Experimental Autoimmune Uveitis Model Research Articles

Effects of rat mesenchymal stem cell-derived exosomes on rat experimental autoimmune uveitis

Objective To observe the effects of exosomes derived from rat mesenchymal stem cells(MSC-exosomes) on the rat experimental autoimmune uveitis (EAU) model. Methods Twelve Lewis rats were randomly divided into experimental group and control group by random number table, with 6 rats in each group. Rats in the experimental group were established with EAU model, 100 μl of MSC-exosomes (50 μg) were periocular injected on the 9th day after modeling while the control rats were injected with the same volume of phosphate buffer. At different time points after modeling, the retinal structure was observed by hematoxylin and eosin (HE) staining, and the clinical and pathological manifestations were evaluated. T cells from the two groups were analyzed by flow cytometry. Immunohistochemical staining was used to observe the expression of macrophage surface marker CD68. The effect of MSC-exosomes on T cells was measured by lymphocyte proliferation assays. And flow cytometry was used to detect Th1, Th17 and regulatory T cells Variety. Electroretinogram (ERG) was used to evaluate the retinal function. Data were compared between the two groups using the t test. Results HE staining showed that the retina structure of the experimental group was more complete than that of the control group on the 15th day after modeling. Immunohistochemical staining showed that the positive expression of CD68 in the experimental group was significantly less than that in the control group. On the 15th day after modeling, the retinal pathological score of the experimental group was lower than that of the control group. On the 9th to 13th day after modeling, compared to the control group, the average clinical scores of the retina in the experimental group were lower, and the difference was statistically significant(t=3.665, 3.21, 3.181, 4.121, 3.227; P 0.05). Conclusions MSC- exosomes can reduce the clinical and pathological manifestations of EAU, protect retinal function, reduce ocular macrophage infiltration, down-regulate the proportion of inflammatory cells in the eye, and inhibit T cell proliferation. Key words: Mesenchymal stem cells; Exosomes; Uveitis/therapy; Animal experimentation

Adenosine receptor activation in the Th17 autoimmune responses of experimental autoimmune uveitis

Th17-type autoreactive T cells contribute to pathogenicity in autoimmune diseases, including autoimmune uveitis. However, the mechanisms of regulation of Th17 cell activities remain unsolved and are likely to be tissue- and disease specific. In this review, we have summarized our studies from the murine model of experimental autoimmune uveitis (EAU). The resultsdemonstrate that γδ T cells have a regulatory effect on Th17 response. The regulatory effects of γδ T cells depend on their action state. Activated γδ T cells express significantly high levels of adenosine receptor A2 (A2AR) but low CD73. Both molecules are crucially involved in adenosine generation, thus modifying T cell responses. While the increased expression of A2AR-allows activated γδ T cells to bind adenosine more effectively than other immune cells, the decreased CD73 restricts their ability to convert AMP to adenosine. Adenosine affects Th1 and Th17 autoimmune responses differently. Its activation of γδ T cells shifts the Th1/Th17 balance towards the Th17 autoreactivity.

CD11bhigh CD11clow Ialow DCs regulate T-cells activation in the experimental autoimmune uveitis

Abstract Uveitis is a serious inflammatory disease that can cause visual disability and blindness. Uveitogenic antigen-specific CD4+ T cells which secrete IFN-γ or IL-17, are believed to be crucial effector cells to infiltrate in the sites of inflammatory eyes to drive inflammation and tissue damage. Recently, dendritic cells (DCs) act as unique antigen presenting cells, are reported to involve into the pathogenic process of uveitis. Regulating the status of DCs could prevent the generation of Ag-specific Th1 and Th17 cells to attenuated EAU. Herein, in a mouse model of experimental autoimmune uveitis (EAU), we found CD11bhighCD11chighIahigh DCs increased at the inflammation stage of EAU, while CD11bhighCD11clowIalowDCs, a regulatory DCs subset, increased at the recovery stage. CD11bhighCD11chighIahigh DCs expressed high level of CD80, CD86 and CD54 to promote T cells secreting IFN-γ, While CD11bhighCD11clowIalowDCs promote T cells to express high level of CTLA-4 and to be CD4+CD25+FoxP3+ T cells. Furthermore, CD11bhighCD11clowIalowDCs were found to form a regulatory immunological synapse with T cells, and CTLA-4 localized at the contact of DC-T to prevent the localization of TCR signaling molecules and the localization of calcium microdomains at the contact of DC-T to inhibit T-cell activation. However, CD11bhighCD11chighIahigh DCs promote the accumulation of TCR signaling molecules in the IS and calcium releasing in T cells. Transferring CD11bhighCD11clowIalowDCs into EAU could relive the symptom of EAU and decrease Ag-specific T cells in EAU. Overall, CD11bhighCD11clowIalowDCs could suppress T-cells activation by forming a regulatory synapse to decrease calcium releasing in T cells to prevent the development of EAU.

