Longdan Xiegan Decoction alleviates experimental autoimmune uveitis in rats by inhibiting Notch signaling pathway activation and Th17 cell differentiation.

This study aimed to investigate the dynamic effects of the traditional Chinese medicine compound Longdan Xiegan Decoction (LXD) on the inhibition of Notch signaling pathway activation and T helper (Th) cell differentiation in rats with experimental autoimmune uveitis (EAU). Based on a network pharmacology strategy, we conducted protein interaction network analysis to construct an active ingredient-disease treatment network. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were further used to screen out the possible signaling pathways regulated by LXD in the treatment of uveitis. In the subsequent functional studies, we established an EAU rat model and investigated the regulatory role of LXD in the Notch signaling pathway and Th cell differentiation in rats with EAU. Female Lewis rats were randomly divided into a normal control (NC) group, an EAU group, and an LXD group. After the induction of EAU, the ocular inflammation and pathological changes in the rats in each group were observed; for documentation, a scanning laser ophthalmoscope (SLO) was used to observe fundus inflammation on day 12 after immunization. Additionally, quantitative polymerase chain reaction (Q-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of Notch1, DLL4, IL-10 and IL-17A in the spleen, lymph nodes and ocular tissues of each group at 0, 6, 9, 12, 15 and 18 days after immunization. In addition, the dynamic frequencies of the CD4+, CD8+, Th17 and Treg cell subsets in the spleen, lymph nodes and ocular tissues were measured by flow cytometry. We found that the Notch signaling pathway was activated and the Th17 frequency was elevated in rats with EAU, leading to disrupted CD4+/CD8+ and Th17/Treg balance. The expression of Notch1, DLL4 and IL-17 mRNA and proteins in the EAU and LXD groups reached a peak on day 12, and then gradually decreased (all P < 0.05), and the ratios of the CD4+/CD8+ and Th17/Treg also peaked on day 12. However, after treatment with LXD, the expression of Notch1, DLL4 and IL-17 mRNA and proteins was significantly decreased (all P < 0.05), and the CD4+/CD8+ and Th17/Treg ratios significantly gradually returns to balance. LXD can efficiently inhibit Th17 cell differentiation, decrease inflammatory cytokine expression, and restore the CD4+/CD8+ and Th17/Treg balance by inhibiting the activation of the Notch signaling pathway in rats with EAU, thus effectively alleviating eye inflammation, protecting eye tissue structures, and positively regulating the immune state of the whole body and the intraocular microenvironment.

Dracocephalum heterophyllum (DH) Exhibits Potent Anti-Proliferative Effects on Autoreactive CD4+ T Cells and Ameliorates the Development of Experimental Autoimmune Uveitis.

Experimental autoimmune uveitis (EAU) is a CD4+ T cell-mediated organ-specific autoimmune disease and has been considered as a model of human autoimmune uveitis. Dracocephalum heterophyllum (DH) is a Chinese herbal medicine used in treating hepatitis. DH suppressed the production of inflammatory cytokines through the recruitment of myeloid-derived suppressor cells (MDSCs) to the liver. However, it remains elusive whether DH can directly regulate CD4+ T cell biology and hence ameliorates the development of CD4+ T cell-mediated autoimmune disease. In the current study, we found that DH extract significantly suppressed the production of pro-inflammatory cytokines by CD4+ T cells. Further study showed that DH didn't affect the activation, differentiation, and apoptosis of CD4+ T cells. Instead, it significantly suppressed the proliferation of conventional CD4+ T cells both in vitro and in vivo. Mechanistic study showed that DH-treated CD4+ T cells were partially arrested at the G2/M phase of the cell cycle because of the enhanced inhibitory phosphorylation of Cdc2 (Tyr15). In addition, we demonstrated that treatment with DH significantly ameliorated EAU in mice through suppressing the proliferation of autoreactive antigen specific CD4+ T cells. Taken together, the current study indicates that DH-mediated suppression of CD4+ T cell proliferation may provide a promising therapeutic strategy for treating CD4+ T cell-mediated diseases.

Tetramethylpyrazine attenuates endotoxin-induced retinal inflammation by inhibiting microglial activation via the TLR4/NF-κB signalling pathway.

