ORIGINAL RESEARCH published: 14 May 2021 doi: 10.3389/fimmu.2021.670279 Medicinal Plant Leaf Extract From Sage and Lemon Verbena Promotes Intestinal Immunity and Barrier Function in Gilthead Seabream (Sparus aurata) Ricardo Salomón 1,2, Felipe E. Reyes-López 3,4,5, Lluis Tort 3, Joana P. Firmino 1,2, Carmen Sarasquete 6, Juan B. Ortiz-Delgado 6, José C. Quintela 7, José M. Pinilla-Rosas 7, Edited by: Samuel A. M. Martin, Eva Vallejos-Vidal 8* and Enric Gisbert 1* University of Aberdeen, 1 Aquaculture Program, Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Centre de Sant Carles de la Ràpita (IRTA- United Kingdom SCR), Sant Carles de la Ràpita, Spain, 2 PhD Program in Aquaculture, Universitat Autònoma de Barcelona, Bellaterra, Spain, Reviewed by: 3 Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Bellaterra, Spain, 4 Facultad Silvia Torrecillas, de Medicina Veterinaria y Agronomı´a, Universidad de Las Américas, Santiago, Chile, 5 Consorcio Tecnológico de Sanidad University of Las Palmas Acuı´cola, Ictio Biotechnologies S.A., Santiago, Chile, 6 Instituto de Ciencias Marinas de Andalucı´a (ICMAN-CSIC), de Gran Canaria, Spain Universidad de Cádiz, Cádiz, Spain, 7 Natac Biotech, Madrid, Spain, 8 Centro de Biotecnologı´a Acuı´cola, Departamento de Marina Machado, Biologı´a, Facultad de Quı´mica y Biologı´a, Universidad de Santiago de Chile, Santiago, Chile University of Porto, Portugal *Correspondence: The inclusion of a medicinal plant leaf extract (MPLE) from sage (Salvia officinalis) and Enric Gisbert enric.gisbert@irta.cat lemon verbena (Lippia citriodora), rich in verbascoside and triterpenic compounds like Eva Vallejos-Vidal ursolic acid, was evaluated in gilthead seabream (Sparus aurata) fed a low fishmeal-based eva.vallejos@usach.cl diet (48% crude protein, 17% crude fat, 21.7 MJ kg-1, 7% fishmeal, 15% fish oil) for 92 Specialty section: days. In particular, the study focused on the effect of these phytogenic compounds on the This article was submitted to gut condition by analyzing the transcriptomic profiling (microarray analysis) and Comparative Immunology, a section of the journal histological structure of the intestinal mucosa, as well as the histochemical properties of Frontiers in Immunology mucins stored in goblet cells. A total number of 506 differentially expressed genes (285 Received: 20 February 2021 up- and 221 down-regulated) were found when comparing the transcriptomic profiling of Accepted: 26 April 2021 the intestine from fish fed the control and MPLE diets. The gut transcripteractome Published: 14 May 2021 revealed an expression profile that favored biological mechanisms associated to the Citation: Salomón R, Reyes-López FE, 1) immune system, particularly involving T cell activation and differentiation, 2) gut integrity Tort L, Firmino JP, Sarasquete C, (i.e., adherens and tight junctions) and cellular proliferation, and 3) cellular proteolytic Ortiz-Delgado JB, Quintela JC, Pinilla-Rosas JM, Vallejos-Vidal E pathways. The histological analysis showed that the MPLE dietary supplementation and Gisbert E (2021) Medicinal promoted an increase in the number of intestinal goblet cells and modified the Plant Leaf Extract From Sage composition of mucins’ glycoproteins stored in goblet cells, with an increase in the and Lemon Verbena Promotes Intestinal Immunity and Barrier staining intensity of neutral mucins, as well as in mucins rich in carboxylated and weakly Function in Gilthead sulfated glycoconjugates, particularly those rich in sialic acid residues. The integration of Seabream (Sparus aurata). Front. Immunol. 12:670279. transcriptomic and histological results showed that the evaluated MPLE from sage and doi: 10.3389/fimmu.2021.670279 lemon verbena is responsible for the maintenance of intestinal health, supporting gut Frontiers in Immunology | www.frontiersin.org 1 May 2021 | Volume 12 | Article 670279 Salomón et al. Phytogenics Promotes Fish Gut Health homeostasis and increasing the integrity of the intestinal epithelium, which suggests that this phytogenic may be considered as a promising sustainable functional additive for aquafeeds. Keywords: cell proliferation, feed additive, gut health, innate immunity, lectin histochemistry, ursolic acid, verbascoside acid, GALT INTRODUCTION key performance indicators (i.e., growth and feed efficiency performances), but also promoted fish systemic immunity. In Aquaculture will supply the majority of aquatic dietary protein particular, an ex vivo study with splenocytes from fish fed this by 2050 (1), playing a relevant role in food security and supply, phytogenic exposed to lipopolysaccharide (LPS) showed an up- and poverty alleviation (2). The sustained growth of aquaculture regulation of genes involved in non-specific immune response, as is highly dependent on the intensification of production (3), well as pro- and anti-inflammatory cytokines, surface T-cell sustainable feed formulations (4) and generating farming marker cd4, and antioxidative stress enzymes (14). However, conditions supporting fish health and welfare (5). Among the the effects of this phytogenic feed additive still needs to be former concepts, disease is considered a main persistent threat to explored in terms of local immune response, especially at the intensive fish farming, which represents an estimated US$6 intestinal level, since optimal health and functionality of the billion loss per annum at a global scale (5). Under this intestinal mucosa is essential for sustainable animal production scenario, aquaculture depends on the use of antibiotics to fight (19, 20). This is of special relevance under the current scenario in against infectious diseases that threatens production (6), with which aquafeeds are formulated with low levels of fishmeal (FM) emerging infectious diseases forecast to increase with warmer (21), since several studies have indicated that low FM diets temperatures (7). However, their use tend to result in the compromised systemic immunity (22–25), as well as gut emergence of antimicrobial resistant bacteria, which may not immune response (26, 27). In this context, intestinal immunity represent a direct threat in terms of aquatic food consumption, is a key factor in maintaining the general health of aquatic animals but they could directly impact production itself by lowering (20). The intestinal mucosa is a complex organ composed of the drug efficacy, decreasing the animal’s immune system and digestive epithelium with its specific structure, the gut-associated selecting more virulent strains of pathogens (7). Considering lymphoid tissue (GALT), and the mucus overlying the epithelium the above-mentioned reasons, along with the increasing public with its commensal microbiota (19). Furthermore, the intestinal awareness regarding food safety issues and the environmental epithelium acts as a selectively permeable barrier for dietary impact linked to antibiotics’ use and animal welfare (7, 8), the nutrients, electrolytes and water, while maintaining an effective development of functional feeds focused on promoting and defense against pathogens and tolerance towards dietary antigens. modulating the host’s immune response has been encouraged Thus, the GALT is reputed for mediating mucosal innate and during the last decade (9–12). adaptive immune responses, as well as being a key element for Functional feeds are recognized for promoting the growth, proper distinction between pathogens and commensal microbiota welfare and health of farmed animals coupled with an inhabiting the intestine (28–30). improvement and/or modulation of their immune system, as Considering the importance of interaction between the diet well as inducing physiological benefits beyond traditional feeding and the gut, in the current study we evaluated