Examinando por Autor "Tafalla, C"

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Analysis of immunostimulatory responses and immune tolerance to β-glucans in rainbow trout cell lines.
(Elsevier, 2021-08-30) Ordás, MC; González- Torres, L; Arense, P; Heavyside, R; Zarza, C; Tafalla, C
β-glucans are widely used in fish diets as immunomodulators to enhance the defensive capacities of farmed animals and improve their resistance to pathogens. Despite their extensive use, the precise effects that β-glucans provoke on immune and intestinal cells in teleost fish have not yet been fully elucidated. In the current work, we have used the RTS11 monocyte-macrophage cell line and the RTgutGC intestinal epithelial cell line to further investigate the effects of β-glucans on rainbow trout (Oncorhynchus mykiss), determining the levels of transcription of a wide range of immune genes that code for pro-inflammatory cytokines/molecules, antimicrobial peptides or adhesion molecules. Our results show that RTS11 monocytes/macrophages are more reactive to β-glucans than intestinal epithelial cells. Despite this, β-glucans were able to enhance some of the transcriptional effects of Aeromonas salmonicida on RTgutGC cells as they did on RTS11 cells, evidencing some immunostimulatory effects on gut epithelial cells. Finally, we have also used these two cell lines to establish whether β-glucans induce tolerance, comparing the effects to those exerted by LPS. Pre-exposure of both cell lines to LPS strongly inhibited a secondary response to LPS that affected the transcription of many of the genes studied, confirming the potential of these cell lines to study mechanisms of immune tolerance. However, when both cell lines were stimulated with β-glucans, only interleukin 6 (IL6) transcription was significantly lower in cells that had been previously exposed to β-glucans in comparison to cells that had not been previously stimulated. These results provide valuable information concerning the immunostimulatory and potential tolerogenic effects exerted by β-glucans in both macrophages and gut epithelial cells.
CK12, a rainbow trout chemokine with lymphocyte chemo-attractant capacity associated to mucosal tissues
(Elsevier, 2011) Montero, J; Ordás, MC; Alejo, A; Sevilla, N; Tafalla, C
Although many chemokine genes have been identified in rainbow trout (Oncorhynchus mykiss) as in other teleost species, almost no studies focused on their biological role have been conducted, despite the fact that no clear inferences as to their functions can be made based on their low similarity to mammalian counterparts. In the current work, we have studied the regulation of mRNA transcription and protein expression of CK12, a rainbow trout CC chemokine previously catalogued within the CCL19/21/25 phylogenetic group. Our studies revealed that CK12 is strongly expressed both at mRNA and protein level in mucosal tissues. Mature lymphocyte populations also express CK12 both at mRNA and protein levels. Concerning its biological activity, a significant chemotatic activity towards purified recombinant CK12 in unfractionated leukocyte populations was observed in the spleen, but not in head kidney or blood. Consequently, a binding assay revealed that the number of leukocytes capable of binding CK12 was much more elevated in spleen populations than in leukocyte populations from other organs. This binding capacity was only observed in small lymphocytes that should account for resident inactivated lymphocytes, in contrast to mature lymphocytes that were responsible for CK12 production. Around 36% of these small lymphocytes were IgM+ cells, of which 40% had a CK12 binding capacity. On the other hand, 10% of thymocytes were also capable of CK12 binding, suggesting that both T and B immature lymphocytes are recruited by CK12. This work constitutes the first description of a mucosal-associated chemokine in fish in which important aspects of its regulation and functionality are revealed. © 2011 Elsevier Ltd.
Differential response of RTGUTGC and RTGILL- W1 rainbow trout epithelial cell lines to viral stimulation.
