Peng Qinghua's Experience in the Staged Treatment of Autoimmune Uveitis / 中医杂志

This paper summarized clinical experience of Professor Peng Qinghua in the staged treatment of autoimmune uveitis （AU）. The pathogenesis of AU is always based on root-cause deficiency and manifestation excess， with weakness of healthy qi as the root， and accumulation of wind， dampness and blood stasis as the minifestation. Syndrome differentiation and treatment is usually carried out according to different stages of disease. During the acute stage， deficiency of healthy qi and stagnation of wind-dampness are the key pathomechanisms， so the treatment is to replenish qi and activate blood， dispel wind and remove dampness， and self-made Yiqi Huoxue Qufeng Chushi Decoction （益气活血祛风除湿方） can be used. During remission stage， deficiency of spleen and kidney is the key mechanism， so the treatment is to tonify the spleen and kidney， replenish the essence and brighten the eyes， and self-made Yijing Mingmu Decoction （益精明目汤） can be used. Meanwhile， it was recommened to treat early， prevent and interrupt the disease， commonly combine with Chinese patent medicine Zhengqing Fengtongning tablets （正清风痛宁缓释片）， and promote regulation of living to prevent recurrence.

Aging weakens Th17 cell pathogenicity and ameliorates experimental autoimmune uveitis in mice

Aging-induced changes in the immune system are associated with a higher incidence of infection and vaccination failure. Lymph nodes, which filter the lymph to identify and fight infections, play a central role in this process. However, careful characterization of the impact of aging on lymph nodes and associated autoimmune diseases is lacking. We combined single-cell RNA sequencing (scRNA-seq) with flow cytometry to delineate the immune cell atlas of cervical draining lymph nodes (CDLNs) of both young and old mice with or without experimental autoimmune uveitis (EAU). We found extensive and complicated changes in the cellular constituents of CDLNs during aging. When confronted with autoimmune challenges, old mice developed milder EAU compared to young mice. Within this EAU process, we highlighted that the pathogenicity of T helper 17 cells (Th17) was dampened, as shown by reduced GM-CSF secretion in old mice. The mitigated secretion of GM-CSF contributed to alleviation of IL-23 secretion by antigen-presenting cells (APCs) and may, in turn, weaken APCs' effects on facilitating the pathogenicity of Th17 cells. Meanwhile, our study further unveiled that aging downregulated GM-CSF secretion through reducing both the transcript and protein levels of IL-23R in Th17 cells from CDLNs. Overall, aging altered immune cell responses, especially through toning down Th17 cells, counteracting EAU challenge in old mice.

Effect and Mechanism of Quercetin on Experimental Autoimmune Uveitis in Rats Based on Wnt/β-catenin Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effects and mechanism of quercetin on experimental autoimmune uveitis （EAU） by regulating the Wnt/β-catenin signaling pathway. MethodThe regulatory relationship between quercetin and EAU was preliminarily determined by network pharmacology. The model was identified by enzyme-linked immunosorbent assay（ELISA） and clinical grading score. The important inflammatory factors， including interleukin（IL）-6 and tumor necrosis factor（TNF）-α in the signaling pathway were detected by ELISA. The binding of important targets in the pathway was verified by molecular docking. The protein and mRNA expression levels of related targets in the signaling pathway were detected by Western blot and Real-time polymerase chain reaction（Real-time PCR）， respectively. ResultAs evaluated by ELISA and the clinical grading score， the model was properly induced. The results of ELISA showed that the levels of IL-6 and TNF-α were the highest in the model group and the dimethyl sulfoxide （DMSO） group， and decreased after medium- and high-dose drug interventions， but the changing tend was the same. Molecular docking showed that the binding energies of quercetin to β-catenin and LRP6 were ideal. Western blot revealed that the expression of β-catenin and LRP6 was the highest in the model group， and decreased with drug intervention at different doses， but the changing trend was the same. Besides， there was no significant difference between the low-dose group and the high-dose group. Real-time PCR results showed that the expression of β-catenin，MYC，AXIN2， and TCF was the highest in the model group，the lowest in the high-dose group， and decreased in the low-dose group. There was no significant difference between the high-dose group and the low-dose group. ConclusionQuercetin can reduce the inflammatory expression of EAU and relieve the pain of patients by regulating the Wnt/β-catenin signaling pathway.

