m. Cnivericin

Keywords:

Oxidative stress

n imchafroint

However, there are several scenarios in which immune privilege is altered and the eye becomes sus-

wrighw to yme a

anterior and posterior segments of the eye. Anterior segment pa-thologies with established immune system intervention include,but may not be limited to, the following: viral and bacterial-induced keratitis, infectious and non-infectious (autoimmune)

al transplantation,t state of immunetina or posterior

nomenon knownby Dutch ophthal-go after observing

that tumor cell inoculums injected in the anterior chamber of theeye successfully proliferated and formed tumors [1]. However, itwas Sir Peter Medawar and his student Ruppert Everett Billinghamwho, several decades later, coined the term immune privilege,after conducting a series of transplant experiments utilizinggenetically disparate rabbit strains. These experiments led to theobservation that, regardless of genetic disparities, skin graftstransplanted into the brain or anterior chamber (AC) of the eyeof recipient rabbits would survive for a longer period of time

* Corresponding author. Ocular Surface Center, Microbiology & Immunology,Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, 163810th Ave NW, Miami, FL 33136, USA.

Contents lists available at

Journal of Aut

els

Journal of Autoimmunity 45 (2013) 7e14E-mail address: Vperez4@med.miami.edu (V.L. Perez).ocular immune responses to allo and autoantigens to dissectpathways of ocular immune regulation. This is of importance to us,as the eye has being considered as an immune privilege site andunderstanding the immune-regulatory pathways of this organcould lead to novel observations in the eld of immunology. Theimmune system is known to be either directly or indirectly impli-cated in a large array of ocular pathologies involving both the

responses that occur following allogeneic cornewhereas the second half will review the currenresponses to auto-antigens found in the resegment.

The rst description pertaining to the phetoday as ocular immune privilege was mademologist J.C. van Dooremaals over a century amammalian immune system in centuries to come. Our laboratoryhas used this window to visualize in real time the development of

nopathy. In line with the interests of our laboratory, the rst half ofthis reviewwill primarily focus on the innate and adaptive immune1. Introduction

The great English poet and playonce said: The Eyes are the windoknow that this organ would beco0896-8411/$ e see front matter 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.jaut.2013.06.011tems is critical in both acute and chronic inammatory responses in the eye, with T cells playing a centralrole in combination with neutrophils and macrophages. In addition, we emphasize the advantage ofusing the eye as a model for in vivo longitudinal imaging of the immune system in action. Through thistechnique, it has been possible to identify functionally distinct intra-graft motility patterns of respondingT cells, as well as the importance of chemokine signaling in situ for T cell activation. The detailed study ofocular autoimmunity could provide novel therapeutic strategies for blinding diseases while alsoproviding more general information on acute versus chronic inammation.

2013 Elsevier Ltd. All rights reserved.

t William Shakespeareour soul. Little did hetrue window to the

uveitis, dry-eye syndromes, oncogenic events such as uveal mela-noma, and corneal allograft rejection following transplantation.Diseases of the posterior segment in which inammation isthought to play a role include age-related macular degeneration(AMD), glaucoma, chorioretinal disorders and autoimmune reti-RetinaInammation related macular degeneration. Interestingly, crosstalk between the innate and adaptive immune sys-Allo-transplantationCornea

ceptible to immune attack. In this review, we highlight the role of the immune system in two clinicalconditions that affect the anterior and posterior segments of the eye: corneal transplantation and age-The eye: A window to the soul of the im

V.L. Perez a,b,*, A.M. Saeed a, Y. Tan a, M. Urbieta a, Fa Laboratory of Ocular Immunology and Transplantation, Bascom Palmer Eye Institute, UbDepartment of Microbiology & Immunology, University of Miami Miller School of Med

a r t i c l e i n f o

Article history:Received 18 June 2013Accepted 18 June 2013

a b s t r a c t

The eye is considered as amolecular and cellular mecornea is mainly protectedretinaeblood barrier is ma

journal homepage: www.All rights reserved.une system

ruz-Guilloty a,b

sity of Miami Miller School of Medicine, Miami, FL 33136, USAe, Miami, FL 33136, USA

mune privileged site, and with good reason. It has evolved a variety ofnisms that limit immune responses to preserve vision. For example, them autoimmunity by the lack of blood and lymphatic vessels, whereas theained in an immunosuppressive state by the retinal pigment epithelium.

