Therapeutic effects of mesenchymal stem cells administered at later phase of recurrent experimental autoimmune uveitis

AIM：To test the therapeutic effects of delayed treatment of mesenchymal stem cells（MSCs） in recurrent experimental autoimmune uveitis（r EAU）.METHODS： The efficacy of different regimens of MSC administration in r EAU were tested by evaluation of clinical and pathological intraocular inflammation,as well as retinal structural and functional integrity using optical coherence tomography（OCT） and electroretinogram（ERG）.The retinal sections were also immunostained with antibodies to glial fibrillary acidic protein（GFAP）and rhodopsin（RHO）. RESULTS： Delayed treatment of MSCs effectively alleviated the severity of intraocular inflammation with relative intact of outer retinal structure and function.Moreover,double therapies with longer interval led to an even better clinical evaluation,as well as a trend of decrease in relapse and amelioration of retinal function.MSC therapies also effectively reduced GFAP expression and increased RHO expression in the retina.CONCLUSION： MSC administration can effectively treat developed diseases of rEAU,and multiple therapies can provide additional therapeutic benefits.

小檗碱调节AMPK/NF-κB信号通路对自身免疫性前列腺炎大鼠Th17/Treg平衡的影响

目的探讨小檗碱调节腺苷酸活化蛋白激酶(AMPK)/核因子-κB(NF-κB)信号通路对自身免疫性前列腺炎(EAP)大鼠辅助性T细胞17(Th17)/调节性T细胞(Treg)平衡的影响。方法将90只SD雄性大鼠随机分为对照组(正常饲养)、模型组、小檗碱低剂量组(25 mg/kg)、小檗碱高剂量组(50 mg/kg)、阳性对照组(40 mg/kg塞来昔布)、抑制剂组(50 mg/kg小檗碱+0.2 mg/kg AMPK抑制剂compound C),每组15只。除对照组外,其余组大鼠均采用盆腔区域及双侧肩胛皮下多点注射完全弗式佐剂与前列腺组织混合悬液构建EAP大鼠模型,建模成功后进行药物处理,每天灌胃给药1次,持续4周。HE染色观察各组大鼠前列腺组织病理情况;免疫组织化学法检测大鼠前列腺组织视黄酸相关核孤儿受体γt(RORγt)、叉头蛋白3(Foxp3)表达;酶联免疫吸附试验(ELISA)法检测大鼠血清中白细胞介素(IL)-17、IL-10水平;流式细胞仪检测大鼠外周血中Th17/Treg细胞比值;通过Western blot法检测大鼠前列腺组织中AMPK/NF-κB信号通路相关蛋白表达。结果与模型组比较,小檗碱低、高剂量组和阳性对照组大鼠前列腺组织腔内有粉色分泌物,腺上皮细胞排列整齐,炎症明显消退,血清IL-17水平、外周血Th17/Treg比值、前列腺组织RORγt和p-NF-κB p65/NF-κB p65表达水平降低,血清IL-10水平、前列腺组织Foxp3、p-AMPK/AMPK和IκBα表达水平升高(P<0.05);加入AMPK抑制剂compound C后,可减弱小檗碱对EAP大鼠Th17/Treg平衡的促进作用。结论小檗碱可通过调节AMPK/NF-κB信号通路促进EAP大鼠Th17/Treg平衡。

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,activatedγδT cells were proved to play a central role in the development of experimental autoimmune uveitis(EAU).However,the mechanism underlyingγδT cell activation in EAU is incompletely known.In this study,we determined the percentage changes in and the phenotypes ofγδT 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 ofγδT cells and DCs obviously increased during the inflammation phase of EAU(days 16-20 of our experiment),and that during this time,γδT cells expressed high levels of CD69 and the integrin lymphocyte function-associated antigen-1(LF A-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 andγδT cells by using flow cytometry and confocal microscopy in order to determine whether DCs affectedγδT-cell activation in vitro.Co-cultures of the two types of cells showed that DCs induced high levels of CD69,LFA-1,and I-17A inγδT cells.Imaging studies revealed contact between the DCs andγδT cells.This interaction was mediated by the accumulation of ICAM-1 and LFA-1 at the interface of DCs-γδT cells.Thus,the activation ofγδT cells in EAU was promoted by DCs interacting withγδT cells.

