https://hdl.handle.net/11000/1459 2026-04-07T20:31:06Z 2026-04-07T20:31:06Z Puente Marín, Sara Nombela Díaz, Iván CHICO GRAS, VERONICA Ciordia, Sergio Mena, María Carmen Pérez, Luis Coll Morales, Julio Ortega-Villaizan Romo, María del Mar https://hdl.handle.net/11000/7007 2025-02-19T08:59:26Z 2021-01-25T10:50:09Z

Título : Potential Role of Rainbow Trout Erythrocytes as Mediators in the Immune Response Induced by a DNA Vaccine in Fish Autor : Puente Marín, Sara; Nombela Díaz, Iván; CHICO GRAS, VERONICA; Ciordia, Sergio; Mena, María Carmen; Pérez, Luis; Coll Morales, Julio; Ortega-Villaizan Romo, María del Mar Resumen : In recent years, fish nucleated red blood cells (RBCs) have been implicated in the response against viral infections. We have demonstrated that rainbow trout RBCs can express the antigen encoded by a DNA vaccine against viral hemorrhagic septicemia virus (VHSV) and mount an immune response to the antigen in vitro. In this manuscript, we show, for the first time, the role of RBCs in the immune response triggered by DNA immunization of rainbow trout with glycoprotein G of VHSV (GVHSV). Transcriptomic and proteomic profiles of RBCs revealed genes and proteins involved in antigen processing and presentation of exogenous peptide antigen via MHC class I, the Fc receptor signaling pathway, the autophagy pathway, and the activation of the innate immune response, among others. On the other hand, GVHSV-transfected RBCs induce specific antibodies against VHSV in the serum of rainbow trout which shows that RBCs expressing a DNA vaccine are able to elicit a humoral response. These results open a new direction in the research of vaccination strategies for fish since rainbow trout RBCs actively participate in the innate and adaptive immune response in DNA vaccination. Based on our findings, we suggest the use of RBCs as target cells or carriers for the future design of novel vaccine strategies

2021-01-25T10:50:09Z Puente Marín, Sara Thwaite, Rosemary Mercado, Luis Coll Morales, Julio Roher, Nerea Ortega-Villaizan Romo, María del Mar https://hdl.handle.net/11000/7005 2025-02-19T08:58:34Z 2021-01-25T10:43:31Z

Título : Fish Red Blood Cells Modulate Immune Genes in Response to Bacterial Inclusion Bodies Made of TNFa and a G-VHSV Fragment Autor : Puente Marín, Sara; Thwaite, Rosemary; Mercado, Luis; Coll Morales, Julio; Roher, Nerea; Ortega-Villaizan Romo, María del Mar Resumen : Fish Red-Blood Cells (RBCs) are nucleated cells that can modulate the expression of different sets of genes in response to stimuli, playing an active role in the homeostasis of the fish immune system. Nowadays, vaccination is one of the main ways to control and prevent viral diseases in aquaculture and the development of novel vaccination approaches is a focal point in fish vaccinology. One of the strategies that has recently emerged is the use of nanostructured recombinant proteins. Nanostructured cytokines have already been shown to immunostimulate and protect fish against bacterial infections. To explore the role of RBCs in the immune response to two nanostructured recombinant proteins, TNFa and a G-VHSV protein fragment, we performed different in vitro and in vivo studies. We show for the first time that rainbow trout RBCs are able to endocytose nanostructured TNFa and G-VHSV protein fragment in vitro, despite not being phagocytic cells, and in response to nanostructured TNFa and G-VHSV fragment, the expression of different immune genes could be modulated.

2021-01-25T10:43:31Z Puente Marín, Sara Nombela Díaz, Iván CHICO GRAS, VERONICA Ciordia, Sergio Mena, María Carmen Coll Morales, Julio Mercado, Luis Ortega-Villaizan Romo, María del Mar https://hdl.handle.net/11000/7004 2025-02-19T08:59:26Z 2021-01-25T10:36:54Z