(Wiley, 2023-01-12) Simón, R; Martínez, P; González, L; Ordás, MC; Tafalla, C
Mucosal surfaces constitute the main route of entry of pathogens into the host. In fish, these mucosal tissues include, among others, the gastrointestinal tract, the gills and the skin. However, knowledge about the mechanisms of regulation of immunity in these tissues is still scarce, being essential to generate a solid base that allows the development of prevention strategies against these infectious agents. In this work, we have used the RTgutGC and RTgill-W1 epithelial-like cell lines, derived from the gastrointestinal tract and the gill of rainbow trout (Oncorhynchus mykiss), respectively, to investigate the transcriptional response of mucosal epithelial cells to a viral mimic, the dsRNA poly I:C, as well as to two important viral rainbow trout pathogens, namely viral haemorrhagic septicaemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV). Additionally, we have established how the exposure to poly I:C affected the susceptibility of RTgutGC and RTgill-W1 cells to both viruses. Our results reveal important differences in the way these two cell lines respond to viral stimuli, providing interesting information on these cell lines that have emerged in the past years as useful tools to study mucosal responses in fish.
First in vivo evidence of pituitary adenylate cyclase-activating polypeptide antiviral activity in teleost.
(Elsevier, 2020) Velázquez, J; Pérez, G; Semple, SL; Rodríguez-Ramos, T; Díaz- Rosales, P; Ordás, MC; Lugo, JM; Dixon, B; Tafalla, C; Estrada, MP; Carpio, Y
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.
Functional nutrition modulates the early immune response against viral haemorrhagic septicaemia virus (VHSV) in rainbow trout.
(Elsevier, 2019-11) Leal, E; Ordás, MC; Soleto, I; Zarza, C; McGurk, C; Tafalla, C
Although viruses represent a major threat for cultured fish worldwide, the commercialization of vaccines capable of providing effective and long-lasting protection is still lacking for most of these viral diseases. In this situation, the use of supplemented diets could be a suitable strategy to increase the immune status of the fish and reduce the impact of viral pathogens. Among possible immunostimulants that could be included in these functional feeds, some studies have previously shown that certain β-glucans can significantly increase certain immune parameters of fish and reduce the impact of viral diseases. However, the mechanisms through which β-glucans exert their activity have not been fully elucidated yet. In the current study, we have studied the immune response of different tissues to viral haemorrhagic septicaemia virus (VHSV) in rainbow trout fed with a non-supplemented control diet as well as in fish fed a commercial functional aquafeed (Protec™, Skretting) containing β-glucans, vitamin C, vitamin E and zinc. For this, after 30 days of feeding the fish with one of the two diets, they were subsequently infected with VHSV by bath or mock-infected. After 2 or 6 days post-infection, fish were sacrificed and the levels of transcription of different immune genes such as IgM, IgT, IgD, Mx, interferon γ (IFN γ) and perforin studied in different tissues (kidney, gut and gills). Additionally, the levels of natural IgMs in serum were also determined. Our results demonstrate that fish fed the functional diet were capable of mounting an increased IgM, IgT, IgD and Mx transcriptional response to the virus. Additionally, these fish also showed increased levels of natural IgMs in serum. These results reveal a previously undescribed effect of functional diets on fish Ig production and point to Protec™ as an adequate diet to be incorporated in holistic programs aimed at mitigating the effect of viral diseases.