Association between islet autoantibodies and the prevalence of autoimmune uveitis

AIM:To evaluate the predictive value of islet autoantibodies for the diagnosis of autoimmune uveitis(AU),as well as to characterize the association bet ween islet autoantibodies and AU.METHODS:Totally 97 patients with AU and 100 healthy persons without any autoimmune diseases as the control group were recruited.Multiple serum islet autoantibodies were measured using commercial enzyme-linked immunosorbent assay kits(ELISA).A supplementary questionnaire was used to complement the subject's demographics and clinical features.The level of glucose concentrations and white blood cells were measured.Conditional logistic regression was performed to estimate odds ratios(ORs),and 95%confidence intervals(CIs)of AU according to islet autoantibodies and to evaluate the predictive value of islet autoantibodies for AU diagnosis.Autoantibodies subgroups and other variables were included into analysis.RESULTS:In AU patients,the prevalence of detecting at least one of the autoantibodies was 31.9%(31/97).The most frequent autoantibody was ZnT8A(30.9%),followed by GADA(11.3%),IA-2A(4.1%),ICA(2.1%)and IAA(2.1%).Islet autoantibodies were found to be correlated positively with AU diagnosis[OR(95%CI):13.86(3.28,58.50),P<0.001].Moreover,Zn-T8A was remarkably correlated with AU diagnosis[OR(95%CI):6.13(1.96,19.17),P<0.001],In contrast,neither GADA nor other islet antibodies(IA-2A,ICA and IAA)showed any association with AU risk under an additive model.CONCLUSION:The prevalence of islet antibodies,especially ZnT8A,in patients with AU is higher.Islet antibodies as well as novel biomarkers should be included in routine evaluation at AU and is a valuable biological marker to classify newly-diagnosed uveitis.

Cytotoxic effect of specific T cells from mice with experimental autoimmune uveitis on murine photoreceptor cells

AIM:To investigate the cytotoxic effect of specific T cells from mice with experimental autoimmune uveitis(EAU)as well as their secreted interferon(IFN)-γand interleukin(IL)-17A on murine photoreceptor(661 W)cells.METHODS:An EAU model was established in female mice by injection of interphotoreceptor retinoid binding protein(IRBP)emulsion supplemented with complete Freund’s adjuvant(CFA)and Mycobacterium tuberculosis(TB).On day 12 after induction of EAU,specific T cells from spleen and lymph node tissues were isolated and cultured for 4 d and the levels of IFN-γand IL-17A in the supernatants were determined by enzyme-linked immunosorbent assays(ELISAs).T cells and their supernatants were added to 661 W cells to observe the alteration of cell morphology;IFN-γand IL-17A were separately added to 661 W cells to observe the effect of IFN-γand IL-17A on cell proliferation.RESULTS:The levels of IFN-γand IL-17A in the T cell supernatants were 1568.64±38.79 pg/m L and 1456.57±46.98 pg/mL,respectively.The supernatants apparently inhibited 661 W cell proliferation(P<0.05).T cells could also attach to the surface of 661 W cells,and IFN-γshowed a more serious cytotoxic effect on 661 W cells than IL-17A,inhibiting cell proliferation(P<0.01).CONCLUSION:IFN-γand IL-17A from T cells of EAU mice model can exert cytotoxic effects on murine photoreceptor cell proliferation,and IFN-γshows more serious cytotoxic effects on murine photoreceptor cells than IL-17A.

High IFN-α expression is associated with the induction of experimental autoimmune uveitis (EAU) in Fischer 344 rat

Th1-response plays a crucial role in determining pathogenesis of organ-specific autoimmune diseases. It is believed that both IL-12 and INF-a are initiators to regulate Th1-response. In our experimental autoimmune uveitis (EAU) model, both Lewis and Fischer 344 rats share the same MHC class II molecules, while Lewis rat is EAU susceptible and Fischer 344 rat is EAU resistant. However, under the same condition of immunization, if pertussis toxin (PTX) was injected intraperitoneally as an additional adjuvant, Fischer 344 rat can develop EAU. In this study we investigate which mechanisms are involved in the induction of EAU in CFA + R16 + PTX treated (CRP- treated) Fischer 344 rats. In vivo and in vitro data demonstrated that Th1-cytokine, IFN- mRNA expression was significantly increased in disease target tissue-eyes and in draining lymph node cells of CRP-treated Fischer 344 rat. When IL-12 and IFN-a mRNA expression were compared in the experimental groups, only IFN-a mRNA expression was associated with EAU development. To distinguish the sources of IFN-a producing cells, it was observed that IFN-a expression was mainly produced by macrophages. It was further confirmed that normal macrophage from Fischer 344 rat was able to produce significant IFN-a in the presence of PTX. The data strongly suggested that IFN-a might be involved in initiating Th1-cell differentiation and in turn contribute to the induction of EAU. High IFN-a expression induced by PTX may represent a novel pathway to initiate Th1 response in Fischer 344 rat.

