The role of chromatin extracellular traps in host defence of fish against pathogens.

Chromatin, a complex of DNA, RNA and protein, is the material that makes up the nucleus of a cell and serves to store genetic information. However, strong evidence has accumulated that it also has a second function, remarkably as a weapon in defence against bacteria and other microbes. In both mammals and invertebrates, some dedicated immune cells involved in inflammation, expel their chromatin in response to the presence of microbes in a controlled and regulated way to the exterior of the cell. It then billows out, like a cloud, to form a mesh that traps pathogens and kills them by virtue of small antibiotic proteins from the cell cytoplasm that become studded on the fibres. Put simply it is a cell death process that enables an immune cell to continue combatting infectious agents even after it has died. The present project aims to investigate this process in fish, as, despite it being widely regarded as an important immune strategy in humans and other mammals, it has not yet been considered in the context of fish health and protection against infection. Disease is a huge problem in fish farming. It causes significant financial losses to the producers and undesirable suffering for the fish. The problem of disease is also likely to worsen as sea temperature rises through climate change. Despite efforts to develop vaccines for fish over recent decades, many do not provide full protection against infections. This is because of strain variability in the pathogens and the structure of the 'memory' component of the fish's immune system that is responsible for producing long-lived antibodies. Instead, fish rely heavily on inflammation, or 'innate' (i.e. inborn) processes, such as phagocytosis (bacteria-eating) and production of natural antibiotic proteins, to protect themselves. Therefore the innate system of fish is an important target for preventing and controlling infection. The proposed research will be directed at Atlantic salmon, as this is the major aquaculture species in the UK, with many fish farms located in Scotland. The work will use in vitro (i.e. cell culture) methods to assess which inflammatory cells in salmon deploy chromatin to kill pathogens, how strong the responses of these cells are and how efficiently pathogens are killed on the chromatin nets. A range of fish pathogens known to cause serious and, for the producers, devastating, diseases in their stock animals will be used in these experiments. These pathogens also include representative types of different microbial groups, so it should be possible to discover if some are able to block, evade or escape chromatin entrapment. To investigate the likely impact of water temperature on this aspect of the fish host defences, the work will compare the strength of the chromatin release response and the efficiency of pathogen killing by cells sampled from fish that have been acclimated to different water temperatures ranging from 9 to 19 degrees Celsius. The highest value in this range covers temperatures experienced by farmed salmon at some sites in Scotland where sea temperatures have already risen, but will be more commonly encountered if coastal water temperatures increase by 2 degrees Celsius, as predicted by some climate change models. This part of the project will help to inform us if farmed fish will become more susceptible to disease under future climatic conditions. The research will generate new information about the importance of a highly novel immune process in fish and thus enable us understand better how pathogens and their host interact. The work may further underpin the development of new compounds, feed additives or strategies to limit the problem of disease in aquaculture.

Atlantic salmon is the major aquaculture species in the UK, worth over £670M annually to the economy. However, it is vulnerable to the devastating effect of transmissible disease that can cost the industry dearly. Many of the available vaccines do not provide full protection so additional strategies are needed. This project aims to investigate a relatively recently discovered immunological process, ETosis, that has not previously been studied or considered in fish health in relation to defence against pathogens. ETosis is part of the innate immune repertoire and entails the controlled release of chromatin from the nucleus of some phagocytic and other inflammatory cells. The extruded material forms extracellular meshes that entrap bacteria and other microbial parasites. These extracellular traps are studded with antimicrobial proteins that kill the ensnared micro-organisms. Using in vitro approaches with highly pure populations of fish blood cells, the project will characterise and quantify the strength of the ETotic response by each of the cell types in salmon, and then determine the efficiency of trapping and killing of selected salmonid pathogens by the ETotically-competent cells. This will provide a measure of the contribution made by this defence strategy to pathogen clearance in vivo. With this information, the project will finally investigate how the environmental temperature experienced by salmon in vivo affects the microbicidal efficiency of the response. This should help our understanding as to how climate change might impact on susceptibility of salmon to microbial infection. The knowledge gained will help inform the development of new feed supplements or drugs to enhance resistance to infection in aquaculture. The techniques employed for the research include: cell culture, flow cytometry, fluorimetry, cell sorting, high-resolution confocal and scanning electron microscopy plus microbiological methodologies.

Who will benefit from this research?

The stakeholders that will benefit from the present research are academics in various fields, industrialists in aquaculture, policy makers and the general public. A series of activities are planned to ensure maximum engagement with the project outputs and the data generated, and these are detailed in the Pathways to Impact document.

How will they benefit from this research?

Economic and societal impact

This proposal aims to increase the efficiency and productivity of the aquaculture industries of the UK, particularly salmon production, by losses caused by transmissible microbial diseases. If successful, this project is expected increase the output of the finfish industry thus supporting the devolved Scottish Government's target of growing these industries by almost 30% by 2020. This target aims to create wealth and employment opportunities across Scotland but this will of course impact advantageously on the rest of the UK. Improvements in UK aquaculture production will allow the global market for UK salmon to continue to expand, and this will meet increased demand especially from Asian countries. However, while this proposal aims to strengthen the prosperity of the UK salmon industry, its findings may lead to interest in the immune process in other finfish, thus impacting on aquaculture species internationally. Still, as pioneers in the research outlined in this proposal, the UK will be well-placed to benefit financially from transfer of these ideas into other species produced in aquaculture systems elsewhere.

This project will contribute to UK policies on sustainable and secured food production, while ensuring the health and well-being of the UK population. The ultimate driver of this research is to permit consumers in the UK to benefit from access to a high-quality and healthy protein product at reasonable cost. The consumption of oily fish, such as salmon, is advised by the UK Government at least once per week due to the health benefits associated particularly with certain vital nutrients contained in these fish, including long-chain polyunsaturated omega-3 fatty acids and vitamin D. Thus, this project will deliver health benefits to society at large through the increased availability of highly nutritious food.

Academic impact

Perhaps one of the most important impacts of this project will be through the training of a PDRA who will gain enhanced knowledge of salmon aquaculture and expertise in cell sorting by fluorescence activated sorters, flow cytometry, cell culture, confocal and electron microscopy, immunocytochemistry and microbiology. All of these techniques will be transferrable to other disciplines. However, the PDRA will become a skilled researcher in fish-pathogen interactions and immunology, which are fields of increasing importance particularly with respect to the burgeoning UK finfish aquaculture industry. Thus, the skills developed by the PDRA will also help to support the development of commercial aquaculture in the UK.

In addition to this, a key aspect of this project is the mentorship of the new investigator APD by AR and VJS, who are experienced academics with strong track records of Research Council funding. This will provide APD with an excellent opportunity to develop further as an independent researcher under the guidance of more experienced colleagues.