SciVerse ScienceDirect

oimmunity

evier .com/locate/ jaut imm

Autocompared to grafts transplanted elsewhere in the body [2].Immunological privilege is thought to have evolved in part toprotect organs such as the brain, the eye, and the testis from loss offunction secondary to overwhelming inammatory responses.Subsequent observations concerning ocular immune privilege ledto the hypothesis that the absence of lymphatic vessels in the AC ofthe eye, as well as in other immune privileged sites, precluded theimmune system from detecting foreign antigens and thereforeinitiating a response [3]. This hypothesis weakened in the late1970s after Kaplan and Streilein documented the presence ofhemagglutinating antibodies in the serum of inbred rats shortlyafter inoculation of allogeneic lymphoid cells in the AC anddemonstrating that intra-ocular antigens do elicit a systemic formof immune-regulation, which was named anterior chamber asso-ciated immune deviation or ACAID [4e6]. Since then, this concepthas evolved and other mechanisms of immune regulation havebeen shown to play a critical role in ocular disorders.

2. Immune responses in the anterior segment of the eye

2.1. Ocular immune responses to allo-antigens after cornealtransplantation

Immune reactions triggered by the presence of histoincompat-ible antigens are not exclusively limited to the protective ACAIDresponses. This is evidenced by the fate of most corneal allograftstransplanted into tissue-incompatible recipients with highly vas-cularized corneal beds, referred to as high-risk recipients. Cornealtransplantation is the most common form of solid organ trans-plantation in the United States and is also the treatment of choice torestore vision in patients with scarred and/or opacied corneas [7].Despite its high rate of success in recipients with non-vascularizedcorneal beds, corneal transplants performed in high-risk (vascu-larized beds) patients have a poor chance of survival [8]. Substantialevidence now exists indicating that these corneal allografts un-dergo immunological rejection, a process in which allospecic Tcells are instrumental [9e11]. Our laboratory has a long standinginterest in utilizing real time imaging of uorescently labeled im-mune cells to elucidate the mechanisms that govern immune cellrecruitment to the site of transplantation, as well as how the tempoand magnitude of cell migration impinge on activation and cornealgraft destruction.

The upregulation and expression of immune cell chemo-attractants at the site of transplantation is a key feature of vascu-larized solid organ transplants [12]. Chemokines are small proteinscharacterized by the presence and particular arrangement of fourconserved cysteine residues (C, CC, CXC, and CX3C) in their N-ter-minal region. More than 50 chemokines have been identied andcharacterized, making this cytokine superfamily one of the largeststudied to date [13,14]. In addition to orchestration of directed cellmigration or chemotaxis, many roles have been ascribed to che-mokines, including their participation in wound repair, inamma-tion, angiogenesis, viral entry, tumor metastasis, and solid allograftrejection [14]. The impact of chemokines in solid organ trans-plantation may extend beyond in-situ production of these mole-cules following transplantation. Studies by Brouard et al.demonstrated that pre-transplant serum levels of specic T cellchemokines such as CXCL9/MIG could be used as biomarkers ofacute allograft rejection [15].

We have previously documented the emergence of a specicchemokine prole following corneal transplant surgery in murinemodels of high-risk corneal transplantation [16]. This chemokineexpression pattern begins early on (days 1e7 post-transplant) withupregulation of CXCL1/KC and CCL2/MCP-1, coinciding with the

V.L. Perez et al. / Journal of8peak of intragraft neutrophil inltration, and culminates with asurge in T cell chemokine expression, namely CXCL9/MIG andCXCL10/IP-10, concomitant with the peak of CD4 T cell inltrationand allograft rejection (days 11e14 post-transplantation) [16]. Thenotion of chemokine regulatory cascades and their involvement inother models of solid organ allograft rejection has been previouslyexamined [17]. Other relevant chemokine studies performed byother groups have demonstrated an important role for chemokinereceptors that control immune cell migration from the site ofinammation to secondary lymphoid organs. A critical example ofsuch receptors is CCR7, which is expressed by resident antigenpresenting cells (APCs) of the cornea. CCR7 has been implicated inpromoting direct alloantigen recognition in the context of high-riskcorneal transplantation by facilitating migration of alloantigen-loaded donor APCs into the draining lymph nodes [18].