Patterns of Eye-Infiltrating Cells between Chronic and Acute Experimental Autoimmune Uveitis

Objective: To characterize the cellular infiltrate of eyes in experimental autoimmune uveitis (EAU) induced in C57BL/6 and B10.RIII strains. Methods: EAU was induced by interphotoreceptor retinoid-binding protein with complete Freund's adjuvant in C57BL/6 and B10.RIII mice. Clinical scores were evaluated by funduscopy and electroretinography. Cells were isolated from inflamed eyes and analyzed by flow cytometry. Results: Different forms of EAU disease were showed with earlier disease onset, peak and higher clinical scores in acute EAU mice than chronic EAU mice [1]. In addition to increase of CD4+ T lymphocytes, percentage of CD11b+ cells were higher in the eyes of acute EAU mice than those in chronic EAU mice, while no differences were showed in CD8+ T cells, natural killer cells or natural killer T cells. Among those infiltrated CD11b+ cells, percentages of macrophages and myeloid-derived suppressor cells were higher in acute EAU mice than those in chronic EAU mice. Conclusions: The different disease pattern in EAU between acute form and chronic form is associated with the increase of infiltrated CD4+ T lymphocytes, macrophages and myeloid-derived suppressor cells.

基于AMPK/SIRT1通路探究祛风明目丸对实验性自身免疫性葡萄膜炎大鼠的作用机制

目的:研究祛风明目丸对实验性自身免疫性葡萄膜炎(EAU)大鼠腺苷酸活化蛋白激酶(AMPK)/SIRT1通路及Th1/Th17细胞的作用机制。方法:SPF级雄性Lewis大鼠70只,随机取19只作为空白组,剩余51只建立EAU模型,造模后行炎症评分。造模成功后随机分为模型组、中药常量组、中药高倍组。中药常量组、中药高倍组每天分别以2.4、4.8 g/kg祛风明目丸药液灌胃,空白组、模型组每天予等量蒸馏水灌胃。14 d后ELISA检测血清γ干扰素(IFN-γ)、白细胞介素(IL)-6、IL-17A、IL-22水平,流式细胞数检测外周Th1/Th17细胞比例,qRT-PCR定量检测视网膜中CCL20、CXCL9、CXCL10、CCR6和CXCR3表达,Western Blot检测视网膜AMPK、P-AMPK、SIRT1蛋白表达。结果:Caspi评分示:自造模后第3天起模型组EAU大鼠眼前节炎症评分显著高于空白组(P<0.01)。ELISA示:与空白组比较,模型组IFN-γ、IL-6、IL-17A、IL-22水平显著升高(P<0.05);与模型组比较,中药常量组、中药高倍组炎症因子表达显著下降(P<0.05)。qRT-PCR示:与空白组比较,模型组CXCL9、CXCL10、CCL20、CCR6和CXCR3水平显著升高(P<0.05);与模型组比较,中药高倍组趋化因子及受体的表达显著下调(P<0.05),中药常量组CXCL10、CCL20、CCR6和CXCR3的表达显著降低(P<0.05)。流式细胞数示:与空白组比较,模型组Th1和Th17细胞亚群比例显著升高(P<0.05);与模型组比较,中药高倍组Th1和Th17细胞比例显著下降(P<0.05),中药常量组Th1细胞比例显著下降(P<0.05)。Western Blot示:与空白组比较,模型组P-AMPK、P-AMPK/AMPK、SIRT1表达显著下调;与模型组比较,中药常量组与高倍组表达显著上调(P<0.05)。结论:祛风明目丸减轻EAU大鼠炎症、抑制Th1/Th17细胞的作用机制可能与AMPK/SIRT1通路激活相关。

Influence of Yanyankang Powder (眼炎康散) on Th1/Th2 in Rats with Experimental Autoimmune Uveitis

Objective： To study the influence of Yanyankang Powder （眼炎康散） on Th1/Th2 in rats with experimental autoimmune uveitis （EAU）. Methods： 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 Thl and Th2 related cytokine mRNA expressions. Results： 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; P=0.01）. The PCR results showed the same tendency. Conclusion： Yanyankang Powder showed favorable effects in the rats with EAU by influencing the function of Th1 and Th2 cells.

