RESUMO
Proliferative kidney disease (PKD), caused by the myxozoan Tetracapsuloides bryosalmonae, is one of the most serious parasitic diseases of salmonids in which outbreaks cause severe economic constraints for the aquaculture industry and declines of wild species throughout Europe and North America. Given that rainbow trout (Oncorhynchus mykiss) is one of the most widely farmed freshwater fish and an important model species for fish immunology, most of the knowledge on how the fish immune response is affected during PKD is from this organism. Once rainbow trout are infected, PKD pathogenesis results in a chronic kidney immunopathology mediated by decreasing myeloid cells and increasing lymphocytes. Transcriptional studies have revealed the regulation of essential genes related to T-helper (Th)-like functions and a dysregulated B-cell antibody type response. Recent reports have discovered unique details of teleost B-cell differentiation and functionality and characterized the differential immunoglobulin (Ig)-mediated response. These studies have solidified the rainbow trout T. bryosalmonae system as a sophisticated disease model capable of feeding key advances into mainstream immunology and have contributed essential information to design novel parasite disease prevention strategies. In our following perspective, we summarize these efforts to evaluate the immune mechanisms of rainbow trout during PKD pathogenesis.
Assuntos
Nefropatias/imunologia , Nefropatias/parasitologia , Myxozoa/imunologia , Oncorhynchus mykiss/imunologia , Doenças Parasitárias em Animais/imunologia , Animais , Linfócitos B/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes , Imunoglobulina D/imunologia , Imunoglobulina M/imunologia , Imunoglobulinas/imunologia , Ativação Linfocitária/imunologia , Myxozoa/genética , Myxozoa/fisiologia , Oncorhynchus mykiss/parasitologia , Doenças Parasitárias em Animais/parasitologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Simultaneous and sequential infections often occur in wild and farming environments. Despite growing awareness, co-infection studies are still very limited, mainly to a few well-established human models. European salmonids are susceptible to both Proliferative Kidney Disease (PKD), an endemic emergent disease caused by the myxozoan parasite Tetracapsuloides bryosalmonae, and Viral Haemorrhagic Septicaemia (VHS), an OIE notifiable listed disease caused by the Piscine Novirhabdovirus. No information is available as to how their immune system reacts when interacting with heterogeneous infections. A chronic (PKD) + acute (VHS) sequential co-infection model was established to assess if the responses elicited in co-infected fish are modulated, when compared to fish with single infections. Macro- and microscopic lesions were assessed after the challenge, and infection status confirmed by RT-qPCR analysis, enabling the identification of singly-infected and co-infected fish. A typical histophlogosis associated with histozoic extrasporogonic T. bryosalmonae was detected together with acute inflammation, haemorrhaging and necrosis due to the viral infection. The host immune response was measured in terms of key marker genes expression in kidney tissues. During T. bryosalmonae/VHSV-Ia co-infection, modulation of pro-inflammatory and antimicrobial peptide genes was strongly influenced by the viral infection, with a protracted inflammatory status, perhaps representing a negative side effect in these fish. Earlier activation of the cellular and humoral responses was detected in co-infected fish, with a more pronounced upregulation of Th1 and antiviral marker genes. These results reveal that some brown trout immune responses are enhanced or prolonged during PKD/VHS co-infection, relative to single infection.
Assuntos
Coinfecção/imunologia , Doenças dos Peixes/imunologia , Nefropatias/veterinária , Oncorhynchus mykiss/imunologia , Imunidade Adaptativa , Animais , Coinfecção/parasitologia , Coinfecção/virologia , Modelos Animais de Doenças , Doenças dos Peixes/parasitologia , Doenças dos Peixes/virologia , Expressão Gênica , Septicemia Hemorrágica Viral/imunologia , Imunidade Inata , Nefropatias/imunologia , Myxozoa/imunologia , Oncorhynchus mykiss/parasitologia , Oncorhynchus mykiss/virologia , Doenças Parasitárias em Animais/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Células Th1/imunologiaRESUMO
The chemokine and chemokine receptor networks regulate leukocyte trafficking, inflammation, immune cell differentiation, cancer and other biological processes. Comparative immunological studies have revealed that both chemokines and their receptors have expanded greatly in a species/lineage specific way. Of the 10 human CC chemokine receptors (CCR1-10) that bind CC chemokines, orthologues only to CCR6, 7, 9 and 10 are present in teleost fish. In this study, four fish-specific CCRs, termed as CCR4La, CCR4Lc1, CCR4Lc2 and CCR11, with a close link to human CCR1-5 and 8, in terms of amino acid homology and syntenic conservation, have been identified and characterized in rainbow trout (Oncorhynchus mykiss). These CCRs were found to possess the conserved features of the G protein-linked receptor family, including an extracellular N-terminal, seven TM domains, three extracellular loops and three intracellular loops, and a cytoplasmic carboxyl tail with multiple potential serine/threonine phosphorylation sites. Four cysteine residues known to be involved in forming two disulfide bonds are present in the extracellular domains and a DRY motif is present in the second intracellular loop. Signaling mediated by these receptors might be regulated by N-glycosylation, tyrosine sulfation, S-palmitoylation, a PDZ ligand motif and di-leucine motifs. Studies of intron/exon structure revealed distinct fish-specific CCR gene organization in different fish species/lineages that might contribute to the diversification of the chemokine ligand-receptor networks in different fish lineages. Fish-specific trout CCRs are highly expressed in immune tissues/organs, such as thymus, spleen, head kidney and gills. Their expression can be induced by the pro-inflammatory cytokines, IL-1ß, IL-6 and IFNγ, by the pathogen associated molecular patterns, PolyIC and peptidoglycan, and by bacterial infection. These data suggest that fish-specific CCRs are likely to have an important role in immune regulation in fish.
Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/imunologia , Receptores CCR/genética , Receptores CCR/imunologia , Sequência de Aminoácidos , Animais , Doenças dos Peixes/microbiologia , Doenças dos Peixes/parasitologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Rim Cefálico/imunologia , Macrófagos/imunologia , Oncorhynchus mykiss/classificação , Filogenia , Receptores CCR/química , Alinhamento de Sequência/veterináriaRESUMO
In this study we show that four arginase isoforms (arg1a, arg1b, arg2a, arg2b) exist in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). We have characterised these molecules in terms of a) sequence analysis, b) constitutive expression in different tissues, and modulated expression following c) stimulation of head kidney macrophages in vitro, or d) vaccination/infection with Yersinia ruckeri and e) parasite infection (AGD caused by Paramoeba perurans and PKD caused by Tetracapsuloides bryosalmonae). Synteny analysis suggested that these arginase genes are paralogues likely from the Ss4R duplication event, and amino acid identity/similarity analyses showed that the proteins are relatively well conserved across species. In rainbow trout constitutive expression of one or both paralogues was seen in most tissues but different constitutive expression patterns were observed for the different isoforms. Stimulation of rainbow trout head kidney macrophages with PAMPs and cytokines also revealed isoform specific responses and kinetics, with arg1a being particularly highly modulated by the PAMPs and pro-inflammatory cytokines. In contrast the type II arginase paralogues were induced by rIl-4/13, albeit to a lesser degree. Vaccination and infection with Y. ruckeri also revealed isoform specific responses, with variation in tissue expression level and kinetics. Lastly, the impact of parasite infection was studied, where down regulation of arg1a and arg1b was seen in two different models (AGD in salmon and PKD in trout) and of arg2a in AGD. The differential responses seen are discussed in the context of markers of type II responses in fish and paralogue subfunctionalization.
Assuntos
Arginase/genética , Doenças dos Peixes/genética , Proteínas de Peixes/genética , Expressão Gênica , Oncorhynchus mykiss , Salmo salar , Yersiniose/veterinária , Animais , Arginase/metabolismo , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Proteínas de Peixes/metabolismo , Rim Cefálico/imunologia , Rim Cefálico/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Especificidade de Órgãos , Análise de Sequência de DNA , Vacinação/veterinária , Yersiniose/genética , Yersiniose/imunologia , Yersiniose/microbiologia , Yersinia ruckeri/fisiologiaRESUMO
Tetracapsuloides bryosalmonae is a myxozoan parasite of freshwater bryozoans and salmonids, causing proliferative kidney disease in the latter. To date, detection of the parasite has required collection of hosts and subsequent molecular or histological examination. The release of infectious spores from both hosts offers an opportunity to detect the parasite in water samples. We developed a novel SYBR® Green quantitative real-time PCR (qPCR) assay for T. bryosalmonae in water samples which provides an estimation of bryozoan malacospore numbers and tested the assay in 3 rivers in southern England (UK) over a period of 5 wk. The assay proved to be both highly sensitive and specific to the parasite, detecting low levels of spores throughout the study period. Larger-volume samples afforded greater detection likelihood, but did not increase the number of spores detected, possibly as a result of low and patchy spore distributions and lack of within-site replication of large-volume samples. Based on point-measurements, temperature was positively associated with the likelihood of detecting spores, possibly reflecting the temperature dependence of spore shedding from bryozoan hosts. The presence of T. bryosalmonae in water samples was predominantly influenced by spatial (sites within rivers, amongst rivers) and temporal (sampling dates) factors, while the latter also influenced quantification cycle (Cq) values and spore abundance. Environmental monitoring for infectious stages can complement traditional methods, providing faster and easier detection and avoiding potentially prolonged searching, collecting and destructive sampling of invertebrate and vertebrate hosts.
