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PURPOSE. Acute anterior uveitis (AAU) is a common intraocular inflammatory disease. AAU occurs in 30% to 50% of patients with ankylosing spondylitis (AS), and both conditions are strongly associated with human leukocyte antigen (HLA)-B27, implying a shared etiology. This study aims to apply genomewide association study (GWAS) to characterize the genetic associations of AAU and their relationship to the genetics of AS. METHODS. We undertook the GWAS analyses in 2752 patients with AS with AAU (cases) and 3836 patients with AS without AAU (controls). There were 7,436,415 single-nucleotide polymorphisms (SNPs) available after SNP microarray genotyping, imputation, and quality-control filtering. RESULTS. We identified one locus associated with AAU at genomewide significance: rs9378248 (P = 2.69 × 10−8, odds ratio [OR] = 0.78), lying close to HLA-B. Suggestive association was observed at 11 additional loci, including previously reported AS loci ERAP1 (rs27529, P = 2.19 × 10−7, OR = 1.22) and NOS2 (rs2274894, P = 8.22 × 10−7, OR = 0.83). Multiple novel suggestive associations were also identified, including MERTK (rs10171979, P = 2.56 × 10−6, OR = 1.20), KIFAP3 (rs508063, P = 5.64 × 10−7, OR = 1.20), CLCN7 (rs67412457, P = 1.33 × 10−6, OR = 1.25), ACAA2 (rs9947182, P = 9.70 × 10−7, OR = 1.37), and 5 intergenic loci. The SNP-based heritability is approximately 0.5 for AS alone, and is much higher (approximately 0.7) for AS with AAU. Consistent with the high heritability, a genomewide polygenic risk score shows strong power in identifying individuals at high risk of either AS with AAU or AS alone. CONCLUSIONS. We report here the first GWAS for AAU and identify new susceptibility loci. Our findings confirm the strong overlap in etiopathogenesis of AAU with AS, and also provide new insights into the genetic basis of AAU.

PURPOSE To investigate the contribution of the gut microbiota to the pathogenesis of uveitis. METHODS Experimental autoimmune uveitis (EAU) in B10.RIII mice was induced using interphotoreceptor binding protein peptide. Mice were treated with oral or intraperitoneal (IP) antibiotics. Effector (Teff) and regulatory (Treg) T lymphocytes were identified using flow cytometry; 16S rRNA gene sequencing and qPCR were performed on gastrointestinal (GI) contents. RESULTS Broad-spectrum (four antibiotics given simultaneously) oral, but not IP, antibiotics reduced mean uveitis clinical scores significantly compared with water-treated animals (0.5 vs. 3.0, P 0.99 for IP). Both oral metronidazole (P = 0.02) and vancomycin (P < 0.0001) alone decreased inflammation, whereas neomycin (P = 0.7) and ampicillin (P = 0.4) did not change mean uveitis scores. Oral broad-spectrum antibiotics increased Tregs in the GI lamina propria of EAU animals at 1 week, and in extraintestinal lymphoid tissues later, whereas Teff and inflammatory cytokines were reduced. 16S sequencing of GI contents revealed altered microbiota in immunized mice compared with nonimmunized mice, and microbial diversity clustering in EAU mice treated with uveitis-protective antibiotics. Experimental autoimmune uveitis mice also demonstrated gut microbial diversity clustering associated with clinical score severity. CONCLUSIONS Oral antibiotics modulate the severity of inducible EAU by increasing Tregs in the gut and extraintestinal tissues, as well as decreasing effector T cells and cytokines. 16S sequencing suggests that there may be protective and, conversely, potentially uveitogenic, gut microbiota. These findings may lead to a better understanding of how uveitis can be treated or prevented by modulating the gut microbiome.