Título : Rainbow Trout Erythrocytes ex vivo Transfection With a DNA Vaccine Encoding VHSV Glycoprotein G Induces an Antiviral Immune Response Autor : Puente Marín, Sara; Nombela Díaz, Iván; CHICO GRAS, VERONICA; Ciordia, Sergio; Mena, María Carmen; Coll Morales, Julio; Mercado, Luis; Ortega-Villaizan Romo, María del Mar Resumen : Fish red blood cells (RBCs), are integral in several biologic processes relevant to immunity, such as pathogen recognition, pathogen binding and clearance, and production of effector molecules and cytokines. So far, one of the best strategies to control and prevent viral diseases in aquaculture is DNA immunization. DNA vaccines (based on the rhabdoviral glycoprotein G [gpG] gene) have been shown to be effective against fish rhabdoviruses. However, more knowledge about the immune response triggered by DNA immunization is necessary to develop novel and more effective strategies. In this study, we investigated the role of fish RBCs in immune responses induced by DNA vaccines. We show for the first time that rainbow trout RBCs express gpG of viral hemorrhagic septicaemia virus (VHSV) (GVHSV) when transfected with the DNA vaccine ex vivo and modulate the expression of immune genes and proteins. Functional network analysis of transcriptome profiling of RBCs expressing GVHSV revealed changes in gene expression related to G-protein coupled receptor (GPCR)-downstream signaling, complement activation, and RAR related orphan receptor a (RORA). Proteomic profile functional network analysis of GVHSV-transfected RBCs revealed proteins involved in the detoxification of reactive oxygen species, interferon-stimulated gene 15 (ISG15) antiviral mechanisms, antigen presentation of exogenous peptides, and the proteasome. Conditioned medium of GVHSV-transfected RBCs conferred antiviral protection and induced ifn1 and mx gene expression in RTG-2 cells infected with VHSV. In summary, rainbow trout nucleated RBCs could be actively participating in the regulation of the fish immune response to GVHSV DNA vaccine, and thus may represent a possible carrier cells for the development of new vaccine approaches.

2021-01-25T10:36:54Z Puente Marín, Sara Nombela Díaz, Iván Ciordia, Sergio Mena, María Carmen CHICO GRAS, VERONICA Coll Morales, Julio Ortega-Villaizan Romo, María del Mar https://hdl.handle.net/11000/7002 2025-02-19T08:59:26Z 2021-01-25T10:28:31Z

Título : In silico functional networks identified in fish nucleated red blood cells by means of transcriptomic and proteomic profiling Autor : Puente Marín, Sara; Nombela Díaz, Iván; Ciordia, Sergio; Mena, María Carmen; CHICO GRAS, VERONICA; Coll Morales, Julio; Ortega-Villaizan Romo, María del Mar Resumen : Nucleated red blood cells (RBCs) of fish have, in the last decade, been implicated in several immune-related functions, such as antiviral response, phagocytosis or cytokine-mediated signaling. RNA-sequencing (RNA-seq) and label-free shotgun proteomic analyses were carried out for in silico functional pathway profiling of rainbow trout RBCs. For RNA-seq, a de novo assembly was conducted, in order to create a transcriptome database for RBCs. For proteome profiling, we developed a proteomic method that combined: (a) fractionation into cytosolic and membrane fractions, (b) hemoglobin removal of the cytosolic fraction, (c) protein digestion, and (d) a novel step with pH reversed-phase peptide fractionation and final Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC ESI-MS/MS) analysis of each fraction. Combined transcriptome- and proteome- sequencing data identified, in silico, novel and striking immune functional networks for rainbow trout nucleated RBCs, which are mainly linked to innate and adaptive immunity. Functional pathways related to regulation of hematopoietic cell differentiation, antigen presentation via major histocompatibility complex class II (MHCII), leukocyte differentiation and regulation of leukocyte activation were identified. These preliminary findings further implicate nucleated RBCs in immune function, such as antigen presentation and leukocyte activation

2021-01-25T10:28:31Z Nombela Díaz, Iván Ortega-Villaizan Romo, María del Mar https://hdl.handle.net/11000/7000 2022-11-23T09:09:29Z 2021-01-25T10:20:26Z

Título : Nucleated red blood cells: Immune cell mediators of the antiviral response Autor : Nombela Díaz, Iván; Ortega-Villaizan Romo, María del Mar Resumen : The involvement of nucleated red blood cells (RBCs) as immune response cell mediators is a novel topic of research. RBCs are the most abundant cell type in the bloodstream and are best known for their roles in gas exchange and respiration. In mammals, mature RBCs are flexible, oval, biconcave disks that lack cell nuclei, organelles, and ribosomes (reviewed in Moras et al. 2017 [1]). In nonmammalian vertebrates, RBCs are oval, flattened, biconvex disks with a cytoskeleton composed of a marginal band of microtubules and a cell nucleus and organelles in their cytoplasm [2], which allow them to de novo synthesize proteins and molecules in response to stress and stimuli. In the recent past, a set of biological processes related to immunity–such as phagocytosis [3], antigen presentation [3], and interleukin-like production [4–7]–have been reported in nucleated RBCs from different species. However, elucidating the role of RBCs during viral infections is an emergent research topic of great interest. Here, we provide a brief overview of the novel role of nucleated RBCs against viral infections

