The aim of this project is to reduce the use of mammalian models in inflammatory lung disease research by establishing zebrafish gill tissue as an alternative model to examine basic mechanisms and pathway biology of severe asthma.
Severe asthma is a disabling non-curable condition that can only be controlled by continuous administration of drugs. Traditional animal models, primarily rodents, have shed light on a number of basic mechanisms involved in severe asthma; however drugs developed against targets within these pathways have not translated to the clinic.
The pathophysiological mechanisms of severe asthma remain unclear. Much attention has focussed on the activation of TH1 and TH2 cells of the adaptive immune response, but it is now understood that there is need to study the interaction between innate immune cells and the epithelial lining of the respiratory system. Recent studies have suggested that environmental factors such as cigarette smoke, combined with viral infection may predispose the respiratory tissue to a higher incidence of asthma.
Although zebrafish do not have lungs, their gills serve the same functions and have a similar tissue structure with a single epithelial layer scattered with immune cells, and a layer of smooth muscle at the base of the lamella. Being exposed directly to the water, the gills can be easily targeted with any waterborne substance without having to use invasive procedures and can be sampled using small swabs in a longitudinal fashion.
Research details and methods
This project will translate approaches used to collect nasal tissue and fluids from human volunteers to gill tissue sampling in zebrafish. Swabs generated from bioengineered substrates will be used to non-invasively sample gill tissue and fluids to allow longitudinal studies in live fish following a short exposure to anaesthetic and analgesia. These studies to detect the presence of specific cells and soluble mediators will be combined with gene transcript analysis and live microscopy imaging to assess the behaviour of fluorescently-labelled immune cells under different conditions, including exposure to cigarette smoke and/or virus. Finally, data generated through these methods will be compared with human data to create hypotheses regarding the involvement of innate immune pathways in human respiratory disease.
Aim 1: Tool development for non-invasive sampling.
We will take advantage of the techniques developed by TH and colleagues for clinical use which uses swabs generated from bioengineered substrates to collect nasal tissue and fluids from human volunteers. We will optimize these techniques for sampling zebrafish gill tissue and fluids to allow non-invasive, longitudinal studies in live fish with only short exposures to anaesthetic (and analgesia).
Aim 2: Cell and pathways analysis.
We will analyse the profile of innate immune markers with our collection of TaqMan Q-PCR assays on cDNA samples of gills exposed to cigarette smoke (CS) using previously optimised protocols. We will optimise exposure of fish to Poly:IC (as a viral mimetic) directly added to the water. Poly:IC and CS will then be combined and their inflammatory effects studied using live imaging over time with our (various) transgenic zebrafish bearing fluorescently tagged neutrophils and macrophages. We will apply findings from aim 1 to sample tissue for longitudinal analysis. Fish will be killed at the end of the study and all tissues collected for histological and end point gene expression analysis. We will also assess mast cell presence and pending our initial findings Ketotifen which targets this cell type will be used to assess the role of mast cells in tissue inflammation and remodeling. We will further assess whether epithelial inflammation mediated by CS and TLR induces smooth muscle impairment and breathing difficulties by monitoring opercular rate with video and by the key histological features (muscle thickness) of asthma.
Aim 3: Data will be compared with human data obtained by others to generate hypotheses regarding the involvement of innate immune pathways in human respiratory disease. If time allows we will target pathways identified in our comparative study using drugs or genetic approaches.
- Further Funding: NC3Rs PhD Studentship, Live imaging of mucosal and vascular inflammation in zebrafish in response to a high cholesterol diet, September 2013, £90,000
- Further Funding: NC3Rs PhD Studentship, Refinement of hematopoietic cell transplantation using optical imaging in zebrafish, October 2016, £90,000
Principal investigatorProfessor Margaret Dallman
InstitutionImperial College London
Co-InvestigatorDr Trevor Hansell
Dr Laurence Bugeon