Ocular inflammation is a common pathological condition of a series of retinal degenerative diseases. Tetramethylpyrazine (TMP), a Chinese herbal extraction, is widely used in the treatment of several ocular diseases in Eastern countries. However, the exact mechanisms correlating the vision protective effects of TMP have not been elucidated. Thus, this study aimed to investigate TMP's molecular targets in anti-inflammatory activity in endotoxin lipopolysaccharide (LPS)-induced retinal inflammation both in vitro and in vivo. The primary cultured retinal microglial cells were pretreated with TMP and then activated by LPS. We found pretreatment with TMP significantly inhibited LPS-induced upregulation of CD68, a marker of mononuclear microglia activation. The morphological changes induced by LPS were also inhibited by the TMP pretreatment. Moreover, Toll like receptor 4 (TLR4), phosphorylation of inhibitor of NF-κB alpha (p-IκB-α) and the translocation of nuclear factor kappa B p65 (NF-κB p65) were significantly downregulated in retinal microglial cells with TMP pretreatment, which indicated that TMP might suppress LPS-induced retinal microglial activation through TLR4/NF-κB signalling pathway. And these results were confirmed in vivo. Pretreatment with TMP inhibited microglial activation, migration and regeneration, especially in ganglion cell layer (GCL). In addition to the inhibition of TLR4, TMP significantly inhibited the translocation of NF-κB p-65 to the nucleus in vivo. The downstream genes of NF-κB, such as the pro-inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß), were significantly downregulated by TMP pretreatment in the retina. Accordingly, the increased expression of cleaved caspase-3 and the decreased ratio of B-cell lymphoma-2 (Bcl-2) to Bcl-2 associated X Protein (Bax) were significantly attenuated by TMP. TUNEL assay also demonstrated that TMP exerted neuroprotective effects in the retina. Therefore, this study elucidated a novel mechanism that TMP inhibits retinal inflammation by inhibiting microglial activation via a TLR4/NF-κB signalling pathway.

Management of Uveitis in Spondyloarthropathy: Current Trends.

Spondyloarthritis is a chronic inflammatory disease predominantly affecting joints of the axial skeleton. However, as many as 50% of patients with this disease may have extra-articular manifestations, which include uveitis; psoriasis; inflammatory bowel disease such as Crohn disease or ulcerative colitis; cardiovascular manifestations in the form of conduction abnormalities, atherosclerosis, or valvular heart disease; pulmonary involvement; and rarely renal involvement. Uveitis occurs in 25% to 40% of patients with spondyloarthritis. Management of uveitis is crucial to prevent morbidity caused by vision loss and secondary complications. Treatment ranges from local therapies to systemic drugs and varies depending on the severity and response to treatment. Categories of medical treatment include nonsteroidal anti-inflammatory agents, corticosteroids, and steroid-sparing agents. Biologic therapies such as antitumor necrosis factor agents act early in the disease process and have revolutionized the field of rheumatology, including management of uveitis. This review will focus on the management of ophthalmic manifestations in spondyloarthropathies.

Silibinin treatment prevents endotoxin-induced uveitis in rats in vivo and in vitro.

Uveitis, an intraocular inflammatory disease, occurs mostly in young people and can result in the loss of socioeconomic capabilities. Silibinin has been shown to exert anti-inflammatory effects in human retinal pigment epithelial (RPE) cells. The present study investigated the anti-inflammatory effect of silibinin pretreatment on endotoxin-induced uveitis (EIU) in rats and the mechanisms by which it exerts these effects. Uveitis was induced via injection of lipopolysaccharides (LPS) into Lewis rats. Twenty-four hours after the LPS injection, histological examination showed that silibinin decreased inflammatory cell infiltration in the anterior segment of the eyes of LPS-treated rats. Analyses of the aqueous humor showed that silibinin decreased cell infiltration, protein concentration, nitric oxide (NO), and prostaglandin (PG)-E2 production. Western blot analysis indicated that silibinin decreased the expression of inducible NO synthase (iNOS), cyclooxygenase (COX-2), and phosphorylated IkB in the iris-ciliary body (ICB). Immunohistochemistry showed that silibinin decreased intercellular adhesion molecule (ICAM-1) expression in the ICB. In addition, western blot analysis showed that silibinin attenuated the expression of iNOS, COX-2, ICAM-1, and nuclear p65 in LPS-treated RAW cells. In conclusion, silibinin pretreatment prevents EIU and the subsequent production of proinflammatory mediators and ICAM-1, at least in part, by blocking the NF-κB-dependent signaling pathway both in vivo and in vitro. These effects may contribute to the silibinin-mediated preventive effects on intraocular inflammatory diseases such as acute uveitis.

