Immune Responses To Ocular Antigens

对眼抗原的免疫反应

• 批准号: 9362355
• 负责人: Igal Gery
• 金额: $ 86.59万
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9362355
• 关键词: Address; Antibodies; Antigens; Autoimmune Diseases; Autoimmune Responses; Autoimmunity; Back; Blindness; Blood Circulation; CD28 gene; CD3 Antigens; CD4 Positive T Lymphocytes; Cells; Data; Development; Disease; Exposure to; Eye; Eye diseases; Female; Flow Cytometry; Gene Expression; Generations; Genes; Genetic screening method; Hen Egg Lysozyme; Home environment; Homing; Immune; Immune response; Immune system; Immunosuppressive Agents; In Vitro; Inferior; Inflammation; Interferons; Interleukin-10; Interleukin-17; Kinetics; Knock-in Mouse; Licensing; Life; Ligands; Light; Liver; Lung; Lymphocyte; Lymphocyte Subset; Manuscripts; Mediating; Messenger RNA; Microarray Analysis; Modeling; Mus; National Institute of Environmental Health Sciences; Nature; New Agents; Organ; Paper; Pathogenicity; Pattern; Physiological Processes; Population; Predisposition; Process; Production; Proliferating; Publications; Published Comment; Regulatory T-Lymphocyte; Reporting; Research; Resistance; Rheumatoid Arthritis; Route; STAT4 gene; Severities; Signal Pathway; Spleen; System; TIS11 protein; Testing; Transcript; Wild Type Mouse; animal facility; autoimmune uveitis; cytokine; in vivo; inhibitor/antagonist; interest; intraperitoneal; lymph nodes; male; men' s group; microbiome; migration; novel strategies; research study; transcription factor; treatment effect; treatment group; Address; Antibodies; Antigens; Autoimmune Diseases; Autoimmune Responses; Autoimmunity; Back; Blindness; Blood Circulation; CD28 gene; CD3 Antigens; CD4 Positive T Lymphocytes; Cells; Data; Development; Disease; Exposure to; Eye; Eye diseases; Female; Flow Cytometry; Gene Expression; Generations; Genes; Genetic screening method; Hen Egg Lysozyme; Home environment; Homing; Immune; Immune response; Immune system; Immunosuppressive Agents; In Vitro; Inferior; Inflammation; Interferons; Interleukin-10; Interleukin-17; Kinetics; Knock-in Mouse; Licensing; Life; Ligands; Light; Liver; Lung; Lymphocyte; Lymphocyte Subset; Manuscripts; Mediating; Messenger RNA; Microarray Analysis; Modeling; Mus; National Institute of Environmental Health Sciences; Nature; New Agents; Organ; Paper; Pathogenicity; Pattern; Physiological Processes; Population; Predisposition; Process; Production; Proliferating; Publications; Published Comment; Regulatory T-Lymphocyte; Reporting; Research; Resistance; Rheumatoid Arthritis; Route; STAT4 gene; Severities; Signal Pathway; Spleen; System; TIS11 protein; Testing; Transcript; Wild Type Mouse; animal facility; autoimmune uveitis; cytokine; in vivo; inhibitor/antagonist; interest; intraperitoneal; lymph nodes; male; men' s group; microbiome; migration; novel strategies; research study; transcription factor; treatment effect; treatment group

