Immune Relevant Models for Ocular Inflammatory Diseases

Immune Relevant Models for Ocular Inflammatory Diseases Abstract Ocular inflammatory diseases, such as dry eye and uveitis, are common, painful, difficult to treat, and may result in vision loss or blindness. Ocular side effects from the use of antiinflammatory drugs (such as corticosteroids or nonsteroidal antiinflammatories) to treat ocular inflammation have prompted development of more specific and safer medications to treat inflammatory and immune-mediated diseases of the eye. To assess the efficacy and safety of these new therapeutics, appropriate immune-relevant animal models of ocular inflammation are needed. Both induced and naturally-occurring models have been described, but the most valuable for translating treatments to the human eye are the animal models of spontaneous, immunologic ocular disease, such as those with dry eye or uveitis. The purpose of this review is to describe common immune-relevant models of dry eye and uveitis with an overview of the immuno-pathogenesis of each disease and reported evaluation of models from small to large animals. We will also review a selected group of naturally-occurring large animal models, equine uveitis and canine dry eye, that have promise to translate into a better understanding and treatment of clinical immune-relevant ocular disease in man. animal models, dry eye, immune-relevant, inflammatory, naturally-occurring, ocular, uveitis Introduction Blindness or low vision affects approximately 1 in 28 Americans older than 40 years of age, the underlying causes of which are commonly noninfectious immune-mediated diseases, including dry eye and uveitis (Acharya et al. 2013; Schaumberg et al. 2003, 2009). Dry eye symptoms are experienced by 20% of adults over 45 years old, and uveitis is a leading cause of blindness in the United States (Acharya et al. 2013; Gritz and Wong 2004; Merrill et al. 1997; Schaumberg et al. 2003, 2009; Suhler et al. 2008). Dry eye and uveitis are also common causes of blindness in domestic animals, and uveitis is the leading cause of blindness in horses worldwide (Deeg et al. 2008; Gerding and Gilger 2015; Gilger et al. 2013c; Kaswan et al. 1989; Moore et al. 2001; Murphy et al. 2011). There are no known cures for immune-mediated ocular diseases, and current treatment regimens are costly, require multiple daily applications, are poorly effective, and have adverse side effects. Therefore, new treatments to address these diseases are needed and for further development, there is a need for accurate and translatable immune-relevant models of ocular disease. The eye, like the brain and the uterus in pregnancy, is considered an immune privileged site (Niederkorn and Wang 2005; Stein-Streilein 2008). An active suppression of the immune response to endogenous and exogenous antigens occurs in the eye, as overt inflammation may compromise vision. The relative lack of antigen-presenting and MHC II-expressing cells and natural tissue barriers (i.e., the blood–ocular barrier) that physically separate ocular tissues from the systemic immune response contribute to the immune tolerance in the eye (Niederkorn 2006). With dry eye and uveitis, the normal ocular tolerance is lost (from several initiating causes) and the physical barriers become disrupted, allowing an influx of inflammatory cells. In addition, proinflammatory mediators induce T-helper cells to proliferate, activate antigen-presenting cells, expand auto-reactive B and T cell populations, and ultimately release proinflammatory and proapoptotic peptides (Caspi 2006; Stern and Pflugfelder 2011). Current treatments for dry eye and uveitis are nonspecific and require frequent use of topical medications that may have severe ocular and systemic side effects (Carnahan and Goldstein 2000; Fraunfelder et al. 2012; Sen et al. 2014). Furthermore, these medications are life-long therapies and patient compliance is commonly poor, leading to treatment failures, worsening of disease, and in some cases, blindness (Uchino and Schaumberg 2013). When testing effectiveness of therapeutics on models of ocular disease, there are two separate but important testing goals. The first question is whether the drug is effective in the ocular disease state that is being studied. For this goal, usually rats or mice are evaluated and dosed by a nonocular route, for example, orally, subcutaneously, or intraperitoneally. These studies help determine pathogenesis of disease-drug mechanisms; therefore, the wide array of reagents and genetically modified mice and rats are a major asset. Determination of the appropriate dose (i.e., dose ranging studies) is usually also performed in these first sets of studies. The second goal is to determine if an appropriate dose can reach the ocular target tissue and be effective in the eye using a dosing route and frequency that is clinically feasible. These studies would determine the pharmacokinetics and pharmacodynamics of a specific route of administration of a drug, typically in a normal eye, then repeated using the optimal dosing and routes in eyes of models of the disease state. For this second group of studies to be clinically valid in most instances, the animal models would have to have eyes anatomically similar to the target species and in the case of humans, use of the rabbit, dog, pig, or primate eye would be most appropriate. Finally, when selecting the appropriate animal model, the target tissue and disease state has to be paired with the most appropriate route of therapy. This determination is important for pharmacokinetic, toxicologic, and efficacy studies. Although there are many disease conditions of the human eye thought to have an immunologic pathogenesis, including allergic conjunctivitis, corneal transplant rejection, and age-related macular degeneration, as examples, the purpose of this review is to describe common immune-relevant models of dry eye and uveitis with an overview and assessment of models from small to large animals. We will also review a selected group of naturally-occurring large animal models, equine uveitis and canine dry eye, which have promise to translate into a better understanding and treatment of clinical immune-relevant ocular disease in man. Review of Commonly Used Animal Models in Inflammatory Ocular Disease Ocular Surface Disease Immune-Relevant Models Dry Eye Disease Dry eye disease (DED) is one of the most common ocular abnormalities and has multiple underlying causes. Dry eye is a disease of the tear film and ocular surface that results in symptoms of discomfort and visual disturbance with potential damage to the ocular surface (DEWS 2007). In one study, nearly one-half of patients claimed to have symptoms of dry eye with a negative effect on quality of life, including ocular pain, decreased activities requiring visual attention (e.g., reading, driving), and reduced productivity in the workplace (Uchino and Schaumberg 2013). Dry eye develops from a deficiency of the aqueous portion of the tear fluid as a result of reduced lacrimal aqueous tear secretion or a result of increased evaporation of tears, such as the result of Meibomian gland deficiencies (Lemp et al. 2012). Decreased aqueous production of the tears results in an increase of tear electrolytes (i.e., increased tear osmolality), proteins, and inflammatory mediators, resulting in damage to the surface ocular tissues, decreased visual acuity, and ocular discomfort. The relative decrease in aqueous tears on the ocular surface in patients with DED causes chronic irritation to ocular surface that disrupts the normal ocular immune tolerance (Barabino et al. 2012). With breakdown of ocular surface tolerance and immune-homeostasis, autoimmunity develops through activation of NK cells and Toll-like receptors, followed by release of proinflammatory factors such as interleukin (IL)-1α, IL-1β, tumor necrosis factor α, and IL-6. These mediators amplify, activating antigen-presenting cells, which internalize autoantigens and migrate to the draining cervical lymph node where autoreactive Th1 cells, Th17 cells, or B cells (i.e., in Sjogren’s syndrome) undergo expansion. Efferent trafficking of these autoreactive T cells to the ocular surface is directed by adhesion molecules (e.g., LFA-1) and chemokine receptors. Autoreactive T-cells in ocular surface tissues potentiate the chronic autoimmune response, resulting in epithelial cell apoptosis, reduced goblet cell density, and squamous metaplasia of epithelium (Barabino et al. 2012; Stern and Pflugfelder 2011; Stern et al. 2010). Current treatments for DED rely on frequently applied artificial tears, punctal plugs, topical tetracycline antibiotic, and omega fatty acids, all of which provide only temporary relief of dry eye (Gayton 2009). Chronic DED is commonly treated with antiinflammatory medications and immunosuppressants, the latter being the mainstay of treatment in the United States (Avunduk et al. 2003; Sall et al. 2000). Topical cyclosporine, an immunosuppressant, used with or without corticosteroids, is effective in DED through inhibition of T-cell activation and reduction of proinflammatory cytokines (Lekhanont et al. 2007). A recently approved topical immunosuppressive for treatment of DED, lifitegrast, is an integrin inhibitor that prevents binding of LFA-1 to ICAM-1, which is upregulated in DED. Lifitegrast thus blocks T-cell efferent recruitment to ocular tissues and reduces inflammatory cytokines (Keating 2017; Sheppard et al. 2014; Tauber et al. 2015). However, both cyclosporine and lifitegrast must be administered indefinitely twice daily by the patient and are associated with burning sensation after application, leading to reduced patient compliance and hence poor treatment efficacy and success. Therefore, an effective, long-term, well-tolerated, and convenient therapy for DED is needed. There are numerous models of ocular surface disease and dry eye, but to be immune relevant, there needs to be evidence of an immuno-pathogenesis in the disease process. There are several mouse models of dry eye disease, the most common of which is a model induced by low humidity and high air flow environments, with or without the additional use of scopolamine (Table 1) (Barabino et al. 2004, 2005; Daull et al. 2016). The extended environmental irritation to the surface of the eye of these mice disrupts the normal ocular immune tolerance and immunohomeostasis (Barabino et al. 2012), as described previously. These mice models have been used to study the immuno-pathogenesis of dry eye and the initial evaluation of therapeutics. Another described model is the use of repeated application of topical benzalkonium chloride to the mouse or rabbit eye. This produces chronic irritation that may develop immunopathology and chronic ocular surface disease (Lin et al. 2011; Xiong et al. 2008). Other induced models of DED in rodents, which may be less immunopathologic in origin, include lacrimal gland excision or injections of toxins or antigens such as botulinum toxin (Zhu et al. 2009) or concanavalin A (Lee et al. 2015). Genetic models, such as the MRL/lpr mouse, manifest multiple autoimmune disorders and can be helpful to study diseases such as systemic lupus erythematosus and Sjorgren’s syndrome (Table 1) (Jabs et al. 1996). Another example of genetic DED are neurturin-deficient mice, which may develop dry eye and serve as models for neurotrophic keratoconjunctivitis sicca, since this model lacks lacrimal innervation (Table 1) (Song et al. 2003). There are numerous other knockout and transgenic mice strains that are commonly studied that may develop DED; however, many of these models do not develop clinical signs of DED observed in large animal models, but instead develop histologic or other features characteristic of human DED (Schrader et al. 2008). Table 1 