STAT3-Specific Single Domain Nanobody Inhibits Expansion of Pathogenic Th17 Responses and Suppresses Uveitis in Mice.

STAT3 activates transcription of genes that regulate cell growth, differentiation, and survival of mammalian cells. Genetic deletion of Stat3 in T cells has been shown to abrogate Th17 differentiation, suggesting that STAT3 is a potential therapeutic target for Th17-mediated diseases. However, a major impediment to therapeutic targeting of intracellular proteins such as STAT3 is the lack of efficient methods for delivering STAT3 inhibitors into cells. In this study, we developed a novel antibody (SBT-100) comprised of the variable (V) region of a STAT3-specific heavy chain molecule and demonstrate that this 15 kDa STAT3-specific nanobody enters human and mouse cells, and induced suppression of STAT3 activation and lymphocyte proliferation in a concentration-dependent manner. To investigate whether SBT-100 would be effective in suppressing inflammation in vivo, we induced experimental autoimmune uveitis (EAU) in C57BL/6J mice by active immunization with peptide from the ocular autoantigen, interphotoreceptor retinoid binding protein (IRBP651-670). Analysis of the retina by fundoscopy, histological examination, or optical coherence tomography showed that treatment of the mice with SBT-100 suppressed uveitis by inhibiting expansion of pathogenic Th17 cells that mediate EAU. Electroretinographic (ERG) recordings of dark and light adapted a- and b-waves showed that SBT-100 treatment rescued mice from developing significant visual impairment observed in untreated EAU mice. Adoptive transfer of activated IRBP-specific T cells from untreated EAU mice induced EAU, while EAU was significantly attenuated in mice that received IRBP-specific T cells from SBT-100 treated mice. Taken together, these results demonstrate efficacy of SBT-100 in mice and suggests its therapeutic potential for human autoimmune diseases.

Induction of antigen-specific Treg cells in treating autoimmune uveitis via bystander suppressive pathways without compromising anti-tumor immunity.

BACKGROUND: Induction of autoantigen-specific Treg cells that suppress tissue-specific autoimmunity without compromising beneficial immune responses is the holy-grail for immunotherapy to autoimmune diseases. METHODS: In a model of experimental autoimmune uveitis (EAU) that mimics human uveitis, ocular inflammation was induced by immunization with retinal antigen interphotoreceptor retinoid-binding protein (IRBP). Mice were given intraperitoneal injection of αCD4 antibody (Ab) after the onset of disease, followed by administration of IRBP. EAU was evaluated clinically and functionally. Splenocytes, CD4+CD25- and CD4+CD25+ T cells were sorted and cultured with IRBP or αCD3 Ab. T cell proliferation and cytokine production were assessed. FINDINGS: The experimental approach resulted in remission of ocular inflammation and rescue of visual function in mice with established EAU. Mechanistically, the therapeutic effect was mediated by induction of antigen-specific Treg cells that inhibited IRBP-driven Th17 response in TGF-ß and IL-10 dependent fashion. Importantly, the Ab-mediated immune tolerance could be achieved in EAU mice by administration of retinal autoantigens, arrestin but not limited to IRBP only, in an antigen-nonspecific bystander manner. Further, these EAU-suppressed tolerized mice did not compromise their anti-tumor T immunity in melanoma model. INTERPRETATION: We successfully addressed a specific immunotherapy of EAU by in vivo induction of autoantigen-specific Treg cells without compromising host overall T cell immunity, which should have potential implication for patients with autoimmune uveitis. FUNDING: This study was supported by the Natural Science Foundation of Guangdong Province and the Fundamental Research Fund of the State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center.

Treatment With FoxP3+ Antigen-Experienced T Regulatory Cells Arrests Progressive Retinal Damage in a Spontaneous Model of Uveitis.