Intraocular DHODH-inhibitor PP-001 suppresses relapsing experimental uveitis and cytokine production of human lymphocytes, but not of RPE cells

BackgroundUveitis is a potentially blinding inflammatory disease of the inner eye with a high unmet need for new therapeutic interventions. Here, we wanted to investigate the suppressive effect of the intraocular application of the small molecule dihydroorotate dehydrogenase (DHODH)-inhibitor PP-001 on experimental relapsing rat uveitis and furthermore determine its effect on proliferation and cytokine secretion of human peripheral blood lymphocytes (PBL) and human retinal pigment epithelial (RPE) cells in vitro.MethodsSpontaneously relapsing uveitis was induced in rats by immunization with interphotoreceptor retinoid-binding protein (IRBP) peptide R14. PP-001 was injected intravitreally after resolution of the primary disease to investigate further relapses. Proliferation and metabolic activity of phytohemagglutinin (PHA)-stimulated human peripheral lymphocytes with and without PP-001 and cytokine secretion were determined by XTT assay and bioplex bead assay. The RPE cell line ARPE-19 as well as primary human RPE cells treated with PP-001 or anti-vascular endothelial growth factor (VEGF) antibody bevacizumab were also investigated for metabolic activity and cytokine/chemokine secretion.ResultsInjection of PP-001 into rat eyes reduced the number of relapses by 70%, from 20 relapses (57% of the rats affected) in the control group to 6 relapses (33% of the rats) in the treatment group. In human PBL cultures, PP-001 reduced the proliferation in a dose-dependent manner. The secretion of several cytokines such as IL-17, IFN-γ, and VEGF was suppressed by PP-001, as previously observed with rat T cells in the experimental autoimmune uveitis (EAU) model. In contrast, human RPE cells were not affected by PP-001, while the anti-VEGF antibody bevacizumab severely impaired the secretion of various cytokines including VEGF.ConclusionsFor the first time, intravitreal injection of PP-001 demonstrated an effective, but transient reduction of relapses in the rat EAU model. In vitro PP-001 suppressed proliferation and cytokine/chemokine secretion of human lymphocytes, while neither human RPE cell line ARPE-19 nor primary RPE cells were affected.

Connection between γδ T-cell- and Adenosine- Mediated Immune Regulation in the Pathogenesis of Experimental Autoimmune Uveitis.

Regulatory effects of γδ T-cells on immune responses have been studied for years. We have investigated the regulatory effect of γδ T-cells on Th1 and Th17 autoimmune responses, and have studied molecular and cellular mechanisms by which γδ T-cells enhance or inhibit immune responses, exploiting a well-characterized murine model of experimental autoimmune uveitis (EAU). Our results show that (1) aberrant γδ T-cell activation is an important pathogenic event in EAU; (2) γδ T-cells have a unique regulatory effect on Th17 autoimmune responses, which is shaped by the activation status of γδ T-cells; and (3) γδ-mediated immunoregulation is closely linked with the extracellular adenosine metabolism. Reciprocal interactions between γδ T-cells and extracellular adenosine partially determine the development of EAU.

