Anterior Segment Immunology Program
The cornea is the part of the eye responsible for allowing the entrance of external images. Corneal clarity and transparency are the unique qualities of this tissue that are critical for visual function. Moreover, the cornea serves as a mechanical barrier against infections and trauma and is considered part of the bio-defense system of the eye. For these reasons, the immune-regulatory mechanisms used by this immune-privileged tissue are key in maintaining a balance between these two important functions of the cornea. Failure of these mechanisms to control inflammation will result in corneal inflammation, vascularization, fibrosis and scarring which can lead to severe functional compromise and even blindness. Common and clinically-relevant examples in which the deregulation of immune-inflammatory responses of the cornea are critical for a good visual outcome include microbial keratitis, herpetic keratitis, contact lens-related ulcers, autoimmune ulcerative keratitis and corneal transplant allograft rejection. The main goal of this laboratory is to develop in vivo models of corneal inflammation to study the mechanisms of immune-regulation of the ocular surface and develop novel immunotherapeutic treatment of these disorders.
Innate and Adaptive Immunological Responses in High-Risk Corneal Transplantation
Corneal transplantation in individuals who have a vascularized corneal bed represents a high risk of graft rejection, opacification and blindness. Despite the use of topical steroids and systemic immunosuppression, the success rate of these transplants is extremely poor. One of the research projects in our laboratory investigates in vivo immune responses after orthotopic corneal allograft transplantation in a murine model of high-risk corneal transplantation. More specifically, we are interested in understanding how early innate immune responses mediated by neutrophils, chemokines and macrophages regulate adaptive T-cell responses to alloantigen. We have developed an in vivo model in which immunological responses during corneal transplantation can be monitored in real time with time-lapsed image analysis. This is being used to analyze inflammatory signals involved in the early recruitment of inflammatory cells.
The laboratory is developing in vivo models where immune responses could be studied as thoroughly and controlled as in vitro. The intimate cellular interactions that occur among inflammatory cells and the endothelium and interstitial space within the microenvironment of different tissues are difficult to mimic in tissue culture systems. Furthermore, new developments in the generation of transgenic and knock-out mice, in addition to other forms of gene manipulation in vivo using gene transfer technology, have given scientists the opportunity to exploit effectively the use of in vivo systems to study ocular immune responses.
In vivo gene delivery and visualization of corneal stromal cells using an adenoviral vector and keratocyte-specific promoter. Carlson EC, Liu CY, Yang X, Gregory M, Ksander B, Drazba J, Perez VL. Invest Ophthalmol Vis Sci. 2004 Jul;45(7):2194-200.
Visualization and characterization of inflammatory cell recruitment and migration through the corneal stroma in endotoxin-induced keratitis. Carlson EC, Drazba J, Yang X, Perez VL. Invest Ophthalmol Vis Sci. 2006 Jan;47(1):241-8.
Our group has utilized some of these techniques to study antigen-specific systemic immune responses to antigens placed in the eye and to describe a new route of antigen processing used by the eye. However, the next level of in vivo biology is the development of models where immune responses can be traced in real time at tissue-specific sites. The eye provides a unique window into the body. Its translucent nature permits the visualization of events, such as cell growth, cell death, migration, and transformation as they occur in vivo, in a non-invasive manner. In order to image, in vivo, the recruitment pattern and track the fate of inflammatory cells during an immune response in the eye, we utilized models that express green fluorescent protein in the majority of their hematopoietic cells (GFP models). To detect the recruitment of inflammatory bone marrow-derived cells into the eye, we used digital in vivo fluorescence microscopy in combination with ex-vivo 3-dimensional reconstruction of corneal whole mount full thickness confocal images. Ongoing experiments utilize this model to understand recruitment, fate and cellular interaction in the corneal stroma in models of keratitis, transplantation, limbal stem cell deficiency and wound healing after refractive surgery.
Gene Delivery to the Cornea
Our initial interest in developing an in vivo model of intra-ocular inflammation led us to pursue ways of expressing exogenous genes of interest in the eye to influence the immunoregulatory mechanisms of this organ and study inflammation. We adopted a method of transfecting and expressing exogenous genes in the stromal cells of the cornea in models by performing intrastromal injections of adenoviral vectors containing the DNA sequence of a gene of interest. These experiments have led us to develop a research program in gene delivery to the cornea. In our recent work, we report that gene delivery can be accomplished in a tissue-specific fashion in the cornea by using the keratocan promoter to drive the expression of any gene of interest.