We specify the clinical features of a spontaneous experimental autoimmune uveitis (EAU) model, in which foreign hen-egg lysozyme (HEL) is expressed in the retina, controlled by the promoter for interphotoreceptor retinol binding protein (IRBP). We previously reported 100% P21 (post-partum day) IRBP:HEL single transgenic (sTg) mice, when crossed to transgenic T cell receptor mice (3A9) generating the double transgenic (dTg) genotype, develop EAU despite profound lymphopenia (thymic HEL-specific T cell deletion). In this work, we characterized the immune component of this model and found conventional dTg CD4+ T cells were less anergic than those from 3A9 controls. Furthermore, prior in vitro HEL-activation of 3A9 anergic T cells (Tan) rendered them uveitogenic upon adoptive transfer (Tx) to sTg mice, while antigen-experienced (AgX, dTg), but not naïve (3A9) T cells halted disease in P21 dTg mice. Flow cytometric analysis of the AgX cells elucidated the underlying pathology: FoxP3+CD25hiCD4+ T regulatory cells (Treg) comprised ∼18%, while FR4+CD73+FoxP3-CD25lo/-CD4+ Tan comprised ∼1.2% of total cells. Further Treg-enrichment (∼80%) of the AgX population indicated FoxP3+CD25hiCD4+ Treg played a key role in EAU-suppression while FoxP3-CD25lo/-CD4+ T cells did not. Here we present the novel concept of dual immunological tolerance where spontaneous EAU is due to escape from anergy with consequent failure of Treg induction and subsequent imbalance in the [Treg:Teffector] cell ratio. The reduced numbers of Tan, normally sustaining Treg to prevent autoimmunity, are the trigger for disease, while immune homeostasis can be restored by supplementation with AgX, but not naïve, antigen-specific Treg.

Single particle cryo-EM of the complex between interphotoreceptor retinoid-binding protein and a monoclonal antibody.

Interphotoreceptor retinoid-binding protein (IRBP) is a highly expressed protein secreted by rod and cone photoreceptors that has major roles in photoreceptor homeostasis as well as retinoid and polyunsaturated fatty acid transport between the neural retina and retinal pigment epithelium. Despite two crystal structures reported on fragments of IRBP and decades of research, the overall structure of IRBP and function within the visual cycle remain unsolved. Here, we studied the structure of native bovine IRBP in complex with a monoclonal antibody (mAb5) by cryo-electron microscopy, revealing the tertiary and quaternary structure at sufficient resolution to clearly identify the complex components. Complementary mass spectrometry experiments revealed the structure and locations of N-linked carbohydrate post-translational modifications. This work provides insight into the structure of IRBP, displaying an elongated, flexible three-dimensional architecture not seen among other retinoid-binding proteins. This work is the first step in elucidation of the function of this enigmatic protein.

Parental Uveitis Influences Offspring With an Increased Susceptibility to the Experimental Autoimmune Uveitis.

Purpose: Previous studies have shown that parental abnormal physiological conditions such as inflammation, stress, and obesity can be transferred to offspring. The purpose of this study was to investigate the impact of parental uveitis on the development and susceptibility to experimental autoimmune uveitis (EAU) in offspring. Methods: Parental male and female B10RIII mice were immunized with interphotoreceptor retinoid binding protein (IRBP) 161-180 in complete Freund's adjuvant and were immediately allowed to mate. Gross examination of the offspring gestated with EAU was performed to determine the influence of parental uveitis on offspring development after birth. Gene expression profiles were analyzed in the affected eyes of offspring under EAU to identify differentially expressed genes (DEGs). Adult offspring were given 5, 25, and 50 µg IRBP161-180 to compare their susceptibility to EAU. Immunized mice were clinically and pathologically evaluated for the development of EAU. Ag-specific T-cell proliferation and IL-17 production from spleens and lymph nodes were evaluated on day 14 or 35 after immunization. Results: Hair loss, delay of eye opening, and swollen spleens in the offspring from parents with uveitis were observed from day 14 to 39 after birth. DEGs were involved in the immune system process, muscle system process, and cell development. The altered antigen processing and presentation, cell adhesion molecules, and phagosome in the eyes of the offspring from uveitis-affected parents were enriched. Offspring gestated with EAU showed a susceptibility to EAU and an earlier onset and higher severity of EAU compared to the control group mice. IRBP-specific lymphocyte proliferation and IL-17 production were observed in the EAU offspring with exposure to parental uveitis. Conclusions: The results suggest that mouse parents with uveitis can increase their offspring's susceptibility to EAU, probably through altering cell adhesion molecules and antigen processing and presentation related to the T-cell proliferation and Th17 response.