[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.

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.

Topically applied standardized aqueous extract of Curcuma longa Linn. suppresses endotoxin-induced uveal inflammation in rats.

Aqueous extract of C. longa when administered 4 h after induction of E. coli lipopolysaccharide-induced uveitis in rats showed significantly suppressed inflammation with a significantly lower mean clinical grade, histopathological grade and aqueous humor (AH) protein level compared to vehicle treated group. Although, prednisolone group showed significantly lower clinical grade, histopathological grades and AH protein levels compared to C. longa group, TNF-alpha levels did not differ significantly. Moreover, when the aqueous extract was administered starting from 3 days before induction of uveitis, the mean clinical and histopathological grade as well as AH protein and TNF-alpha levels were comparable to C. longa group when treatment was administered 4 h after induction of uveitis. It is concluded that topically applied standardized aqueous extract of C. longa suppresses endotoxin-induced uveitis in rats by reducing TNF-alpha activity.

Effects of Japanese herbal medicine Sairei-to on murine experimental autoimmune uveitis.

PURPOSE: It has been suggested thatSairei-to (TJ114), a traditional Japanese herbal medicine, has immunomodulatory activities. To evaluate the effects of TJ114 on uveitis, we examined the effectiveness of oral administration in a murine model of experimental autoimmune uveitis (EAU). METHODS: Murine EAU was induced by subcutaneous injection of human inter-photoreceptor retinoid-binding protein (IRBP) peptide mixed with complete Freund's adjuvant. In the TJ114-treated group, 2 g/kg was administrated orally from 0 to 20 days after immunization. Clinical scoring, histopathological scoring of EAU, cell proliferation, cytokine assessment, and adoptive transfer experiment of splenic T cells into naïve mice were performed. RESULTS: EAU development occurred in 32 of 38 mice (86 %) in the untreated group and 12 of 33 (36 %) in the TJ114-treated group. The clinical scores for EAU in the vehicle-treated and TJ114-treated groups were 1.56 ± 1.65 and 0.59 ± 0.63 respectively, at 14 days after immunization (p < 0.01, Mann-Whitney U-test), and 2.26 ± 1.56 and 0.75 ± 1.31 respectively at 21 days (p < 0.001, Mann-Whitney U-test), while the histopathological scores at 21 days were 1.47 ± 1.42 and 0.54 ± 0.84 respectively (p < 0.01, Mann-Whitney U-test). Interferon (IFN)-γ and tumor necrosis factor (TNF)-α production by cervical lymph node cells obtained from the TJ114-treated group were significantly reduced as compared with those from the vehicle-treated group (p < 0.01, Student's unpaired t-test). Moreover, the levels of C-C motif chemokine 2 (CCL2) and IFN-γ were significantly reduced in splenocytes of TJ114-treated mice as compared with the vehicle-treated group (p < 0.01, Student's unpaired t-test). Mice that received adoptive transfer of splenic T cells from TJ114-treated EAU mice caused significantly lower severity of EAU compared to those that received from vehicle-treated EAU mice. CONCLUSION: Oral administration of TJ114 has an inhibitory effect on a murine model of EAU, possibly via reduction in cytokine production by helper type-1 T cells.

Mimicry epitope from Ehrlichia canis for interphotoreceptor retinoid-binding protein 201-216 prevents autoimmune uveoretinitis by acting as altered peptide ligand.

We report here identification of novel mimicry epitopes for interphotoreceptor retinoid-binding protein (IRBP) 201-216, a candidate ocular antigen that causes experimental autoimmune uveoretinitis (EAU) in A/J mice. One mimicry epitope from Ehrlichia canis (EHC), designated EHC 44-59, induced cross-reactive T cells for IRBP 201-216 capable of producing T helper (Th)1 and Th17 cytokines, but failed to induce EAU in A/J mice. In addition, animals first primed with suboptimal doses of IRBP 201-216 and subsequently immunized with EHC 44-59 did not develop EAU; rather, the mimicry epitope prevented the disease induced by IRBP 201-216. However, alteration in the composition of EHC 44-59 by substituting alanine with valine at position 49, similar to the composition of IRBP 201-216, enabled the mimicry epitope to acquire uveitogenicity. The data provide new insights as to how microbes containing mimicry sequences for retinal antigens can prevent ocular inflammation by acting as naturally occurring altered peptide ligands.

