Novel zebrafish approaches to investigate leaderless protein secretion in vivo

Lead Research Organisation: University of Sheffield
Department Name: Infection Immunity & Cardiovasc Disease

Abstract

All cells within the body traffic their proteins either to compartments within the cell, or to the cell surface. At the cell surface they can either remain attached to the cell or they are secreted so that these proteins can be "seen" outside the cell, and away from the cell. The vast majority of surface or secreted proteins have a specific signal directing them to automatically traffic there. Recently it has been found that a small number of proteins, which play a role outside the cell but were assumed to be externalised only upon cell death, are in fact secreted under certain circumstances. One group of secreted proteins are inflammatory cytokines (messengers), critical in signaling to blood cells the location of an injury or infection and so essential to maintain a healthy immune system throughout life.

We now know that activation of the damage-sensing "P2X7" cell surface receptor causes release (secretion) of inflammatory mediators (specifically "interleukin-1) into the bloodstream. Interestingly, when interleukin-1 exits the cell, it is enveloped in small, protected membrane packets, that we have termed "microvesicles". These packets are likely to prevent this highly inflammatory cytokine from acting at random sites within the body, and may provide a means of targeting it to particular cells where the microvesicles can dock.

The cellular machinery coordinating microvesicle formation and cytokine secretion is complex. Several pathways involved in this process have been proposed, based on studies using a single type of cell grown in culture. We have developed a new system where we can study fluorescent cytokine in a whole live organism, namely the transparent zebrafish larva. For the first time, this offers us the opportunity to view the cells that secrete interleukin-1, and to see where interleukin-1 is targeted in a live organism. In this project we will optimise computational approaches to quantify vesicle formation, tracking and targeting in a whole organism using our fluorescent zebrafish lines. We will then test which cellular components are responsible for interleukin-1 secretion by adding inhibitors to these components and measuring changes in response to an infection. We will also use these fluorescent zebrafish to screen for small molecules that are effective in blocking inflammation, so that we can identify the critical parts of the cell that are required to for a healthy immune response. In the future, beyond this project, this will help us to develop new therapies to fight disease.

Technical Summary

Secretion of leaderless proteins is important in a range of contexts, but poorly understood. Specific proteins have been identified lacking a leader sequence and secreted independently of the conventional ER-Golgi route including fibroblast growth factors, interleukins and galectins. The importance of this unconventional secretion pathway is underlined by the tight regulation seen in the secretion of the best-studied example, the pro-inflammatory cytokine, interleukin-1 (IL-1).

Whole animal models have been used to evaluate the requirement of specific proteins in permitting leaderless protein secretion whilst cell culture studies have allowed us to observe whether these pathways are vesicular or otherwise. It has not so far been possible to combine the key features of such models to determine, in an intact live organism, whether leaderless protein secretion is vesicular and how this enables IL-1 targeting. We have recently characterised the zebrafish as a model of vesicular IL-1 secretion and targeting in response to injury and pathogens, and have generated specific transgenic lines that permit novel studies of leaderless protein secretion mechanisms in vivo.

Our work has led us to hypothesise that the key to controlling IL-1 function is the regulation of vesicular release of IL-1 via components of the vesicular budding pathway and disruption of cell membrane lipid diffusion. We will use our zebrafish lines to establish this as a widely applicable model to study IL-1 processing, leaderless protein secretion more generally, and to identify the in vivo extracellular targeting of these proteins. We will generate rapid, high content quantitative assays of IL-1 function in vivo for application to downstream objectives & investigate whether components of the vesicle budding pathway or of membrane lipid diffusion are required for the release of leaderless proteins in vivo, with important consequences for the regulation of fundamental processes such as inflammation.

Planned Impact

The proposal addresses fundamental cell biology questions of how leaderless proteins are secreted from the cell. There are less than 40 known leaderless proteins within the cell, a significant proportion of these play a critical role in inflammatory responses to injury or infection. The discoveries from this project will enhance the knowledge economy with new scientific advancement, as described in 'academic beneficiaries".

There is considerable interest from the pharmaceutical industry in discovering regulators of leaderless protein secretion which are fundamental in inflammation and become dysregulated and chronic in a number of ageing diseases. We anticipate that the unique nature of these secretion mechanisms will make them attractive targets for anti-inflammatory therapeutics. Protection of IP for these targets as they are discovered will bring significant economic gains to UK plc. Integration with the Pharma industry will allow rapid drug development, building on existing collaborations such as Renshaw's MRC Industry Partnership Award with GSK.

The project will establish and validate assays for the future identification of new small molecular inhibitors of leaderless protein secretion and/or targeting in inflammation. This leads to obvious longer-term commercial opportunities to develop the technology to a higher throughput level, and to engage industrial partners in developing new therapies. Via existing and new links, we will encourage Pharma investment in this programme, and develop IP sharing arrangements to ensure mutual benefit from emerging knowledge and know how. The advances in knowledge, and potential for driving drug development will ultimately impact on quality of life, health and well-being. Avoidance of dysregulated inflammation is a prerequisite for healthy ageing.

The project uses cross-disciplinary approaches from mammalian cell biology, zebrafish models and systems engineering. These approaches will be used to develop and make use of innovative systems technologies to identify, track and quantify accumulation of secreted leaderless proteins, in vivo. The project will contribute to new expertise in developing these unique tools to address biological questions by a systematic, and ultimately, high throughput approach. The project will strengthen links between these different disciplines and forge a greater understanding of how we can engage, complement and enhance research for the future.
This proposal will deliver highly trained researchers offering unique skills. The researcher will combine skills in fish models and state of the art in vivo microscopy, in parallel with mammalian cell based assays. The researcher will develop distinctive skills in generating new computational software to provide a systems approach to understanding biological responses. This expertise will provide transferable skills to other non-academic beneficiaries, but will also be used to train researchers from other groups in our methodologies. The new software developed will be made freely available to academic community, but may contain novel elements, which will provide additional opportunities in knowledge transfer and commercialisation.