Selected immune-models of dry eye disease Animal Method Advantages Disadvantages Reference Rodent Mice Environmental chambers ± scopolamine patch Reproducible, economical Small eye, anatomic differences Keating (2017) Botulinum toxin injection into lacrimal gland Reproducible, economical Above, and toxin present Lekhanont et al. (2007), Zhu et al. (2009) Intraorbital injection of concanavalin A Above, possible orbital inflammation Lee et al. (2015) Topical administration of benzalkonium chloride Economical Above, time to develop, may not be immunologic Lin et al. (2011) Extraorbital lacrimal gland excision ± scopolamine Surgically induced desiccating irritation Initiates an abnormal Th1/Th17 T cell response, exogenous antigens Guzmán et al. (2016), Stevenson et al. (2014) Neurturin-deficient (NRTN(−/−)) Song et al. (2003) MRL/lpr Sjogrens-like dry eye, autoimmune pathogenesis Chronic model, systemic disease Gao et al. (2004), Jabs et al. (1996), Jie et al. (2010), Ma et al. (2014) Rat Extraorbital lacrimal gland excision ± scopolamine May not be immunologic Fujihara et al. (2001), Meng et al. (2015) Rabbit Activated autologous lymphocytes injected into lacrimal gland Sjögren’s-like autoimmune dacryoadenitis Specialized, difficult model Thomas et al. (2010) Topical administration of benzalkonium chloride Economical Above, time to develop, may not be immunologic Xiong et al. (2008) Lacrimal gland excision May not be immunologic Gilbard et al. (1988) Canine Naturally-occurring Immunologic, translatable Availability Gilger et al. (2013c), Kaswan et al. (1989), Moore et al. (2001) Animal Method Advantages Disadvantages Reference Rodent Mice Environmental chambers ± scopolamine patch Reproducible, economical Small eye, anatomic differences Keating (2017) Botulinum toxin injection into lacrimal gland Reproducible, economical Above, and toxin present Lekhanont et al. (2007), Zhu et al. (2009) Intraorbital injection of concanavalin A Above, possible orbital inflammation Lee et al. (2015) Topical administration of benzalkonium chloride Economical Above, time to develop, may not be immunologic Lin et al. (2011) Extraorbital lacrimal gland excision ± scopolamine Surgically induced desiccating irritation Initiates an abnormal Th1/Th17 T cell response, exogenous antigens Guzmán et al. (2016), Stevenson et al. (2014) Neurturin-deficient (NRTN(−/−)) Song et al. (2003) MRL/lpr Sjogrens-like dry eye, autoimmune pathogenesis Chronic model, systemic disease Gao et al. (2004), Jabs et al. (1996), Jie et al. (2010), Ma et al. (2014) Rat Extraorbital lacrimal gland excision ± scopolamine May not be immunologic Fujihara et al. (2001), Meng et al. (2015) Rabbit Activated autologous lymphocytes injected into lacrimal gland Sjögren’s-like autoimmune dacryoadenitis Specialized, difficult model Thomas et al. (2010) Topical administration of benzalkonium chloride Economical Above, time to develop, may not be immunologic Xiong et al. (2008) Lacrimal gland excision May not be immunologic Gilbard et al. (1988) Canine Naturally-occurring Immunologic, translatable Availability Gilger et al. (2013c), Kaswan et al. (1989), Moore et al. (2001) Table 1 Selected immune-models of dry eye disease Animal Method Advantages Disadvantages Reference Rodent Mice Environmental chambers ± scopolamine patch Reproducible, economical Small eye, anatomic differences Keating (2017) Botulinum toxin injection into lacrimal gland Reproducible, economical Above, and toxin present Lekhanont et al. (2007), Zhu et al. (2009) Intraorbital injection of concanavalin A Above, possible orbital inflammation Lee et al. (2015) Topical administration of benzalkonium chloride Economical Above, time to develop, may not be immunologic Lin et al. (2011) Extraorbital lacrimal gland excision ± scopolamine Surgically induced desiccating irritation Initiates an abnormal Th1/Th17 T cell response, exogenous antigens Guzmán et al. (2016), Stevenson et al. (2014) Neurturin-deficient (NRTN(−/−)) Song et al. (2003) MRL/lpr Sjogrens-like dry eye, autoimmune pathogenesis Chronic model, systemic disease Gao et al. (2004), Jabs et al. (1996), Jie et al. (2010), Ma et al. (2014) Rat Extraorbital lacrimal gland excision ± scopolamine May not be immunologic Fujihara et al. (2001), Meng et al. (2015) Rabbit Activated autologous lymphocytes injected into lacrimal gland Sjögren’s-like autoimmune dacryoadenitis Specialized, difficult model Thomas et al. (2010) Topical administration of benzalkonium chloride Economical Above, time to develop, may not be immunologic Xiong et al. (2008) Lacrimal gland excision May not be immunologic Gilbard et al. (1988) Canine Naturally-occurring Immunologic, translatable Availability Gilger et al. (2013c), Kaswan et al. (1989), Moore et al. (2001) Animal Method Advantages Disadvantages Reference Rodent Mice Environmental chambers ± scopolamine patch Reproducible, economical Small eye, anatomic differences Keating (2017) Botulinum toxin injection into lacrimal gland Reproducible, economical Above, and toxin present Lekhanont et al. (2007), Zhu et al. (2009) Intraorbital injection of concanavalin A Above, possible orbital inflammation Lee et al. (2015) Topical administration of benzalkonium chloride Economical Above, time to develop, may not be immunologic Lin et al. (2011) Extraorbital lacrimal gland excision ± scopolamine Surgically induced desiccating irritation Initiates an abnormal Th1/Th17 T cell response, exogenous antigens Guzmán et al. (2016), Stevenson et al. (2014) Neurturin-deficient (NRTN(−/−)) Song et al. (2003) MRL/lpr Sjogrens-like dry eye, autoimmune pathogenesis Chronic model, systemic disease Gao et al. (2004), Jabs et al. (1996), Jie et al. (2010), Ma et al. (2014) Rat Extraorbital lacrimal gland excision ± scopolamine May not be immunologic Fujihara et al. (2001), Meng et al. (2015) Rabbit Activated autologous lymphocytes injected into lacrimal gland Sjögren’s-like autoimmune dacryoadenitis Specialized, difficult model Thomas et al. (2010) Topical administration of benzalkonium chloride Economical Above, time to develop, may not be immunologic Xiong et al. (2008) Lacrimal gland excision May not be immunologic Gilbard et al. (1988) Canine Naturally-occurring Immunologic, translatable Availability Gilger et al. (2013c), Kaswan et al. (1989), Moore et al. (2001) In rats, the most commonly described dry eye model is the extraorbital lacrimal gland excision model (with or without use of scopolamine) (Fujihara et al. 2001; Meng et al. 2015). Like other models of induced dry eye, this rat lacrimal-excision model likely does not develop, substantially, an immunologic pathogenesis and therefore may not be as effective for evaluation of immunosuppressive therapies as naturally-occurring models of dry eye (Barabino et al. 2004; Meng et al. 2015). All of these rodent models of dry eye are similar in that they can be used to determine proof of principal of therapeutic response to a drug, but all have similar disadvantages of having orbital and lacrimal anatomy and eye size that differs from the human eye. Rabbit or dog models are more commonly used to evaluate dry eye signs and response to therapy, because they have easily measured decreased tear production and develop ocular surface changes (Schrader et al. 2008). Therefore, larger animal dry eye models are needed (see later description of canine dry eye). Rabbits are commonly used as models of ocular disease and for pharmacokinetic studies because of their relatively large eye, compared to rodents, while still being a common and economical laboratory animal. However, there are few true immune-relevant models of DED in rabbits. Most described models induce dry eye signs, but not likely an immunopathogenesis, by use of a topical irritant, such as benzalkonium chloride, or by short-term reduction in lacrimal secretion using parasympathomimetic drug, such as atropine (Burgalassi et al. 1999; Li et al. 2012; Xiong et al. 2008). A very promising model of autoimmune dacryoadenitis in rabbits that produces a Sjögren’s-like keratoconjunctivitis is created by an intra-lacrimal or subcutaneous injection of autologous peripheral blood lymphocytes activated by purified rabbit lacrimal epithelial cells (Thomas et al. 2008; Zhu et al. 2003). This rabbit autoimmune dacryoadenitis model has been used to effectively evaluate immunomodulatory treatments for dry eye, including topical cyclosporine and lacrimal gland adeno-associated virus (AAV) mediated-IL-10 gene therapy (Thomas et al. 2009, 2010). Naturally-Occurring Keratoconjunctivitis Sicca in Canines Domestic canines develop spontaneous dry eye that clinically and immunopathologically is similar to dry eye in humans (Table 1) (Kaswan et al. 1989). Not only do dogs spontaneously develop dry eye symptoms of ocular discomfort, conjunctival hyperemia, and corneal scarring, these symptoms correlate directly with reduced aqueous tear production, a reduction readily measured using a standard Schirmer tear test strip (Figure 1). Furthermore, dogs with dry eye have a reduced tear breakup time and increased corneal staining, all abnormalities also observed in humans with DED (Kaswan et al. 1989). Like humans, canine dry eye is typically bilateral, develops in middle age, is more common in female dogs and in certain breeds, such as the American Cocker spaniel, Bulldog, and West Highland white terrier (Sanchez et al. 2007). The pathogenesis of dry eye in dogs appears similar to that of humans, where an apparent immunologic inflammation occurs with progressive lymphocytic infiltration and damage to the lacrimal gland with subsequent decreased production of the aqueous tear film (Izci et al. 2015; Kaswan et al. 1984). With chronicity, the ocular surface becomes progressively more dessicated and inflamed, the cornea vascularizes and scars, and ultimately the dog may lose vision (Gilger et al. 2013c; Sanchez et al. 2007). Initial proof of concept of commonly used immunosuppressive eye drops was first demonstrated to be effective in this spontaneous dog model, including topical cyclosporine, tacrolimus, and LTF-1 inhibitors (Barachetti et al. 2015; Berdoulay et al. 2005; Gilger et al. 2013c; Kaswan et al. 1989; Murphy et al. 2011). Figure 1 View largeDownload slide Naturally-occurring dry eye in a dog. (A) Moderate dry eye disease in a dog resulting in conjunctival hyperemia, corneal vascularization, and corneal opacity. (B) Chronic dry eye disease in a dog with mucopurulent ocular discharge, hyperpigmented cornea, and conjunctival hyperemia. Figure 1 View largeDownload slide Naturally-occurring dry eye in a dog. (A) Moderate dry eye disease in a dog resulting in conjunctival hyperemia, corneal vascularization, and corneal opacity. (B) Chronic dry eye disease in a dog with mucopurulent ocular discharge, hyperpigmented cornea, and conjunctival hyperemia. Uveitis Disease Models Uveitis is inflammation of the iris, ciliary body, and choroid and is associated with both infectious and noninfectious causes. Uveitis is estimated to be the third leading cause of preventable blindness worldwide (Siddique et al. 2013). In the United States, the incidence of uveitis was estimated to be approximately 58 to 69 cases/100,000 people (Acharya et al. 2013; Suhler et al. 2008); however, another study estimated that the rate of uveitis, especially anterior uveitis, was approximately 3 times higher and it increased with increasing age of patients (Gritz and Wong 2004). The most common causes of uveitis in humans are human leukocyte antigen (HLA)-B27 related uveitis, acute anterior uveitis in herpes zoster disease, toxoplasmosis, sarcoidosis, and pars planitis (Jabs 2008). Uveitis results from several causes. The uveal tract supplies blood to the eye and is in direct contact with peripheral vasculature; therefore, diseases of the systemic circulation (e.g., septicemia, bacteremia, infection, activated lymphocytes, immune diseases, etc.) will disrupt the blood-ocular barrier (Generali et al. 2015; Levitt et al. 2015). The blood-ocular barrier prevents large molecules and cells from entering the eye and thus limits the immune response to intraocular antigens. With trauma or inflammation, this barrier can be disrupted, allowing blood products and cells to enter the eye, resulting in the clinical signs typical of uveitis, such as flare, cell accumulation, and vitreous haze. Disruption of the barrier enables activation of various host immune responses, including antibody production to self-antigens that are not normally recognized by the immune system, as well as antibody production to foreign antigens inside the eye. As a result of the blood-ocular barrier, lack of lymphatics, and the presence of limited numbers of resident leukocytes, the eye is considered to have immune privilege. Naïve T cells cannot cross the normal blood-retinal barrier due to the lack of fenestration in the retinal vessels and the lack of appropriate adhesion molecules (Caspi 2011). Expression of chemokines in inflammation and activated T cells in the ciliary epithelium may play a role in recruitment and activation of leukocytes in diseased eyes (Gilger et al. 2002). As in other autoimmune disorders, infections may trigger events, either by antigenic mimicry with a pathogen’s antigen or as a bystander effect due to the general systemic or local immune stimulation by the pathogen. Uveitogenic retinal proteins documented in experimental animals include retinal arrestin, interphotoreceptor retinoid-binding protein (IRBP), rhodopsin, recoverin, phosducin, and retinal pigment epithelium derived RPE-65 (Deeg 2008; Deeg et al. 2001, 2006b; Siddique et al. 2013). Irrespective of the eliciting antigen, available experimental evidence suggests that the immunological mechanisms driving the resultant disease are similar (Caspi 2006). Following disruption of the blood-ocular barrier, large amounts of predominantly CD4+ T cells enter the eye and secrete proinflammatory cytokines such as IL-2 and interferon γ (Gilger et al. 1999). Auto-reactive effector CD4+ T cells have been associated with the pathogenesis of inflammatory and autoimmune disorders including uveitis. Naıve CD4+ T cells differentiate into effector subsets depending on the nature of the environment in which exposure to the antigen occurs (Caspi 2011). Several T cell effector phenotypes have been defined, known as T helper 1 (TH1), TH2, or TH17. Early studies suggested that the interferon-γ-producing TH1 and IL-17-releasing TH17 subsets are responsible for the pathology of uveitis, with the latter being associated with development of autoimmune disease (Caspi 2006). Additionally, clinical uveitis frequently develops spontaneous recurrent or relapsing bouts of inflammation, likely from T cells recognizing additional autoantigens in the ocular tissue (Deeg et al. 2006a). Resolution of uveitis is dependent on the presence of T regulatory cells (Tregs) that are labeled as CD4+Foxp3+ cells. When Foxp3+ T cell percentages in uveitis increase to approximately 10% of the total CD4+ cells, the acute inflammation rapidly resolves. Therefore, Foxp3+ Tregs are important to induce spontaneous resolution and in maintaining remission of uveitis (Silver et al. 2015). Multiple models have been developed to evaluate the immuno-pathogenesis of uveitis and recurrent uveitis, including identification of autoantigens. Most of these models are rodent based. Other models, including those that are acute, chronic, and recurrent in nature, have been developed to evaluate therapeutics (Table 2). Large animal models, such as uveitis induced in rabbits and pigs, have been evaluated to test therapeutics in larger eyes to help translate these treatments to humans (Table 2). Table 2. Models of uveitis Type of uveitis Species/Strains Agents Type of inflammation Advantages Disadvantages Reference Rodent EIU Lewis rat: Harlan Sprague Dawley Mice: (C3H and other strains) Endotoxin Anterior uveitis Rapid onset, predictable Nonimmunologic Cousins et al. (1984), Li et al. (1995) EAU Rat Melanin from bovine RPE Tyrosinase-related proteins 1 and 3 Anterior uveitis Immunologic Agarwal et al. (2012) Experimental autoimmune uveoretinitis Mice Rat Retinal arrestin (S-Ag), IRBP, recoverin, phosducin, rhodopsin/opsin Posterior segment Immunologic Chen et al. (2013, 2015), Shao et al. (2006), Silver et al. (2015) Spontaneous Mice RBP T cell receptor transgenic mice (R161H) Autoimmune Regulator (AIRE)(−/−) mice Adoptive transfer Retinal degeneration and persistent cellular infiltrates and lymphoid aggregation, multi-focal infiltrates and severe choroidal inflammation. Chen et al. (2013), Shao et al. (2006) Rabbit EIU NZW Dutch belted Endotoxin Anterior and posterior segment Rapid onset, predictable Nonimmunogenic Fleisher et al. (1990), Goldblum et al. (2007) Recurrent uveitis NZW Mycobacterium tuberculosis H37Ra antigen Ovalbumin Anterior (intracameral) or posterior (intravitreal) segment Immunologic and recurrent Ang et al. (2014), Edmond et al. (2016), Ghosn et al. (2011), Neumann et al. (1993) Cytokine induced uveitis NZW IL-1 TNF-alpha Acute onset (6 hours) Nonimmunologic Fleisher et al. (1990), Tilden et al. (1990) Porcine EIU Various Endotoxin Posterior Large eye Nonimmunogenic Gilger et al. (2013b) Horse Recurrent uveitis Various Spontaneous Anterior and posterior Large eye; immunologic; recurrent Nonstandard research animal Cost Deeg (2008), Deeg et al. (2008), Gerding and Gilger (2016) Type of uveitis Species/Strains Agents Type of inflammation Advantages Disadvantages Reference Rodent EIU Lewis rat: Harlan Sprague Dawley Mice: (C3H and other strains) Endotoxin Anterior uveitis Rapid onset, predictable Nonimmunologic Cousins et al. (1984), Li et al. (1995) EAU Rat Melanin from bovine RPE Tyrosinase-related proteins 1 and 3 Anterior uveitis Immunologic Agarwal et al. (2012) Experimental autoimmune uveoretinitis Mice Rat Retinal arrestin (S-Ag), IRBP, recoverin, phosducin, rhodopsin/opsin Posterior segment Immunologic Chen et al. (2013, 2015), Shao et al. (2006), Silver et al. (2015) Spontaneous Mice RBP T cell receptor transgenic mice (R161H) Autoimmune Regulator (AIRE)(−/−) mice Adoptive transfer Retinal degeneration and persistent cellular infiltrates and lymphoid aggregation, multi-focal infiltrates and severe choroidal inflammation. Chen et al. (2013), Shao et al. (2006) Rabbit EIU NZW Dutch belted Endotoxin Anterior and posterior segment Rapid onset, predictable Nonimmunogenic Fleisher et al. (1990), Goldblum et al. (2007) Recurrent uveitis NZW Mycobacterium tuberculosis H37Ra antigen Ovalbumin Anterior (intracameral) or posterior (intravitreal) segment Immunologic and recurrent Ang et al. (2014), Edmond et al. (2016), Ghosn et al. (2011), Neumann et al. (1993) Cytokine induced uveitis NZW IL-1 TNF-alpha Acute onset (6 hours) Nonimmunologic Fleisher et al. (1990), Tilden et al. (1990) Porcine EIU Various Endotoxin Posterior Large eye Nonimmunogenic Gilger et al. (2013b) Horse Recurrent uveitis Various Spontaneous Anterior and posterior Large eye; immunologic; recurrent Nonstandard research animal Cost Deeg (2008), Deeg et al. (2008), Gerding and Gilger (2016) EAU, experimental autoimmune anterior uveitis; EIU, endotoxin-induced uveitis; IL, interleukin; NZW, Zealand White. Table 2. Models of uveitis Type of uveitis Species/Strains Agents Type of inflammation Advantages Disadvantages Reference Rodent EIU Lewis rat: Harlan Sprague Dawley Mice: (C3H and other strains) Endotoxin Anterior uveitis Rapid onset, predictable Nonimmunologic Cousins et al. (1984), Li et al. (1995) EAU Rat Melanin from bovine RPE Tyrosinase-related proteins 1 and 3 Anterior uveitis Immunologic Agarwal et al. (2012) Experimental autoimmune uveoretinitis Mice Rat Retinal arrestin (S-Ag), IRBP, recoverin, phosducin, rhodopsin/opsin Posterior segment Immunologic Chen et al. (2013, 2015), Shao et al. (2006), Silver et al. (2015) Spontaneous Mice RBP T cell receptor transgenic mice (R161H) Autoimmune Regulator (AIRE)(−/−) mice Adoptive transfer Retinal degeneration and persistent cellular infiltrates and lymphoid aggregation, multi-focal infiltrates and severe choroidal inflammation. Chen et al. (2013), Shao et al. (2006) Rabbit EIU NZW Dutch belted Endotoxin Anterior and posterior segment Rapid onset, predictable Nonimmunogenic Fleisher et al. (1990), Goldblum et al. (2007) Recurrent uveitis NZW Mycobacterium tuberculosis H37Ra antigen Ovalbumin Anterior (intracameral) or posterior (intravitreal) segment Immunologic and recurrent Ang et al. (2014), Edmond et al. (2016), Ghosn et al. (2011), Neumann et al. (1993) Cytokine induced uveitis NZW IL-1 TNF-alpha Acute onset (6 hours) Nonimmunologic Fleisher et al. (1990), Tilden et al. (1990) Porcine EIU Various Endotoxin Posterior Large eye Nonimmunogenic Gilger et al. (2013b) Horse Recurrent uveitis Various Spontaneous Anterior and posterior Large eye; immunologic; recurrent Nonstandard research animal Cost Deeg (2008), Deeg et al. (2008), Gerding and Gilger (2016) Type of uveitis Species/Strains Agents Type of inflammation Advantages Disadvantages Reference Rodent EIU Lewis rat: Harlan Sprague Dawley Mice: (C3H and other strains) Endotoxin Anterior uveitis Rapid onset, predictable Nonimmunologic Cousins et al. (1984), Li et al. (1995) EAU Rat Melanin from bovine RPE Tyrosinase-related proteins 1 and 3 Anterior uveitis Immunologic Agarwal et al. (2012) Experimental autoimmune uveoretinitis Mice Rat Retinal arrestin (S-Ag), IRBP, recoverin, phosducin, rhodopsin/opsin Posterior segment Immunologic Chen et al. (2013, 2015), Shao et al. (2006), Silver et al. (2015) Spontaneous Mice RBP T cell receptor transgenic mice (R161H) Autoimmune Regulator (AIRE)(−/−) mice Adoptive transfer Retinal degeneration and persistent cellular infiltrates and lymphoid aggregation, multi-focal infiltrates and severe choroidal inflammation. Chen et al. (2013), Shao et al. (2006) Rabbit EIU NZW Dutch belted Endotoxin Anterior and posterior segment Rapid onset, predictable Nonimmunogenic Fleisher et al. (1990), Goldblum et al. (2007) Recurrent uveitis NZW Mycobacterium tuberculosis H37Ra antigen Ovalbumin Anterior (intracameral) or posterior (intravitreal) segment Immunologic and recurrent Ang et al. (2014), Edmond et al. (2016), Ghosn et al. (2011), Neumann et al. (1993) Cytokine induced uveitis NZW IL-1 TNF-alpha Acute onset (6 hours) Nonimmunologic Fleisher et al. (1990), Tilden et al. (1990) Porcine EIU Various Endotoxin Posterior Large eye Nonimmunogenic Gilger et al. (2013b) Horse Recurrent uveitis Various Spontaneous Anterior and posterior Large eye; immunologic; recurrent Nonstandard research animal Cost Deeg (2008), Deeg et al. (2008), Gerding and Gilger (2016) EAU, experimental autoimmune anterior uveitis; EIU, endotoxin-induced uveitis; IL, interleukin; NZW, Zealand White. Rodent Models of Uveitis Endotoxin-induced uveitis A commonly used model of induced uveitis in rodents is the endotoxin-induced uveitis (EIU) model (Table 2) (Altinsoy et al. 2011; Cousins et al. 1984; Li et al. 1995). The uveitis in this model is primarily an acute anterior uveitis (i.e., iris, ciliary body) that is thought to be driven by the innate immune system (Table 2) (Agarwal et al. 2012; Caspi 2006). Following intraparentoneal, subcutaneous, or hind footpad injection of endotoxin (lipopolysaccharide; 100 μg or 500 μg) in Lewis or Sprague-Dawley rats or various mouse strains (C3H) (Caspi 2006), ocular inflammation develops within hours of injection characterized by a breakdown of the blood-aqueous barrier and the development of clinical disease. Clinical and histopathologic abnormalities peak at 24 hours and resolve by 48 to 72 hours (Cousins et al. 1984; Li et al. 1995). Experimental