Effect of interleukin-23R overexpression on Th17/Treg balance in experimental autoimmune uveitis mice / 中华眼底病杂志

Objective:To investigate the effect of interleukin (IL)-23 receptor (IL-23R) overexpression on the balance of T helper 17 (Th17 cells)/regulatory T cells (Treg cells) in experimental autoimmune uveitis (EAU) mice.Methods:Twelve 8-week-old female C57BL/6J mice were randomly divided into LV-Ctrl group and LV-IL-23R group, with 6 mice in each group. Two groups of mice were injected with LV-Ctrl and LV-IL-23R lentiviruses through the tail vein, respectively; 7 days after injection, the EAU mouse model was established by active immunization with vitamin A-binding protein 1-20 between photoreceptors. Starting from 13 days after immunization, the fundus of the mice was observed by indirect ophthalmoscopy every 2 days and clinical scores were performed; 30 days after immunization, hematoxylin-eosin staining was used to observe the histopathological changes of mouse retina. The levels of IL-17 in serum of the two groups of mice were detected by enzyme-linked immunosorbent assay; the proportion of Th17 cells and Treg cells was detected by flow cytometry. The relative mRNA expression of IL-23R, IL-17, retinoic acid-related orphan receptor γt (RORγt), IL-10 and forkhead transcripyion factor p3 (Foxp3) were detected by real-time quantitative polymerase chain reaction. Comparisons between groups were performed using repeated measures analysis of variance, independent samples Mann-Whitney U test, and independent samples t test. Results:Compared with the LV-Ctrl group, the retinal inflammatory reaction of the LV-IL-23R group was more severe. At 13 days after immunization, there was no significant difference in fundus inflammation scores between LV-IL-23R group and LV-Ctrl group ( t=-2.001, P=0.058); 15-29 days after immunization. The fundus inflammation scores of LV-IL-23R group were higher than those of LV-Ctrl group, and the difference was statistically significant ( t=-4.429,-6.578, -7.768, -10.183, -6.325, -7.304, -4.841, -6.872; P<0.001). Histopathological examination showed that the infiltration of inflammatory cells in the fundus increased, the retinal structure was damaged more seriously, and the histopathological score was significantly increased, and the difference was statistically significant ( t=-4.339, P=0.001). Compared with the LV-Ctrl group, the relative expression of IL-23R mRNA in the spleen of the LV-IL-23R group was significantly increased, and the difference was statistically significant ( Z=2.087, P=0.037). The relative expression of IL-17 and RORγt mRNA increased, while the relative expression of IL-10 and Foxp3 mRNA decreased, and the differences were statistically significant ( t=-6.313,-5.922, 4.844, 7.572; P=0.003, 0.004, 0.008, 0.002). Compared with the LV-Ctrl group, the level of IL-17 in the serum of the mice in the LV-IL-23R group was significantly increased, and the difference was statistically significant ( t=-5.423, P=0.002); the proportion of Th17 cells in the spleen and lymph nodes was significantly increased, whereas, the proportion of Treg cells was significantly reduced, and the difference was statistically significant ( t=-4.290, 3.700; P=0.002, 0.006). Conclusion:IL-23R overexpression can promote Th17/Treg imbalance in EAU mice, and aggravate the clinical and pathological manifestations of EAU.

Study on the effect of miR-223-3p regulating transcription factor Rbpj on the differentiation of Th1 and Th17 cells in rats with experimental autoimmune uveitis / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the regulatory effect of miR-223-3p on the expression of transcription factor Rbpj and on the differentiation of Th1 and Th17 cells in experimental autoimmune uveitis(EAU)rats.<p>METHODS: The regulatory role of miR-223-3p in Rbpj gene expression was investigated by a dual luciferase expression reporter system. In the present study, 24 female Lewis rats were randomly divided into EAU model group, normal control(NC)group and blank control(BC)group, and each group included 8 rats. The EAU model group was injected with interphotoreceptor retinoid-binding protein(IRBP)emulsion containing Mycobacterium tuberculin H37RA and complete Freund's adjuvant to induce uveitis, while the NC group was injected with an equal volume of emulsion without IRBP peptide. The rats in the BC group received the same volume of sterile saline solution. At 12d after immunization, the spleen, lymph node and eye tissues in both groups were aseptically isolated, and the expression levels of miR-223-3p and Rbpj RNAs were detected by real-time quantitative PCR(Q-PCR); Meanwhile, the expression levels of Rbpj, IFN-γ and IL-17 proteins were detected by ELISA, and the levels of Th1 and Th17 cell lineages in each tissue from each groups were detected by flow cytometry. <p>RESULTS: The results of dual fluorescein assay indicated that Rbpj was the target gene which regulated by miR-223-3p. At 12d after immunization, compared with NC group, the relative expression levels of miR-223-3p in spleen, lymph node and eye tissues from EAU model rats were 0.33±0.29, 0.11±0.12 and 0.18±0.11, respectively, accompanied by the down-regulated expression, and the differences were statistically significant(all <i>P</i><0.05); Rbpj mRNA levels were 3.00±0.06, 1.52±0.12 and 3.01±0.34, respectively, and were all up-regulated, while the differences were statistically significant(all <i>P</i><0.05). Moreover, the differences in miR-223-3p and Rbpj mRNA levels in spleen, lymph node and eye tissues of rats in the blank control group were not statistically significant compared with those in the NC group(<i>P</i>>0.05); ELISA results revealed that the expression levels of RBPJ, IFN-γ and IL-17 proteins in all tissues from EAU rats at 12d after immunization were significantly higher than those in the NC group( all <i>P</i><0.05), and there was no statistically significant difference in the expression levels of Rbpj, IFN-γ and IL-17 protein in all tissues of rats in the blank control group compared with the NC group(<i>P</i>>0.05); Meanwhile, flow cytometry results showed that the proportions of Th1 and Th17 cell lineages in all tissues from EAU model group were significantly higher than those from the NC group at 12d after immunization, and the differences were statistically significant(all <i>P</i><0.05). Furthermore,there was no significant change in the proportion of Th1 and Th17 cells in each tissue in the BC and NC groups(all <i>P</i> >0.05). <p>CONCLUSION: The miR-223-3p can negatively regulate the expression of the transcription factor Rbpj of Notch signaling pathway. The down-regulated miR-223-3p expression in EAU rats can increase the expression levels of Rbpj gene and protein, and aggravate the differentiation of Th1 and Th17 cells and the expression levels of related molecules IFN-γ and IL-17, which in turn affect the development of uveitis.