the transcriptomic practices (13). In this sense, by preventive health management profile and histochemical properties of mucins stored in goblet through the diet, fish can divert more energy to somatic growth cells of the intestine in juvenile gilthead seabream (Sparus and reduce biological energy reserves needed to fight disease or aurata) fed a functional diet containing a medicinal plant leaf stress resistance (14). Furthermore, they can be used in addition extract (MPLE) from sage and lemon verbena. This species is to chemotherapeutic agents and vaccines (15). Among the long recognized as the most important Mediterranean aquaculture list of feed additives used in animal production (9, 16), fish species in terms of volume and economic value (31). For this phytogenics derived from herbs, spices, medicinal or aromatic purpose, we focused on the modulation of the intestinal mucosa plants are residue-free, unlike synthetic antibiotics, and are safe functionality and health by the above-mentioned phytogenics ingredients for sustainable feeds (11, 17, 18). Although the mode when included in a diet with low fishmeal levels. of action of most phytogenic feed additives has not yet been fully elucidated, they are well-known for their antimicrobial, immunomodulatory, antioxidative, and growth-promoting MATERIAL AND METHODS effects in livestock (17) and aquatic animals (9, 18). In a recent study from our research group, we showed that a Diets, Fish and Rearing Conditions phytogenic feed additive from sage (Salvia officinalis, Lamiaceae) Two isoproteic (48% crude protein), isolipidic (17% crude fat) and lemon verbena (Lippia citriodora, Verbenaceae) is an and isoenergetic (21.7 MJ kg -1 ) experimental diets were effective additive for aquafeeds since its inclusion at 0.1% in formulated with a low FM content (7% FM) as described in diets with low fishmeal (FM) content not only improved some Salomó n et al. (14). Diets, named as control and MPLE, only Frontiers in Immunology | www.frontiersin.org 2 May 2021 | Volume 12 | Article 670279 Salomón et al. Phytogenics Promotes Fish Gut Health differed in their content of the feed additive evaluated, the MPLE A total of 300 gilthead seabream (body weight, BW = 5.0 ± obtained from sage and verbena leaves, which was included in 0.2 g; mean ± standard deviation) were obtained from a the MPLE diet at 0.1% (Table 1). The MPLE was obtained by commercial fish farm, Piscicultura Marina Mediterrá nea SL NATAC Biotech SL using water/ethanol extraction (plant leaf (Andromeda Group, Burriana, Spain) and transported to the extract ratio 5:1) and characterized as described in Arthur et al. experimental facilities of IRTA in Sant Carles de la Ràpita (32) and Wó jciak-Kosior et al. (33). The tested extract (Tarragona, Spain). Then, fish were acclimatized for three (proximate composition: 73% carbohydrates, 2% crude weeks in 450 L tanks connected to a water recirculation system lipids, <1% crude proteins, 5% salts and 4% water) contained (IRTAmar™) at an initial density of 2 kg m-³. Acclimation was 10%, ursolic acid, 3% other triterpenic compounds, 2% conducted in the same experimental tanks (450 L) where the verbascoside and <1% polyphenols. Thus, the content in plant- nutritional experiment was carried out. Just before the start of derived bioactive compounds in the MPLE diet was 100 ppm the trial, all animals were gently anesthetized (50 mg l-1 MS-222, ursolic acid, 30 ppm other triterpenic compounds, 60 ppm Sigma-Aldrich, Madrid, Spain), individually measured in BW verbascoside and <10 ppm polyphenols. This phytogenic was (26.0 ± 0.2 g) and distributed homogeneously among the eight incorporated in the mixture prior to extrusion. In brief, all experimental tanks (n = 35 fish per tank; 4 replicate tanks per powder ingredients were mixed in a double-paddle mixer experimental diet). During the trial that lasted 92 days, fish were (model RM90L, Mainca, Spain) and ground (below 250 μm) in fed at the daily rate of 3.0% of the stocked biomass, which a micropulverizer hammer mill (model SH1, Hosokawa-Alpine, approached apparent satiation as described in Salomó n et al. Germany). Diets were manufactured with a twin-screw extruder (14). Feed ration was regularly adjusted by means of intermediate (model BC45, Clextral, France) with a screw diameter of samplings along the trial. At the end of the trial, all fish were 55.5 mm. Extruded pellets were dried in a vibrating fluid bed anesthetized as previously indicated and measured for individual dryer (model DR100, TGC Extrusion, France). Oils were added BW. Fish performance in terms of survival (S control diet = 98.0 ± by vacuum coating (model PG-10VCLAB, Dinnissen, The 1.0%; S MPLE diet = 99.0 ± 0.8%), growth (BW control diet = 173.8 ± Netherlands). Immediately after coating, diets were packed in 8.2 g; BW MPLE diet = 189.6 ± 5.0g) and feed conversion ratio sealed plastic buckets and shipped by road to the facilities at (FCR control diet = 1.25 ± 0.04; FCR MPLE diet = 1.10 ± 0.04) was IRTA Sant Carles de la Ràpita, Spain. Both extruded diets (pellet already published in Salomó n et al. (14). Twelve fish from each size: 2 mm) used in this trial were manufactured by SPAROS experimental diet (n total= 3 fish per tank replicate) were Lda. (Portugal) and kept at 4°C until their administration. randomly selected and sacrificed with an overdose of Proximate composition of the extract and experimental diets anesthetic (300 mg l-1 MS-222) for gut transcriptomic and have been described in Salomó n et al. (14). histological analyses. Sacrificed animals were eviscerated and mid-anterior intestine samples (1-1.5 cm length per specimen) TABLE 1 | List of ingredients and proximal composition of experimental diets; were put in RNAlater™ (Invitrogen, Thermo Fisher Scientific, control and a basal diet supplemented with a medicinal plant leaf extract (MPLE) Lithuania), incubated overnight at 4°C, and stored at -80°C until obtained from sage (Salvia officinalis) and lemon verbena (Lippia citriodora). RNA extraction. This region of the intestine (mid-anterior section) was chosen due to its specialized immunological Ingredients, % Control diet MPLE diet functionality when compared with other intestinal sections Fishmeal LT70 7.0 7.0 (34). In addition, a similar piece of tissue was also dissected Soy protein concentrate 21.0 21.0 and fixed in 10% v/v neutral formaldehyde (pH: 7.2 ± 0.01) Pea protein concentrate 12.0 12.0 buffered with sodium phosphate (0.1M) for histological and Wheat gluten 12.0 12.0 Corn gluten 12.0 12.0 histochemical purposes. Soybean meal 48 5.0 5.0 Water temperature (25.1 ± 1.5°C, range: 22-27°C), oxygen Wheat meal 10.4 10.4 (6.1 ± 0.2 mg l-1) (OXI330, Crison Instruments), and pH (7.5 ± Fish oil (SAVINOR) 15.0 15.0 0.01) (pHmeter 507, Crison Instruments, Barcelona, Spain) were Vitamin and mineral Premix PV01 1.0 1.0 daily controlled. Salinity (35‰) (MASTER-20 T; ATAGO Co. Soy lecithin – Powder 1.0 1.0 Binder (guar gum) 1.0 1.0 Ltd), ammonia (0.13 ± 0.1 mg NH4+ l-1) and nitrite (0.18 ± 0.1 MCP 2.0 2.0 mg NO- l-1) levels (HACH DR9000 Colorimeter, Hach®, Spain) L-Lysine 0.3 0.3 were weekly monitored. The trial was run under natural L-Tryptophan 0.1 0.1 photoperiod according to the season of the year (August to DL-Methionine 0.2 0.2 MPLE – 0.1 November; 40°37’41” N). Proximate composition Crude protein, % 48.37 48.37 Transcriptional Analysis Crude fat, % 17.19 17.21 RNA Isolation and Quality Control Fiber, % 1.52 1.52 Total RNA was extracted from mid-anterior intestine of Ash, % 5.88 5.88 individual fish (n = 12 fish per dietary treatment) using the Gross Energy, MJ/kg 21.62 21.62 RNeasy® Mini Kit (Qiagen, Germany). Total RNA from each MPLE, medicinal plant leaf extract obtained from sage (Salvia officinalis) and