Assuntos
Myxozoa/genética , Myxozoa/fisiologia , Rios/parasitologia , Animais , DNA/genética , Inglaterra , Reação em Cadeia da Polimerase/métodos , Sensibilidade e EspecificidadeRESUMO
IL-12 is a heterodimeric cytokine composed of an α-chain (p35) and a ß-chain (p40). Primarily produced by APCs, IL-12 induces IFN-γ production in T, B and NK cells. IL-12 drives Th1-cell differentiation and IFN-γ secretion to promote cell-mediated immunity, which is essential in the defence against intracellular pathogens. The importance of IL-12 in Th1 responses is echoed by its targeted suppression by intracellular pathogens evading cell-mediated immunity. IL-12 subunits have been identified recently in fish, although reported bioactivities are limited to higher vertebrates. Here, we report the cloning of a p35 gene and two divergent p40 genes (p40b and p40c), capable of producing two functional IL-12 isoforms (p35/p40b and p35/p40c) in rainbow trout. Trout IL-12 isoforms possess distinct bioactivities with respect to the induction of IFN-γ, IL-10 and p40c expression. Trout IL-12 isoforms are differentially expressed and modulated in vivo, exhibiting specific gene expression profiles in bacterial, viral and parasitic infection models, and in vitro in stimulated macrophage and leucocyte cultures. These data imply that alternative or additional pathogen-specific Th-like cell populations may exist in fish. This study will facilitate a broader understanding of the evolutionary processes driving host-pathogen interactions and Th1-like immune responses in lower vertebrates.
Assuntos
Proteínas de Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Subunidade p35 da Interleucina-12/imunologia , Subunidade p40 da Interleucina-12/imunologia , Oncorhynchus mykiss/imunologia , Células Th1/imunologia , Animais , Feminino , Doenças dos Peixes/imunologia , Doenças dos Peixes/metabolismo , Doenças dos Peixes/patologia , Proteínas de Peixes/biossíntese , Imunidade Celular , Interferon gama/biossíntese , Interferon gama/imunologia , Subunidade p35 da Interleucina-12/biossíntese , Subunidade p40 da Interleucina-12/biossíntese , Masculino , Oncorhynchus mykiss/metabolismo , Células Th1/metabolismo , Células Th1/patologiaRESUMO
Atypical chemokine receptors (ACKRs) have emerged as key components of the chemokine system, with an essential regulatory function in innate and adaptive immune responses and inflammation. In mammals ACKR2 is a 'scavenging' receptor for inflammatory CC chemokines and plays a central role in the resolution of in vivo inflammatory responses. An ACKR2 like gene has been identified and cloned in rainbow trout (Teleostei) in the present study, enabling the further identification of this molecule in another group of ray-finned teleost fish (Holostei), in a lobe-finned fish (Sarcopterygii-coelacanth), and in reptiles. The identity of these ACKR2 molecules is supported by their conserved structure, and by phylogenetic tree and synteny analysis. Trout ACKR2 is highly expressed in spleen and head kidney, suggesting a homeostatic role of this receptor in limiting the availability of its potential ligands. Trout ACKR2 expression can be modulated in vivo by bacterial and parasitic infections, and in vitro by PAMPs (poly I:C and peptidoglycan) and cytokines (IL-6, TNF-α, IFN-γ and IL-21) in a time dependent manner. These patterns of expression and modulation suggest that trout ACKR2 is regulated in a complex way and has an important role in control of the chemokine network in fish as in mammals.
