The Ancient Cytokine BAFF- and APRIL-like Molecule Regulates the Functionality of Teleost IgM+ B Cells Similarly to BAFF and APRIL.

TNF superfamily (TNFSF) members, such as BAFF and a proliferation-inducing ligand (APRIL), emerged in vertebrates as key regulators of B cell homeostasis and activation. Many cartilaginous and teleost fish contain an additional gene, designated as BAFF- and APRIL-like molecule (BALM), of unknown function and lost in tetrapods. In this study, we have performed a wide characterization of the functions of BALM on naive B cells for the first time, to our knowledge, in teleosts using rainbow trout (Oncorhynchus mykiss) as a model. Similar to BAFF and APRIL, BALM increased the survival and promoted the proliferation of peripheral blood IgM+ B cells and cooperated with BCR cross-linking to increase the proliferation rate of IgM+ B cells. BALM also seemed to be a differentiating factor for trout IgM+ B cells, as it increased IgM secretion and increased cell size. Additionally, BALM appeared to increase the Ag-presenting properties of IgM+ B cells, augmenting MHC class II surface expression and upregulating the phagocytic capacity of these cells. Finally, the fact that there was no synergy between BALM and BAFF/APRIL in any of these functions strongly suggests that BALM signals through the same receptors as BAFF and APRIL to carry out its functions. This hypothesis was further supported in competitive BALM binding assays. The results presented provide relevant information for understanding how these TNFSF members cooperate in teleost fish to regulate B cell functionality, helping us to interpret the evolutionary relations between molecules of this family.

Search for effective oral adjuvants for rainbow trout (Oncorhynchus mykiss).

Disease prevention by vaccination is, on economic, environmental and ethical grounds the most appropriate method for pathogen control currently available to the aquaculture sector. However, vaccine administration in aquatic animals faces obvious technical problems not encountered in other land animals. Thus, oral vaccines are highly demanded by the aquaculture sector that requests alternatives to the labor-intensive injectable vaccines that require individual handling of fish, provoking stress-related immunosuppression and handling mortalities. Despite this, most previous attempts to obtain effective oral vaccines have failed both in fish and mammals. This could be a consequence of very restricted tolerance mechanisms in the intestine given the fact that this mucosa is at the frontline upon antigen encounter and has to balance the delicate equilibrium between tolerance and immunity in a microbe rich aquatic environment. In this context, the search for an optimal combination of antigen and adjuvant that can trigger an adequate immune response able to circumvent intestinal tolerance is needed for each pathogen. To this aim, we have explored potential of molecules such as ß-glucans, flagellin, CpG and bacterial lipopolysacharide (LPS) as oral adjuvants. For this, we have determined the effects of these adjuvants ex vivo in rainbow trout intestine tissue sections, and in vitro in leucocytes isolated from rainbow trout spleen and intestine. The effects were evaluated by analyzing the levels of transcription of different genes related to the innate and adaptive immune response, as well as evaluating the number of IgM-secreting cells. LPS seems to be the molecule with stronger immunostimulatory potential, and could safely be used as a mucosal adjuvant in rainbow trout. Moreover, the designed strategy provides a fast methodology to screen adjuvants that are suitable for oral vaccination, providing us with valuable information about how the intestinal mucosa is regulated in fish.

Immunostimulant properties of full-length and truncated Marinobacter algicola flagellins, and their effects against viral hemorrhagic septicemia virus (VHSV) in trout.

Adjuvants that would help optimize fish vaccines against bacterial and viral pathogens are highly demanded by the aquaculture sector. Flagellin has been proposed as an immunostimulant and an adjuvant for more than a decade. However, the adjuvant ability of flagellins with hypervariable region deleted is still unclear in fish. In this study, we evaluated the immune-stimulating capacity of two recombinant flagellins, the wild-type flagellin F from Marinobacter algicola and a version with the hypervariable region deleted (FredV2), to induce the transcription of a wide range of immune genes using two rainbow trout cell lines: a monocyte/macrophage-cell line (RTS-11) and an epithelial cell line from intestine (RTgutGC). Additionally, we studied the capacity of both flagellins to limit the replication of viral hemorrhagic septicemia virus (VHSV) on the RTgutGC cell line. Our results demonstrated that both recombinant flagellins can significantly increase the transcription of IL-1ß1, IL-6, and IL-8 in both cell lines. However, other cytokines such as IFNγ1, and TNFα or antimicrobial peptides such as hepcidin were induced by both flagellins in RTgutGC but not in RTS-11 cells. Furthermore, both flagellins were capable of reducing the replication of VHSV in RTgutGC cells. Although the immunostimulatory and the antiviral capacities exerted by F were slightly more potent than those obtained with FredV2, the effects were retained after losing the hypervariable region. Our results provide new information on the immunostimulating and antiviral capacities of flagellins that point to their potential as suitable adjuvants for the future optimization of vaccines in aquaculture.

