Teleost Immune System Research Articles

A Novel Antigen-Sampling Cell in the Teleost Gill Epithelium With the Potential for Direct Antigen Presentation in Mucosal Tissue.

In mammals, M cells can take up antigens through mucosal surfaces of the gut and the respiratory tract. Since M cells are deficient of lysosomes and phagosomes, the antigens are directly delivered to the mucosa-associated lymphoid tissue (MALT) without degradation. In teleost fish, the entire body surface (gills, skin, and intestinal system) is covered by mucus; however, specific antigen-sampling cells have not yet been identified in their mucosal tissues. Here, we show that two phenotypes of antigen-sampling cells take up antigens through epithelial surfaces of the rainbow trout gill. One phenotype of antigen-sampling cells has features of monocyte/macrophage/dendritic cell-type cells; they have large vacuoles in the cytoplasm and express PTPRC (CD45), CD83, IL-1β, and IL-12p40b. The second phenotype exhibits similar characteristics to mammalian M cells; the corresponding cells bind the lectin UEA-1 but not WGA and show expression of M cell marker gene Anxa5. In contrast to mammalian M cells, teleost M-type cells were found to exhibit small vacuoles in their cytoplasm and to express almost all genes related to the “phagosome”, “lysosome,” and “antigen processing and presentation” pathways. Furthermore, MHC class II was constitutively expressed on a fraction of M-type cells, and this expression was significantly increased after antigen uptake, suggesting that the MHC class II is inducible by antigen stimulation. Here, we suggest that teleost M-type cells play a role in the phylogenetically primitive teleost immune system, similar to bona-fide M cells. In addition, the presence of MHC class II expression suggests an additional role in antigen presentation in the gills, which are an organ with high T cell abundance, especially in interbranchial lymphoid tissue. The present results suggest an unconventional antigen presentation mechanism in the primitive mucosal immune system of teleosts, which generally lack highly organized lymphoid tissues. Moreover, the results of this work may be valuable for the development of mucosal vaccines that specifically target M-type cells; mucosal vaccines significantly reduce working costs and the stress that is usually induced by vaccination via injection of individual fish.

Analysis of the Long-Lived Responses Induced by Immunostimulants and Their Effects on a Viral Infection in Zebrafish (Danio rerio).

In recent years, the innate immune response has gained importance since evidence indicates that after an adequate priming protocol, it is possible to obtain some prolonged and enhanced immune responses. Nevertheless, several factors, such as the timing and method of administration of the immunostimulants, must be carefully considered. An inappropriate protocol can transform the treatments into a double-edged sword for the teleost immune system, resulting in a stressful and immunosuppressive state. In this work, we analyzed the long-term effects of different stimuli (β-glucans, lipopolysaccharide, and polyinosinic:polycytidylic acid) on the transcriptome modulation induced by Spring Viremia Carp Virus (SVCV) in adult zebrafish (Danio rerio) and on the mortality caused by this infection. At 35 days post-immunostimulation, the transcriptome was found to be highly altered compared to that of the control fish, and these stimuli also conditioned the response to SVCV challenge, especially in the case of β-glucans. No protection against SVCV was found with any of the stimuli, and non-significant higher mortalities were even observed, especially with β-glucans. However, in the short term (pre-stimulation with β-glucan and infection after 7 days), slight protection was observed after infection. The transcriptome response in the zebrafish kidney at 35 days posttreatment with β-glucans revealed a significant response associated with stress and immunosuppression. The identification of genes that were differentially expressed before and after the infection seemed to indicate a high energy cost of the immunostimulation that was prolonged over time and could explain the lack of protection against SVCV. Differential responses to stress and alterations in lipid metabolism, the tryptophan–kynurenine pathway, and interferon-gamma signaling seem to be some of the mechanisms involved in this response, which represents the end of trained immunity and the beginning of a stressful state characterized by immunosuppression.

Original Ligand for LTβR Is LIGHT: Insight into Evolution of the LT/LTβR System.

