Modulation of conjunctival goblet cell differentiation by immunoregulatory cells

描述（由申请方提供）：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途径对结膜上皮稳态的作用以及结膜上皮对干眼干燥应激的反应。我们假设干眼症导致结膜中CD 8+和34 T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（如角质形成细胞生长因子）水平降低。这有利于干扰素-γ（IFN-？）产生CD 4 + T细胞进入结膜上皮，导致细胞因子失衡，其有利于结膜上皮产生皮质包膜，而不是杯状细胞分化。在目的1中，将通过免疫染色表征正常小鼠眼睛结膜活检中的主要34 T细胞亚群，并在体外扩增后，将通过多重免疫珠测定、实时PCR和微阵列评价34和CD 8 IEL的细胞因子和生长因子谱。目的二是探讨IEL对结膜杯状细胞在干燥应激后存活的作用。为了实现这一点，将在C57 BL/6小鼠和TCR-/-小鼠（34 T细胞敲除）、CD 8-/-小鼠和显性阴性成纤维细胞生长因子受体2（dnFGFR 2）转基因小鼠中，在实验性干眼之前和之后5天和10天评价CD 8+和34 IEL的驻留密度。将评价KGF和TGF-22表达的时间过程。将通过双标记免疫组织化学完成这些因子对34 T和CD 8 + T细胞的共定位。通过评价正常和干眼症小鼠结膜切片中杯状细胞和CD 4+细胞密度以及杯状细胞粘蛋白MUC 5AC和化生标志物小脯氨酸富集残基2蛋白（SPRR-2）的表达，证实34 T和CD 8 + T细胞以及KGF和TGF-22损失对结膜稳态的影响。KGF和TGF-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损失。 公共卫生相关性。本研究将探讨眼表结膜免疫调节细胞在维持内环境稳定和应对干燥环境压力方面的功能。这些研究将提供有关控制眼表炎症的自然机制的新信息，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.