ORIGINAL RESEARCH published: 06 July 2021 doi: 10.3389/fimmu.2021.693613 GAS1: A New b-Glucan Immunostimulant Candidate to Increase Rainbow Trout Edited by: (Oncorhynchus mykiss) Resistance to Brian Dixon, University of Waterloo, Canada Bacterial Infections With Aeromonas Reviewed by: Seyed Hossein Hoseinifar, salmonicida achromogenes Gorgan University of Agricultural Sciences and Natural Resources, Iran Valérie Cornet 1‡, Trinh Dinh Khuyen 1†‡, Syaghalirwa. N. M. Mandiki 1, Stéphane Betoulle 2, David Huyben, Peter Bossier 3, Felipe E. Reyes-López 4,5,6, Lluis Tort 4 and Patrick Kestemont 1* University of Guelph, Canada 1Research Unit in Environmental and Evolutionary Biology (URBE), Research Institute of Life, Earth & Environment, University *Correspondence: of Namur (UNamur), Namur, Belgium, 2 UMR-INERIS 02 SEBIO StressEnvironnementaux et Biosurveillance des milieux Patrick Kestemont aquatiques, Plateau technique mobile en cytométrie environnementale MOBICYTE, UFR Sciences Exactes et Naturelles, patrick.kestemont@unamur.be Université de Reims Champagne-Ardenne, Reims, France, 3 Laboratory of Aquaculture & Artemia Reference Center, Faculty † Present address: of Bioscience Engineering, Ghent University, Ghent, Belgium, 4 Department of Cell Biology, Physiology and Immunology, Trinh Dinh Khuyen, Universitat Autònoma de Barcelona, Bellaterra, Spain, 5 Consorcio Tecnológico de Sanidad Acuı´cola, Ictio Biotechnologies Aquatic Genetic and Selective S.A., Santiago, Chile, 6 Facultad de Medicina Veterinaria y Agronomı´a, Universidad de Las Américas, Providencia, Chile Breeding Unit, Department of Aquaculture, Faculty of Fisheries, Vietnam National University of b-glucans are prebiotic and/or food additives used by the aquaculture industry to enhance Agriculture, Hanoi, Vietnam the immune response of fish. Their efficiency may vary according to their origin and ‡ These authors have contributed structure. In this study, the immunostimulant effects of two b-glucan types extracted from equally to this work and share first authorship wild-type baker’s yeast (Saccharomyces cerevisiae) and its null-mutant Gas1 were investigated. Gas1 has a beta-1,3-glucanosyltransferase activity necessary for cell wall Specialty section: assembly. Using a positive (commercial product MacroGard®) and a negative control This article was submitted to Comparative Immunology, (a diet without glucans), we evaluated the immune responses and disease resistance of a section of the journal rainbow trout juveniles (mean weight, ~44 g) fed control, low (0.2%) and high (0.5%) doses Frontiers in Immunology of Macrogard®, Gas1, and Wild type-b-glucan after a short-term (15 days, D15) or mid- Received: 11 April 2021 term (36 days, D36) feeding periods. We found that b-glucan supplemented diets did not Accepted: 18 June 2021 Published: 06 July 2021 affect growth performance, mortality, splenic index, or leukocyte respiratory burst activity Citation: on D15 nor D36. However, each b-glucan triggered different immune effectors, depending Cornet V, Khuyen TD, of the doses or length of exposure compared to others and/or the negative control. Mandiki SNM, Betoulle S, Bossier P, Reyes-López FE, Tort L Indeed, high dose of MacroGard® significantly increased lysozyme activities at D15 and Kestemont P (2021) GAS1: compared with the control and other diets (p<0.05). At D36, MacroGard b-glucan A New b-Glucan Immunostimulant enhanced the production of lymphocytes in comparison with the control diet (p<0.05). Candidate to Increase Rainbow Trout (Oncorhynchus mykiss) Resistance to Regarding WT b-glucan, at D36, WT-b-glucan, especially the high dose, provided the Bacterial Infections With Aeromonas highest enzymatic activities (lysozyme and ACH50) and Ig level (p<0.01). Furthermore, on salmonicida achromogenes. Front. Immunol. 12:693613. D36, Gas1 also increased lysozyme activity, Ig proportion, and some immune genes doi: 10.3389/fimmu.2021.693613 (mcsfra, hepcidin) compared with MacroGard® (p<0.05). Besides, both doses of Gas1-b- Frontiers in Immunology | www.frontiersin.org 1 July 2021 | Volume 12 | Article 693613 Cornet et al. GAS1: A New b-Glucan Immunostimulant Candidate glucans increased the resistance of juveniles to bacterial infection highlighted by a higher survival rate at 14 days post-challenge compared with the control and other types and doses of b-glucans (p<0.05). In conclusion, our results suggest that Gas1-b-glucan could represent a promising immunostimulant that would help to prevent diseases in aquaculture even more efficiently than other b-glucans already in use. Mode of action and particular efficiency of this new Gas1 mutant are debated. Keywords: immune gene expression, immunostimulant, rainbow trout, bacterial challenge, beta-glucan INTRODUCTION the development of adaptive immunity and elimination and killing of microorganisms (11, 17–19). A wide range of studies described Diseases are major constraints to sustainable aquaculture numerous immunostimulant effects of b-glucans in several fish production, especially for intensive aquaculture systems (1–3). species with different levels of immune stimulation, depending on Indeed, fish might often be exposed to stressful conditions, fish species, b-glucans structure, dose, and duration of eventually becoming more susceptible to microbial infections administration (5, 20–23). (4). For the past few decades, the traditional strategy for fish In the rainbow trout, the potential immunomodulatory of b- disease control in aquaculture relied on the use of antibiotics and glucans has been reported in several publications. Djordjevic et chemical disinfectants. However, application of antibiotics can al. (24) showed that dietary b-1,3/1,6-glucans (level 0.2% and lead to the development of antibiotic-resistant bacterial strains 0.4% diets for 37 days) decreased the expression of genes and cause many other problems, such as environmental hazards, involved in acute inflammatory reactions to the inflammatory food safety problems, and increasing resistance of pathogens (5). agent, whereas major parts of the immune response remained The use of antibiotics can also adversely affect the health status of un-changed. Also, Lauridsen & Buchmann (25) reported that the fish (6, 7). Further, sub-therapeutic doses of antibiotic have dietary b-1,3/1,6-glucans (0.2% diets, for 46 days) fed rainbow been often added to aquatic feeds to promote growth, and this trout increased resistance against challenge with Ichthyophthirius has further contributed to drug resistance (8). Therefore, the multifiliis (white spot disease). Moreover, Skov et al. (26) prophylactic use of antibiotic treatments in aquaculture has reported that dietary b-1,3-glucans from Euglena gracilis at 1% resulted in a very limited use or a ban of most compounds in diets, 1% biomass day−1 for 84 consecutive days down-regulated top aquaculture production countries and stringent regulations the expression of pro-inflammatory genes, whereas no effect of on the application of antibiotics worldwide (9). This situation b-1,3-glucans diets on survival after Yersinia ruckeri challenge. and recent restrictions on the use of antibiotics have promoted However, the effects of b-glucans on fish immune system the use of prebiotics, probiotics (or a combination of both named functions can be variable, depending on the frequency, location, synbiotics), and immunostimulant compounds (such as glucans) and length of the side-chains, which may play a role in as preventive strategies to limit and control fish diseases (10–13). immunomodulation. Differences in molecular weight, shape, b-glucan is a heterogeneous group of glucose polysaccharides and structure of b-glucans explain not only the differences in consisting of a backbone of b(1,3)-link b-D-glucopyranosyl units biological activity (15) but also fish species and administration with b-(1,6)-link side chains of varying length and distribution route. Indeed, diets supplemented by a commercial b-glucans (14) and are a major structural component of