Functional Feeds to Tackle Meagre (Argyrosomus regius) Stress: Physiological Responses under Acute Stressful Handling Conditions.

Marine algae are recognised sources of bioactive compounds that have attracted great interest as nutritional supplements for aquaculture fish. Intensive rearing conditions often expose fish to husbandry-related stressors, rendering fish more susceptible to disease and reducing production yields. The present work evaluated the potential of two marine algae extracts (Fucus vesiculosus and Nannochloropsis gaditana) as nutritional supplements to mitigate stress effects in meagre (Argyrosomus regius) exposed to an acute handling stress (AS). A plant-based diet was used as a control, and three other diets were prepared, which were similar to the control diet but supplemented with 1% of each algal extract or a combination of the two extracts (0.5% each). The effects of supplemented diets on stress biomarkers, antioxidant enzyme activities, and immune response were analysed in fish exposed to AS after 4 weeks of feeding. Supplemented diets did not affect growth performance but the inclusion of F. vesiculosus promoted higher feed efficiency, as compared to the control group. Dietary algal extracts supplementation reduced plasma glucose levels, increased white blood cell counts, and reduced the expression of pro-inflammatory genes when compared with the control. N. gaditana supplementation led to a reduction in hepatic antioxidant enzyme activity and glutathione levels, while F. vesiculosus supplementation increased muscle glutathione reductase activity and reduced lipid peroxidation. These findings support the potential of algal extracts as nutraceuticals in aquafeeds to enhance the ability of fish to cope with husbandry-related stressful conditions and ultimately improve fish health and welfare.

Potential of the Escherichia coli LT(R192G/L211A) toxoid as a mucosal adjuvant for rainbow trout (Oncorhynchus mykiss).

Oral vaccines are highly demanded by aquaculture sector that requires alternatives to injectable vaccines, involving fish handling, stress-related immunosuppression and mortalities. However, most previous attempts to obtain effective oral vaccines have failed due to a restricted tolerance mechanisms in intestine, whose mucosa is at the frontline of antigen encounter and has to balance the equilibrium between tolerance and immunity in a microbe-rich environment. Thus, the search for oral adjuvants that could augment immune responses triggered by antigens allowing them to circumvent intestinal tolerance is of great relevance. The present work focuses on the adjuvant potential of the Escherichia coli LT(R192G/L211A) toxoid (dmLT). To undertake an initial screening of the potential that dmLT has as an oral adjuvant in rainbow trout (Oncorhynchus mykiss), we have analyzed its transcriptional effects alone or in combination with Aeromonas salmonicida subsp. salmonicida or viral hemorrhagic septicemia virus (VHSV) on rainbow trout intestinal epithelial cell line RTgutGC and gut explants. Our results show that although dmLT provoked no significant effects by itself, it increased the transcription of pro-inflammatory cytokines and antimicrobial genes induced by the bacteria. In contrast, when combined with VHSV, dmLT only increased the transcription of Mx and the intracellular adhesion molecule 1 (ICAM1). Therefore, the protocol designed is an effective method to initially evaluate the effects of potential oral adjuvants, and points to dmLT as an effective adjuvant for oral antibacterial vaccines.