Assuntos
Regulação da Expressão Gênica/imunologia , Rim Cefálico/metabolismo , Oncorhynchus mykiss/metabolismo , Receptores CCR10/genética , Receptores CCR10/metabolismo , Baço/metabolismo , Análise de Variância , Animais , Sequência de Bases , Clonagem Molecular , Citocinas/metabolismo , Primers do DNA/genética , Dados de Sequência Molecular , Oncorhynchus mykiss/microbiologia , Oncorhynchus mykiss/parasitologia , Peptidoglicano/metabolismo , Filogenia , Poli I-C/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Sintenia , Receptor D6 de QuimiocinaRESUMO
Lower vertebrates have been found to possess genes that have similar homology to both interleukin (IL)-17A and IL-17F, which have been termed IL-17A/F. In fish species, several of these genes can be present, but, to date, very little is known about their functional activity. This article describes the discovery and sequence analysis of a rainbow trout (Oncorhynchus mykiss) IL-17A/F2 molecule and an IL-17RA receptor. In addition, the bioactivity of the trout IL-17A/F2 is investigated for the first time in any species. The predicted IL-17A/F2 and IL-17RA proteins consist of 146 and 966 amino acids (aa), respectively, with both molecules containing conserved family motifs. Expression analysis revealed high constitutive expression of trout IL-17A/F2 in mucosal tissues from healthy fish, suggesting a potential role in mucosal immunity. When the modulation of IL-17A/F2 and IL-17RA in vitro was analyzed, it was observed that the two molecules were similarly affected. The expression of IL-17A/F2 was also induced in head kidney during bacterial, parasitic, and viral infections, revealing a possible function in defense against such pathogens. However, downregulation of IL-17RA was seen in some tissues and infections. The recombinant IL-17A/F2 protein was produced in Escherichia coli and was found to affect the expression of an antimicrobial peptide and the proinflammatory cytokines IL-6 and IL-8 in splenocytes. Consistent with mammalian IL-17 homologues, our expression and bioactivity results imply that trout IL-17A/F2 plays an important role in promoting inflammatory and host innate immune responses directed against different pathogen groups.
Assuntos
Interleucina-17/genética , Interleucina-17/imunologia , Oncorhynchus mykiss/genética , Receptores de Interleucina-17/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular/métodos , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Escherichia coli/genética , Imunidade Inata/genética , Imunidade Inata/imunologia , Interleucina-17/metabolismo , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/imunologia , Interleucina-8/metabolismo , Dados de Sequência Molecular , Oncorhynchus mykiss/imunologia , Oncorhynchus mykiss/metabolismo , Receptores de Interleucina-17/imunologia , Receptores de Interleucina-17/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
The myxozoan Tetracapsuloides bryosalmonae is the causative agent of Proliferative Kidney Disease (PKD) targeting primarily the kidney of infected fish where it causes a chronic lymphoid immunopathology. Although known to be associated with suppression of some cellular aspects of innate immunity and a prominent lymphocytic hyperplasia, there remains a considerable knowledge gap in our understanding of the underlying immune mechanisms driving PKD pathogenesis. To provide further insights, the expression profiles of a panel of innate/inflammatory and adaptive immune molecules were examined in rainbow trout Oncorhynchus mykiss following a natural exposure to the parasite. Relative to controls, fish with early to advanced stages of kidney pathology exhibited up-regulation of the inflammatory cytokines interleukin (IL)-6 and IL-11, although remaining refractory towards genes indicative of macrophage activity. Antimicrobial peptides (AMPs) and anti-inflammatory markers, including cathelicidin (CATH) and IL-10 were markedly up-regulated during clinical disease. Up-regulation of adaptive immune molecules, including cell markers and antibody genes reflect the lymphocytic dominance of this disease and the likely importance of lymphocyte subsets in PKD pathogenesis. Up-regulation of T helper (TH) cell-like response genes and transcription factors implies that T. bryosalmonae may elicit a complex interplay between TH cell subsets. This work, for the first time in the study of fish-myxozoan interactions, suggests that PKD pathogenesis is shaped by an anti-inflammatory phenotype, a profound B cell/antibody response and dysregulated TH cell-like activities. A better understanding of the functional roles of fish immune cells and molecules in PKD pathogenesis may facilitate future development of control measures against this disease.
Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Nefropatias/veterinária , Rim/imunologia , Oncorhynchus mykiss , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Doenças dos Peixes/genética , Doenças dos Peixes/parasitologia , Proteínas de Peixes/metabolismo , Perfilação da Expressão Gênica/veterinária , Regulação da Expressão Gênica , Rim/parasitologia , Rim/patologia , Nefropatias/imunologia , Nefropatias/parasitologia , Myxozoa/isolamento & purificação , Myxozoa/fisiologia , Reação em Cadeia da Polimerase/veterináriaRESUMO
Myxozoans are enigmatic endoparasitic organisms sharing morphological features with bilateria, protists and cnidarians. This, coupled with their highly divergent gene sequences, has greatly obscured their phylogenetic affinities. Here we report the sequencing and characterization of a minicollagen homologue (designated Tb-Ncol-1) in the myxozoan Tetracapsuloides bryosalmonae. Minicollagens are phylum-specific genes encoding cnidarian nematocyst proteins. Sequence analysis revealed a cysteine-rich domain (CRD) architecture and genomic organization similar to group 1 minicollagens. Homology modelling predicted similar three-dimensional structures to Hydra CRDs despite deviations from the canonical pattern of group 1 minicollagens. The discovery of this minicollagen gene strongly supports myxozoans as cnidarians that have radiated as endoparasites of freshwater, marine and terrestrial hosts. It also reveals novel protein sequence variation of relevance to understanding the evolution of nematocyst complexity, and indicates a molecular/morphological link between myxozoan polar capsules and cnidarian nematocysts. Our study is the first to illustrate the power of using genes related to a taxon-specific novelty for phylogenetic inference within the Metazoa, and it exemplifies how the evolutionary relationships of other metazoans characterized by extreme sequence divergence could be similarly resolved.
Assuntos
Cnidários/classificação , Cnidários/genética , Colágeno/genética , Myxozoa/classificação , Myxozoa/genética , Sequência de Aminoácidos , Animais , Colágeno/química , Evolução Molecular , Dados de Sequência Molecular , Myxozoa/ultraestrutura , Nematocisto/metabolismo , Nematocisto/ultraestrutura , Filogenia , Dobramento de Proteína , Proteínas/genética , Proteínas/metabolismoRESUMO
A novel IL-1 family member (nIL-1F) has been discovered in fish, adding a further member to this cytokine family. The unique gene organization of nIL-1F, together with its location in the genome and low homology to known family members, suggests that this molecule is not homologous to known IL-1F. Nevertheless, it contains a predicted C-terminal beta-trefoil structure, an IL-1F signature region within the final exon, a potential IL-1 converting enzyme cut site, and its expression level is clearly increased following infection, or stimulation of macrophages with LPS or IL-1beta. A thrombin cut site is also present and may have functional relevance. The C-terminal recombinant protein antagonized the effects of rainbow trout rIL-1beta on inflammatory gene expression in a trout macrophage cell line, suggesting it is an IL-1beta antagonist. Modeling studies confirmed that nIL-1F has the potential to bind to the trout IL-1RI receptor protein, and may be a novel IL-1 receptor antagonist.
Assuntos
Interleucina-1/isolamento & purificação , Sequência de Aminoácidos , Animais , Citocinas/isolamento & purificação , Peixes , Componentes do Gene , Infecções/imunologia , Interleucina-1/genética , Interleucina-1/fisiologia , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/farmacologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos , Ligação Proteica , Conformação Proteica , Regulação para Cima/imunologiaRESUMO
The myxozoan parasite, Tetracapsuloides bryosalmonae has a two-host life cycle alternating between freshwater bryozoans and salmonid fish. Infected fish can develop Proliferative Kidney Disease, characterised by a gross lymphoid-driven kidney pathology in wild and farmed salmonids. To facilitate an in-depth understanding of T. bryosalmonae-host interactions, we have used a two-host parasite transcriptome sequencing approach in generating two parasite transcriptome assemblies; the first derived from parasite spore sacs isolated from infected bryozoans and the second from infected fish kidney tissues. This approach was adopted to minimize host contamination in the absence of a complete T. bryosalmonae genome. Parasite contigs common to both infected hosts (the intersect transcriptome; 7362 contigs) were typically AT-rich (60-75% AT). 5432 contigs within the intersect were annotated. 1930 unannotated contigs encoded for unknown transcripts. We have focused on transcripts encoding proteins involved in; nutrient acquisition, host-parasite interactions, development, cell-to-cell communication and proteins of unknown function, establishing their potential importance in each host by RT-qPCR. Host-specific expression profiles were evident, particularly in transcripts encoding proteases and proteins involved in lipid metabolism, cell adhesion, and development. We confirm for the first time the presence of homeobox proteins and a frizzled homologue in myxozoan parasites. The novel insights into myxozoan biology that this study reveals will help to focus research in developing future disease control strategies.