autoimmune uveitis (or uveoretinitis) Experimental autoimmune uveitis (EAU) is a primarily posterior uveitis (or panuveitis [i.e., inflammation of the iris, ciliary, and choroid]) that is induced by immunizing susceptible rodents with retinal antigens (e.g., S-antigen [S-ag], IRBP, recoverin, rhodopsin/opsin); while experimental melanin–protein induced uveitis, a predominantly anterior uveitis, is elicited by immunization with melanin (from RPE) or tyrosinase-related proteins 1 and 2 (Table 2) (Caspi 2006). The predominant animal model is the Lewis rat, but other animals such as the guinea pig or mice have also been described (Agarwal et al. 2012; Wacker 1972). Injection of autoantigens into rodents, combined with bacterial adjuvants, results in EAU; EAU does not develop without the use of adjuvants. The use of complete Freund’s adjuvant (Mycobacterium cell wall product) or pertussis toxin is necessary to stimulate the innate immune response and develop inflammation (Agarwal et al. 2012; Chen et al. 2015; Silver et al. 2015) that ultimately generates activated antigen-presenting cells capable of presenting the injected autoantigen with the coactivation factors required to activate T cells capable of recognizing the antigen. Severe EAU was induced in B6 mice by adoptive transfer of IRBP-specific T cells (Shao et al. 2006). Most of the rodent experimental models of uveitis are not recurrent. They often elicit a single, albeit chronic course of uveitis that eventually resolves. Therefore, the immunologic pathways involved in the development of these rodent models may not be the same as in naturally occurring uveitis. Spontaneous uveitis has been observed in various mouse models, including IRBP T cell receptor transgenic mice (R161H) and autoimmune regulator (AIRE)(-/-) mice (Chen et al. 2015). These mouse models have a gradual onset of chronic ocular inflammation that ultimately leads to retinal degeneration (Chen et al. 2015). Despite limitations, these rodent experimental models offer great insight into the pathogenesis and immunopathogenesis of uveitis. These models have been critical in evaluation of therapies, particularly broader immunosuppressive therapies, for treating uveitis. Rabbit Models of Uveitis Two rabbit models of uveitis have been most commonly evaluated, including the acute uveitis induced by injection of endotoxin (Allen et al. 1996; Ghosn et al. 2011; Nussenblatt et al. 2012; Rafie et al. 2010) and the recurrent uveitis induced by tuberculosis antigen (Ang et al. 2014; Ghosn et al. 2011; Mruthyunjaya 2006). Other uveitis models in rabbits include those following intravitreal injection of human interleukin 1 alpha (Tilden et al. 1990), TNF-alpha (Fleisher et al. 1990), or ovalbumin in animals previously ovalbumin-immunized (Neumann et al. 1993), among others (Table 2). An advantage of rabbit models of uveitis over rodent models is that the rabbit eye is more similar in size to the human eye, and therefore, more pharmacologically valid when evaluating routes of therapy. In the endotoxin-induced uveitis, after 10 to 100 ng of LPS is injected intracamerally (Nussenblatt et al. 2012) or intravitreally (Fleisher et al. 1990; Goldblum et al. 2007), aqueous flare and iridal hyperemia develop within 6 hours, suggesting rapid disruption of the blood–aqueous barrier (Allen et al. 1996). The LPS induces inflammation by activating a Toll-like receptor 4-initiated signaling cascade. The inflammatory response peaks at approximately 24 hours after injection, then rapidly declines (Nussenblatt et al. 2012). Like the rat model of EIU, this endotoxin rabbit model of uveitis is not considered to have a predominantly immunopathogenesis. Experimental uveitis can be induced by unilateral intravitreal or intracameral injection of Mycobacterium tuberculosis H37Ra antigen (50 μg; 1 μg/L) in preimmunized rabbits, typically 7 to 14 days after initial subcutaneous injection (Ang et al. 2014; Edmond et al. 2016; Ghosn et al. 2011; Jaffe et al. 1998). To simulate chronic recurrent inflammation, eyes are re-challenged with intravitreal antigen every 14 to 21 days (Jaffe et al. 2006). This model has advantages similar to the endotoxin model; however, it is predominantly a T-cell lymphocyte-mediated uveitis that can be induced to be recurrent and therefore, more closely simulate endogenous human uveitis (Jaffe et al. 1998). Porcine Models of Uveitis The pig has been used as a large animal model of uveitis (Table 2), which, similar to the rabbit model, has an eye similar in size to the human eye; however, unlike the rabbit, it has a retinal vascular anatomy similar to humans (Gilger et al. 2013a). An acute model of uveitis has been used in the pig to evaluate novel therapeutics and routes of administration. In this model, similar to endotoxin uveitis in rabbits, endotoxin is injected intravitreally and the eye is monitored for up to 72 hours following injection (Gilger et al. 2013b). Like rodent EIU and endotoxin uveitis in rabbits, the endotoxin porcine model of uveitis is not considered to have an immunopathogenesis. Naturally-Occurring Uveitis Models Equine Recurrent Uveitis Horses spontaneously develop severe, immunologic uveitis called equine recurrent uveitis (ERU) that is frequently recurrent and chronic (Figure 2) (Wollanke et al. 2004). ERU is the most common cause of blindness in horses (Deeg et al. 2008; Gerding and Gilger 2015; Wollanke et al. 2004). Spontaneous bouts of uveitis develop and blindness may occur after multiple recurrent episodes of uveitis. The immunopathology of ERU has been extensively studied and has demonstrated that T cells are the predominant mononuclear inflammatory cells infiltrating ocular tissues in horses with naturally occurring chronic uveitis, with a significant number of CD4+ cells (Deeg et al. 2006a; Gilger et al. 1999; Kleinwort et al. 2016). Recruitment of proinflammatory cells as well as autoreactive lymphocytes may be in part driven by the expression of the chemokine RANTES in the ciliary body (Gilger et al. 2002). Figure 2 View largeDownload slide Naturally-occurring uveitis in a horse. (A) Acute active uveitis in a horse, with a miotic pupil and anterior chamber opacity. (B) Chronic uveitis in a horse, from equine recurrent uveitis, with dyscoria, synechiae, and cataract. Figure 2 View largeDownload slide Naturally-occurring uveitis in a horse. (A) Acute active uveitis in a horse, with a miotic pupil and anterior chamber opacity. (B) Chronic uveitis in a horse, from equine recurrent uveitis, with dyscoria, synechiae, and cataract. Study of this common, spontaneous uveitis in horses has helped understand the pathogenesis of uveitis in humans, especially the identification of autoantigens and how recurrence of uveitis develops immunologically (Deeg 2009; Deeg et al. 2001, 2006a, 2006b, 2007, 2008; Degroote et al. 2014; Zipplies et al. 2009). Several potential autoantigens have been identified in horses that could play a role in the development of autoimmune uveitis. T cells isolated from the eyes of horses with ERU proliferate in response to two common autoantigens in rodents: retinal S-Ag and IRBP (Deeg et al. 2001). In addition, several additional potential autoantigens were identified by analyzing antibodies in the sera of ERU horses that reacted with retinal proteins. These include recoverin, cellular retinaldehyde-binding protein, and malate dehydrogenase (Deeg et al. 2007). While all these potential autoantigens are capable of inducing experimental uveitis in rodent models, only cellular retinaldehyde-binding protein and IRBP consistently produce uveitis in outbred horses (Deeg 2009; Deeg et al. 2006b). Additionally, studies of horses with ERU have also helped elucidate how Leptospira infections induce immunological uveitis, specifically autoimmune uveitis (Lucchesi and Parma 1999; Lucchesi et al. 2002; Verma et al. 2005). Field studies of horses in the 1950s after an outbreak of acute leptospirosis caused by L. interrogans serogroup Pomona demonstrated that one of the six horses (17%) developed intraocular inflammation during acute leptospiral disease, and all horses developed ERU 18 to 24 months after the initial infection. Subsequent studies demonstrated cross-reactivity between equine ocular tissues and Leptospira antigens (Lucchesi and Parma 1999; Lucchesi et al. 2002), and horses with uveitis associated with Leptospira interrogans infections had high levels of IgA and IgG in their intraocular fluids that reacted to two Leptospira lipoproteins, LruA and LruB (Verma et al. 2005, 2010). These antibodies were also subsequently discovered in the serum of human leptospiral uveitis patients (Verma et al. 2008). Studies of spontaneous ERU have helped elucidate the immunopathogenesis of recurrent uveitis. In autoimmune disease, several autoantigens, or epitopes, participate in the immunopathogenesis; epitope spreading is accountable for disease induction, progression, and inflammatory relapses (Deeg et al. 2006a). Epitope spreading is defined as the diversification of epitope specificity from the initial focused, dominant, epitope-specific immune response, directed against a self or foreign protein to cryptic epitopes on that protein (intramolecular spreading) or other proteins (intermolecular spreading) (Deeg et al. 2006a). The shifts in immunoreactivity, or epitope spreading, have been documented in ERU and are thought to be responsible for the recurring character of ERU (Deeg et al. 2006a). ERU, as a model of spontaneous immune-mediated uveitis, has also led to the study of promising therapeutics. For example, several sustained release ocular implants have shown much promise in the treatment of ERU (Gilger et al. 2000, 2001). Evaluation of drug delivery to the suprachoroidal space has been shown to control ERU and prevent recurrences (Gilger et al. 2006, 2010). Triamcinolone injections into the suprachoroidal space are currently under development for treatment of human uveitis (Moisseiev et al. 2016). Further study of ERU and its treatment will translate well to improving the understanding and treatment of human autoimmune uveitis. Next Steps As further therapeutics are developed that more specifically target immune-mediated diseases, evaluation of these treatments in spontaneous or naturally-occurring models of ocular disease will be needed to provide proof of concept and help translate these therapies to humans. Excellent examples of developing, targeted therapies include gene therapy (especially gene addition therapy) and stem cell therapy. Our laboratory and collaborators at the Gene Therapy Center at the University of North Carolina have developed AAV delivery of immunosuppressive proteins, such as HLA-G, for suppression of ocular surface inflammation and vascularization (Hirsch et al. 2017). Target ocular surface diseases for AAV-HLA-G gene therapy are DED and for prevention of corneal graft rejection (Hirsch et al. 2017). Autologous stem cell therapy, or use of stem cell supernatant extracts, also shows much promise for immunomodulation in the eye. Effectiveness of topical ocular mesenchymal stem cell therapy was initially demonstrated in dry eye models in mice (Lee et al. 2015). Locally injected fat-derived mesenchymal stem cells near the lacrimal glands of dogs with advanced dry eye demonstrated clinical improvement and increased tear production (Villatoro and Fernández 2014). These results in a naturally-occurring model provides evidence of possible clinical translation to humans with severe dry eye. 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Immune Relevant Models for Ocular Inflammatory Diseases

ILAR Journal , Volume Advance Article – Feb 21, 2018

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the National Academy of Sciences. All rights reserved. For permissions, please email: journals.permissions@oup.com