First in vivo evidence of pituitary adenylate cyclase-activating polypeptide antiviral activity in teleost.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide belonging to the glucagon/secretin superfamily. In teleost fish, PACAP has been demonstrated to have an immunomodulatory role. Although previous studies have shown that viral/bacterial infections can influence the transcription of PACAP splicing variants and associated receptors in salmonids, the antiviral activity of PACAP has never been studied in teleost. Thus, in the present work, we investigated in vitro the influence of synthetic Clarias gariepinus PACAP-38 on the transcription of genes related to viral immunity using the rainbow trout monocyte/macrophage-like cell line RTS11 as a model. Positive transcriptional modulation of interferon gamma (IFNγ), interferon alpha (FNα1,2), interleukin 8 (IL-8), Mx and Toll-like receptor 3 (TLR3) genes was found in a dose and time dependent manner. We also explored how a pre-treatment with PACAP could enhance antiviral immune response using poly (I:C) as viral mimic. Interferons and IL-8 transcription levels were enhanced when PACAP was added 24 h previous to poly (I:C) exposure. With these evidences, we tested in vivo how PACAP administration by immersion bath affected the survival of rainbow trout fry to a challenge with viral hemorrhagic septicemia virus (VHSV). After challenge, PACAP-treated fish had increased survival compared to non-treated/challenge fish. Furthermore, PACAP was able to decrease the viral load in spleen/kidney and stimulate the transcription of IFNs and Mx when compared to untreated infected fish. Altogether, the results of this work provide valuable insights regarding the role of teleost PACAP in antiviral immunity and point to a potential application of this peptide to reduce the impact of viral infections in aquaculture.

Taurine modulates hepatic oxidative status and gut inflammatory markers of European seabass (Dicentrarchus labrax) fed plant feedstuffs-based diets.

This study aimed to evaluate the effect of taurine (tau) supplementation to low fishmeal (FM) diets on growth performance, oxidative status, and immune response of European seabass juveniles. Four isoproteic (46% crude protein) and isolipidic (19% crude lipid) diets were formulated to contain either 25 or 12.5% FM and a mixture of plant feedstuffs, supplemented or not with 1% tau. Twelve groups of 20 fish (IBW = 9.4 g) were fed each diet for 9 weeks. Reduction of dietary FM from 25 to 12.5% impaired growth performance, feed efficiency, and protein efficiency ratio but had no effect on nitrogen retention (% N intake). Independently of FM level, dietary tau supplementation improved growth performance and nitrogen retention without affecting feed efficiency. Dietary FM level reduction increased liver G6PDH activity, but did not affect lipid peroxidation or activities of redox key enzymes. Contrarily, dietary tau supplementation decreased hepatic G6PDH and GPX activities and lipid peroxidation. Gene expression COX-2 was not affected either by FM or tau levels but TNF-α increased with the reduction of FM level but not with the tau level. Dietary tau supplementation decreased Casp3 and Casp9 expression regardless of dietary FM level. Overall, this study evidenced that dietary tau supplementation improved growth performance and antioxidant response and reduced intestine inflammatory and apoptosis processes.

Role of teleost B cells in viral immunity.

Teleost fish possess all the necessary elements to mount an adaptive immune response. Despite this, the important physiological and structural differences between the mammalian and the teleost fish immune system, anticipate significant changes regarding how this response is coordinated and executed. B cells are key players in adaptive immune responses through the production of antibodies. However, recent studies performed in mammals and other species including fish point to many additional functions of B cells within both the adaptive and the innate immune system, in many occasions taking part in the crosstalk between these two arms of the immune response. Furthermore, it should be taken into account that fish B cells share many functional and phenotypical features with innate B cell populations from mammals, which will surely condition their response to antigens. Concerning viral infections, although most studies undertaken to date in fish have been focused on characterizing antibody production, some recent studies have demonstrated that fish B cells are able to interact with viruses at different levels. In this sense, in the current review, we have tried to provide an overview of what is currently known regarding the role of teleost B cells in antiviral immunity.

Identification of CD8α+ dendritic cells in rainbow trout (Oncorhynchus mykiss) intestine.

Dendritic cells (DCs) are professional antigen presenting cells located at mucosal surfaces and lymphoid tissues. Their main role is to present antigens to T cells and thus regulate the initiation of the acquired immune response and modulate tolerance mechanisms towards self-antigens. Despite their relevance, not many studies have addressed the identification and characterization of specific DC subsets in teleost fish. Previous studies in our group identified a DC subpopulation co-expressing CD8α and major histocompatibility complex II (MHC II) on the cell surface in rainbow trout (Oncorhynchus mykiss) skin and gills. A complete functional and phenotypical characterization of these cell subsets was then undertaken, unequivocally recognizing them as DCs (CD8+ DCs). In the current study, we report the identification of a homologous population in rainbow trout intestinal lamina propria (LP). We have studied the main features of these intestinal CD8+ DCs, comparing them to those of CD8+ DCs from another mucosal tissue (gills). Interestingly, intestinal CD8+ DCs exhibited significant phenotypical and functional differences when compared to gill CD8+ DCs, suggesting that the location of DCs strongly conditions their activation state. These results will contribute to further expand our knowledge on how intestinal immune responses are regulated in fish, helping us to rationally design oral vaccines in the future.