2021-01-25T10:20:26Z Nombela Díaz, Iván Requena Platek, Ricardo Morales Lange, Byron CHICO GRAS, VERONICA Puente Marín, Sara Ciordia, Sergio Mena, María Carmen Coll Morales, Julio Pérez, Luis Mercado, Luis Ortega-Villaizan Romo, María del Mar https://hdl.handle.net/11000/6997 2025-02-19T08:59:26Z 2021-01-25T10:14:01Z

Título : Rainbow Trout Red Blood Cells Exposed to Viral Hemorrhagic Septicemia Virus Up-Regulate Antigen-Processing Mechanisms and MHC I&II, CD86, and CD83 Antigen-presenting Cell Markers Autor : Nombela Díaz, Iván; Requena Platek, Ricardo; Morales Lange, Byron; CHICO GRAS, VERONICA; Puente Marín, Sara; Ciordia, Sergio; Mena, María Carmen; Coll Morales, Julio; Pérez, Luis; Mercado, Luis; Ortega-Villaizan Romo, María del Mar Resumen : Nucleated teleost red blood cells (RBCs) are known to express molecules from the major histocompatibility complex and peptide-generating processes such as autophagy and proteasomes, but the role of RBCs in antigen presentation of viruses have not been studied yet. In this study, RBCs exposed ex vivo to viral hemorrhagic septicemia virus (VHSV) were evaluated by means of transcriptomic and proteomic approaches. Genes and proteins related to antigen presentation molecules, proteasome degradation, and autophagy were up-regulated. VHSV induced accumulation of ubiquitinated proteins in ex vivo VHSV-exposed RBCs and showed at the same time a decrease of proteasome activity. Furthermore, induction of autophagy was detected by evaluating LC3 protein levels. Sequestosome-1/p62 underwent degradation early after VHSV exposure, and it may be a link between ubiquitination and autophagy activation. Inhibition of autophagosome degradation with niclosamide resulted in intracellular detection of N protein of VHSV (NVHSV) and p62 accumulation. In addition, antigen presentation cell markers, such as major histocompatibility complex (MHC) class I & II, CD83, and CD86, increased at the transcriptional and translational level in rainbow trout RBCs exposed to VHSV. In summary, we show that nucleated rainbow trout RBCs can degrade VHSV while displaying an antigen-presenting cell (APC)-like profile

2021-01-25T10:14:01Z Nombela Díaz, Iván López Lorigados, Marina Salvador Mira, María Elizabhet Puente Marín, Sara CHICO GRAS, VERONICA Ciordia, Sergio Mena, María Carmen Mercado, Luis Ortega-Villaizan Romo, María del Mar https://hdl.handle.net/11000/6996 2025-02-19T08:59:26Z 2021-01-25T10:04:58Z

Título : Integrated Transcriptomic and Proteomic Analysis of Red Blood Cells from Rainbow Trout Challenged with VHSV Point Towards Novel Immunomodulant Targets Autor : Nombela Díaz, Iván; López Lorigados, Marina; Salvador Mira, María Elizabhet; Puente Marín, Sara; CHICO GRAS, VERONICA; Ciordia, Sergio; Mena, María Carmen; Mercado, Luis; Ortega-Villaizan Romo, María del Mar Resumen : Integrated Transcriptomic and Proteomic Analysis of Red Blood Cells from Rainbow Trout Challenged with VHSV Point Towards Novel Immunomodulant Targets

2021-01-25T10:04:58Z Nombela Díaz, Iván Carrión Pérez, Lourdes Aurora Puente Marín, Sara CHICO GRAS, VERONICA Mercado, Luis Pérez, Luis Coll Morales, Julio Ortega-Villaizan Romo, María del Mar https://hdl.handle.net/11000/6993 2025-02-19T08:58:34Z 2021-01-25T09:55:16Z