NEW INFORMATION ON THE "LICENSING" PROCESS This project, dealing with the migration and licensing for pathogenicity of Th lymphocytes, has been extended in FY 2016 to address comments of reviewers (J. Immunol) on our manuscript ("Shedding new light on the process of licensing for pathogenicity by Th lymphocytes"). In the experimental system used in these experiments, lymphocytes transgenically expressing TCR against hen egg lysozyme (HEL) are adoptively transferred into recipients in which HEL is transgenically expressed in the eyes. The transferred cells initiate inflammation in the recipient eye only when they are activated and in the experiments carried out in FY 2016, we examined systems in which the transferred cells were activated (i) in vitro, by exposure to the antigen (HEL), or (ii) in vivo, by exposure to HEL and TLR ligands. The transferred cells of the two systems require 2-3 days of licensing in organs other than the target organ (the eye), during which they proliferate AND acquire the capacity to initiate the ocular inflammation. New information was collected on three issues: (1) Donor cells activated in vitro and injected by the intraperitoneal (i.p.) route migrate with similar kinetics to the lung and the parathymic lymph nodes). Importantly, following the licensing process, the total number of donor cells in the parathymic lymph nodes was approximately x10 higher than that in the lung. Furthermore, the total number of donor cells in the spleen in these recipient mice was approximately x100 larger than that in the lung. These observations thus provide additional new data to indicate that the lung is not the organ where licensing takes place, as claimed in a recent Nature paper (Odoardi et al., 2012). (2) We also investigated the homing of donor cells to the recipients liver. The liver is the organ where dead and dying cells are eliminated, but yet, we found that small numbers of the live transferred donor cells home to the liver and undergo in this organ the licensing process, before migrating back into the circulation. This observation further supports our notion, that the licensing process takes place in multiple organs, rather than only in the lung, as claimed by Odoardi et al., 2012. (3) We used the microarray technology to compare between donor cells that acquire pathogenicity following activation in vitro and those that are activated in vivo, in their patterns of transcript expression of major inflammation-related genes prior to their invasion of the eyes. Similarities were found between the two cell populations in the patterns of expression changes by the majority of the tested genes. Since the system in which donor cells are activated in vivo could be considered a model for the physiological process of initiation of pathogenic autoimmunity, our data thus provide new information to support the notion concerning the licensing phenomenon in this physiological process. NEW INFORMATION CONCERNING THE IMMUNOSUPPRESSIVE AGENT TMP778 New information collected in FY 2016 include: (i) Treatment with TMP778 inhibits the development of EAU and its effect is attributed to the selective inhibition of the Th17 lymphocyte subpopulation. Yet, we found in new experiments in FY 2016 that treatment with TMP778 also reduces the production of interferon (IFN)-, the signature cytokine for Th1 lymphocytes. This unexpected finding of reduced levels of IFN- in lymphocytes from TMP778-treated mice was demonstrated by both the reduced levels of IFN- released by cells from treated mice, as well as by flow cytometry, showing that the proportion of spleen cells expressing intracellular IFN- was smaller among