fungi, bacteria, (MacroGard®) administrated to channel catfish (Ictalurus plants, algae, yeast, and mushroom cell walls. Although the cell punctatus) for 4 weeks did not significantly affect several wall b-glucans of yeast and fungi is mostly composed of 1,3-b immune parameters, such as plasma lysozyme, bactericidal and linked glycopyranosyl residues with small numbers of 1,6 b- hemolytic complement activities, respiratory burst of phagocytes, linked branches, oat and barley cell walls are formed of and the number of lymphocytes found, survival rate in fish unbranched b-glucans with 1,3 and 1,4 b-linked glycopyranosyl infected with Edwardsiella ictaluri (27). Positive dose-related residues, bacteria have unbranched 1,3 b-linked glycopyranosyl effects of b-1,3-glucans extracted from by Saccharomyces residues (15). Because of some similar structure with fungal or cerevisiae have been reported on some immune functions in bacterial gram-negative polysaccharides, the recognition of b- rainbow trout (28). Besides, Douxfils et al. (29) found that glucan by host’s cell pattern recognition receptors can trigger overdoses of b-glucans (MacroGard ® ) and/or prolonged inflammatory cascade, leading to the enhancement of the medication can lead to a non-reactive physiological status and, immune system (16). consequently, to a poor immune response in rainbow trout. In a Particularly, it has been reported that b-glucans bind to specific previous study, Han et al. (30) demonstrated that Gas1, a b-1,3- cell surface receptors of macrophages and neutrophilic granulocytes glucans produced by the null-mutant yeasts Gas1 of that promote the enhancement of an organism’s protective activity Saccharomyces cerevisiae provided the best protection to A. against infection through the activation of leukocytes, phagocytic franciscana against Vibrio harveyi in comparison with wild- activity, inflammatory cytokines, chemokines, reactive oxygen free type (WT) Saccharomyces cerevisiae and commercial b-1,3- radicals, increase the activity of antioxidant enzymes, and initiate glucans. The authors partly explained this observation by a Frontiers in Immunology | www.frontiersin.org 2 July 2021 | Volume 12 | Article 693613 Cornet et al. GAS1: A New b-Glucan Immunostimulant Candidate different structure of the Gas1 b-1,3-glucans that has a lower During this period, water temperature was maintained at 13.9 ± degree of branching and a shorter side chain length in 1.2°C by a cooling system, oxygen level averaged 11.6 ± 0.7 mg/L comparison with the two others. (aeration applied), constant photoperiod (light/dark ratio = 12:12), In our study, we aimed to evaluate if Gas1 b-1,3-glucans could and fish were fed 1.5% of fish biomass, twice daily (at 9:00 AM and also improve fish resistance in comparison with the WT b-1,3- 5:00 PM) with a specific trout diet (Coppens TROCO SUPREME-16, glucans of Saccharomyces cerevisiae and to a commercial one, The Netherlands, crude protein = 48%, crude fat = 15%). MacroGard® that effects are already well described in literature. In that purpose, we conducted a comprehensive evaluation of short- Fish Diet and Experimental Design and mid-term (D15 and D36) and dose effects (low dose at 0.2% After acclimation, fish (mean body weight, 44.5 ± 3.0 g) were fed diet vs high dose at 0.5% diet) of dietary administration of different by hand either a control diet (no b-glucans) or diets containing types b-glucans extracted from yeast wild or cell-wall mutants three types of b-glucans: a commercial mixture of b-glucans, MO Gas1 with different particle size and chemical structure (WT- and (MacroGard ® , Biorigin, Brazil), and two yeast b-glucan Gas1-b-glucan) extracted by the laboratory of Aquaculture and products, namely GAS1-b-glucans (GAS1) and wild type b- Artemia Reference Centre (ARC, UGent) compared to a glucans (WT) extracted by the Laboratory of Aquaculture & commercial b-glucan (MacroGard®) on the immune functions Artemia Reference Center (ARC) of Ghent University, Belgium and resistance to pathogens of rainbow trout juveniles. In that as described in Han et al. (30). For each type of b-glucans, two purpose, blood leukocyte cells, humoral immune parameters, and doses were tested (0.2% or 0.5% of the diet) for 36 days at 1% of immune-related gene expression in relevant immune organs, such fish biomass/day that are called further in the text: G0.2%, G0.5% as spleen and kidneys, will be evaluated. Moreover, the disease for Gas1 glucan, M0.2%, M0.5% for MacroGard® glucan, resistance of trout juveniles was tested by applying a bacterial W0.2%, W0.5% for wild-type glucan. All diets were formulated challenge test based on the intraperitoneal injection of Aeromonas and pelleted in the laboratory of the University of Namur salmonicida achromogenes. (Table 1). Three replicate tanks were used for each experimental diet. After 15 days (short-term nutrient test, D15) and 36 days (mid-term nutrient test, D36) of feeding, six MATERIALS AND METHODS fish per tank (18 fish per experimental diet) were anesthetized in buffered (pH 7) ethyl 3-aminobenzoate methane sulfonic acid Experimental Fish salt (98% purity, MS-222, Sigma) solution (120 mg/L). Blood was Feeding trial and bacterial challenge were carried out in obtained by caudal vein puncture using a heparinized syringe agreement with the European and Belgian national legislation and stored on ice in heparinized tubes. Fish were then euthanized on animal welfare (Protocol number: 13197 KE). Rainbow trout by overdose of buffered MS-222 (240 mg/L) before decapitation. juveniles (n = 315) were transported from a local fish farm Spleen and head kidney were dissected; a part of spleen was (Hatrival, Belgium) to the University of Namur (in facilities of immediately homogenized after dissection to prepare for spleen the research unit of environmental and evolutive biology) and respiratory burst activity analysis. The remaining spleen and distributed into nine fiberglass tanks of 100 L (35 fish/100 L/ kidney samples were immediately snap-frozen in liquid nitrogen tank) in a recirculation system. Fish were allowed to acclimate to and finally stored at −80°C until analysis (RT-qPCR immune- the new environment for 21 days. related genes). Heparinized blood was immediately analyzed for TABLE 1 | Ingredients and proximate composition of the experimental pelleted diet. Ingredients Diet* (g/kg) Cod fish meal a 350.0 Blood mealb 70.0 Wheat glutenc 134.0 Cod fish oild 128.0 Starche 223.6 Carboxylmethylcellulosee 20.0 a- cellulosee 42.4 Mineral mixf 10.0 Vitamin mixg 10.0 Betainee 10.0 BHA 1.0 BHT 1.0 Analyzed diet composition Crude protein (%) Crude fat (%) Dry matter (%) Ash (%) Energy (MJ/kg) 48.00 14.75 92.16 14.30 19.8 *b-glucan doses were added in respective % of the diet reducing the levels of a-cellulose. aCod fish meal (CP-crude protein = 89%, CF-crude fat = 4.0%) provided by SNICK euroingredient NV, Ruddervoorde (Belgium). bBlood meal (CP = 87.6%, CF = 0.0) as Actipro Hemoglobin, Zwevezele (Belgium). cWheat Gluten (CP = 80.0, CF = 6.0%), Roquette Freres, Lestrem (France). dSigma-Aldrich, Saint-Louis, MO, (USA). eMosselman SA, Chlin (Belgium). fMineral mix (g kg–1 of mix) was prepared in the lab, from (CaHPO4)2H2O, 727.77; (MgSO4)7H2O, 127.50; NaCl, 60.00; KCl, 50.00; (FeSO4)7H2O, 25.00; (ZnSO4)7H2O, 5.50; (MnSO4)4H2O, 2.54; (CuSO4)5H2O, 0.78; (CoSO4)7H2O, 0.48; (CaIO3)6H2O, 0.29; (CrCl3)6H2O, 0.13 g. g Vitamin mix was provided by INVE Aquaculture Company. BHA (butylated hydroxyanisole) and BHT (butylated hydroxyl toluene) were provided by Sigma-Aldrich-Belgium. Frontiers in Immunology | www.frontiersin.org 3 July 2021 | Volume 12 | Article 693613 Cornet et al. GAS1: A New b-Glucan Immunostimulant Candidate leukocyte populations by flow cytometry, and the remaining of freshly prepared Micrococcus luteus (Sigma-Aldrich, Saint- volume of blood was then centrifuged at 7,500g for 10 min to Louis, USA) solution (0.6 mg/ml of Na2HPO4 0.05 M, pH 6.2) collect plasma stored at −80°C until subsequent analyses in triplicate. Absorbance corresponding to Micrococcus