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Effect of β-glucans on rainbow trout (Oncorhynchus mykiss) IgM+ B cells
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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.
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 naïve 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 increase, while that of mitochondrial genes decrease. 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.
The immune response of the adipose tissue (AT) has been neglected in most animal models until investigations in human and mice linked obesity to chronic inflammation, highlighting the immune nature of this tissue. Despite this, in teleost fish, only a few studies have addressed the immune role of the AT. These studies have mostly focused on reporting transcriptional changes in the AT in response to diverse intraperitoneally delivered stimuli. Although the presence of B cells within the AT was also previously revealed, these cells have never been phenotypically or functionally characterized and this is what we have addressed in the current study. Initially, the B cell populations present in the rainbow trout (Oncorhynchus mykiss) AT were characterized in comparison to B cells from other sources. As occurs in other rainbow trout tissues, IgM⁺IgD⁺, IgM⁺IgD⁻ and IgD⁺IgM⁻ B cell subsets were identified in the AT. Interestingly, AT IgM⁺IgD⁻ B cells showed a transcriptional profile that agrees with that of cells that have committed to plasmablasts/plasma cells, being this profile much more pronounced towards a differentiation state than that of blood IgM⁺IgD⁻ B cells. Accordingly, the IgM-secreting capacity of AT B cells is significantly higher than that of blood B cells. Additionally, AT IgM⁺IgD⁺ B cells also showed specific phenotypic traits when compared to their counterparts in other tissues. Finally, we established how these B cell subsets responded when rainbow trout were intraperitoneally injected with a model antigen. Our results demonstrate that the AT hosts plasmablasts/plasma cells that secrete specific IgMs, as happens in the peritoneal cavity and systemic immune tissues. Although the presence of these antigen-specific IgM-secreting cells was more abundant in the peritoneal cavity, these specific differentiated B cells were detected in the AT for long time periods at levels similar to those of spleen and head kidney. Our results provide new evidence regarding the immune role of the teleost AT, indicating that it functions as a secondary lymphoid organ that promotes immunity to peritoneal antigens.
In the absence of class switch recombination and germinal centers, the mechanisms through which B cells from teleost fish mount extrafollicular immunoglobulin M (IgM) memory responses remains mostly unexplored. In this report, we demonstrate that teleost IgM⁺ B cells respond to CD40L, a thymus-dependent activation signal, similarly to mammalian B2 cells. However, when stimulated with different types of antigens, fish IgM⁺ B cells only reach a general activation state in response to antigens cataloged as thymus-independent 1 (TI-1) in mammals, as established through both functional assays and RNA sequencing. Interestingly, fish IgM⁺ B cells remained completely unresponsive to TI-2 antigens, suggesting that the engagement of innate receptors provided by TI-1 antigens is required for the activation of teleost B cells. Finally, a synergy between CD40L and TI-1 antigens was also demonstrated, further supporting that there is no clear dichotomy between thymus-dependent and TI responses in teleost fish.
β-glucan is a non-starch soluble polysaccharide widely present in yeast, mushrooms, bacteria, algae, barley, and oat. β-Glucan is regarded as a functional food ingredient due to its various health benefits. The high molecular weight (Mw) and high viscosity of β-glucan are responsible for its hypocholesterolemic and hypoglycemic properties. Thus, β-glucan is also used in the food industry for the production of functional food products. The inherent gel-forming property and high viscosity of β-glucan lead to the production of low-fat foods with improved textural properties. Various studies have reported the relationship between the molecular structure of β-glucan and its functionality. The structural characteristics of β-glucan, including specific glycosidic linkages, monosaccharide compositions, Mw, and chain conformation, were reported to affect its physiochemical and biological properties. Researchers have also reported some chemical, physical, and enzymatic treatments can successfully alter the molecular structure and functionalities of β-glucan. This review article attempts to review the available literature on the relationship of the molecular structure of β-glucan with its functionalities, and future perspectives in this area.
Oligodeoxynucleotides (ODN) containing unmethylated CpG motifs have been widely postulated as vaccine adjuvants both in mammals and teleost fish. However, to date, the effects that CpGs provoke on cells of the adaptive immune system remain mostly unexplored in fish. Given that rainbow trout (Oncorhynchus mykiss) IgM⁺ B cells from spleen and blood transcribe high levels of toll like receptor 9 (TLR9), the receptor responsible for CpG detection in mammals, in the current work, we have investigated the effects of CpGs on both spleen and blood IgM⁺ B cells from this species. CpGs were shown to exert strong proliferative effects on both spleen and blood IgM⁺ B cells, also increasing their survival. The fact that CpGs increase the size of IgM⁺ B cells, reduce the expression of surface IgM and IgD and up-regulate the number of IgM-secreting cells strongly suggest that IgM⁺ B cells differentiate to plasmablasts/plasma cells in response to CpG stimulation. Additionally, CpGs were shown to modulate the antigen presenting capacities of trout IgM⁺ B cells through an increased surface MHC II expression and transcriptional up-regulation of co-stimulatory molecules, although in this case, significant differences were observed between the effects exerted on spleen and blood cells. Similarly, differences were observed between spleen and blood IgM⁺ B cells when CpG stimulation was combined with B cell receptor (BCR) cross-linking. Finally, CpGs were also shown to affect innate functions of teleost IgM⁺ B cells such as their phagocytic capacity. These results demonstrate that CpGs regulate many adaptive and innate functions of teleost B cells, supporting their inclusion as adjuvants in novel vaccine formulations.