Preventive effect of chrysin on experimental autoimmune uveitis triggered by injection of human IRBP peptide 1-20 in mice

Uveitis is a common cause of blindness worldwide.Experimental autoimmune uveitis(EAU)is an animal model of noninfectious uveitis.Chrysin(5,7-dihydroxyflavone)is a member of the flavonoid family and has anti-inflammatory effects.We immunized C57BL/6J mice with human interphotoreceptor retinoid-binding protein peptide 1–20 to induce EAU.Chrysin was administered intragastrically at 25 mg/kg daily to the chrysin-treated mice from 3 days before immunization to 21 days after immunization.Vehicle was administered to the mice in the control group according to the same protocol.Lower clinical and histopathological scores,increased integrity of the blood–retinal barrier(BRB)and higher expression of tight junction proteins were observed in the chrysin-treated mice.Chrysin significantly decreased the proportions of Th1,Th17 and CD4^(+)CD3^(+)CD62L^(+)Th0 cells,and increased the proportion of Treg cells.Both macrophage infiltration and the expression of inducible nitric oxide synthase in the retina were efficiently inhibited by chrysin treatment.In chrysin-treated mice,the expression of interferon-γ,interleukin(IL)-17A,IL-6,IL-1βand tumor necrosis factor-αwas reduced in the retina,whereas higher levels of transforming growth factor-βwere detected.Furthermore,NF-κBp65 was downregulated after chrysin treatment.In conclusion,as an anti-inflammatory molecule,chrysin exerts a preventive effect on EAU by modulating the balance among helper T-cell subsets and suppressing ocular inflammation,thereby maintaining the integrity of the BRB.

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 GMCSF 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.

Activation of AMPKα1 is essential for regulatory T cell function and autoimmune liver disease prevention

Regulatory T cells(Treg cells)are crucial for maintaining immune tolerance.Compromising the regulatory function of Treg cells can lead to autoimmune liver disease.However,how Treg cell function is regulated has not been fully clarified.Here,we report that mice with AMP-activated protein kinase alpha 1(AMPKα1)globally knocked out spontaneously develop immune-mediated liver injury,with massive lymphocyte infiltration in the liver,elevated serum alanine aminotransferase levels,and greater production of autoantibodies.Both transplantation of wild-type bone marrow and adoptive transfer of wild-type Treg cells can prevent liver injury in AMPKα1-KO mice.In addition,Treg cell-specific AMPKα1-KO mice display histological features similar to those associated with autoimmune liver disease,greater production of autoantibodies,and hyperactivation of CD4^(+)T cells.AMPKα1 deficiency significantly impairs Treg cell suppressive function but does not affect Treg cell differentiation or proliferation.Furthermore,AMPK is activated upon T cell receptor(TCR)stimulation,which triggers Foxp3 phosphorylation,suppressing Foxp3 ubiquitination and proteasomal degradation.Importantly,the frequency of Treg cells and the phosphorylation levels of AMPK at T172 in circulating blood are significantly lower in patients with autoimmune liver diseases.Conclusion:Our data suggest that AMPK maintains the immunosuppressive function of Treg cells and confers protection against autoimmune liver disease.