Matairesinol ameliorates experimental autoimmune uveitis by suppression of IRBP-specific Th17 cells.

We investigated the effects of matairesinol (MAT) in the experimental autoimmune uveitis (EAU), a classical animal model of uveitis. We found that treatment with MAT could alleviate intraocular inflammation of EAU. Notably, Th17 cells in eyes of EAU mice could be predominantly restrained by MAT. Furthermore, MAT could inhibit Th17 differentiation in vitro. In addition, MAT inhibited the signaling of MAPK and ROR-γt, a pivotal transcription factor for Th17 cell differentiation in vitro and in vivo. Taken together, these results suggested that MAT had immune-suppressive effects on autoimmune inflammation through Th17 cells.

Aminooxy-Acetic Acid Inhibits Experimental Autoimmune Uveitis by Modulating the Balance between Effector and Regulatory Lymphocyte Subsets.

PURPOSE: A small molecular compound, aminooxy-acetic acid (AOA), has been shown to modulate experimental autoimmune encephalomyelitis (EAE). The current study was designed to investigate whether AOA has a similar effect on the development of experimental autoimmune uveitis (EAU) and to further explore underlying mechanisms of this drug. METHODS: EAU was induced in C57BL/6J mice by immunization with interphotoreceptor retinoid-binding protein peptide 651-670 (IRBP 651-670). AOA (500µg or 750µg) or vehicle was administered by intraperitoneal injection from day 10 to 14 after EAU induction. The severity was assessed by clinical and histological scores. The integrity of the blood retinal barrier was detected with Evans Blue. Frequencies of splenic Th1, Th17 and Foxp3+ Treg cells were examined by flow cytometry. The production of cytokines was tested by ELISA. The mRNA expression of IL-17, IFN-γ and IL-10 was detected by RT-PCR. The expression of p-Stat1 and NF-κB was detected by Western Blotting. RESULTS: AOA was found to markedly inhibit the severity of EAU, as determined by clinical and histopathological examinations. AOA can relieve the leakage of blood retinal barrier (BRB). Functional studies found a decreased frequency of Th1 and Th17 cells and an increased frequency of Treg cells in EAU mice as compared with controls. Further studies showed that AOA not only downregulated the production of the proinflammatory cytokines including IFN-γ and IL-17 but also upregulated the expression of an anti-inflammatory cytokine such as IL-10, which might be caused by inhibiting the expressions of p-Stat1 and NF-κB. CONCLUSION: This study shows that AOA inhibits the severity and development of EAU by modulating the balance between regulatory and pathogenic lymphocyte subsets.

Effect of berberine on spleen transcriptome and gut microbiota composition in experimental autoimmune uveitis.

BACKGROUND: Berberine (BBR) was reported to have immunoregulatory and anti-inflammatory properties. In this study, we investigated whether BBR could exert its effects on the development of experimental autoimmune uveitis (EAU), and if so, what was the underlying mechanism? METHODS: EAU was induced in B10R.III mice by immunization with IRBP 161-180, followed by 100 mg/kg/d BBR intragastric administration. Disease severity was assessed by evaluation of clinical and histopathological scores. Blood-retinal barrier (BRB) breakdown was tested by Evans blue. Effector and regulatory T (Treg) cell balance was evaluated by quantitative real-time PCR and flow cytometry. Spleen transcriptome was characterized by RNA sequencing (RNA-seq). Gut microbiota composition was investigated by 16S rRNA analysis. RESULTS: BBR treatment significantly blocked EAU as shown by the decrease of the clinical and histological scores, as well as the inhibition of BRB breakdown. The frequency of splenic Th1 and Th17 cells was decreased, whereas Treg cells were increased in the BBR-treated group. RNA-seq of the spleen revealed 476 differentially expressed genes (DEGs) between the EAU and EAU-BBR group. GO functional classification, as well as KEGG analysis demonstrated that BBR treatment markedly influences genes belonging to chromatin remodeling and immune-related pathways. Intervention with BBR modified the gut microbiome in EAU mice, increasing the number of bacteria with immunomodulatory capacity. Depletion of gut microbiota affected the efficacy of BBR on EAU. Moreover, the altered bacterial strains showed a significant correlation with the expression of histones. CONCLUSIONS: BBR inhibited IRBP induced EAU, which was associated with a significant change in the spleen transcriptome and intestinal microbial composition.