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1084-2020
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1930-6180
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10.1093/ilar/ily002
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Abstract

Abstract Ocular inflammatory diseases, such as dry eye and uveitis, are common, painful, difficult to treat, and may result in vision loss or blindness. Ocular side effects from the use of antiinflammatory drugs (such as corticosteroids or nonsteroidal antiinflammatories) to treat ocular inflammation have prompted development of more specific and safer medications to treat inflammatory and immune-mediated diseases of the eye. To assess the efficacy and safety of these new therapeutics, appropriate immune-relevant animal models of ocular inflammation are needed. Both induced and naturally-occurring models have been described, but the most valuable for translating treatments to the human eye are the animal models of spontaneous, immunologic ocular disease, such as those with dry eye or uveitis. The purpose of this review is to describe common immune-relevant models of dry eye and uveitis with an overview of the immuno-pathogenesis of each disease and reported evaluation of models from small to large animals. We will also review a selected group of naturally-occurring large animal models, equine uveitis and canine dry eye, that have promise to translate into a better understanding and treatment of clinical immune-relevant ocular disease in man. animal models, dry eye, immune-relevant, inflammatory, naturally-occurring, ocular, uveitis Introduction Blindness or low vision affects approximately 1 in 28 Americans older than 40 years of age, the underlying causes of which are commonly noninfectious immune-mediated diseases, including dry eye and uveitis (Acharya et al. 2013; Schaumberg et al. 2003, 2009). Dry eye symptoms are experienced by 20% of adults over 45 years old, and uveitis is a leading cause of blindness in the United States (Acharya et al. 2013; Gritz and Wong 2004; Merrill et al. 1997; Schaumberg et al. 2003, 2009; Suhler et al. 2008). Dry eye and uveitis are also common causes of blindness in domestic animals, and uveitis is the leading cause of blindness in horses worldwide (Deeg et al. 2008; Gerding and Gilger 2015; Gilger et al. 2013c; Kaswan et al. 1989; Moore et al. 2001; Murphy et al. 2011). There are no known cures for immune-mediated ocular diseases, and current treatment regimens are costly, require multiple daily applications, are poorly effective, and have adverse side effects. Therefore, new treatments to address these diseases are needed and for further development, there is a need for accurate and translatable immune-relevant models of ocular disease. The eye, like the brain and the uterus in pregnancy, is considered an immune privileged site (Niederkorn and Wang 2005; Stein-Streilein 2008). An active suppression of the immune response to endogenous and exogenous antigens occurs in the eye, as overt inflammation may compromise vision. The relative lack of antigen-presenting and MHC II-expressing cells and natural tissue barriers (i.e., the blood–ocular barrier) that physically separate ocular tissues from the systemic immune response contribute to the immune tolerance in the eye (Niederkorn 2006). With dry eye and uveitis, the normal ocular tolerance is lost (from several initiating causes) and the physical barriers become disrupted, allowing an influx of inflammatory cells. In addition, proinflammatory mediators induce T-helper cells to proliferate, activate antigen-presenting cells, expand auto-reactive B and T cell populations, and ultimately release proinflammatory and proapoptotic peptides (Caspi 2006; Stern and Pflugfelder 2011). Current treatments for dry eye and uveitis are nonspecific and require frequent use of topical medications that may have severe ocular and systemic side effects (Carnahan and Goldstein 2000; Fraunfelder et al. 2012; Sen et al. 2014). Furthermore, these medications are life-long therapies and patient compliance is commonly poor, leading to treatment failures, worsening of disease, and in some cases, blindness (Uchino and Schaumberg 2013). When testing effectiveness of therapeutics on models of ocular disease, there are two separate but important testing goals. The first question is whether the drug is effective in the ocular disease state that is being studied. For this goal, usually rats or mice are evaluated and dosed by a nonocular route, for example, orally, subcutaneously, or intraperitoneally. These studies help determine pathogenesis of disease-drug mechanisms; therefore, the wide array of reagents and genetically modified mice and rats are a major asset. Determination of the appropriate dose (i.e., dose ranging studies) is usually also performed in these first sets of studies. The second goal is to determine if an appropriate dose can reach the ocular target tissue and be effective in the eye using a dosing route and frequency that is clinically feasible. These studies would determine the pharmacokinetics and pharmacodynamics of a specific route of administration of a drug, typically in a normal eye, then repeated using the optimal dosing and routes in eyes of models of the disease state. For this second group of studies to be clinically valid in most instances, the animal models would have to have eyes anatomically similar to the target species and in the case of humans, use of the rabbit, dog, pig, or primate eye would be most appropriate. Finally, when selecting the appropriate animal model, the target tissue and disease state has to be paired with the most appropriate route of therapy. This determination is important for pharmacokinetic, toxicologic, and efficacy studies. Although there are many disease conditions of the human eye thought to have an immunologic pathogenesis, including allergic conjunctivitis, corneal transplant rejection, and age-related macular degeneration, as examples, the purpose of this review is to describe common immune-relevant models of dry eye and uveitis with an overview and assessment of models from small to large animals. We will also review a selected group of naturally-occurring large animal models, equine uveitis and canine dry eye, which have promise to translate into a better understanding and treatment of clinical immune-relevant ocular disease in man. Review of Commonly Used Animal Models in Inflammatory Ocular Disease Ocular Surface Disease Immune-Relevant Models Dry Eye Disease Dry eye disease (DED) is one of the most common ocular abnormalities and has multiple underlying causes. Dry eye is a disease of the tear film and ocular surface that results in symptoms of discomfort and visual disturbance with potential damage to the ocular surface (DEWS 2007). In one study, nearly one-half of patients claimed to have symptoms of dry eye with a negative effect on quality of life, including ocular pain, decreased activities requiring visual attention (e.g., reading, driving), and reduced productivity in the workplace (Uchino and Schaumberg 2013). Dry eye develops from a deficiency of the aqueous portion of the tear fluid as a result of reduced lacrimal aqueous tear secretion or a result of increased evaporation of tears, such as the result of Meibomian gland deficiencies (Lemp et al. 2012). Decreased aqueous production of the tears results in an increase of tear electrolytes (i.e., increased tear osmolality), proteins, and inflammatory mediators, resulting in damage to the surface ocular tissues, decreased visual acuity, and ocular discomfort. The relative decrease in aqueous tears on the ocular surface in patients with DED causes chronic irritation to ocular surface that disrupts the normal ocular immune tolerance (Barabino et al. 2012). With breakdown of ocular surface tolerance and immune-homeostasis, autoimmunity develops through activation of NK cells and Toll-like receptors, followed by release of proinflammatory factors such as interleukin (IL)-1α, IL-1β, tumor necrosis factor α, and IL-6. These mediators amplify, activating antigen-presenting cells, which internalize autoantigens and migrate to the draining cervical lymph node where autoreactive Th1 cells, Th17 cells, or B cells (i.e., in Sjogren’s syndrome) undergo expansion. Efferent trafficking of these autoreactive T cells to the ocular surface is directed by adhesion molecules (e.g., LFA-1) and chemokine receptors. Autoreactive T-cells in ocular surface tissues potentiate the chronic autoimmune response, resulting in epithelial cell apoptosis, reduced goblet cell density, and squamous metaplasia of epithelium (Barabino et al. 2012; Stern and Pflugfelder 2011; Stern et al. 2010). Current treatments for DED rely on frequently applied artificial tears, punctal plugs, topical tetracycline antibiotic, and omega fatty acids, all of which provide only temporary relief of dry eye (Gayton 2009). Chronic DED is commonly treated with antiinflammatory medications and immunosuppressants, the latter being the mainstay of treatment in the United States (Avunduk et al. 2003; Sall et al. 2000). Topical cyclosporine, an immunosuppressant, used with or without corticosteroids, is effective in DED through inhibition of T-cell activation and reduction of proinflammatory cytokines (Lekhanont et al. 2007). A recently approved topical immunosuppressive for treatment of DED, lifitegrast, is an integrin inhibitor that prevents binding of LFA-1 to ICAM-1, which is upregulated in DED. Lifitegrast thus blocks T-cell efferent recruitment to ocular tissues and reduces inflammatory cytokines (Keating 2017; Sheppard et al. 2014; Tauber et al. 2015). However, both cyclosporine and lifitegrast must be administered indefinitely twice daily by the patient and are associated with burning sensation after application, leading to reduced patient compliance and hence poor treatment efficacy and success. Therefore, an effective, long-term, well-tolerated, and convenient therapy for DED is needed. There are numerous models of ocular surface disease and dry eye, but to be immune relevant, there needs to be evidence of an immuno-pathogenesis in the disease process. There are several mouse models of dry eye disease, the most common of which is a model induced by low humidity and high air flow environments, with or without the additional use of scopolamine (Table 1) (Barabino et al. 2004, 2005; Daull et al. 2016). The extended environmental irritation to the surface of the eye of these mice disrupts the normal ocular immune tolerance and immunohomeostasis (Barabino et al. 2012), as described previously. These mice models have been used to study the immuno-pathogenesis of dry eye and the initial evaluation of therapeutics. Another described model is the use of repeated application of topical benzalkonium chloride to the mouse or rabbit eye. This produces chronic irritation that may develop immunopathology and chronic ocular surface disease (Lin et al. 2011; Xiong et al. 2008). Other induced models of DED in rodents, which may be less immunopathologic in origin, include lacrimal gland excision or injections of toxins or antigens such as botulinum toxin (Zhu et al. 2009) or concanavalin A (Lee et al. 2015). Genetic models, such as the MRL/lpr mouse, manifest multiple autoimmune disorders and can be helpful to study diseases such as systemic lupus erythematosus and Sjorgren’s syndrome (Table 1) (Jabs et al. 1996). Another example of genetic DED are neurturin-deficient mice, which may develop dry eye and serve as models for neurotrophic keratoconjunctivitis sicca, since this model lacks lacrimal innervation (Table 1) (Song et al. 2003). There are numerous other knockout and transgenic mice strains that are commonly studied that may develop DED; however, many of these models do not develop clinical