The lymphotoxin (LT)/LTβ receptor (LTβR) axis is crucial for the regulation of immune responses and development of lymphoid tissues in mammals. Despite the importance of this pathway, the existence and function of LT and LTβR remain obscure for nonmammalian species. In this study, we report a nonmammalian LTβR and its ligand. We demonstrate that TNF-New (TNFN), which has been considered orthologous to mammalian LT, was expressed on the cell surface as a homomer in vitro. This different protein structure indicates that TNFN is not orthologous to mammalian LTα and LTβ. Additionally, we found that LTβR was conserved in teleosts, but the soluble form of recombinant fugu LTβR did not bind to membrane TNFN under the circumstance tested. Conversely, the LTβR recombinant bound to another ligand, LIGHT, similar to that of mammals. These findings indicate that teleost LTβR is originally a LIGHT receptor. In the cytoplasmic region of fugu LTβR, recombinant fugu LTβR bound to the adaptor protein TNFR-associated factor (TRAF) 2, but little to TRAF3. This difference suggests that teleost LTβR could potentially activate the classical NF-κB pathway with a novel binding domain, but would have little ability to activate an alternative one. Collectively, our results suggested that LIGHT was the original ligand for LTβR, and that the teleost immune system lacked the LT/LTβR pathway. Acquisition of the LT ligand and TRAF binding domain after lobe-finned fish may have facilitated the sophistication of the immune system and lymphoid tissues.

Interactions between microbiota and the teleost immune system in health and disease

Abstract The symbiosis between metazoans and prokaryotes is one of the most ancient and successful relationships found in nature. Mucosal surfaces are the main interface between animal hosts and their environment, and these surfaces are colonized by unique microbial communities that shape every physiological system of the host. Teleost fish have both an innate and adaptive immune system and are the most ancient vertebrates with a dedicated mucosal immunoglobulin (Ig), known as IgT/IgZ. IgT is secreted via the polymeric Ig receptor across teleost mucosal epithelia, including the gills, skin, nose and gut. Recent studies have shed light on the interactions between mucosal Igs and microbiota in teleosts both during health and disease. Teleost fish symbiotic bacteria are coated by both secretory Igs (sIgT and sIgM) as well as pIgR. Using two different models, a stress model and an IgT depletion model, we provide evidence that any alterations in the levels of mucosal Igs result in different states of dysbiosis in rainbow trout (Oncorhynchus mykiss). Dysbiosis in trout is characterized by losses of certain taxa and subsequent invasion of new species in several mucosal sites. Unlike in mammalian models of IgA deficiency, where IgM compensatory responses occur, no compensatory IgM or IgD responses were observed in IgT-depleted rainbow trout. These results demonstrate that teleost fish, similar to mammals, utilize specialized mucosal Igs to maintain harmonic relationships with symbiotic bacteria.

Impacts of Low Temperature on the Teleost Immune System.

As poikilothermic vertebrates, fish can experience changes in water temperature, and hence body temperature, as a result of seasonal changes, migration, or efflux of large quantities of effluent into a body of water. Temperature shifts outside of the optimal temperature range for an individual fish species can have negative impacts on the physiology of the animal, including the immune system. As a result, acute or chronic exposure to suboptimal temperatures can impair an organisms’ ability to defend against pathogens and thus compromise the overall health of the animal. This review focuses on the advances made towards understanding the impacts of suboptimal temperature on the soluble and cellular mediators of the innate and adaptive immune systems of fishes. Although cold stress can result in varying effects in different fish species, acute and chronic suboptimal temperature exposure generally yield suppressive effects, particularly on adaptive immunity. Knowledge of the effects of environmental temperature on fish species is critical for both the optimal management of wild species and the best management practices for aquaculture species.

CXCR3.1 and CXCR3.2 Differentially Contribute to Macrophage Polarization in Teleost Fish.

The study of multiple copies of chemokine receptor genes in various teleosts has long appealed to investigators seeking to understand the evolution of the immune system. The CXCR CXCR3 gene has two isoforms, CXCR3.1 and CXCR3.2, which are both expressed in macrophages. The distinct roles of teleost CXCR3s have not been identified previously. In this article, we found that CXCR3.1 and CXCR3.2 differentially contributed to macrophage polarization in the teleosts: ayu (Plecoglossus altivelis), grass carp (Ctenopharyngodon idella), and spotted green pufferfish (Tetraodon nigroviridis). In ayu macrophages, the P. altivelis CXCR3.1 (PaCXCR3.1) gene was constitutively expressed, whereas the P. altivelis CXCR3.2 (PaCXCR3.2) gene was induced postinfection with Escherichia coli Upon E. coli infection, PaCXCR3.1+ and PaCXCR3.2+ macrophages showed an M1 and an M2 phenotype, respectively. CXCL9-11-like proteins mediated M1 and M2 polarization by interacting with the PaCXCR3.1 and PaCXCR3.2 proteins on macrophages, respectively. The transcription factors P. altivelis STAT1 and P. altivelis STAT3 were activated in PaCXCR3.1+ and PaCXCR3.2+ macrophages, respectively. Furthermore, the prognosis of septic ayu adoptively transferred with PaCXCR3.2+ macrophages was improved. Our data reveal a previously unknown mechanism for macrophage polarization, suggesting that redundant genes may regulate crucial functions in the teleost immune system.