lemon individual sample was eluted in a final volume of 35 mL nuclease- free water and treated with DNAse using the DNA-free™ DNA verbena (Lippia citriodora). Proximal compositions of the diets were according to Salomon et al. (14), following the AOAC guidelines. Removal Kit according to manufacturer’s instructions Frontiers in Immunology | www.frontiersin.org 3 May 2021 | Volume 12 | Article 670279 Salomón et al. Phytogenics Promotes Fish Gut Health (Invitrogen, Thermo Fisher Scientific, Lithuania). Total RNA Transcripteractome concentration and purity were quantified using a Nanodrop- The Search Tool for the Retrieval of Interacting Genes (STRING) 2000® spectrophotometer (Thermo Scientific, USA) and stored public repository version 11.0 (https://string-db.org) was used in at -80°C until analysis. Prior to hybridization with microarrays order to obtain the gut transcripteractome for those differentially (3 pooled RNA per dietary condition), four individual RNA expressed genes (DEGs) from fish fed the MPLE diet in comparison samples were pooled by mixing a volume of 1.5 mL [(RNA) = to the control group (P- value < 0.05) (36). This functional network 133.33 ng/μL] per individual sample [final volume = 6 μL; analysis has gained increasing attention because of the association (RNA) = 133.33 ng/μL] and checked for integrity using an between different genes sorted in different clusters that may have Agilent 2100 Bioanalyzer (Agilent Technologies, Spain). Pooled complementary functions into a biological context of response (35). RNA analyzed in this study were selected by the criteria of a RIN A protein-protein interaction (PPI) network of DEGs was value > 8.5 (Supplementary Figure 1). This methodological conducted with a high-confidence interaction score (0.9) using approach of pooling RNA from four different specimens in Homo sapiens as model organism. Furthermore, to confirm each replicate (n = 3 microarray replicates) allowed authors matches of the genes acronyms tag between both H. sapiens and evaluating population’s variability (n = 4 replicate tanks; 1 fish gilthead seabream species Genecards (37) and Uniprot (38) per tank in each pooled RNA; N = 12 animals); however, the databases were used. In order to confirm match of gene acronyms information regarding individual variability was lost with between both H. sapiens and gilthead seabream species, human this choice. orthology identification based on gene/protein name was accessed through the Genecards (www.genecards.org) (37) and Uniprot Microarray Hybridization and Analysis (www.uniprot.org) (UniProt, 2019) databases. Additionally, Transcriptional analysis was carried out using the Aquagenomics protein-protein BLAST (BLASTp) were analyzed (E-value < 10-7; Sparus aurata oligonucleotide microarray v2.0 (4 x 44 K) (SAQ) query cover > 95%). Gene ontology (GO) enrichment analysis for platform. The detailed information about the platform and the the DEGs were also performed by STRING (P < 0.05). transcriptomic raw data for all samples included in this current analysis is available through the public repository Gene Histological and Histochemical Analyses Expression Omnibus (GEO) at the US National Centre for Samples (n = 12 fish per experimental diet) were embedded in Biotechnology Information (NCBI) (accession number paraffin and sagitally sectioned (5-6 μm). A total of 576 sections GPL13442 and GSE166558, respectively). (2 per each sample x 24 samples x 12 techniques) were used for Transcriptomic analysis from both experimental groups were histological and histochemical purposes. Two sections per each conducted as described by Reyes-Ló pez et al. (35). Briefly, 200 ng sample were stained with hematoxylin–VOF for descriptive of total RNA from each sample pool was reverse transcribed purposes; the rest were used for evaluating the histochemical along with Agilent One-Color RNA spike-in kit (Agilent properties of epithelial and mucous cells. In brief, Schiff, Periodic Technologies, USA). Then, total RNA was used as template for Acid Schiff (PAS), diastase-PAS and Alcian Blue (AB) pH 2.5, Cyanine-3 (Cy3) labelled cRNA synthesis and amplification with 1 and 0.5 (carboxylated and sulphated glycoconjugates/ the Quick Amp Labelling kit (Agilent Technologies). cRNA glycoproteins) techniques were used for studying carbohydrate samples were purified using the RNeasy micro kit (Qiagen). distribution. Furthermore, several horseradish peroxidase (HRP) Dye incorporation and cRNA yield were checked with the conjugated lectins (Sigma-Aldrich, Spain) were used for proper NanoDrop ND-2000® spectrophotometer. Then, 1.5 mg of characterization of different glucidic residues bound to the Cy3-labeled cRNA with specific activity > 6.0 pmol Cy3/mg glycoconjugates; in particular, Canavalia ensiformes/ConA cRNA were fragmented at 60°C for 30 min, and then the samples (Mannose and/or Glucose), Ulex europeus/UEA-I (L-Fucose), were mixed with hybridization buffer and hybridized to the array Triticum vulgaris/WGA (N-acetyl-D-glucosamine and/or N- (ID 025603, Agilent Technologies) at 65°C for 17 h using acetylneuraminic acid, NeuNAc/sialic acid/NANA), Glycine the Gene expression hybridization kit (Agilent Technologies). max/SBA (a-N-acetyl-D-galactosamine) and Sambucus nigra/ The microarray washes were conducted as recommended by the SNA (NeuNAc/sialic acid/NANA). Lectin concentrations ranged manufacturer using Gene expression wash buffers (Agilent between 15 μg ml-1 to 30 μg ml-1. Regarding negative controls, Technologies) and stabilization and drying solution (Agilent omission of the respective lectin, substitution of lectin-HPR Technologies). Microarrays slides were scanned with an conjugates by TBS and treatments with different enzymes were Agilent Technologies Scanner (model G2505B); spot intensities performed according to Sarasquete et al. (39). The peroxidase and other quality control features were extracted with Agilent’s activity was visualized with 3,3-diaminobenzidine tetra Feature Extraction software version 10.4.0.0 (Agilent hydrochloride/DAB and hydrogen peroxide (0.05%). All the Technologies). Quality reports were checked for each array. techniques were performed according to Pearse (40) and Although validation by mean of qPCR is required when there following proper standardized techniques and protocols (41). is a high risk of obtaining paralog genes and unspecific All reagents were purchased from Sigma Chemical Co. St Louis, hybridization; in our study, we used an oligonucleotide-based MO, USA. microarray, which has probes with a reduced number of bases Histological images were taken with a Leitz Wetzlar and high affinity, which avoids the necessity of conducting the microscope with a built-in SPOT Insight Color camera (Ernst validation of gene expression results by qPCR validation. Leitz Wetzlar GmbH, Germany). Results were manually Frontiers in Immunology | www.frontiersin.org 4 May 2021 | Volume 12 | Article 670279 Salomón et al. Phytogenics Promotes Fish Gut Health registered using a semi-quantitative assessment scoring based Furthermore, a specific affinity for WGA, SNA and SBA lectins on color intensity scores (0, negative; 1, weak; 2, moderate; was detected in the mucinous content of goblet cells (Table 2 and 3, intense; 4, very intense) from four independent observers, Figures 3A–L). Moreover, no variations were detected in the comparing the sections of the control with the experimental diet. distribution of mucosal cell glycoconjugates between the upper The mucous cell count was determined in four different sites of and the bottom areas of the intestinal folds. When comparing each histological section, and the number of cells expressed per both dietary groups, the dietary administration of the MPLE length unit of the