luteus lysis (lysozyme activity, alternative complement activity, total was measured at 450 nm for 60 min at regular intervals (5 min). immunoglobulin content). One unit (U) of lysozyme was determined as an absorbance decrease of 0.001 per min. Bacterial Challenge To evaluate whether b-glucan has a beneficial effect on disease Plasma Hemolytic Activity of Alternative resistance, rainbow trout juveniles were experimentally infected Complement Activity with a virulent strain of Aeromonas salmonicida achromogenes Plasma hemolytic activity of alternative complement activity provided by the CER group (Centre d’Economie Rurale, (ACH50) was assayed following Oriol Sunyer and Tort (36), Laboratoire de Pathologie des Poissons, Belgium). Bacteria were later modified by Milla et al. (37). Briefly, 10 µl of rabbit red cultured in sterile Brain Heart Infusion (BHI; Sigma Aldrich, Saint- blood cell suspension (RRBC, Biomerieux, Mary-l’Etoile, France) Louis, MO, USA) and incubated at 28°C for 24 h. A preliminary test suspended at 3% in veronal buffer was mixed with serial dilutions infection, including various bacterial doses was performed to of plasma (70 µl of total volume). Hemolysis (100%) was determine the LD50 CFU of the targeted rainbow trout obtained by adding 60 µl of distillate water to 10 µl of RRBC. population (LD50 = 3.1 × 107 CFU/100 g fish body weight). Negative control (fresh water) was obtained by adding 60 µl of On D37, a total of 30 fish from each dietary condition (10 fish × 3 veronal buffer to 10 µl of RRBC. Samples were incubated 100 min replicate tanks) were anesthetized. Then, the fish were at 27°C and centrifuged (3,000g, 5 min, 4°C). Then, 35 µl of intraperitoneally injected with a weight-adjusted dose (3.1 × 107 supernatant was transferred to a new microplate to measure the CFU/100 g of fish body weight) of the freshly prepared A. absorbance at 405 nm. The ACH50 value was defined as the salmonicida achromogenes culture and equally distributed into reciprocal of the plasma dilution that induced the hemolysis of three 50-L tanks. The fish were confined at the animal facility 50% RRBC. (Biosafety level 2) along the infection assay. They were starved 1 day ahead of infection as well as the day of bacterial injection, and then Plasma Total Immunoglobulin Assay fed the respective experimental diets until the end of the challenge Analysis of total plasma immunoglobulin content (Ig) was based test. At 35 h post-injection (D39), a total of nine fish from each on a spectrophotometric technique described by Siwicki and dietary condition (3 fish × 3 replicate tanks) were anesthetized, and Anderson and later adapted by Milla et al. (37) with some blood was sampled for subsequent immunological assays (lysozyme modifications. Immunoglobulin was precipitated using 10,000 and alternative complement pathway activity). Levels of plasma kDa polyethylene glycol (PEG, Sigma). Plasma was mixed with total Ig content and spleen RBA were not determined because of an equal volume of 12% PEG solution and shake 150 rpm for 2 h limitations in this experiment. Fish were then euthanized, and at room temperature. After centrifugation at 1,000g for 10 min, spleen and kidney were sampled and immediately snap-frozen until the supernatant was collected and assayed for its protein immune gene expression analysis (RT-qPCR). concentration by the method of Bradford (38). Plasma total immunoglobulin content was calculated by subtracting this value Blood Leukocyte Populations from the total protein concentration in the plasma before Blood cell populations were analyzed at D15 and D36 of dietary precipitation with PEG. treatment by flow cytometry (Flow Activated Cell Sorter Calibur; Flow Cytometry System) according to Inoue et al. (31), later adapted Spleen Leukocyte Respiratory by Mathieu et al. (32). Briefly, 10 µl of fresh heparinized blood were Burst Activity mixed with 1950 µl of Hanks Balanced Salt Solution (HBSS, Sigma) Just after collection, spleen samples were conditioned in L15 and 40 µl of fluorochrome DiOC6 (3,3-dihexyloxacarbocyanine, medium (Sigma-Aldrich) at 4°C, and gently pressed through Molecular Probes, Eugene) diluted 1:10 in ethanol. The tube was sterilized nylon mesh (40 µm, Dutscher) to obtain leukocyte mixed gently and incubated at room temperature (RT) for 10 min. suspensions. Then, the L15 medium-diluted samples were loaded The FACS was calibrated with True Count Beads diluted in HBSS, onto Ficoll gradient (Healthcare, GE). After centrifugation (2500 Sigma-Aldrich, Steinheim, Germany). Each blood cell population rpm, 20 min, at 4°C), leukocyte suspensions were collected and was identified by its typical location in an FL-1 v. SSC (FITC filter washed twice in L15 medium and again centrifuged (1200 rpm, versus Side-scattered light) and FSC vs. SSC (Forward Scatter versus 5 min, 4°C). They were re-suspended in L15 medium and viable Side-scattered light) according to Inoue et al. (31) and Pierrard et al. leukocytes were adjusted at 106 cells/ml before classification of (33). Four clusters were identified, thrombocyte and lymphocyte leukocyte populations (lymphocyte, macrophage, and cells were gathered in the same cluster according to Pierrard granulocyte) by flow cytometry. RBA analysis was performed et al. (33). using the flow cytometry method as previously described by Chilmonczyk and Monge (39) with some modifications of Jolly Plasma Lysozyme Activity et al. (40). The RBA test corresponded to an evaluation of Lysozyme activity assay was performed by the turbidimetric intracellular hydrogen peroxide production following cell method of Siwicki and Studnicka (34), later adapted by activation or not with phorbol 12-myristate 13-acetate (PMA). Mathieu et al. (35). Briefly, 7 ml of plasma were added to 130 ml The fluorescence levels of unstimulated and PMA-stimulated Frontiers in Immunology | www.frontiersin.org 4 July 2021 | Volume 12 | Article 693613 Cornet et al. GAS1: A New b-Glucan Immunostimulant Candidate cells were determined after 30 min of cell incubation (18°C in the treatment (n = 3 fish per each pool). On day 37 of feeding, fish were dark) with 2′-7′dichlorofluorescin diacetate (DCFH-DA (5 µM) challenged against A. salmonicida achromogenes and 35 h post- and DCFH-DA plus PMA (2 mg ml−1), respectively. The spleen challenge (D39 of treatment) three fish of three different tanks for respiratory burst activities were expressed in a stimulation index each treatment (n = 9 fish) were also collected. Samples were also as the ratio between the mean fluorescence measured in taken using the same strategy from fish fed with basal diet and from stimulated cells (DCFH-DA + PMA) and the basal mean non-infected fish (n = 3 fish per each pool). As a housekeeping gene, fluorescence of control (DCFH-DA only). elongation factor 1 alpha (ef-1a) that exhibited the most stable expression (compared to b-actin and 18S genes) between samples RNA Precipitation and Complementary was amplified from all the evaluated samples. The gene expression DNA Synthesis of pro-inflammatory (il-1), macrophages (mcsfra), antibacterial Three pools of each experimental condition and time-point were (Lysozyme, Hepcidin), humoral (mIgM), membrane protein collected to compare their gene expression profiles. Total RNA (TLR2), neutrophils (mpo), Cathelicidin (CATH1), T-helper (cd4- was extracted individually from the spleen and head kidney) by 2b), and anti-inflammatory responses (il-10, tgf-b1) were evaluated. grounding 100 mg of the organ with a pestle until full The list of specific primers used for gene expression analysis is homogenization in TriReagent (Sigma-Aldrich, St. Louis, MO). given in Table 2, and those were designed according to Cornet et al. The next steps were performed according to the manufacturer’s (41) and Khuyen et al. (42). Real-time PCR reactions were carried instructions. The pellet was dried and resuspended in 200 µl of out with iTaq Universal SYBR Green Supermix (Bio-Rad RNase-free water. Total RNA concentration was determined by Laboratories) using a 1:40 