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

Periodic Reporting for period 3 - BLOODCELLSCROSSTALK (The Crosstalk Between Red And White Blood Cells: The Case Of Fish)

Reporting period: 2018-04-01 to 2019-09-30

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 confer
We have carried out during this period the study of the implication of rainbow trout red blood cells (RBCs) in the immune response to viral infection, by means of transcriptomic and proteomic evaluation, in vitro and in vivo. As well, we have evaluated the crosstalk between red blood cells (RBCs) and other immune cells, such as leukocytes and stromal cells, in the scenario of fish viral infections. The work carried out includes objective 1, tasks 1.1 1.2 1.3 and 1.4 which have been achieved, with a few items added to the initial plan. In addition, task 1.5 has been added to the initial plan, due to the striking discovery of a possible novel red blood cell stage.
On the other hand, we have prioritised to continue in this reporting period with objective 2, motivated by the results obtained in objective 1, in order to evaluate at the same time the implication of rainbow trout red blood cells (RBCs) in the immune response to DNA vaccines with the viral antigen as well as to the virus itself. Therefore, task 2.1 and 2.2 which include the transcriptomic and proteomic evaluation of RBCs after DNA vaccination, in vitro and in vivo, have been nearly achieved in this reporting period, with a few items still on-going. Moreover, task 2.3 has been added to the initial plan motivated by the results obtained after DNA vaccination and with the aim of investigating the role of RBCs under other vaccine types, bacterial-based vaccines.
In consequence, the most significant deviations for this reporting period are task 1.2 has been postponed to year 4, tasks 2.1 and 2.2 have been advanced, and task 1.5 and 2.3 have been added to the initial plan.
Finally, the future plans for the next reporting period include finishing tasks 1.2 1.5 2.2 and 2.3.
The results obtained until now have unveiled a previously unobserved but important function for fish nucleated RBCs in the contribution to immune defenses against viral aggression. The results showed that rainbow trout RBCs might have the capacity of inactivating internalized VHSV and IPNV viruses by means of endogenous antiviral proteins. Besides, RBCs-stimulated cytokines might be implicated in the modulation of other immune cells, and/or in the inflammatory response. The results suggested that RBCs may act as Antigen Presenting Cells (APCs) and participate in the adaptive immunity response against viral infections. Besides, RBCs immune response to a DNA vaccine and to a bacterial-based vaccine encoding the viral antigen showed an immune response mediated by chemokines, interferon stimulated proteins, antimicrobial peptides and antigen presentation. Furthermore, we have unveiled the striking capacity of rainbow trout nucleated red blood cells to derive to a novel cell stage, which we have termed shape-shifted red blood cells (shRBCs). shRBCs demonstrated a role in the antiviral response to VHSV as well as in the mediation of the immune response.