Recent advances of exosomes in immune-mediated eye diseases / 国际眼科杂志(Guoji Yanke Zazhi)

@#Immune-mediated eye diseases(IMED)represent a kind of autoimmune eye diseases, such as thyroid-associated ophthalmopathy, autoimmune uveitis and Sjögren syndromes. Owning to its complicated pathophysiological mechanisms, the efficacies of current therapeutic strategies for IMED are unsatisfactory, which may ultimately result in severe visual impairment. Exosomes are lipid bilayer vesicles secreted by cells that play an important role in cell-to-cell communication and immune regulation. Recent studies have shown that exosomes secreted by cells under pathological conditions are closely associated with the development and progression of IMED, and the exosomes derived from certain cells(such as mesenchymal stem cells)are deemed as promising therapy for IMED. Hence, we review the recent advances of exosomes in IMED.

Effects of intravenous injection of human umbilical cord mesenchymal stem cells-derived small extracellular vesicles on experimental autoimmune uveitis in mice / 中华实验眼科杂志

Objective:To explore the therapeutic effects of intravenous injection of small extracellular vesicles (sEVs) derived from human umbilical cord mesenchymal stem cells (MSCs) on experimental autoimmune uveitis (EAU) in mice.Methods:MSCs from human umbilical cord were cultured and the supernatant was collected.The sEVs were isolated by ultracentrifugation method and a NanoSight instrument was used to analyze the particle size.The expression of surface markers sEVs, CD9, CD81 and CD63 was determined via Western blot.The morphology of sEVs was observed with a transmission electron microscope.Forty-eight 7-week-old female C57BL/6 mice were seclected to establish the EAU model through immunization with interphotoreceptor retinoid-binding protein peptide 651-670 (IRBP 651-670). The mice were divided into sEVs treatment group and phosphate buffer solution (PBS) control group using a random number table, with 24 mice in each group.The mice in the sEVs treatment group were injected with 50 μg of MSCs-derived sEVs via tail vein on the 11th day after modeling.In the PBS control group, the mice were injected with the same volume of PBS.Six mice in each group were randomly selected to observe the inflammation of the retina after mydriasis with an ophthalmoscope every other day from 8th day following modeling and the inflammation scores were evaluated.Six mice were randomly selected and sacrificed on the 14th day and 6 on the 18th day following modeling in each group, and both eyeballs of the mice were enucleated.Retinal tissue sections of the 6 mice sacrificed on the 18th day were stained with hematoxylin-eosin and the pathological scores were evaluated.The infiltration of helper T 1 (Th1) cells and Th17 cells in the eyeballs of the 6 mice sacrificed on the 18th day following modeling was detected by flow cytometry.T cells were isolated from spleen and lymph nodes of the 6 mice sacrificed on the 14th day, and the proliferation of T cells under different concentrations of IRBP 651-670 (0, 1, 10 and 20 μg/ml) was detected using a 5-bromodeoxyuridine (BrdU) method.To further study the effects of MSCs-derived sEVs on Th1/Th17 cells differentiation, naive T cells of spleen from another 3 normal mice were isolated by magnetic bead negative sorting and incubated with 10 μg/ml MSCs-derived sEVs or 10 μg/ml PBS, and then were cultured under Th1/Th17 cell differentiation conditions, respectively.Flow cytometry was used to measure the differentiation of naive T cells into Th1/Th17 cells.This study protocol complied with the regulations of the care and use of laboratory animals in China and was approved by an Ethics Committee of Tianjin Medical University (No.TJYY2019103022). Results:The isolated human MSCs-derived sEVs was with an average diameter of (102.4±33.6) nm and showed a double-layer membrane vesicle structure under the transmission electron microscope.The CD9, CD63 and CD81 proteins were highly expressed in sEVs.The inflammation scores of the sEVs treatment group were significantly lower than those in the control group on day 14, 16, 18, 20 and 22 after modeling (all at P<0.05). The pathological score of mice in the sEVs treatment group was significantly lower than that of PBS control group on the 18th day following modeling ( P<0.05). The flow cytometry results showed that on day 18 after modeling, the proportions of Th1 and Th17 cells in eyeballs in the sEVs treatment group were (15.55±2.03)% and (15.67±2.15)%, respectively, which were significantly lower than (21.35±0.72)% and (20.90±1.10)% in the PBS control group ( t=6.58, 5.31; both at P<0.01). BrdU results showed that when the IRBP 651-670 concentration was 20 μg/ml, the T cell proliferation ability in the sEVs treatment group was inhibited obviously compared with the control group ( P<0.05). The proportions of naive T cells differentiated into Th1 cells and Th17 cells in the sEVs treatment group were (28.15±1.32)% and (11.60±2.23)% respectively, which were significantly lower than (31.58±1.75)% and (23.52±1.76)% of the PBS control group, and the differences were statistically significant ( t=3.93, 10.26; both at P<0.05). Conclusions:Intravenous injection of human umbilical cord MSCs-derived sEVs can reduce the inflammation in EAU mice.The mechanism may be related to inhibiting the differentiation of naive T cells to Th1 and Th17 cells, and reducing the infiltration of Th1 and Th17 cells in the eyeballs.