Under Pressure: Interactions between Commensal Microbiota and the Teleost Immune System.

Commensal microorganisms inhabit every mucosal surface of teleost fish. At these surfaces, microorganisms directly and indirectly shape the teleost immune system. This review provides a comprehensive overview of how the microbiota and microbiota-derived products influence both the mucosal and systemic immune system of fish. The cross talk between the microbiota and the teleost immune system shifts significantly under stress or disease scenarios rendering commensals into opportunists or pathogens. Lessons learnt from germ-free fish models as well as from oral administration of live probiotics to fish highlight the vast impact that microbiota have on immune development, antibody production, mucosal homeostasis, and resistance to stress. Future studies should dissect the specific mechanisms by which different members of the fish microbiota and the metabolites they produce interact with pathogens, with other commensals, and with the teleost immune system.

Linking species habitat and past palaeoclimatic events to evolution of the teleost innate immune system.

Host-intrinsic factors as well as environmental changes are known to be strong evolutionary drivers defining the genetic foundation of immunity. Using a novel set of teleost genomes and a time-calibrated phylogeny, we here investigate the family of Toll-like receptor (TLR) genes and address the underlying evolutionary processes shaping the diversity of the first-line defence. Our findings reveal remarkable flexibility within the evolutionary design of teleost innate immunity characterized by prominent TLR gene losses and expansions. In the order of Gadiformes, expansions correlate with the loss of major histocompatibility complex class II (MHCII) and diversifying selection analyses support that this has fostered new immunological innovations in TLRs within this lineage. In teleosts overall, TLRs expansions correlate with species latitudinal distributions and maximum depth. By contrast, lineage-specific gene losses overlap with well-described changes in palaeoclimate (global ocean anoxia) and past Atlantic Ocean geography. In conclusion, we suggest that the evolvability of the teleost immune system has most likely played a prominent role in the survival and successful radiation of this lineage.

A Review of Immune System Components, Cytokines, and Immunostimulants in Cultured Finfish Species

Aquaculture is a rapidly growing global agriculture sector and the importance of fish health has become of upmost importance as production levels and stocking densities increase. Over the past few decades, there have been a large number of immunological investigations on commonly cultured finfish species. Further, new technologies and strategies that embody use of fish immunostimulants, probiotics, and vaccinology rely heavily upon a comprehensive understanding of teleost immune system mechanics. The teleost immune system works in concert to properly recognize, control, and clear aquatic pathogens. Recent findings have exemplified the cooperative efforts of the nonspecific and adaptive branches, and have put forth an emphasis on the importance of the mucosal immune response in all aspects of a mounted immune response. This review provides a generalized overview of the innate and adaptive arms of the fish immune system, and provides highlights of recently published work in the areas of signaling networks and mucosal immune interactions.

Di(2-ethylhexyl) phthalate inhibits B cell proliferation and reduces the abundance of IgM-secreting cells in cultured immune tissues of the rainbow trout