Local regulation of immune genes in rainbow trout (Oncorhynchus mykiss) naturally infected with Flavobacterium psychrophilum.

Flavobacterium psychrophilum is the etiological agent of bacterial cold water disease (BCWD), also referred to as rainbow trout fry syndrome (RTFS), a disease with great economic impact in salmonid aquaculture. Despite this, to date, not many studies have analyzed in depth how the immune system is regulated during the course of the disease. In the current study, we have studied the transcription of several immune genes related to T and B cell activity in the skin of rainbow trout (Oncorhynchus mykiss) naturally infected with F. psychrophilum in a farm located in Lake Titicaca (Peru). The levels of expression of these genes were tested and compared to those obtained in asymptomatic and apparently healthy rainbow trout. In the case of symptomatic fish, skin samples containing characteristic ulcerative lesions were taken, as well as skin samples with no lesions. Our results pointed to a significant local up-regulation of IgD, CD4, CD8, perforin and IFNγ within the ulcerative lesions. On the other hand, no differences between the levels of expression of these genes were visible in the spleen. To confirm these results, the distribution of IgD+ and CD3+ cells was studied through immunohistochemical techniques in the ulcerative lesions. Our results demonstrate a strong local response to F. psychrophilum in rainbow trout in which IgD and T cells seem to play a major role.

Humoral and mucosal immune responses in meagre (Argyrosomus regius) juveniles fed diets with varying inclusion levels of carob seed germ meal.

Many studies have assessed the effects of incorporation of plant feedstuffs in fish diets on growth performance, whereas few studies have addressed the effects of fish meal replacement by plant protein sources on fish immune parameters. Thus, the aim of this study was to evaluate the effects on immune response of different inclusion levels of carob seed germ meal (CSGM) as partial replacement for fish meal in diets for meagre (Argyrosomus regius) juveniles. Fish were fed four experimental diets with increased CSGM inclusion levels [0% (control), 7.5% (CSGM7.5), 15% (CSGM15) and 22.5% (CSGM22.5)]. After 1, 2, and 8 weeks of feeding fish were sampled to determine haematological profile and several humoral parameters in plasma and intestine. Results showed that dietary inclusion of CSGM did not negatively affect the immune parameters of meagre. In addition, total numbers of red and white blood cells, as well as thrombocytes, lymphocytes, monocytes, and neutrophils counts were not affected by dietary treatments. All parameters evaluated in plasma were unaffected by dietary CSGM inclusion after 1 and 2 weeks of feeding, with only the haemolytic complement activity showing an increase in fish fed diets with CSGM after 1 week and in fish fed CSGM22.5 diet after 2 weeks. Regarding the innate immune parameters analysed in the intestine, it could be highlighted the increase in alkaline phosphatase and antiprotease activities in fish fed the diet with the higher inclusion of CSGM at 8 weeks. Overall, results suggest that high dietary CSGM inclusion do not compromise immune status or induce an inflammatory response in meagre juveniles.

Effects of fish oil replacement by vegetable oil blend on digestive enzymes and tissue histomorphology of European sea bass (Dicentrarchus labrax) juveniles.

The impact of replacing circa 70% fish oil (FO) by a vegetable oil (VO) blend (rapeseed, linseed, palm oils; 20:50:30) in diets for European sea bass juveniles (IBW 96 ± 0.8 g) was evaluated in terms of activities of digestive enzymes (amylase, lipase, alkaline phosphatase, trypsin and total alkaline proteases) in the anterior (AI) and posterior (PI) intestine and tissue morphology (pyloric caeca-PC, AI, PI, distal intestine-DI and liver). For that purpose, fish were fed the experimental diets for 36 days and then liver and intestine were sampled at 2, 6 and 24 h after the last meal. Alkaline protease characterization was also done in AI and PI at 6 h post-feeding. Dietary VO promoted higher alkaline phosphatase activity at 2 h post-feeding in the AI and at all sampling points in the PI. Total alkaline protease activity was higher at 6 h post-feeding in the PI of fish fed the FO diet. Identical number of bands was observed in zymograms of alkaline proteases of fish fed both diets. No alterations in the histomorphology of PC, AI, PI or DI were noticed in fish fed the VO diets, while in the liver a tendency towards increased hepatocyte vacuolization due to lipid accumulation was observed. Overall, and with the exception of a higher intestine alkaline phosphatase activity, 70% FO replacement by a VO blend in diets for European sea bass resulted in no distinctive alterations on the postprandial pattern of digestive enzyme activities and intestine histomorphology.