Our current hypothesis sustains that early phase chemokinessuch as CXCL1/KC are responsible for the recruitment of neutrophilsinto the graft; once activated by the inammatorymilieuwithin thegraft, these cells are capable of interferon gamma (IFNg) produc-tion, which would result in the upregulation of late phase che-mokines (CXCL9/MIG and CXCL10/IP-10) responsible foralloreactive T cell recruitment. We have found ample evidence insupport of this hypothesis. Experiments involving in vivo neutrali-zation of CXCL1/KC with rabbit anti-sera resulted in prolongedallograft survival in high-risk recipients. In line with our hypothe-sis, the same anti-KC treated animals showed signicantlydecreased numbers of CD4 T cell inltrates upon histological ex-amination of corneal allografts [16]. Future studies in our laboratorywill explore the possibility of promoting long-term corneal allo-graft survival by administering anti-chemokine combination ther-apies aimed at reducing the number of innate and adaptiveimmune cell inltrates.

2.2. The eye as the perfect setting for in vivo imaging of the immunesystem

In vivo imaging has emerged as an indispensable tool in bio-logical research, and a variety of imaging techniques have beendeveloped to noninvasively monitor tissues under living condi-tions. By virtue of its easy access, avascular and transparent struc-ture, the cornea has been used as a natural body window fornoninvasive imaging to study the physiology of pancreatic isletstransplanted into the AC of the mouse eye in vivo [19]. Morerecently, intraocular transplantation was used to noninvasivelystudy immune responses with single cell-resolution during rejec-tion of islet allografts in the living animal [20].

Although corneal graft rejection is multifactorial where variousmechanisms and cell types are likely to be involved, it is wellaccepted that CD4 T-cells play a crucial role in this process. In vivodepletion of CD4 T-cells resulted in improved corneal allograftsurvival [11,21e23]. Adoptive transfer of activated CD4 T-cells intoimmune compromised mice induced rejection in 100% of cornealallografts [23]. We reasoned that using high-resolution in vivoimaging to study T-cell motility within corneal allografts couldprovide a cellular mechanism that contributes to effector T-cellfunction during allorejection. In order to visualize T cells in vivo, weemployed Bonzo mice as recipients for corneal transplantation.Bonzo or CXCR6 (also known as CD186, STRL33, or TYMSTR) is aputative chemokine receptor with unclear biologic function [24].However, it is preferentially expressed on activated and memory T-cells [25e27]. In Bonzo mice, expression of the green uorescentprotein (GFP) is driven by the Bonzo gene promoter, allowing forin vivo visualization of T cells [28,29]. Our group successfully useduorescence confocal microscopy to obtain longitudinally imagesafter corneal transplantation. As early as post-operative day (POD)

immunity 45 (2013) 7e141, Bonzo recipient GFP (both CD4 and CD8) T-cells appeared in

the host cornea of both syngeneic and allogeneic recipients, but notwithin the grafts [30]. While syngeneic grafts remained relativelyclear of inltrating GFP T-cells throughout the follow up period, T-cells progressively inltrated the allogeneic grafts starting on POD7and signicantly increased between POD14 and POD21 [30]. Asshown in Fig. 1, T cells can be seen throughout the ocular surface,from host to the grafts, at POD21. Furthermore, 3-D time-lapserecordings (20 min) enabled us to understand the dynamicbehavior of the inltrating T cells, which display different pheno-types (round, elongated, and rufed) with distinct morphologicaland dynamic features [30]. Round cells appeared predominantlyspherical with low net translational movement. Fast moving elon-gated cells displayed ameboidal-type movement with a largeleading edge and a thin, long trailing tail (uropod) and traveled forlong distances (30 mm/20 min). Rufed cells, however, movedvividly within shorter distances and tended to form clusters as theyengaged in simultaneous contacts with neighboring T-cells. Insyngeneic grafts, T-cells appear predominantly round with mark-edly lowmotility, while we found all cell types in allografts (Fig. 2).

novel effect could only be studied by the in vivo imaging techniqueswe used, which cannot be readily modeled in vitro. Therefore, theorchestration of chemokine production is a critical feature of ocularimmune regulation of adaptive immune responses to allo-antigens.

3. Immune responses in the posterior segment of the eye

3.1. Inammation and the innate immune system in age-relatedmacular degeneration

Moving away from the anterior portion of the eye, the posteriorsegment of the eye also houses an immunologically unique struc-ture: the retina. The retina contains photoreceptor cells whichdetect light and generate electrical impulses that eventually formvision. Like the cornea, the retina also exhibits mechanisms ofimmune regulation. Many mechanisms contribute toward retinalimmune protection, but relevant for our discussion are thoseassociated with a very important cell layer in the retina known asthe retinal pigment epithelium (RPE). RPE cells have a variety of

nltr

V.L. Perez et al. / Journal of Autoimmunity 45 (2013) 7e14 9The round and ruffed cells actively moved and continuouslychanged shape as they migrated within the allograft tissue [30].