Enhanced effect of microRNA-338-3p in bone marrow-derived dendritic cells on IRBP 1-20-specific T helper 17 cells activation in experimental autoimmune uveitis / 中华实验眼科杂志

Objective:To investigate the role of microRNA-338-3p (miR-338-3p) in regulating the generation and function of interphotoreceptor retinoid-binding protein (IRBP) 1-20-specific T helper 17 (Th17) cells in experimental autoimmune uveitis (EAU). Methods:Bone marrow cells were flushed from the femurs and tibiae of wild-type C57BL/6 mice and cultured in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4)to differentiate into bone marrow-derived dendritic cells (BMDCs). On day 5 after induction, immature BMDCs were collected and divided into miR-338-3p mimics transfection group and mimics negative control transfection group, then transfected with miR-338-3p mimics or negative mimics according to grouping.Twenty-four hours after transfection, the BMDCs were stimulated with 100 ng/ml of lipopolysaccharide to mature.Relative expression levels of miR-338-3p, IL-6, IL-23 and IL-1β mRNA in BMDCs of the two groups were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The EAU model was established with IRBP 1-20, incomplete Freund adjuvant and mycobacterium tuberculosis (H37Ra) in mice.On day 13 after modeling, T cells were isolated from the mice spleen or draining lymph nodes and co-cultured with miR-338-3p mimics or negative control mimics-transfected BMDCs under Th17-polarizing conditions.Concentration of IL-17 in the supernatant was detected by ELISA.Relative expression levels of retinoic acid receptor-related orphan nuclear receptor γt (RORγt) and IL-17 mRNA were analyzed by qRT-PCR.The proportion of IL-17 + cells among T cells co-cultured with BMDCs was assessed by flow cytometry.To further verify the role of miR-338-3p in dendritic cells on Th17 cells, BMDCs transfected with miR-338-3p inhibitor or control inhibitor were co-cultured with T cells isolated from spleen or draining lymph nodes of EAU mice.Concentration of IL-17 in the supernatant was detected by ELISA.The use and care of the animals complied with Regulations for the Administration of Affairs Concerning Experiment Animals by State Science and Technology Commission.The study protocol was approved by the Institutional Animal Care and Use Committee of Tianjin Medical University (No.TJYY2019110117). Results:Relative expression level of miR-338-3p in BMDCs was significantly increased in the miR-338-3p mimics transfection group than the mimics negative control group ( t=6.861, P=0.002). In T cells co-cultured with miR-338-3p mimics-transfected BMDCs, the relative expression levels of RORγt and IL-17 mRNA were 1.34±0.16 and 1.33±0.16, which were significantly higher than 1.00±0.01 and 1.00±0.01 in the mimics negative control group ( t=3.632, P=0.022; t=3.681, P=0.021). ELISA showed that the concentration of IL-17 in the supernatant was (5 941.00±452.40)pg/ml in the miR-338-3p mimics transfection group, which was significantly higher than (4 299.00±348.30)pg/ml in the mimics negative control group ( t=4.979, P=0.008), and IL-17 concentration in the supernatant was (3 092.00±200.90)pg/ml in the miR-338-3p inhibitor transfection group, which was lower than (4 063.00±131.50)pg/ml in the inhibitor negative control group ( t=7.005, P=0.002). The proportion of IL-17 + cells among T cells was (8.03±1.35)% in the miR-338-3p mimics transfection group, which was significantly higher than (4.52±0.73)% in the mimics negative control group ( t=3.968, P=0.017). The relative expression levels of IL-6, IL-23, and IL-1β mRNA were 2.23±0.21, 2.21±0.56, 2.32±0.43, respectively in the miR-338-3p mimics transfection group, which were significantly higher than 1.00±0.06, 1.00±0.07, 1.01±0.15 in the mimics negative control group ( t=10.290, P=0.001; t=3.747, P=0.020; t=5.280, P=0.006). Conclusions:Overexpression of miR-338-3p in BMDCs can promote the IRBP 1-20-specific Th17 cell response by increasing the expression of Th17-polarizing cytokines including IL-6, IL-1β and IL-23.