Dietary carbohydrate and lipid sources affect differently the oxidative status of European sea bass (Dicentrarchus labrax) juveniles.

This study aimed to evaluate the effects of dietary lipid source and carbohydrate content on the oxidative status of European sea bass (Dicentrarchus labrax) juveniles. For that purpose, four diets were formulated with fish oil (FO) and vegetable oils (VO) as the lipid source and with 20 or 0 % gelatinised starch as the carbohydrate source, in a 2×2 factorial design. Liver and intestine antioxidant enzyme activities (catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD)), hepatic and intestinal lipid peroxidation (LPO), as well as hepatic oxidative stress index (OSI), were measured in fish fed the experimental diets for 73 d (n 9 fish/diet). Carbohydrate-rich diets promoted a decrease in hepatic LPO and OSI, whereas the lipid source induced no changes. Inversely, dietary lipid source, but not dietary carbohydrate concentration, affected LPO in the intestine. Lower intestinal LPO was observed in VO groups. Enzymes responsive to dietary treatments were GR, G6PD and CAT in the liver and GR and GPX in the intestine. Dietary carbohydrate induced GR and G6PD activities and depressed CAT activity in the liver. GPX and GR activities were increased in the intestine of fish fed VO diets. Overall, effects of diet composition on oxidative status were tissue-related: the liver and intestine were strongly responsive to dietary carbohydrates and lipid sources, respectively. Furthermore, different metabolic routes were more active to deal with the oxidative stress in the two organs studied.

Dietary tryptophan and methionine as modulators of European seabass (Dicentrarchus labrax) immune status and inflammatory response.

Amino acids regulate key metabolic pathways important to immune responses and their nutritional supply may increase synthesis of immune-related proteins. The present study aimed to evaluate the effects of dietary supplementation of tryptophan and methionine on European seabass (Dicentrarchus labrax) cellular and humoral status. The immunomodulatory effects of tryptophan and methionine during an inflammatory insult was also evaluated after intraperitoneal injection with inactivated Photobacterium damselae subsp. piscicida (Phdp). A practical isonitrogenous (45% crude protein) and isolipidic (16% crude fat) diets was formulated to include fish meal and a blend of plant feedstuffs as protein sources and fish oil as the main lipid source (CRL diet). Two other diets were formulated similar to the control but including L-tryptophan or L-methionine at ×2 the requirement level (diets TRP and MET, respectively). European seabass weighing 275 g were fed the experimental diets for a period of 15 days before being sampled (trial 1). Then, fish were subjected to a peritoneal inflammation by intraperitoneally injecting UV killed Phdp (10(6) colony forming units ml(-1)) and sampled following 4 and 24 h post-injection (trial 2). Fish injected with a saline solution served as control. The haematological profile, peripheral cell dynamics and several plasma immune parameters were determined in trials 1 and 2, whereas cell migration to the inflammatory focus was also determined in trial 2. MET positively affected European seabass immune status by improving the peripheral leucocyte response, complement activity and bactericidal capacity, a stronger cellular recruitment to the inflammatory focus, and higher plasma peroxidase and bactericidal activities. TRP also seemed to improve immunostimulation, as there was a trend to augment both cell-mediated immunity and humoral capacity. However, TRP failed to improve an inflammatory response, verified by a decrease in blood phagocyte numbers and lack of immune cells recruitment. In summary, it is confirmed that MET has a pronounced influence on the innate immune response to inflammation, which is more evident than TRP, and raises its potential to incorporate in functional feeds to be used in prophylactic strategies against predictable unfavourable events.

Immunomodulatory properties of Bacillus subtilis extracellular vesicles on rainbow trout intestinal cells and splenic leukocytes.