Ly6Chi monocytes are required for mesenchymal stem/stromal cell-induced immune tolerance in mice with experimental autoimmune uveitis

The cells of the innate immune system, in addition to their capacity to elicit immunity, play a substantial role in immune tolerance induction. Our group has recently shown that a distinct subset of MHC IIhiB220hiCD11bmid suppressive macrophages is increased in the lung by intravenous (IV) administration of mesenchymal stem/stromal cells (MSC) and induces immune tolerance. Herein, we demonstrate that circulating CD11bhiLy6Chi monocytes are precursors to MHC IIhiB220hiCD11bmid macrophages in the lung and required for MSC-induced tolerance in a mouse model of experimental autoimmune uveitis (EAU). Analysis revealed that IV MSC induced an increase in IL-10-expressing MHC IIhiB220hiCD11bmid macrophages in the lung with a concomitant decrease in CD11bhiLy6Chi monocytes. Selective depletion of circulating CD11bhiLy6Chi cells abrogated the effects of MSC in the induction of IL-10hiMHC IIhiB220hiCD11bmid macrophages and immune tolerance in EAU mice. Similarly, an increase in CD4+CD25+Foxp3+ Tregs by MSCs was also reversed by CD11bhiLy6Chi cell depletion. These results suggest that CD11bhiLy6Chi monocytes are critical for MSC-induced immune tolerance.

Short chain fatty acids ameliorate immune-mediated uveitis partially by altering migration of lymphocytes from the intestine

Short chain fatty acids (SCFA) are metabolites of intestinal bacteria resulting from fermentation of dietary fiber. SCFA are protective in various animal models of inflammatory disease. We investigated the effects of exogenous administration of SFCAs, particularly propionate, on uveitis using an inducible model of experimental autoimmune uveitis (EAU). Oral SCFA administration attenuated uveitis severity in a mouse strain-dependent manner through regulatory T cell induction among lymphocytes in the intestinal lamina propria (LPL) and cervical lymph nodes (CLN). SCFA also suppressed effector T cell induction in the CLN and mesenteric lymph nodes (MLN). Alterations in intestinal morphology and gene expression demonstrated in the EAU model prior to the onset of uveitis were blunted by oral SCFA administration. Using a Kaede transgenic mouse, we demonstrated enhanced leukocyte trafficking between the intestine and the eye in EAU. Propionate suppressed T effector cell migration between the intestine and the spleen in EAU Kaede mice. In conclusion, our findings support exogenous administration of SCFAs as a potential treatment strategy for uveitis through the stabilization of subclinical intestinal alterations that occur in inflammatory diseases including uveitis, as well as prevention of trafficking of leukocytes between the gastrointestinal tract and extra-intestinal tissues.

Behavioral Conditioning of Immune Responses with Cyclosporine A in a Murine Model of Experimental Autoimmune Uveitis

Objective: We examined the role of behavioral conditioning of immune responses with cyclosporine A (CsA) on the development of Th1/Th17-driven experimental autoimmune uveoretinitis (EAU). Methods: Mice received a 0.2% w/v saccharin solution as conditioned stimulus combined with CsA (20 mg/kg) in 6 association trials at 72-h intervals. For evocation periods, conditioned mice were reexposed to saccharin, whereas the conditioned but not reexposed group received water only. Animals were immunized with human interphotoreceptor-retinoid-binding protein peptide 161-180 (hIRBP_p161-180) peptide in complete Freund adjuvant (CFA) and a concomitant injection of pertussis toxin. Results: In naïve mice subjected to the behavioral conditioning regimen, mitogen-induced interleukin (IL)-2 production was decreased in conditioned mice compared to conditioned but not reexposed animals. Incidence and severity of EAU were not significantly lower in behaviorally conditioned and immunized mice. ELISA analysis of splenocytes revealed a reduced interferon (IFN)-γ/IL-17 ratio in CsA-treated, conditioned but not reexposed, and conditioned animals. The adoptive transfer of antigen-specific splenocytes from animals behaviorally conditioned with CsA to naïve mice decreased the severity of EAU in recipient mice compared to the control group. In vitro activation of splenocytes isolated from immunized mice with agonists targeting TLR2 and NOD2 together with β₂-adrenergic activation (induced by epinephrine, norepinephrine, or salbutamol) resulted in decreased IFN-γ but increased IL-17 immune responses. The β₂-adrenergic antagonist propranolol could restore IFN-γ production, whereas only the norepinephrine-induced increase in IL-17 production was abrogated. Conclusions: We conclude that CsA conditioning in the EAU model mitigates Th1 but enhances Th17 immune responses, and does not ameliorate disease. The results imply that in EAU the mechanism of immune conditioning interacts with CFA components during active immunization, most likely via the TLR2/NOD2 pathway, and induces differentiation of Th17 cells that drive autoimmune diseases.

