RESUMO
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide belonging to the glucagon/secretin superfamily. In teleost fish, PACAP has been demonstrated to have an immunomodulatory role. Although previous studies have shown that viral/bacterial infections can influence the transcription of PACAP splicing variants and associated receptors in salmonids, the antiviral activity of PACAP has never been studied in teleost. Thus, in the present work, we investigated in vitro the influence of synthetic Clarias gariepinus PACAP-38 on the transcription of genes related to viral immunity using the rainbow trout monocyte/macrophage-like cell line RTS11 as a model. Positive transcriptional modulation of interferon gamma (IFNγ), interferon alpha (FNα1,2), interleukin 8 (IL-8), Mx and Toll-like receptor 3 (TLR3) genes was found in a dose and time dependent manner. We also explored how a pre-treatment with PACAP could enhance antiviral immune response using poly (I:C) as viral mimic. Interferons and IL-8 transcription levels were enhanced when PACAP was added 24 h previous to poly (I:C) exposure. With these evidences, we tested in vivo how PACAP administration by immersion bath affected the survival of rainbow trout fry to a challenge with viral hemorrhagic septicemia virus (VHSV). After challenge, PACAP-treated fish had increased survival compared to non-treated/challenge fish. Furthermore, PACAP was able to decrease the viral load in spleen/kidney and stimulate the transcription of IFNs and Mx when compared to untreated infected fish. Altogether, the results of this work provide valuable insights regarding the role of teleost PACAP in antiviral immunity and point to a potential application of this peptide to reduce the impact of viral infections in aquaculture.
Assuntos
Antivirais/imunologia , Peixes-Gato/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Imunidade Inata , Oncorhynchus mykiss , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Animais , Proteínas de Peixes/imunologia , Novirhabdovirus/fisiologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/imunologia , Poli I-C/farmacologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterináriaRESUMO
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a regulatory neuropeptide that belongs to the secretin/glucagon superfamily, of which some members have shown antimicrobial activities. Contrasting to mammals, published studies on the action of PACAP in non-mammalian vertebrate immune system remain scarce. Some of our recent studies added this peptide to the growing list of mediators that allow cross-talk between the nervous, endocrine and immune systems in teleost fish. Regulation of PACAP and expression of its receptor genes has been demonstrated during an immune response mounted against acute bacterial infection in fish, though the direct effect of PACAP against fish pathogenic bacteria has never been addressed. Current work provides evidence of antimicrobial activity of Clarias gariepinus PACAP against a wide spectrum of Gram-negative and Gram-positive bacteria and fungi of interest for human medicine and aquaculture, in which computational prediction studies supported the putative PACAP therapeutic activity. Results also indicated that catfish PACAP not only exhibits inhibitory effects on pathogen growth, but also affects the proliferation of human non-small cell lung cancer cell line H460 in a dose-dependent manner. The observed cytotoxic activity of catfish PACAP against human tumor cells and pathogenic microorganisms, but not healthy fish and mammalian erythrocytes support a potential physiological role of this neuropeptide in selective microbial and cancer cell killing. All together, our findings extend the mechanisms by which PACAP could contribute to immune responses, and open up new avenues for future therapeutic application of this bioactive neuropeptide.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Peixes-Gato/imunologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Aeromonas hydrophila/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Bactérias/patogenicidade , Candida albicans/efeitos dos fármacos , Candida albicans/patogenicidade , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Peixes-Gato/microbiologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Peptídeos Penetradores de Células/farmacologia , Eritrócitos/efeitos dos fármacos , Hemólise , Humanos , Neoplasias Pulmonares/tratamento farmacológicoRESUMO
Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and PACAP-Related Peptide (PRP) are structurally similar peptides encoded in the same transcripts. Their transcription has been detected not only in the brain but also in a wide range of peripheral tissues, even including organs of the immune system. PACAP exerts pleiotropic activities through G-protein coupled membrane receptors: the PACAP-specific PAC-1 and the VPAC-1 and VPAC-2 receptors that exhibit similar affinities for the Vasoactive Intestinal Peptide (VIP) and PACAP. Recent findings added PACAP and its receptors to the growing list of mediators that allow cross-talk between the nervous, endocrine and immune systems in fish. In this study the expression of genes encoding for PACAP and PRP, as well as VIP/PACAP receptors was studied in laboratory-reared brown trout (Salmo trutta) after septicaemic infections. Respectively Viral Haemorrhagic Septicaemia Virus (VHSV-Ia) or the Gram-negative bacterium Yersinia ruckeri (ser. O1 - biot. 2) were used in infection challenges. Kidney and spleen, the teleost main lymphopoietic organs, were sampled during the first two weeks post-infection. RT-qPCR analysis assessed specific pathogens burden and gene expression levels. PACAP and PRP transcription in each organ was positively correlated to the respective pathogen burden, assessed targeting the VHSV-glycoprotein or Y. ruckeri 16S rRNA. Results showed as the transcription of PACAP splicing variants and VIP/PACAP receptors is modulated in these organs during an acute viral and bacterial septicaemic infections in brown trout. These gene expression results provide clues as to how the PACAP system is modulated in fish, confirming an involvement during active immune responses elicited by both viral and bacterial aetiological agents. However, further experimental evidence is still required to fully elucidate and characterize the role of PACAP and PRP for an efficient immune response against pathogens.
Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Septicemia Hemorrágica Viral/imunologia , Fragmentos de Peptídeos/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Precursores de Proteínas/genética , Receptores de Peptídeo Intestinal Vasoativo/genética , Truta , Yersiniose/veterinária , Animais , Doenças dos Peixes/genética , Doenças dos Peixes/microbiologia , Doenças dos Peixes/virologia , Proteínas de Peixes/metabolismo , Septicemia Hemorrágica Viral/genética , Septicemia Hemorrágica Viral/virologia , Rim/microbiologia , Rim/virologia , Dados de Sequência Molecular , Novirhabdovirus/fisiologia , Fragmentos de Peptídeos/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Precursores de Proteínas/metabolismo , Receptores de Peptídeo Intestinal Vasoativo/metabolismo , Análise de Sequência de DNA/veterinária , Organismos Livres de Patógenos Específicos , Baço/microbiologia , Baço/virologia , Transcriptoma , Yersinia/fisiologia , Yersiniose/genética , Yersiniose/imunologia , Yersiniose/microbiologiaRESUMO
The high conservation of the pituitary adenylate cyclase activating polypeptide (PACAP) sequence indicates that this peptide fulfills important biological functions in a broad spectrum of organisms. However, in invertebrates, little is known about its presence and its functions remain unclear. Up to now, in non-mammalian vertebrates, the majority of studies on PACAP have focused mainly on the localization, cloning and structural evolution of this peptide. As yet, little is known about its biological functions as growth factor and immunomodulator in lower vertebrates. Recently, we have shown that PACAP, apart from its neuroendocrine role, influences immune functions in larval and juvenile fish. In this work, we isolated for the first time the cDNA encoding the mature PACAP from a crustacean species, the white shrimp Litopenaeus vannamei, corroborating its high degree of sequence conservation, when compared to sequences reported from tunicates to mammalian vertebrates. Based on this, we have evaluated the effects of purified recombinant Clarias gariepinus PACAP administrated by immersion baths on white shrimp growth and immunity. We demonstrated that PACAP improves hemocyte count, superoxide dismutase, lectins and nitric oxide synthase derived metabolites in treated shrimp related with an increase in total protein concentration and growth performance. From our results, PACAP acts as a regulator of shrimp growth and immunity, suggesting that in crustaceans, as in vertebrate organisms, PACAP is an important molecule shared by both the endocrine and the immune systems.
Assuntos
Proteínas de Artrópodes/genética , Penaeidae/genética , Penaeidae/imunologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Escherichia coli/genética , Larva/genética , Larva/crescimento & desenvolvimento , Larva/imunologia , Dados de Sequência Molecular , Penaeidae/crescimento & desenvolvimento , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Reação em Cadeia da Polimerase/veterinária , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência/veterináriaRESUMO
There are different studies concerning the immune functions of pituitary adenylate cyclase-activating polypeptide (PACAP), however information of its source in lymphoid organs is still scarce. Although the occurrence of the PACAP receptors PAC1, VPAC1 and VPAC2 in the immune system of mammals is known, only limited studies have reported the presence of some of these receptors in lymphoid organs in fish. In this work, we have studied both the expression of the two PACAP transcriptional variants (PRP/PACAP and PACAP) together with their receptors in diverse lymphoid organs of the rainbow trout (Oncorhynchus mykiss). Our results demonstrate for the first time in fish the presence of both transcripts in spleen, in which immunohistochemistry confirmed the production of PACAP by lymphocyte-like cells. In contrast, PACAP but not PRP/PACAP mRNA was detected in gills. Additionally, we observed a differential expression pattern of the PAC1, the PACAP specific receptor, with respect to VPAC1 and VPAC2 in lymphoid organs of fish. All receptors were detected in brain, intestine and spleen. By contrast, PAC1 and VPAC1 receptors but not VPAC2 were found in peripheral blood and in RTS11 rainbow trout monocyte/macrophage cells. Besides, in gills and skin, PAC1 and VPAC2 but not VPAC1 were observed, whereas in head kidney, the PAC1 receptor was the only one detected. In general, our finding added PACAP and its receptors to the list of neuroendocrine molecules present in the fish immune system, suggesting a direct autocrine/paracrine mechanism of PACAP action to mediate immune function in fish.