Extracellular vesicles (EVs) are cell-derived membrane-surrounded vesicles that carry bioactive molecules. Among EVs, outer membrane vesicles (OMVs), specifically produced by Gram-negative bacteria, have been extensively characterized and their potential as vaccines, adjuvants or immunotherapeutic agents, broadly explored in mammals. Nonetheless, Gram-positive bacteria can also produce bilayered spherical structures from 20 to 400 nm involved in pathogenesis, antibiotic resistance, nutrient uptake and nucleic acid transfer. However, information regarding their immunomodulatory potential is very scarce, both in mammals and fish. In the current study, we have produced EVs from the Gram-positive probiotic Bacillus subtilis and evaluated their immunomodulatory capacities using a rainbow trout intestinal epithelial cell line (RTgutGC) and splenic leukocytes. B. subtilis EVs significantly up-regulated the transcription of several pro-inflammatory and antimicrobial genes in both RTgutGC cells and splenocytes, while also up-regulating many genes associated with B cell differentiation in the later. In concordance, B. subtilis EVs increased the number of IgM-secreting cells in splenocyte cultures, while at the same time increased the MHC II surface levels and antigen-processing capacities of splenic IgM+ B cells. Interestingly, some of these experiments were repeated comparing the effects of B. subtilis EVs to EVs obtained from another Bacillus species, Bacillus megaterium, identifying important differences. The data presented provides evidence of the immunomodulatory capacities of Gram-positive EVs, pointing to the potential of B. subtilis EVs as adjuvants or immunostimulants for aquaculture.

Functional characterization of a nonmammalian IL-21: rainbow trout Oncorhynchus mykiss IL-21 upregulates the expression of the Th cell signature cytokines IFN-gamma, IL-10, and IL-22.

In mammals, IL-21 is a common γ chain cytokine produced by activated CD4(+) T cells and NKT cells that acts on multiple lineages of cells. Although IL-21 has also been discovered in birds, amphibians, and fish, to date, no functional studies have been reported for any nonmammalian IL-21 molecule. We have sequenced an IL-21 gene (tIL-21) in rainbow trout, which has a six-exon/five-intron structure, is expressed in immune tissues, and is induced by bacterial and viral infection and the T cell stimulant PHA. In contrast to mammals, calcium ionophore and PMA act synergistically to induce tIL-21. Recombinant tIL-21 (rtIL-21) induced a rapid and long-lasting (4-72 h) induction of expression of IFN-γ, IL-10, and IL-22, signature cytokines for Th1-, Th2-, and Th17-type responses, respectively, in head kidney leukocytes. However, rtIL-21 had little effects on the expression of other cytokines studied. rtIL-21 maintained the expression of CD8α, CD8ß, and IgM at a late stage of stimulation when their expression was significantly decreased in controls and increased the expression of the Th cell markers CD4, T-bet, and GATA3. Intraperitoneal injection of rtIL-21 confirmed the in vitro bioactivity and increased the expression of IFN-γ, IL-10, IL-21, IL-22, CD8, and IgM. Inhibition experiments revealed that the activation of JAK/STAT3, Akt1/2, and PI3K pathways were responsible for rtIL-21 action. This study helps to clarify the role of IL-21 in lower vertebrates for the first time, to our knowledge, and suggests IL-21 is a likely key regulator of T and B cell function in fish.

Teleost IgD+IgM- B cells in gills and skin have a plasmablast profile, but functionally and phenotypically differ from IgM+IgD- B cells in these sites.

Although most B cells in teleost systemic compartments co-express IgM and IgD on the surface, cells exclusively expressing either of the two Igs are common in fish mucosal tissues, providing us with a unique opportunity to further characterize IgD+IgM- B cells, an intriguing B cell subset. Hence, we compared the phenotype of IgD+IgM- cells to that of IgM+IgD- B cells in rainbow trout gills and skin, also establishing the response of these subsets to immune stimulation. The transcriptional profile and secreting capacity of IgD+IgM- B cells corresponded to that of cells that have started a differentiation program toward plasmablasts, similarly to IgM+IgD- B cells. Yet, IgM+IgD- B cells retained high levels of surface MHC II and antigen-processing abilities, while these were much lower in IgD+IgM- cells, suggesting important differences in their antigen-presenting capacities. Our findings contribute to a deeper understanding of the enigmatic role of IgD in mucosal surfaces.