Inhibitory effects of Longdan Xiegan Decoction on Notch signaling in rats with experimental autoimmune uveitis / 眼科新进展

Objective To investigate the effects of Longdan Xiegan Decoction on the expression of Notch signaling-related genes in rats with experimental autoimmune uveitis (EAU).Methods Totally 24 Lewis rats were randomly divided into normal control group,EAU model group and Longdan Xiegan Decoction treatment group.A rat model of EAU was established in the EAU model group and Longdan Xiegan Decoction treatment group,followed by intragastrical administration of Longdan Xiegan Decoction in the latter group after successful modeling.On day 12 after different treatments,the spleen and lymph nodes were isolated from rats of all the three groups for the collection of CD4 + T ceils,and the levels of both Th17 and Treg cells were analyzed by flow cytometry to calculate the ratio of Th17 to Treg cells.Meanwhile the levels of Notch1,Notch2,Notch3,Notch4 mRNAs expression were monitored by qRT-PCR.Moreover,the expressions of Notch signaling-related proteins were further detected using ELISA technique.Results Flow cytometry showed increased Th17 cell level but decreased Treg cell level in the EAU model group when compared with the normal control group.However,after treatment with Longdan Xiegan Decoction,the Th17 level was decreased,whereas the Treg level was increased,and the ratio of Th17 to Treg gradually restored to equilibrium when compared to EAU model group.qRT-PCR results showed that the expression levels of Notch1,Notch2 and Notch4 mRNAs in Longdan Xiegan Decoction treatment group were significantly higher than those in the normal control group (all P ＜ 0.01),but lower than those in EAU model group on day 12 after treatment (all P ＜ 0.01),without Notch 3 expression in the spleen and lymph nodes.In addition,Notch 1,Notch 2 and Notch 4 protein levels of the spleen and lymph nodes in Longdan Xiegan Decoction treatment group showed the similar tendency as compared to those of Notch 1,Notch 2 and Notch 4 mRNAs (all P ＜ 0.01),and the expression of Notch 3 protein was still not observed.Conclusion Longdan Xiegan Decoction can effectively relieve the imbalance of Th17/Treg cells in EAU rats,and its mechanism may involve the differentiation of naive CD4+ T cells into Th17 and Treg cells,which is regulated by Notch signaling.

Dynamic expression and mechanism of γδ T cells in the spleen of mouse with experimental autoimmune uveitis / 中华实验眼科杂志

Background Previous studies showed that the pathogenesis of uveitis is related to γδ T cells.However,it remains unclear that how these cells are involved in experimental autoimmune uveitis (EAU).Objective This study aimed to observe the dynamic changes of γδ T cells in EAU and explore the role of γδ T cells in the pathological process of EAU.Methods Forty-five C57BL/6(B6) mice were assigned to the normal control group (six mice) and EAU model group (thirty-nine mice).The mice were immunized subcutaneously at 6 spots on the footpads,tail base,and flank with emulsion containing human interphotoreceptor retinoid binding protein1-20 (IRBP1-20) emulsified in complete Freund's adjuvant.After immunization,the mice were examined for clinical signs of EAU by using a Genesis-D camera.The changes of histopathology were compared by hematoxylin and eosin staining.Mouse lymphocytes were isolated and purified from the spleens of IRBP1-20-immunized or normal B6 mice by using a γδ T-cell isolation kit.Flow cytometry was used to detect the changes of intracellular expression of interleukin-17A (IL-17A),and then transferred the activated γδ T cells into EAU models to analyze the changes of clinical signs and histopathology of EAU.Experimental study program as well as the use and feeding of the animals were authorized by the Animal Management and Use Committee of Shandong Traditional Chinese Medicine University.Results The inflammatory symptoms in mouse eyes appeared on day 12 after modeling.The initial changes were fundal blood vessel thickening and minimal inflammatory cell infiltration.Then,multifocal chorioretinal lesions,serious vasculitis and linear lesions were observed on days 16-20,along with abundant lymphocyte infiltration in the vitreous and retinal disorganization.The inflammation symptom scores and the pathological inflammation scores at different time points after modeling had statistically significant differences (F =51.399,P =0.000;F =47.342,P =0.000).The inflammatory symptoms in the eyes began to abate from day 28 onwards.The number of γδ T cells was obviously increased during the inflammation phase of EAU at day 16-20 after modeling,with the number of γδ T cells was (5.67 ±-0.49) ％ and (5.78 ±±0.55) ％,respectively,which was significantly higher than (1.53 ± 0.14) ％ before modeling,with significant differences between them (both at P＜0.05),meanwhile CD69 levels and the integrin lymphocyte function-associated antigen-1 (LFA-1) and secreted IL-17A were elavated.The secretion level of IL-17A was (13.40±0.50)％ and (17.80±2.37)％ on day 16 and day 20 after modeling,respectively,which was significantly higher than (1.53 ± 0.19) ％ before modeling,with significant differences between them (P =0.000,0.001).The activated γδ T cells were transferred into EAU model,the inflammation symptom scores were 1.00 (1.00,2.00) after activated γδ T cells were transferred into EAU model,which was significantly higher than 0.75 (0.05,1.00) of the untransferred group (Z =27.00,P =0.03),and the symptoms of EAU were aggravated.Conclusions The proportion of γδ T cells reaches peak in inflammation of EAU,and the cells are activated.The activated γδ T cells in the EAU model play a immune regulation role by secreting IL-17A.