[Study on inhibitory effect of lycium pigment on lipopolysaccharide-induced uveitis in rats and its mechanism].

OBJECTIVE: To investigate the inhibitory effect of lycium pigment on lipopolysaccharide (LPS)-induced uveitis in rats and its mechanism. METHOD: The rat uveitis model was established by 30-day oral administration of lycium pigment (50, 100, 200 mg x kg(-1)) and footpad injection of LPS. Ocular tissues were collected for a histopathological inspection. The protein, nitric oxide and ADMA in aqueous humor, level of inducible nitric oxide synthase (iNOS) in retina, activities of serum total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), and content of malondialdehyde (MDA) were determined by using Western blot, ELISA and biochemical methods. RESULT: According to the pathological study, lycium pigment (50, 100, 200 mg x kg(-1)) could notably reduce the inflammatory cell infiltration around corpus ciliare matrix of uveitis rats, and the concentration of protein and nitric oxide, and increased ADMA in aqueous humor. Lycium pigment (100, 200 mg x kg(-1)) could significantly inhibit the expression of iNOS in ocular tissues. In addition, lycium pigment (100, 200 mg x kg(-1)) also decrease the activities of serum T-AOC, SOD, GSH-PX, and the content of lipid peroxide MDA. CONCLUSION: Lycium pigment has the inhibitory effect on LPS-induced uveitis in rats. Its mechanism is related to the regulation of nitric oxide/ADMA pathway and the improvement of oxidation resistance.

Prevention of endotoxin-induced uveitis in rats by plant sterol guggulsterone.

PURPOSE: To investigate the anti-inflammatory effects of guggulsterone, an antioxidant and antitumor agent, in endotoxin-induced uveitis (EIU) in rats and to elucidate the underlying molecular mechanism or mechanisms related to ocular inflammation. METHODS: EIU was induced by subcutaneous injection of lipopolysaccharide (LPS; 150 µg) into Lewis rats treated with guggulsterone (30 mg/kg body weight, intraperitoneally) or its carrier. After 24 hours the rats were killed, eyes were enucleated, and aqueous humor (AqH) was collected. Numbers of infiltrating cells and levels of matrix metalloproteinase-2 (MMP-2), nitric oxide (NO), and prostaglandin E(2) (PGE(2)) were determined in AqH by specific ELISAs. An antibody array was used to measure the expression of various inflammatory cytokines in AqH. The expression of MMP-2, iNOS, Cox-2, phospho-IκB, and phospho-NF-κB was determined immunohistochemically. Human primary nonpigment ciliary epithelial cells (HNPECs) were used to determine the in vitro efficacy of guggulsterone on the LPS-induced inflammatory response. RESULTS: Compared with control, the EIU rat eye AqH had a significantly higher number of infiltrating cells, total protein, and inflammatory markers, such as MMP-2, NO, and PGE(2), and the treatment of guggulsterone prevented EIU-induced increases. Guggulsterone also prevented the expression of MMP-2, iNOS, and Cox-2 proteins and of IκB and NF-κB in various eye tissues. Moreover, in cultured HNPECs, guggulsterone inhibited LPS-induced expression of inflammatory proteins. CONCLUSIONS: These results for the first time demonstrate that the plant sterol guggulsterone suppresses ocular inflammation in EIU, suggesting that the supplementation of guggulsterone could be a novel approach for the treatment of ocular inflammation.

Protective effects of bilberry ( Vaccinium myrtillus L.) extract against endotoxin-induced uveitis in mice.

Endotoxin-induced uveitis (EIU), a useful animal model of ocular inflammation, is induced by injection of lipopolysacharide (LPS). These experiments showed that the nitric oxide (NO) level significantly increased in the whole eye homogenate of BALB/C mice 24 h after footpad injection of LPS at a dosage of 100 mg/mouse. However, the elevated NO level was significantly reduced by oral administration of bilberry extract (containing 42.04% anthocyanins) at dosages of 50, 100, and 200 mg/kg/day for 5 days before the LPS injection. In addition, bilberry extract decreased malondialdehyde (MDA) level and increased oxygen radical absorbance capacity (ORAC) level, glutathione (GSH) level, vitamin C level, and total superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities. Moreover, bilberry extract increased expression of copper/zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), and GPx mRNA. Taken together, bilberry extract showed protective effects against EIU, whereas the effects of bilberry extract (100 and 200 mg/kg/day, 5 days) were dose-dependent. In conclusion, these results provide new evidence to elucidate the beneficial effects of bilberry extract on eye health.