The gene encoding IgH δ has been found in all species of teleosts studied to date. However, catfish (Ictalurus punctatus) is the only species of fish in which a secretory form of IgD has been characterized, and it occurs through the use of a dedicated δ-secretory exon, which is absent from all other species examined. Our studies have revealed that rainbow trout (Oncorhynchus mykiss) use a novel strategy for the generation of secreted IgD. The trout secretory δ transcript is produced via a run-on event in which the splice donor site at the end of the last constant domain exon (D7) is ignored and transcription continues until a stop codon is reached 33 nt downstream of the splice site, resulting in the production of an in-frame, 11-aa secretory tail at the end of the D7 domain. In silico analysis of several published IgD genes suggested that this unique splicing mechanism may also be used in other species of fish, reptiles, and amphibians. Alternative splicing of the secretory δ transcript resulted in two δ-H chains, which incorporated Cμ1 and variable domains. Secreted IgD was found in two heavily glycosylated isoforms, which are assembled as monomeric polypeptides associated with L chains. Secretory δ mRNA and IgD(+) plasma cells were detected in all immune tissues at a lower frequency than secretory IgM. Our data demonstrate that secretory IgD is more prevalent and widespread across taxa than previously thought, and thus illustrate the potential that IgD may have a conserved role in immunity.
Over the past decades, it has become evident that immune-modulation of fish with β-glucans, using injection, dietary or even immersion routes of administration, has stimulating but presumed short-lived effects on both intestinal and systemic immunity and can increase protection against a subsequent pathogenic challenge. Although the exact effects can be variable depending on, among others, fish species and administration route, the immune-stimulating effects of β-glucans on the immune system of fish appear to be universal. This review provides a condensed update of the most recent literature describing the effects of β-glucans on the teleost fish immune system. We shortly discuss possible mechanisms influencing immune-stimulation by β-glucans, including microbial composition of the gut, receptor recognition and downstream signalling. Of interest, in mammalian monocytes, β-glucans are potent inducers of trained immunity. First, we screened the literature for indications of this phenomenon in fish. Criteria that we applied include indications for at least one out of three features considered characteristic of trained immunity; (i) providing protection against a secondary infection in a T- and B-lymphocyte independent manner, (ii) conferring increased resistance upon re-infection and, (iii) relying on key roles for innate immune cell types such as natural killer cells and macrophages. We conclude that several indications exist that support the notion that the innate immune system of teleost fish can be trained. Second, we screened the literature for indications of long-lived effects on innate immunity of fish after administering β-glucans, a criterion which could help to identify key roles for macrophages on resistance to infection. We discuss whether β-glucans, as well-known immune-stimulants, are able to train the immune system of fish and argue in favour of further studies designed to specifically investigate this phenomenon in fish.
Sea lice (Lepeophtheirus salmonis) are the most economically important ectoparasites affecting Atlantic salmon (Salmo salar) culture worldwide. In recent years the efficacy of historically successful treatments has been on the decline. As such, a new management strategy for controlling lice infections is a high priority for the salmon farming industry. In this study, we tested the ability of three orally administered immunostimulants to decrease the number of lice successfully infecting Atlantic salmon post-smolts. It was found that the β-glucan (ProVale) fed group actually maintained more sea lice than did the control group (24% increase). However, both the CpG ODN (31–46%) and AllBrew NuPro (11–31%) fed groups showed decreased infection levels when compared to the control group. Histopathological and differential gene expression analyses indicate that localized and systemic inflammatory mechanisms may be transiently altered by these immunostimulatory feeds and may result in increased host resistance to sea lice.
B lymphocytes differentiate into antibody-secreting cells under the antigen-specific control of follicular helper T cells (TFH cells). Here we demonstrate that isotype-switched plasma cells expressed major histocompatibility complex (MHC) class II, the costimulatory molecules CD80 and CD86, and the intracellular machinery required for antigen presentation. Antigen-specific plasma cells accessed, processed and presented sufficient antigen in vivo to induce multiple helper T cell functions. Notably, antigen-primed plasma cells failed to induce interleukin 21 (IL-21) or the transcriptional repressor Bcl-6 in naive helper T cells and actively decreased these key molecules in antigen-activated TFH cells. Mice lacking plasma cells showed altered TFH cell activity, which provided evidence of this negative feedback loop. Hence, antigen presentation by plasma cells defines a previously unknown layer of cognate regulation that limits the antigen-specific TFH cell program that controls ongoing B cell immunity.
Twenty years after the proposal that pattern recognition receptors detect invasion by microbial pathogens, the field of immunology
has witnessed several discoveries that have elucidated receptors and signaling pathways of microbial recognition systems and
how they control the generation of T and B lymphocyte–mediated immune responses. However, there are still many fundamental
questions that remain poorly understood, even though sometimes the answers are assumed to be known. Here, we discuss some
of these questions, including the mechanisms by which pathogen-specific innate immune recognition activates antigen-specific
adaptive immune responses and the roles of different types of innate immune recognition in host defense from infection and
injury.
The immunomodulator Betafectin(R) PGG-glucan is a homopolymer of glucose derived from yeast cell walls which has been demonstrated to enhance leukocyte anti-infective activity in vitro and in vivo, without the induction of proinflammatory cytokines. We report here the purification of a PGG-glucan-binding element from human leukocytes and its identification as lactosylceramide, a major glycosphingolipid of neutrophils, which includes the CDw17 epitope. The binding of radiolabeled PGG-glucan to purified lactosylceramide was saturable, specific, and time- and temperature-dependent. Lactosylceramides from human leukocytes were fractionated by high performance liquid chromatography in order to analyze the effect of ceramide structure on binding. A variety of fatty acid chain lengths with varying degrees of unsaturation were found to support binding to radiolabeled PGG-glucan. However, DL-lactosylceramides containing dihydrosphingosine did not bind. Radiolabeled PGG-glucan bound several other neutral glycosphingolipids with a terminal galactose, including galactosylceramide, globotriaosylceramide, and gangliotetraosylceramide. The binding of radiolabeled PGG-glucan to lactosylceramide was not inhibited by glycogen, dextran, mannan, pustulan, laminarin, or a low molecular weight beta-(1-3)-glucan, but was inhibited by high molecular weight beta-(1-3)-glucans and by a monoclonal antibody to lactosylceramide. Although this glycosphingolipid has been shown in numerous reports to bind various microorganisms, this represents the first report of lactosylceramide binding to a macromolecular carbohydrate.