dilution of the cDNA for target genes Nano Drop-2000 spectrophotometer (Thermo Scientific), and or 1:1000 dilutions for ef-1a. the integrity was checked by Experion RNA Std Sens analysis Primers for target genes were used at 500 nM. The thermal (Bio-Rad Laboratories, Hercules, CA). Total RNA (1 µg) was conditions used were 3 min at 95°C of pre-incubation, followed by used to synthesize cDNA with iScript cDNA synthesis kit (Bio- 40 cycles at 95°C for 30 s, and 60°C for 30 s. All reactions were Rad Laboratories) following this protocol: priming for 5 min at performed using ABIprism 7300 (Applied Biosystem), and 25°C, reverse transcription for 20 min at 46°C, RT inactivation quantification was done according to the Pfaffl method (43) for 1 min at 95°C as recommended by the manufacturer. corrected for efficiency of each primer set. Values for each sample were expressed as normalized relative expression (NRE), calculated Gene Expression Analysis in relation to values of the control group, and normalized against Real-time PCR assay was carried out to analyze the expression those of the housekeeping gene ef-1a. The results are expressed as pattern of different genes of immunological relevance in spleen an average of values obtained in all pools from D15 and D36 of and kidney from rainbow trout stimulated with b-glucan. feeding, and D39 (after bacterial challenge test). Samples were taken randomly from three fish of three different tanks for each treatment (n = 9) at D15 and D36 days of feeding Statistical Analysis with different doses and types of b-glucan. Samples of three Results are presented as box plot representing the quartile randomly selected fish from each tank were pooled for each distribution of the data. Statistical analyses were performed using TABLE 2 | Primers used for each gene expression analysis by real-time RT. Genes Primer sequence (5’-3’) Accession number Amplicon size Efficiency ef-1a Fw: 5’-ATGCCCCCAAGTTCCTGAAG-3’ NM_001124339.1 140 1.95 Rv: 5’-AACAGCAACAGTCTGCCTCA-3’ il-1 Fw: 5’-TGAGAACAAGTGCTGGGTCC-3’ NM_001124347.2 148 2.02 Rv: 5’-GGCTACAGGTCTGGCTTCAG-3’ lysozyme Fw: 5’-TGCCTGTCAAAATGGGAGTC-3’ NM_001124716.1 152 1.89 Rv: 5’-CAGCGGATACCACAGACGTT-3’ mIgM Fw: 5’-AAAGCCTACAAGAGGGAGACCGAT-3’ X65263.1 128 2 Rv: 5’-AGAGTTATGAGGAAGAGTATGATGAAGGTG-3’ mcsf-ra Fw: 5’-ATCTCCACTCATGGCGACACA-3’ AB091826 177 2 Rv: 5’-CATCGCACTGGGTTTCTGGTA-3’ mpo Fw: 5’-GCAGAGTCACCAATGACACCA-3’ GBTD01119227 68 2 Rv: 5’-ATCCACACGGGCATCACCTG-3’ il-10 Fw: 5’-CCGCCATGAACAACAGAACA-3’ NM_001245099.1 105 1.98 Rv: 5’-TCCTGCATTGGACGATCTCT-3’ tgf-b1 Fw: 5’-GCCAAGGAGGTCCACAAGTT-3’ NM_001281366.1 146 2.06 Rv: 5’-GTGGTTTTGATGAGCAGGCG-3’ cd4-2b Fw: 5’-AAGCCCCTCTTGCCGAGGAA-3’ AY899932 108 2 Rv: 5’-CTCAACGCCTTTGGTACAGTGA-3’ vapA Fw: 5’-ATTAGCCCGAACGACAACAC-3’ KP184543.1 148 2.02 Rv: 5’-CCAACACAATGAAACCGTTG-3’ Primers were designed according to Cornet et al. (41) and Khuyen et al. (42). Frontiers in Immunology | www.frontiersin.org 5 July 2021 | Volume 12 | Article 693613 Cornet et al. GAS1: A New b-Glucan Immunostimulant Candidate Rstudio software (R version 4.0.3). For each timepoint, which was Concerning lysozyme parameters, on D15, M0.5% treated independently, the effects of the concentration, type of significantly improved its activity compared to the control diet glucan, and their interaction were analyzed using a generalized and M0.2%. On the contrary, for GAS1 and wild type b-glucan, linear model with the following code: glm (variable ~ type of the lowest doses G0.2% and W0.2% were better to improve glucan × concentration, family = Gaussian, data = timepoint). Post lysozyme activity in comparison with the control (p<0.0001) and hoc comparisons (Tukey’s test) at a 5% significant level were their respective high dose (Figure 1C). At D36, we only observed performed on the factor (type of glucan or concentration) or a type of glucan effect, where WT exhibited a significant highest their interaction (type of glucan × concentration) with general lysozyme activity, followed by Gas1 and then MacroGard linear hypotheses and multiple comparisons for parametric models (p<0.0001). After the bacterial infection, an effect of the (multcomp R package) and Benjamin-Hochberg (BH) correction concentration of b-glucan was highlighted, with a higher was applied. When two groups (from either the concentration, type activity with 0.2% in comparison with 0.5% of b-glucan (p<0.05). of glucans or their interaction depending on the results of the glm) Eventually, levels of respiratory burst activity (RBA) of spleen do not differ from each other, they will share the same letter lymphocytes (as for macrophage cells) did not significantly differ annotation (for example “a”), and when two groups are whatever the b-glucan administration doses and types on D15, and significantly different, they will be identified by different letters an effect of the concentration of b-glucan was highlighted, with a (“a” and “b”). As in this study, the interaction of two factors is higher RBA in 0.2% compared to 0.5% (p<0.05) (Figure 1D). considered and that seven groups (control, 3 types b-glucans × 2 concentrations) are compared, it is possible that a group is Blood Leukocyte Cell Proportions annotated with more than one letter (for example “ab”). Blood leukocytes were composed of high percentages of lymphocytes followed by those of neutrophils, monocytes, and basophils (Figures 2A–D). Lymphocyte proportion was not RESULTS modified at D15; however, on D36, lymphocyte proportions were higher with MacroGard b-glucan in comparison with the Specific Growth Rate, Mortality, control (p<0.01). and Splenic Index On D15, the type of b-glucan significantly influenced the Fish fed different b-glucan types showed no significant proportion of basophils with the highest quantity in fish fed differences of SGR in comparison with the control diet either MacroGard and control diets in comparison with Gas1 (p<0.05). on day 15 (D15) or day 36 (D36) (Table 3). Mortality rate did Finally, the type of b-glucan also significantly affected not differ between controls and b-glucan treatments (Table 3). neutrophils proportion of fish as MacroGard diet reduced it in Furthermore, no significant changes in SI values were observed comparison of WT and the control diet. on D15 and D36 in all b-glucan diets compared with control diet (Table 3). Immune Gene Expression Pro- and Anti-Inflammatory Gene Expressions Humoral Immune Variables Expression levels of both pro-inflammatory (il-1b) and anti- On D36, the high dose of wild type b-glucan (W0.5%) significantly inflammatory (tgf-b1, il-10) genes were evaluated in spleen and stimulated ACH50 level in comparison with M0.5% and G0.2%, kidney organs of fish sampled after short-term (D15) or mid- whereas G0.2% significantly reduced it compared to the M0.2% term b-glucan administration, also the expression of some (p<0.01, Figure 1A). At D15 and 2 days after bacterial injection immune genes was recorded on D39 after bacterial challenge (D39), there was no significant difference of plasma ACH50 level (Figures 3A–F). between treatment and control groups. In the spleen, no significant effect was observed at D15, On D36, G0.2%, W0.2%, and W0.5% significantly elevated whereas only the type of b-glucan affected the expression of il- the levels of plasma total Ig (p < 0.001) compared to the control 10, with a significant decrease in MacroGard and WT diets in diet (Figure 1B) and M0.2%. comparison of the control diet (p < 0.05, Figure 3E). TABLE 3 | Mean ( ± SD) values for specific growth rate (SGR), mortality and splenic index (SI) of trout juveniles fed with low (0.2%) and high (0.5%) doses of different beta glucan types on day 15 and day 36 of the feeding trial. Times (Days) Variables Control Macrogard GAS1 Wild type F-values P-values M0.2% M0.5% G0.2% G0.5% W0.2% W0.5% 15 SGR (%/day) 1.53 ± 0.33 1.46 ± 0.26 1.72 ± 0.32 1.56 ± 0.32 1.32 ± 0.37 1.80 ± 0.01 1.60 ± 0.18 1.497 0.254 Mortality (%) 3.8 ± 6.6 4.8 ± 8.2 2.9 ± 4.9 4.8 ± 5.9 1.0 ± 1.6 4.8 ± 5.9 1.0 ± 1.6 0.233 0.958 Splenic index (%) 0.11± 0.02 0.11 ± 0.02 0.13 ± 0.04 0.12 ± 0.03 0.12 ± 0.02 0.11 ± 0.01 0.11 ± 0.03 0.214 0.966 36 SGR (%/day) 1.58 ± 0.06 1.54 ± 0.10 1.73 ± 0.35 1.51 ± 0.08 1.75 ± 0.34 1.36 ± 0.34 1.54 ± 0.06 0.224 0.965 Mortality (%) 3.3 ± 5.8 0.0 ± 0.0 1.1 ± 1.9 1.3 ± 2.2 1.1 ± 1.9 3.6 ± 3.5 1.1 ± 2.0 0.586 0.736 Splenic index (%) 0.16 ± 0.03 0.16 ± 0.04 0.18 ± 0.03 0.18 ± 0.02 0.17 ± 0.01 0.16 ± 0.04 0.17 ± 0.00 0.438 0.841 Statistical differences between dietary treatments are indicated by different lower-case letters (p < 0.05). Splenic index = weight of spleen (g)/weight of body(g). (n= 3 for SGR and mortality and n=9 for SI). Frontiers in Immunology | www.frontiersin.org 6 July 2021 | Volume 12 | Article 693613 Cornet et al. GAS1: A New b-Glucan Immunostimulant Candidate A B C D FIGURE 1 | Effects of dietary beta glucan administration on humoral parameters of fish sampled during the nutrient test on D15 and D36, or 2 days after bacterial infection (D39). (A) Plasma hemolytic alternative complement activity. (B) Plasma total immunoglobulin’s content. (C) Plasma lysozyme activity. (D) Respiratory burst activity (RBA) of spleen macrophage cells. Values are expressed as mean ± SD of 18 fish/diet/day on D15, D36 and 9 fish/diet on D39. For spleen respiratory burst activity (RBA), plasma hemolytic alternative complement activity (ACH50) data are expressed as mean ± SD of 9 fish/diet/day on D15, D36. Statistical differences between groups are indicated by different letters Statistical differences, highlighted by letters, are indicated for glucan concentrations in front of 0, 0.2, and 0.5% next to the corresponding plot, for glucan type by letter above the horizontal bar grouping the two doses of the same glucan, for glucan type × concentrations interactions above each box. P values are shown at the bottom of each graph at a given timepoint (D15, D36, or D39). A B C D FIGURE 2 | Effects of dietary beta glucan administration on blood leukocyte cell proportions of fish sampled during the nutrient test on D15 and D36. (A) Lymphocyte + thrombocyte cells proportions. (B) Monocyte cells proportion. (C) Neutrophil cells proportion and (D) Basophil cells proportion in total blood leukocyte cells population. Values are expressed as mean ± SD of 18 fish/diet/day on D15, D36. Statistical differences, highlighted by letters, are indicated for glucan concentrations in front of 0, 0.2, and 0.5% next to the corresponding plot, for glucan type by letter above the horizontal bar grouping the two doses of the same glucan, for glucan type × concentrations interactions above each box. P values are shown at the bottom of each graph at a given time point (D15, D36, or D39). Frontiers in Immunology | www.frontiersin.org 7 July 2021 | Volume 12 | Article 693613 Cornet et al. GAS1: A New b-Glucan Immunostimulant Candidate A B C D E F FIGURE 3 | Effects of dietary b-glucan on pro-inflammatory gene expressions in spleen and kidney of fish sampled during the nutrient test on D15 and D36, or 2 days after the bacterial challenge (D39). (A–F) are expression levels of il-1b, tgf-b1, and il-10 in spleen and kidney, respectively. Relative transcript (mRNA) levels were determined by real-time RT-PCR and normalized by the arithmetic mean of ef-1a expression. Values are expressed as mean ± SD of three pool samples per experimental condition (3 fish per pool sample). Statistical differences, highlighted by letters, are indicated for glucan concentrations in front of 0%, 0.2%, and 0.5% next to the corresponding plot, for glucan type by letter above the horizontal bar grouping the two doses of the same glucan, for glucan type × concentrations interactions above each box. P values are shown at the bottom of each graph at a given time point (D15, D36, or D39). On D39, after bacterial challenged, G0.2% and G0.5% diets fish-fed Gas1 and WT diets compared with the control group significantly reduced il-1b gene expression compared to the (p<0.05, Figures 4A, E). Similarly, Gas1 and Macrogard diets control (p<0.05, Figure 3A). Meanwhile, MacroGard diet up- enhanced the expression of mIgM in comparison to WT diet at regulated tgf-b1 gene expression in comparison with the Gas1 D15. In addition, Gas 1 diet also significantly raised the and WT diet (p<0.05, Figure 3C). expression of C3a in comparison with MG and WT diets In kidneys, MacroGard and WT diets significantly reduced il- (Figure 5A). On D36, only mcsfra was modulated by the type 10 gene expression in comparison of the control diet on D36 of b-glucan with an increase of its expression with Gas1 and WT (p<0.05; Figure 3F). After bacterial challenge, only the type of b- diets (p < 0.05, Figure 5C). After bacterial injection (D39), only glucan affected tgf-b1 and il-10 gene expression compared to the tlr2 was affected by the type of b-glucan as fish-fed Gas1 diet had control (p<0.05, Figures 3D, F). Indeed, MacroGard diet a significant higher expression level compared with MacroGard significantly enhanced the expression of tgf-b1 and il-10 and WT diets (p<0.05, Figure 4E). compared, respectively, to WT and Gas1 diets. In kidneys, WT diet significantly repressed cd4-2b gene expression compared with the control on D15 (p < 0.05; Expression of T-Helper, mIgM, Complements C3, Figure 4B) on D15. On D36, no gene expression was mcsfra, and TLR2 Genes modulated by the diets, while after bacterial infection, only igm The expression levels of T-helper genes (cd4-2b), membrane and mcsfra expression was changed by the type of b-glucan in the immunoglobulin M (mIgM), complement C3, macrophage diet. Indeed, WT diet significantly increase igm expression colony-stimulating factor receptor a (mcsfra), and Toll like compared with the control (p < 0.05, Figure 4D), whereas it receptor (tlr2) genes were determined in spleen and kidney repressed mcsfra expression in comparison of control and MG samples (Figure 4). diets (p < 0.05, Figure 5D). There was no change of tlr2 and C3a In spleen, after 15 days, cd4-2b and tlr2 gene expressions were gene expression in kidneys on D15, D36, and D39 after b-glucan modified by the type of b-glucan with a repression observed in treatments (Figure 5B). Frontiers in Immunology | www.frontiersin.org 8 July 2021 | Volume 12 | Article 693613 Cornet et al. GAS1: A New b-Glucan Immunostimulant Candidate A B C D E F FIGURE 4 | Effects of dietary beta glucan on T-helper (cd4-2b), humoral (mIgM) and membrane protein (TLR2) genes expressions in the spleen and kidney of fish sampled during the nutrient test on D15 and D36, or 2 days after the bacterial challenge (D39). (A–F) are expression levels of cd4-2b, mIgM, TLR2 in spleen and kidney, respectively. Relative transcript (mRNA) levels were determined by real-time RT-PCR and normalized by the arithmetic mean of ef-1a expression. Values are expressed as mean ± SD of 3 pool samples per experimental condition (3 fish per pool sample). Statistical differences, highlighted by letters, are indicated for glucan concentrations in front of 0, 0.2, and 0.5% next to the corresponding plot, for glucan type by letter above the horizontal bar grouping the two doses of the same glucan, for glucan type × concentrations interactions above each box. P values are shown at the bottom of each graph at a given time point (D15, D36, or D39). Antibacterial Gene Expressions of infection, except for fish fed with low dose of GAS1 b-glucan Expression of antibacterial genes (C-type lysozyme, Hepcidin, (G0.2%) (Figure 8). The mortality of fish fed with G0.2% was Cathelicidin, and Myeloperoxidase) were evaluated in the kidneys reduced from day 5 and stopped on the 10th day after bacterial and spleen on D15, D36, and D39 (Figures 6A–D and 7A–D). injections, whereas other b-glucan diets were stopped later In spleen at D15, Gas1 and WT diets caused a significant between the 10th and 12th day post-infection. The survival decrease of Hepcidin gene expression compared with the control rates were significantly higher in fish fed G0.2% (43.3%) and diet (p=0.015, Figure 6C). On the contrary, at D36, hepcidin G0.5% (30.0%) than in controls (16.7%) 14 