T cells are essential for the development of autoimmune uveitis. Although the costimulatory molecule OX40 promotes T-cell function and expansion, it is unclear whether OX40 is implicated in ocular inflammation. The purpose of this study was to examine the role of OX40 in uveitis.Experimental autoimmune uveitis (EAU) was induced in B10.RIII mice by subcutaneous injection of interphotoreceptor retinoid-binding protein peptide 161-180 (IRBP(161-180)). Some mice received an intravenous administration of OX40-activating antibody on days 0 and 4 after IRBP(161-180) sensitization or on days 10 and 14 of uveitis onset. The severity of EAU was evaluated by histology at different time points. In addition, ocular inflammatory cytokine expression was determined by real time-PCR, and peripheral activated CD4(+)CD44(+)CD62L(-) T cells and IL-7Rα expression were analyzed by flow cytometry. The activated CD4(+)CD44(+) lymphocytes were rechallenged with IRBP(161-180) in vitro to assess their antigen recall response.The authors demonstrated a marked OX40 expression by infiltrating lymphocytes in enucleated human eyes with end-stage inflammation. In addition, the administration of OX40-activating antibody prolonged and exacerbated the disease course of EAU. Moreover, activation of OX40 not only increased CD4(+)CD44(+)CD62L(-) lymphocyte number, it upregulated IL-7Rα expression in the activated T-cell population. Lastly, these cells exhibited a stronger interferon-γ response to IRBP(161-180) restimulation in vitro.The results reveal a pathogenic role of OX40 in uveitis. Furthermore, the upregulation of IL-7R in CD4(+)CD44(+) lymphocytes suggests that the activation of OX40 promotes the generation or expansion of uveitogenic memory T cells.

The innate immune receptor NOD2 is a genetic cause of uveitis (Blau syndrome). Intriguingly, in the intestine where polymorphisms of NOD2 predispose to Crohn's disease, NOD2 reportedly suppresses inflammation triggered by the bacterial cell wall component, peptidoglycan (PGN). Whether NOD2 exerts a similar capacity in the regulation of ocular inflammation to PGN has not been explored.NOD2, NOD1, or MyD88 knockout (KO) mice and their wild-type (WT) controls were administered an intravitreal injection of PGN (a metabolite of which is the NOD2 agonist, muramyl dipeptide), or synthetic TLR2/1 and TLR2/6 agonists, Pam₃CSK4 and FSL-1. Ocular inflammation was assessed by intravital microscopy and histopathology. Cytokine production in eye tissue homogenates was measured by ELISA.PGN triggered uveitis in mice. This inflammation was abolished in the absence of the TLR signaling mediator MyD88. NOD2 exerted a negative regulatory role because PGN-triggered eye inflammation was exacerbated in NOD2 KO mice. Increased intravascular response coincided with enhanced leukocytes within the aqueous and vitreous humors. The enhanced susceptibility of NOD2 KO mice to PGN uveitis coincided with increased cytokine production of IL-12p40, IL-17, and IL-23 but not IL-12p70, TNFα, or IFNγ. NOD1 deficiency did not result in the same sensitivity to PGN. Ocular inflammation induced by synthetic TLR2 agonists required MyD88 but not NOD2 or NOD1.NOD2 may serve differential roles in the eye to promote inflammation while also tempering cell responses to PGN akin to what has been reported in colitis.

Purpose To determine associations between polymorphic genes that encode KIRs and their HLA class I ligands in patients with HLA-B27-associated acute anterior uveitis (AAU), with and without axial spondyloarthropathy (axial SpA). Methods Molecular DNA typing methods were used to define the frequencies of variable KIR genes and their relevant HLA class I ligands in HLA-B27(+) (B27(+)) Caucasian subjects with AAU and 429 healthy Caucasian control subjects. The patients were evaluated for axial SpA based on their histories using published criteria. Results Of 143 Caucasian subjects with AAU, 71 (49.6%) had features of axial SpA. The only difference between cases and controls in KIR gene frequencies was a trend toward fewer activating KIRs in subjects with AAU with axial SpA, which reached statistical significance for 2DS5 (P = 0.025, corrected P [P(c)] = 0.05; odds ratio [OR], 0.48; 95% CI, 0.25-0.90). The 3DL1+Bw4(T80) combination implicated in weak inhibition was more frequent in subjects with AAU than in control subjects (P = 2.73 x 10(-28), P(c) = 8.2 x 10(-27); OR, 13.5; 95% CI, 7.73-23.68). The 2DL1+HLA-C2 combination was decreased in subjects with axial SpA compared with subjects with AAU without axial SpA (P = 0.022; P(c) = NS; OR, 0.43; 95% CI, 0.21-0.88). Conclusions Evidence was found of a role for KIR-HLA combinations that trigger weaker inhibition in subjects with AAU. Furthermore, there was a trend toward fewer KIR3DS1, -2DS1, and -2DS5 in AAU patients with axial SpA, which have been implicated in NK cell activation. HLA-B27(+) without KIR2DS3 (and -2DS1 and -3DS1) may fail to trigger an early NK cell response to clear antigenic stimuli, which may in part contribute to disease pathogenesis.