Ligand binding properties of two Brugia malayi fatty acid and retinol (FAR) binding proteins and their vaccine efficacies against challenge infection in gerbils.

Parasitic nematodes produce an unusual class of fatty acid and retinol (FAR)-binding proteins that may scavenge host fatty acids and retinoids. Two FARs from Brugia malayi (Bm-FAR-1 and Bm-FAR-2) were expressed as recombinant proteins, and their ligand binding, structural characteristics, and immunogenicities examined. Circular dichroism showed that rBm-FAR-1 and rBm-FAR-2 are similarly rich in α-helix structure. Unexpectedly, however, their lipid binding activities were found to be readily differentiated. Both FARs bound retinol and cis-parinaric acid similarly, but, while rBm-FAR-1 induced a dramatic increase in fluorescence emission and blue shift in peak emission by the fluorophore-tagged fatty acid (dansyl-undecanoic acid), rBm-FAR-2 did not. Recombinant forms of the related proteins from Onchocerca volvulus, rOv-FAR-1 and rOv-FAR-2, were found to be similarly distinguishable. This is the first FAR-2 protein from parasitic nematodes that is being characterized. The relative protein abundance of Bm-FAR-1 was higher than Bm-FAR-2 in the lysates of different developmental stages of B. malayi. Both FAR proteins were targets of strong IgG1, IgG3 and IgE antibody in infected individuals and individuals who were classified as endemic normal or putatively immune. In a B. malayi infection model in gerbils, immunization with rBm-FAR-1 and rBm-FAR-2 formulated in a water-in-oil-emulsion (®Montanide-720) or alum elicited high titers of antigen-specific IgG, but only gerbils immunized with rBm-FAR-1 formulated with the former produced a statistically significant reduction in adult worms (68%) following challenge with B. malayi infective larvae. These results suggest that FAR proteins may play important roles in the survival of filarial nematodes in the host, and represent potential candidates for vaccine development against lymphatic filariasis and related filarial infections.

Cyclosporine A-Loaded Nanocarriers for Topical Treatment of Murine Experimental Autoimmune Uveoretinitis.

In the present study, tissue distribution and the therapeutic effect of topically applied cyclosporine A (CsA)-loaded methoxy-poly(ethylene-glycol)-hexyl substituted poly(lactic acid) (mPEGhexPLA) nanocarriers (ApidSOL) on experimental autoimmune uveitis (EAU) were investigated. The CsA-loaded mPEGhexPLA nanocarrier was tolerated well locally and showed no signs of immediate toxicity after repeated topical application in mice with EAU. Upon unilateral CsA treatment, CsA accumulated predominantly in the corneal and sclera-choroidal tissue of the treated eye and in lymph nodes (LN). This regimen reduced EAU severity in treated eyes compared to PBS-treated controls. This improvement was accompanied by reduced T-cell count, T-cell proliferation, and IL-2 secretion of cells from ipsilateral LN. In conclusion, topical treatment with CsA-loaded mPEGhexPLA nanocarriers significantly improves the outcome of EAU.

Comparative Analysis of the Interphotoreceptor Retinoid Binding ProteinInduced Models of Experimental Autoimmune Uveitis in B10.RIII versus C57BL/6 Mice.