The interaction of dendritic cells and γδ T cells promotes the activation of γδ T cells in experimental autoimmune uveitis

Uveitis is a severe inflammatory disease that can cause visual impairment. Recently, activated [Formula: see text] T cells were proved to play a central role in the development of experimental autoimmune uveitis (EAU). However, the mechanism underlying [Formula: see text] T-cell activation in EAU is incompletely known. In this study, we determined the percentage changes in and the phenotypes of [Formula: see text] T cells and dendritic cells (DCs) obtained from the spleens of immunized C57BL/6 (B6) mice, an animal model of EAU. We found that the number of [Formula: see text] T cells and DCs obviously increased during the inflammation phase of EAU (days 16–20 of our experiment), and that during this time, [Formula: see text] T cells expressed high levels of CD69 and the integrin lymphocyte function–associated antigen-1 (LFA-1) and secreted high levels of interleukin (IL)-17A. Moreover, DCs obtained during this phase expressed high levels of CD80, CD83, CD86, and intracellular cell adhesion molecule-1 (ICAM-1). Furthermore, we studied the interaction between DCs and [Formula: see text] T cells by using flow cytometry and confocal microscopy in order to determine whether DCs affected [Formula: see text] T-cell activation in vitro. Co-cultures of the two types of cells showed that DCs induced high levels of CD69, LFA-1, and IL-17A in [Formula: see text] T cells. Imaging studies revealed contact between the DCs and [Formula: see text] T cells. This interaction was mediated by the accumulation of ICAM-1 and LFA-1 at the interface of DCs-[Formula: see text] T cells. Thus, the activation of [Formula: see text] T cells in EAU was promoted by DCs interacting with [Formula: see text] T cells.

Pharmacological Inhibition of Bromodomain Proteins Suppresses Retinal Inflammatory Disease and Downregulates Retinal Th17 Cells.

Experimental autoimmune uveitis (EAU), in which CD4+ Th1 and/or Th17 cells are immunopathogenic, mimics various clinical features of noninfectious uveitis in humans. The impact of bromodomain extraterminal (BET) inhibitors on Th17 cell function was studied in a mouse model of EAU in vivo and in mouse and human Th17 cells in vitro. Two BET inhibitors (GSK151 and JQ1) were able to ameliorate the progression of inflammation in EAU and in mouse CD4+ T cells in vitro, downregulating levels of Th17 cells. Additionally, the uveitogenic capacity of Th17 cells to transfer EAU was abrogated by BET inhibitors in an adoptive transfer model. In human CD4+ T cells, a 5-d exposure to BET inhibitors was accompanied by a significant downregulation of Th17-associated genes IL-17A, IL-22, and retinoic acid-related orphan receptor γt. However, in vitro, the inhibitors had no effect on already polarized Th17 cells. The key finding is that, in response to BET inhibitors, Th17-enriched cultures developed a regulatory phenotype, upregulated FOXP3 expression and IL-10 secretion, and lost pathogenicity in vivo. We conclude that BET targeting of Th17 cells is a potential therapeutic opportunity for a wide range of inflammatory and autoimmune diseases, including uveitis.

Notch signalling suppresses regulatory T-cell function in murine experimental autoimmune uveitis.

Autoimmune uveitis is an intraocular inflammatory disorder in developed countries. Understanding the mechanisms underlying the development and modulation of immune reaction in uveitic eyes is critical for designing therapeutic interventions. Here we investigated the role of Notch signalling in regulatory T-cell (Treg cell) function during experimental autoimmune uveitis (EAU). Using the Foxp3-GFP reporter mouse strain, the significance of Notch signalling for the function of infiltrating Treg cells was characterized in an EAU model. We found that infiltrating Treg cells substantially expressed Notch-1, Notch-2, JAG1 and DLL1 in uveitic eyes. Activation of Notch signalling, represented by expression of HES1 and HES5, was enhanced in infiltrating Treg cells. Treatment with JAG1 and DLL1 down-regulated Foxp3 expression and immunosuppressive activity of isolated infiltrating Treg cells in vitro, whereas neutralizing antibodies against JAG1 and DLL1 diminished Notch ligand-mediated negative effects on Treg cells. To investigate the significance of Notch signalling for Treg cell function in vivo, lentivirus-derived Notch short hairpin RNAs were transduced into in vitro expanded Treg cells before adoptive transfer of Treg cells into EAU mice. Transfer of Notch-1-deficient Treg cells remarkably reduced pro-inflammatory cytokine production and inflammatory cell infiltration in uveitic eyes. Taken together, Notch signalling negatively modulates the immunosuppressive function of infiltrating Treg cells in mouse EAU.