signs of DED observed in large animal models, but instead develop histologic or other features characteristic of human DED (Schrader et al. 2008). Table 1 Selected immune-models of dry eye disease Animal Method Advantages Disadvantages Reference Rodent Mice Environmental chambers ± scopolamine patch Reproducible, economical Small eye, anatomic differences Keating (2017) Botulinum toxin injection into lacrimal gland Reproducible, economical Above, and toxin present Lekhanont et al. (2007), Zhu et al. (2009) Intraorbital injection of concanavalin A Above, possible orbital inflammation Lee et al. (2015) Topical administration of benzalkonium chloride Economical Above, time to develop, may not be immunologic Lin et al. (2011) Extraorbital lacrimal gland excision ± scopolamine Surgically induced desiccating irritation Initiates an abnormal Th1/Th17 T cell response, exogenous antigens Guzmán et al. (2016), Stevenson et al. (2014) Neurturin-deficient (NRTN(−/−)) Song et al. (2003) MRL/lpr Sjogrens-like dry eye, autoimmune pathogenesis Chronic model, systemic disease Gao et al. (2004), Jabs et al. (1996), Jie et al. (2010), Ma et al. (2014) Rat Extraorbital lacrimal gland excision ± scopolamine May not be immunologic Fujihara et al. (2001), Meng et al. (2015) Rabbit Activated autologous lymphocytes injected into lacrimal gland Sjögren’s-like autoimmune dacryoadenitis Specialized, difficult model Thomas et al. (2010) Topical administration of benzalkonium chloride Economical Above, time to develop, may not be immunologic Xiong et al. (2008) Lacrimal gland excision May not be immunologic Gilbard et al. (1988) Canine Naturally-occurring Immunologic, translatable Availability Gilger et al. (2013c), Kaswan et al. (1989), Moore et al. (2001) Animal Method Advantages Disadvantages Reference Rodent Mice Environmental chambers ± scopolamine patch Reproducible, economical Small eye, anatomic differences Keating (2017) Botulinum toxin injection into lacrimal gland Reproducible, economical Above, and toxin present Lekhanont et al. (2007), Zhu et al. (2009) Intraorbital injection of concanavalin A Above, possible orbital inflammation Lee et al. (2015) Topical administration of benzalkonium chloride Economical Above, time to develop, may not be immunologic Lin et al. (2011) Extraorbital lacrimal gland excision ± scopolamine Surgically induced desiccating irritation Initiates an abnormal Th1/Th17 T cell response, exogenous antigens Guzmán et al. (2016), Stevenson et al. (2014) Neurturin-deficient (NRTN(−/−)) Song et al. (2003) MRL/lpr Sjogrens-like dry eye, autoimmune pathogenesis Chronic model, systemic disease Gao et al. (2004), Jabs et al. (1996), Jie et al. (2010), Ma et al. (2014) Rat Extraorbital lacrimal gland excision ± scopolamine May not be immunologic Fujihara et al. (2001), Meng et al. (2015) Rabbit Activated autologous lymphocytes injected into lacrimal gland Sjögren’s-like autoimmune dacryoadenitis Specialized, difficult model Thomas et al. (2010) Topical administration of benzalkonium chloride Economical Above, time to develop, may not be immunologic Xiong et al. (2008) Lacrimal gland excision May not be immunologic Gilbard et al. (1988) Canine Naturally-occurring Immunologic, translatable Availability Gilger et al. (2013c), Kaswan et al. (1989), Moore et al. (2001) Table 1 Selected immune-models of dry eye disease Animal Method Advantages Disadvantages Reference Rodent Mice Environmental chambers ± scopolamine patch Reproducible, economical Small eye, anatomic differences Keating (2017) Botulinum toxin injection into lacrimal gland Reproducible, economical Above, and toxin present Lekhanont et al. (2007), Zhu et al. (2009) Intraorbital injection of concanavalin A Above, possible orbital inflammation Lee et al. (2015) Topical administration of benzalkonium chloride Economical Above, time to develop, may not be immunologic Lin et al. (2011) Extraorbital lacrimal gland excision ± scopolamine Surgically induced desiccating irritation Initiates an abnormal Th1/Th17 T cell response, exogenous antigens Guzmán et al. (2016), Stevenson et al. (2014) Neurturin-deficient (NRTN(−/−)) Song et al. (2003) MRL/lpr Sjogrens-like dry eye, autoimmune pathogenesis Chronic model, systemic disease Gao et al. (2004), Jabs et al. (1996), Jie et al. (2010), Ma et al. (2014) Rat Extraorbital lacrimal gland excision ± scopolamine May not be immunologic Fujihara et al. (2001), Meng et al. (2015) Rabbit Activated autologous lymphocytes injected into lacrimal gland Sjögren’s-like autoimmune dacryoadenitis Specialized, difficult model Thomas et al. (2010) Topical administration of benzalkonium chloride Economical Above, time to develop, may not be immunologic Xiong et al. (2008) Lacrimal gland excision May not be immunologic Gilbard et al. (1988) Canine Naturally-occurring Immunologic, translatable Availability Gilger et al. (2013c), Kaswan et al. (1989), Moore et al. (2001) Animal Method Advantages Disadvantages Reference Rodent Mice Environmental chambers ± scopolamine patch Reproducible, economical Small eye, anatomic differences Keating (2017) Botulinum toxin injection into lacrimal gland Reproducible, economical Above, and toxin present Lekhanont et al. (2007), Zhu et al. (2009) Intraorbital injection of concanavalin A Above, possible orbital inflammation Lee et al. (2015) Topical administration of benzalkonium chloride Economical Above, time to develop, may not be immunologic Lin et al. (2011) Extraorbital lacrimal gland excision ± scopolamine Surgically induced desiccating irritation Initiates an abnormal Th1/Th17 T cell response, exogenous antigens Guzmán et al. (2016), Stevenson et al. (2014) Neurturin-deficient (NRTN(−/−)) Song et al. (2003) MRL/lpr Sjogrens-like dry eye, autoimmune pathogenesis Chronic model, systemic disease Gao et al. (2004), Jabs et al. (1996), Jie et al. (2010), Ma et al. (2014) Rat Extraorbital lacrimal gland excision ± scopolamine May not be immunologic Fujihara et al. (2001), Meng et al. (2015) Rabbit Activated autologous lymphocytes injected into lacrimal gland Sjögren’s-like autoimmune dacryoadenitis Specialized, difficult model Thomas et al. (2010) Topical administration of benzalkonium chloride Economical Above, time to develop, may not be immunologic Xiong et al. (2008) Lacrimal gland excision May not be immunologic Gilbard et al. (1988) Canine Naturally-occurring Immunologic, translatable Availability Gilger et al. (2013c), Kaswan et al. (1989), Moore et al. (2001) In rats, the most commonly described dry eye model is the extraorbital lacrimal gland excision model (with or without use of scopolamine) (Fujihara et al. 2001; Meng et al. 2015). Like other models of induced dry eye, this rat lacrimal-excision model likely does not develop, substantially, an immunologic pathogenesis and therefore may not be as effective for evaluation of immunosuppressive therapies as naturally-occurring models of dry eye (Barabino et al. 2004; Meng et al. 2015). All of these rodent models of dry eye are similar in that they can be used to determine proof of principal of therapeutic response to a drug, but all have similar disadvantages of having orbital and lacrimal anatomy and eye size that differs from the human eye. Rabbit or dog models are more commonly used to evaluate dry eye signs and response to therapy, because they have easily measured decreased tear production and develop ocular surface changes (Schrader et al. 2008). Therefore, larger animal dry eye models are needed (see later description of canine dry eye). Rabbits are commonly used as models of ocular disease and for pharmacokinetic studies because of their relatively large eye, compared to rodents, while still being a common and economical laboratory animal. However, there are few true immune-relevant models of DED in rabbits. Most described models induce dry eye signs, but not likely an immunopathogenesis, by use of a topical irritant, such as benzalkonium chloride, or by short-term reduction in lacrimal secretion using parasympathomimetic drug, such as atropine (Burgalassi et al. 1999; Li et al. 2012; Xiong et al. 2008). A very promising model of autoimmune dacryoadenitis in rabbits that produces a Sjögren’s-like keratoconjunctivitis is created by an intra-lacrimal or subcutaneous injection of autologous peripheral blood lymphocytes activated by purified rabbit lacrimal epithelial cells (Thomas et al. 2008; Zhu et al. 2003). This rabbit autoimmune dacryoadenitis model has been used to effectively evaluate immunomodulatory treatments for dry eye, including topical cyclosporine and lacrimal gland adeno-associated virus (AAV) mediated-IL-10 gene therapy (Thomas et al. 2009, 2010). Naturally-Occurring Keratoconjunctivitis Sicca in Canines Domestic canines develop spontaneous dry eye that clinically and immunopathologically is similar to dry eye in humans (Table 1) (Kaswan et al. 1989). Not only do dogs spontaneously develop dry eye symptoms of ocular discomfort, conjunctival hyperemia, and corneal scarring, these symptoms correlate directly with reduced aqueous tear production, a reduction readily measured using a standard Schirmer tear test strip (Figure 1). Furthermore, dogs with dry eye have a reduced tear breakup time and increased corneal staining, all abnormalities also observed in humans with DED (Kaswan et al. 1989). Like humans, canine dry eye is typically bilateral, develops in middle age, is more common in female dogs and in certain breeds, such as the American Cocker spaniel, Bulldog, and West Highland white terrier (Sanchez et al. 2007). The pathogenesis of dry eye in dogs appears similar to that of humans, where an apparent immunologic inflammation occurs with progressive lymphocytic infiltration and damage to the lacrimal gland with subsequent decreased production of the aqueous tear film (Izci et al. 2015; Kaswan et al. 1984). With chronicity, the ocular surface becomes progressively more dessicated and inflamed, the cornea vascularizes and scars, and ultimately the dog may lose vision (Gilger et al. 2013c; Sanchez et al. 2007). Initial proof of concept of commonly used immunosuppressive eye drops was first demonstrated to be effective in this spontaneous dog model, including topical cyclosporine, tacrolimus, and LTF-1 inhibitors (Barachetti et al. 2015; Berdoulay et al. 2005; Gilger et al. 2013c; Kaswan et al. 1989; Murphy et al. 2011). Figure 1 View largeDownload slide Naturally-occurring dry eye in a dog. (A) Moderate dry eye disease in a dog resulting in conjunctival hyperemia, corneal vascularization, and corneal opacity. (B) Chronic dry eye disease in a dog with mucopurulent ocular discharge, hyperpigmented cornea, and conjunctival hyperemia. Figure 1 View largeDownload slide Naturally-occurring dry eye in a dog. (A) Moderate dry eye disease in a dog resulting in conjunctival hyperemia, corneal vascularization, and corneal opacity. (B) Chronic dry eye disease in a dog with mucopurulent ocular discharge, hyperpigmented cornea, and conjunctival hyperemia. Uveitis Disease Models Uveitis is inflammation of the iris, ciliary body, and choroid and is associated with both infectious and noninfectious causes. Uveitis is estimated to be the third leading cause of preventable blindness worldwide (Siddique et al. 2013). In the United States, the incidence of uveitis was estimated to be approximately 58 to 69 cases/100,000 people (Acharya et al. 2013; Suhler et al. 2008); however, another study estimated that the rate of uveitis, especially anterior uveitis, was approximately 3 times higher and it increased with increasing age of patients (Gritz and Wong 2004). The most common causes of uveitis in humans are human leukocyte antigen (HLA)-B27 related uveitis, acute anterior uveitis in herpes zoster disease, toxoplasmosis, sarcoidosis, and pars planitis (Jabs 2008). Uveitis results from several causes. The uveal