Assuntos
Regulação da Expressão Gênica , Sistema Imunitário/imunologia , Oncorhynchus mykiss , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Processamento Alternativo , Animais , Linhagem Celular , Perfilação da Expressão Gênica , Tecido Linfoide/imunologia , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/imunologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/imunologia , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/imunologiaRESUMO
To date, published in-vivo studies on the action of the PACAP in fish are few and these are concerned with reproduction, brain development and feeding behavior. Recently, we demonstrated for the first time that PACAP, apart from its neuroendocrine role, influences immune functions in fish larvae. In this work, we have evaluated the effects of recombinant Clarias gariepinus PACAP administration by intraperitoneal injection on important immune parameters in juvenile fish. We observed that a single injection of the recombinant peptide (0.1 microg per g of body weight) was able to increase the nitric oxide synthase-derived metabolites (NOS) and total immunoglobulin M (IgM) concentration in serum of juvenile catfish C. gariepinus and tilapia Orechromis niloticus respectively, after 24 h of its administration. In addition, our results showed that recombinant PACAP increases IgM, NOS and lysozyme in serum correlated with its ability to enhance growth performance in juvenile fish. Finally, the PACAP mRNA expression and PACAP immunoreactivity detected in peripheral blood leucocytes from juvenile catfish suggest a direct autocrine or/and paracrine mechanism of regulation of this peptide to mediate immune functions in fish.
Assuntos
Peixes-Gato/imunologia , Sistema Imunitário/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Animais , Peso Corporal , Peixes-Gato/sangue , Peixes-Gato/crescimento & desenvolvimento , Ciclídeos/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Crescimento/efeitos dos fármacos , Imunoglobulina M/sangue , Leucócitos/imunologia , Tecido Linfoide/imunologia , Muramidase/sangue , Óxido Nítrico Sintase/sangue , Óxido Nítrico Sintase/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
To date, many technologies have been developed to increase efficiency in aquaculture, but very few successful biotechnology molecules have arrived on the market. In this context, marine biotechnology has an opportunity to develop products to improve the output of fish in aquaculture. Published in vivo studies on the action of the pituitary adenylate cyclase-activating polypeptide (PACAP) in fish are scarce. Recently, our group, for the first time, demonstrated the biological role of this neuropeptide administrated by immersion baths in the growth and development of larval fish. In this work, we have evaluated the effects of recombinant Clarias gariepinus PACAP administration by intraperitoneal injection on growth performance and feeding behavior in juvenile fish. Our results showed the physiological role of this peptide for growth control in fish, including the juvenile stage, and confirm that its biological functions are well conserved in fish, since C. gariepinus PACAP stimulated growth in juvenile tilapia Oreochromis niloticus. In addition, we have observed that the growth-promoting effect of PACAP in juvenile tilapia was correlated with higher GH concentration in serum. With regard to the neuroendocrine regulation of growth control by PACAP, it was demonstrated that PACAP stimulates food intake in juvenile tilapia. In general, PACAP appears to act in the regulation of the growth control in juvenile fish. These findings propose that PACAP is a prominent target with the potential to stimulate fish growth in aquaculture.
Assuntos
Comportamento Alimentar/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Animais , Peixes-Gato/crescimento & desenvolvimento , Ingestão de Alimentos/efeitos dos fármacos , Feminino , Crescimento/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/isolamento & purificação , Coelhos/imunologia , Tilápia/crescimento & desenvolvimentoRESUMO
UNLABELLED: Quantization of gene expression requires that an accurate measurement of a specific transcript is made. In this paper, a quantitative reverse transcription-polymerase chain reaction (RT-PCR) by competition for tilapia growth hormone receptor type I is designed and validated. This experimental procedure was used to determine the abundance of growth hormone receptor type I transcript in different tilapia tissues. The results obtained with this developed competitive RT-PCR were similar to real-time PCR results reported recently. This protocol provides a reliable alternative, but less expensive than real-time PCR to quantify specific genes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (DOI:10.1007/s12575-009-9002-3) contains supplementary material, which is available to authorized users.