Plasticizer di(2-ethylhexyl) phthalate (DEHP) and its active metabolite MEHP have important immunotoxic effects in mammalian species, including inhibition of cell proliferation, inflammation inhibition, lowering of the antibody response, and apoptosis. Virtually nothing is known about the potential detrimental effects of DEHP/MEHP on the teleost immune system, although phthalates are a likely threat to fish health. Here we investigated whether short-term in vitro DEHP exposure would affect B lineage cells in the rainbow trout, using cultured immune tissues. Cell culture conditions, evidence of cellular incorporation of DEHP, and possible effects of DEHP on immune genes were first established using the mouse pre-B cell line PD31 and data confirmed a dose-dependent cellular uptake of DEHP using liquid chromatography-coupled ion trap mass spectrometry. Effects of in vitro DEHP exposure on trout B cell proliferation were tested by flow cytometry. Significant, dose-dependent inhibition was evident in both anterior and posterior kidney cultures after 24 h exposure to ≥4 μM DEHP. DEHP-induced cell death was not significant for the range of DEHP tested. Further, the abundance of IgM-secreting plasmablasts and plasma cells was significantly reduced after in vitro exposure of ≥16 μM DEHP for 2 or 7 days. Finally, in vitro DEHP exposure significantly lowered the levels of secreted HCmu transcripts in a dose-dependent manner. B lineage cells from posterior kidney were more sensitive to effects of in vitro DEHP exposure than those from anterior kidney. Together, the data support a model where DEHP modifies the normal B cell activation pathways in rainbow trout, promoting B cell differentiation while suppressing plasmablast expansion, resulting in fewer IgM-secreting plasma cells. Insufficient production of protective antibody make fish more susceptible to infection, and increases their risk for disease and mortality in polluted waters.

Spatio-temporal expression of blunt snout bream (Megalobrama amblycephala) mIgD and its immune response to Aeromonas hydrophila.

The function of IgD in fish and mammals has not been fully understood since its discovery. In this study, we have isolated and characterized the cDNA that encodes membrane-bound form of the immunoglobulin D heavy chain gene (mIgD) of blunt snout bream (Megalobrama amblycephala) using RT-PCR and rapid amplification of cDNA ends (RACE). The full-length cDNA of mIgD consisted of 3313 bp, encoding a putative protein of 943 amino acids. The structure of blunt snout bream mIgD is VDJ-μ1-δ1-δ2-δ3-δ4-δ5-δ6-δ7-TM. Multiple alignment and phylogenetic analyses indicated that blunt snout bream mIgD clusters with the homologues of cyprinid fish and that its highest identity is with that of C. idella (82%). The mIgD expression in early different developmental stages showed that the level of mIgD mRNA decreased dramatically from the unfertilized egg stage to the 32-cell stage, suggesting that mIgD mRNA was maternally transferred. As cell differentiation initially took place in the blastula stage, the mIgD expression increased significantly from the blastula stage to prelarva, which might be attributed to embryonic stem cell differentiation processes. Compared with juvenile fish, the expression and tissue distribution patterns of mIgD in adult individuals exhibited considerable variation. After the injection of Aeromonas hydrophila, mIgD expression was up-regulated in various tissues, reaching the peak expression at 5 d, 14 d or 21 d (depending on the tissue type). The present study provides a theoretical basis for further research of the teleost immune system.

Molecular cloning of NCCRP-1 gene from orange-spotted grouper (Epinephelus coioides) and characterization of NCCRP-1+ cells post Cryptocaryon irritans infection

Nonspecific cytotoxic cells (NCCs) are an important cytotoxic cell population in the innate teleost immune system. The receptor designated “NCC receptor protein 1” (NCCRP-1) has been reported to be involved in the recognition and activation of NCCs. In this study, the full-length cDNA of Epinephelus coioides NCCRP-1 (ecnccrp-1) was cloned. The open reading frame (ORF) of ecnccrp-1 is 699bp, encoding a 232 amino acid protein that includes proline-rich motifs at the N-terminus and is related to the F-box associated family. Although a bioinformatics analysis showed that EcNCCRP-1 had no signal peptide or transmembrane helices, a polyclonal antibody directed against recombinant EcNCCRP-1 efficiently labeled a membrane protein in the head kidney, detected with Western blot analysis, which indicated that the protein localized to the cell surface. RT-PCR showed that the constitutive expression of ecnccrp-1 was higher in the lymphoid organs, such as the trunk kidney, spleen, head kidney, and thymus, and lower in brain, heart, fat, liver, muscle, and skin. After infection with Cryptocaryon irritans, the transcription of ecnccrp-1 was analyzed at the infected sites (skin and gills) and in the systemic immune organs (head kidney and spleen). At the infected sites, especially the skin, ecnccrp-1 expression was upregulated at 6h post infection, reaching peak expression on day 3 post the primary infection. However, the expression patterns differed in the systemic immune organs. In the spleen, ecnccrp-1 was gradually increased in the early infection period and decreased sharply on day 3 post the primary infection, whereas in the head kidney, the transcription of ecnccrp-1 was depressed during almost the whole course of infection. An immunohistochemical analysis showed that EcNCCRP-1+ cells accumulated at the sites of infection with C. irritans. These results suggested that NCCs were involved in the process of C. irritans infection in E. coioides.