Assuntos
Doenças dos Peixes/genética , Perfilação da Expressão Gênica , Interações Hospedeiro-Parasita/genética , Nefropatias/genética , Nefropatias/parasitologia , Transcriptoma/genética , Animais , Briozoários/genética , Briozoários/parasitologia , DNA/genética , Receptores Frizzled/metabolismo , Regulação da Expressão Gênica , Ontologia Genética , Genes Controladores do Desenvolvimento , Genes Homeobox , Genoma , Anotação de Sequência Molecular , Parasitos/fisiologiaRESUMO
The evolutionary aspects of cystatins are greatly underexplored in early-emerging metazoans. Thus, we surveyed the gene organization, protein architecture, and phylogeny of cystatin homologues mined from 110 genomes and the transcriptomes of 58 basal metazoan species, encompassing free-living and parasite taxa of Porifera, Placozoa, Cnidaria (including Myxozoa), and Ctenophora. We found that the cystatin gene repertoire significantly differs among phyla, with stefins present in most of the investigated lineages but with type 2 cystatins missing in several basal metazoan groups. Similar to liver and intestinal flukes, myxozoan parasites possess atypical stefins with chimeric structure that combine motifs of classical stefins and type 2 cystatins. Other early metazoan taxa regardless of lifestyle have only the classical representation of cystatins and lack multi-domain ones. Our comprehensive phylogenetic analyses revealed that stefins and type 2 cystatins clustered into taxonomically defined clades with multiple independent paralogous groups, which probably arose due to gene duplications. The stefin clade split between the subclades of classical stefins and the atypical stefins of myxozoans and flukes. Atypical stefins represent key evolutionary innovations of the two parasite groups for which their origin might have been linked with ancestral gene chimerization, obligate parasitism, life cycle complexity, genome reduction, and host immunity.
RESUMO
The polarization of naïve CD4+ T cells to T helper (Th)1 or Th2 cells is specified by two master transcription factors, T-bet and GATA3, and is an essential feature of mammalian adaptive immune responses to pathogens and the development of long-lasting immunity. We report here the cloning of rainbow trout Oncorhynchus mykiss T-bet and GATA3, to allow the future evaluation of the existence of Th1 and Th2 cells in salmonid fish. The trout T-bet translation shares high amino acid identities to other fish T-bet molecules (71-72%) but low identities to mammalian T-bet genes (41-42%), although the middle T-box DNA binding domain is highly conserved among all the T-bet proteins from fish and mammals. The trout GATA3 has high amino acid sequence identities (73-88%) to all known vertebrate molecules, with two highly conserved zinc finger motifs. The identity of the trout T-bet and GATA3 molecules was confirmed by phylogenetic tree analysis. A comparable expression level of T-bet and GATA3 was seen in the spleen, head kidney and muscle in healthy trout, but a higher expression level of GATA3 was seen in the gills, brain, skin and intestine relative to that of T-bet. T-bet and GATA3 expression was modulated by different stimulants. The T cell stimulant PHA up-regulated the expression of both T-bet and GATA3 in splenocytes, suggesting that they may be mainly expressed by activated T cells. The expression of T-bet and GATA3 in the spleen was increased by acute stress, but their expression was inhibited by bacterial (Yersinia ruckeri) infection. In a parasitic infection model, Tetracapsuloides bryosalmonae infection induced a biased gene expression profile where a large increase in the expression of T-bet, IFN-gamma and IL-2 was seen, suggesting that a Th1-like response is likely induced by this disease. A better understanding of pathogen modulated expression of T-bet and GATA3, and the potential underlying host immune responses elicited as a consequence of their expression, may allow novel future control measures against disease in fish to be developed.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Doenças dos Peixes/genética , Doenças dos Peixes/imunologia , Fator de Transcrição GATA3/genética , Oncorhynchus mykiss , Doenças Parasitárias em Animais/imunologia , Proteínas com Domínio T/genética , Yersiniose/veterinária , Animais , Sequência de Bases , Linfócitos T CD4-Positivos/metabolismo , Clonagem Molecular , Análise por Conglomerados , Primers do DNA/genética , Doenças dos Peixes/metabolismo , Fator de Transcrição GATA3/metabolismo , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Myxozoa , Doenças Parasitárias em Animais/genética , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência , Especificidade da Espécie , Proteínas com Domínio T/metabolismo , Yersiniose/genética , Yersiniose/imunologia , Yersinia ruckeriRESUMO