Uveitis in autoimmune hepatitis:A case report

在这份病案报告，我们第一次描述在自体免疫的肝炎(AIH ) 和眼色素层炎之间的一个协会，没有关于另外的可能的病原学的任何疑问，例如 HCV，自从在为 HCV 相关的肝炎的测试能是可得到的以前，所有旧报告与虹膜睫状体炎描述 AIH 的协会。有反常的肝功能测试和球结膜的充血的一个 38 岁的商人进入医院。在承认，与坚持的发烧介绍的病人，关节痛，结膜充血，白细胞增多和增加的 ESR 前的六年，指了尖锐风湿热。系统病的存在，通常与眼色素层炎联系的大多数，没有结果，被调查，病人然后与热门 corticosteroids 被对待。他解决的症状。为反原子的抗体的测试是阳性的，在 1:320 的一个效价，与一个有斑点、核仁的染色的模式。肝超声与肝的增加的回响结构显示出温和肝肿大。经皮的肝活体检视在超声帮助下面被执行。组织学的检查在门，仙子门和小叶片的区域上显示出 necroinflammation，纤维变性门道与仙子门纤维变性和偶尔的 portal-to-portal bridgings，但是未经触动的肝的建筑学。某 hepatocytes 在小叶片的区域显示出含铁血黄素的恰好看得清的小粒。胆小管没有任何重要词法改变。METAVIR 分数是 A2-F3，根据修改 HAI 等级 / 纤维变性阶段。病人被诊断与温和活动和纤维变性有 AIH 并且在 25 mg 泼尼松上被解除，进入临床、生物化学的宽恕，进一步的证实诊断。在分泌物以后，病人继续在 5 mg 的剂量作为一个门诊病人与 corticosteroids 有治疗。在 2002 年 1 月，病人被重新接纳到医院。为反原子的抗体的测试是阳性的，在 1:320 的一个效价，与一个有斑点、核仁的染色的模式。反光滑的肌肉抗体测试也是阳性的(1:160 ) ，当 anti-LKM 抗体是否定的时。眼科学检查在角膜在左眼，和基质损害的前房揭示了煽动性的房间和 proteinaceous 闪光。他在抑制免疫力的治疗(加为二个星期的热门抗菌素治疗的 5 mg 泼尼松) 上被维持然后排出。症状的完全的宽恕在后续上被登记。目前(2005 年 7 月) ，病人在泼尼松(5 mg ) 上并且没有症状。肝功能测试也在正常范围以内。

芍药苷调节RhoA/ROCK信号通路对实验性自身免疫性葡萄膜炎小鼠Th17/Treg免疫平衡的影响

目的探讨芍药苷调节Ras同源基因家族蛋白A(RhoA)/Rho相关卷曲螺旋形成蛋白激酶(ROCK)信号通路对实验性自身免疫性葡萄膜炎(experimental autoimmune uveitis,EAU)小鼠辅助性T淋巴细胞17(Th17)/调节性T细胞(Treg)免疫平衡的影响。方法随机选出10只小鼠作为正常对照组,其余小鼠构建EAU模型,将造模成功小鼠随机平分为模型组、芍药苷组(10 mg·kg^(-1)芍药苷)、RhoA/ROCK信号通路激活剂溶血磷脂酸(LPA)组(25μmol·L^(-1)LPA)、芍药苷+LPA组(10 mg·kg^(-1)芍药苷+25μmol·L^(-1)LPA),每组10只。模型组和正常对照组给予等量生理盐水,给药每天1次,持续14 d。眼前节临床表现评分评估炎症严重程度;HE染色观察眼球组织病理形态;ELISA法检测血清中炎性因子转化生长因子-β(trarsforming growth factor beta,TGF-β)、白细胞介素10(IL-10)、白细胞介素17(IL-17)、白细胞介素23(IL-23)水平;流式细胞术检测脾脏Th17/Treg细胞水平;Western Blot法检测维甲酸相关核孤儿受体γt(RORγt)、叉头状转录因子3(Foxp3)以及RhoA/ROCK信号通路蛋白表达。结果正常对照组小鼠无虹膜、结膜血管舒张充血,与正常对照组比,模型组小鼠出现虹膜血管舒张充血,前房、虹膜、睫状体和视网膜内均有大量炎性细胞膨胀,眼前节临床表现评分、IL-17和IL-23含量、Th17细胞水平、Th17/Treg、RORγt、RhoA、ROCK1蛋白水平明显升高(P<0.05),IL-10和TGF-β含量、Treg细胞水平、Foxp3水平明显下降(P<0.05)。与模型组比,芍药苷组小鼠虹膜血管充血症状明显缓解,眼前节临床表现评分、IL-17和IL-23含量、Th17细胞水平、Th17/Treg、RORγt、RhoA、ROCK1蛋白水平明显降低(P<0.05),IL-10和TGF-β含量、Treg细胞水平、Foxp3水平明显升高(P<0.05);而LPA加重了眼部损伤(P<0.05),逆转了芍药苷对EAU小鼠的改善效果(P<0.05)。结论芍药苷可通过下调RhoA/ROCK信号通路调节EAU小鼠Th17/Treg免疫平衡减轻EAU小鼠葡萄膜炎。