Establishment of a leukocyte cell line derived from peritoneal macrophages of fish, Labeo rohita (Hamilton, 1822)

A continuous leukocyte cell line with phagocytic activity was established from peritoneal macrophages of rohu, Labeo rohita (LRPM). LRPM was initiated from adherent mononuclear leukocytes isolated from peritoneal cavity of rohu, without use of any growth factors or feeder cells. These cells exhibited maximum growth at 30°C in L-15 medium containing 20% foetal bovine serum, and has been subcultured for more than 60 passages till date. The cells showed 85% viability after 6months of storage in liquid nitrogen. The species of origin of the LRPM was confirmed by the amplification and sequencing of 655bp fragment of cytochrome oxidase subunit I of mitochondrial DNA. Functionally, LRPM showed phagocytic activity of yeast cells and fluorescent latex beads as evaluated by phase contrast and scanning electron microscopy, respectively. Immuno-modulators such as bacterial lipopolysaccharide and phorbol myristate acetate resulted in functional activation of LRPM; and enhanced their microbicidal activity through release of reactive oxygen species and nitric oxide. Culture supernatant from activated cells also revealed lysozyme activity. Cells of LRPM were positive for alpha-naphthyl acetate esterase enzyme indicating macrophage lineage. Our results indicate that this cell line can be a useful in vitro tool to study the role of macrophages in teleost immune system and to evaluate the effects of new aquaculture drugs. The LRPM cell line represents the first reported leukocyte cell line of peritoneal origin from any freshwater species of fish.

Fishing for mammalian paradigms in the teleost immune system.

Recent years have witnessed a renaissance in the study of fish immune systems. Such studies have greatly expanded the knowledge of the evolution and diversification of vertebrate immune systems. Several findings in those studies have overturned old paradigms about the immune system and led to the discovery of novel aspects of mammalian immunity. Here I focus on how findings pertaining to immunity in teleost (bony) fish have led to major new insights about mammalian B cell function in innate and adaptive immunity. Additionally, I illustrate how the discovery of the most ancient mucosal immunoglobulin described thus far will help resolve unsettled paradigms of mammalian mucosal immunity.

Differential compartmentalization of memory B cells versus plasma cells in salmonid fish

The disposition of teleost memory and plasma cells (PCs) has essentially been un-explored. As the organization of the teleost immune system differs significantly from that of mammals (i.e. no bone marrow or lymph nodes, hematopoietic anterior kidney), this disposition could be essential in understanding how comparable functions are achieved. To address this question, the primary and secondary antibody-secreting cell, B memory cell, and antibody responses to T-independent and T-dependent antigens were analyzed in trout. Although the TI and TD antibody responses did not differ substantively from one another, the secondary responses to both were significantly prolonged compared with the primary responses. Logarithmic increases in titer and affinity were achieved for both antigens during the primary, with only modest increases during the secondary response. Antibody-secreting cells, both PCs and plasmablasts, quantitatively paralleled antibody production, with antibody-secreting cells skewing to the hematopoietic anterior kidney for both antigens. However, the enhanced antigen-inducible response of immune fish (indicative of the memory pool) skewed to the peripheral blood and spleen. This pattern of memory versus PC disposition parallels that observed in mammals even though the organization of the respective immune systems differs considerably.

Characterization of Foxp3 gene from grass carp (Ctenopharyngodon idellus): A rapamycin-inducible transcription factor in teleost immune system

In this study, we cloned grass carp foxp3 (gcfoxp3) gene including 5′ flanking region and determined its expression profiles in vivo under immunosuppressive conditions. Sequence analysis revealed that the promoter of gcfoxp3 contains AP-1, AML-1/Runx1, NF-κb and GATA-3 binding sites, which positively or negatively regulate mammalian foxp3 expression. In addition, the intron II of gcfoxp3 contains some putative binding sites including AP-1, NFAT, Smad3, RAR, CREB/ATF and FOXO1, which are corresponding to their locations in the proximal intronic enhancers of human foxp3. In an in vivo model of grass carp, an immunosuppressive agent rapamycin was showed to stimulate gcfoxp3 mRNA expression in thymus, gill, head kidney and spleen after bacterial challenge. Moreover, rapamycin increased gcFoxp3 protein levels with an additive manner in the infected fish. These findings support the involvement of fish Foxp3 in immune response and highlight a possible signaling pathway that regulates teleost Foxp3 expression.