tract supplies blood to the eye and is in direct contact with peripheral vasculature; therefore, diseases of the systemic circulation (e.g., septicemia, bacteremia, infection, activated lymphocytes, immune diseases, etc.) will disrupt the blood-ocular barrier (Generali et al. 2015; Levitt et al. 2015). The blood-ocular barrier prevents large molecules and cells from entering the eye and thus limits the immune response to intraocular antigens. With trauma or inflammation, this barrier can be disrupted, allowing blood products and cells to enter the eye, resulting in the clinical signs typical of uveitis, such as flare, cell accumulation, and vitreous haze. Disruption of the barrier enables activation of various host immune responses, including antibody production to self-antigens that are not normally recognized by the immune system, as well as antibody production to foreign antigens inside the eye. As a result of the blood-ocular barrier, lack of lymphatics, and the presence of limited numbers of resident leukocytes, the eye is considered to have immune privilege. Naïve T cells cannot cross the normal blood-retinal barrier due to the lack of fenestration in the retinal vessels and the lack of appropriate adhesion molecules (Caspi 2011). Expression of chemokines in inflammation and activated T cells in the ciliary epithelium may play a role in recruitment and activation of leukocytes in diseased eyes (Gilger et al. 2002). As in other autoimmune disorders, infections may trigger events, either by antigenic mimicry with a pathogen’s antigen or as a bystander effect due to the general systemic or local immune stimulation by the pathogen. Uveitogenic retinal proteins documented in experimental animals include retinal arrestin, interphotoreceptor retinoid-binding protein (IRBP), rhodopsin, recoverin, phosducin, and retinal pigment epithelium derived RPE-65 (Deeg 2008; Deeg et al. 2001, 2006b; Siddique et al. 2013). Irrespective of the eliciting antigen, available experimental evidence suggests that the immunological mechanisms driving the resultant disease are similar (Caspi 2006). Following disruption of the blood-ocular barrier, large amounts of predominantly CD4+ T cells enter the eye and secrete proinflammatory cytokines such as IL-2 and interferon γ (Gilger et al. 1999). Auto-reactive effector CD4+ T cells have been associated with the pathogenesis of inflammatory and autoimmune disorders including uveitis. Naıve CD4+ T cells differentiate into effector subsets depending on the nature of the environment in which exposure to the antigen occurs (Caspi 2011). Several T cell effector phenotypes have been defined, known as T helper 1 (TH1), TH2, or TH17. Early studies suggested that the interferon-γ-producing TH1 and IL-17-releasing TH17 subsets are responsible for the pathology of uveitis, with the latter being associated with development of autoimmune disease (Caspi 2006). Additionally, clinical uveitis frequently develops spontaneous recurrent or relapsing bouts of inflammation, likely from T cells recognizing additional autoantigens in the ocular tissue (Deeg et al. 2006a). Resolution of uveitis is dependent on the presence of T regulatory cells (Tregs) that are labeled as CD4+Foxp3+ cells. When Foxp3+ T cell percentages in uveitis increase to approximately 10% of the total CD4+ cells, the acute inflammation rapidly resolves. Therefore, Foxp3+ Tregs are important to induce spontaneous resolution and in maintaining remission of uveitis (Silver et al. 2015). Multiple models have been developed to evaluate the immuno-pathogenesis of uveitis and recurrent uveitis, including identification of autoantigens. Most of these models are rodent based. Other models, including those that are acute, chronic, and recurrent in nature, have been developed to evaluate therapeutics (Table 2). Large animal models, such as uveitis induced in rabbits and pigs, have been evaluated to test therapeutics in larger eyes to help translate these treatments to humans (Table 2). Table 2. Models of uveitis Type of uveitis Species/Strains Agents Type of inflammation Advantages Disadvantages Reference Rodent EIU Lewis rat: Harlan Sprague Dawley Mice: (C3H and other strains) Endotoxin Anterior uveitis Rapid onset, predictable Nonimmunologic Cousins et al. (1984), Li et al. (1995) EAU Rat Melanin from bovine RPE Tyrosinase-related proteins 1 and 3 Anterior uveitis Immunologic Agarwal et al. (2012) Experimental autoimmune uveoretinitis Mice Rat Retinal arrestin (S-Ag), IRBP, recoverin, phosducin, rhodopsin/opsin Posterior segment Immunologic Chen et al. (2013, 2015), Shao et al. (2006), Silver et al. (2015) Spontaneous Mice RBP T cell receptor transgenic mice (R161H) Autoimmune Regulator (AIRE)(−/−) mice Adoptive transfer Retinal degeneration and persistent cellular infiltrates and lymphoid aggregation, multi-focal infiltrates and severe choroidal inflammation. Chen et al. (2013), Shao et al. (2006) Rabbit EIU NZW Dutch belted Endotoxin Anterior and posterior segment Rapid onset, predictable Nonimmunogenic Fleisher et al. (1990), Goldblum et al. (2007) Recurrent uveitis NZW Mycobacterium tuberculosis H37Ra antigen Ovalbumin Anterior (intracameral) or posterior (intravitreal) segment Immunologic and recurrent Ang et al. (2014), Edmond et al. (2016), Ghosn et al. (2011), Neumann et al. (1993) Cytokine induced uveitis NZW IL-1 TNF-alpha Acute onset (6 hours) Nonimmunologic Fleisher et al. (1990), Tilden et al. (1990) Porcine EIU Various Endotoxin Posterior Large eye Nonimmunogenic Gilger et al. (2013b) Horse Recurrent uveitis Various Spontaneous Anterior and posterior Large eye; immunologic; recurrent Nonstandard research animal Cost Deeg (2008), Deeg et al. (2008), Gerding and Gilger (2016) Type of uveitis Species/Strains Agents Type of inflammation Advantages Disadvantages Reference Rodent EIU Lewis rat: Harlan Sprague Dawley Mice: (C3H and other strains) Endotoxin Anterior uveitis Rapid onset, predictable Nonimmunologic Cousins et al. (1984), Li et al. (1995) EAU Rat Melanin from bovine RPE Tyrosinase-related proteins 1 and 3 Anterior uveitis Immunologic Agarwal et al. (2012) Experimental autoimmune uveoretinitis Mice Rat Retinal arrestin (S-Ag), IRBP, recoverin, phosducin, rhodopsin/opsin Posterior segment Immunologic Chen et al. (2013, 2015), Shao et al. (2006), Silver et al. (2015) Spontaneous Mice RBP T cell receptor transgenic mice (R161H) Autoimmune Regulator (AIRE)(−/−) mice Adoptive transfer Retinal degeneration and persistent cellular infiltrates and lymphoid aggregation, multi-focal infiltrates and severe choroidal inflammation. Chen et al. (2013), Shao et al. (2006) Rabbit EIU NZW Dutch belted Endotoxin Anterior and posterior segment Rapid onset, predictable Nonimmunogenic Fleisher et al. (1990), Goldblum et al. (2007) Recurrent uveitis NZW Mycobacterium tuberculosis H37Ra antigen Ovalbumin Anterior (intracameral) or posterior (intravitreal) segment Immunologic and recurrent Ang et al. (2014), Edmond et al. (2016), Ghosn et al. (2011), Neumann et al. (1993) Cytokine induced uveitis NZW IL-1 TNF-alpha Acute onset (6 hours) Nonimmunologic Fleisher et al. (1990), Tilden et al. (1990) Porcine EIU Various Endotoxin Posterior Large eye Nonimmunogenic Gilger et al. (2013b) Horse Recurrent uveitis Various Spontaneous Anterior and posterior Large eye; immunologic; recurrent Nonstandard research animal Cost Deeg (2008), Deeg et al. (2008), Gerding and Gilger (2016) EAU, experimental autoimmune anterior uveitis; EIU, endotoxin-induced uveitis; IL, interleukin; NZW, Zealand White. Table 2. Models of uveitis Type of uveitis Species/Strains Agents Type of inflammation Advantages Disadvantages Reference Rodent EIU Lewis rat: Harlan Sprague Dawley Mice: (C3H and other strains) Endotoxin Anterior uveitis Rapid onset, predictable Nonimmunologic Cousins et al. (1984), Li et al. (1995) EAU Rat Melanin from bovine RPE Tyrosinase-related proteins 1 and 3 Anterior uveitis Immunologic Agarwal et al. (2012) Experimental autoimmune uveoretinitis Mice Rat Retinal arrestin (S-Ag), IRBP, recoverin, phosducin, rhodopsin/opsin Posterior segment Immunologic Chen et al. (2013, 2015), Shao et al. (2006), Silver et al. (2015) Spontaneous Mice RBP T cell receptor transgenic mice (R161H) Autoimmune Regulator (AIRE)(−/−) mice Adoptive transfer Retinal degeneration and persistent cellular infiltrates and lymphoid aggregation, multi-focal infiltrates and severe choroidal inflammation. Chen et al. (2013), Shao et al. (2006) Rabbit EIU NZW Dutch belted Endotoxin Anterior and posterior segment Rapid onset, predictable Nonimmunogenic Fleisher et al. (1990), Goldblum et al. (2007) Recurrent uveitis NZW Mycobacterium tuberculosis H37Ra antigen Ovalbumin Anterior (intracameral) or posterior (intravitreal) segment Immunologic and recurrent Ang et al. (2014), Edmond et al. (2016), Ghosn et al. (2011), Neumann et al. (1993) Cytokine induced uveitis NZW IL-1 TNF-alpha Acute onset (6 hours) Nonimmunologic Fleisher et al. (1990), Tilden et al. (1990) Porcine EIU Various Endotoxin Posterior Large eye Nonimmunogenic Gilger et al. (2013b) Horse Recurrent uveitis Various Spontaneous Anterior and posterior Large eye; immunologic; recurrent Nonstandard research animal Cost Deeg (2008), Deeg et al. (2008), Gerding and Gilger (2016) Type of uveitis Species/Strains Agents Type of inflammation Advantages Disadvantages Reference Rodent EIU Lewis rat: Harlan Sprague Dawley Mice: (C3H and other strains) Endotoxin Anterior uveitis Rapid onset, predictable Nonimmunologic Cousins et al. (1984), Li et al. (1995) EAU Rat Melanin from bovine RPE Tyrosinase-related proteins 1 and 3 Anterior uveitis Immunologic Agarwal et al. (2012) Experimental autoimmune uveoretinitis Mice Rat Retinal arrestin (S-Ag), IRBP, recoverin, phosducin, rhodopsin/opsin Posterior segment Immunologic Chen et al. (2013, 2015), Shao et al. (2006), Silver et al. (2015) Spontaneous Mice RBP T cell receptor transgenic mice (R161H) Autoimmune Regulator (AIRE)(−/−) mice Adoptive transfer Retinal degeneration and persistent cellular infiltrates and lymphoid aggregation, multi-focal infiltrates and severe choroidal inflammation. Chen et al. (2013), Shao et al. (2006) Rabbit EIU NZW Dutch belted Endotoxin Anterior and posterior segment Rapid onset, predictable Nonimmunogenic Fleisher et al. (1990), Goldblum et al. (2007) Recurrent uveitis NZW Mycobacterium tuberculosis H37Ra antigen Ovalbumin Anterior (intracameral) or posterior (intravitreal) segment Immunologic and recurrent Ang et al. (2014), Edmond et al. (2016), Ghosn et al. (2011), Neumann et al. (1993) Cytokine induced uveitis NZW IL-1 TNF-alpha Acute onset (6 hours) Nonimmunologic Fleisher et al. (1990), Tilden et al. (1990) Porcine EIU Various Endotoxin Posterior Large eye Nonimmunogenic Gilger et al. (2013b) Horse Recurrent uveitis Various Spontaneous Anterior and posterior Large eye; immunologic; recurrent Nonstandard research animal Cost Deeg (2008), Deeg et al. (2008), Gerding and Gilger (2016) EAU, experimental autoimmune anterior uveitis; EIU, endotoxin-induced uveitis; IL, interleukin; NZW, Zealand White. Rodent Models of Uveitis Endotoxin-induced uveitis A commonly used model of induced uveitis in rodents is the endotoxin-induced uveitis (EIU) model (Table 2) (Altinsoy et al. 2011; Cousins et al. 1984; Li et al. 1995). The uveitis in this model is primarily an acute anterior uveitis (i.e., iris, ciliary body) that is thought to be driven by the innate immune system (Table 2) (Agarwal et al. 2012; Caspi 2006). Following intraparentoneal, subcutaneous, or hind footpad injection of endotoxin (lipopolysaccharide; 100 μg or 500 μg) in Lewis or Sprague-Dawley rats or various mouse strains (C3H) (Caspi 2006), ocular inflammation develops within hours of injection characterized by a breakdown of the blood-aqueous barrier and the development of