Immunomodulatory effects of Longdan Xiegan Tang on related inflammatory cytokines in rats with experimental autoimmune uveitis / 眼科新进展

Objective To study the immunomodulatory effects of Longdan Xiegan Tang on related inflammatory cytokines in rots with experimental autoimmune uveitis (EAU).Methods Lewis rats were randomly divided into control group (6 rats),EAU group (18 rats) and LXT group (18 rats).Rats in EAU and LXT groups were immunized with interphotoreceptor retinoid-binding protein (IRBP) emulsion.The expressions of IFN-γ,IL-17,IL-10 and TNF-α were investigated by quantitative real-time PCR and ELISA,respectively.Results In blood of LXT group,the mRNA level of IFN-γ on day 8 was higher than that in control group (P =0.000),but obviously lower than that in EAU group (P =0.000);The mRNA level of IL-17 was peaked on day 12,but was lower than EAU group (P=0.000);The mRNA level of TNF-α on day 12 was higher than that in control group (P =0.000),but obviously lower than that in EAU group (P =0.000);The mRNA level of IL-10 on day 16 was higher than that in EAU group (P =0.042).In lymph node and spleen of LXT group,the mRNA levels of IFN-γ,IL-17 and TNF-α on day 12 were lower than those in EAU group (all P=0.000);The mRNA level of IL-10 was peaked on day 12,but was lower than EAU group (P =0.000).In serum of LXT group,the mRNA levels of IFN--γ,IL-17 and TNF-α on day 12 and day 16 were lower than those in EAU group;The level of IL-10 in EAU and LXT group on day 12 were higher than that in control group,peaked on day 16,but the LXT group was the highest.Conclusion Longdan Xiegan Tang can reduce the expressions of proinflammatory cytokines including IFN-γ,IL-17 and TNF-α.Meanwhile,it can also promote the production of IL-10 and further accelerate the recovery of EAU,indicating that Longdan Xiegan Tang can play a significant role in treating uveitis.

Influence of Yanyankang Powder on Th1/Th2 in rats with experimental autoimmune uveitis / 中国结合医学杂志

<p><b>OBJECTIVE</b>To study the influence of Yanyankang powder on Th1/Th2 in rats with experimental autoimmune uveitis (EAU).</p><p><b>METHODS</b>The EAU models were induced in Lewis rats by immunization with interphotoreceptor retinoid binding protein (IRBP) 1177-1191 in complete Freund's adjuvant. The rats were randomly divided into 3 groups: a model control group, a Yanyankang group, and a prednisone group, 9 rats in each group. The model control group was intervened with saline solution by gavage. The Yanyankang group was intervened with Yanyankang powder 4 g/(kg day) by gavage. The prednisone group were intervened with prednisone acetate tablets 5 mg/(kg d) by gavage. All groups were intervened after immunization once every 2 days for 18 days and monitored by slit-lamp biomicroscopy daily until day 18. The levels of gamma interferon (INF-γ) and interleukin-10 (IL-10) in the supernatants of T cells were determined by enzyme-linked immunosorbent assay. Polymerase chain reaction (PCR) technology was used for measuring Th1 and Th2 related cytokine mRNA expressions.</p><p><b>RESULTS</b>Slighter intraocular inflammation was found in the Yanyankang group and the prednisone group than the control group. The levels of the IFN-γ and IL-10 in the supernatants of the spleen lymph node cells were 382.33±6.30, 155.87±4.46 μg/L in the Yanyankang group and 270.93±7.76, 265.32±11.88 μg/L in the prednisone group. Both had significant differences compared with the control group (941.53±8.59, 20.67±4.65 μg/L; =0.01). The PCR results showed the same tendency.</p><p><b>CONCLUSION</b>Yanyankang powder showed favorable effects in the rats with EAU by influencing the function of Th1 and Th2 cells.</p>

Experimental autoimmune uveitis and other animal models of uveitis: An update.

Over the past several decades, animal models of autoimmune uveitis directed at eye‑specific antigens (Ags) have been developed. These have allowed researchers to understand the basic mechanisms that lead to these diseases and also recently helped the researchers in translational research for therapeutic interventions. Experimental autoimmune uveitis (EAU) is an animal disease model of human endogenous uveitis and can be induced in susceptible animals by immunization with retinal Ags. Ever since the first description of EAU in mice in 1988, several animal models of uveitis has been described by researchers. Disease‑specific model for cytomegalovirus retinitis and tubercular uveitis has evolved our understanding of these complex entities. Endotoxin induced uveitis is another useful model for anterior uveitis, which is not an autoimmune process and is triggered by injection of bacterial endotoxin (lipopolysaccharides) resulting in a rapid short lasting uveitis. The current article will give an insight into the various EAU animal models and their current implications in translational research. The article will also highlight the different grading systems for EAU in the animal model.