Since our previous work demonstrated an orchestrated pro-duction of chemokines after transplantation, we tested the hy-pothesis that CCL5/RANTES, CXCL9/MIG, and CXCL10/IP-10 directleukocyte migration and trafcking into the ocular tissue and shapeimmune responses to allo-antigens by neutralizing them withsystemic administration of TAK-779, a highly selective antagonist ofCCR5 and CXCR3 [31,32]. Corneal allograft survival was signicantlyimproved [30]. To determine the acute and local effects of CCR5/CXCR3 blockade on cellular motility, we injected TAK-779 directlyinto the stroma of corneal allografts during ongoing rejection. LocalTAK treatment resulted in signicant phenotypic and dynamicchanges in graft-inltrating T-cells; the majority of cells convertedfrom the predominant rufed to the round phenotype [30]. Suchchanges associated with signicantly reduced displacement andvelocity of the cells (Fig. 3). More importantly, injection of a CXCL9/CXCL10 mix subsequent to TAK-779 increased the proportion ofelongated and rufed T-cells and recovered the movement dy-namics of the overall population to a higher motility state [30].

Our ndings demonstrate that, in addition to their involvementin immune cell recruitment into corneal allografts, specic che-mokines played an important role in mediating T-lymphocyte localmotility patterns, which signicantly inuenced T-cell activationand effector function, and impacted on corneal graft survival. This

Fig. 1. In vivo imaging of T cell responses in allogeneic versus syngeneic cornea grafts. I

the center of the grafts. Many GFP T cells migrated into the allografts, displaying distincsyngeneic grafts at POD14.functions that maintain the health of the retina and its preciousphotoreceptor cells (e.g., phagocytosis of damaged photoreceptors,supply and transport of nutrients, recycling of retinol, etc.). The RPEalso aids in the retinas immune privilege by active and passivemechanisms. The RPE is responsible for forming the blooderetina-barrier (BRB), which physically sequesters away retinal antigensand keeps the systemic immune system from entering the retinalspace. The RPE also actively inhibits immune cells throughexpression of surface ligands and secretion of cytokines that inhibit,for instance, T cells and macrophages [33,34]. Thus, the healthyretina is inherently averse to inammation.

However, the aging process disrupts the normal functioning ofthe retina and especially the RPE. One the most prevalent diseasesof the retina is age-related macular degeneration (AMD), which isthe leading cause of blindness in the US and other industrializedcountries, largely due to a rise in the aging population. As its nameimplies, AMD is a disease of the macula. The macula, the centralportion of the retina, contains the highest concentration ofphotoreceptor cells and is responsible for central vision. A keyclinical nding in patients with AMD or in patients at-risk for AMDdevelopment is drusen, which is photoreceptor cellular debris thataccumulates below the RPE and clinically manifests as small yellowclumps during a funduscopic examination. Drusen is the biggestrisk factor that predicts AMD progression. Mechanistically, drusendevelops because with age, RPE cells have an impaired ability to

ating T cells in the corneal grafts at post-operative day (POD) 14. Images were taken in

t morphological and dynamic phenotypes. Only a few GFP T cells were observed in

t PO

V.L. Perez et al. / Journal of Auto10phagocytose damaged photoreceptor cells and cellular debris ac-cumulates as a result. It has been recognized that RPE celldysfunction is closely associated with AMD [35].

The etiologic mechanisms behind AMD are not fully understood,

Fig. 2. T cell distribution within allografts. Images were taken during ongoing rejection acells with different morphological and functional patterns were seen throughout.but mounting evidence supports a role involving retinal inam-mation and autoimmunity, at least in a subset of AMD patients. Thisis best evidenced by the presence of complement proteins in dru-sen and the fact that polymorphisms in complement genes arehighly associated with AMD [35]. A striking 50e70% of AMD casesare associated with single-nucleotide polymorphisms in comple-ment factor H, factor B, or C2 genes [36e39]. Evidence for a role ofautoimmunity in AMD comes from the presence of autoantibodiesagainst retinal proteins detected in the serum of AMD patients[35,40,41].