Glucans are cell wall constituents of fungi and bacteria that bind to pattern recognition receptors and modulate innate immunity, in part, by macrophage activation. We used surface plasmon resonance to examine the binding of glucans, differing in fine structure and charge density, to scavenger receptors on membranes isolated from human monocyte U937 cells. Experiments were performed at 25 degrees C using a biosensor surface with immobilized acetylated low density lipoprotein (AcLDL). Inhibition of the binding by polyinosinic acid, but not polycytidylic acid, confirmed the interaction of scavenger receptors. Competition studies showed that there are at least two AcLDL binding sites on human U937 cells. Glucan phosphate interacts with all sites, and the CM-glucans and laminarin interact with a subset of sites. Polymer charge has a dramatic effect on the affinity of glucans with macrophage scavenger receptors. However, it is also clear that human monocyte scavenger receptors recognize the basic glucan structure independent of charge.
Effects of beta-glucan administration on survival and immune modulations were studied in Cyprinus carpio against the bacterial pathogen, Aeromonas hydrophila. Beta-glucan was extracted from Saccharomyces cervisiae and purified. A virulent strain of the pathogen A. hydrophila was collected from infected fish. Different concentrations of beta-glucan were administered to test animals on day 1, 3 and 5 through different routes (intraperitoneal injection (ip), bathing and oral administration). Control and test animals were challenged by ip injection of LD50 concentration of A. hydrophila on day 7 and mortality was observed and Relative Percent Survival (RPS) was calculated. Intraperitoneal injection of 500 microg of glucan significantly enhanced the RPS; bathing and oral administration of glucan did not influence the RPS. On day 7, test animals injected with 100, 500 and 1000 microg of glucan had a significant increase in total blood leucocyte counts and an increase in the proportion of neutrophils and monocytes. Superoxide anion production by kidney macrophages was also elevated. RT-PCR and northern blot analysis of interleukin-1 mRNA showed elevated expression in kidney on day 7 in fish injected with glucan. Glucan had an adjuvant effect on antibody production as pretreatment by injection of 100-1000 microg glucan/fish resulted in the highest antibody titer against A. hydrophila following vaccination. Classical and alternative complement pathways were not affected by glucan administration by any of the three routes.
Mushroom glucan and bovine lactoferrin (Lf), known for their immunostimulatory potential, were used as adjuvant in conjunction with a formalin-killed Aeromonas hydrophila vaccine in catla, Catla catla. In vitro antigen-specific responsiveness of catla leucocytes and protective responses against experimental challenge with homologous antigen were monitored following immunization. Antigen-specific proliferation, 'macrophage activating factor' (MAF) production and antibody production were significantly higher in fish injected with glucan adjuvanted vaccine. Lf adjuvanted preparations showed a weak proliferative response and MAF production, although the antibody production was significantly higher than the controls. A good degree of protection was achieved with the glucan adjuvanted vaccine. However, in spite of producing significant anti-A. hydrophila antibody, Lf adjuvanted vaccine did not confer any protection following challenge with A. hydrophila. The potential of adjuvanticity of mushroom glucan and bovine Lf in intraperitoneal vaccination is discussed.
Combined effects of beta-glucan and lipopolysaccharide (LPS) on survival and immune response were studied in Cyprinus carpio that were challenged with the pathogen Aeromonas hydrophila. beta-Glucan from Saccharomyces cervisiae and LPS from a virulent strain of A. hydrophila were used in this study. Different concentrations of beta-glucan+LPS mixture were administered on days 1, 7, and 14 through different routes (intraperitoneal injection, bathing, and oral administration). Control and test fish were challenged by intraperitoneal injection of LD50 concentration of A. hydrophila on day 16 and subsequently, mortality and relative percent survival (RPS) were recorded. Intraperitoneal injection elicited 100% RPS even at the lowest concentration (100 microg beta-glucan+10 microg LPS); whereas, oral administration improved RPS rate of carps at higher concentration (1% beta-glucan+0.25% LPS). Bathing did not improve the RPS. Test animals injected with even the minimum dose of the immunomodulators (100 microg beta-glucan+10 microg LPS/fish) had a significant increase in total blood leukocyte counts and an increase in the proportion of neutrophils and monocytes. Superoxide anion production by macrophages was also elevated, which presumably aided the efficient killing of bacterial pathogen. Lower concentration of beta-glucan+LPS had an adjuvant effect on antibody production as pretreatment by injection of 100 microg beta-glucan+10 microg LPS/fish resulted in higher antibody titer against A. hydrophila following vaccination. RT-PCR analyses showed that the expression of interleukin-1beta mRNA did not increase in test fish when compared with the control. Classical and alternative complement pathways were not affected by either the dose or the route of administration of the compounds. It may be concluded that intraperitoneal injection and oral administration, and not the bathing, of beta-glucan+LPS mixture in carp could enhance resistance to challenge by A. hydrophila through changes in several non-specific and specific immune responses.