Transcriptional Analysis of Nod-Like Receptors in a Mouse Model of Experimental Autoimmune Uveitis

PURPOSE: To evaluate the transcription pattern of Nod-like receptors (NLRs), the intracellular sensors, to detect danger signals in murine eyes with experimental autoimmune uveitis (EAU). METHODS: EAU was induced in B6 (C57BL/6) mice by subcutaneous injection of human interphotoreceptor retinoid binding protein and intraperitoneal injection of pertussis toxin. At 1, 2, and 3 weeks post-immunization, the eyeballs were extracted and subjected to histological and molecular assays using real-time reverse transcription polymerase chain reaction. RESULTS: The levels of nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain 1 (NLRP1), NLRP3, nucleotide-binding oligomerization domain-containing protein 1 (NOD1), and NOD2 transcripts were increased at 2 weeks and gradually reduced thereafter. Notably, NLRP3 showed the highest expression in the eyes with EAU. Similarly, the transcript level of pro-inflammatory cytokine, interleukin-1beta, increased and reached a peak at 2 weeks post-immunization. The retinal structure was severely damaged by inflammation at 3 weeks post-immunization. CONCLUSIONS: Among NLRs, NLRP3 may induce inflammation in eyes after EAU immunization.

The inflammatory characteristics of experimental autoimmune uveitis / 中华实验眼科杂志

Background Lewis rat is a commonly used specie in experimental autoimmune uveitis(EAU).However,the characteristics of EAU,especially ocular uhrastructural change,are rarely reported.Objective This study was to investigate the inflammatory characteristics and ocular uhrastructure of EAU models in Lewis rat.Methods EAU models were induced in 12 SPF female Lewis rats(6-8 weeks old)by injection of complete Freund adjuvant(CFA) containing interphotoreceptor retinoid-binding protein (IRBP,1177-1191) and tuberculin (TB) into footpads,napes and back of the body,and 6 normal matched rats were used as normal controls.The diet and drinking,temperature and behavior acts of the rats were recorded during the observational duration.Ocular manifestations were examined under the slit lamp biomicroscope after modeling and scored.Eyeballs were obtained in 12 days after modeling for histopathological examination,and the ultrastructures of eyeballs were observed under the scanning electron microscope and transmission electron microscope.The use and care of the animals complied with Statement of ARVO.Results The food-intake was (190.00± 18.03)g in the model group,and that in the control group was (285.33 ±28.02) g,showing a significant difference between them (t =4.955,P =0.012).The drinking-water volume of rats was (241.67±t 18.56)ml in the model group,which was significantly less than (289.67± 18.18)ml in the control group,showing a significant difference between them (t =3.201,P =0.033).In addition,elevated temperature and tiredness were found in the model rats.Anterior chamber empyema,iris hyperemia and occlusion of the pupil appeared in the models on the 6th day and peaked on the 12nd day after immunized,with the inflammatory scores of 3.83±0.41.The infiltration of inflammatory cells were seen in anterior chamber,ciliary body and vitreous cavity under the optical microscope in the model rats.Scanning electron microscopy found uneven iris texture,coarse ciliary surface and loosen villi of retinal pigment epithelial (RPE) cells in the model rats.Under the transmission electron microscope,the infiltration of macrophagocytes on the iris,wrinkle sparse of ciliary epithelium,myeloid bodies in retinal Müller cells and vacuolus in mitochondria of RPE cells were exhibited.No obvious abnormality was found in the control rats.Conclusions Lewis rats are autoimmune status following injection of CFA with IRBP(1177-1191) and TB.The morphology and ultrastructure of eyeballs in the EAU rats can explain the finding of eyes.

Inducible costimulatory molecule and autoimmune uveitis / 中华实验眼科杂志

Inducible costimulatory molecule (ICOS) is one of the costimulatory molecules CD28 superfamily and is upregulated on the surface of T cells following T cell activation.ICOS initiates costimulatory signals after binds with its ligand (ICOSL) and ICOS/ICOSL promote activation of T cells proliferation,differentiation and cytokine production.Clinical and laboratory researches documented that ICOS/ICOSL plays significant roles in various autoimmune diseases,similarly,it plays critical effects on the outcome and evolution of autoimmune uveitis (AIU) and Behcet disease.To further study the influence and mechanism of ICOS/ICOSL pathway on AIU is of helpful for finding out a new therapeutic approach of AIU.This paper reviewed the basic concept of ICOS,the relationship between ICOS/ICOSL pathway and effector cell,the outlook of ICOS/ICOSL in the treatment of AIU.