Contribution of CD4+CD25+ T cells to the regression phase of experimental autoimmune uveoretinitis.

PURPOSE: To investigate the role of CD4(+)CD25(+) Treg cells in the development of experimental autoimmune uveoretinitis (EAU). METHODS: EAU was induced in B10RIII mice by immunization with IRBP(161-180) in complete Freund's adjuvant and evaluated clinically and pathologically on days 0, 7, 14, 21, and 28. Lymphocytes from draining lymph nodes (LNs) were subjected to flow cytometry to analyze the frequency of CD4(+)CD25(+) Treg cells. CD4(+)CD25(+) Treg cells and CD4(+)CD25(-) T cells were separated by means of magnetic-assisted cell sorting and cocultured or crossover cultured for 3 days. Proliferation of CD4(+)CD25(-) T cells was measured using a modified MTT assay. The levels of IFN-gamma and IL-17 in the supernatants were determined by enzyme-linked immunosorbent assay. RESULTS: Clinical and histopathologic results showed a severe intraocular inflammation in the immunized mice. The frequency of CD4(+)Foxp3(+) T cells and CD4(+)CD25(+)Foxp3(+) T cells in the draining LN lymphocytes was increased on day 7, reached its peak on day 14, and maintained a high level up to day 42. CD4(+)CD25(+) Treg cells obtained from mice on days 14 and 28 after immunization showed a stronger inhibitory effect on the proliferation of CD4(+)CD25(-) T cells and the production of IFN-gamma by CD4(+)CD25(-) T cells compared with those obtained from control mice. CD4(+)CD25(+) Treg cells did not affect IL-17 production. Transfer of CD4(+)CD25(+) Treg cells obtained from EAU mice was able to suppress EAU induction by IRBP(161-180) that was not observed after transfer of cells from mice that had received CFA alone, suggesting antigen specificity of the Treg response. CONCLUSIONS: A significantly increased frequency and immunoregulatory action of CD4(+)CD25(+) Treg cells is associated with the development and regression of EAU, suggesting that CD4(+)CD25(+) Treg cells are induced during EAU and may be involved in its regression.

Prevention of endotoxin-induced uveitis in rats by benfotiamine, a lipophilic analogue of vitamin B1.

PURPOSE: To study the amelioration of ocular inflammation in endotoxin-induced uveitis (EIU) in rats by benfotiamine, a lipid-soluble analogue of thiamine. METHODS: EIU in Lewis rats was induced by subcutaneous injection of lipopolysaccharide (LPS) followed by treatment with benfotiamine. The rats were killed 3 or 24 hours after LPS injection, eyes were enucleated, aqueous humor (AqH) was collected, and the number of infiltrating cells, protein concentration, and inflammatory marker levels were determined. Immunohistochemical analysis of eye sections was performed to determine the expression of inducible-nitric oxide synthase (iNOS), cyclooxygenase (Cox)-2, protein kinase C (PKC), and transcription factor NF-kappaB. RESULTS: Infiltrating leukocytes, protein concentrations, and inflammatory cytokines and chemokines were significantly elevated in the AqH of EIU rats compared with control rats, and benfotiamine treatment suppressed these increases. Similarly increased expression of inflammatory markers iNOS and Cox-2 in ciliary body and retinal wall was also significantly inhibited by benfotiamine. The increased phosphorylation of PKC and the activation of NF-kappaB in the ciliary body and in the retinal wall of EIU rat eyes were suppressed by benfotiamine. CONCLUSIONS: These results suggest that benfotiamine suppresses oxidative stress-induced NF-kappaB-dependent inflammatory signaling leading to uveitis. Therefore, benfotiamine could be used as a novel therapeutic agent for the treatment of ocular inflammation, especially uveitis.

Down-regulation of inducible co-stimulator (ICOS) by intravitreal injection of small interfering RNA (siRNA) plasmid suppresses ongoing experimental autoimmune uveoretinitis in rats.