basal lamina of the mucosal epithelium (1 mm) modified the composition of glycoproteins of mucins produced according to Yamamoto et al. (42). by goblet cells, with an increase in the staining intensity of neutral mucins, as well as in mucins rich in carboxylated and Ethics Statement weakly sulphated glycoconjugates (Table 2 and Figures 1C, D). All animal experimental procedures were complied with the In addition, an increase in affinity for WGA and SBA lectins and Guiding Principles for Biomedical Research Involving Animals a decrease in the affinity for the SNA lectin was found in the (EU2010/63), the guidelines of the Spanish laws (law 32/2007 mucinous content of goblet cells, whereas no changes were and RD 1201/2015), and authorized by the Ethical Committee of detected regarding ConA and UEA-I lectins (Table 2 and the Institute for Research and Technology in Food and Figures 3A–L). Agriculture (IRTA, Spain) for the use of laboratory animals. Microarrays and Gut Transcripteractome Statistics A total number of 506 DEGs were found when comparing the Extracted raw data from microarrays were imported and transcriptomic profiling of the intestine from gilthead seabream analyzed with Genespring version 14.5 GX software (Agilent fed the control and MPLE diets (P < 0.05; Figure 4A and Technologies). The 75% percentile normalization was used to Supplementary Table 1). Common segregation among the standardize arrays for comparisons and data were filtered by pool samples within the same dietary treatment was observed expression. An unpaired t-test was conducted without correction in the hierarchical clustering for the gut transcriptomic response to identify those DEGs between fish fed control and MPLE diets. based in similitude patterns of the DEGs response (P < 0.05; The Principal Component Analysis (PCA) was carried out using Figure 4B). The observed segregation among dietary treatments GeneSpring software, four eigenvectors were calculated to is supported by the PCA analysis for the analyzed samples describe the aggrupation of the MPLE and control groups in a (Figure 4C); in particular, four eigenvectors were calculated 3D plot. Venn diagram and the hierarchical heatmap were all and three principal components were plotted, explaining the obtained also with Genespring (version 14.5 GX software, 83.2% of the total variability [component 1 (X-axis): 45.5%; Agilent). Changes in the number of mucous cells between component 2 (y-axis): 24.4%; component 3 (z-axis): 13.3%]. The experimental diets were analyzed by means of an unpaired t- detailed analysis of gene absolute fold-change (AFC) revealed test assuming data homoscedasticity (Barlett’s test). All the that genes were mostly up-regulated in fish fed the MPLE diet analysis were performed using GraphPad PRISM 7.00. The (56.3% of DEGs), while its modulation was moderate in terms of level of significance was set at P < 0.05 for all statistical tests. AFC intensity (Figure 4). In particular, 285 of the above- mentioned DEGs were up-regulated with 219 of them within the 1.0 ≤ AFC ≤ 1.5 interval, 58 DEGs were grouped within the RESULTS 1.5 ≤ AFC ≤ 2.0, and the last 9 DEGs up-regulated were grouped 2.0 ≤ AFC ≤ 3.0. In contrast, 221 genes were significantly down- Organization of the Intestine and Mucins’ regulated and grouped in the range -1.0 ≤ AFC ≤ -3.0. (P < 0.05). Histochemistry Produced by Goblet Cells In particular, 164 DEGs were mainly concentrated in the -1.0 ≤ In both experimental groups, the intestinal mucosa was lined by AFC ≤ -1.5 range. Among them, 42 DEGs were grouped a simple columnar epithelium with basal nuclei, basophilic within the -1.5 ≤ AFC ≤ -2.0 interval, 10 DEGs were felt cytoplasm and prominent brush border with scattered goblet within the -2.0 ≤ AFC ≤ -3.0 category, while only 5 were cells. The organization of the lamina propria-submucosa and found to have an AFC higher than -3 (Figure 4). muscular layers was normal. No signals of histological alterations From the whole set of DEGs, a functional network analysis associated to inflammatory processes in the intestine were was performed. The transcripteractome showed 244 genes with observed in fish fed the MPLE diet compared to the control 552 interactions (edges). The remaining 264 DEGs were group (Figures 1A, B). Fish fed the MPLE diet showed a higher classified as unknown genes; thus, they were excluded from the density of goblet cells along the intestinal epithelium compared analysis. According to GO results and their respective annotation to fish fed the control diet (Figure 2). hierarchy, three main representative groups of genes were Regarding the histochemical properties of mucins in goblet identified in the transcripteractome among the totality of cells from the anterior intestine, results showed a variable biological processes obtained from the enrichment analysis richness of neutral glycoproteins (PAS and diastase-PAS (Supplementary Table 2): (1) immune system processes, positive) (Table 2). In addition, mucins from goblet cells (2) cellular development and organization, and (3) cellular showed a mixture of carboxylated (AB pH = 2.5) and catabolism (Supplementary Tables 3–5). Table 3 summarizes sulphated acidic groups (weak and strongly ionized; AB pH = the most relevant DEGs in fish fed the MPLE diet in relation to 1.0 and pH 0.5, respectively) (Table 2 and Figure 1C). the above-mentioned biological processes. Frontiers in Immunology | www.frontiersin.org 5 May 2021 | Volume 12 | Article 670279 Salomón et al. Phytogenics Promotes Fish Gut Health FIGURE 1 | Histological organization in the intestine of gilthead seabream (Sparus aurata) fed a control diet (A), and medicinal plant leaf extract (MPLE)- supplemented diet (B). Numbers indicate the different intestinal layers: (1) mucosa; (2) lamina propria-submucosa; (3) circular muscle layer; (4) longitudinal muscle layer. bb: brush border; mc: mucous cells; sm: serous membrane. Histochemical properties of mucins secreted by intestinal goblet cells with regard to their content on carboxylated and/or sulphated acidic groups (Alcian Blue pH = 2.5) from fish the control diet (C) and the MPLE-supplemented diet (D). mc, mucous cells. The MPLE included in the basal diet was obtained from sage (Salvia officinalis) and lemon verbena (Lippia citriodora). Staining: hematoxylin-VOF (A, B), Alcian Blue pH = 2.5/PAS (C, D). Scale bar = 50 µm. Regarding the dietary regulation of biological processes 7 up-regulated genes; 2 down-regulated genes) (Figure 5 and related to gut immunity, 18 genes were up-regulated and 14 Supplementary Table 3). genes down-regulated in fish fed the MPLE diet. In particular, Furthermore, the MPLE diet promoted the regulation of several relevant GOs related to immunity were obtained such biological processes associated with cellular development and as “T cell activation” (GO:0042110; 7 up-regulated genes; organization with 19 up-regulated and 14 down-regulated genes. 