days after bacterial expression was higher in Gas1 and WT diet in comparison with injection (F = 6.5, p = 0.0019; Figure 8), followed by M0.5% the respective levels of fish fed MG diet (p<0.05, Figure 6C). (26.7%). The higher mortality rates were observed in M0.2% After the bacterial infection, only lysozyme and mpo genes were and W0.5%. affected by the type of b-glucan with an increase of their expression with Gas1 in comparison with MG diet (p < 0.05; Figures 6A and 7C). DISCUSSION In kidneys, there was no change of lysozyme and cathelicidin gene expression on D15, D36, and D39 after b-glucan treatments In this study, rainbow trout juveniles were fed with a control diet or (Figures 6B and 7B). Regarding hepcidin gene expression, it was enriched diets with Macrogard-b-glucan, GAS1- b-glucan, and only modulated on D15 by the concentration of the compound WT-b-glucan at low (0.2% diet) and high doses (0.5% diet), and with a higher expression of the gene with the lowest dose (p < fish were sampled after short-term (15 days) and mid-term (36 0.05; Figure 6D). days) feedings. After 15 and 36 days of treatment, b-glucan diets did not significantly modify specific growth rate (SGR), mortality, or Survival Rate splenic index of treated fish in comparison with the control group. The mortality rate increased earlier and more rapidly in trout fed This result may be due to the duration of the experiment that was with the control and most of b-glucan diets during the first week not long enough to induce a significant modification of growth Frontiers in Immunology | www.frontiersin.org 9 July 2021 | Volume 12 | Article 693613 Cornet et al. GAS1: A New b-Glucan Immunostimulant Candidate A B C D FIGURE 5 | Effects of dietary beta glucan on C3a complement (c3a) and macrophage colony-stimulating factor receptor a (mcsfra) genes expressions in the spleen and kidney of fish sampled during the nutrient test on D15 and D36, or 2 days after the bacterial challenge (D39). (A–D) are expression levels of c3a and mcsfra in spleen and kidney, respectively. Relative transcript (mRNA) levels were determined by real-time RT-PCR and normalized by the arithmetic mean of ef-1a expression. Values are expressed as mean ± SD of three pool samples per experimental condition (3 fish per pool sample). Statistical differences, highlighted by letters, are indicated for glucan concentrations in front of 0, 0.2, and 0.5% next to the corresponding plot, for glucan type by letter above the horizontal bar grouping the two doses of the same glucan, for glucan type × concentrations interactions above each box. P values are shown at the bottom of each graph at a given time point (D15, D36, or D39). A B C D FIGURE 6 | Effects of dietary beta glucans on C-type lysozyme and Hepcidin genes expression in the spleen or kidney of fish sampled during the nutrient test on D15 and D36, or 2 days after the bacterial challenge (D39). (A) lysozyme gene expression in spleen. (B) lysozyme gene expression in kidney. (C) Hepcidin gene expression in spleen. (D) Hepcidin gene expression in kidney. Relative transcript (mRNA) levels were determined by real-time RT-PCR and normalized by the arithmetic mean of ef-1a gene expressions. Values are expressed as mean ± SD of three pool samples per experimental condition (3 fish per pool sample). Statistical differences, highlighted by letters, are indicated for glucan concentrations in front of 0%, 0.2%, and 0.5% next to the corresponding plot, for glucan type by letter above the horizontal bar grouping the two doses of the same glucan, for glucan type × concentrations interactions above each box. P values are shown at the bottom of each graph at a given time point (D15, D36, or D39). Frontiers in Immunology | www.frontiersin.org 10 July 2021 | Volume 12 | Article 693613 Cornet et al. GAS1: A New b-Glucan Immunostimulant Candidate A B C D FIGURE 7 | Effects of dietary beta glucans on Cathelicidin and Myeloperoxidase (MPO) genes expression in spleen or kidney of fish sampled during the nutrient test on D15 and D36, or 2 days after the bacterial challenge (D39). (A) CATH1 gene expression in spleen. (B) CATH1 gene expression in kidney. (C) MPO gene expression in spleen. (D) MPO gene expression in kidney. Relative transcript (mRNA) levels were determined by real-time RT-PCR and normalized by the arithmetic mean of ef-1a gene expressions. Values are expressed as mean ± SD of 3 pool samples per experimental condition (3 fish per pool sample). Statistical differences, highlighted by letters, are indicated for glucan concentrations in front of 0%, 0.2%, and 0.5% next to the corresponding plot, for glucan type by letter above the horizontal bar grouping the two doses of the same glucan, for glucan type × concentrations interactions above each box. P values are shown at the bottom of each graph at a given time point (D15, D36, or D39). performances. However, this was not the targeted effect in this study studies have shown that Macrogard b-glucan could stimulate as apart from the effects on growth performance, b-glucans are lysozyme activity. Particularly, dietary with this b-glucan at 0.2% mainly used for their immunostimulant capacities and previous and 0.3% increased lysozyme activity on Persian sturgeon juvenile studies highlighted the importance of the quality and structures of (47) or sea bass-fed Macrogard (0.1%) diet for 30 days had a glucans in the activation of immune cells (22, 23, 44, 45). significant increase of serum lysozyme activity (46). In a previous paper, Han et al. (30) already demonstrated that However, most of significant immune modulations induced by Gas1 glucan, which was produced in their laboratory, could this b-glucan were observed on D36 and D39 (after bacterial induce a higher survival of Artemia franciscana against Vibrio infection) and were independent of the doses, except for M0.2% harveyi infection. The authors hypothesize that the structural that significantly increased plasma alternative complement activity features of b-glucans could be responsible for this protection, as (ACH50) on D36 compared with G0.2%. This alternative Gas1 had lower degree of branching and shorter side chain complement activity plays a major role in the innate immune length than WT glucan. In the present study, the capacity to response by destroying the cell surface membranes of pathogens trigger different immune responses of the two extracted b- by creating pores and opsonizing the pathogens for destruction by glucans in the Ghent’s laboratory (GAS1 and WT) and the increased phagocyte uptake, through ligand-receptor interactions commercial control (MacroGard®) was demonstrated. between the two cell surfaces (49). In addition, on D36, MacroGard b-glucan enhanced the production of lymphocytes in comparison MacroGard: The Well-Known with the control diet. Siwicki et al. (50) already demonstrated the Commercial b-Glucans proliferative response of pronephros lymphocytes that was shown MacroGard is a well-known commercial b-glucan that was to be stimulated by mitogens. already proven to exert several immune stimulation in fish (29, Adversely, on D36, some immune parameters, such as lysozyme 46, 47). In the present study, we showed that the high dose (0.5%) activity, immunoglobulin production, neutrophils proliferation, of Macrogard b-glucan (M0.5%) was better than the lower dose and genes involved in macrophage production (mcsfra) or in (M0.2%) to protect fish against bacterial infection but also to antibacterial activity (hepcidin) were down-regulated by the induce some effectors of the innate immune system. Indeed, on supplementation with MacroGard in comparison with Gas1/WT D15, M0.5% significantly stimulated plasma lysozyme activity. b-glucans or the control. Falco et al. (51) reported that treatment Lysozyme is an enzyme that possesses a lytic activity against both with dietary MacroGard b-glucan administered daily to carp for 14 Gram-positive bacteria and Gram-negative bacteria and is known days prior to infection in generally down-regulated the expression to activate