NOD2 plays an important role in the recognition of intracellular bacteria through its ability to sense the components of bacterial peptidoglycan (PGN), namely muramyl dipeptide (MDP) and muramyl tripeptide (MTP). Specific mutations in the human NOD2 gene cause Blau syndrome, an autosomal dominant form of uveitis, arthritis, and dermatitis. As a first step toward understanding the role of NOD2 in the pathogenesis of uveitis, the authors developed a mouse model of MDP-dependent uveitis.BALB/c mice and mice deficient in L-selectin or NOD2 received intravitreal injection of MDP, MTP, or PGN. The intravascular response within the iris and cellular infiltration was quantified by intravital microscopy and histologic assessment.MDP induced an acute, ocular inflammatory response, wherein rolling and adhering leukocytes within the vasculature were significantly increased within 6 hours after MDP treatment. A minor increase in cellular infiltration occurred at 12 hours after MDP treatment. The adhesion molecule L-selectin participated in MDP-induced vascular inflammation because L-selectin knockout mice showed a significant decrease in the number of rolling cells. Importantly, NOD2 plays an essential role in ocular inflammation induced by MDP, as indicated by the fact that uveitis did not develop in Nod2 knockout mice in response to MDP. Nod2 knockout mice also showed abolished ocular inflammation in response to MTP but not to PGN treatment.These findings demonstrate a novel mouse model of uveitis, wherein NOD2 plays an essential role in inflammation induced by the minimal components of PGN. Thus, innate immune responses mediated by NOD2 may participate in the development of uveitis in response to bacterial products.

PURPOSE. Identification of transcription factors that regulate the transcription of the vascular endothelial growth factor (VEGF) gene may facilitate understanding of the etiology and progression of ocular neovascular diseases. The purpose of this study was to determine whether transcriptional enhancer factor 1-related (RTEF-1) was present within ocular vascular endothelial cells and whether it played a role in the control of the transcription of the VEGF gene. METHODS. Primary cultures of human retinal vascular endothelial cells (RVECs) were maintained under normoxic or hypoxic conditions before isolation of mRNA. RT-PCR was performed to detect RTEF-1 transcripts. Amplified products were cloned into an expression plasmid. Human VEGF promoter and deletion constructs were cloned into a pSEAP reporter vector. Various RTEF-1 isoforms and VEGF promoter constructs were coelectroporated into human cells, and reporter expression levels were determined. Retinal tissue from a mouse model of retinopathy of prematurity (ROP) was analyzed by RT-PCR for the presence of RTEF-1 transcripts. RESULTS. Full-length 1305-bp and novel 936-bp RTEF-1 transcripts were identified in cultured human RVECs under normoxic conditions. A novel 447-bp isoform was present in cells maintained in a hypoxic environment. Four of the 11 translated exons predicted to code for the 1305-bp product were spliced out of the 936-bp transcript. The 1305-bp product enhanced expression from the VEGF promoter 4-fold greater than background, whereas the 936-bp and the 447-bp isoforms enhanced expression 3X and 12X, respectively. Analysis with deletion promoter constructs determined that all isoforms required the presence of Spl elements for efficient activation and that the hypoxia response element (HRE) was not essential for enhancement. Transcripts for novel RTEF-1 isoforms were also identified in neural retinal tissue of mice. Different murine-specific isoforms were present at different stages of postnatal development. CONCLUSIONS. Novel RTEF-1 transcripts are present within human ocular vascular endothelial cells and mouse neural retina during normal and ROP development, and alternatively spliced products are produced under hyperoxic and hypoxic conditions. Alternative spliced variants of human RTEF-1 transcripts are able to potentiate expression from the VEGF 5' proximal promoter region.

