Modulation of conjunctival goblet cell differentiation by immunoregulatory cells

描述(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，以及结膜上皮对干燥性干眼压力的反应。我们假设干眼导致结膜中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(例如转化生长因子-22)和杯状细胞支持因子(例如角质细胞生长因子)水平降低。这有助于干扰素-γ(IFN-？)的迁移。产生CD4+T细胞进入结膜上皮，导致细胞因子失衡，有利于角化包膜的产生，而不是结膜上皮对杯状细胞的分化。在目标1中，对正常小鼠结膜活检组织中主要的34T细胞亚群进行免疫染色和体外扩增，并用多重免疫珠法、实时荧光定量聚合酶链式反应和基因芯片检测34和CD8IEL的细胞因子和生长因子的变化。目的2研究IEL在干燥应激后结膜杯状细胞存活中的作用。为了实现这一目标，我们将在C57BL/6小鼠、TCR-/-小鼠(34个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)转基因小鼠中，在实验性干眼5天和10天之前和之后评估CD8+和34个IEL的驻留密度。观察KGF和TGF-22表达的时程变化。这些因子与34T和CD8+T细胞的共定位将通过双标记免疫组织化学完成。通过检测正常小鼠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含残基2蛋白(SPRR-2)的表达，证实34T和CD8+T细胞、KGF和转化生长因子-22的缺失对结膜动态平衡的影响。KGF和转化生长因子-22将使用腺病毒载体过表达，目的是防止干眼症中发生的杯状细胞丢失。与公共卫生相关。这项建议将研究眼表结膜中免疫调节细胞在维持内环境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视角。

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.