We believe this work will lay the groundwork for the development of gene therapy as a novel therapy for corneal disorders.
Posterior Segment Immunology Program
Oxidative damage and inflammation are postulated to be involved in age-related macular degeneration (AMD). However, the molecular signal(s) linking oxidation to inflammation in this late-onset disease is unknown. Here we describe AMD-like lesions in models after immunization with serum albumin adducted with carboxyethylpyrrole, a unique oxidation fragment of docosahexaenoic acid that has previously been found adducting proteins in drusen from AMD donor eye tissues1 and in plasma samples2 from individuals with AMD. Immunized modles develop antibodies to this hapten, fix complement component-3 in Bruch’s membrane, accumulate drusen below the retinal pigment epithelium during aging, and develop lesions in the retinal pigment epithelium mimicking geographic atrophy, the blinding end-stage condition characteristic of the dry form of AMD. We hypothesize that these models are sensitized to the generation of carboxyethylpyrrole adducts in the outer retina, where docosahexaenoic acid is abundant and conditions for oxidative damage are permissive. This new model provides a platform for dissecting the molecular pathology of oxidative damage in the outer retina and the immune response contributing to AMD.
Oxidative damage-induced inflammation initiates age-related macular degeneration. Hollyfield JG, Bonilha VL, Rayborn ME, Yang X, Shadrach KG, Lu L, Ufret RL, Salomon RG, Perez VL. Nat Med. 2008 Feb;14(2):194-8.
Autoimmunity and immunotherapy in age-related macular degeneration
Life-expectancy for humans keeps steadily increasing and with it also grows a number of diseases and ailments associated with aging. A prime example of this type of disease is age-related macular degeneration (AMD), which represents the leading cause of legal blindness in the elderly population of the United States; almost two-thirds of people over 80 years old are afflicted by AMD to some degree. One of the AMD clinical signs long-recognized by clinicians is the accumulation of debris (called drusen) below the retinal pigment epithelium (RPE) in the macula of aging eyes. Another factor that strongly correlates with AMD progression is oxidative damage (such as the oxidative stress caused by smoking). Recently, it has been shown that AMD donor eyes contained carboxyethylpyrrole (CEP)-modified proteins in drusen and that CEP-adducted proteins were present at 40% higher levels in the plasma of AMD patients compared to age-matched controls. CEP is an adduct resulting from an oxidation fragment of docosahexaenoic acid (DHA), the most oxidizable of all long-chain polyunsaturated fatty acids that is highly concentrated in the outer retina. Interestingly, CEP auto-antibodies were more abundant in the plasma samples of AMD patients relative to controls, suggesting that immune recognition of these adducts occurs and that adaptive immune responses could play a critical role in the pathogenesis of AMD. To directly test this hypothesis, our laboratory immunized models with CEP-adducted serum albumin (CEP-MSA) and produced a novel murine model with dry AMD-like pathology. Our model demonstrates that an adaptive immune response against ?oxidation-induced? products in the retina plays an important role in the pathogenesis of AMD.
The main goal of this project is to test the hypothesis that adaptive immune responses generated against retinal products adducted with CEP are the initiating step in the development of AMD. We are currently testing the novel concept that antigen specific T and B cell responses are crucial in the targeting of complement-mediated retinal damage in AMD and their regulation can be modulated to prevent or reverse disease. We will first utilize our murine model of immune-mediated retinal degeneration to directly test the role of CEP-specific T cells and anti-CEP auto-antibodies in the initiation of AMD, and to identify the major immunological pathways involved in this process. In addition, we will develop a model of immune-mediated AMD, which will be crucial to confirm the data from our murine model and to test new immune and non-immune therapies to prevent or treat dry AMD. The major significance of these experiments will be to obtain data that can be expanded and integrated into immunological studies of patients with AMD in the search for novel therapeutic schemes.
Laboratory funding provided in part by:
- NIH/NEI 1 RO1 EY018624-02
- Edward N. & Della L. Thome Memorial Foundation
- Research to Prevent Blindness
- Stanley J. Glaser Foundation
- Alcon Research LTD