The co-investigators are actively involved in public engagement and broader dissemination, with regular school visits and high level involvement with public exhibitions such as Royal Society Summer Science exhibition, and the University of Sheffield Festival of the Mind.
 
Description Secretion of leaderless proteins, is important in a range of contexts, but poorly understood. This unconventional secretion pathway is involved in the release of the pro-inflammatory cytokine, interleukin-1 (IL-1), the first line cytokine in the host response and resistance to pathogens. In this project we have developed the zebrafish as a model of vesicular IL-1beta secretion and targeting in response to injury and pathogens. The project has generated novel tools including a new zebrafish line (TgBAC(il-1b:GFP)SH445), to assess IL-1beta expression in real-time following injury or infection, as well as zebrafish deficient in IL-1b expression (il-1bSH446/ il-1bSH446), to assess the role of IL-1b in injury and pathogen responses. We took advantage of these transgenic zebrafish lines to understand the dynamics and cell specificity of IL-1b production in inflammation and infection, at the early stages of the innate immune response. Specifically, we assessed leukocyte specific activation of IL-1b in response to injury and mycobacterial (TB) infection, demonstrating that first neutrophils and then macrophages migrate to and upregulate IL-1b at the site of injury or infection. We also showed that HIF1alpha driven nitric oxide production, critical in the inflammatory response to TB, is dependent on IL-1b. This work has been published in the Journal of Immunology (doi: 10.4049/jimmunol.1801139).

We have additionally assessed microvesicle production in vivo, using live zebrafish microscopy imaging, and have identified cell membrane molecular mechanisms involved in targeting IL-1b distal the site of injury, demonstrating that microvesicles provide a delivery mechanism for activating inflammation via the circulation (publication in progress). Our testing of the role of the vesicle budding pathway in IL-1b release was limited as inhibition or mutation of these entities was not tolerated in the zebrafish.
The above outcomes have shown that we have: (a) established the zebrafish as a widely applicable model for the study of IL-1 processing and release (objective 1); (b) generated assays of IL-1 function in vivo, including their application in injury and pathogen responses (objectives 2 & 3); and (c) shown that membrane protein targeting of microvesicles occurs in vivo during the inflammatory response (objective 4, with some deviation).

We are currently progressing these findings by comparing these in mammalian systems, including in human macrophages using cell culture models to assess responses to infection. This work forms part of a wider programme to understanding the cellular mechanisms regulating inflammation and the response to infection, and where this becomes dysregulated in disease.
Exploitation Route In the future our findings may allow us to identify new targets for combating diseases related to chronic inflammation and ageing. Our work helps to reveal mechanisms of leaderless proteins secretion, allowing others to understand important principles in a number of biological processes in development and homeostasis.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description The project has generated novel tools including a new zebrafish line TgBAC(il-1b:GFP)SH445, to assess IL-1beta expression in real-time following injury or infection, as well as zebrafish deficient in IL-1b expression (il-1bSH446/ il-1bSH446). As well as being used in our own research groups these tools have been disseminated amongst the zebrafish community investigating inflammation and host-pathogen responses. During the project the post-holder developed new cross-disciplinary skills in using zebrafish models and computational technologies to track and quantify membrane labelled cells and microvesicles, in vivo, using Lightsheet and spinning disk microscopy. The post-holder was also actively involved in public engagement (these events are listed separately), with impact beyond the scientific community.
First Year Of Impact 2016
Sector Education
Impact Types Societal

 
Title IL-1:GFP zebrafish line 
Description This is a new zebrafish line developed within this project where the IL-1 promoter is linked to a GFP reporter. The activity of this reporter is specifically induced upon injury or infection and so is a good assay of inflammatory and immune responses. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2015 
Provided To Others? Yes  
Impact No impact at this stage as other researchers have only recently begun to use this tool. 
 
Description TAM protein collaboration 
Organisation University of Edinburgh
Department MRC Centre for Inflammation Research
Country United Kingdom 
Sector Public 
PI Contribution We have collaborated with Ian Dransfield who is an expert in TAM receptor tyrosine kinases. We have made use of reagents he has provided (see below) and in return we envisage that our zebrafish models will be helpful as an in vivo model in which to determine the role of these proteins in immune responses. We anticipate this collaboration moving forward to application for further RCUK funding towards the end of 2016.
Collaborator Contribution Ian Dransfield has provided us with ligands that act upon this receptor complex which has helped us refine our hypothesis with respect to microvesicle formation within the zebrafish in response to injury and infection. This work will form part of our publication which is currently in preparation.
Impact We are currently preparing a research manuscript for publication output where part of the work is based on this collaboration. This is not a multi-disciplinary collaboration
Start Year 2015
 
Description Festival of Medicine, Dentistry and Health (Zebrafish, Univ Sheffield) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Demonstrated zebrafish research showing fluorescent zebrafish to the public and discussing why these offer a good model in studying developmental and disease pathways. Many medical students attended this session, although this was open to the public.

We plan on repeating this activity on an annual basis.
Year(s) Of Engagement Activity 2014
 
Description Festival of the Mind (University of Sheffield, Zebrafish demonstrations) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Members of the public were introduced to our zebrafish research. We displayed fluorescent zebrafish for the public to view and discussed why zebrafish are a good model in studying developmental and disease pathways, how we modified them, advantages over other models and the ethical issues involved, as well as providing school children with a basic understanding of what is involved in a career in science or in studying science.

This was a recent activity so impact has yet to be reported.
Year(s) Of Engagement Activity 2014