrane receptors in a
mammals.
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non-self-recognition and binding toward foreign particles, similar to that of macrophages in
Recognition and phagocytosis of foreign particles in mollusks might involve lectins
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or lectin-like receptors with specific carbohydrate-binding properties [5], [13], [36]. The involvement of humoral lectins that function as opsonins has been demonstrated for the
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phagocytosis of yeast by hemocytes of common mussel Mytilus edulis [37] and freshwater snail Biomphalaria glabrata [13]. A galectin (CvGal) located on the surface of C. virginica hemocytes was found to recognize and phagocytize a variety of microbe pathogens [6]. Recently, a new type of phagocytic receptor, CgNimC, was reported as strongly binding to LPS and able to promote phagocytosis of Vibrio species by C. gigas hemocytes [7]. In addition, peptidoglycan recognition protein (PGRP) and β-1,3-glucan-binding protein (LGBP) gene was found in the scallop Chlamys farreri, and these proteins were inferred to be 18
ACCEPTED MANUSCRIPT a humoral recognition molecule or a phagocytic receptor [8], [9]. S. cerevisiae (HK yeast), zymosan and zymocel have similar sizes and shapes but differ in the composition of polysaccharides on their surface: S. cerevisiae has a large amount
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of mannan and only a trace amount of chitin; zymosan contains a large amount of β-glucan and low amounts of mannan and chitin; and zymocel is almost entirely composed of β-glucan. Therefore, we proposed that if differences occurred among the three particles in terms of their
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respective phagocytosis by agranulocytes and granulocytes in C. gigas hemocytes, this would be a result of differential recognition of the polysaccharides on the surface.
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Laminarin, a soluble algal product, is structurally and chemically similar to yeast glucan [38] and it is well used as an inhibitor of β-1,3-glucan receptors [39]. In the present study, laminarin partly inhibited ingestion of zymosan and zymocel by Gs, but did not inhibit that of HK yeast. In addition, soluble mannan did not block ingestion of any of the tested
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particle types. These results indicate that the Gs of C. gigas hemocytes might possess β-glucan receptors for phagocytosis on their cell surface. We concluded that β-glucan
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receptors are important for phagocytosis of foreign particles by oyster hemocytes. In fact, there are microorganisms, fungi [40,41] and oomycetes [41], that possess β-glucan on their surface.
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However, the residual phagocytosis, of about 50%, that was not blocked by laminarin suggests that β-1,3-glucan-independent modes of phagocytosis by Gs exist. Laminarin strongly inhibited phagocytosis of unopsonized, autoclave-killed yeast by B. glabrata hemocytes [13]. In this study, however, phagocytosis of HK yeast by both AGs and Gs was unaffected by pretreatment with either α-mannan or laminarin, which contrasts to the situation in mammals and in B. glabrata. The findings of the present study suggest that C. gigas has multiple types of hemocytes with phagocytic ability, some of which depend on 19
ACCEPTED MANUSCRIPT β-glucan receptors for phagocytosis. In conclusion, the results show that both AGs and Gs, as the two subpopulations of C. gigas hemocytes, exerted strong phagocytic ability for baker’s yeast and its cell-walls
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derivatives, zymosan and zymocel. Especially, zymosan was greatly phagocytized by a portion of the hemocytes. Furthermore, the present findings suggest that C. gigas has multiple types of hemocytes with phagocytic ability, some of which depend on β-glucan
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receptors for phagocytosis. Although we did not obtain evidence for this, we speculate that Gs and AGs contain subpopulations that possess different receptors for phagocytosis. The
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β-glucan receptors might mediate the phagocytosis of these particles by Gs, if not all the particles. Apart from these exceptional Gs, however, other types of hemocytes with similarly
Acknowledgements
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high phagocytic ability might phagocytize foreign particles through different mechanisms.
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JP16K07866.
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This work was supported in part by JSPS KAKENHI Grant Numbers 17380112 and
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zymocel
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ACCEPTED MANUSCRIPT Table 1 Purity of hemocyte sub-populations in layers separated by Percoll density
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Fig. 1 Phagocytosis of HK yeast, zymosan, and zymocel by agranulocytes and granulocytes
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of C. gigas: (a) phagocytic rate (i.e., percentage of phagocytosis-positive cells); (b) phagocytic index (i.e., mean number of foreign particles ingested by one phagocytic hemocyte). The data are expressed as mean ± SD in columns having different alphabetical
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designations to show significant differences using Tukey’s multiple comparison test (P <
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0.05; n = 26).
Fig. 2 Frequency of the phagocytic index values (in increments of 5: 1–5, 6–10, 11–15, and ≥16) for the granulocytes (a) and agranulocytes (b) in response to HK yeast, zymosan and zymocel. The data are expressed as mean ± SD in columns having different alphabetical
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0.05; n = 26).
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designations to show significant differences using Tukey’s multiple comparison test (P <
Fig. 3 Effects of soluble α-mannan or laminarin on the phagocytosis of HK yeast, zymosan
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and zymocel by granulocytes: (a) phagocytic rate; (b) phagocytic index. *Significantly lower than values for treatment with oyster balanced salt solution (BSS) as a control, as determined by Dunnett’s multiple comparison test (P < 0.05; n = 15).
Fig. 4 Dose dependency of the inhibitory effects of laminarin on the phagocytosis of zymosan by granulocytes. The data are expressed as mean ± SD. *Significantly lower than the values for treatment with oyster balanced salt solution (0 µg/ml of laminarin) as a control, as 32
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determined by Williams’ multiple comparison test (P < 0.05; n = 6).
33
ACCEPTED MANUSCRIPT Highlights
1. Two sub-populations, AGs and Gs, could be separated from the hemocytes of C. gigas.
phagocytes. 3. A part of Gs showed markedly high PI for zymosan.
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2. Both AGs and Gs exhibited phagocytic ability, especially Gs functioned much active
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4. Soluble laminarin partly inhibited ingestion of zymosan and zymocel by Gs.
5. C. gigas has multiple types of hemocytes with phagocytic ability, some of which depend
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on β-glucan receptors for phagocytosis.