OBJECTIVE: Experimental autoimmune uveitis (EAU) represents autoimmune uveitis in humans, among which B10.RIII and C57BL/6 are the frequently used strains in mice, but to date, no study has been reported to compare EAU disease between the two strains. Here we compared the differences in morphology, pathology, visual function of the retinal inflammation and Th1/Th17 immune responses in the EAU models induced by the interphotoreceptor retinoid binding protein (IRBP) between the B10.RIII and C57BL/6 strains, using fundus and histological examinations, optical coherence tomography, electroretinography and immunoassays. METHOD: EAU induced in B10.RIII mice exhibited a shortterm severe inflammation with massive ocular infiltrates of inflammatory cells and extensive destruction of the retina that culminated in rapid degeneration of the retina and permanent loss of visual function. In contrast, C57BL/6 mice developed chronic inflammation with recurring and persistent retinitis for several months, highlighting moderate scores of disease severity and visual signal in comparison with those in B10.RIII mice. Consistent with the clinical manifestations, increased Th1/Th17 effector responses were detected in the uveitic eyes of B10.RIII strain than those in C57BL/6 strain. These data demonstrate distinguishing features of retinal inflammation and T-cell immune responses involved in IRBP-induced EAU between B10.RIII and C57BL/6 strains. CONCLUSION: Our findings suggest that the persistent-recurring EAU model induced in C57BL/6 mice may serve as a better tool to represent distinct aspects of human uveitis.

Antigen-specificity of antiretinal antibodies in patients with noninfectious uveitis.

OBJECTIVE: Antiretinal antibodies (ARAs) have previously been described in noninfectious uveitis. However, the antigen specificity of these ARAs has not been investigated. The purpose of this study was to identify antigen-specific ARAs in noninfectious uveitis. METHODS: A total of 18 patients with noninfectious uveitis were enrolled. Surface plasmon resonance was used to measure binding responses of patient and control sera against several uveitogenic proteins: recoverin, S-antigen, interphotoreceptor retinoid binding (IRBP), retinal-pigment-epithelium-specific 65-kDa protein (RPE65), tyrosinase-related protein 1 (TRYP1), and tyrosinase-related protein 2 (TRYP2). RESULTS: The frequency of ARA positivity against S-antigen, IRBP, RPE65, TYRP1, and TYRP2 in patients with uveitis did not differ significantly from that of normal controls. However, ARA positivity for recoverin was more frequently observed in patients with uveitis (p = 0.002). A total of 10 patients in the uveitis cohort had birdshot chorioretinopathy, and all 10 were positive for anti-recoverin ARAs. CONCLUSIONS: Patients with noninfectious uveitis have increased frequency of ARA positivity against recoverin. This ARA deserves further investigations as a potential biomarker and pathogenic agent in noninfectious uveitis, especially in birdshot chorioretinopathy.

TLR7 Engagement on Dendritic Cells Enhances Autoreactive Th17 Responses via Activation of ERK.

In this study, we showed that TLR7 activation significantly promoted interphotoreceptor retinoid-binding protein (IRBP)-specific Th17 responses by upregulating RORγt, IL-17, GM-CSF, and IL-23R expression in experimental autoimmune uveitis mice. In vivo administration of CL097 activated dendritic cells (DCs) and endowed them with an increased ability to activate IRBP-specific Th17 cells. CL097-treated DCs (CL097-DCs) formed a cytokine milieu that favored the generation and maintenance of Th17 cells by stimulating IL-1ß, IL-6, and IL-23 expression. Furthermore, IRBP-specific T cells from immunized mice injected with CL097-DCs produced more IL-17 and transferred more severe experimental autoimmune uveitis than did those from mice injected with DCs. The enhanced immunostimulatory activities of CL097-DCs depended on JNK, ERK, and p38 activation. Blockade of ERK, but not p38 or JNK, completely abolished the Th17 responses induced by CL097-DCs. Collectively, our findings suggest that CL097 treatment significantly promotes autoreactive IL-17+ T cell responses through enhancing DC activation, which is mediated, at least in part, via the activation of ERK signaling.

LYN- and AIRE-mediated tolerance checkpoint defects synergize to trigger organ-specific autoimmunity.

