RED FLAG. Salmonid red blood cells as sensors of stress and infection

Red blood cells (RBC) serve the essential function of transporting oxygen to the organs of the body, and keep us alive. In contrast to human red blood cells that lack a nucleus and DNA, red blood cells in fish, reptiles and birds have the cell nucleus intact and can respond to changes in blood plasma components that reflect the state of health, and this may again affect their natural role as oxygen carriers. Salmonid aquaculture give rise to healthy and nutritious food that are transported all over the world. Ensuring that farmed salmonids are in good health is very important for sustainability of the industry. One still lack effective tools for monitoring salmonid health, and understanding of salmonid biology is still incomplete. The RED FLAG Project aims to provide and share information on how salmonid red blood cells sense danger - like stress and infection, and on how these danger signals affects gas exchange and fish survival. Based on the integration of signals in red blood cells, a small blood sample will be tested as source of information on the state of health in salmon and rainbow trout. Salmonid red blood cells can be infected by Piscine orthoreovirus (PRV-1), whereas no known cell lines from salmonids are susceptible. The RED FLAG group have studied the transcriptome response of red blood cells in contact with PRV-1 in cell culture, compared with two salmonid cell lines. We show that genes involved in antiviral signaling pathways, including interferon response factor subtypes (IRFs), are differencially expressed between the cell types both initially and after PRV-1 exposure. In vivo in Atlantic salmon we study how the blood cell transcriptome reflects differences between infection with the PRV-variant that lead to Heart and skeletal muscle Inflammation (HSMI) in farmed salmon (PRV-1), and a variant that do not lead to disease - but immunological cross-protection (PRV-3). We show that mechanisms regulating virus entry mechanisms - and the power and timing of antiviral responses - may be involved. We have optimized our ex vivo culture model to study how stress and infection responses are integrated, and show that different stress hormones (adrenalin/epinephrine and cortisol) affects the response to viruses in very different ways. We have identified specific target genes signalling stress hormone effects that represent promising biomarker candidates in vivo, and can help us better understand the mechanisms of interaction between stress and infection. The RED FLAG project group has had significant international collaboration, and arranged a workshop with partners from NMBU, UBC -Canada and UMH - Spain in June 2023, which also contained an open/online RED FLAG seminar. In Oct-Nov 2023 the RED FLAG project group had a visit from a post doc researcher from the UBC partner lab, studying how algea toxins affect red blood cell survival and gene expression in salmonid red blood cells. From June 2023 (until Feb 2024), an exchange PhD has been visiting from the University of Valdivia, Chile on a project comparing effects of PRV-3 infection in RBC from Coho salmon and Atlantic salmon. The project group has also worked on establishing transfection of mRNA and siRNA in RBC, and has recruited an NMBU master student on siRNA knock down of genes involved in antiviral response pathways, in collaboration with the NMBU partner.

RED FLAG Salmonid red blood cells as sensors of internal health The RED FLAG project aims to clarify the ability of nucleated pluripotent red blood cells (RBCs) of salmonids to sense “danger” like stress and infection. The initial focus is on how the signals elicited by acute/chronic stress hormones and viral infections impact red blood cell fate, regulation of gas Exchange, immunological functions, and thereby fish health and survival. The project will establish and optimize red blood cell ex vivo models for mechanistic studies, and determine blood cell fate in vivo through use of cell tracers. The project covers exploration of the RBC intracellular signalling pathways and transcriptional/translational control, cell signalling crosstalk and effects on gene expression, cellular functions and differentiation potential. The new knowledge on RBC responses will be used to introduce a minimal blood sample as the source of biomarkers of previous exposure to infection and stress. RED FLAG will represent a starting point of establishing red blood cells as sensors and information providers, with the expanding potential of using RBC responses to also evaluate effects of environmental conditions, chemical treatments, and different composition of feed– i.e. fish RBCs as the source of integrated information on internal Health.