Hemocitos de mejillón. Producción de radicales de oxígeno y nitrógeno
(Consejo Superior de Investigaciones Científicas, 2007) Novoa, B; Tafalla, C; Ordás, Camino; Figueras, A
Identification of a novel CCR7 gene in rainbow trout with differential expression in the context of mucosal or systemic infection
(Elsevier, 2012) Ordás, MC; Castro, R; Dixon, B; Sunyer, JO; Bjork, S; Bartholomew, J; Korytar, T; Köllner, B; Cuesta, A; Tafalla, C
In mammals, CCR7 is the chemokine receptor for the CCL19 and CCL21 chemokines, molecules with a major role in the recruitment of lymphocytes to lymph nodes and Peyer's patches in the intestinal mucosa, especially naïve T lymphocytes. In the current work, we have identified a CCR7 orthologue in rainbow trout (Oncorhynchus mykiss) that shares many of the conserved features of mammalian CCR7. The receptor is constitutively transcribed in the gills, hindgut, spleen, thymus and gonad. When leukocyte populations were isolated, IgM+ cells, T cells and myeloid cells from head kidney transcribed the CCR7 gene. In blood, both IgM+ and IgT+ B cells and myeloid cells but not T lymphocytes were transcribing CCR7, whereas in the spleen, CCR7 mRNA expression was strongly detected in T lymphocytes. In response to infection with viral hemorrhagic septicemia virus (VHSV), CCR7 transcription was down-regulated in spleen and head kidney upon intraperitoneal infection, whereas upon bath infection, CCR7 was up-regulated in gills but remained undetected in the fin bases, the main site of virus entry. Concerning its regulation in the intestinal mucosa, the ex vivo stimulation of hindgut segments with Poly IC or inactivated bacteria significantly increased CCR7 transcription, while in the context of an infection with Ceratomyxa shasta, the levels of transcription of CCR7 in both IgM+ and IgT+ cells from the gut were dramatically increased. All these data suggest that CCR7 plays an important role in lymphocyte trafficking during rainbow trout infections, in which CCR7 appears to be implicated in the recruitment of B lymphocytes into the gut.
L- methionine supplementation modulates IgM+ cell responses in rainbow trout.
(Frontiers Media, 2023-10-10) Martín, D; Ordás, MC; Carvalho, I; Díaz- Rosales, P; Núñez- Ortiz, N; Vicente- Gil, S; Arrogante, A; Zarza, C; Machado, M; Costas, B; Tafalla, C
The interest in dietary amino acids (AAs) as potential immunomodulators has been growing the recent years, since specific AAs are known to regulate key metabolic pathways of the immune response or increase the synthesis of some immune-related proteins. Methionine, tryptophan and lysine are among the ten essential AAs for fish, meaning that they cannot be produced endogenously and must be provided through the diet. To date, although dietary supplementation of fish with some of these AAs has been shown to have positive effects on some innate immune parameters and disease resistance, the effects that these AAs provoke on cells of the adaptive immune system remained unexplored. Hence, in the current study, we have investigated the effects of these three AAs on the functionality of rainbow trout (Oncorhynchus mykiss) IgM+ B cells. For this, splenic leukocytes were isolated from untreated adult rainbow trout and incubated in culture media additionally supplemented with different doses of methionine, tryptophan or lysine in the presence or absence of the model antigen TNP-LPS (2,4,6-trinitrophenyl hapten conjugated to lipopolysaccharide). The survival, IgM secreting capacity and proliferation of IgM+ B cells was then studied. In the case of methionine, the phagocytic capacity of IgM+ B cells was also determined. Our results demonstrate that methionine supplementation significantly increases the proliferative effects provoked by TNP-LPS and also up-regulates the number of cells secreting IgM, whereas tryptophan or lysine have either minor or even negative effects on rainbow trout IgM+ B cells. This increase in the number of IgM-secreting cells in response to methionine surplus was further verified in a feeding experiment, in which the beneficial effects of methionine on the specific response to anal immunization were also confirmed. The results presented demonstrate the beneficial effects of dietary supplementation with methionine on the adaptive immune responses of fish.