Characterization of an anti-rainbow trout (Oncorhynchus mykiss) CD3ɛ monoclonal antibody

This study characterizes a monoclonal antibody (mAb) produced against the cytoplasmic tail region of the epsilon chain of the CD3 (CD3ɛ) transmembrane protein found on T lymphocytes of rainbow trout (Oncorhynchus mykiss). Flow cytometry and fluorescent microscopy conducted on trout leukocytes with the anti-trout CD3ɛ mAb showed a distinctive population of IgM− CD3e+ lymphocytes fitting the expected profile of T-cells. Immunoprecipitation of lysates derived from trout lymphocytes revealed a 19kDa protein and peptide analysis confirmed its specificity for CD3ɛ. In vitro proliferation assays with T-cell mitogens, ConA and PHA, resulted in a 3 fold increase in the percentage of CD3ɛ+ lymphocytes compared to LPS and control cultures. The mAb characterized in this study will be useful in further elucidation for both the role and distribution of T lymphocytes in the teleost immune system.

Plasmablasts and plasma cells: Reconsidering teleost immune system organization

Comparative immunologists have expended extensive efforts in the characterization of early fish B cell development; however, analysis of the post-antigen induction stages of antibody secreting cell (ASC) differentiation has been limited. In contrast, work with murine ASCs has resolved the physically and functionally distinct cells known as plasmablasts, the short-lived plasma cells and long-lived plasma cells. Teleost ASCs are now known to also possess comparable subpopulations, which can greatly differ in such basic functions as lifespan, antigen sensitivity, antibody secretion rate, differentiative potential, and distribution within the body. Understanding the mechanisms by which these subpopulations are produced and distributed is essential for both basic understanding in comparative immunology and practical vaccine engineering.

GATA3 mRNA in ginbuna crucian carp ( Carassius auratus langsdorfii): cDNA cloning, splice variants and expression analysis

GATA3, a transcriptional activator, plays a critical role in the development of T-cells and differentiation to T helper type 2 cells. To date, no information is available on the role of GATA3 in the teleost immune system. We identified full-length cDNA and alternatively spliced variants of ginbuna crucian carp GATA3 (gbGATA3). The gbGATA3 gene is transcribed into multiple splice variants lacking either one or both zinc finger domains, although the sequences of both domains are fully conserved between ginbuna and other vertebrates. We found that alternative splice site and stop codon in gbGATA3 intron 3, located between exons that separately encode the two zinc finger domains, are conserved among teleosts, suggesting that teleost GATA3 gene can be translated into multiple isoforms. RT-PCR analysis revealed that the gbGATA3 is strongly expressed in the brain, thymus and gill of unstimulated fish. Moreover, gbGATA3 expression was detected in surface-IgM-negative lymphocytes among kidney cells sorted by FACS. Real-time PCR demonstrated that expression levels of full-length gbGATA3 and the splice variants differed with tissue type, but full length was always the predominantly expressed form. These results suggest that gbGATA3, including its splice variants, is involved in teleost T-cell function.

Inflammation in Fish As Seen from A Morphological Point of View with Special Reference to the Vascular Compartment

Modern bony fishes or teleosts are increasingly being used as model organisms for human diseases. Ambitious mapping programmes have revealed parts of or entire genomes of several species. This information suggests that there are several similarities between the mammalian and teleost immune systems, but also important differences. These differences are especially evident in the anatomical and functional constructions. However, compared to mammals, morphological studies of the immune system and in particular the inflammatory responses in fish are scarce, much due to a general lack of good cell markers. This review seeks to give an overview of the current knowledge of the teleost immune system related to inflammation and morphological research. The emphasis is placed on coronary changes which may be observed in salmonids over the size of 10 cm. Here, the immunopathological picture has some resemblance to that observed in similar changes in humans.