the complement system and phagocytes and its activity of several measured genes when compared to their corresponding reflects the activation of innate immunity of fish (48). Several control. In our study, the decreased were observed mostly in Frontiers in Immunology | www.frontiersin.org 11 July 2021 | Volume 12 | Article 693613 Cornet et al. GAS1: A New b-Glucan Immunostimulant Candidate FIGURE 8 | Survival rate profile in rainbow trout juveniles fed with different types of beta glucan at low (0.2% diets) or high doses (0.5% diets) and challenged with Aeromonas salmonicida achromogenes (3.1 × 107 CFU/100g fish body weight) for 14 days. Statistical differences, highlighted by letters, are indicated for glucan concentrations in front of 0, 0.2, and 0.5% next to the corresponding plot, for glucan type by letter above the horizontal bar grouping the two doses of the same glucan, for glucan type x concentrations interactions above each box. P values are shown at the bottom of each graph at a given time point (D15, D36, or D39). comparison with Gas1 and WT b-glucans potentially suggesting (isogenic deletion strains derived from baker’s yeast strain BY that MacroGard could induced a weakening of the immune system 4741). This b-glucan possesses a lower degree of branching and after a shorter period of supplementation. Nevertheless, a bacterial shorter side chain length and exerted the most prominent V. challenge was essential to validate this hypothesis. harveyi-protective effects in A. franciscana (30). Similarly, in our After bacterial infection on D39, M0.5% diet slightly but not study, low and high doses of Gas1-b-glucan (G0.2% and G0.5% significantly improved fish survival after the infection to the diet) significantly increase trout immune system with different pathogen A. salmonicida achromogenes in comparison to the cellular or molecular immune effectors stimulated at the three control (26% vs 16%) while the survival in fish fed M0.2% was timepoints (i.e., D15, D36, and D39). worse than the control diet. Although some immune genes involved On D15, Gas1 b-glucan significantly decreased basophils in anti-inflammatory responses (i.e., Tgfb and il-10) were enhanced proportions after a short-term exposure compared to control and in comparison with Gas1 and/or WT b-glucans, several other MacroGard diets. In previous studies, the modulation capacity of essential in antibacterial activity (lysozyme, mpo) and Toll-NFkb the leukocyte production by b-glucans was already observed (53– pathway (tlr2) were repressed. Altogether, those results could 55). In mammals, basophils are immune cells known to be involved support the hypothesis of a weakening of the innate immune in allergic process and antiparasitic immunity, and release system when the organism is exposed to such a high dose of b- inflammatory mediators, such as histamine, by degranulation and glucan for a long period. Indeed, it has been reported that overdoses induce inflammation (56). In Teleost, although their function of b-glucan and/or prolonged medication can lead to non-reaction remains poorly described, basophils were proved to be activated physiological status of rainbow trout (29). The decrease of several in an antibody-dependent manner (bounding with IgM induced immune genes expression in this study may be explained by the high degranulation) and the released granules exhibited the capacity to doses of b-glucan used in this experiment leading to high levels of induce the migration of various leukocytes (57). Furthermore, the butyric acid production in the guts by the fermentation of expression of cd4 coding for a co-receptor of the T cell receptor bifidobacterium and lactic acid producing bacteria. On the other (TCR) that assists in the communication with antigen-presenting hand, butyric acid is also a histone deacetylase (HDAC) inhibitor, cells and tlr2 were also repressed in fish fed Gas1. This repression of such as HDAC1, HDAC2, HDAC3, and HDAC8, which inhibits basophils proliferation and cd4 with Gas1 could alter inflammatory the function of histone deacetylase enzymes leading to deacetylase processes, but further investigation would be necessary to evaluate (52). The decrease of HDCA causes the loss of structures of to which extent. Nevertheless, Gas1 diet stimulated the activity of chromatin due to the decrease of electrostatic attraction between lysozyme and the expression of C3a involved in the alternative histone and DNA (52). complement pathways in comparison to MacroGard and WT b- Therefore, our results suggest that MacroGard b-glucan glucan potentially indicating a better response of innate immune supplementation in trout should be at least 0.5% of the diet system on D15. and should be restricted to 15 days to avoid immune weakening On D36, Gas1 also increased lysozyme activity, Ig proportion and improve fish survival in case of bacterial infection. and some immune genes involved in macrophage production (mcsfra) and antibacterial activity (hepcidin) in comparison with GAS1: The Best b-Glucan to Improve MacroGard b-glucans. As already described for MacroGard b- Resistance to A. salmonicida glucan, those results suggest the induction of some effectors of achromogenes the immune system but triggered by different molecular As described by Han et al. (30), Gas1 is a b-glucan produced by pathways. Surprisingly, in another study, brood rainbow trout the null-mutant yeasts Gas1 of Saccharomyces cerevisiae fed 0.2% MacroGard b-glucan diet for two months also exhibited Frontiers in Immunology | www.frontiersin.org 12 July 2021 | Volume 12 | Article 693613 Cornet et al. GAS1: A New b-Glucan Immunostimulant Candidate a significant increase of lysozyme activity and total Ig (54). Here, branching. Our results of measured parameters highlighted only Gas1 modified those immune parameters but this could be substantial differences in the modulation of the immune explained by the duration of feeding or the age of the fish at the systems at the three timepoints. First, on D15, W0.5% moment of the experiment. Besides, the increase of plasma total Ig exhibited the lowest lysozyme activity compared with other was also observed with both low and high doses of Gas1 b-glucan on diets. In addition, this b-glucan significantly reduced the D36. Immunoglobulins are complex glycoproteins that play critical expression of cd4 and hepcidin in comparison with the control functions in innate and adaptive immunity thanks to their capacity diet. On D36, W0.5% increased ACH50 and lysozyme activities, of recognition, binding, and fixation of cells (58). Ig production, and repressed one immune gene (il-10). In the context of a bacterial intrusion, those Ig could help to Surprisingly, although the stimulation of these immune cope with the infection, and could be one of the immune effectors features appeared even more enhanced in WT-b-glucan than responsible for the results obtained on rainbow trout juvenile in Gas1, W0.2% and W0.5% provided no protection against A. resistance to A. salmonicida pathogens at D39. Indeed, as already salmonicida achromogenes in comparison with the control or observed in Han et al. (30), low and high doses of Gas1-b-glucan MacroGard b-glucan at both doses. Other studies already (G0.2% and G0.5% diet) exhibited the best protective effect reported that dietary b-glucans (0.1%, 0.2%, and 0.3% in diets) against the pathogen. Regarding the cumulative survival rate had no significant effect on survival of juvenile Persian sturgeon 10 days after bacterial challenge, fish fed with G0.2% displayed (47) or that the administration of dietary b-1,3-glucan from the highest survival rate with 43.3%, followed by G0.5% with 30% Eulena gracilis (1% in diet) had no effect on the survival rate of and fish fed control diet at 16.7%. Meanwhile, there was no unvaccinated or vaccinated rainbow trout with Y. ruckeri after 70 difference in survival rate for fish fed low and high doses of or 84 days of treatments (26). In the present experiment, the Macrogard b-glucan, Wild type (WT)-b-glucan diets in