Modulation of conjunctival goblet cell differentiation by immunoregulatory cells

描述（由申请人提供）：免疫调节细胞对结膜杯状细胞分化的调节：本项目将研究局部免疫调节通路在结膜上皮稳态中的作用以及结膜上皮对干眼症干燥应激的反应。我们假设干眼症导致结膜内CD8+和34T上皮内淋巴细胞（IEL）数量减少，导致免疫调节细胞因子（如TGF-22）和杯状细胞支持因子（如角化细胞生长因子）水平下降。这促进了产生干扰素- γ (IFN- γ)的CD4+ T细胞迁移到结膜上皮，导致细胞因子失衡，有利于结膜形成，而不是结膜上皮的杯状细胞分化。在Aim 1中，正常小鼠眼睛结膜活检中34T细胞的主要亚群将通过免疫染色进行表征，体外扩增后，34和CD8 IEL的细胞因子和生长因子谱将通过多重免疫珠测定、实时PCR和微阵列进行评估。目的2是研究IEL对干燥应激后结膜杯状细胞存活的影响。为了实现这一目标，我们将在C57BL/6小鼠、TCR-/-小鼠（34 T细胞敲除）、CD8-/-小鼠和显性阴性成纤维细胞生长因子受体2 （dnFGFR2）转基因小鼠的干眼实验前和实验后5天和10天评估CD8+和34个IELs的常驻密度。评估KGF和TGF-22表达的时间过程。将这些因子共定位到34T和CD8+ T细胞将通过双标记免疫组织化学完成。通过评估正常和干眼小鼠结膜切片中杯状细胞和CD4+细胞密度以及杯状细胞粘蛋白MUC5AC和化生标志物小脯氨酸富残基2蛋白（SPRR-2）的表达，可以证实34T和CD8+ T细胞以及KGF和TGF-22缺失对结膜稳态的影响。利用腺病毒载体将KGF和TGF-22过表达，目的是防止干眼症中发生的杯状细胞丢失。公共卫生相关性。本研究将探讨眼表结膜中免疫调节细胞在维持体内平衡和应对干燥环境压力方面的功能。这些研究将为控制眼表炎症的自然机制提供新的信息，并可能为治疗眼表炎症性疾病提供新的见解。

DESCRIPTION (provided by applicant): Modulation of conjunctival goblet cell differentiation by immunoregulatory cells: This project will investigate the role of local immunoregulatory pathways on conjunctival epithelial homeostasis and the response of the conjunctival epithelium to the desiccating stress of dry eye. We hypothesize that dry eye causes a decrease in the number of CD8+ and 34T intraepithelial lymphocytes (IEL) in the conjunctiva, resulting in decreased levels of immunoregulatory cytokines (e.g. TGF-22) and goblet-cell supporting factors (e.g. keratinocyte growth factor). This facilitates migration of interferon-gamma (IFN-?) producing CD4+ T cells into the conjunctival epithelium resulting in a cytokine imbalance that favors cornified envelope production, rather than goblet cell differentiation by the conjunctival epithelium. In Aim 1, the predominant subset of 34T cells in conjunctival biopsies from normal mouse eyes will be characterized by immunostaining and following in vitro expansion, the cytokine and growth factor profile of 34 and CD8 IEL will be evaluated by a multiplex immunobead assay, real-time PCR and microarray. The objective of Aim 2 is to investigate the role of IEL on survival of conjunctiva goblet cells after desiccating stress. To accomplish this, the resident density of CD8+ and 34 IELs will be evaluated prior to and after 5 and 10 days of experimental dry eye in C57BL/6 mice and in TCR-/- mice (34 T cell knock-out), CD8-/- mice and dominant negative fibroblast growth factor receptor 2 (dnFGFR2) transgenic mice. The time course of expression of KGF and TGF-22 will be evaluated. Co-localization of these factors to 34T and CD8+ T cells will be accomplished by dual label immunohistochemistry. The consequences of loss of 34T and CD8+ T cells and KGF and TGF-22 on conjunctival homeostasis will be confirmed by evaluating goblet cell and CD4+ cell density and expression of the goblet cell mucin MUC5AC and metaplasia marker small proline rich residue 2 protein (SPRR-2) in conjunctival sections from normal and dry eye mice. KGF and TGF-22 will be overexpressed using adenoviral vectors with the intent of preventing the goblet cell loss that occurs in dry eye. PUBLIC HEALTH RELEVANCE. This proposal will investigate the function of immuno-regulatory cells in the conjunctiva of the eye surface with regard to maintaining homeostasis and responding to desiccating environmental stress. These studies will provide new information about natural mechanisms to control inflammation on the ocular surface and may provide new insight into treating ocular surface inflammatory diseases.