雷公藤红素在实验性自身免疫性葡萄膜炎(EAU)小鼠眼组织中的抗炎作用及其对小胶质细胞极化的影响

目的探讨雷公藤红素在实验性自身免疫性葡萄膜炎(EAU)小鼠眼组织中的抗炎作用及其对小胶质细胞极化的影响。方法取健康6~8周龄B10.RIII小鼠36只,随机分为正常对照组、EAU溶剂对照组、雷公藤红素干预组,每组12只。将光感受器间维生素A类结合蛋白161-180和完全弗氏佐剂充分乳化混合后,注射于EAU溶剂对照组和雷公藤红素干预组小鼠双侧大腿和尾部皮下,总体积200μL,每只小鼠注射光感受器间维生素A类结合蛋白161-180的量为50μg。免疫后第7-14天,雷公藤红素干预组小鼠每天接受0.5 mg·kg^(-1)雷公藤红素腹腔注射治疗,EAU溶剂对照组小鼠注射同等剂量的无菌PBS缓冲溶液。免疫后第14天,通过裂隙灯显微镜观察各组小鼠眼前节表现及行组织切片HE染色,并参照Caspi分级标准对各组小鼠进行临床评分及组织病理学评分;免疫荧光染色观察小鼠眼内小胶质细胞活化情况;Western blot检测小鼠视网膜中诱导型一氧化氮合酶(iNOS)、精氨酸酶-1(Arg1)蛋白表达水平;qRT-PCR检测小鼠视网膜中炎症因子肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β、IL-6 mRNA相对表达量。采用Graphpad Prism 9.0统计软件进行数据分析。结果免疫后第14天,通过裂隙灯显微镜观察发现,EAU溶剂对照组小鼠眼前节可见虹膜血管扩张充血明显、角膜水肿、前房渗出;而雷公藤红素干预组小鼠眼前节炎症明显减轻,可见虹膜血管轻度充血。与正常对照组相比,EAU溶剂对照组与雷公藤红素干预组小鼠临床评分均升高,差异均有统计学意义(均为P<0.05)。雷公藤红素干预组小鼠临床评分低于EAU溶剂对照组,差异有统计学意义(P<0.05)。HE染色结果显示,免疫后第14天,EAU溶剂对照组小鼠出现严重的视网膜皱褶和脱离,炎症细胞弥漫浸润;而雷公藤红素干预组小鼠视网膜结构破坏轻微,可见少量炎症细胞浸润。与正常对照组相比,EAU溶剂对照组与雷公藤红素干预组小鼠组织病理学评分均升高,差异均有统计学意义(均为P<0.05)。雷公藤红素干预组小鼠组织病理学评分低于EAU溶剂对照组,差异有统计学意义(P<0.05)。正常对照组、EAU溶剂对照组、雷公藤红素干预组小鼠眼内Iba1+细胞密度分别为(1.00±0.12)%、(36.07±4.57)%、(1.83±0.36)%,与正常对照组相比,EAU溶剂对照组及雷公藤红素干预组小鼠眼内Iba1+细胞数增加,差异均有统计学意义(均为P<0.05);与EAU溶剂对照组相比,雷公藤红素干预组小鼠眼内Iba1+细胞数明显减少,差异有统计学意义(P<0.05)。与正常对照组相比,EAU溶剂对照组小鼠视网膜中iNOS、Arg1蛋白表达水平均升高,差异均有统计学意义(均为P<0.01);与EAU溶剂对照组相比,雷公藤红素干预组小鼠视网膜中iNOS蛋白表达水平降低,差异有统计学意义(P<0.01)。与正常对照组相比,EAU溶剂对照组小鼠视网膜中TNF-α、IL-1β、IL-6 mRNA相对表达量均升高,差异均有统计学意义(均为P<0.05);与EAU溶剂对照组相比,雷公藤红素干预组小鼠视网膜中TNF-α、IL-1β、IL-6 mRNA相对表达量均下降,差异均有统计学意义(均为P<0.05)。结论雷公藤红素可抑制M1型小胶质细胞活化,减少EAU小鼠视网膜炎症因子TNF-α、IL-1β、IL-6的产生,减轻炎症反应。