L-methionine supplementation modulates IgM+ B cell responses in rainbow trout.

The interest in dietary amino acids (AAs) as potential immunomodulators has been growing the recent years, since specific AAs are known to regulate key metabolic pathways of the immune response or increase the synthesis of some immune-related proteins. Methionine, tryptophan and lysine are among the ten essential AAs for fish, meaning that they cannot be produced endogenously and must be provided through the diet. To date, although dietary supplementation of fish with some of these AAs has been shown to have positive effects on some innate immune parameters and disease resistance, the effects that these AAs provoke on cells of the adaptive immune system remained unexplored. Hence, in the current study, we have investigated the effects of these three AAs on the functionality of rainbow trout (Oncorhynchus mykiss) IgM+ B cells. For this, splenic leukocytes were isolated from untreated adult rainbow trout and incubated in culture media additionally supplemented with different doses of methionine, tryptophan or lysine in the presence or absence of the model antigen TNP-LPS (2,4,6-trinitrophenyl hapten conjugated to lipopolysaccharide). The survival, IgM secreting capacity and proliferation of IgM+ B cells was then studied. In the case of methionine, the phagocytic capacity of IgM+ B cells was also determined. Our results demonstrate that methionine supplementation significantly increases the proliferative effects provoked by TNP-LPS and also up-regulates the number of cells secreting IgM, whereas tryptophan or lysine have either minor or even negative effects on rainbow trout IgM+ B cells. This increase in the number of IgM-secreting cells in response to methionine surplus was further verified in a feeding experiment, in which the beneficial effects of methionine on the specific response to anal immunization were also confirmed. The results presented demonstrate the beneficial effects of dietary supplementation with methionine on the adaptive immune responses of fish.

Immunomodulatory effects of bacteriocinogenic and non-bacteriocinogenic Lactococcus cremoris of aquatic origin on rainbow trout (Oncorhynchus mykiss, Walbaum).

Lactic Acid Bacteria (LAB) are a group of bacteria frequently proposed as probiotics in aquaculture, as their administration has shown to confer positive effects on the growth, survival rate to pathogens and immunological status of the fish. In this respect, the production of antimicrobial peptides (referred to as bacteriocins) by LAB is a common trait thoroughly documented, being regarded as a key probiotic antimicrobial strategy. Although some studies have pointed to the direct immunomodulatory effects of these bacteriocins in mammals, this has been largely unexplored in fish. To this aim, in the current study, we have investigated the immunomodulatory effects of bacteriocins, by comparing the effects of a wild type nisin Z-expressing Lactococcus cremoris strain of aquatic origin to those exerted by a non-bacteriocinogenic isogenic mutant and a recombinant nisin Z, garvicin A and Q-producer multi-bacteriocinogenic strain. The transcriptional response elicited by the different strains in the rainbow trout intestinal epithelial cell line (RTgutGC) and in splenic leukocytes showed significant differences. Yet the adherence capacity to RTgutGC was similar for all strains. In splenocyte cultures, we also determined the effects of the different strains on the proliferation and survival of IgM+ B cells. Finally, while the different LAB elicited respiratory burst activity similarly, the bacteriocinogenic strains showed an increased ability to induce the production of nitric oxide (NO). The results obtained reveal a superior capacity of the bacteriocinogenic strains to modulate different immune functions, pointing to a direct immunomodulatory role of the bacteriocins, mainly nisin Z.

Endoplasmic reticulum expansion throughout the differentiation of teleost B cells to plasmablasts.

The differentiation of B cells into antibody-secreting cells is fundamental for the generation of humoral immunity. In mammals, this process involves a series of metabolic and intracellular changes, not studied to date in teleost fish, where a clear distinction between naive B cells and plasmablasts/plasma cells (PCs) is still missing. Thus, in the current study, we have established that upon activation, teleost B cells undergo an expansion of the endoplasmic reticulum (ER) but experience no significant changes in mitochondria content. In parallel, the transcription of genes implicated in B cell differentiation increases, while that of mitochondrial genes decreases. In this context, ER monitoring has allowed us to distinguish between small cells with low amounts of ER (FSCloERlo B cells), that correspond to undifferentiated cells, and large cells with expanded ER (FSChiERhi B cells), characterized as plasmablasts. The results shed new light on the B cell differentiation process in teleosts and provide us with novel tools to study B cell function in these species.