PURPOSE. Consistent with clinical observations that posterior uveitis frequently involves the retinal vasculature and recent recognition of vascular heterogeneity, the hypothesis for this study was that retinal vascular endothelium was a cell population of unique molecular phenotype. METHODS. Donor-matched cultures of primary retinal and choroidal endothelial cells from six human cadavers were incubated with either Toxoplasma gondii tachyzoites (10:1, parasites per cell) or Escherichia coli lipopolysaccharide (100 ng/ mL); control cultures were simultaneously incubated with medium. Gene expression profiling of endothelial cells was performed using oligonucleotide arrays containing probes designed to detect 8746 human transcripts. After normalization, differential gene expression was assessed by the significance analysis of microarrays, with the false-discovery rate set at 5%. For selected genes, differences in the level of expression between retinal and choroidal cells were evaluated by real-time RT-PCR. RESULTS. Graphic descriptive analysis demonstrated a strong correlation between gene expression of unstimulated retinal and choroidal endothelial cells, but also highlighted distinctly different patterns of expression that were greater than differences noted between donors or between unstimulated and stimulated cells. Overall, 779 (8.9%) of 8746 transcripts were differentially represented. Of note, the 330 transcripts that were present at higher levels in retinal cells included a larger percentage of transcripts encoding molecules involved in the immune response. Differential gene expression was confirmed for 12 transcripts by RT-PCR. CONCLUSIONS. Retinal and choroidal vascular endothelial cells display distinctive gene expression profiles. The findings suggest the possibility of treating posterior uveitis by targeting specific interactions between the retinal endothelial cell and an infiltrating leukocyte. (Invest Ophthalmol Vis Sci. 2007;48: 2676‐2684) DOI:10.1167/iovs.06-0598

PURPOSE Ephrin ligands and their Eph receptors are key regulators of endothelial cell (EC) proliferation, migration, adhesion, and repulsion during mammalian vascular development. The hypothesis was that these molecules also play a role in pathologic neovascularization (NV) in the mouse model of oxygen-induced retinopathy. METHODS C57BL/6 mice at postnatal day (P)7 were exposed to 75% oxygen (O(2)) for 5 days (until P12) and allowed to recover in room air to induce retinal NV. Retinas from unexposed and hyperoxia-exposed mice between P7 to P24 were analyzed specifically for EphrinB2 and EphB4 transcript expression by RT-PCR. Phospho-Eph (p-Eph) receptor was evaluated during active EC proliferation at P15 and P17 by immunohistology. Some hyperoxia-exposed mice had one eye injected intravitreally with 150 ng/1.5 microL of soluble EphrinB2/Fc or EphB4/Fc chimeras during transition from high O(2) to room air (P12) and injected again on P14. Contralateral eyes were injected with human IgG as the control. Preretinal nuclei and retinal blood vessels were quantified at peak disease (P17). RESULTS EphrinB2 mRNA was constitutively expressed in the developing retina and was unchanged by hyperoxia. In contrast, EphB4 mRNA expression was modulated during normal retinal development and was altered by hyperoxia. Furthermore, p-Eph was detected in developing preretinal tufts, thus implying that Ephrin/Eph signaling system is active in this experimental model. Intravitreal injection of soluble versions of these molecules significantly reduced pathologic neovascularization. The number of preretinal nuclei in hyperoxia-treated mice was reduced by 66% (P < 0.05) in EphrinB2-injected eyes, whereas EphB4 treatment yielded a 69% reduction (P < 0.05), compared with control injections. Intraretinal vessel development was not altered by the injections. CONCLUSIONS These results support the hypothesis that endogenous EphrinB2 and EphB4 are regulators of retinal NV during oxygen-induced retinopathy and may be useful targets for therapeutic intervention.

PURPOSE Retinochoroidal infection with the protozoan parasite Toxoplasma gondii is the most common cause of posterior uveitis worldwide. Tachyzoites spread throughout the body through the blood stream and lymphatics, but preferentially encyst in the eye and other parts of the central nervous system (CNS). It is unknown whether T. gondii penetrates the CNS selectively or whether these sites of immune privilege have limited capacity to eradicate the parasite. METHODS Human vascular endothelial cell lines, including retinal (three lines from three different donors), aortic, umbilical vein, and dermal microvascular endothelium, as well as human foreskin fibroblasts, were grown to confluence in 24-well plates. Cells were incubated with RH-strain T. gondii tachyzoites in the presence of [(3)H]-uracil. Trichloroacetic acid-insoluble radioactivity was measured as an index of T. gondii proliferation, because tachyzoites, but not human cells, incorporate uracil directly through pyrimidine salvage. RESULTS Tachyzoites showed higher [(3)H]-uracil incorporation after incubation with retinal vascular endothelial cells in comparison with aortic (55% more), umbilical vein (33% more) and dermal (34% more) endothelial cells. In eight separate assays, significantly greater radioactivity was measured for tachyzoites cultured with retinal versus other cell subtypes (P < 0.05), except for one assay in which differences reached only borderline significance (P