BACKGROUND: RNA interference (RNAi) is now being exploited as a powerful tool for gene knockdown. Recently, we had shown that inducible co-stimulator (ICOS) was up-regulated in experimental autoimmune uveoretinitis (EAU). The aim of this study was to investigate whether intravitreal injection of small interfering RNA (siRNA) plasmid, targeting ICOS, suppresses the ongoing experimental autoimmune uveoretinitis (EAU) in rats. METHODS: Oligonucleotide targeting ICOS was cloned into linearized pRNAT-U6.1/Neo eukaryotic expression vector to construct the recombinant plasmid (pRNAT-U6.1/Neo-ICOS). After transfecting activated rat T cells with the recombinant plasmid, ICOS mRNA and protein expression levels were determined by real-time RT-PCR and Western blot analysis respectively. Rats were immunized with IRBP R16 peptide emulsified in complete Freund's adjuvant (CFA) and given an intravitreal injection of pRNAT-U6.1/Neo-ICOS on day 6 after immunization. After 13days of immunization, the ICOS protein expression and CD4(+) ICOS (+) T cells were identified in retinae through Western blot analysis and flow cytometry respectively. Intraocular inflammation was assessed by the scores of the clinical and histological appearances. Delayed-type hypersensitivity (DTH) and lymphocyte proliferation were detected to evaluate the systemic effect of intravitreal injection of pRNAT-U6.1/Neo-ICOS. RESULT: The recombinant plasmid (pRNAT-U6.1/Neo-ICOS) for the ICOS siRNA was successfully constructed. In vitro studies using the recombinant plasmid has showed the down-regulation of ICOS gene expression both at the mRNA and protein levels. Clinical and pathological scores showed that ocular inflammation of pRNAT-U6.1/Neo-ICOS-treated eyes was markedly less than that of vehicle-treated eyes. The expression of ICOS protein and the amount of CD4(+) ICOS(+) T cells in retinae significantly decreased by intravitreal injection of the recombinant plasmid, whereas delayed-type hypersensitivity response and lymphocyte proliferation were not impaired in rats treated with the recombinant plasmid. CONCLUSION: Intravitreal injection of siRNA plasmid targeting ICOS effectively down-regulated the expression of ICOS, and was highly effective in suppressing the ongoing process of EAU without any side-effects on systemic cellular immunity.

LX211 (voclosporin) suppresses experimental uveitis and inhibits human T cells.

PURPOSE: To test the therapeutic effectiveness of voclosporin against experimental autoimmune uveoretinitis (EAU) in rats and to evaluate its effect on human T cells. METHODS: EAU was induced by immunization with a uveitogenic protein. Voclosporin administration, by subcutaneous injection, began on day (d) 0 or d7 after immunization. Treatment effectiveness was evaluated in vivo using clinical EAU scoring (d7-d13) and histopathologic evaluation of enucleated eyes after experimental termination. Rodent lymphocytes were harvested from lymph nodes on d14 for antigen-specific proliferation assays. The effect of voclosporin on human T-cell proliferation and cytokine secretion was examined in vitro. RESULTS: Voclosporin prevented EAU development in rats receiving medium and high preventive doses, whereas high-dose voclosporin administration effectively treated EAU. Lymphocytes from animals treated with voclosporin had decreased antigen-specific proliferation in vitro compared with lymphocytes from untreated animals. No evidence of abnormal ocular histopathology was found in the eyes from animals that received high doses of therapeutic voclosporin. Using human T cells, voclosporin inhibited human T-cell proliferation up to 100-fold. Furthermore, voclosporin treatment of human T cells significantly reduced pan T-cell effector responses. CONCLUSIONS: Voclosporin effectively suppressed uveoretinitis in an animal model that imitates the human inflammatory ocular disease by inhibiting lymphocyte proliferation. In addition, voclosporin effectively inhibited human T-cell proliferation and function in vitro. The authors report the first evidence supporting the application of voclosporin to treat intraocular inflammation.

Either a Th17 or a Th1 effector response can drive autoimmunity: conditions of disease induction affect dominant effector category.