Mammalian interferon regulatory factor (IRF)4 (PIP, LSIRF, and ICSAT) and IRF8 (ICSBP) are known to be critical in regulating a spectrum of functional and developmental processes in lymphomyeloid cell lineages either through direct binding to IRF-E motifs in target gene promoters or indirectly by binding to composite motifs recognized by Ets family members, PU.I and Sp.I. Here we report, for the first time in fish, the sequencing and characterization of full-length cDNA homologues of rainbow trout (rt) IRF4 and rtIRF8. The rtIRF4 molecule consists of 1848 bp with a 45 bp 5' UTR and a predicted 378 bp 3' UTR translating into a 474 aa protein. RtIRF8 consists of 1951 bp with a 52 bp 5' UTR and a 564 bp 3' UTR translating into a 444 aa protein. Each gene possesses a putative DNA binding domain (DBD) containing the tryptophan pentad-repeat domain found in all IRF family members. Both molecules also possess a well conserved IRF association domain (IAD). The presence of these domains along with phylogenetic analysis places the two genes in the IRF4 subfamily. Both genes were detected in a range of trout tissues where IRF8 was the overall predominant transcript. Consistent with mammalian studies, the highest expression levels of IRF4 and IRF8 were observed in the lymphomyeloid-rich fish tissues, spleen, head kidney and gills. IRF8 expression in stimulated trout splenocytes was significantly up-regulated by polyinosinic:polycytidylic acid (poly I:C), trout recombinant (r)IL-15, phorbol 12-myristate 13-acetate (PMA), and phytohaemagglutinin (PHA) treatment whilst remaining refractory towards lipopolysaccharide (LPS) treatment. IRF4 was significantly down-regulated by LPS stimulation and remained refractory towards poly I:C, trout rIL15, and PHA. PMA stimulation elicited a significant upregulation of IRF4 expression. Overall, these data support the premise that these IRFs are likely to play important roles in the functional and developmental processes occurring in fish lymphomyeloid tissues.
Assuntos
Regulação da Expressão Gênica/imunologia , Fatores Reguladores de Interferon/imunologia , Tecido Linfoide/imunologia , Oncorhynchus mykiss/imunologia , Filogenia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Etiquetas de Sequências Expressas , Fatores Reguladores de Interferon/genética , Dados de Sequência Molecular , Oncorhynchus mykiss/genética , Poli I-C/imunologia , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Ativação Transcricional/imunologiaRESUMO
Basic leucine zipper transcription factor ATF-like (BATF) -3 is a member of the activator protein 1 (AP1) family of transcription factors and is known to play a vital role in regulating differentiation of antigen-presenting cells in mammals. In this study, two BATF3 homologues (termed BATF3a and BATF3b) have been identified in rainbow trout (Oncorhynchus mykiss). Both genes were constitutively expressed in tissues, with particularly high levels of BATF3a in spleen, liver, pyloric caecae and head kidney. BATF3a was also more highly induced by PAMPs and cytokines in cultured cells, with type II IFN a particularly potent inducer. In rIL-4/13 pre-stimulated cells, the viral PAMPS polyI:C and R848 had the most pronounced effect on BATF3 expression. BATF3 expression could also be modulated in vivo, following infection with Yersinia ruckeri, a bacterial pathogen causing redmouth disease in salmonids, or with the rhabdovirus IHNV. The results suggest that BATF3 may be functionally conserved in regulating the differentiation and activation of immune cells in lower vertebrates and could be explored as a potential marker for comparative investigation of leucocyte lineage commitment across the vertebrate phyla.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/imunologia , Proteínas de Peixes/imunologia , Oncorhynchus mykiss/imunologia , Sequência de Aminoácidos , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Citocinas/imunologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Rim Cefálico/imunologia , Rim Cefálico/microbiologia , Rim Cefálico/virologia , Oncorhynchus mykiss/microbiologia , Oncorhynchus mykiss/virologia , Filogenia , Rhabdoviridae/imunologia , Alinhamento de Sequência , Yersiniose/imunologia , Yersiniose/microbiologia , Yersinia ruckeri/imunologiaRESUMO
Proliferative kidney disease (PKD) is a widespread disease caused by the endoparasite Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea). Clinical disease, provoked by the proliferation of extrasporogonic parasite stages, is characterized by a chronic kidney pathology with underlying transcriptional changes indicative of altered B cell responses and dysregulated T-helper cell-like activities. Despite the relevance of PKD to European and North American salmonid aquaculture, no studies, to date, have focused on further characterizing the B cell response during the course of this disease. Thus, in this work, we have studied the behavior of diverse B cell populations in rainbow trout (Oncorhynchus mykiss) naturally infected with T. bryosalmonae at different stages of preclinical and clinical disease. Our results show a clear upregulation of all trout immunoglobulins (Igs) (IgM, IgD, and IgT) demonstrated by immunohistochemistry and Western blot analysis, suggesting the alteration of diverse B cell populations that coexist in the infected kidney. Substantial changes in IgM, IgD, and IgT repertoires were also identified throughout the course of the disease further pointing to the involvement of the three Igs in PKD through what appear to be independently regulated mechanisms. Thus, our results provide strong evidence of the involvement of IgD in the humoral response to a specific pathogen for the first time in teleosts. Nevertheless, it was IgT, a fish-specific Ig isotype thought to be specialized in mucosal immunity, which seemed to play a prevailing role in the kidney response to T. bryosalmonae. We found that IgT was the main Ig coating extrasporogonic parasite stages, IgT+ B cells were the main B cell subset that proliferated in the kidney with increasing kidney pathology, and IgT was the Ig for which more significant changes in repertoire were detected. Hence, although our results demonstrate a profound dysregulation of different B cell subsets during PKD, they point to a major involvement of IgT in the immune response to the parasite. These results provide further insights into the pathology of PKD that may facilitate the future development of control strategies.