In addition to inammatory processes, oxidative stress has longbeen recognized to play a pathological role in AMD. For example,

Fig. 3. Acute inhibition of chemokine receptor signaling alters T cell motility patters incorneal allografts. TAK-779 (which inhibits CCR5 and CXCR3) was administeredthrough local injection in the cornea stroma at POD21. Flower plot representation ofmovement trajectories of individual cells tracked within the allografts before and14 min after intrastromal TAK-779 injection during ongoing rejection.cigarette smoking and light exposure increase the risk of AMD,while zinc and antioxidant vitamins are known to be protectivefactors for AMD [42]. The aging process in general is associatedwiththe accumulation of oxidative damage. The retina, in particular, is

D21 spanning from central graft to the host bed, including the graft interphase. GFP T

immunity 45 (2013) 7e14subjected to high levels of oxidative stress because of its exposureto light, high oxygenation and continual RPE phagocytic activity[43,44]. Thus, the retina likely accumulates a great deal of oxidativeinsults over time and a persons intrinsic or environmental expo-sures will impact the degree of oxidative damage, dictating theirprobability of developing AMD.

Oxidative stress, inammation, and autoimmunity are notseparate etiologic factors in AMD pathogenesis. In fact, work fromour group and others has shown that these factors are actuallylinked in the context of AMD. Two examples involving lipid per-oxidation products are highlighted: malondialdehyde (MDA) andcarboxyethylpyrrole (CEP). MDA and CEP have pro-inammatoryactivity and interact with both the innate and the adaptive im-mune systems, although there are specic differences for each ofthese immune responses in the process of AMD development.

As oxidative stress builds up in the retina, lipids found in the cellmembrane undergo lipid peroxidation. Oxidation of phosphati-dylcholine, for instance, gives rise to the reactive degradationproduct MDA, which is actually found in drusen [45]. Weismannet al. recently discovered that MDA interacts with complementfactor H (CFH) [46]. CFH is centrally important in AMD and aparticular CFH polymorphism (yielding a missense mutation[Y402H]) is highly associated with the AMD disease state [36e38].Interestingly, this mutant version of CFH has an impaired ability tobind MDA. This has very important immunological consequenceswith respect to retinal inammation. As inammation and lightexposure induce retinal oxidative stress over decades, levels ofMDA become elevated. This MDA serves as a docking site for (wild-type) CFH, which acts to minimize inammation, and lessens tissuedamage. Mutant CFH, on the other hand, cannot localize to theseareas of oxidative stress and as a result inammation and tissue

damage proceed unchecked. The discovery of the interaction of CFHand MDA is a striking example of where oxidative stress and im-munity intersect and this interaction has a profound clinicalimplication since a homozygotic CFH mutation carries a 6-fold in-crease of AMD development [47].

Oxidative damage to retinal tissues induces inammation and iscapable of creating novel self-antigens, which trigger a pathologicalautoimmuneprocess targeting themacula/retina. In termsof possibleself-antigens, our lab has previously explored the role of oxidativelymodied lipideprotein adducts as potential initiating signals. Evi-dence for oxidatively modied proteins in AMD pathogenesis stemsfrom the analysis of drusen. Crabb et al. analyzed drusen from AMDpatient donor eyes with mass spectrometry and identied over ahundred different proteins [48]. Interestingly, a signicant number of

these mice also develop retinal lesions and RPE damage. The pa-thology observed in CEP-immunized mice mirrors that of humanAMD pathology. Furthermore, it was found that the degree ofretinal pathology directly correlated with the degree of CEP anti-body production [52].

In addition to retinopathy, CEP-immunized mice were charac-terized by retinal deposition of complement proteins (C3d) and theappearance of retinal inltrating macrophages [52]. The role ofmacrophages in the pathogenesis of AMD remains controversial,due to contradictory ndings from transgenic and retinal injury-mediated animal models [53]. However, emerging evidencepoints towards a mechanism by which macrophages accumulatewithin the retina with aging. Progression towards AMD does notdepend on the simple absence or presence of macrophages, but

rophand

V.L. Perez et al. / Journal of Autoimmunity 45 (2013) 7e14 11the proteins had oxidative protein modications. In particular, pro-teins were covalently cross-linked with a reactive lipid moiety calledcarboxyethylpyrrole (CEP). CEP is formed from its precursor docosa-hexanoic acid (DHA) under conditions of oxidative stress [49]. Oncegenerated from DHA, CEP then condenses with amino acids of pro-teins (lysine, cysteine, histidine) forming covalent adducts. Interest-ingly, DHA-containing lipids are highly abundant in the RPE andphotoreceptor cells and DHA also happens to be the most oxidizablefatty acid in the body [49]. Since the retina is inherently a highlyoxidizing environment, it provides an optimized setting for thegeneration of CEP-adducted proteins. Over time, the aging processlikely drives the accumulation of CEP-adducts.

