Investigating sub-functionalization of CD4+ T cells in the model teleost zebrafish

Lead Research Organisation: University of Manchester
Department Name: School of Medical Sciences

Abstract

Many of us enjoy a fish supper, and this has been made more affordable and sustainable through the development of the fish farming industry. Farmed fish provide an important source of healthy, nutritious food, and is a growth industry that now employs several thousand people and contributes significantly to the UK economy. But can we guarantee the future of farmed fish when, like many intensively reared animals, they are susceptible to infection? Vaccination is a well-established and safe method for protecting livestock (and indeed humans) from infectious disease. Effective vaccines generate an immune response that is appropriate for the pathogen: cells infected with virus need to be killed by so called killer T cells, while blood-borne bacteria need to be soaked up by antibodies. Different subsets of a type of white blood cell known as helper T cells are essential for co-ordinating the behaviour of those immune cells capable of neutralizing pathogens. While helper cells and associated genes have been identified in fish, their function has yet to be investigated. As such, it is currently unknown how similar fish and mammalian immune responses are. This lack of understanding is hampering the development of fish vaccines. We propose to study the immune system of zebrafish, a fish now used widely in research, to learn more about the immune system of fish generally. Unlike the fish we typically eat, zebrafish can be genetically modified and studied under the microscope. We aim to vaccinate normal zebrafish but also zebrafish with mutations in genes whose function in mammalian immunity is well recognised and study how helper cells in these different groups of zebrafish respond to the vaccines. We hope also to be able to visualise the individual cells responding to the vaccine. Ultimately, we hope our research and the future research it inspires will translate into the development of effective vaccines that protect economically valuable farmed fish from infectious disease.

Technical Summary

From humans and mammalian models, CD4+ T cells are known to perform essential roles in immunity, including the activation of CD8+ T-cells, innate immune cells, and B-lymphocytes to fend off pathogens. CD4+ T cells are also required for self-tolerance thereby preventing auto-immune disease. The central position of these cells in coordinating immunity has made understanding them a priority, particularly with a view to manipulating the immune system towards an efficacious humoral or cellular response, following challenge with specific immunogens delivered in vaccines. However, our knowledge of CD4+ T-cells in fish remains markedly limited in comparison and this is hampering the development of fish vaccines that could help protect these valuable creatures, which are increasingly being intensively farmed. Farmed fish constitute an important source of healthy food as well as a significant source of revenue for the UK economy. Zebrafish are a teleost species being used extensively for biomedical research due to a constellation of features and the development of methodologies that allow rapid and efficient interrogation of gene and cellular function. In this application, we propose utilising zebrafish mutants and transgenics to accelerate acquisition of understanding of the regulation of fish immunity. Specifically, we want to test the hypothesis that fish immunity is co-ordinated by CD4+ T cells comparably to mammals, with subsets of Th1-like (IFN-gamma producing) or Th2-like (IL-4 producing) cells being induced in response to immunisation. Moreover, we will assess the effect of inactivating or forcing the expression of the fish homologues of these cytokines on the ability of CD4+ cells to manifest a Th1- or Th2-like expression profile, as well as directly visualise differentiation through the construction of transgenic reporter animals.

Planned Impact

Industry
Aquaculture is an important economic sector in Scotland, supporting more than 6000 jobs in rural areas. Scotland currently produces nearly 200,000 tonnes of Atlantic salmon and 7,000 tonnes of rainbow trout and is the third largest producer of farmed salmon in the world. The salmon industry is estimated to be worth >£400M annually, with a retail value estimated at >£1 billion. Diseases pose a constant threat to the industry and some cause severe economic loss. Having an understanding of fish adaptive immune responses will be crucial in developing effective vaccines. This project aims to gain an in-depth understanding of the regulatory mechanisms involved in the adaptive immunity of fish, using the zebrafish as a model. Thus, this work will have a potential future beneficial impact on the aquaculture industry by aiding vaccine development, which require the correct type of adaptive immune response to be produced. Zebrafish can also be used to model infection with fish pathogens and thus could become the 'preclinical' model of choice for developing immunotherapeutics intended for farmed fish. Professor Chris Secombes has developed significant long-standing links with the aquaculture and allied industries and has worked successfully with industry partners to develop vaccines. His involvement as co-investigator will maximise the potential of the research to be translated ultimately and he will communicate our findings to existing industry partners with a view to attracting funding to sustain and translate this research effort.

Human and animal health
Zebrafish is now established as an important model for investigating development, physiology and disease mechanisms that are relevant to man and other vertebrates. Our knowledge of the immune system in zebrafish is, however, still in its infancy, impeding the application of this model to furthering our understanding of the role of immunity in maintaining health and causing disease. Efforts like ours will begino unlock the potential of zebrafish in this domain of research, with, in the long run, potential health benefits to man and other animals.

NC3Rs
Depending on the extent of concordance between zebrafish and mammalian immune regulation, zebrafish could potentially replace mouse as a model organism for studying the immune system. This would be considered a refinement as fish have lower neurological complexity than mouse.

Public engagement/outreach
The Hurlstone lab participates in the annual community open day in the faculty, where members of the public talk to lab staff and the PI about the lab's research and get to view fluorescent and translucent zebrafish embryos.

Training and career development
All investigators on the grant will benefit from broadening their knowledge and skill base. The research co-investigator Dr Christopher Dee has helped draft this proposal and thus has already begun his induction in fish immunology and furthered his experience in grant writing. Dr Dee has a 14 month contract only, to cover maternity leave, but would like to remain in the Hurlstone lab and extend the research he is currently pursuing. Subsequently, he can use the theoretical and practical expertise he would acquire through pursuing this grant to establish an independent line of research. A technician currently funded in the lab would also be employed on this grant and acquire experience of a new discipline, extending her repertoire and scope for future employment. Typically the lab supervises three undergraduate project students in a year and two MSc students. It is expected that staff employed on this project would assist in the training and supervision of several of these students over the life-time of the grant.

Publications

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Description We have developed a reagent for visualising specific immune cells in zebrafish and performed a molecular characterisation of those cells. This has allowed us to conclude that a protein called CD4, conserved in mammals including humans, is expressed in zebrafish T cells and mononuclear phagocytes. Further, we conclude that the T cells in zebrafish specialise in ways similar to mammals including humans to perform functions appropriate to the microorganisms they encounter. Thus T cells in the gill where gaseous exchange occurs and that are exposed to the aqueous environment express cytokines il4 and il13 which promote antibody production (humoral immunity), mucous secretion and tissue integrity. In contrast T cells in the gut express cytokines il10 and tgf-beta which suppress immune responses and thereby prevent allergic reactions to food and gut-resident bacteria.
Exploitation Route To date our work has been cited by another 23 scientific articles covering a wide range of disciplines and interests. We have shared the transgenic reporter line we made with seven other institutions in America and Europe, including world-leading biomedical research centres and a contract research organisation who are evaluating its potential for toxicity studies.
Our findings and our reagents support research into fish T cells, with broad implications for fish health and potentially the development of fish vaccines. As zebrafish is also used for biomedical research, we anticipate our findings and our reagents supporting research into leukaemia, infection and immunity.
Sectors Pharmaceuticals and Medical Biotechnology