Specific regulation of the chemokine response to viral hemorrhagic septicemia virus (VHSV) at the entry site
(American Society for Microbiology, 2011) Montero, J; García, J; Ordás, MC; Casanova, I; González, A; Coll, J; Tafalla, C
The fin bases constitute the main portal of rhabdovirus entry into rainbow trout (Oncorhynchus mykiss), and replication in this first site strongly conditions the outcome of the infection. In this context, we studied the chemokine response elicited in this area in response to viral hemorrhagic septicemia virus (VHSV), a rhabdovirus. Among all the rainbow trout chemokine genes studied, only the transcription levels of CK10 and CK12 were significantly upregulated in response to VHSV. As the virus had previously been shown to elicit a much stronger chemokine response in internal organs, we compared the effect of VHSV on the gills, another mucosal site which does not constitute the main site of viral entry or rhabdoviral replication. In this case, a significantly stronger chemokine response was triggered, with CK1, CK3, CK9, and CK11 being upregulated in response to VHSV and CK10 and CK12 being down-modulated by the virus. We then conducted further experiments to understand how these different chemokine responses of mucosal tissues could correlate with their capacity to support VHSV replication. No viral replication was detected in the gills, while at the fin bases, only the skin and the muscle were actively supporting viral replication. Within the skin, viral replication took place in the dermis, while viral replication was blocked within epidermal cells at some point before protein translation. The different susceptibilities of the different skin layers to VHSV correlated with the effect that VHSV has on their capacity to secrete chemotactic factors. Altogether, these results suggest a VHSV interference mechanism on the early chemokine response at its active replication sites within mucosal tissues, a possible key process that may facilitate viral entry.
Viral hemorrhagic septicaemia virus (VHSV) up- regulates the cytotoxic activity and the perforin/ granzyme pathway in the rainbow trout RTS11 cell line.
(Elsevier, 2011) Ordás, MC; Cuesta, A; Mercado, L; Bols, NC; Tafalla, C
A survey of immune-relevant genes that might be up-regulated in response to viral hemorrhagic septicaemia virus (VHSV) in the rainbow trout monocyte-macrophage cell line, RTS11, unexpectedly revealed an increased expression of perforin (PRF) and granzyme (GRZ) genes, which represent components of the major cytotoxic pathway. The natural killer-enhancing factor (NKEF), also known to modulate cytotoxic activity, was up-regulated at the gene but strikingly down-regulated at protein level. The expression of these genes was not affected in head kidney leukocytes (HKLs) infected with VHSV, leading us to evaluate the potential cytotoxic activity of RTS11 and HKLs. For the first time, the cytotoxic activity of RTS11 against xenogeneic targets has been demonstrated, although this was modest relative to HKLs. Yet the activity in RTS11 was significantly increased by VHSV, as in HKLs. This cytotoxic activity elicited by viral infection appeared to require viral gene expression because inactivated VHSV failed to increase RTS11 cytotoxic activity. As for other immune functions, RTS11 cells provide a model for further studying cytotoxic activities of fish monocyte-macrophages.
Yersinia ruckeri infection activates local skin and gill B cell responses in rainbow trout.
(Elsevier, 2023-08-06) Herranz- Jusdado, JG; Morel, E; Ordás, MC; Martín, D; Docando, F; González, L; Sanjuán, E; Díaz- Rosales, P; Saura, M; Fouz, B; Tafalla, C
Teleost fish lack organized structures in mucosal tissues such as those of mammals, but instead contain dispersed B and T cells with the capacity to respond to external stimuli. Nonetheless, there is still a great lack of knowledge regarding how B cells differentiate to plasmablasts/plasma cells in these mucosal surfaces. To contribute to a further understanding of the mechanisms through which fish mucosal B cells are activated, in the current study, we have studied the B cell responses in the skin and gills of rainbow trout (Oncorhynchus mykiss) exposed to Yersinia ruckeri. We have first analyzed the transcription levels of genes related to B cell function in both mucosal surfaces, and in spleen and kidney for comparative purposes. In a second experiment, we have evaluated how the infection affects the presence and size of B cells in both skin and gills, as well as the presence of plasmablasts secreting total or specific IgMs. The results obtained in both experiments support the local differentiation of B cells to plasmablasts/plasma cells in the skin and gills of rainbow trout in response to Y. ruckeri. Interestingly, these plasmablasts/plasma cells were shown to secrete specific IgMs as soon as 5 days after the exposure. These findings contribute to a further understanding of how B cells in the periphery respond to immune stimulation in teleost fish.