CD73 Pathway Contributes to the Immunosuppressive Ability of Mesenchymal Stem Cells in Intraocular Autoimmune Responses.

Mesenchymal stem cells (MSCs) exhibit a potent immunomodulatory capacity and have been applied to treat diseases such as graft versus host disease and severe autoimmune diseases. However, the mechanism underlying their immunosuppressive effect is not yet completely understood. Here, we investigated the role of the CD73/adenosine pathway in immune modulation by MSCs using a mouse model of experimental autoimmune uveitis (EAU). Moreover, we examined the in vitro modulatory effect of MSCs mediated through the CD73/adenosine pathway in human and mouse T cells. We found that the severity of EAU was significantly attenuated by MSCs; however, most therapeutic effects of MSCs were lost by pretreatment with a CD73 inhibitor. The inhibitory mechanism of MSCs might be contributed by CD73 on MSCs that cooperated with CD39 and CD73 on activated T cells to produce adenosine, resulting in inhibition of T-cell proliferation. Furthermore, MSCs increased the expression of CD73 on CD4(+) T cells, and transforming growth factor-β1 (TGF-β1) was the only tested cytokine that contributed to upregulation of CD73. Hence, our study demonstrates that the CD73/adenosine pathway involves the immunomodulatory function of MSCs in autoimmune responses.

2-Methoxyestradiol Alleviates Experimental Autoimmune Uveitis by Inhibiting Lymphocytes Proliferation and T Cell Differentiation.

Purpose. To investigate the effect of 2-Methoxyestradiol (2ME2) on experimental autoimmune uveitis (EAU) and the mechanism. Method. C57BL/6 male mice were used to establish the EAU model. 2ME2 was abdominal administrated in D0–D13, D0–D6, and D7–D13 and control group was given vehicle from D0–D13. At D14, pathological severity was scored. Lymphocyte reaction was measured using MTT assay. T cell differentiation in draining lymph nodes and eye-infiltrating cells was tested by flow cytometry. Proinflammatory cytokines production from lymphocytes was determined by ELISA. Result. The disease scores from 2ME2 D0–D13, 2ME2 D0–D6, 2ME2 D7–D13, and vehicle groups were 0.20 ± 0.12, 1.42 ± 0.24, 2.25 ± 0.32, and 2.42 ± 0.24. Cells from all 2ME2 treated groups responded weaker than control (p < 0.05). The inhibitory effect of 2ME2 on lymphocyte proliferation attenuated from 2ME2 D0–D13 to 2ME2 D0–D6 and to 2ME2 D7–D13 groups (p < 0.05). 2ME2 treated mice developed fewer Th1 and Th17 cells both in draining lymph nodes and in eyes than control (p < 0.05). Lymphocytes from 2ME2 group secreted less IFN-γ and IL-17A than those from control (p < 0.05). Conclusion. 2ME2 ameliorated EAU progression and presented a better effect at priming phase. The possible mechanism could be the inhibitory impact on IRBP specific lymphocyte proliferation and Th1 and Th17 cell differentiation.

Modulating of ocular inflammation with macrophage migration inhibitory factor is associated with notch signalling in experimental autoimmune uveitis.