clinical disease. Clinical and histopathologic abnormalities peak at 24 hours and resolve by 48 to 72 hours (Cousins et al. 1984; Li et al. 1995). Experimental autoimmune uveitis (or uveoretinitis) Experimental autoimmune uveitis (EAU) is a primarily posterior uveitis (or panuveitis [i.e., inflammation of the iris, ciliary, and choroid]) that is induced by immunizing susceptible rodents with retinal antigens (e.g., S-antigen [S-ag], IRBP, recoverin, rhodopsin/opsin); while experimental melanin–protein induced uveitis, a predominantly anterior uveitis, is elicited by immunization with melanin (from RPE) or tyrosinase-related proteins 1 and 2 (Table 2) (Caspi 2006). The predominant animal model is the Lewis rat, but other animals such as the guinea pig or mice have also been described (Agarwal et al. 2012; Wacker 1972). Injection of autoantigens into rodents, combined with bacterial adjuvants, results in EAU; EAU does not develop without the use of adjuvants. The use of complete Freund’s adjuvant (Mycobacterium cell wall product) or pertussis toxin is necessary to stimulate the innate immune response and develop inflammation (Agarwal et al. 2012; Chen et al. 2015; Silver et al. 2015) that ultimately generates activated antigen-presenting cells capable of presenting the injected autoantigen with the coactivation factors required to activate T cells capable of recognizing the antigen. Severe EAU was induced in B6 mice by adoptive transfer of IRBP-specific T cells (Shao et al. 2006). Most of the rodent experimental models of uveitis are not recurrent. They often elicit a single, albeit chronic course of uveitis that eventually resolves. Therefore, the immunologic pathways involved in the development of these rodent models may not be the same as in naturally occurring uveitis. Spontaneous uveitis has been observed in various mouse models, including IRBP T cell receptor transgenic mice (R161H) and autoimmune regulator (AIRE)(-/-) mice (Chen et al. 2015). These mouse models have a gradual onset of chronic ocular inflammation that ultimately leads to retinal degeneration (Chen et al. 2015). Despite limitations, these rodent experimental models offer great insight into the pathogenesis and immunopathogenesis of uveitis. These models have been critical in evaluation of therapies, particularly broader immunosuppressive therapies, for treating uveitis. Rabbit Models of Uveitis Two rabbit models of uveitis have been most commonly evaluated, including the acute uveitis induced by injection of endotoxin (Allen et al. 1996; Ghosn et al. 2011; Nussenblatt et al. 2012; Rafie et al. 2010) and the recurrent uveitis induced by tuberculosis antigen (Ang et al. 2014; Ghosn et al. 2011; Mruthyunjaya 2006). Other uveitis models in rabbits include those following intravitreal injection of human interleukin 1 alpha (Tilden et al. 1990), TNF-alpha (Fleisher et al. 1990), or ovalbumin in animals previously ovalbumin-immunized (Neumann et al. 1993), among others (Table 2). An advantage of rabbit models of uveitis over rodent models is that the rabbit eye is more similar in size to the human eye, and therefore, more pharmacologically valid when evaluating routes of therapy. In the endotoxin-induced uveitis, after 10 to 100 ng of LPS is injected intracamerally (Nussenblatt et al. 2012) or intravitreally (Fleisher et al. 1990; Goldblum et al. 2007), aqueous flare and iridal hyperemia develop within 6 hours, suggesting rapid disruption of the blood–aqueous barrier (Allen et al. 1996). The LPS induces inflammation by activating a Toll-like receptor 4-initiated signaling cascade. The inflammatory response peaks at approximately 24 hours after injection, then rapidly declines (Nussenblatt et al. 2012). Like the rat model of EIU, this endotoxin rabbit model of uveitis is not considered to have a predominantly immunopathogenesis. Experimental uveitis can be induced by unilateral intravitreal or intracameral injection of Mycobacterium tuberculosis H37Ra antigen (50 μg; 1 μg/L) in preimmunized rabbits, typically 7 to 14 days after initial subcutaneous injection (Ang et al. 2014; Edmond et al. 2016; Ghosn et al. 2011; Jaffe et al. 1998). To simulate chronic recurrent inflammation, eyes are re-challenged with intravitreal antigen every 14 to 21 days (Jaffe et al. 2006). This model has advantages similar to the endotoxin model; however, it is predominantly a T-cell lymphocyte-mediated uveitis that can be induced to be recurrent and therefore, more closely simulate endogenous human uveitis (Jaffe et al. 1998). Porcine Models of Uveitis The pig has been used as a large animal model of uveitis (Table 2), which, similar to the rabbit model, has an eye similar in size to the human eye; however, unlike the rabbit, it has a retinal vascular anatomy similar to humans (Gilger et al. 2013a). An acute model of uveitis has been used in the pig to evaluate novel therapeutics and routes of administration. In this model, similar to endotoxin uveitis in rabbits, endotoxin is injected intravitreally and the eye is monitored for up to 72 hours following injection (Gilger et al. 2013b). Like rodent EIU and endotoxin uveitis in rabbits, the endotoxin porcine model of uveitis is not considered to have an immunopathogenesis. Naturally-Occurring Uveitis Models Equine Recurrent Uveitis Horses spontaneously develop severe, immunologic uveitis called equine recurrent uveitis (ERU) that is frequently recurrent and chronic (Figure 2) (Wollanke et al. 2004). ERU is the most common cause of blindness in horses (Deeg et al. 2008; Gerding and Gilger 2015; Wollanke et al. 2004). Spontaneous bouts of uveitis develop and blindness may occur after multiple recurrent episodes of uveitis. The immunopathology of ERU has been extensively studied and has demonstrated that T cells are the predominant mononuclear inflammatory cells infiltrating ocular tissues in horses with naturally occurring chronic uveitis, with a significant number of CD4+ cells (Deeg et al. 2006a; Gilger et al. 1999; Kleinwort et al. 2016). Recruitment of proinflammatory cells as well as autoreactive lymphocytes may be in part driven by the expression of the chemokine RANTES in the ciliary body (Gilger et al. 2002). Figure 2 View largeDownload slide Naturally-occurring uveitis in a horse. (A) Acute active uveitis in a horse, with a miotic pupil and anterior chamber opacity. (B) Chronic uveitis in a horse, from equine recurrent uveitis, with dyscoria, synechiae, and cataract. Figure 2 View largeDownload slide Naturally-occurring uveitis in a horse. (A) Acute active uveitis in a horse, with a miotic pupil and anterior chamber opacity. (B) Chronic uveitis in a horse, from equine recurrent uveitis, with dyscoria, synechiae, and cataract. Study of this common, spontaneous uveitis in horses has helped understand the pathogenesis of uveitis in humans, especially the identification of autoantigens and how recurrence of uveitis develops immunologically (Deeg 2009; Deeg et al. 2001, 2006a, 2006b, 2007, 2008; Degroote et al. 2014; Zipplies et al. 2009). Several potential autoantigens have been identified in horses that could play a role in the development of autoimmune uveitis. T cells isolated from the eyes of horses with ERU proliferate in response to two common autoantigens in rodents: retinal S-Ag and IRBP (Deeg et al. 2001). In addition, several additional potential autoantigens were identified by analyzing antibodies in the sera of ERU horses that reacted with retinal proteins. These include recoverin, cellular retinaldehyde-binding protein, and malate dehydrogenase (Deeg et al. 2007). While all these potential autoantigens are capable of inducing experimental uveitis in rodent models, only cellular retinaldehyde-binding protein and IRBP consistently produce uveitis in outbred horses (Deeg 2009; Deeg et al. 2006b). Additionally, studies of horses with ERU have also helped elucidate how Leptospira infections induce immunological uveitis, specifically autoimmune uveitis (Lucchesi and Parma 1999; Lucchesi et al. 2002; Verma et al. 2005). Field studies of horses in the 1950s after an outbreak of acute leptospirosis caused by L. interrogans serogroup Pomona demonstrated that one of the six horses (17%) developed intraocular inflammation during acute leptospiral disease, and all horses developed ERU 18 to 24 months after the initial infection. Subsequent studies demonstrated cross-reactivity between equine ocular tissues and Leptospira antigens (Lucchesi and Parma 1999; Lucchesi et al. 2002), and horses with uveitis associated with Leptospira interrogans infections had high levels of IgA and IgG in their intraocular fluids that reacted to two Leptospira lipoproteins, LruA and LruB (Verma et al. 2005, 2010). These antibodies were also subsequently discovered in the serum of human leptospiral uveitis patients (Verma et al. 2008). Studies of spontaneous ERU have helped elucidate the immunopathogenesis of recurrent uveitis. In autoimmune disease, several autoantigens, or epitopes, participate in the immunopathogenesis; epitope spreading is accountable for disease induction, progression, and inflammatory relapses (Deeg et al. 2006a). Epitope spreading is defined as the diversification of epitope specificity from the initial focused, dominant, epitope-specific immune response, directed against a self or foreign protein to cryptic epitopes on that protein (intramolecular spreading) or other proteins (intermolecular spreading) (Deeg et al. 2006a). The shifts in immunoreactivity, or epitope spreading, have been documented in ERU and are thought to be responsible for the recurring character of ERU (Deeg et al. 2006a). ERU, as a model of spontaneous immune-mediated uveitis, has also led to the study of promising therapeutics. For example, several sustained release ocular implants have shown much promise in the treatment of ERU (Gilger et al. 2000, 2001). Evaluation of drug delivery to the suprachoroidal space has been shown to control ERU and prevent recurrences (Gilger et al. 2006, 2010). Triamcinolone injections into the suprachoroidal space are currently under development for treatment of human uveitis (Moisseiev et al. 2016). Further study of ERU and its treatment will translate well to improving the understanding and treatment of human autoimmune uveitis. Next Steps As further therapeutics are developed that more specifically target immune-mediated diseases, evaluation of these treatments in spontaneous or naturally-occurring models of ocular disease will be needed to provide proof of concept and help translate these therapies to humans. Excellent examples of developing, targeted therapies include gene therapy (especially gene addition therapy) and stem cell therapy. Our laboratory and collaborators at the Gene Therapy Center at the University of North Carolina have developed AAV delivery of immunosuppressive proteins, such as HLA-G, for suppression of ocular surface inflammation and vascularization (Hirsch et al. 2017). Target ocular surface diseases for AAV-HLA-G gene therapy are DED and for prevention of corneal graft rejection (Hirsch et al. 2017). Autologous stem cell therapy, or use of stem cell supernatant extracts, also shows much promise for immunomodulation in the eye. Effectiveness of topical ocular mesenchymal stem cell therapy was initially demonstrated in dry eye models in mice (Lee et al. 2015). Locally injected fat-derived mesenchymal stem cells near the lacrimal glands of dogs with advanced dry eye demonstrated clinical improvement and increased tear production (Villatoro and Fernández 2014). These results in a naturally-occurring model provides evidence of possible clinical translation to humans with severe dry eye. 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ILAR JournalOxford University Press

Published: Feb 21, 2018

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