Glucans are (1→3)-β-D-glucose polymers that are found in the cell wall of fungi, bacteria and plants. Glucans are known to stim ulate humoral and cell-mediated immunity in humans and animals. In addition to the potent immune stimulatory effects of (1→3)-β-D-glucans, there are a number of toxicological effects associated with exposure to the water-insoluble, microparticulate form of the polymer. Recent investigations have suggested a potential role for (1→3)-β-D-glucans in inhalational toxicity. Specifically, (1→3)-β-D-glucans have been implicated in the symptomatology associated with ‘sick building’ syndrome. The mechanisms by which
(1→3)-β-D-glucans mediate immune stimulation and, perhaps, toxicity are currently under investigation. It is now established that (1→3)-β-D-glucans are recognized by macrophages and, perhaps, neutrophils and natural killer cells via a (1→3)-β-glucan specific receptor. Following receptor binding, glucan modulates macrophage cytokine expression. Here we review the chemistry, immunobiology and toxicity of (1→3)-β-D-glucans and how it may relate to effects caused by inhalation.
The use of immunostimulants has received increased attention due to the discovery of Toll-like receptors (TLR) or/and pattern recognition receptors (PRR). These receptors have been found to bind molecules from a range of pathogens including self-molecules. When cell damage has occurred many of the released molecular structures act as so-called "danger" signals possessing pathogen-associated molecular patterns (PAMP). These danger signals often consist of repeating molecular moieties yielding high molecular weight compounds. Examples are ß-glucans and CpG containing DNA, but some danger signals possess low molecular weight structures. It has been found that the PRR bind unit structures of PAMP, and that PAMP-binding involves several other humoral and cell membrane proteins, exemplified by the more or less simultaneous LPS recognition displayed by MD-2, CD-14 and TLR4 on the cell membrane. Also, the binding of ß-glucans has been shown to include several different cell membrane receptors. Several immunostimulants are commercially exploited in aquaculture as feed additives. This applies to ß-glucans, alginates and nucleotides. Despite their use as feed additives no targeted approach has been conducted to include PAMP as adjuvants in fish vaccines. Interestingly, most of the PAMP studied activate antigen-presenting cells together with naïve T cells into dendritic cells and Th1 or Th2 cells [1]. In turn, this may activate Th1 and Th2 immune responses with production of Th1 or Th2 signature molecules such as IFN-gamma and IL-4, respectively [2-4]. This review will mainly focus on binding characteristics of ß-glucans, their effects on T helper cell differentiation, effects on functional levels, gene expression profiles and application of the commonly used ß-glucan in the aquaculture sector. In addition, ß-glucans show promises in shrimp aquaculture by inducing disease resistance, this review will also highlight the use and the effects of ß-glucans in experimental models.
Teleost fish lack organized structures in mucosal tissues such as those of mammals, but instead contain dispersed B and T cells with the capacity to respond to external stimuli. Nonetheless, there is still a great lack of knowledge regarding how B cells differentiate to plasmablasts/plasma cells in these mucosal surfaces. To contribute to a further understanding of the mechanisms through which fish mucosal B cells are activated, in the current study, we have studied the B cell responses in the skin and gills of rainbow trout (Oncorhynchus mykiss) exposed to Yersinia ruckeri. We have first analyzed the transcription levels of genes related to B cell function in both mucosal surfaces, and in spleen and kidney for comparative purposes. In a second experiment, we have evaluated how the infection affects the presence and size of B cells in both skin and gills, as well as the presence of plasmablasts secreting total or specific IgMs. The results obtained in both experiments support the local differentiation of B cells to plasmablasts/plasma cells in the skin and gills of rainbow trout in response to Y. ruckeri. Interestingly, these plasmablasts/plasma cells were shown to secrete specific IgMs as soon as 5 days after the exposure. These findings contribute to a further understanding of how B cells in the periphery respond to immune stimulation in teleost fish.
In the present study, we studied the effect of β-glucan on the activation of antiviral immune responses against nervous necrosis virus (NNV) taking into consideration the role of innate immune training. Sevenband grouper primary macrophages showed an attenuated proinflammatory response and elevated antiviral response to NNV infection. In vitro, priming of β-glucan enhanced macrophage viability against NNV infection which is associated with the activation of sustained inflammatory cytokines gene expression. Observations were clear to understand that NLR Family CARD Domain Containing 3 (NLRC3) and caspase-1 activation and subsequent IL-1β production were reduced in β-glucan–primed macrophages. Subsequent markers for training including Lactate and abundance of HIF-1α were elevated in the cells following training. However, the lactate dehydrogenase (LDH) concentrations remained stable among the β-glucan stimulated infected and uninfected groups suggesting similar macrophage health in both groups. In vivo, the NNV-infected fish primed with β-glucan had a higher survival rate (60%) than the control NNV-infected group (40%). Our findings demonstrate that β-glucan induced protective responses against NNV infection and studies are underway to harness its potential applicability for prime and boost vaccination strategies.
Juvenile common carp Cyprinus carpio L. (5.52 ± 1.66 cm, TL) were fed on four diets containing either beta-glucan (MacroGard, 1 g kg −1), nucleotides (Optimûn, 0.2 g kg - 1), chitosan (deacetylated chitin ≥75% shrimp shells, 10 g kg −1) or a basal control diet for 35 days to test whether these so-called “immunostimulants” could affect eye fluke Diplostomum spp, infection success. The immunostimulants diets reduced the number of eye fluke infecting the eyes of C. carpio, with significantly higher infections in the control diet (4.78 ± 1.27) compared with the chitosan (2.08 ± 0.87), nucleotide (2.98 ± 1.01), and β-glucan (1.41 ± 0.79) diets. To our knowledge, this is the first study to provide evidence that β-glucan, nucleotides, and chitosan diets can act as an aid against a Diplostomum infection and provides valuable preliminary knowledge on the use of immunostimulants to ameliorate parasitic infections.
