Novel insights into pathogenesis and response to treatment of a variety of diseases depend increasingly on our ability to image biologic processes in vivo. Here we propose to use novel imaging technology in genetically engineered zebrafish to tackle the widespread problem of human autoimmunity.
T cell development in the thymus generates a virtually unlimited number of different receptors capable of recognizing antigens in the context of MHC molecules. This is one of the principal mechanisms our body relies on for protection against microbial infections. Recognition and destruction of "self' is prevented by negative selection of auto-reactive effector T cells (arTeffs) in the thymus (central tolerance). "Escapee" arTeffs are tolerized in the periphery by regulatory T cells (Tregs), which are characterized by expression of the transcription factor Foxp3. If the number of arleffs is overwhelming (defective central tolerance), or Tregs are absent or inefficient (defective peripheral tolerance),T cell infiltration into and destruction of tissues can result, a process termed autoimmunity (Al).
Al affects approximately one in five Americans, and ranges from relatively harmless vitiligo to debilitating type I diabetes, multiple sclerosis (MS), and fatal Immuno dysregulation. Polyendocrinopathy and Enteropathy, X-linked syndrome (IPEX). The genetic basis (causative and disease-modifying genes) for the majority of autoimmune diseases remains unresolved. Furthermore, while T cell infiltration precedes disease manifestations, pre-clinical noninvasive disease detection is problematical in humans and mice. Finally, while conventional immunosuppressive drugs are fraught with side effects, high-throughput testing for novel compounds is difficult to achieve in mammals. A simple, genetically tractable small-animal model of Al is therefore desirable, where disease onset, progression, and modification by drugs can be visually monitored.
Zebrafish represent a powerful genetic vertebrate model system, endowed with an immune system that is closely related to that of mammals. A number of zebrafish models for human diseases have already been established successfully. Availability of transgenic lines with tissue-specific fluorochrome expression, coupled with small size and transparency of skin, make in vivo imaging of biologic processes and high-throughput drug testing feasible in zebrafish. One of the main impediments of successful fluorescent in vivo imaging is tissue auto-fluorescence that can significantly decrease sensitivity and specificity of GFP-detection. The aim of this proposal is to adapt multispectral imaging (MSI) for use in zebrafish to detect and quantify multiple fluorescent colors, approaching the genetics and treatment of Al by in vivo imaging.
MSI is the acquisition of a high resolution optical spectrum at every pixel of an image over a large range of the optical spectrum in 10nm intervals, for GFP typically from 400 to 700nm. For image capture MSI relies on a liquid crystal tunable filter attached to a sensitive scientific-qrade, cooled megapixel CCD. The camera is coupled with algorithms to discern different signals based on their unique emission spectra. Its increased spectral resolution compared to RGB can be used for distinction of GFP from auto-fluorescence by unmixing of the pure GFP spectrum. We have pioneered this new technology in live zebrafish and have dramatically increased sensitivity and specificity of GFP detection in the p55lck promoter-GFP (L+) transgenic line we have previously created, where all T cells are green fluorescent.
Neither Tregs nor Foxp3 have been previously identified in any fish species. We have cloned and characterized the zebrafish Foxp3 gene. To explore presence and functionality of Tregs in zebrafish, we propose to generate a transgenic line, where the zebrafish Foxp3 promoter drives expression of DsRed (FD+). This will allow fluorescent tagging of Tregs, which are characterized by Foxp3 expression. Mating of L+ and FD+ lines will allow us to investigate tissue distribution and co-localization of Teffs and Tregs by in vivo MSl in physiologic and disease states (see below). We have previously demonstrated that GFP signals from thymocytes in d6 L+ larvae can be extinguished by adding dexamethasone to water. On this basis we will perform high-throughput screening of 48,000 small molecule compounds (DIVERSet, Chembridge Corp., San Diego, CA) for similar activity in the L+ line. MSI quantification of fluorescent signals is possible, and will be critical when partial effects of compounds need to be detected.
To further explore functionality of Tregs in zebrafish, we will establish a model for T cell mediated autoimmunity. In order to achieve this aim, we propose to use Targeted Induced Local Lesions In Genomes (TILLING) to inactivate Foxp3, whose absence in humans results in IPEX. We propose to sequence the exons of the zebrafish Foxp3 gene to identify inactivating mutations among 8,000 ENU mutagenized F1 individuals represented in the TILLlNG library in collaboration with Dr. Yi Zhou (Children's Hospital, Boston MA). A mutant line will then be established by in vitro fertilization of eggs with sperm of individuals identified as mutant in the library. This heterozygous Foxp3+/- line will then be mated to the L+ line. lncrossing will then establish a Foxp3-I-lck-GFP (F-/L'*) line.
We hypothesize that by analogy to other vertebrates, the F-IL,+ line will be prone to developing Al, and we propose to test this hypothesis by in vivo fluorescent imaging to reveal abnormal GFP signals as surrogate markers for T cell infiltration. We propose to inspect F-/L+ individuals for disease manifestations visually and by MSI for abnormal GFP signals as surrogate markers for T cell infiltration into tissues, and verify T cell-induced tissue destruction histologically. Susceptibility of invading T cells to known immunosuppressive drugs, such as dexamethasone or to small molecules identified in the screen (see above), will be monitored by in vivo MSI microscopy. We will then attempt to suppress autoimmunity in F-IL+ individuals with adoptively transferred Tregs from the transgenic FD+ line, obtained by fluorescence activated cell sorting.
This zebrafish model will represent a paradigm for autoimmunity in a lower vertebrate and will be a unique tool to further our insight into this debilitating disease complex. It represents a platform for new therapeutic approaches through identification of small molecule compounds that may be as active, but fraught with fewer side effects than conventional immuno-suppressive agents. In the future we propose to use enhancer/suppressor mutagenesis screens in this model to identify "modifier" genes, which may represent new targets for more directed treatments.