Work from Gu et al. further implicates CEP-adducted proteinswith AMD. They observed greater amounts of CEP-adducts in theRPE and photoreceptor cells of AMD patient donor eyes, whencompared to healthy donor eyes [50]. Furthermore, they found CEP-adducted proteins in the blood of AMD patients and found higherlevels of CEP auto-antibodies in AMD patient sera (versus age-matched healthy controls) [50,51]. Importantly, this last resultdemonstrates that CEP-adducts are immunogenic and the hostmounts an adaptive immune response against CEP. Our groupdemonstrated that CEP is not only immunogenic, but that CEP caninduce an immune-mediated attack upon the retina [52]. Younghealthy mice were immunized with CEP-adducted self protein(albumin) and their eyes were analyzed to determine if AMD-likelesions developed (Fig. 4). The rationale for this approach is thatCEP-immunization would greatly enhance the sensitivity of theimmune system to react to endogenously generated CEP. This highsensitivity could conceivably promote immune recognition of evensmall, basal levels of CEP-adducts that may be present in the eyes ofotherwise healthy mice. Therefore, immunized mice wouldgenerate a pathological auto-immune response targeting theretina. The results from these experiments show that CEP-immunized mice develop an antibody immune response. Notonly do CEP-immunized mice develop anti-CEP antibodies, but

Fig. 4. Immunization with CEP-adducted mouse serum albumin (CEP-MSA) leads to macday 150 post-immunization from CEP-MSA immunized (versus nave) C57BL/6 mice,

inltrating cells (left image), whereas CEP immunization results in the recruitment of macropouter segments (right image). The RPE is located at the lower part of each image.depends on the type of macrophages present. Macrophages canadopt different polarization states with distinct effector functionsand this may dictate disease outcome. Data from our laboratorydemonstrated that the macrophages observed in the CEP-immunization model are inammatory (IL-12 and TNF-a-produc-ing) macrophages [54]. Inammatory (also known as M1) macro-phages have a tissue destructive role, as opposed to other types ofmacrophages (like tissue remodeling M2 macrophages) [55]. Insupport of this, Cao et al. demonstrated a general accumulation ofretinal macrophages associated with age [56]. Interestingly, theyfound an increased M1 to M2 ratio in patients with AMD, whencompared with retinal samples from age-matched controls [56]. Itis possible that these M1 macrophages (perhaps in response tocomplement deposition or chemokine-regulated recruitment)mediate RPE-injury.

Retinal inltrating macrophages could be initiating destructionor responding to the tissue damage. Evidence from our lab suggeststhat these macrophages might be actually inducing the observedretinal damage. For instance, it is possible that the observed com-plement deposition on the RPE may prime these cells for opsoni-zation by macrophages. Additional evidence for macrophagesinducing/causing damage is that macrophage inltration tempo-rally precedes lesion development [54]. Furthermore, mice withimpaired macrophage-recruitment ability (CCR2 knockout mice),failed to develop CEP-induced retinopathy [54]. Therefore, macro-phages seem to have a causative role in retinal injury in conditionsassociated with M1 polarization.

3.2. The adaptive immune system in age-related maculardegeneration

Overall, the CEP model of AMD points to the conclusion thatAMD is an autoimmune disease in which a lipid peroxidationproduct coordinates both a pathological innate (as evidenced bycomplement and macrophages) and adaptive (as evidenced by

age inltration into the retina and AMD-like pathology in mice. Eyes were harvested athistology was performed as described [52]. Nave retinas look normal and devoid of

hages to the outer retina (center image) and focal lesions of the RPE and photoreceptor

Autoantibody production) immune response directed against the retina.Additional evidence strengthens the idea that AMD is an autoim-mune disease and stems from work in both animal models andclinical studies implicating T-cells in the pathogenesis of AMD.