The main objective of this study was to determine the effect of two feeding additives (cMOS: Actigen®, Alltech Inc., USA) and a phytogenic specifically designed for reducing ectoparasite incidence, combining garlic and labiatae herbal extracts (HERB; Delacon, Austria) on growth performance, stress and immune response of greater amberjack (Seriola dumerili) before and after an experimental parasitization with the skin ectoparasite Neobenedenia girellae, a monogenean with high incidence on Seriola cultured in sea cages. Two parallel trials were conducted in order to determine the effect of 70 days of functional diets supplementation (0.2% cMOS and 200 ppm HERB) on greater amberjack: a) Growth performance and stress resistance and b) disease resistance against N. girellae. Additionally, in order to evaluate the effects of the dietary supplements pre- and post- parasitization challenge against N. girellae, the expression of some immune-related genes was evaluated. The relative expression of Il-1β, piscidin, hepcidin, IgT, tnf-α, muc-2, cathelicidin, complement c3, cd8, and caspase-3 was determined in skin. The activity of protease and lysozyme in serum and skin mucus were also evaluated, as well as parasite load and parasite total size.
The use of both functional additives did not affect fish growth performance. However, the dietary supplementation of HERB significantly (p<0.05) reduced circulating cortisol levels after the stress challenge, whereas dietary cMOS significantly (p<0.05) reduced the parasite load and total parasite size. When comparing pre and post parasitization challenge, skin mucus protease activity was significantly (p<0.05) higher in all the treatments before parasitization, while the use of both functional additives increased skin mucus lysozyme activity after challenge. From the eleven immune genes studied, only piscidin was significantly (p<0.05) up-regulated by HERB supplementation before challenge, whereas after parasitization, muc-2 and piscidin were significantly (p<0.05) up-regulated by HERB and tnf-α, il1-β, hep, c3, cd8 and casp3 were up-regulated by both functional additives. Moreover, hep, IgT, cath, c3 and cd8 expressions were negatively correlated with the parasite load.
In general, greater amberjack supplementation with cMOS and HERB at the dose used in the present study was effective for reducing stress and for increasing the skin associated lymphoid tissue (SALT) immune response against N. girellae during the infection process.
The innate immune response is able to ward off pathogens and remember previous infections using different mechanisms; this kind of immune reaction has been called "trained immunity". Changes in cellular metabolism (aerobic glycolysis) have been observed during training with some immunostimulants like β-glucans or during viral and bacterial infections. We hypothesize that β-glucans can induce metabolic changes used by the host to fight pathogens. Accordingly, we evaluated changes in metabolic parameters in turbot that could affect their survival after a previous intraperitoneal treatment with β-glucans and subsequent administration of Viral Hemorrhagic Septicemia Virus (VHSV) or bacteria (Aeromonas salmonicida subsp. salmonicida). The results obtained support that β-glucans, VHSV and A. salmonicida induce changes in lactate, glucose and ATP levels in plasma, head kidney and liver and in the mRNA expression of enzymes related to glucose and fatty acid metabolism in head kidney. Additionally, the metabolic changes induced by β-glucans are beneficial for VHSV replication, but they are harmful to A. salmonicida, resulting in reduced mortality. β-glucans appear to have great therapeutic potential and can induce trained immunity against bacterial disease but not against viral disease, which seems to take advantage of β-glucan metabolic alterations.
β-glucans, a group of polysaccharides exist in many organism species such as mushrooms, yeasts, oats, barley, seaweed, but not mammalians, have a variety of biological activities and applications in drugs and other healthcare products. In recent years, β-glucans have been studied as adjuvants in anti-infection vaccines as well as immunomodulators in anti-cancer immunotherapy. β-glucans can regulate immune responses when administered alone and can connect innate and adaptive immunity to improve immunogenicity of vaccines. When β-glucans act as immunostimulants or adjuvants, a set of receptors have been revealed to recognize β-glucans, including dectin-1, complement receptor 3 (CR3), CD5, lactosylceramide, and so on. Therefore, this review is mainly focused on the application of β-glucans as immune adjuvants, the receptors of β-glucans, as well as their structure and activity relationship which will benefit future research of β-glucans.
In teleost fish, IgM(+) B cells are one of the main responders against inflammatory stimuli in the peritoneal cavity, as IgM(+) B cells dominate the peritoneum after intraperitoneal stimulation, also increasing the levels of secreted IgM. BAFF, a cytokine known to play a major role in B cell biology, has been shown to be up-regulated along with its receptors in the peritoneum of rainbow trout upon antigenic exposure, however, the regulatory mechanisms underneath this response remain unclear. In this study, we have identified two different IgM(+) B cell types residing in the peritoneal cavity of previously vaccinated rainbow trout (Oncorhynchus mykiss): IgD(+)IgM(hi)MHCII(hi) cells, resembling naïve B cells, and IgD(-)IgM(lo)MHCII(lo) cells, resembling antibody-secreting cells. Based on their membrane IgM levels, these cell types were named IgM(hi) and IgM(lo) B cells, respectively. As each of these B cell populations showed a distinct expression pattern for the different BAFF receptors, we studied the effect of BAFF individually on each cell subset. Recombinant BAFF promoted the survival of IgM(lo) but not IgM(hi) B cells in vitro, resulting in increased levels of IgM-secreting cells. In contrast, BAFF increased the levels of membrane MHC II only on IgM(hi) B cells, suggesting different functions on these B cell subsets. Moreover, we also showed that peritoneal IgM(hi) B cells expressed BAFF at levels comparable to those seen on myeloid cells. These results point to BAFF as a main regulator of B cell homeostasis in the peritoneal cavity, suggesting that this cytokine can trigger different signals on different peritoneal B cell subsets in a specific manner.
