Establishing a Model of T Cell Mediated Autoimmunity in Zebrafish

Nikolaus S. Trede, M.D., Ph.D.

University of Utah

Funded in December, 2005: $300000 for 3 years
LAY SUMMARY . ABSTRACT . HYPOTHESIS . SELECTED PUBLICATIONS .

LAY SUMMARY

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Studying Errant (Autoimmune) T Cells in Zebrafish May Yield Clues to Human Autoimmune Disease

The researchers will apply a new molecular imaging technique to see if it can help advance efforts to detect, treat, and identify the genetic bases for an autoimmune disease in zebrafish that is similar to autoimmunity in humans.

Autoimmune diseases occur when errant immune T cells, which mistake the body’s own tissues as foreign and attack them, are not eliminated by regulatory immune cells during development or when the errant T cells first enter the blood stream.  Our understanding of autoimmunity may be enhanced significantly by studies of autoimmune diseases in zebrafish.  The zebrafish is small, its skin is transparent and permits visualization of internal organs (targets of autoimmunity), disease-causing genetic mutations are easy to induce, and human autoimmune disease models already have been established.  The investigators, therefore, will create a zebrafish model of a deadly autoimmune disease called IPEX.  In humans and mice, IPEX occurs when a factor called FoxP3 is inactivated and therefore fails to identify and kill off errant T cells.

After inactivating the FoxP3 factor in zebrafish, the researchers will create a strain of these zebrafish in which all of their immune T cells will fluoresce green when examined under a fluorescent microscope.  Then, the investigators will image the T cells in the zebrafish to see how they attack organs.  The researchers will use an imaging technique they have developed, called “multi-spectral imaging” (MSI).  This technique eliminates the problem of “auto-fluorescence,” in which animal tissues often obscure detection of the fluorescent signals.  Using MSI, the investigators will visualize the activities of the errant T cells, and then verify that the T cells have infiltrated and destroyed organ tissue by sacrificing the zebrafish and examining their tissue under the microscope.  Finally, the investigators will try to identify potential therapies that suppress the T cells without producing major side effects.

Significance: This imaging approach is anticipated to provide vital new information about how autoreactive T cells attack organs. It also may become a means for evaluating potential therapies that block the destructive actions of T cells in autoimmune diseases.  

ABSTRACT

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Establishing a Model of T Cell Mediated Autoimmunity in Zebrafish

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.

 

HYPOTHESIS

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Hypothesis:
This project will employ in vivo multispectral imaging (MSI) to study the problem of human autoimmune disease (AI) in genetically engineered green fluorescent zebrafish. The main hypothesis of this proposal holds that inactivation of the zebrafish Foxp3 gene will lead to uncontrolled autoimmunity and that in vivo MSI will allow visualization of organ infiltration of fluorescently labeled T cells. Thus, disease onset and progression as well as effects of therapeutic interventions can be monitored in vivo.

Goals:
To adapt in vivo multispectral imaging (MSI) for high-throughput screening of embryos and adult zebrafish, for simultaneous detection of two fluorochromes, and for detection of quantitative differences in fluorescent signals. To create a Foxp3 promoter-DsRed transgenic line to characterize regulatory T cells in zebrafish. To perform a high-throughput small molecule screen to identify novel immunosuppressive compounds. To create genetic inactivation of the Foxp3 gene in a transgenic zebrafish line where T cells are GFP tagged, and monitor T cell infiltration into organs by in vivo MSI, and validate this zebrafish model of AI by using a number of known and novel immunosuppressive compounds to modify disease expression.

Methods:

With MSI technology, spectral datasets are acquired by imaging a sample through a liquid crystal tunable filter that can be set to allow only light of a narrow bandpass to reach the coupled CCD camera. The peak position of this bandpass can be rapidly switched to any other position within milliseconds with about 1-nm precision. A series of images (typically 10 to 20) of a particular field can thus be rapidly acquired at different wavelengths to create a spectral data "cube", in which the 3 dimensions are x, y and wavelength. In this cube, a spectrum is associated with every pixel. Since fluorescent light emissions combine linearly, mixtures of signals can be mathematically disentangled (or "unmixed"), as long as the spectrum of the desired signal(s) and that of autofluorescence (AF) are known or can be extracted from the data. Since AF has a spectral signature that differs from GFP, the resulting data can be used to identify, separate and remove the contribution of AF in analyzed images.

This procedure results in images reflecting the abundance of each of the components isolated from the others. The entire process can be completed in a matter of seconds, which allows recovery of examined fish that have to be anesthetized for the procedure. Using MSI, we will quantify the signal emanating from T cells in the zebrafish thymus by integrating the unmixed GFP signal intensity over the area of the thymus from different individuals at the same magnification and light intensity. In this way signal intensities can be easily compared. MSI has the capacity for "multiplexing," i.e., the simultaneous capture of up to five different fluorescent signals. For our studies of AI suppression, we will need to be able to capture and differentiate GFP and DsRed signals. AF is present throughout the visible range, and MSI will be essential, not only for cleanly separating GFP from DsRed signals, but also for removal of AF in both these channels.

 

SELECTED PUBLICATIONS

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Langenau D.M., Ferrando A.A., Traver D., Kutok J.L., Hezel J.P., Kanki J.P., Zon L.I., Look A.T., and Trede N.S.   In vivo tracking of T-cell development, ablation, and engraftment in transgenic zebrafish. Proc Natl Acad Sci U S A. 2004 May 11;101(19):7369-74.

Trede N.S., Langenau D.M., Traver D., Look A.T., and Zon L.I.   The use of zebrafish to understand immunity. Immunity. 2004 Apr;20(4):367-79.