母体炎性暴露对子代小鼠受IRBP抗原诱导的葡萄膜炎的影响

目的 探讨母体葡萄膜炎暴露对子代小鼠受光感受器间视黄醇结合蛋白(interphotoreceptor retinoid-binding protein, IRBP)诱导的实验性自身免疫性葡萄膜炎(experimental autoimmune uveitis, EAU)免疫效应的影响。方法 从本课题组前期获得的受双亲EAU暴露的子代C57BL/6J小鼠眼球的RNA测序转录组数据中,筛选免疫相关差异表达基因(differentially expressed genes, DEGs)。利用基因本体论(Gene Ontology, GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes, KEGG)的富集分析,确定这些免疫相关的差异表达基因生物学途径。通过蛋白质互作网络(protein-protein interaction networks, PPI)分析交集基因以此鉴定出枢纽基因。进一步通过荧光定量聚合酶链反应(quantitative real-time PCR,qPCR)确定筛选出的异常表达的枢纽基因是否也在受母体葡萄膜炎影响的子代小鼠中差异表达,最后通过视网膜抗原IRBP_(651-670)诱导受母体活动期炎症影响的子代以评价母体葡萄膜炎对子代患葡萄膜炎的易感性以及相关免疫效应的影响。结果 从受双亲葡萄膜炎影响的子代小鼠眼球组织中鉴定出72个免疫相关差异基因。这些基因主要富集参与Toll样受体信号通路、MAPK信号通路以及B细胞受体信号通路等。进一步进行PPI网络互作分析,筛选出proteasome 26S subunit, ATPase 5 (Psmc5)、proteasome 26S subunit, ATPase 3 (Psmc3)、proteasome 26S subunit ubiquitin receptor, non-ATPase 4 (Psmd4)和proteasome 26S subunit, non-ATPase 8 (Psmd8)这4个枢纽基因。qPCR检测证实受母体葡萄膜炎影响的子代小鼠中也上调表达这4个枢纽基因。而且与健康子代相比,150μg IRBP抗原诱导可增加受活动期炎症影响的子代EAU临床表现的严重程度和病理组织学损伤(P=0.0087,P=0.0410);另外,受母体活动期炎症影响的子代在IRBP诱导的EAU中脾T细胞增殖功能增强,其血清中产生的细胞因子IL-17和IL-6增加(P=0.0450,P=0.0300)。结论 母体活动性炎症暴露加剧子代小鼠受IRBP诱导的EAU疾病的严重性,增强抗原特异性T细胞增殖以及血清中IL-17、IL-6的产生,可能与受影响的子代上调表达的4个枢纽基因Psmc5、Psmc3、Psmd4、Psmd8相关。

衣康酸二甲酯对树突状细胞分泌促炎因子以及实验性自身免疫性葡萄膜炎小鼠辅助性T细胞17的影响

目的探究衣康酸二甲酯(DMI)对树突状细胞(DCs)分泌促炎因子的作用,并初步研究其对实验性自身免疫性葡萄膜炎(EAU)小鼠光感受器间维生素A类结合蛋白1-20(IRBP_(1-20))特异性辅助性T细胞17(Th17)的影响。方法分离C57BL/6J小鼠双侧股骨和胫骨得到骨髓细胞,利用粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素(IL)-4将其定向诱导分化为骨髓来源的DCs。诱导分化6 d,将细胞分为DMI组和PBS组,分别用250μmol·L^(-1)DMI及等量的磷酸盐缓冲液(PBS)预处理3 h后,每组加入100μg·L^(-1)脂多糖(LPS)刺激24 h。采用实时荧光定量PCR(qRT-PCR)检测两组DCs中促炎因子IL-6、IL-1β和IL-23 mRNA相对表达量。采用IRBP_(1-20)、弗氏不完全佐剂及结核分枝杆菌H37RA主动免疫小鼠构建EAU模型,免疫后13 d,将分离的EAU小鼠脾脏及淋巴结T细胞与PBS或DMI处理的DCs在含有IRBP_(1-20)的培养基中共培养,并向Th17方向极化。流式细胞仪检测共培养细胞中Th17细胞比例。ELISA检测共培养上清液中IL-17水平。qRT-PCR检测共培养细胞中维甲酸相关核孤儿受体γt(RORγt)、IL-17、IL-23R和GM-CSF mRNA相对表达量。结果qRT-PCR检测结果显示,DMI组DCs中IL-6、IL-1β和IL-23 mRNA相对表达量均明显低于PBS组,差异均有统计学意义(均为P<0.05)。流式细胞仪检测结果显示,DMI组共培养细胞中Th17细胞比例明显低于PBS组,差异有统计学意义(P<0.05)。ELISA检测结果显示,DMI组共培养上清液中IL-17水平明显低于PBS组,差异有统计学意义(P<0.05)。DMI组共培养细胞中IL-17、RORγt、IL-23R和GM-CSF mRNA相对表达量均明显低于PBS组,差异均有统计学意义(均为P<0.05)。结论DMI能够抑制DCs中促炎因子IL-6、IL-1β和IL-23的表达,进而负向调控IRBP_(1-20)特异性Th17细胞反应。