Glucans are cell wall constituents of fungi and bacteria that bind to pattern recognition receptors and modulate innate immunity, in part, by macrophage activation. We used surface plasmon resonance to examine the binding of glucans, differing in fine structure and charge density, to scavenger receptors on membranes isolated from human monocyte U937 cells. Experiments were performed at 25degreesC using a biosensor surface with immobilized acetylated low density lipoprotein (AcLDL). Inhibition of the binding by polyinosinic acid, but not polycytidylic acid, confirmed the interaction of scavenger receptors. Competition studies showed that there are at least two AcLDL binding sites on human U937 cells. Glucan phosphate interacts with all sites, and the CM-glucans and laminarin interact with a subset of sites. Polymer charge has a dramatic effect on the affinity of glucans with macrophage scavenger receptors. However, it is also clear that human monocyte scavenger receptors recognize the basic glucan structure independent of charge.
B lymphocytes play an essential regulatory role in the adaptive immune response through Ab production during infection. A less known function of B lymphocytes is their ability to respond directly to infectious Ags through stimulation of pattern recognition receptors expressed on their surfaces. β-Glucans are carbohydrates present in the cell wall of many pathogenic fungi that can be detected in the peripheral blood of patients during infection. They have been shown to participate in the innate inflammatory response, as they can directly activate peripheral macrophages and dendritic cells. However, their effect as direct stimulators of B lymphocytes has not been yet fully elucidated. The aim of this study was to examine the molecular mechanisms and cytokine profiles generated following β-glucan stimulation of B lymphocytes, compared with the well-established TLR-9 agonist CpG oligodeoxynucleotide (CpG), and study the participation of β-glucan-stimulated B cells in the innate immune response. In this article, we demonstrate that β-glucan-activated B lymphocytes upregulate proinflammatory cytokines (TNF-α, IL-6, and IL-8). Of interest, β-glucan, unlike CpG, had no effect on B lymphocyte proliferation or IgM production. When compared with CpG (TLR9 agonist), β-glucan-activated cells secreted significantly higher levels of IL-8. Furthermore, IL-8 secretion was partially mediated by Dectin-1 and required SYK, MAPKs, and the transcription factors NF-κB and AP-1. Moreover, we observed that conditioned media from β-glucan-stimulated B lymphocytes elicited neutrophil chemotaxis. These studies suggest that β-glucan-activated B lymphocytes have an important and novel role in fungal innate immune responses.
Although fish constitute the most ancient animal group in which an acquired immune system is present, the presence of dendritic cells (DCs) in teleosts has been addressed only briefly, and the identification of a specific DC subset in teleosts remained elusive because of the lack of specific Abs. In mice, DCs expressing CD8α(+) in lymphoid tissues have the capacity to cross-present extracellular Ags to T cells through MHC I, similarly to tissue-derived CD103(+) DCs and the human CD141(+) DC population. In the current study, we identified a large and highly complex subpopulation of leukocytes coexpressing MHC class II and CD8α. This CD8α(+) MHC II(+) DC-like subpopulation constituted ∼1.2% of the total leukocyte population in the skin, showing phenotypical and functional characteristics of semimature DCs that seem to locally regulate mucosal immunity and tolerance in a species lacking lymph nodes. Furthermore, we identified trout homologs for CD141 and CD103 and demonstrated that, in trout, this skin CD8(+) DC-like subpopulation expresses both markers. To our knowledge, these results provide the first evidence of a specific DC-like subtype in nonimmune tissue in teleosts and support the hypothesis of a common origin for all mammalian cross-presenting DCs.
Copyright © 2015 by The American Association of Immunologists, Inc.
Administration of glucans through immersion, dietary inclusion or injection has been found to enhance many types of immune responses, resistance to bacterial and viral infections and to environmental stress in many fish species. Although the efficacy of the glucan varies with types and administration, glucan used as an immunomodulatory and mostly immunostimulatory additive has been found satisfactory in eliciting immunity in commercial aquaculture. Development of more efficient administration methods will facilitate the routine and prophylactic use of glucans as natural immunostimulants of fish. Using a PubMed search, this review has an extensive literature on glucan in fish immunity.
The effect of M-Glucan, a β-1,3- and β-1,6-linked glucan from Saccharomyces cerevisiae , as an adjuvant in vaccines against furunculosis in Atlantic salmon (Salmo salar L.) was studied. Vaccines consisted of formalin-killed cultures of Aeromonas salmonicida and in one case of a mixture of formalin-killed cultures of A. salmonicida and Vibrio salmonicida . M-Glucan was mixed with the vaccines to provide an administration dose of 0·5 mg/fish. Vaccines with or without glucan were injected into Atlantic salmon presmolts of 20-50 g, and the fish were challenged with A. salmonicida by a cohabitation method 3-46 weeks after vaccination. In all cases, vaccines supplied with M-glucan induced significantly higher protection against furunculosis than vaccines without M-Glucan (P<0·015). When M-Glucan was injected alone, no protection was observed at 11 weeks after injection. Serum antibodies against A-layer protein, lipopolysaccharide, whole cells and extracellular products from A. salmonicida were measured by an ELISA-procedure. The non-adjuvanted vaccines induced antibodies to all antigens in Atlantic salmon. M-Glucan significantly enhanced the level of antibody against A-layer protein (P=0·028), but not against lipopolysaccharide.