RESUMO
Immunoglobulin molecules play an important role in the immune defense system in all jawed vertebrates, by protecting the organism from a wide variety of pathogens. Nile tilapia (Oreochromis niloticus) is extensively cultivated worldwide, with a strong established market demand. It constitutes one of the model species for the study of fish immunology and its genome is currently fully sequenced. The presence of the immunoglobulin M gene in this species is well documented, as well as its major role in systemic immunity. To date, the IgT gene from O. niloticus has not been identified and, therefore, no information is available on the role of this immunoglobulin isotype in the immune response in tilapia. In the present work, novel secreted and membrane immunoglobulin T isotypes and a fragment of IgM were isolated from tilapia head kidney lymphocytes. Their transcriptional profiles were analyzed by quantitative PCR in larval development and in different tissues of healthy or lipopolysaccharide/Edwardsiella tarda-challenged tilapia adults. The presence of IgT and IgM were detected in early stages of larval development. Additionally, these genes exhibited differential expression profiles in basal conditions and after E. tarda infection in adult tilapia, in accord with the proposed effector functions of these immunoglobulins in the systemic and mucosal compartments. Our results suggest the potential involvement of this new Ig in mucosal immunity in tilapia.
Assuntos
Ciclídeos/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/imunologia , Imunidade nas Mucosas , Imunoglobulinas/imunologia , Animais , Biomarcadores , Edwardsiella tarda/imunologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/veterinária , Infecções por Enterobacteriaceae/virologia , Doenças dos Peixes/microbiologia , Proteínas de Peixes/genética , Proteínas de Peixes/isolamento & purificação , Perfilação da Expressão Gênica , Rim Cefálico/citologia , Imunoglobulinas/genética , Imunoglobulinas/isolamento & purificação , Larva/imunologia , Lipopolissacarídeos/imunologia , Linfócitos/metabolismo , FilogeniaRESUMO
There are several studies that clearly indicate a close bidirectional communication between the neuroendocrine and immune systems. In this sense, hypothalamic releasing hormones, besides their neuroendocrine role, have been shown to influence immune functions. Despite studies developed in mammals, there is, as yet, no information available about the role of the pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP-related peptide (PRP) in the fish innate immune system. The present study has evaluated the effect of PACAP and PRP administered by bath immersion, on important parameters of innate immunity and antioxidant defenses in African catfish (Clarias gariepinus) fry. We have shown, for the first time, that administration of recombinant C. gariepinus PACAP not only promotes growth but also increases lysozyme, nitric oxide synthase-derived metabolites and antioxidant defenses in treated fry. From our results, PACAP appears to act as a regulator of the teleostean immune system, in addition to its physiological role in controlling growth of fish.
Assuntos
Adjuvantes Imunológicos/farmacologia , Peixes-Gato/imunologia , Imunidade Inata/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Proteínas Recombinantes/farmacologia , Animais , Catalase/metabolismo , Peixes-Gato/crescimento & desenvolvimento , Glutationa/metabolismo , Larva/efeitos dos fármacos , Larva/enzimologia , Larva/crescimento & desenvolvimento , Larva/imunologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Superóxido Dismutase/metabolismoRESUMO
Nowadays, the studies of pituitary adenylate cyclase-activating polypeptide (PACAP)-related peptide (PRP) and PACAP in non-mammalian vertebrates, especially in fish, have paid attention mainly to the localization, cloning, and structural evolution of the peptides, but very little is known about its biological functions as growth-promoting factors in low vertebrates. In this work, we have cloned and characterized the PRP/PACAP cDNA from the commercially important North African catfish Clarias gariepinus. The sequence obtained agrees with the higher conservation of PACAP than of PRP peptide sequences. We have reported for the first time the recombinant expression of fish PRP and PACAP in mammalian cells and bacteria and also demonstrated that the growth rate of fish is enhanced by both PRP and PACAP recombinant peptides. The results obtained in vivo in three different fish species, catfish (C. gariepinus), tilapia (Oreochromis niloticus), and carp (Cyprinus carpio) support the finding that PACAP rather than PRP plays a primordial role in growth control in teleost fish. This finding could help to elucidate the neuroendocrine axis proposed to explain the hypothalamic regulation of growth in non-mammalian vertebrates.