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The Crosstalk Between Red And White Blood Cells: The Case Of Fish

Periodic Reporting for period 4 - BLOODCELLSCROSSTALK (The Crosstalk Between Red And White Blood Cells: The Case Of Fish)

Reporting period: 2019-10-01 to 2020-10-31

What is the issue being addressed?

Fish are the phylogenetically oldest vertebrate group with an immune system with clear similarities to the immune system of mammals. However, it is an actual matter of fact that the current knowledge of the fish immune system seems to lack the key piece to complete the puzzle.
In 1953 Nelson described a new role of human red blood cells (RBCs) which would go beyond the simple transport of O2 to the tissues. This new role, involved in the defence against microbes, described the antibody and complement-dependent binding of microbial immune complexes to RBCs. Regardless of the importance of this finding in the field of microbial infection, this phenomenon has been poorly evaluated. Just recently, a set of biological processes relevant to immunity has been described in the RBCs of a diverse group of organisms, which include: pathogen recognition, pathogen binding and clearance and cytokines production.
Furthermore, it has been demonstrated that nucleated erythrocytes from fish and avian species develop specific responses to different pathogen-associated molecular patterns and produce soluble factors that modulate leukocyte activity.
In the light of these pieces of evidence, and in an attempt to improve the knowledge of the immune mechanism(s) responsible for fish protection against viral infections, we raised the question: could nucleated fish erythrocytes be the key mediators of the antiviral responses? To answer this question we decided to focus our project on the evaluation of the crosstalk between red and white blood cells in the scenario of fish viral infections and prophylaxis. For that a working model composed of the rainbow trout and the viral haemorrhagic septicaemia virus (VHSV) was chosen, being the objectives of the project to evaluate: i) the implication trout RBCs in the clearance of VHSV, and ii) the involvement of RBCs in the blood transportation of the glycoprotein G of VHSV (GVHSV), the antigen encoded by the DNA vaccine.

Why is it important for society?

The results obtained from this project will have a strong impact in the field of fish immunology, since they will contribute to understand the function of the RBCs in fish in relation to viral infections and DNA vaccinations. This will allow the design of novel vaccines and therapies to reinforce fish immunity and to find novel drug delivery systems. Both will have a considerable impact on the aquaculture industry, since they will permit the production of healthier fish, leading to an increased and eco-sustainable aquaculture. And, on the other hand, the results could be translated to humans, and they will help to discover new roles for RBCs in mammals.

What are the overall objectives?

A great deal of the success of a vaccine in piscine species depends on the knowledge of the fish immune system. For that, the global objective of the present project is to elucidate the role of the nucleated fish RBCs in the immune response to viral infections and related DNA vaccines. Next specific interdisciplinary objectives, comprising the fields of immunology, virology, genomics, transcriptomics and proteomics, are being addressed:

1.-Evaluate the implication of trout RBCs in VHSV binding, antiviral signalling and viral clearance, comprising: i) in vitro characterization of the association between VHSV infected trout RBCs and trout leukocytes; ii) characterization of the complement pathways involved in RBCs/VHSV immune adherence; iii) involvement of RBCs in the blood clearance of VHSV virus after rainbow trout VHSV infection; and iv) characterization of the immune system receptor- response networks implicated in the RBCs response to VHSV infection, by means of transcriptome and proteome evaluation.

2.- Involvement of trout RBCs in the blood transportation of the glycoprotein G of VHSV (GVHSV), the antigen encoded by the DNA vaccine against VHSV, in order to demonstrate the implication of trout RBCs on the protection conferred by the DNA vaccine, by means of: i) in vitro characterization of the immune system networks implicated in the RBCs response to GVHSV gene transfection by transcriptome and proteome analysis; and ii) in vivo involvement of RBCs in the transportation of the GVHSV DNA vaccine and evaluation of the crosstalk between GVHSV transfected trout RBCs and trout leukocytes.
In this project we have carried out the investigation of the implication of rainbow trout red blood cells (RBCs) in the immune response to a viral infection and to its prophylaxis, in vitro and in vivo, by means of transcriptomic, proteomic, and functional assays evaluation. We have completed all the tasks planned and moreover tasks were added to the initial plan motivated by the results obtained.
The results obtained in the project have unveiled a previously unobserved but important function for fish nucleated RBCs in the contribution to immune defences against viral aggression. The results, based on transcriptomic, proteomic, and functional assays analyses, carried out in vitro and in vivo, have shown that rainbow trout RBCs have the capacity of mounting an antiviral response against viral hemorraghic septicaemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV), by means of endogenous antiviral proteins and modulation of complement decay factors. Besides, RBCs stimulated cytokines are implicated in the modulation of other immune cells, and/or in the inflammatory response. On the other hand, using another working model, the Korean rock bream and the rock bream iridovirus (RBIV), rock bream RBCs happened to be permissive to RBIV infection and triggered the stimulation of proteins related antigen presentation and apoptosis. The results also pointed out that RBCs may act as Antigen Presenting Cells (APCs), by means of processing and presenting viral antigens, and therefore participating in the adaptive immune response against viral infections. On the other hand, IFIT5, an antiviral protein found in rainbow trout RBCs to decrease VHSV infection. On the other hand, RBCs have shown an immune response to a DNA vaccine and to bacterial-based vaccines encoding the viral antigen mediated by chemokines, interferon stimulated proteins, antimicrobial peptides, antigen presentation, and participation in antigen-specific antibody production, evaluated by means of transcriptomic, proteomic, and functional assays analyses, in vitro and in vivo. Separately, we have unveiled the striking capacity of rainbow trout nucleated red blood cells to derive into a novel cell stage, which we have termed shape-shifted red blood cells (shRBCs). shRBCs have demonstrated a role in the antiviral response to VHSV as well as in the mediation of the immune response and possible participation in immune cell differentiation and renewal.
This project outcomes revealed the previously unknown function of fish RBCs in the antiviral response against VHSV and IPNV viruses, leading to halt viral replication inside RBCs by means of several mechanisms such as interferon stimulation and antigen processing and presentation. Besides, we have demonstrated that fish RBCs are also capable of enhancing the immune response elicited by different vaccines strategies. These outcomes are a breakthrough in the field of fish immunology since they constitute the first statement of fish RBCs as mediators of the host antiviral immune response.
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