The aim of this study was to examine whether macrophage migration inhibitory factor (MIF) could exaggerate inflammatory response in a mouse model of experimental autoimmune uveitis (EAU) and to explore the underlying mechanism. Mutant serotype 8 adeno-associated virus (AAV8) (Y733F)-chicken β-actin (CBA)-MIF or AAV8 (Y733F)-CBA-enhanced green fluorescent protein (eGFP) vector was delivered subretinally into B10.RIII mice, respectively. Three weeks after vector delivery, EAU was induced with a subcutaneous injection of a mixture of interphotoreceptor retinoid binding protein (IRBP) peptide with CFA. The levels of proinflammatory cytokines were detected by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Retinal function was evaluated with electroretinography (ERG). We found that the expression of MIF and its two receptors CD74 and CD44 was increased in the EAU mouse retina. Compared to AAV8.CBA.eGFP-injected and untreated EAU mice, the level of proinflammatory cytokines, the expression of Notch1, Notch4, delta-like ligand 4 (Dll4), Notch receptor intracellular domain (NICD) and hairy enhancer of split-1 (Hes-1) increased, but the ERG a- and b-wave amplitudes decreased in AAV8.CBA.MIF-injected EAU mice. The Notch inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) reduced the expression of NICD, Hes-1 and proinflammatory cytokines. Further, a MIF antagonist ISO-1 attenuated intraocular inflammation, and inhibited the differentiation of T helper type 1 (Th1) and Th17 in EAU mice. We demonstrated that over-expression of MIF exaggerated ocular inflammation, which was associated with the activation of the Notch signalling. The expression of both MIF and its receptors are elevated in EAU mice. Over-expression of MIF exaggerates ocular inflammation, and this exaggerated inflammation is associated with the activation of the Notch signalling and Notch pathway. Our data suggest that the MIF-Notch axis may play an important role in the pathogenesis of EAU. Both the MIF signalling pathways may be promising targets for developing novel therapeutic interventions for uveitis.

The critical points in induction of experimental autoimmune uveitis

Autoimmune uveitis is a leading cause of visual impairment in developed countries in patients of working age. Animal models of experimental autoimmune uveitis (EAU) have been established to serve as a useful template for novel therapeutic approaches. Experimental autoimmune uveitis is induced in C57BL/6 mice by subcutaneous application of interphotoreceptor retinoid binding protein in complete Freund's adjuvant and pertussis toxin. Clinical and histological grading is used to assess the inflammation intensity of EAU. The protocol of induction of EAU in mice hides several important aspects, which are crucial for developing the disease. These details have to be addressed to ensure reproducible disease induction. We describe our experience in establishing the model by pointing out the critical steps in EAU protocol which we found important. The mouse model of EAU has practical value for preclinical studies, is robust and well established. However, the induction of inflammation of the eye can be quite challenging when important details of the protocol are not recognized and adhered to.

