Nucleated Red Blood Cells Contribute to the Host Immune Response Against Pathogens

It has recently come to light that nucleated red blood cells (RBCs) of fish, amphibians, reptiles and birds are multifunctional cells, because in addition to being involved in gas exchange and transport, it has also been reported that they respond to pathogens by means of (i) phagocytosis, (ii) antigen presentation, (iii) production of cytokines and antimicrobial peptides, (iv) regulation of complement system, and (v) exerting paracrine molecular communication with other immune cells and modulating their functions. Similarly, human cord blood nucleated RBCs have been shown to exert a regulatory function in the innate immune response, by means of the suppression of the production of inflammatory cytokines. This chapter comprises the study of the implications of nucleated RBCs as mediators of both branches of immune system (innate and adaptive immune responses)

Fish Red Blood Cells Modulate Immune Genes in Response to Bacterial Inclusion Bodies Made of TNFa and a G-VHSV Fragment

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.This work was supported by the European Research Council fund to MO-V (ERC Starting Grant GA639249)and by grants from the Spanish Ministry of Science, European commission and AGAUR funds to NR (AGL2015-65129-R MINECO/FEDERand 2014SGR- 345 AGAUR). RT holds a pre-doctoral scholarship from AGAUR (Spain)

The immunogenicity of viral haemorragic septicaemia rhabdovirus (VHSV) DNA vaccines can depend on plasmid regulatory sequences

A plasmid DNA encoding the viral hemorrhagic septicaemia virus (VHSV)-G glycoprotein under the control of 5′ upstream sequences (enhancer/promoter sequence plus both non-coding 1st exon and 1st intron sequences) from carp β-actin gene (pAE6-GVHSV) was compared to the vaccine plasmid usually described the gene expression is regulated by the human cytomegalovirus (CMV) immediate-early promoter (pMCV1.4-GVHSV). We observed that these two plasmids produced a markedly different profile in the level and time of expression of the encoded-antigen, and this may have a direct effect upon the intensity and suitability of the in vivo immune response. Thus, fish genetic immunisation assays were carried out to study the immune response of both plasmids. A significantly enhanced specific-antibody response against the viral glycoprotein was found in the fish immunised with pAE6-GVHSV. However, the protective efficacy against VHSV challenge conferred by both plasmids was similar. Later analysis of the transcription profile of a set of representative immune-related genes in the DNA immunized fish suggested that depending on the plasmid-related regulatory sequences controlling its expression, the plasmid might activate distinct patterns of the immune system. All together, the results from this study mainly point out that the selection of a determinate encoded-antigen/vector combination for genetic immunisation is of extraordinary importance in designing optimised DNA vaccines that, when required for inducing protective immune response, could elicit responses biased to antigen-specific antibodies or cytotoxic T cells generation

The Megalocytivirus RBIV Induces Apoptosis and MHC Class I Presentation in Rock Bream (Oplegnathus fasciatus) Red Blood Cells

The Megalocytivirus RBIV Induces Apoptosis and MHC Class I Presentation in Rock Bream (Oplegnathus fasciatus) Red Blood CellsThis research was supported by the European Research Council (ERC Starting Grant GA639249)and by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2015R1C1A1A01053685The proteomic analysis was performed in the Proteomics Facility of The Spanish National Center for Biotechnology (CNB-CSIC) that belongs to ProteoRed, PRB3-ISCIII, supported by grant PT17/001

In Silico Functional Networks Identified in Fish Nucleated Red Blood Cells by Means of Transcriptomic and Proteomic Profiling

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 activationThis work was supported by the European Research Council (ERC Starting Grant GA639249The proteomic analyses were performed in the Proteomics Facility of The Spanish National Center for Biotechnology (CNB-CSIC) that belongs to ProteoRed, PRB2-ISCIII, supported by Grant PT13/000

Autophagy-inducing peptides from mammalian VSV and fish VHSV rhabdoviral G glycoproteins (G) as models for the development of new therapeutic molecules

It has not been elucidated whether or not autophagy is induced by rhabdoviral G glycoproteins (G) in vertebrate organisms for which rhabdovirus infection is lethal. Our work provides the first evidence that both mammalian (vesicular stomatitis virus, VSV) and fish (viral hemorrhagic septicemia virus, VHSV, and spring viremia carp virus, SVCV) rhabdoviral Gs induce an autophagic antiviral program in vertebrate cell lines. The transcriptomic profiles obtained from zebrafish genetically immunized with either Gsvcv or Gvhsv suggest that autophagy is induced shortly after immunization and therefore, it may be an important component of the strong antiviral immune responses elicited by these viral proteins. Pepscan mapping of autophagy-inducing linear determinants of Gvhsv and Gvsv showed that peptides located in their fusion domains induce autophagy. Altogether these results suggest that strategies aimed at modulating autophagy could be used for the prevention and treatment of rhabdoviral infections such as rabies, which causes thousands of human deaths every year

The Megalocytivirus RBIV Induces Apoptosis and MHC Class I Presentation in Rock Bream (Oplegnathus fasciatus) Red Blood Cells

Rock bream iridovirus (RBIV) causes severe mass mortality in Korean rock bream (Oplegnathus fasciatus) populations. To date, immune defense mechanisms of rock bream against RBIV are unclear. While red blood cells (RBCs) are known to be involved in the immune response against viral infections, the participation of rock bream RBCs in the immune response against RBIV has not been studied yet. In this study, we examined induction of the immune response in rock bream RBCs after RBIV infection. Each fish was injected with RBIV, and virus copy number in RBCs gradually increased from 4 days post-infection (dpi), peaking at 10 dpi. A total of 318 proteins were significantly regulated in RBCs from RBIV-infected individuals, 183 proteins were upregulated and 135 proteins were downregulated. Differentially upregulated proteins included those involved in cellular amino acid metabolic processes, cellular detoxification, snRNP assembly, and the spliceosome. Remarkably, the MHC class I-related protein pathway was upregulated during RBIV infection. Simultaneously, the regulation of apoptosis-related proteins, including caspase-6 (CASP6), caspase-9 (CASP9), Fas cell surface death receptor (FAS), desmoplakin (DSP), and p21 (RAC1)-activated kinase 2 (PAK2) changed with RBIV infection. Interestingly, the expression of genes within the ISG15 antiviral mechanism-related pathway, including filamin B (FLNB), interferon regulatory factor 3 (IRF3), nucleoporin 35 (NUP35), tripartite motif-containing 25 (TRIM25), and karyopherin subunit alpha 3 (KPNA3) were downregulated in RBCs from RBIV-infected individuals. Overall, these findings contribute to the understanding of RBIV pathogenesis and host interaction

Integrated Transcriptomic and Proteomic Analysis of Red Blood Cells from Rainbow Trout Challenged with VHSV Point Towards Novel Immunomodulant Targets

Integrated Transcriptomic and Proteomic Analysis of Red Blood Cells from Rainbow Trout Challenged with VHSV Point Towards Novel Immunomodulant TargetsThis work was supported by the European Research Council (ERC Starting Grant GA639249).The proteomic analysis was performed in the Proteomics Facility of the Spanish National Center for Biotechnology (CNB-CSIC) belonging to ProteoRed, PRB3-ISCIII, supported by grant PT17/001