Assuntos
Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , Imunoglobulinas/metabolismo , Oncorhynchus mykiss/imunologia , Doenças Parasitárias em Animais/imunologia , Animais , Aquicultura , Doenças dos Peixes/imunologia , Proteínas de Peixes/metabolismo , Imunidade nas Mucosas , Nefropatias/imunologia , Ativação Linfocitária , Myxozoa/imunologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Proliferative kidney disease (PKD) is a parasitic infection of salmonid fish characterized by hyper-secretion of immunoglobulins in response to the presence of the myxozoan parasite, Tetracapsuloides bryosalmonae. In this context, we hypothesized that the BAFF/APRIL axis, known to play a major role in B cell differentiation and survival in mammals, could be affected by the parasite and consequently be involved in the apparent shift in normal B cell activity. To regulate B cell activity, BAFF and APRIL bind to transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA), whereas BAFF also binds to BAFF receptor (BAFF-R). In teleost fish, although some BAFF and APRIL sequences have been reported, their receptors have not been identified. Thus, as a first step in the current work, we have identified homologues to mammalian TACI, BCMA and BAFF-R in rainbow trout (Oncorhynchus mykiss), that constitute the first report of BAFF and APRIL receptor sequences in fish. Subsequently we studied the transcriptional modulation of BAFF, APRIL, and the fish-specific related cytokine, BALM and their putative receptors in fish naturally exposed to T. bryosalmonae. Finally, to gain further insights on the functional role that these cytokines play during the course of PKD, we have studied their effect on the survival of kidney IgM+ B cells and on immunoglobulin transcription. Our results support the premise that the BAFF / APRIL axis could play an important role during PKD, which may open the possibility of new therapeutic treatments against the disease.
Assuntos
Receptor do Fator Ativador de Células B/metabolismo , Antígeno de Maturação de Linfócitos B/metabolismo , Doenças dos Peixes/patologia , Nefropatias/patologia , Oncorhynchus mykiss/parasitologia , Doenças Parasitárias em Animais/patologia , Proteína Transmembrana Ativadora e Interagente do CAML/metabolismo , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Animais , Linfócitos B/imunologia , Sequência de Bases , Doenças dos Peixes/parasitologia , Regulação da Expressão Gênica , Nefropatias/parasitologia , Myxozoa , Doenças Parasitárias em Animais/parasitologia , Análise de Sequência de DNA , Proteína Transmembrana Ativadora e Interagente do CAML/genéticaRESUMO
The endangered Chinese giant salamander (Andrias davidianus) is the largest extant amphibian species. Disease outbreaks represent one of the major factors threatening A. davidianus populations in the wild and the viability of artificial breeding programmes. Development of future immune therapies to eliminate infectious disease in A. davidianus is dependent on a thorough understanding of the immune mechanisms elicited by pathogen encounters. To this end we have undertaken, for the first time in amphibians, differential transcriptome analysis of the giant salamander response to Aeromonas hydrophila, one of the most devastating pathogens affecting amphibian populations. Out of 87,204 non-redundant consensus unigenes 19,216 were annotated, 6834 of which were upregulated and 906 down-regulated following bacterial infection. 2058 unigenes were involved with immune system processes, including 287 differentially expressed unigenes indicative of the impact of bacterial infection on several innate and adaptive immune pathways in the giant salamander. Other pathways not directly associated with immune-related activity were differentially expressed, including developmental, structural, molecular and growth processes. Overall, this work provides valuable insights into the underlying immune mechanisms elicited during bacterial infection in amphibians that may aid in the future development of disease control measures in protecting the Chinese giant salamander. With the unique position of amphibians in the transition of tetrapods from aquatic to terrestrial habitats, our study will also be invaluable towards the further understanding of the evolution of tetrapod immunity.