Rapamycin ameliorates experimental autoimmune uveoretinitis by inhibiting Th1/Th2/Th17 cells and upregulating CD4+CD25+ Foxp3 regulatory T cells

· AIM: To determine the effects of rapamycin on experimental autoimmune uveoretinitis(EAU) and investigate of role of rapamycin on T cell subsets in the disease.·METHODS: EAU was induced in rats using peptides1169 to 1191 of the interphotoreceptor binding protein(IRBP). Rapamycin(0.2 mg/kg/d) was administrated by intraperitoneal injection for a consecutive 7d after immunization. Th1/Th2/Th17 cytokines, TGF-β1, and IL-6produced by lymphocyteswere measured by ELISA, while Th17 cells and CD4 +CD25 + regulatory T cells(Tregs)from rat spleen were detected by flow cytometry.·RESULTS: Intraperitoneal treatment immediately after immunization dramatically ameliorated the clinical course of EAU. Clinical responses were associated with reduced retinal inflammatory cell infiltration and tissue destruction. Rapamycin induced suppression of Th1/Th2/Th17 cytokines, including IFN-γ, IL-2, IL-17, IL-4, and IL-10 release from T lymphocytes of EAU rats, in vitro.Rapamycin also significantly increased TGF-β1production but had no effect on IL-6 productionof T lymphocytes from EAU rats in vitro. Furthermore,rapamycin decreased the ratio of Th17 cells/CD4 +T cells and upregulated Tregs in EAU, as detected by flow cytometry.·CONCLUSION: Rapamycin effectively interferes with T cell mediated autoimmune uveitis by inhibiting antigen-specific T cell functions and enhancing Tregs in EAU.Rapamycin is a promising new alternative as an adjunct corticosteroid-sparing agent for treating uveitis.

Human umbilical cord-derived mesenchymal stem cells treatment for refractory uveitis: a case series

AIM:To evaluate therapeutic outcomes of human umbilical cord-derived mesenchymal stem cells(HUC-MSCs)treatment in patients with refractory uveitis.METHODS:A retrospective and noncomparative review was performed on four patients with refractory uveitis from December 2013 to December 2017.HUC-MSCs were administered intravenously at a dose of 1×106 cells/kg.Clinical response,relapse rate,change of visual acuity,and other metrics were evaluated.RESULTS:All four patients presented with responses to HUC-MSCs treatment,with three males and one female.The numbers of uveitis attacks per year after the HUCMSCs treatment(0,2,0,0 respectively)all decreased compared with the numbers before the treatment(3,6,4,4 respectively).The oral steroid and immunosuppressive agents were tapered in all patients without recrudescence of ocular inflammation,and three patients discontinued their oral medicine at the last visit.The best corrected visual acuity(BCVA)of 3 patients was improved to varying degrees,and the BCVA of 1 patient remained at 20/20(Snellen chart)from the first to the last consultation.CONCLUSION:The study provides an effective therapy of HUC-MSCs in maintaining remission in patients affected by uveitis refractory to previous immunosuppressant treatments.

Lymphocyte Proliferation Response to S Antigen in Patients with Uveitis and Optic Neuritis

Purpose:To evaluate the autoimmunity which may play a major role in the etiolo-gy of certain forms of uveitis and optic neuritis.Methods:lymphocyte proliferation response to retinal soluble antigen in vitro by gy of certain forms of uveitis and optic neuritis.Methods:Lymphocyte proliferation response toretinal soluble antigen in vitro by incoperation^3H-thymidine withDNA was tested in 115patients with anterior u-veitis,posterior/pan-uveitis,optic neuritis,and 50volunteers with unrelated diseases such as congenital ptosis,strabismus,or completely healthy persons as control.Results:The positive rate of lymphocyte stimulation was34%(18/53)in anteri-or uveitis,41.5%(17/41)in posterior/pan-uveitis,and57.1%(12/21)in optic euritis,The results in the experimental groups were significantly different from those of the control group(x^2=14.76,P<0.05,x^2=19.14P<0.005,x^2=26.38,P<0.005,respectively).Conclusion:The autoimmunity plays a role in the patogenesis in certain forms of uveitis and optic neuritis,Such immune responses may be secondary to the expo-sition or release of retinal antigens by various causes,leading to activation or augmentation of meager or low-affinity S antigen specific lymphocytes which may preexist in the circulation and starting the pathogenic autoimmune process.Eye Science 1995;11:120-123.