Studies of the genetic factors associated with human autoimmune disease suggest a multigenic origin of susceptibility; however, how these factors interact and through which tolerance pathways they operate generally remain to be defined. One key checkpoint occurs through the activity of the autoimmune regulator AIRE, which promotes central T cell tolerance. Recent reports have described a variety of dominant-negative AIRE mutations that likely contribute to human autoimmunity to a greater extent than previously thought. In families with these mutations, the penetrance of autoimmunity is incomplete, suggesting that other checkpoints play a role in preventing autoimmunity. Here, we tested whether a defect in LYN, an inhibitory protein tyrosine kinase that is implicated in systemic autoimmunity, could combine with an Aire mutation to provoke organ-specific autoimmunity. Indeed, mice with a dominant-negative allele of Aire and deficiency in LYN spontaneously developed organ-specific autoimmunity in the eye. We further determined that a small pool of retinal protein-specific T cells escaped thymic deletion as a result of the hypomorphic Aire function and that these cells also escaped peripheral tolerance in the presence of LYN-deficient dendritic cells, leading to highly destructive autoimmune attack. These findings demonstrate how 2 distinct tolerance pathways can synergize to unleash autoimmunity and have implications for the genetic susceptibility of autoimmune disease.

The Microbiota Determines Susceptibility to Experimental Autoimmune Uveoretinitis.

The microbiota is a crucial modulator of the immune system. Here, we evaluated how its absence or reduction modifies the inflammatory response in the murine model of experimental autoimmune uveoretinitis (EAU). We induced EAU in germ-free (GF) or conventionally housed (CV) mice and in CV mice treated with a combination of broad-spectrum antibiotics either from the day of EAU induction or from one week prior to induction of disease. The severity of the inflammation was assessed by fundus biomicroscopy or by histology, including immunohistology. The immunophenotyping of T cells in local and distant lymph nodes was performed by flow cytometry. We found that GF mice and mice where the microbiota was reduced one week before EAU induction were protected from severe autoimmune inflammation. GF mice had lower numbers of infiltrating macrophages and significantly less T cell infiltration in the retina than CV mice with EAU. GF mice also had reduced numbers of IFN-γ and IL-17-producing T cells and increased numbers of regulatory T cells in the eye-draining lymph nodes. These data suggest that the presence of microbiota during autoantigen recognition regulates the inflammatory response by influencing the adaptive immune response.

Photoreceptor IRBP prevents light induced injury.

Interphotoreceptor retinoid-binding protein (IRBP) is a classic inducer of experimental autoimmune uveoretinitis (EAU). Although IRBP causes neuronal loss in susceptible animals, resistant animals such as Sprague-Dawley (SPD) rats can benefit from the evoked protective autoimmune responses. The aim of the present study was to analyze the neuroprotective effects of IRBP against light-induced photoreceptor degeneration. We immunized 75 male SPD rats with IRBP and the rats were then exposed to blue light for 24 hours (IRBP group). Seventy five rats were included in the control group. We found that the number of apoptotic cells in the outer nuclear layer (ONL) peaked on 1 day after light exposure, and the ONL thickness decreased significantly on day 3. OX42-positive cells appeared in the ONL immediately after light exposure, and their number peaked on day 3, and changed from resting ramified cells to activated amoeboid cells. Compared with the control group (n=75), the IRBP group showed less apoptotic cells, a thicker ONL, and reduced expression of tumor necrosis factor-alpha. These outcomes indicate the IRPB might protect retinal photoreceptors against light-induced injury.

CD73 Expressed on γδ T Cells Shapes Their Regulatory Effect in Experimental Autoimmune Uveitis.

γδ T cells can either enhance or inhibit an adaptive immune response, but the mechanisms involved are not fully understood. Given that CD73 is the main enzyme responsible for conversion of AMP into the immunosuppressive molecule adenosine, we investigated its role in the regulatory function of γδ T cells in experimental autoimmune uveitis (EAU). We found that γδ T cells expressed different amounts of CD73 during the different stages of EAU and that low CD73 expression on γδ T cells correlated with enhanced Th17 response-promoting activity. Functional comparison of CD73-deficient and wild-type B6 (CD73+/+) mice showed that failure to express CD73 decreased both the enhancing and suppressive effects of γδ T cells on EAU. We also demonstrated that γδ T cells expressed different amounts of CD73 when activated by different pathways, which enabled them to either enhance or inhibit an adaptive immune response. Our results demonstrate that targeting CD73 expression on γδ T cells may allow us to manipulate their pro- or anti-inflammatory effect on Th17 responses.