7 down-regulated genes), “T cell differentiation” (GO:0030217; Among them, we found the terms “regulation of adherens 6 up-regulated genes; 5 down-regulated genes), “T cell lineage junction organization” (GO:1903391; 3 up-regulated genes; commitment” (GO:0002360; 3 up-regulated genes; 2 down- 3 down-regulated genes), “regulation of anatomical structure regulated genes), “leukocyte differentiation” (GO:0002521; 8 up- morphogenesis” (GO:0022603; 14 up-regulated genes; 12 down- regulated genes; 7 down-regulated genes), “lymphocyte activation” regulated genes), “negative regulation of cell size” (GO:0045792; (GO:0046649; 7 up-regulated genes; 9 down-regulated genes), 2 up-regulated genes; 1 down-regulated gene), “establishment of “leukocyte activation” (GO:0045321; 15 up-regulated genes; endothelial barrier” (GO:0061028; 3 up-regulated genes; 1 down- 12 down-regulated genes), “lymphocyte differentiation” regulated gene), “regulation of cell junction assembly” (GO:0030098; 6 up-regulated genes; 6 down-regulated genes), (GO:1901888; 4 up-regulated genes; 2 down-regulated genes), “CD4+ or CD8+, a-b T cell lineage commitment” (GO:0043369; “regulation of cell-substrate adhesion” (GO:0010810; 4 up- 2 up-regulated genes; 2 down-regulated genes), “T cell selection” regulated genes; 5 down-regulated genes), “negative regulation of (GO:0045058; 3 up-regulated genes; 2 down-regulated genes) cell-substrate adhesion” (GO:0010812; 3 up-regulated genes; and “intracellular receptor signaling pathway” (GO:0030522; 2 down-regulated genes), “regulation of focal adhesion Frontiers in Immunology | www.frontiersin.org 6 May 2021 | Volume 12 | Article 670279 Salomón et al. Phytogenics Promotes Fish Gut Health genes; 3 down-regulated genes) and “ubiquitin-dependent protein catabolic process” (GO:0006511; 14 up-regulated genes; 3 down- regulated genes) were obtained (Figure 7 and Supplementary Table 5). DISCUSSION Considering the close relationship between diet and gut condition and the consequences on the organism and overall health, evaluating the interactions between dietary ingredients and the intestine is of special relevance due to the wide array of functions that have been associated to the gastrointestinal tract (34, 43). This is of special relevance when evaluating functional feed additives that are supposed to promote health and nutrition in farmed animals (9, 44). In this context, the present study aimed to evaluate the effects of a MPLE from sage and lemon verbena on the transcriptomic profiling, histological FIGURE 2 | Goblet cell density in the intestine of gilthead seabream (Sparus organization and lectin histochemistry of the intestine. Extracts aurata) fed a control or a medicinal plant leaf extract (MPLE)-supplemented from these medicinal plants are reputed for their beneficial diet. The MPLE included in the basal diet was obtained from sage (Salvia pharmacological activities, including antiseptic, antioxidant, officinalis) and lemon verbena (Lippia citriodora). The asterisk indicates statistically significant differences among dietary treatments (t-test; P < 0.05). and anti-inflammatory properties among others (45–47). Moreover, in gilthead seabream we demonstrated that in addition to act as growth promoters, they modulated systemic TABLE 2 | Histochemical characteristics and lectin affinity of mucins produced by immunity when splenocytes were incubated with a bacterial-type goblet cells from anterior intestine of gilthead seabream (Sparus aurata) fed the pathogen-associated molecular pattern (PAMP) like LPS (14). control and this basal diet supplemented with a medicinal plant leaf extract (MPLE) obtained from sage (Salvia officinalis) and lemon verbena (Lippia citriodora). MPLE From Sage and Lemon Verbena Control diet MPLE diet Exert Controlled Immune and Pro- General histochemistry Inflammatory Responses in GALT Neutral glycoproteins 1-2 2-3 Fish possess innate and adaptive immune defense systems. The Carboxylated glycoproteins 1-3 3 innate parameters are at the forefront of immune defense and are Weakly inonised sulphated glycoconjugates 2-3 3 a crucial factor in disease resistance. The adaptive response of Strongly ionised sulphated glycoconjugates 2-3 2-3 fish is essential for long-lasting immunity and is considered as a Lectin histochemistry key factor in improving prophylactic strategies, and the use of ConA (Man/Glu) 0 0 WGA (bGlcNAc>>NeuNAc/sialic acids/NANA) 2-3 3 functional feeds with immunomodulatory properties (20, 48). T SNA (Neur5Aca2; sialic acids/NANA) 1-3 0 cells are one of the main players of the adaptive immune SBA (a/b GalNAc) 0-3 1-3 response, being the intestine of teleosts an important site of T UEA-I (Fuc) 0 0 cells production among mucosal tissues (49, 50). In particular, Semi-quantitative assessment scoring based on color intensity scores: 0, negative (non T cells represent the major leucocyte population within the detected); 1, weak; 2, moderate; 3, intense; 4, very intense. teleost gut (51) with relevant cytotoxic activity (52), as well as playing an important role in foreign antigen recognition and assembly” (GO:0051893; 3 up-regulated genes; 2 down-regulated gut homeostasis (53, 54). Under the current experimental genes), “negative regulation of adherens junction organization” conditions, MPLE from sage and lemon verbena promoted T (GO:1903392; 1 up-regulated genes; 1 down-regulated genes), and cell activation, differentiation and selection in gilthead seabream “cell aging” (GO:0007569; 3 up-regulated genes; 1 down-regulated gut. Thus, the MPLE-supplemented diet induced up-regulation gene) (Figure 6 and Supplementary Table 4). of the mammalian target of rapamycin (mtor) gene in the gut of The tested functional feed additive resulted in the positive gilthead seabream. The mTOR signaling pathway has been regulation of biological processes related to cellular proteolytic reported to promote the production of anti-inflammatory processes, showing 35 up-regulated and 11 down-regulated cytokines by myeloid immune cells with an important ability genes. In particular, “cellular macromolecule catabolic process” to limit the pro-inflammatory mediators (55) thus, playing a (GO: 0044265; 23 up-regulated genes; 6 down-regulated genes), crucial role in intestine inflammation, epithelial morphogenesis “proteolysis” (GO:0006508; 27 up-regulated genes; 8 down- (56, 57), as well as being an important central regulator of regulated genes), “protein catabolic process” (GO:0030163; 20 immune responses (58, 59). The increase in mtor expression up-regulated genes; 3 down-regulated genes), “proteolysis involved may be associated to the down-regulation of nlrc3, which is a in cellular protein catabolic process” (GO:0051603; 17 up-regulated negative regulator of the PI3K/AKT/mTOR pathway (60), Frontiers in Immunology | www.frontiersin.org 7 May 2021 | Volume 12 | Article 670279 Salomón et al. Phytogenics Promotes Fish Gut Health FIGURE 3 | Histochemical localization of glycoconjugates containing sugar residues in the intestine of gilthead seabream (Sparus aurata) fed a control or a medicinal plant leaf extract (MPLE)-supplemented diet. Presence of glycoconjugates containing N-acetyl-D-glucosamine and/or N-acetylneuraminic acid residues in mucous cells of S. aurata fed a control (A) (B: negative control) or a MPLE-supplemented diet (C). Note the increase in affinity for WGA lectin in the mucinous content of goblet cells from MPLE diet. Glycoconjugates containing a-N-acetyl-D-galactosamine residues in mucous cells of S. aurata fed a control (D) (E: negative control) or a MPLE-supplemented diet (F). Results denote a moderate increase in affinity for the SBA lectin in the mucous cells from MPLE diet. Histochemical detection of glycoconjugates containing N-acetylneuraminic acid/sialic acid residues in the intestine from control (G) (H: negative control) or MPLE group (I). Note the decrease in affinity for the SNA lectin in the intestinal epithelium of S. aurata fed a MPLE-supplemented diet. Glycoconjugates containing a-mannose/a-glucose residues in intestine from S. aurata fed a control (J) (K: negative control) or a MPLE-supplemented diet (L). Observe the increase in affinity for the ConA lectin in the intestinal epithelium of S. aurata fed a MPLE-supplemented diet. Mucous cells were negative for ConA lectin in both control and MPLE groups. e: epithelium; mc: mucous cells. The MPLE included in the basal diet was obtained from sage (Salvia officinalis) and lemon verbena (Lippia citriodora). Scale bar = 50 µm. thus suggesting a balanced inflammatory dietary-induced inflammatory mechanisms