While appreciably little is known about the potential role ofadaptive immunity, specically T cells, in the initiation or regula-tion of AMD pathology, this is a current area of increased interest inthe eld. Most of the evidence for adaptive immunity in AMDcomes from the presence of anti-retinal autoantibodies in AMDpatients, which is recapitulated in our CEP mouse model [52,57,58].Despite the fact that autoantibodies are associated with AMD, it isstill unknown whether they cause or protect from damage, or ifthey simply arise as secondary effects and have no specic role indisease pathogenesis. On the other hand, increased IgG ratios inAMD patients could point to a productive immune response thatincludes antigen-specic T cells [58]. In this context, oxidationspecic epitopes (OSEs), such as MDA and CEP, could providethe signals from the outer retina that initiate inammationand possibly involve the adaptive immune system in AMDpathogenesis.

T cells constitute the main effector cells of the adaptive immuneresponse, providing the antigen specicity lacking in innate im-munity. One of the primary jobs carried out by T cells is theorchestration of appropriate responses by recruiting other immunecells and shaping the type of response based on the cytokines theyproduce. The study of experimental autoimmune uveitis (EAU), ananimal model in mice and rats of the inammatory human disease,has greatly inuenced our understanding of T cell-mediated auto-immune responses in the retina. EAU is induced by immunizationwith retinal antigens and susceptibility depends on the geneticbackground of the host: C57BL/6 (B6) mice are susceptible whileBALB/c mice are resistant. It has been shown that both Th1 andTh17 cells can mediate EAU pathology depending on the context ofinitial antigen exposure, suggesting a role for antigen presentingcells (APCs) in dictating pathology outcomes [59]. Further compli-cating the picture is the fact that retinal antigens can induce reg-ulatory T cell (Treg) peripheral tolerance [60]. As for the role of Tcells in AMD, surprisingly little information is available in theliterature. Robert Nussenblatts group showed that complementcomponent 5a (C5a) can induce IL-22 and IL-17 expression in hu-man CD4 T cells and that elevated levels of these cytokines werepresent in AMD patients [61]. Furthermore, they also showed thatthe IL-17RC promoter region is preferentially hypomethylated inAMD patients [62]. Taken together, these data show the ability of Tcells to respond to retinal antigens, to affect tissue integrity and topotentially be involved in AMD.

In addition to our published data showing that M1macrophagesplay a role in the onset of disease [54], additional data stronglysuggest that CEP-specic T cells play a leading role in the initiationof AMD in our model (FCG, VLP, unpublished observations). MDA-specic T cells have been reported and shown to be involved inthe pathology of atherosclerosis [63], which proves that antigen-specic T cells can indeed recognize lipid peroxidation productsthat modify proteins and serve as haptens. This antigen specicityis provided by unique T cell receptors (TCR) on the surface of T cells,but to date not a single TCR against lipid modications of proteinshas been isolated. In the future, the analysis of CEP and/or MDA-specic T cell clones will shed light on relevant pathways (Th1/T-bet; Th2/Stat6; Th17/Ror-gt) that mediate responses againstoxidative damage, in a context-dependent manner. Interestingly,MDA-specic T cells adopt a Th2 phenotype [63], whereas CEP-specic T cells produce IFNg and IL-17 (FCG, VLP, unpublished ob-servations). In the case of AMD, pro-inammatory cytokine pro-duction by antigen-specic T cells may contribute to the

V.L. Perez et al. / Journal of12polarization of macrophages toward theM1 phenotype, providing apossible link between adaptive and innate immunity in patho-genesis. However, the precise mechanisms of T cellemacrophageinteractions in chronic inammation (as opposed to acute infectionmodels) remain to be determined.

In the original publication of our model, we reported that CEP-induced AMD-like pathology does not develop in RAG/ mice,which lack an adaptive immune system [52]. Therefore, a majorquestion in our model is the role of autoantibodies (produced by Bcells) in the generation of retinal lesions or whether the modelrepresents a T cell-mediated disease. To address this issue, C57BL/6mice decient in mature B cells (mMT/ mice) were immunizedwith our protocol. Although no CEP antibodies were detectable,splenic T cells from immunized mice were activated in response toin vitro stimulationwith CEP and, importantly, strong retinal lesionswere observed in these B cell-decient mice (FCG, VLP, unpublishedobservations), indicating that the observed pathology is indepen-dent of B cells (and their antibodies). This result clearly points to Tcells as the leading players within the adaptive immune systemassociatedwith AMD in ourmodel. However, it does not necessarilymean that antibodies are not involved, in some cases and at somecapacity, in the AMD disease process. It is still possible that anti-retinal antibodies can x complement in the outer retina orcontribute to macrophage-mediated destruction of retinal struc-tures. Alternatively, at least a proportion of autoantibodies mayactually serve a protective role. For example, autoantibodies againstCFH were detected at lower levels in AMD patients compared toage-matched controls [64]. Regardless of their individual activities,autoantibodies are still themost likely and accessible candidates forthe development of AMD biomarkers, although each specicity willrequire detailed functional analysis to uncover disease-related ef-fects. Proling autoantibody signatures, as opposed to single anti-bodies, may prove useful in this regard.