Effects of leflunomide on experimental autoimmune uveitis in Lewis rats

Objectives To investigate the protective efficacy of leflunomide on the Lewis rats with experimental autoimmune uveitis (EAU). Methods Complete randomized controlled trials research. Lewis rats were immunized with interphotoreceptor retinoid-binding peptide (IRBP) in order to build the model of EAU. Rats were randomized assigned into four groups, that is control group as A, model group without leflunomide as B, model group with leflunomide administrations as C, and model group with cyclosporine A as D. Rats in group C received intragastric administration of three doses of leflunomide at 3mg/kg/d; 6mg/kg/d; 12mg/kg/d. Rats in group D received 10 mg/kg cyclosporin A were considered as a positive control. Each group has 6 to 8 rats. At the second day of immunization with IRBP, rats were intragastric administrated one time every day till day 13. Rats were investigated for EAU symptoms under slit lamp. Enucleated eyes were collected for sections with HE staining as histopathological evidences at the peak point of disease activity day 14. Treatment effectiveness was evaluated referred by Agarwal standard for clinical EAU and histopathological scoring. The expression of IL-17 in ocular sections was detected by immunohistochemistry (SP method). The expression levels of IL-17 and IFN-γ in the serum were quantified by ELISA. Intracellular expression of IL-17 in the activated CD4 + T cells was assessed by flow cytometry. Ocular of rats were harvested and mRNA expression of IL-17 and IFN-γ were quantified through RT-PCR. Continuous variables were reported as mean ± SD. The comparison among groups was done by using analysis of students't test. Nonparametric test was used in Hierarchical data comparison and multiple comparison method was Bonferroni. Results The model of EAU disease was built successfully in Lewis rats. With giving IRBP for 14 days, the clinic EAU scores were lower in model rats than those without leflunomide. Moreover, the effects of leflunomide on the clinic EAU scores was dose-dependent. Comparing to vehicle-treated eyes, treatment with leflunomide significantly prevented the onset of EAU-induced ocular inflammation [1.5 (1,2) vs. 3 (3,4), P=0.0006, P<α', α'=0.05/15]. The pathological examination showed model rats eye characterized by severe inflammatory cells infiltration and all layers of retina damaged. The pathologic grade was significant higher in model group than in medium dose leflunomide. [3(3, 4) vs. 2(1,3), P=0.0014, P<α', α'= 0.05/15]. IL-17 was positively expressed in iris, ciliary and retina in model group. While, it was markedly reduced in leflunomide-treated eyes. Flow cytometry detection found that compared with normal group, Th17 cells rates in rats' spleen of model group also increased significantly (8.5%±1.3% vs. 0.5%±0.2%; t=8.057, P=0.000, P<α', α'=0.05/15). Compared with model group, Th17 cells in spleen of rats in leflunomide groups showed a decreased number by flow cytometry. And it showed dosage dependent. It was significant different between different doses leflunomide treated group compared with control group. The results showed as below, in low dose group (4.1%±0.6% vs. 8.5%±1.3%; t=6.372, P=0.01, P<α', α'=0.05/15), in medium dose group (2.8%±0.2% vs. 8.5%±1.3%; t=4.49, P=0.002, P<α', α'=0.05/15) and in high dose group (1.8%±0.2% vs. 8.5%±1.3%; t=5.743, P=0.000, P<α', α'=0.05/15). Gene expression of IL-17 and IFN-γ were markedly reduced in leflunomide-treated eyes. Leflunomide significantly decreased the serum levels of IL-17 and IFN-γ. Compared with model group, in leflunomide-treated low dosage group (0.603±0.03 vs. 0.787±0.104; t= 0.183, P=0.002, P<α', α'=0.05/15), medium dosage group (0.535±0.048 vs. 0.787±0.104; t=0.252, P=0.000, P<α', α'=0.05/15) and high dosage group (0.374±0.051 vs. 0.787±0.104; t=0.412, P=0.000, P<α', α'=0.05/15), IL-17 mRNA showed lower expression. Moreover, IFN-γ mRNA in the tissue of EAU eyes were suppressed by medium dosage leflunomide group (0.375±0.018 vs. 0.427±0.056;t=0.69, P=0.001, P<α', α'= 0.05/15) and high leflunomide dosage group respectively (0.367±0.018 vs. 0.427±0.056; t=0.077, P=0.000, P<α', α'=0.05/15) . The difference was statistically significant. All the results suggested that IL-17, which was secreted by Th17 cell, a subtype of T lymphocytes, might play an important role in the pathogenesis of uveitis. Conclusions Oral administration of leflunomide effectively suppressed IRBP-induced uveitis in rats, not only reduced exudation in iris but also alleviated the infiltration damage of inflammation cells in fundus. It might be ascribed to the effect that leflunomide could treat inflammation by down-regulating the expressions of IL-17 and IFN-γ. Therefore, it suggested that leflunomide had protective effects against EAU in Lewis rats. (Chin J Ophthalmol, 2015, 51:754-761) Key words: Uveitis; Autoimmune diseases; Isoxazoles; Th17 cells; Rats

Sarilumab for the treatment of posterior segment non‐infectious uveitis (NIU): The SATURN (SARIL‐NIU) study

PurposeInterleukin‐6 (IL‐6) and/or its soluble receptor are detected in the vitreous and aqueous humors of patients with uveitis. Inhibition of IL‐6 signaling in a murine model of experimental autoimmune uveitis suppresses the development of uveitis. We designed an exploratory Phase 2 study to evaluate the efficacy and safety of sarilumab, a human monoclonal antibody directed against the alpha subunit of the IL‐6 receptor complex in the management of posterior segment NIU.MethodsSATURN is a 52‐week, multicenter, double‐masked, placebo‐controlled, parallel arm, randomized trial to evaluate the efficacy and safety of sarilumab (200 mg) administered subcutaneously every 2 weeks in patients with posterior NIU, who are treated with systemic steroids (as single therapy or with methotrexate). Efficacy and safety are assessed at each visit. The study primary endpoints are reduction from baseline in vitreous haze or systemic‐steroid sparing effects, both measured at week 16. Other key endpoints assessed at week 16 include the change from baseline in: central retinal thickness, best‐corrected visual acuity, and percentage of patients with retinal vessel leakage on fluorescein angiography.ResultsThe study has completed enrollment. As of April 6, 2015, 58 patients have been randomized and treated.ConclusionsThe SATURN study may help clarify the role of IL‐6 in the pathogenesis of NIU and the potential for IL‐6 inhibition in the management of posterior segment NIU.