related to the NF-kB signaling response in the gut of fish fed the MPLE diet. pathway (68, 69), which involved the production of pro- The NF-kB pathway is involved in the transcriptional inflammatory cytokines (70). In this sense, histological data regulation of several cytokines, chemokines, transcription allow discarding the hypothesis that the administration of the factors, antimicrobial peptides, and interferon-stimulated MPLE caused an exacerbated gut inflammation, which would genes, playing a critical role in regulating the survival, have been potentially detrimental for the health and condition of activation and differentiation of innate and adaptive immune the intestinal mucosa of the gilthead seabream. cells (61); thus, playing a key role in regulating gut inflammation The Caspase‐8 is a crucial executor for apoptotic initiation in (62). In this way, several genes associated with the NF-kB fish (71). In our study, casp8 was significantly up-regulated in the pathway (cyld, clec4e, nlrc3, chi3l1, bcl2) were differentially intestine of fish fed the MPLE-supplemented diet. Several studies regulated by the dietary supplementation of MPLE. Although have reported the advantageous outcomes of having the casp8 scarce information is available for teleost fish, CYLD identified in expressed in a controlled way, demonstrating the essential role of rainbow trout (Oncorhynchus mykiss) would have a similar this gene in maintaining the gut barrier in response to mucosal function as in mammals (63). In our study, cyld was up- pathogens by permitting inflammatory shedding and preventing regulated, which may indicate that in fish fed the MPLE diet necroptosis of infected cells (72, 73). In addition, stat3 (Signal this gene acted as an inhibitor of the NF-kB signaling pathway, transducer and activator of transcription 3) was down-regulated, thus acting as a negative regulator of gut inflammation (63, 64). which suggests a healthy intestinal mucosa as this gene is Accordingly, the up-regulation of ubiquitin specific peptidase 4 positively regulated by pro-inflammatory cytokines under (usp4) and down-regulation of Ubiquitin specific peptidase 7 mucosal damage (74). Furthermore, our enrichment analysis (usp7) may be also associated to the inhibition of the activation showed regulation of biological processes associated with cellular of NF-kB and mitogen-activated protein kinases stimulated proteolysis, in particular several genes coding for de- and by innate immune receptor (65–67). Additionally, chi3l1 was ubiquitination (cyld, uba1, ube2a, ube2g1, ube2d2, usp4, usp7) also up-regulated in fish fed the MPLE diet, promoting cell and proteasome (cops4, psmb2, psmd1, psmb5, psmd5, sqstm1) survival and proliferation, but not acting as a first inflammation- that may be indirectly involved in NF-kB pathway. In addition, responsive factor. Thus, the differential regulation of the above- degradation of a protein via the ubiquitin-proteasome pathway mentioned genes provide evidence that MPLE might induced and subsequent proteolysis are important mechanisms in the Frontiers in Immunology | www.frontiersin.org 8 May 2021 | Volume 12 | Article 670279 Salomón et al. Phytogenics Promotes Fish Gut Health A B C FIGURE 4 | Differential expression analysis of the gilthead seabream (Sparus aurata) mid-anterior intestine transcriptomic response to MPLE diet. (A) Distribution of the differential expressed genes (DEGs) obtained from the microarray-based transcriptomic analysis. The absolute fold change (AFC) indicates the fold-change magnitude interval of response. (B) Hierarchical clustering of the gilthead seabream mid-anterior intestine transcriptomic response for the control and MPLE diet, based in similitude patterns of the differentially expressed genes (DEGs) detected from three sample pools per dietary group. Data of the six microarrays are depicted, one for each representing the pooled RNA. Genes from each replicate are ordered from lower to higher AFC intensities using the left sample (randomly chosen) as a reference for ordering the other five samples (GeneSpring version 14.5 GX software; Agilent Technologies). Both increased and decreased gene expression pattern is shown in green and red, respectively, according to the color range (bottom). All transcripts represented are statistically significant (P < 0.05). (C) Principal component analysis (PCA) of the DEGs for the gilthead seabream intestine in response to the control (yellow node) and MPLE-supplemented diet (red node). The MPLE included in the basal diet was obtained from sage (Salvia officinalis) and lemon verbena (Lippia citriodora). regulation of cell cycle, cell growth, tissue regeneration, signal and function (77). Additionally, we found down-regulation of transduction, and gene transcription (75). the NOD-like family CARD domain containing 3 (nlrc3), which A successful immune response involves the tight control of a plays an important role in modulating T cell responsiveness and wide repertoire of processes including activators and regulators inhibition of the pro-inflammatory mechanism (78). Besides, at cellular and molecular level. In this context, another up- galectin 1 (lgals1) was also down-regulated in the intestine of fish regulated gene that deserves attention is the Chitinase 3 like 1 fed the MPLE diet. LGALS1 is considered a master regulator of (chi3l1). This gene is reputed for regulating the AKT1 signaling homeostatic signals to shut off T-cell effector functions (79), pathway (76), which controls innate immune cell development acting as an immunoregulator and in turn modulating the Frontiers in Immunology | www.frontiersin.org 9 May 2021 | Volume 12 | Article 670279 Salomón et al. Phytogenics Promotes Fish Gut Health TABLE 3 | List of the most relevant DEGs related to three main representative biological processes identified by the transcripteractome (1, immune system processes; 2, cellular development and organization; 3, cellular catabolism) in fish fed the MPLE diet. Gene description Gene acronym Biological processes AFC P-value CYLD lysine 63 deubiquitinase cyld 1 2,374 0,027 Actinin Alpha 4 actn4 2 1,631 0,022 Proteasome 26S subunit, non-ATPase 1 psmd1 1, 3 1,582 0,015 Ubiquitin like modifier activating enzyme 1 uba1 1, 3 1,570 0,020 Chitinase 3 Like 1 chi3l1 1 1,549 0,046 Proteasome 26S subunit, non-ATPase 5 psmd5 1, 3 1,537 0,041 Erythrocyte membrane protein band 4.1 epb41 2 1,476 0,032 Caspase 8 casp8 1 1,472 0,004 Sequestosome 1 sqstm1 1, 3 1,413 0,031 Proteasome subunit beta 5 psmb5 1, 3 1,408 0,020 Zinc and ring finger 3 znrf3 3 1,336 0,038 RAP1B, member of RAS oncogene family rap1b 2, 3 1,321 0,004 LIM domain binding 1 ldb1 2, 3 1,311 0,019 COP9 signalosome subunit 4 cops4 1, 3 1,255 0,001 Ubiquitin specific peptidase 4 usp4 1, 3 1,231 0,037 Ras homolog family member A rhoa 2 1,222 0,047 Fibroblast growth factor 18 fgf18 3 1,206 0,041 Mechanistic target of rapamycin kinase mtor 1, 3 1,176 0,021 Proteasome subunit beta 2 psmb2 1, 3 1,167 0,023 Phosphoglycerate kinase 1 pgk1 3 1,164 0,046 Ubiquitin conjugating enzyme E2 A ube2a 1, 3 1,102 0,039 Ubiquitin conjugating enzyme E2 G1 ube2g1 1, 3 -1,100 0,001 C-Type lectin domain family 4 member e clec4e 1 -1,154 0,013 NLR family card domain containing 3 nlrc3 1 -1,315 0,020 Signal transducer and activator of transcription 3 stat3 1, 3 -1,367 0,0005 BCL2, apoptosis regulator bcl2 1 -1,378 0,038 Ubiquitin specific peptidase 7 ups7 1, 3 -1,385 0,044 Galectin 1 lgals1 1, 3 -1,452 0,020 Ubiquitin conjugating enzyme E2 D2 ube2d2 1, 3 -1,457 0,021 pro-inflammatory cytokines secretion (80). At receptor level, the producing a thick layer of mucus that forms the front line of C-type lectin domain family 4 member e (clec4e, also known as innate host defense, among other