spleen cells from mice treated with TMP778 than those from the control (vehicle-treated) mice. (ii) The effect of treatment with TMP778 on the immune system was further examined by analyzing the expression of genes known to regulate the immune response by spleen cells from mice treated with TMP778 and their controls, treated with the vehicle. So far, we found that treatment with TMP778 reduces in spleen cells the expression of RORt, Tbet, RUNX3, STAT4 and Eomes, but moderately enhances the expression of IL-10. (iii) The possible involvement of increased proportion of T-regulatory (Treg) cells in the reduced immune responses in TMP778-treated mice was examined by flow cytometry and ruled out: the proportion of these cells, identified by their expression of the transcription factor FoxP3, was the same in mice treated with TMP778 and their controls, treated with the vehicle (15% of total Th cells). INHIBITORY EFFECTS OF TOFACITINIB IN VIVO AND IN VITRO Another compound with immunosuppressive capacity we examined during FY 2016 is Tofacitinib. This synthetic molecule is an inhibitor of JAK-STAT signaling pathways and has been found to efficiently inhibit the pathogenic processes of rheumatoid arthritis and similar diseases. We tested the effects of Tofacitinib on development of EAU and related immune responses in mice. The major observations include: (i) Treatment with Tofacitinib inhibited moderately the development of EAU, analyzed by histological analysis; (ii) Lymphocytes from mice treated with Tofacitinib were inferior to their controls (mice treated with the vehicle) in their production of IFN-, but not of IL-17. The differences were demonstrated by comparing both the release of the cytokines into culture supernatants and by the proportions of lymphocytes expressing the cytokines intracellularly, determined by flow cytometry. (iii) The selective inhibitory effects of Tofacitinib on generation of Th1 and Th17 was also observed in an in vitro system in which nave CD4 cells were activated by antibodies against CD3 and CD28. Adding Tofacitinib to these cultures selectively inhibited the production of IFN-, but not of IL-17. EFFECTS OF TRISTETRAPROLIN (TTP) ON THE DEVELOPMENT OF EAU AND RELATED IMMUNE RESPONSES A recent publication in PNAS (113:1865, 2016) reported that mice in which the TTP mRNA is stabilized ("KI" mice) are resistant to experimental autoimmune diseases. The "KI" mice and their wild type (WT) controls were provided to us by Perry Blackshear, NIEHS, Research Triangle Park, NC (who generated the "KI mice), to be tested for their capacity to develop EAU and related immune responses. The results were remarkably unequivocal: all the WT mice developed EAU, while essentially no disease developed in the "KI" mice. Likewise, both the cellular and humoral immune responses of the "KI" mice were strikingly lower than those of the WT mice. Thus, our data shed new light on the extent of inhibition of the autoimmune response in the "KI" mice and support the notion that TTP could be used as an immunosuppressive agent. This study also yielded convincing data showing the higher susceptibility of female mice to autoimmunity as compared to male mice. Thus, whereas the mean level of EAU severity of the female mice (determined by histological