Dietary Omega-3 Fatty Acids Suppress Experimental Autoimmune Uveitis in Association with Inhibition of Th1 and Th17 Cell Function.

Omega (ω)–3 long-chain polyunsaturated fatty acids (LCPUFAs) inhibit the production of inflammatory mediators and thereby contribute to the regulation of inflammation. Experimental autoimmune uveitis (EAU) is a well-established animal model of autoimmune retinal inflammation. To investigate the potential effects of dietary intake of ω-3 LCPUFAs on uveitis, we examined the anti-inflammatory properties of these molecules in comparison with ω-6 LCPUFAs in a mouse EAU model. C57BL/6 mice were fed a diet containing ω-3 LCPUFAs or ω-6 LCPUFAs for 2 weeks before as well as after the induction of EAU by subcutaneous injection of a fragment of human interphotoreceptor retinoid-binding protein emulsified with complete Freund’s adjuvant. Both clinical and histological scores for uveitis were smaller for mice fed ω-3 LCPUFAs than for those fed ω-6 LCPUFAs. The concentrations of the T helper 1 (Th1) cytokine interferon-γ and the Th17 cytokine interleukin-17 in intraocular fluid as well as the production of these cytokines by lymph node cells were reduced for mice fed ω-3 LCPUFAs. Furthermore, the amounts of mRNAs for the Th1- and Th17-related transcription factors T-bet and RORγt, respectively, were reduced both in the retina and in lymph node cells of mice fed ω-3 LCPUFAs. Our results thus show that a diet enriched in ω-3 LCPUFAs suppressed uveitis in mice in association with inhibition of Th1 and Th17 cell function.

Mouse Models of Experimental Autoimmune Uveitis: Comparative Analysis of Adjuvant-Induced vs Spontaneous Models of Uveitis.

Mouse models of experimental autoimmune uveitis (EAU) mimic unique features of human uveitis, and serve as a template for preclinical study. The "classical" EAU model is induced by active immunization of mice with the retinal protein IRBP in adjuvant, and has proved to be a useful tool to study basic mechanisms and novel therapy in human uveitis. Several spontaneous models of uveitis induced by autoreactive T cells targeting on IRBP have been recently developed in IRBP specific TCR transgenic mice (R161H) and in AIRE(-/-) mice. The "classical" immunization-induced EAU exhibits acute ocular inflammation with two distinct patterns: (i) severe monophasic form with extensive destruction of the retina and rapid loss of visual function, and (ii) lower grade form with an acute onset followed by a prolonged chronic phase of disease. The spontaneous models of uveitis in R161H and AIRE(-/-) mice have a gradual onset and develop chronic ocular inflammation that ultimately leads to retinal degeneration, along with a progressive decline of visual signal. The adjuvant-dependent model and adjuvant-free spontaneous models represent distinct aspects and/or various forms of human uveitis. This review will discuss and compare clinical manifestations, pathology as well as visual function of the retina in the different models of uveitis, as measured by fundus imaging and histology, optical coherence tomography (OCT) and electroretinography (ERG).

The Role of αA-Crystallin in Experimental Autoimmune Uveitis.

Uveitis refers to a group of ocular inflammatory diseases that can lead to blindness. For years, researchers have been trying to decipher the underlying mechanisms and develop therapeutic strategies using the model of experimental autoimmune uveitis (EAU). Recently, αA-crystallin has been found to be upregulated in EAU and can even ameliorate its severity through different mechanisms, suggesting its use as a potent therapeutic factor against uveitis. Here we review the protective role of αA-crystallin and discuss its functional mechanisms in EAU.