Purpose To develop a technology to image cell death in the eye of a live mouse and to apply that technology to characterize the role of apoptosis and necrosis in the evolution of endotoxin-induced uveitis (EIU), a standard model of intraocular inflammation. Methods To induce EIU, 250 ng Escherichia coli 055:B5 lipopolysaccharide was injected into the vitreous body of BALB/c mice. At 0, 6, 10, 16, 20, 24, 48, 72, and 96 hours and on day 7 after endotoxin injection, annexin V and propidium iodide were injected into the anterior chamber of these mice, and labeled cells were observed by using intravital epifluorescence video microscopy. Iris and corneal wholemounts isolated from the mice were also evaluated with standard and confocal fluorescence microscopy. TUNEL staining was performed on iris wholemounts taken from additional mice similarly injected with endotoxin, to confirm the in vivo results. Results Uveitis was induced in all the mice that received an endotoxin injection. The percentages of annexin V(+) propidium(-) cells, annexin V(+) propidium(+) cells, and annexin V(-) propidium(+) cells in the iris tissues visible by intravital microscopy were comparable to those observed by TUNEL staining in vitro. In addition, intravital microscopy allowed observation of labeled cells in the aqueous humor and on the surface of the lens. Both the number and the pattern of labeled cells changed dramatically over time. The cells stained with annexin V had a variety of morphologies, including small and round, round with a lobulated or a kidney-shaped nucleus, dendriform, and irregular. Conclusions A technique was developed to image cell death in the anterior segment of the eye in vivo and used to demonstrate that the number and proportion of early apoptotic (annexin V(+) propidium(-)) and late apoptotic or necrotic (annexin V(+) propidium(+) and annexin V(-) propidium(+)) cells change over the course of EIU. A variety of inflammatory cells and resident cells undergo apoptosis, or possibly necrosis, which may contribute to the rapid resolution of EIU. This in vivo technique will be a valuable tool for future studies on the resolution of ocular inflammation.

PURPOSE Fractalkine (FKN) is a dual-adhesion molecule-chemokine that plays a role in inflammation but has not been explored in the eye. In the current study, constitutive expression of FKN was identified in human iris and retina, and its regulation by various cytokines in endothelial cells (ECs) and stromal cells from human iris, retina, and choroid was investigated. METHODS Human iris and retina explants were evaluated for FKN mRNA and protein expression using RT-PCR and immunohistochemistry, respectively. Cultured ocular ECs and stromal cells were stimulated with various inflammatory mediators (endotoxin; TNFalpha; interferon-gamma; interleukin (IL)-1alpha, -4, -10, -13, -17, and -18; and/or CD40 ligand, or combinations thereof), with FKN mRNA being subsequently evaluated by cDNA array and/or RT-PCR and FKN protein by enzyme-linked immunoculture assay (ELICA) and/or by Western blot analysis. RESULTS Iris and retina explants constitutively expressed FKN protein in microvascular ECs and also in several stromal cell types. Iris and retina both express FKN mRNA. TNFalpha upregulated FKN in iris explants. All ocular microvascular ECs and stromal cultures expressed low FKN mRNA and/or protein levels, which were variably upregulated by endotoxin, TNFalpha, interferon-gamma, IL-1alpha, and/or CD40 ligand, but not by IL-18. In ECs, the Th2 cytokines IL-4 and -13, but not IL-10, reduced TNFalpha-induced FKN protein. IL-17, usually considered proinflammatory, reduced TNFalpha-induced FKN protein in ocular ECs. CONCLUSIONS FKN is expressed in various ocular tissues and cells. Inflammatory mediator modulation of ocular FKN expression suggests that this adhesive chemokine may play important roles in regulating leukocyte efflux in inflammatory eye diseases, such as anterior uveitis and retinochoroiditis.