analysis) was 2.5 +/- 0.31, that of the males was 0.95 +/- 0.21 (p =0.006). In addition, the differences between cellular immune responses of PARENT and their WT controls were remarkably more apparent between the female mouse groups than between the male groups. Further, this study provides an additional piece of interesting information: the WT (C57Bl/6) mice from the animal facility in Research Triangle Park developed EAU more consistently and with levels clearly higher than those we are routinely observing with the C57Bl/6 mice provided by Jackson Lab. A likely explanation is that the microbiome of the mice from the facility in Research Triangle Park is different from that of the mice from Jackson Lab and it better promotes the pathogenic process of EAU.

有关“许可”过程的新信息 该项目涉及淋巴细胞致病性的迁移和许可，已在2016财年扩展，以解决审稿人（J. Immunol）对我们的手稿的评论（“为Themphocopytes提供了有关致病性许可的新灯，”）。在这些实验中使用的实验系统中，针对鸡蛋溶菌酶（HEL）的转基因表达TCR的淋巴细胞被顺从地转移到受体中，其中HEL在眼睛中转基因表达。转移的细胞只有在受体激活时才引发炎症，并且在2016财年进行的实验中，我们检查了通过暴露于抗原（HEL）或（II）在体内通过暴露于HEL和TLR配体暴露于抗原（HEL）或（II）的系统。两个系统的转移电池需要在目标器官（眼睛）以外的器官中进行2-3天的许可，在此期间它们扩散并获得启动眼部炎症的能力。 在三个问题上收集了新信息： （1）在体外激活并被腹膜内（i.p.）路线注射的供体细胞以与肺和副淋巴结相似的动力学迁移。重要的是，在许可过程之后，副淋巴结中的供体细胞总数大约高于肺部。此外，这些受体小鼠的脾脏中的供体细胞总数大约比肺大约x100。 因此，这些观察结果提供了其他新数据，以表明肺不是发生许可的器官，如最近的《自然论文》中所述（Odoardi等，2012）。 （2）我们还调查了将供体细胞归为受体肝脏。肝脏是消除死亡和垂死细胞的器官，但是，我们发现，在迁移回到循环之前，我们发现少数活的活细胞转移到了肝脏中并在此器官中经历了许可过程。这一观察结果进一步支持了我们的观念，即许可过程发生在多个器官，而不仅仅是在肺中进行，正如Odoardi等人所说，2012年。 （3）我们使用微阵列技术比较了在体外激活后获得致病性的供体细胞与在体内激活的细胞，其在其侵袭眼睛之前的转录本表达的模式。大多数测试基因在两个细胞种群之间发现了表达变化的模式的相似之处。由于可以将体内激活的供体细胞的系统视为生理过程的生理过程模型，因此我们的数据提供了新的信息，以支持有关此生理过程中有关许可现象的概念。 有关免疫抑制剂TMP778的新信息 2016财年收集的新信息包括： （i）用TMP778处理抑制EAU的发展，其作用归因于对Th17淋巴细胞亚群的选择性抑制。然而，我们在2016财年的新实验中发现，用TMP778的治疗也降低了干扰素（IFN） - Th1淋巴细胞的签名细胞因子的产生。通过TMP778处理的小鼠的淋巴细胞中IFN水平降低的这种出乎意料的发现，这两种细胞释放的IFN水平降低，通过处理的小鼠释放的IFN水平以及流式细胞仪，以及流式细胞仪，表明，在spleen ifn- spleentular ifn-的脾细胞比例中，来自spleen细胞的脾细胞的比例是从TMPERED的spleen细胞中较小的。 （ii）通过分析已知的基因表达来调节用TMP778治疗的小鼠的脾细胞及其对照，并用车辆处理的对照，进一步研究了用TMP778治疗对免疫系统的影响。到目前为止，我们发现用TMP778的治疗在脾细胞中降低了RORT，TBET，RUNX3，STAT4和EOMES的表达，但适度增强了IL-10的表达。 （iii）通过流式细胞术检查了TMP778处理的小鼠中T调节（TREG）细胞中增加比例增加的免疫反应的参与，并排除了：这些细胞的比例通过TMP778及其对照组的THER中的小鼠的表达来鉴定，这些细胞的表达是相同的，该小鼠与对照组相同。 Tofacitib在体内和体外的抑制作用 我们在2016财年期间检查的另一种具有免疫抑制能力的化合物是Tofacitinib。该合成分子是JAK-STAT信号通路的抑制剂，已发现可以有效抑制类风湿关节炎和类似疾病的致病过程。我们测试了Tofacitinib对小鼠EAU和相关免疫反应的发展的影响。主要观察结果包括：（i）通过组织学分析分析的tofacitinib治疗EAU的中等发展； （ii）在产生IFN-17的IFN-生产时，用Tofacitinib治疗的小鼠的淋巴细胞不如其对照（用媒介物处理的小鼠）。通过将细胞因子释放到培养上清液中以及通过流式细胞仪确定的细胞内表达细胞因子的淋巴细胞的比例来证明差异。 （iii）在体外系统中还观察到了to依替尼对Th1和Th17产生的选择性抑制作用，在该系统中，通过针对CD3和CD28的抗体激活了中含CD4细胞。在这些培养物中添加tofacitib添加了tofacitib，选择性抑制了IFN-的产生，但不能抑制IL-17的产生。 Tristraprolin（TTP）对EAU和相关免疫反应的发展的影响 最近在PNAS（2016年113：1865，2016）发表的一份出版物报道，TTP mRNA稳定的小鼠（“ Ki”小鼠）对实验性自身免疫性疾病具有抗药性。佩里·黑皮（Perry Blackshear，Niehs），北卡罗来纳州研究三角公园（生成“ Ki小鼠），为我们发展EAU和相关免疫反应的能力，对我们提供了非常不可取的症状：所有的WT小鼠，所有的疾病都在ki ki中，ki ki and ki ki and ki ki is ki ki and ki ki is ki is ki is ki y no ki is ki is ki is ki and，“ ki”小鼠及其野生型（wt）的控制均由北卡罗来纳州研究的三角形公园（生成“ Ki小鼠））提供给我们。 “ Ki”小鼠的免疫反应明显低于WT小鼠的免疫反应。 这项研究还产生了令人信服的数据，表明与雄性小鼠相比，雌性小鼠对自身免疫性的敏感性更高。因此，尽管雌性小鼠的EAU严重程度的平均水平（通过组织学分析确定）为2.5 +/- 0.31，但男性的平均水平为0.95 +/- 0.21（p = 0.006）。 此外，母小鼠组之间的父母的细胞免疫反应及其WT对照之间的差异比男性组之间的差异更为明显。 此外，这项研究还提供了其他有趣的信息：研究三角公园中动物设施的WT（C57BL/6）小鼠更加一致地开发了EAU，并且水平明显高于杰克逊实验室提供的C57BL/6小鼠。一个可能的解释是，研究三角形公园设施中的小鼠的微生物组与杰克逊实验室的小鼠不同，它更好地促进了EAU的致病过程。