Experimental autoimmune uveitis (EAU) represents autoimmune uveitis in humans. We examined the role of the interleukin (IL)-23-IL-17 and IL-12-T helper cell (Th)1 pathways in the pathogenesis of EAU. IL-23 but not IL-12 was necessary to elicit disease by immunization with the retinal antigen (Ag) interphotoreceptor retinoid-binding protein (IRBP) in complete Freund's adjuvant. IL-17 played a dominant role in this model; its neutralization prevented or reversed disease, and Th17 effector cells induced EAU in the absence of interferon (IFN)-gamma. In a transfer model, however, a polarized Th1 line could induce severe EAU independently of host IL-17. Furthermore, induction of EAU with IRBP-pulsed mature dendritic cells required generation of an IFN-gamma-producing effector response, and an IL-17 response by itself was insufficient to elicit pathology. Finally, genetic deficiency of IL-17 did not abrogate EAU susceptibility. Thus, autoimmune pathology can develop in the context of either a Th17 or a Th1 effector response depending on the model. The data suggest that the dominant effector phenotype may be determined at least in part by conditions present during initial exposure to Ag, including the quality/quantity of Toll-like receptor stimulation and/or type of Ag-presenting cells. These data also raise the possibility that the nonredundant requirement for IL-23 in EAU may extend beyond its role in promoting the Th17 effector response and help provide a balance in the current Th1 versus Th17 paradigm.

Labrafil--a new adjuvant for peptide-specific oral tolerance in rat experimental autoimmune uveitis.

Application of soluble antigen via the oral route results in systemic antigen-specific tolerance, a therapeutic approach that has already been used for uveitis patients. In the Lewis rat experimental autoimmune uveitis (EAU) can be induced by active immunisation with retinal antigens such as retinal soluble antigen (S-Ag) or interphotoreceptor retinoid-binding protein (IRBP) and peptides thereof. These normally pathogenic antigens can also be used to induce oral tolerance. In order to optimize oral tolerance induction we analysed the effect of Labrafil M 2125 CS, an orally administrable composition for pharmaceutical use, consisting of fatty acid esters and glycerides and capable of forming micro emulsions. Feeding peptide emulsified in Labrafil M 2125 CS/PBS prior to immunisation significantly improved oral tolerance compared to feeding peptide in PBS only. We observed a delayed onset of disease, reduced intraocular inflammation and less retinal destruction. Application of Labrafil M 2125 CS without tolerogen had no effect. Combined feeding of peptide with Labrafil M 2125 CS even allowed 10-fold reduction of the tolerogenic peptide dose. Furthermore, the effect of Labrafil M 2125 CS upon oral tolerance was dose-dependent, a peptide emulsion containing 0.5-2% Labrafil M 2125 CS achieved a maximal enhancement of oral tolerance induction, suggesting that Labrafil M 2125 CS might be a useful adjuvant to enhance therapeutic use of oral tolerance.

Role of T-cell receptor V beta 8.3 peptide vaccine in the prevention of experimental autoimmune uveoretinitis.

BACKGROUND: T-cell receptor (TCR) plays an important role in the development of autoimmune diseases. Recently, it was reported that immunization of animals with TCR peptide derived from the pathogenic cells could prevent autoimmune diseases. The aim of this study was to investigate whether vaccination with a synthetic peptide from the hypervariable region of TCR V(beta) 8.3, an experimental autoimmune uveoretinitis (EAU)-associated gene, was able to prevent the disease. METHODS: EAU was induced in Lewis rats by immunization with IRBP R16 peptide emulsified in complete Freund's adjuvant (CFA). The clinical and histological appearances were scored. Delayed type hypersensitivity (DTH) and lymphocyte proliferation were detected. Cytokine levels of aqueous humour, supernatants of cells from spleen and draining lymph nodes were measured by enzyme linked immunosorbent assay (ELISA). Gene expression of TCR V(beta) 8.3 on CD(4)(+) T cells was examined by real time quantitative polymerase chain reaction (PCR). RESULTS: After vaccination, the intraocular inflammation was significantly mitigated, antigen specific DTH and lymphocyte proliferation responses were suppressed, interleukin (IL)-2 in aqueous humour, interferon (IFN)-gamma and IL-2 produced by the spleen and draining lymph node cells were significantly decreased, whereas the production of IL-4 and IL-10 were increased. The response of draining lymph node cells to TCR V(beta) 8.3 peptide was enhanced after vaccination. Inoculation with CFA alone did not affect the severity of EAU and the above parameters. The suppression of EAU was much stronger in the group of four fold inoculations than the group of two fold inoculations. The expression of TCR V(beta) 8.3 gene was significantly reduced in the group of fourfold inoculations. CONCLUSION: Vaccination with the synthetic TCR V(beta) 8.3 peptide could remarkably inhibit the development of EAU.