The similarities between human AMD patients and the CEPimmunized mice support the notion that our model recapitulatesessential features of AMD pathogenesis and progression. Therefore,it provides new avenues to study the onset factors involved in AMD.Many important questions remain to fully elucidate the T cellpathways relevant to the onset and progression of disease. Trans-lation of ndings in our animal model into clinically relevantstrategies in human AMD patients will be crucial. Will immuno-logical intervention be successful for novel prevention and/ortreatment protocols for AMD? Answers to these questionsregarding ocular disease from an immunological viewpoint couldlead to innovative treatments for this prevalent condition.

4. Conclusion

In conclusion, the eye is the window to the soul (of the immunesystem) and can be successfully used to understand the mecha-nisms behind immune responses in transplantation and autoim-munity. Furthermore, the eye is an easily accessible organ toevaluate and treat with therapies that will be developed from theunderstanding of pathways that lead to ocular immune regulation.

5. Final comments

This paper is dedicated to Abul Abbas from Victor L. Perez:A great mentor, teacher and friend. It is part of this dedicated issuethat addresses and recognizes unique people from the perspectiveof teaching, research, public service and their implications forautoimmunity and the patients who suffer from autoimmune dis-ease [65e67]. Many people are lucky enough to have a good sci-entic mentor, but there are only a few that have a mentor who isnot only a great teacher, but a true friend as well. I am one of those

immunity 45 (2013) 7e14few individuals. Thanks to Abul, I developed the tools to become an

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[9] Sonoda Y, Sano Y, Ksander B, Streilein JW. Characterization of cell-mediatedimmune responses elicited by orthotopic corneal allografts in mice. InvestOphthalmol Vis Sci 1995;36:427e34.

[10] Boisgerault F, Liu Y, Anosova N, Ehrlich E, Dana MR, Benichou G. Role of CD4and CD8 T cells in allorecognition: lessons from corneal transplantation.J Immunol 2001;167:1891e9.

[11] Yamada J, Kurimoto I, Streilein JW. Role of CD4 T cells in immunobiology oforthotopic corneal transplants in mice. Invest Ophthalmol Vis Sci 1999;40:2614e21.grafts transplanted to the brain, to subcutaneous tissue, and to the anteriorchamber of the eye. Br J Exp Pathol 1948;29:58e69.

[3] Streilein JW. Anterior chamber associated immune deviation: the privilege ofimmunity in the eye. Surv Ophthalmol 1990;35:67e73.

[4] Streilein JW. Immune regulation and the eye: a dangerous compromise. FASEBJ 1987;1:199e208.

[5] Kaplan HJ, Streilein JW. Immune response to immunization via the anteriorchamber of the eye. I. F. lymphocyte-induced immune deviation. J Immunol1977;118:809e14.

[6] Stein-Streilein J, Streilein JW. Anterior chamber associated immune deviationimmunologist, tools that allowed me to fulll my dream of being aclinician scientist that can use my laboratory to develop new con-cepts to treat blind patients and improve their quality of life.Moreover, when I have doubts regarding what to do with any issuein my life, I ask myself what would Abul do? and the path be-comes very clear. Thanks Abul, for your inuence in my life andgiving me the opportunity to love and enjoy immunology.

Financial support

This work was supported by the National Eye Institute, NationalInstitutes of Health R01 EY018624-04 (VLP), The Edward N. & DellaL. Thome Memorial Foundation Bank of America N.A. TrusteeAward Program in Macular Degeneration Research (VLP), NIHP30EY14801 (Center Grant), Research to Prevent Blindness (Unre-stricted Grant to the Bascom Palmer Eye Institute). A.M.S. ac-knowledges partial support and assistance from the Sheila andDavid Fuente Graduate Program in Cancer Biology, SylvesterComprehensive Cancer Center. FCG is a Howard Hughes MedicalInstitute Fellow of the Life Sciences Research Foundation.

Acknowledgments

We thank members of the Perez lab, past and present, for theircontributions to this work. We also thank the active ocularimmunology community for applying the principles of autoim-munity to the eye to shed light on major clinical problems affectingvision.

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