Interaction between mouse retinal astrocyte and antigen specific Th1 and Th17 Cells / 中华实验眼科杂志

Background C57BL/6andB10R Ⅲareroutinemurinespeciesusedinexperimental autoimmune uveitis (EAU).The inflammation is light for mouse after immunization whereas it is prominent for B10R Ⅲ.ObjectiveThis study was to observe the killing effect of interphotoreceptor retinoid binding protein (IRBP) 1-20-specific T cells on mouse retinal astrocyte.Th1 and Th17 cells effect in the EAU mechanism was discussed.MethodsB10RllⅢ mice and C57BL/6 mice were immunized with IRBP 161-180 and IRBP 1-20 in complete Freund adjuvant (CFA).The infiltrating cells of diseased B10R Ⅲ eyes were analyzed by flow cytometry.IRBP 1-20-specific T cells were isolated from the drainage lymph node and spleen and cultured in IL-2 or IL-23 for Th1 and Th17 cells polarization,respectively.Th1 and Th17 cells cultured for 5 days were seeded on the mouse retinal astrocyte monolayer pretreated with gamma interferon.Cell interaction was observed and the quantity of TNF-α was tested by ELISA.Every test was repeated 6 times and the mean was calculated.The maintenance of experimental animals complied with the Statement of ARVO.ResultsThere were lots of infiltrating cells in the eyes of B10Rm mice after immunization,including 9.5％ IFNγ+ cells,5.1％ IL-17+cells and 41.4％ CD45+ cells.Six days after IRBP1-20 stimulation and cultured by IL-2 and IL-23,44.0％ and 8.0％ cells were IFNγ+,and 1.0％ and 26.0％ cells were IL17+.Twentyfour hours after the interaction between Th1 or Th17 and retinal astrocyte,retinal astrocyte died and detached.The killing effect of Th17 was stronger than Th1.48 hours after co-culture of Th1 or Th17T cells with astrocytes,the concentrations of TNF-α were ( 500± 10 ) and ( 801 ±24 μg/L) μg/L,respectively,with a significant statistical difference (t =-20.36,P =0.00).ConclusionsBoth Th1 and Th17 can kill retinal astrocyte,but Th17 plays a key role in the EAU pathogenesis process.The killing effect is caused by intercellular contact and interaction under the induction of cytokines.

Immunopathology in ocular toxoplasmosis: facts and clues

Although parasite-mediated host cell lysis is deemed to be an important cause of tissue destruction in ocular toxoplasmosis (OT), the severity of the disease is probably correlated with hypersensitivity and inflammation. Notwithstanding, the mechanisms that regulate the inflammatory process in recurrent OT are poorly understood. Recent evidence has identified interleukin (IL) 17 as a marker for disease severity. The ocular and cerebral presence of this cytokine is generally associated with the induction of autoimmune responses in the brain and the eye. Indeed, there are indications that autoimmunity may contribute to clinical variability in the activity of OT. IL-23, which induces the proliferation of IL-17-producing cells and IL-27, which is a counterplayer to IL-17, may regulate T(H)-1-cell-mediated responses in OT. The importance of these cytokines in experimental models of uveitis and encephalitis has been recently reported. CD25(+) regulatory T-cells may control the local inflammatory response and protect the host against collateral inflammatory tissue damage. The responses of these cells to OT may be suitably tailored to cope with either an acquired or a congenital aetiology. Knowledge relating to immunoreactivity in OT has grown impressively during the past few years. Its characteristic and variable features have been identified and the potential relevance of autoimmunity has been assessed. In light of this knowledge, potential future treatment options have been considered.

Sympathetic Ophthalmia: Mangement and Role of Immunosuppressants.

Presented here is a case of sympathetic ophthalmia that provided us an opportunity to evaluate the eficacay of immunosuppressive drugs with steroids in reduced doses and their outcome in improving thevisual loss in a young patient who had fast deterioration in his visual acuity.

Experimental Autoimmune Uveitis induced by Bovine Iris and Ciliary body in Lewis Rat

This study was conducted to develop an animal model of uveitis resembled anterior uveitis in humans after immunization with iris-ciliary body antigen. Male Lewis rats were immunized with the buffer-and detergent insoluble bovine iris-ciliary body mixed with Complete Freund`s adjuvant (CFA) and Pertussis toxin(PTX). A soluble fraction derived from bovine melanin associated antigen(BMAA) after digestion with the proteolytic enzyme V8 protease was prepared and this soluble fraction of BMAA also induced an experimental autoimmune uveitis (EAU). On gel eletrophoresis for soluble fraction of BMAA, prominent bands between 29 kDa and 43 kDa were clealy observed. In this model, clinical anterior uveitis was induced around 2 weeks, peaked at 18 days and disappeared later than 4 weeks after immunization. Histopathological results of EAU disclosed an infiltration of inflammatory cells, mainly lymphocytes and macrophages, into iris and ciliary body as well as in part the choroid, not retina. In conclusion, we developed a model of EAU with Lewis rats after immunization with BMAA subcutaneously and confirmed the immune mediated inflammation was focused mainly on iris and ciliary body and in part on choroid as well as found that MAA might be soluble after V8 protease treatment.