functions (83). The increase in mincle) was down-regulated in the intestine of fish from the intestinal goblet cell number in fish fed MPLE-supplemented diet MPLE group. Mincle is a transmembrane germline-encoded would benefit fish by providing an effective immune barrier pattern recognition receptors (PRRs) with a pivotal role in the against potentially pathogenic gut bacteria. In addition to activation of the immune response (81, 82). Thus, when changes in their number, goblet cells also changed their considering the overall expression patterns of DEGs, the histochemical composition. Particularly, we observed an dietary-induced activation of the immune function by the increment in staining intensity of neutral and acid carboxylated tested MPLE seems to be a well-controlled process, according and sulphated glycoproteins, results that are of special relevance to the observed balance between the up- and down-regulated since O-glycosylation pattern of mucin glycoproteins and their DEGs (18 and 14 genes, respectively) and also the functional role polypeptide backbone structure in combination with the depth of the DEGs related to T cells. As mentioned above, these results and viscosity of the mucus layer, are important factors that are in agreement with those recently published by Salomó n et al. regulate gut health and condition (84). In this sense, the (14) where the same feed additive showed a tightly controlled increase in acid carboxylated (AB pH = 2.5) and sulphated systemic immune response in an ex vivo assay using splenocytes (weak and strongly ionized; AB pH = 1.0 and pH 0.5, stimulated by LPS. respectively) mucin glycoproteins produced by goblet cells from It is also of interest to illustrate that fish fed the supplemented- fish fed the MPLE-supplemented diet would be associated to an MPLE diet resulted in an increase of goblet cell population in the increment in the viscous properties of the mucus (85, 86). Our intestinal epithelium. These secretory cells are responsible for results are in agreement with those reported in poultry where the Frontiers in Immunology | www.frontiersin.org 10 May 2021 | Volume 12 | Article 670279 Salomón et al. Phytogenics Promotes Fish Gut Health FIGURE 5 | Immune system process related Protein-Protein Interactions Network (PPI) network of the differentially expressed genes (DEGs) in the mid-anterior intestine of juvenile gilthead seabream (Sparus aurata) fed the mixture of MPLE obtained from sage (S. officinalis) and lemon verbena (L. citriodora) (see also Supplementary Table 3). Nodes colors indicate the biological processes for each DEG represented. Gene Ontology (GO) definitions, count of DEGs within each biological processes and respective false discovery rate are described in the graphical figure legend. ▲ nodes represent up-regulated genes and ▼ nodes represent down-regulated genes. Graphic keys and network stats are indicated in the graphical figure legend. Network Stats: number of nodes: 243; number of edges: 550; average node degree: 4.53; avg. local clustering coefficient: 0.416; expected number of edges: 423; PPI enrichment p-value: 1.98e-09. The MPLE included in the basal diet was obtained from sage (Salvia officinalis) and lemon verbena (Lippia citriodora). administration of phytogenics from sage promoted the secretion WGA lectin affinity, as they indicate an increase in the of neutral and acidic mucins in gut as well as mucus layer presence of sialic acid residues. Sialic acids are involved in thickness in the ileum (87). Regarding lectin histochemistry, recognition processes, and they participate in defense mucins from fish fed the MPLE-supplemented diet showed an mechanisms against microorganisms (88). These results from increase in affinity for the WGA and SBA lectins, while for SNA lectin histochemistry reinforce the hypothesis that the MPLE- lectin affinity was diminished. These results indicated that the supplementation varied the composition of mucus produced by tested feed additive changed the glycosylation patterns of goblet cells; thus, modulating an effective immune barrier against intestinal mucins, being of special relevance the increase in potentially pathogenic gut bacteria. Frontiers in Immunology | www.frontiersin.org 11 May 2021 | Volume 12 | Article 670279 Salomón et al. Phytogenics Promotes Fish Gut Health FIGURE 6 | Cellular development and organization related Protein-Protein Interactions Network (PPI) network of the differentially expressed genes (DEGs) in the mid- anterior intestine of juvenile gilthead seabream (Sparus aurata) fed the mixture of MPLE obtained from sage (S. officinalis) and lemon verbena (L. citriodora) (see also Supplementary Table 4). Nodes colors indicate the biological processes for each DEG represented. Gene Ontology (GO) definitions, count of DEGs within each biological processes and respective false discovery rate are described in the graphical figure legend. ▲ nodes represent up-regulated genes and ▼ nodes represent down-regulated genes. Graphic keys and network stats are indicated in the graphical figure legend. Network Stats: number of nodes: 243; number of edges: 550; average node degree: 4.53; avg. local clustering coefficient: 0.416; expected number of edges: 423; PPI enrichment p-value: 1.98e-09. The MPLE included in the basal diet was obtained from sage (Salvia officinalis) and lemon verbena (Lippia citriodora). MPLE From Sage and Lemon an uncontrollable immune reaction in the intestinal micro Verbena Promote Gut Integrity environment, increasing the translocation of bacterial antigens and Cell Proliferation and stimulating inflammation in the intestine (90). Thus, the maintenance of the integrity of the gut barrier is essential The homeostatic balance between epithelial cell proliferation to counteract such imbalances and guarantee fish growth, health and apoptosis is essential for the maintenance of the epithelial and welfare (91). Dietary MPLE from sage and lemon verbena function, including regulation of epithelial permeability, the modulated the expression of genes involved in biological inflammatory response and the absorption of nutrients (89). processes related to cellular development and organization. An imbalance in the intestinal barrier structure can trigger In particular, Ras homolog family member A (rhoa) was Frontiers in Immunology | www.frontiersin.org 12 May 2021 | Volume 12 | Article 670279 Salomón et al. Phytogenics Promotes Fish Gut Health FIGURE 7 | Proteolysis related Protein-Protein Interactions Network (PPI) network of the differentially expressed genes (DEGs) in the mid-anterior intestine of juvenile gilthead seabream (Sparus aurata) fed the mixture of MPLE obtained from sage (S. officinalis) and lemon verbena (L. citriodora) (see also Supplementary Table 5). Nodes colors indicate the biological processes for each DEG represented. Gene Ontology (GO) definitions, count of DEGs within each biological processes and respective false discovery rate are described in the graphical figure legend. ▲ nodes represent up-regulated genes and ▼ nodes represent down-regulated genes. Graphic keys and network stats are indicated in the graphical figure legend. Network Stats: number of nodes: 243; number of edges: 550; average node degree: 4.53; avg. local clustering coefficient: 0.416; expected number of edges: 423; PPI enrichment p-value: 1.98e-09. The MPLE included in the basal diet was obtained from sage (Salvia officinalis) and lemon verbena (Lippia citriodora). up-regulated in the intestine of fish fed the MPLE-diet. In particular, the Erythrocyte membrane protein band RhoA belongs the small GTPase protein family, thus having a 4.1 (epb41) was another up-regulated gene in the intestine functional role in regulating many cellular events, including