Regulation of Adenosine Deaminase on Induced Mouse Experimental Autoimmune Uveitis.

Adenosine is an important regulator of the immune response, and adenosine deaminase (ADA) inhibits this regulatory effect by converting adenosine into functionally inactive molecules. Studies showed that adenosine receptor agonists can be anti- or proinflammatory. Clarification of the mechanisms that cause these opposing effects should provide a better guide for therapeutic intervention. In this study, we investigated the effect of ADA on the development of experimental autoimmune uveitis (EAU) induced by immunizing EAU-prone mice with a known uveitogenic peptide, IRBP1-20. Our results showed that the effective time to administer a single dose of ADA to suppress induction of EAU was 8-14 d postimmunization, shortly before EAU expression; however, ADA treatment at other time points exacerbated disease. ADA preferentially inhibited Th17 responses, and this effect was γδ T cell dependent. Our results demonstrated that the existing immune status strongly influences the anti- or proinflammatory effects of ADA. Our observations should help to improve the design of ADA- and adenosine receptor-targeted therapies.

Vorinostat Modulates the Imbalance of T Cell Subsets, Suppresses Macrophage Activity, and Ameliorates Experimental Autoimmune Uveoretinitis.

The purpose of the study was to investigate the anti-inflammatory efficiency of vorinostat, a histone deacetylase inhibitor, in experimental autoimmune uveitis (EAU). EAU was induced in female C57BL/6J mice immunized with interphotoreceptor retinoid-binding protein peptide. Vorinostat or the control treatment, phosphate-buffered saline, was administrated orally from 3 days before immunization until euthanasia at day 21 after immunization. The clinical and histopathological scores of mice were graded, and the integrity of the blood-retinal barrier was examined by Evans blue staining. T helper cell subsets were measured by flow cytometry, and the macrophage functions were evaluated with immunohistochemistry staining and immunofluorescence assays. The mRNA levels of tight junction proteins were measured by qRT-PCR. The expression levels of intraocular cytokines and transcription factors were examined by western blotting. Vorinostat relieved both clinical and histopathological manifestations of EAU in our mouse model, and the BRB integrity was maintained in vorinostat-treated mice, which had less vasculature leakage and higher mRNA and protein expressions of tight junction proteins than controls. Moreover, vorinostat repressed Th1 and Th17 cells and increased Th0 and Treg cells. Additionally, the INF-γ and IL-17A expression levels were significantly decreased, while the IL-10 level was increased by vorinostat treatment. Furthermore, due to the reduced TNF-α level, the macrophage activity was considerably inhibited in EAU mice. Finally, transcription factors, including STAT1, STAT3, and p65, were greatly suppressed by vorinostat treatment. Our data suggest that vorinostat might be a potential anti-inflammatory agent in the management of uveitis and other autoimmune inflammatory diseases.

Spontaneous Ocular Autoimmunity in Mice Expressing a Transgenic T Cell Receptor Specific to Retina: A Tool to Dissect Mechanisms of Uveitis.

The "classical" EAU model induced by immunization of mice with the retinal protein IRBP or its peptides has been very useful to study basic mechanisms of ocular inflammation, but is inadequate for some types of studies due to the need for active immunization in the context of strong bacterial adjuvants. We generated transgenic (Tg) mice on the B10.RIII background that express a T cell receptor (TCR) specific for IRBP161-180. Three strains of TCR Tg mice were established. Spontaneous uveitis developed in two of the three strains by 2-3 months of age. Susceptibility correlated with a higher copy number of the transgenic TCR and a higher proportion of TCR Tg T cells in the peripheral repertoire. Even in mice with uveitis, peripheral IRBP-specific CD4(+) T cells displayed mostly a naïve phenotype. In contrast, T cells infiltrating uveitic eyes mostly showed an effector/memory phenotype, and included Th1, Th17 as well as T regulatory cells. These mice thus provide a new and distinct model of uveitis from the "classical" EAU, and may represent some types of uveitis more faithfully. Importantly, this new transgenic model of uveitis can serve as a template for therapeutic manipulations, and as a source of naïve retina-specific T cells for a variety of basic and pre-clinical studies. Several examples of such studies will be discussed.