results might indicate that the stimulation of ACH50 and comparison to those of the control. Numerous studies have lysozyme activities, Ig and monocyte production was not in reported the immunomodulation potential of b-glucan in fish itself sufficient in this case to protect the fish against bacterial disease resistance and the available results vary greatly depending infection. This lack of protection could be due, as already on the fish species, doses, and administration modes (14, 28, 59– suggested for Macrogard b-glucan, to an overstimulation of the 61). In the present study, in addition to higher amount of Ig, the immune system, that would be too weak after 36 days of feeding immune protection of fish fed G0.2% and G0.5% may be related trial to respond properly to an infection. Nevertheless, it could to a differential expression of several innate immune genes also be due to mpo in neutrophils or other bactericidal enzymes (lysozyme, tlr2). Particularly, on D39, myeloperoxidase gene that activities would be more efficient in fish fed Gas1 diet than was still enhanced after 35 h post-infection. This enzyme fish fed other b-glucan diets. Further studies should be present in neutrophils was shown to exert a bactericidal performed to exclude the influence of the doses and duration activity by producing hypochlorous acid (HOCl) that oxidizes on the absence of protection by testing lower and higher doses key components of the invading pathogens (62). Therefore, the (for example 0.1 and 1%) with a bacterial challenge at 15 days of higher mpo gene expression could have induced a higher feeding. However, as previously suggested by Han et al. (30), one production of the enzyme leading to a higher oxidant capacity explanation to this huge difference in fish protection could be to deal with the bacterial infection in fish fed Gas1 diet. because of the size and degree of branching. Bohn et al. (45) Surprisingly, we also observed that several genes related to indicate that the activation of the immune cells needs the inflammatory process (il1b, il10, tgfb) were repressed after 35 h presence of some structural features, such as b-1,3-linkages in of infection with the low dose of Gas1, and could be explained by the main chain and b-1,6 or b-1,4 branch of the glucan. Finally, it a modified dynamic of gene expression over time. Indeed, in a is possible that their structure influences their absorption in the previous study, we have shown that infection of zebrafish by A. intestine and the binding to the receptor, and this should be salmonicida achromongenes stimulated the expression of il6 at further investigated to understand why Gas1 is such more 6 h post-infection (hpi) but was already repressed after 24 hpi, efficient to protect fish against pathogen infection. and this decrease depended on the previous infections applied on the zebrafish (63). Altogether, our results suggest that Gas1 is an efficient b-glucan that should be used at a dose of 0.2% of the diet and can be given for a month although it could have provided an CONCLUSIONS even better protection against pathogen after 15 days of feeding. In this study, the diet supplemented with 0.2% of GAS1-b-glucans The high dose at 0.5% can also be used but it exhibited some was the most effective to protect rainbow trout juveniles against a immune weakening as the survival was reduced compared to the bacterial infection with A. salmonicida achromogenes. After short- low dose at 0.2%. term b-glucan feeding (D15), the immune system of rainbow trout juveniles differentially responded to the two b-glucan doses tested WT-b-Glucans: Similar Immune depending on the type of b-glucans. Regarding lysozyme enzyme, Stimulation Than Gas1, but Worst low doses of Gas1 and WT (G0.2% and W0.2%) were better to Resistance to Pathogens stimulates its activity, while M0.5% exhibited the highest activity This last b-glucans produced from wild-type yeast S. cerevisiae in comparison with M0.2%. On D36, independently of the doses, differs from Gas1 by its longer size and higher degree of MacroGard supplementation resulted in the down-regulation of Frontiers in Immunology | www.frontiersin.org 13 July 2021 | Volume 12 | Article 693613 Cornet et al. GAS1: A New b-Glucan Immunostimulant Candidate several immune parameters such as lysozyme activity, AUTHOR CONTRIBUTIONS immunoglobulin production, neutrophils proliferation, and immune genes (mcsfra, hepcidin) in comparison with Gas1 diet Ideas: SM, PB, LT, and PK. In vivo experiment: VC, TK, SM, SB, potentially suggesting a weakening of the immune system after a and FR-L. Data generation: TK, VC, FR-L, and SB. Data analysis: shorter period of supplementation confirmed by the low survival VC and TK. Manuscript preparation: VC, TK, SM, SB, PB, FR-L, rates of fish fed with this diet. Besides, considering the difference of LT, and PK. All authors contributed to the article and approved survival rate between Gas1 and WT fed fish, our results suggest the submitted version. that the structure of the b-glucan with a lower degree of branching and shorter side chain length could potentially play a substantial role in the efficiency of the immuno-stimulation. To conclude, in FUNDING the context of a mid-term feeding (36 days), it appeared that Gas1 b-glucan represents the best immunostimulant as MacroGard The authors thank to Belgian Science Policy Office (Belspo) for already exhibited some immune weakening. financial supporting through the Inter-University Attraction Poles (IAP) “AquaStress” project. TK thanks Vietnamese Government for financial supporting through Vietnamese DATA AVAILABILITY STATEMENT Students Postgraduate in Foreign Countries Programme (VIED). FR-L thanks Fondecyt regular grant (project number The raw data supporting the conclusions of this article will be 1211841; ANID; Government of Chile). made available by the authors, without undue reservation. ETHICS STATEMENT ACKNOWLEDGMENTS The animal study was reviewed and approved by Ethical The authors thank Enora Flamion, Dr. David Parra, and commitee of Namur University, Protocol number: 13197 KE. Sé bastien Baekelandt for their help in this experiment. REFERENCES 11. Ringø E, Erik Olsen R, Gonzalez Vecino JL, Wadsworth S. Use of Immunostimulants and Nucleotides in Aquaculture: A Review. J Mar Sci 1. Soltanian S, Franç ois JM, Dhont J, Arnouts S, Sorgeloos P, Bossier P. Res Dev (2011) 02:1–22. doi: 10.4172/2155-9910.1000104 Enhanced Disease Resistance in Artemia by Application of Commercial b- 12. 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Dietary Administration of Beta-Mercapto-Ethanol Treated Saccharomyces Cerevisiae The remaining authors declare that the research was conducted in the absence of Enhanced the Growth, Innate Immune Response and Disease Resistance of any commercial or financial relationships that could be construed as a potential the Rainbow Trout, Oncorhynchus Mykiss. Fish Shellfish Immunol (2011) conflict of interest. 30:923–8. doi: 10.1016/j.fsi.2011.01.016 61. Lin S, Pan Y, Luo L, Luo L. Effects of Dietary b-1,3-Glucan, Chitosan or Copyright © 2021 Cornet, Khuyen, Mandiki, Betoulle, Bossier, Reyes-Loṕ ez, Tort and Raffinose on the Growth, Innate Immunity and Resistance of Koi (Cyprinus Kestemont. This is an open-access article distributed under the terms of the Creative Carpio Koi). Fish Shellfish Immunol (2011) 31:788–94. doi: 10.1016/ Commons Attribution License (CC BY). The use, distribution or reproduction in other j.fsi.2011.07.013 forums is permitted, provided the original author(s) and the copyright owner(s) are 62. Klebanoff SJ, Kettle AJ, Rosen H, Winterbourn CC, Nauseef WM. credited and that the original publication in this journal is cited, in accordance with Myeloperoxidase: A Front-Line Defender Against Phagocytosed accepted academic practice. No use, distribution or reproduction is permitted which Microorganisms. J Leukoc Biol (2013) 93:185–98. doi: 10.1189/jlb.0712349 does not comply with these terms. Frontiers in Immunology | www.frontiersin.org 16 July 2021 | Volume 12 | Article 693613