Título : Infectious pancreatic necrosis virus triggers antiviral immune response in rainbow trout red blood cells, despite not being infective [version 2; referees: 2 approved] Autor : Nombela Díaz, Iván; Carrión Pérez, Lourdes Aurora; Puente Marín, Sara; CHICO GRAS, VERONICA; Mercado, Luis; Pérez, Luis; Coll Morales, Julio; Ortega-Villaizan Romo, María del Mar Resumen : Background: Some fish viruses, such as piscine orthoreovirus and infectious salmon anemia virus, target red blood cells (RBCs), replicate inside them and induce an immune response. However, the roles of RBCs in the context of infectious pancreatic necrosis virus (IPNV) infection have not been studied yet. Methods: Ex vivo rainbow trout RBCs were obtained from peripheral blood, Ficoll purified and exposed to IPNV in order to analyze infectivity and immune response using RT-qPCR, immune fluorescence imaging, flow cytometry and western-blotting techniques. Results: IPNV could not infect RBCs; however, IPNV increased the expression of the INF1-related genes ifn-1, pkr and mx genes. Moreover, conditioned media from IPNV-exposed RBCs conferred protection against IPNV infection in CHSE-214 fish cell line. Conclusions: Despite not being infected, rainbow trout RBCs could respond to IPNV with increased expression of antiviral genes. Fish RBCs could be considered as mediators of the antiviral response and therefore targets of new strategies against fish viral infections. Further research is ongoing to completely understand the molecular mechanism that triggers this antiviral response in rainbow trout RBCs.

2021-01-25T09:55:16Z Nombela Díaz, Iván Puente Marín, Sara CHICO GRAS, VERONICA Villena, Alberto Carracedo, Begoña Ciordia, Sergio Mena, María Carmen Mercado, Luis Pérez, Luis Coll Morales, Julio Estepa Pérez, Amparo Ortega-Villaizan Romo, María del Mar https://hdl.handle.net/11000/6990 2025-02-19T08:59:26Z 2021-01-25T09:45:42Z

Título : Identification of diverse defense mechanisms in rainbow trout red blood cells in response to halted replication of VHS virus [version 2; referees: 2 approved, 1 approved with reservations] Autor : Nombela Díaz, Iván; Puente Marín, Sara; CHICO GRAS, VERONICA; Villena, Alberto; Carracedo, Begoña; Ciordia, Sergio; Mena, María Carmen; Mercado, Luis; Pérez, Luis; Coll Morales, Julio; Estepa Pérez, Amparo; Ortega-Villaizan Romo, María del Mar Resumen : Background: It has been described that fish nucleated red blood cells (RBCs) generate a wide variety of immune-related gene transcripts when viruses highly replicate inside them and are their main target cell. The immune response and mechanisms of fish RBCs against viruses targeting other cells or tissues has not yet been explored and is the objective of our study. Methods: Rainbow trout RBCs were obtained from peripheral blood, ficoll purified and exposed to Viral Haemorrhagic Septicaemia virus (VHSV). Immune response was evaluated by means of RT-qPCR, flow cytometry, immunofluorescence and isobaric tag for relative and absolute quantification (iTRAQ) protein profiling. Results: VHSV N gene transcripts incremented early postexposure and were drastically decreased after 6 hours postexposure (hpe). The expression of type I interferon ( ifn1) gene was significantly downregulated at early postexposure (3 hpe), together with a gradual downregulation of interferon-inducible mx and pkr genes until 72 hpe. Type I IFN protein was downregulated and interferon-inducible Mx protein was maintained at basal levels. Co-culture assays of RBCs, previously exposed to UV-inactivated VHSV, and TSS (stromal cell line from spleen) revealed IFN crosstalk between both cell types. On the other hand, anti-microbial peptide β-defensin 1 and neutrophil chemotactic factor interleukin 8 were slightly upregulated in VHSV-exposed RBCs. iTRAQ profiling revealed that VHSV exposure can induce a global protein downregulation in rainbow trout RBCs, mainly related to RNA stability and proteasome pathways. Antioxidant/antiviral response is also suggested to be involved in the response of rainbow trout RBCs to VHSV. Conclusions: A variety of mechanisms are proposed to be implicated in the antiviral response of rainbow trout RBCs against VHSV halted infection. Ongoing research is focused on understanding the mechanisms in detail.

2021-01-25T09:45:42Z Jung, Myung-Hwa CHICO GRAS, VERONICA Ciordia, Sergio Mena, María Carmen Jung, Sung-Ju Ortega-Villaizan Romo, María del Mar https://hdl.handle.net/11000/6988 2025-02-19T08:59:26Z 2021-01-25T09:32:31Z

Título : The Megalocytivirus RBIV Induces Apoptosis and MHC Class I Presentation in Rock Bream (Oplegnathus fasciatus) Red Blood Cells Autor : Jung, Myung-Hwa; CHICO GRAS, VERONICA; Ciordia, Sergio; Mena, María Carmen; Jung, Sung-Ju; Ortega-Villaizan Romo, María del Mar Resumen : The Megalocytivirus RBIV Induces Apoptosis and MHC Class I Presentation in Rock Bream (Oplegnathus fasciatus) Red Blood Cells

2021-01-25T09:32:31Z