cell of fish fed the MPLE-diet. EPB41 regulates cell proliferation migration, organization of the cytoskeleton, cell cycle progression and adhesion (96), as well as the integrity of the AJ (95) and and cell adhesion (92). In this sense, rhoa expression has been plays an important role in the organization and function of associated to the assembly, regulation and maintenance of the TJs and AJs by establishing a link between the TJ and adherens (AJs) and tight (TJs) junctions (93, 94). Adherens actin in the cytoskeleton (97). Furthermore, other DEGs junctions are cadherin/catenin-containing adhesive structures related to the formation and stabilization of AJs and TJs (f11r, located below the tight junctions on the bilateral membrane (95). actn4, rap1b) were also up-regulated in the gut of fish fed the Frontiers in Immunology | www.frontiersin.org 13 May 2021 | Volume 12 | Article 670279 Salomón et al. Phytogenics Promotes Fish Gut Health MPLE-supplemented diet. The F11 receptor (F11R) also known CONCLUSIONS as the Junctional adhesion molecule (JAM)-A is a cell-cell adhesion molecule of the immunoglobulin superfamily, which The present study shows that phytogenics obtained from sage is expressed by a variety of tissues, regulating diverse processes and lemon verbena included at 0.1% in diets with a low FM such as epithelial and endothelial barrier formation, among content (7%), promoted transcriptional innate and adaptive others (98). Our results are in agreement with those found in immune responses in gut, especially through the modulation of gilthead seabream fed a phytogenic feed additive derived from those processes involved in T cell activation, differentiation and olive oil (89). Alpha-actinin-4 (ACTN4) is a member of the selection. Furthermore, the evaluated feed additive increased the superfamily of actin-binding, which is localized at AJs and more number of intestinal goblet cells and modified the glycosylation specifically as a component of the zonula occludens of the TJ (99) properties of lectins from mucins. These changes resulted in a and/or belt desmosomes in the zonula adherens (100) in moderate increase in sialic acid residues, which also supports the connection with a-catenin, regulating the actin cytoskeleton idea that phytogenics from sage and common verbena might and increasing cellular motility (101, 102). Another gene enhance gut immunity. Overall, this study shows that the that was up-regulated by the dietary administration of MPLE evaluated phytogenic can be used as a safe feed additive for from sage and lemon verbena was the rap1b. This gene is a gilthead seabream, since its immunomodulatory properties were member of RAS oncogene family being necessary for normal observed without compromising gut homeostasis and integrity of human endothelial cell function (103), as well as having a crucial the intestinal epithelium. role for T cell homeostasis in the intestine (104). Regarding the gut integrity barrier, rap1b controls epithelial permeability probably by regulation of PI3K/Akt and correct nectin localization (94). DATA AVAILABILITY STATEMENT The homoeostasis of the constantly renewing intestinal The datasets presented in this study can be found in online epithelium relies on an integrated control of proliferation, repositories. The names of the repository/repositories and accession differentiation and apoptosis, as well as on the functional number(s) can be found in the article/Supplementary Material. architecture of the epithelial cells. In this sense, different DEGs indicated that the MPLE-supplemented diet regulated mostly processes involved cell proliferation (fgf18, pgk1, ldb1, znrf3, mtor, lgals1, stat3) and de- and ubiquitination (cyld, uba1, ube2a, ETHICS STATEMENT ube2g1, ube2d2, usp4, usp7) and proteasome (cops4, psmb2, The animal study was reviewed and approved by Guiding psmd1, psmb5, psmd5, sqstm1) pathways. The Fibroblast Principles for Biomedical Research Involving Animals growth factor 18 (FGF18) is a pleiotropic growth factor which (EU2010/63), the guidelines of the Spanish laws (law 32/2007 stimulates the proliferation of mesenchymal and epithelial cells and RD 1201/2015), and authorized by the Ethical Committee of and tissues (105); thus, its up-regulation in the tissue of gilthead the Institute for Research and Technology in Food and seabream fed the MPLE-supplemented diet suggests that the tested Agriculture (IRTA, Spain). phytogenic feed additive might promote cell proliferation. Similar conclusions may be drawn when considering the up-regulation ldb1 (LIM domain binding 1) and znrf3 (Zinc and ring finger 3), since these genes regulate cell proliferation in intestinal crypts by AUTHOR CONTRIBUTIONS means of the regulation of the Wnt/ß-catenin signaling (106, 107). In addition, the up-regulation of mtor (mammalian Target of Conceptualization, EG. Methodology, EV-V, FER-L, CS, JO-D, Rapamycin) and pgk1 (Phosphoglycerate kinase 1) indicate the RS. Formal analysis, RS, FER-L, JF, EV-V. Resources, EG, CS. regulation of intestinal cell proliferation by means of controlling Writing original draft, RS. Writing review and editing, EV-V, RS, cell metabolism (108, 109). These results are supported by the FER-L, EG, JF, LT. Visualization, RS, FER-L, EV-V, JO-D, EG. down-regulation of lgals1 (galectin 1), since this gene product may Supervision, EG, EV-V. Project administration, EG. Funding act as a negative growth factor that regulates cell proliferation acquisition, EG. All authors contributed to the article and (110). Differential expression of genes involved in de- and approved the submitted version. ubiquitination and proteasome pathways supports the above- mentioned idea that the tested MPLE-supplemented diet promotes and regulates cell cycle associated to intestinal cell FUNDING proliferation (75) as well in cell junctions (111). All these results together suggested that the MPLE from sage and lemon This work has been financially supported by the project verbena might promote a positive effect on intestinal cell “Nutritional strategies for the improvement of productive proliferation, a process that is essential for the maintenance performance: the use of functional feeds and health diets in of the integrity and health of the gastrointestinal tract (112). In aquaculture (DIETAplus)”, funded by JACUMAR (Ministry of line with this idea, no signs of tissue damage were observed Agriculture, Fisheries and Environment of Spain, MAPAMA) at histological level either pathways involved in cell apoptosis and FEMP (EU), as well as by the ERC (European Research were found in these animals. Council) in MedAID project (Mediterranean Aquaculture Frontiers in Immunology | www.frontiersin.org 14 May 2021 | Volume 12 | Article 670279 Salomón et al. Phytogenics Promotes Fish Gut Health Integrated Development; Grant Agreement Nb. 727315). FR-L improvement of fish larvae production in Ibero-America” thanks the support of Fondecyt Regular grant (Nb. 1211841; (117RT0521) funded by the Ibero-American Program of ANID; Government of Chile). RS is supported by a PhD grant Science and Technology for Development (CYTED, Spain). from the government of Paraguay (BECAL). JF have been subsidized by the Industrial PhD program of Generalitat de Catalunya and TECNOVIT-FARMFAES S.L. (Nb. 2017 DI 017). SUPPLEMENTARY MATERIAL EV-V was financially granted with DICYT-USACH Postdoctoral fellowship (Nb. 022043IB). Collaboration between Ibero- The Supplementary Material for this article can be found online American researchers has been done under the framework of at: https://www.frontiersin.org/articles/10.3389/fimmu.2021. the network LARVAplus “Strategies for the development and 670279/full#supplementary-material 14. 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