Toll like receptors (TLRs) are present in many different fish families from several different orders, including cyprinid, salmonid, perciform, pleuronectiform and gadiform representatives, with at least some conserved properties among these species. However, low conservation of the leucine-rich repeat ectodomain hinders predictions of ligand specificities of fish TLRs based on sequence information only. We review the presence of a TLR genes, and changes in their gene expression profiles as result of infection, in the context of different fish orders and fish families. The application of RT-qPCR and availability of increasing numbers of fish genomes has led to numerous gene expression studies, including studies on TLR gene expression, providing the most complete dataset to date. Induced changes of gene expression may provide (in)direct evidence for the involvement of a particular TLR in the reaction to a pathogen. Especially when findings are consistent across different studies on the same fish species or consistent across different fish species, up-regulation of TLR gene expression could be a first indication of functional relevance. We discuss TLR1, TLR2, TLR4, TLR5 and TLR9 as presumed sensors of bacterial ligands and discuss as presumed sensors of viral ligands TLR3 and TLR22, TLR7 and TLR8. More functional studies are needed before conclusions on ligands specific to (groups of) fish TLRs can be drawn, certainly true for studies on non-mammalian TLRs. Future studies on the conservation of function of accessory molecules, in conjunction with TLR molecules, may bring new insight into the function of fish TLRs.
β-glucans are glucose polymers that are found in the cell walls of plants, bacteria, certain fungi, mushrooms and the cell wall of baker's yeast. In mammals, myeloid cells express several receptors capable of recognizing β-glucans, with the C-type lectin receptor dectin-1 in conjunction with Toll-like receptor 2 (TLR2), considered key receptors for recognition of β-glucan. In our studies to determine the possible involvement of these receptors on carp macrophages a range of sources of β-glucans were utilised including particulate β-glucan preparations of baker's yeast such as zymosan, which is composed of insoluble β-glucan and mannan, and MacroGard®, a β-glucan-based feed ingredient for farmed animals including several fish species. Both preparations were confirmed TLR2 ligands by measuring activation of HEK293 cells transfected with human TLR2 and CD14, co-transfected with a secreted embryonic alkaline phosphatase (SEAP) reporter gene. In addition, dectin-1-specific ligands in mammals i.e. zymosan treated to deplete the TLR-stimulating properties and curdlan, were monitored for their effects on carp macrophages by measuring reactive oxygen and nitrogen radicals production, as well as cytokine gene expression by real time PCR. Results clearly show the ability of carp macrophages to strongly react to particulate β-glucans with an increase in the production of reactive oxygen and nitrogen radicals and increase in cytokine gene expression, in particular il-1β, il-6 and il-11. We identified carp il-6, that was previously unknown. In addition, carp macrophages are less, but not unresponsive to selective dectin-1 agonists, suggesting recognition of β-glucans by multiple pattern recognition receptors that could include TLR but also non-TLR receptors. Candidate receptors for recognition of β-glucans are discussed.
A monoclonal antibody to trout serum IgM was tested by immunofluorescence analysis with lymphocytes from thymus, spleen and head kidney. By visual examination, the antibody reacted with only a subpopulation of lymphocytes. The mean values ± SE for positive cells were 5.2 ± 2.3% in the thymus, 30.3 ± 7.9% in the spleen and 12.4 ± 3.0% in the head kidney. Flow cytofluorometric analysis revealed evidence of heterogeneity by size among the membrane IgM-positive cells of the head kidney and spleen. Depletion of head kidney cells positive for surface IgM by an immune affinity adherence technique of panning, using monoclonal anti-IgM, significantly reduced the mitogenic response to lipopolysaccharide but not to concanavalin A. It is suggested that this information supports the existence of distinct subpopulations of fish lymphocytes that may be homologous in certain respects to mammalian T and B type cells.
An experiment was performed to determine the effects of stress on non-specific defence mechanisms in rainbow trout fed diets containing different doses of glucan. Fish were fed with 0, 0.1, 0.5 and 1.0% glucan concentration in food. After 4 weeks of feeding, fish were stressed by 2 h transportation. The effect of stress was investigated by measuring changes in blood levels of cortisol, glucose, total protein and composition of their leukocyte population, as well as by changes in non-specific defence mechanisms of the fish. After 4 weeks feeding with glucan, elevated phagocytosis and oxidative radical production were observed in treated fish, but the levels did not correlate with the different doses of glucan. Stress induced by 2 h transportation caused high cortisol levels in plasma and hyperglycaemia in all groups, but the lowest level of glucose was measured in the group fed the low (0.1%) dose of glucan. Respiratory burst activity, phagocytosis, serum protein and lyzosyme levels were found to be significantly reduced by stress. The most dramatic reduction was observed in the control group, but the changes were not affected by glucan doses. One week post stress, hyperglycaemia was still observed in control and fish fed medium and high doses of glucan. Further reductions of total protein and intracellular oxidative radical production, were measured in all groups, but in fish fed with low dose of glucan the changes were less dramatic. The phagocytosis ratio increased in all groups, but did not attain the levels measured before stress in control and in the group fed the high concentration of glucan. A spontaneous infection with Flexibacter columnaris caused mortality in all groups except the group fed the low level of glucan. The results of the present study show that feeding of glucan in low doses several weeks before transportation can help to prevent negative effects of stress.
Six hundred and forty Nile tilapia (Oreochromis niloticus) weighing 80-100g were randomly allocated into eight equal groups (80 each). The first group acts as control. Groups S, B and L were fed on a ration supplemented with Saccharomyces cerevisiae, beta-glucans and laminaran, respectively for 21 days. Groups M, MS, MB and ML were subjected throughout the experiment to sublethal concentration of mercuric chloride (0.05 ppm). Gps. MS, MB and ML were fed on a ration containing S. cerevisiae, beta-glucan and laminaran respectively for 21 days. Fish were challenged with Aeromonas hydrophila (0.4 x 10(7) cells mL(-1)) via intra-peritoneal injection and the mortality rate was recorded up to 10 day post-challenge. The non-specific defense mechanisms, cellular and humoral immunity, beside the total and differential leukocytic count were determined. Lymphocyte transformation index, phagocytic activity percent, phagocytic index, total lymphocyte count, serum bactericidal activity and nitric oxide as well as the survival rate were insignificantly changed after 21 day in gps. MS&ML, when compared with mercuric chloride immune depressed group M. These parameters as well as the neutrophil adhesion, serum nitric oxide and survival rate were significantly increased in gp. MB when compared with gp. M. Meanwhile the cellular and humoral immunity beside the survival rate were significantly increased in groups S, B, L when compared with control group. It could be concluded that the whole yeast S. cerevisiae, beta-glucan and laminaran can be used as immunostimulants for the farmed Nile tilapia. The beta-glucans could be used in farmed Nile tilapia, under immune depressive stressful condition to increase their resistance to diseases.
The beta 2 integrin CR3 is a leukocyte adhesion heterodimeric glycoprotein which functions both as receptor for iC3b and in several cell-cell and cell-substrate adhesion interactions. In order to elucidate the molecular evolution of the CR3 receptor, here we report the cloning and characterization of its beta2 (CD18) and aM (CD11b) subunits in rainbow trout (Oncorhynchus mykiss). The predicted polypeptide sequences of trout CD18 and CD11b-like exhibit 50, 49, or 61% and 25, 25, or 30% identity with human, mouse, and zebrafish orthologs, respectively. The 'domain' architecture of trout CD18 and CD11b-like subunits retains several characteristics of the mammalian ortholog proteins, such as cysteine-rich regions, N-linked glycosylation sites and several proposed domains and signal sequences (von Willebrand factor type A, Integrin alpha, Integrin B tail, EGF, and Transmembrane domain). The tissue expression profiles of trout CR3 subunits diverge from those of mammalian counterparts, showing the kidney as the main source of the trout CD18 and CD11b-like mRNA transcripts. This is the first report of cloning and characterization of the CR3 receptor in low vertebrates.
Here, Charles Janeway argues that the requirement for two signals to initiate the adaptive immune response may reflect the evolutionary history of host defences. Early phases of host defence involve receptors and ligands that may have controlled immune responses prior to the development of clonally-distributed receptors encoded in rearranging genes. The former receptors persist in contemporary vertebrates both to trigger innate or nonclonal responses and to signal to lymphocytes that a particular antigen is associated with a microorganism.
We have prepared casein conjugates of two BODIPY dyes for use as fluorogenic protease substrates in homogeneous assays. Both conjugates are labeled to such an extent that the dyes are efficiently quenched in the protein, yielding virtually nonfluorescent substrate molecules. These fluorogenic substrates release highly fluorescent BODIPY dye-labeled peptides upon protease digestion, with fluorescence increases proportional to enzyme activity. These quenched substrates are suitable for the continuous assay of enzymatic activity using standard fluorometers, filter fluorometers, or fluorescence microplate readers using either fluorescein excitation and emission wavelengths to measure BODIPY FL casein hydrolysis or Texas Red wavelengths to detect proteolysis of BODIPY TR-X casein. Most current techniques for detecting protease activity, such as the fluorescein thiocarbamoyl casein (FTC-casein) protease assay, require extensive manipulation, including separation steps, and are therefore labor intensive and error-prone. In comparison, we found the BODIPY dye-labeled casein protease assays to be simple and precise and to have greater sensitivity and a broader dynamic range of detection than the FTC-casein assay. We were able to sensitively detect the activities of a wide variety of enzymes with these new substrates, including serine, acid, sulfhydryl, and metalloproteases. We also found the assay suitable for quantitating protease inhibitor concentrations and for real-time analysis of proteolysis.
The carbohydrate polymers known as beta-1,3-d-glucans exert potent effects on the immune system - stimulating antitumour and antimicrobial activity, for example - by binding to receptors on macrophages and other white blood cells and activating them. Although beta-glucans are known to bind to receptors, such as complement receptor 3 (ref. 1), there is evidence that another beta-glucan receptor is present on macrophages. Here we identify this unknown receptor as dectin-1 (ref. 2), a finding that provides new insights into the innate immune recognition of beta-glucans.
Plasma cells are the terminally differentiated, non-dividing effector cells of the B-cell lineage. They are cellular factories devoted to the task of synthesizing and secreting thousands of molecules of clonospecific antibody each second. To respond to microbial pathogens with the necessary specificity and rapidity, B cells are exquisitely regulated with respect to both development in the bone marrow and activation in the periphery. This review focuses on the terminal differentiation of B cells into plasma cells, including the different subsets of B cells that become plasma cells, the mechanism of regulation of this transition, the transcription factors that control each developmental stage and the characteristics of long-lived plasma cells.
PGG-glucan, and lactosylceramide of human leukocytes
- Immunomodulator -Glucan
-glucan immunomodulator, PGG-glucan, and lactosylceramide of human
leukocytes, J. Biol. Chem. 273 (34) (1998) 22014-22020.
B cells as antigen presenting cells
- Rodriguez-Pinto
D. Rodriguez-Pinto, B cells as antigen presenting cells, Cell. Immunol. 238 (2)
(2005) 67-75.
Plasma cells negatively regulate the follicular helper T cell program
- Pelletier