Effect of SS-associated cytokines on salivary gland dysfunction

描述（由申请人提供）：干燥综合征（SS）是一种自身免疫性疾病，其特征在于炎症和唾液腺功能障碍，导致分泌功能受损。促炎细胞因子肿瘤坏死因子-1（TNF 1）、干扰素-3（IFN 3）、IL-12、白细胞介素-6（IL-6）、白细胞介素-18（IL-18）和白细胞介素-12（IL-12）的水平在SS患者的唾液腺中升高，尽管对这些细胞因子对唾液腺上皮细胞紧密连接（TJ）的影响知之甚少，这是建立驱动唾液分泌的跨上皮离子梯度所必需的。我们已经证明，长期暴露极化大鼠腮腺（Par-C10）上皮细胞单层TNF 1和/或IFN 3降低跨上皮阻力（TER）和跨上皮阴离子分泌诱导卡巴胆碱，毒蕈碱胆碱能受体激动剂，或UTP，P2 Y2核苷酸受体激动剂。相反，TNF 1和/或IFN 3对Par-C10细胞中激动剂诱导的细胞内钙浓度[Ca 2 +]i增加没有影响，表明单个细胞信号传导不受细胞因子的影响。我们的研究表明，在TJ中，claudin-1被TNF 1和/或IFN 3选择性下调。在用TNF 1处理的细胞中，细胞在无细胞因子培养基中孵育24小时后，紧密连接蛋白-1下调恢复到正常水平.在这些条件下，claudin-1表达的恢复与TER和激动剂诱导的短路电流（Isc）的增加相对应。我们已经获得了新的初步数据，表明封闭蛋白-1表达的沉默降低了Par-C10单层中的TER。此外，TJ蛋白闭合蛋白和ZO-1的细胞分布通过Par-C10三维（3D）腺泡球的TNF 1和/或IFN 3处理而改变。基于这些初步研究结果，我们将检查总体假设，即细胞因子诱导的TJ蛋白表达和/或分布的变化影响Par-C10细胞单层中TJ的完整性，这与完整腮腺中与细胞因子产生相关的唾液分泌减少一致。此外，我们建议使用SS的体外和体内模型来确定潜在的姜黄素诱导的TJ完整性破坏的细胞机制。在特定目标1的研究将表征潜在的机制，马槟榔诱导的claudin-1表达的减少。特定目标2中的研究将确定TNF 1和IFN 3介导的TJ蛋白磷酸化的变化以及从Par-C10细胞中TJ复合物的去除。与野生型小鼠相比，特定目标3的研究将表征SS的C57 BL/6. NOD-Aec 1Aec 2小鼠模型和过表达TNF 1的小鼠的腮腺中TJ mRNA和蛋白表达、TJ形态和TJ细胞分布。这些研究将提供一个更好的了解唾液分泌减少的机制，在唾液腺疾病的促炎阶段，一个重要的初始步骤，在定义研究不足的信号通路，调节TJ结构完整性腮腺腺泡。 公共卫生相关性：干燥综合征（SS）是一种以唾液腺功能障碍为特征的自身免疫性疾病，可导致严重的口腔干燥。促炎细胞因子在SS患者的血浆和唾液腺中上调，然而，很少有人知道它们在唾液腺功能障碍中的作用。我们建议确定促炎细胞因子在唾液腺紧密连接完整性导致唾液分泌减少中的作用。

DESCRIPTION (provided by applicant): Sjogren's syndrome (SS) is an autoimmune disorder characterized by inflammation and dysfunction of salivary glands, resulting in impaired secretory function. Levels of the pro-inflammatory cytokines tumor necrosis factor-1 (TNF1), interferon-3 (IFN3), IL-12, interleukin-6 (IL-6), interleukin-18 (IL-18) and interleukin-12 (IL-12) are elevated in salivary glands of patients with SS, although little is known about the effects of these cytokines on salivary epithelial cell tight junction (TJ) integrity which is necessary to establish transepithelial ion gradients that drive saliva secretion. We have demonstrated that chronic exposure of polarized rat parotid gland (Par-C10) epithelial cell monolayers to TNF1 and/or IFN3 decreases transepithelial resistance (TER) and transepithelial anion secretion induced by carbachol, a muscarinic cholinergic receptor agonist, or UTP, a P2Y2 nucleotide receptor agonist. In contrast, TNF1 and/or IFN3 had no effect on agonist-induced increases in the intracellular calcium concentration [Ca2+]i in Par-C10 cells indicating that individual cell signaling is unaffected by cytokines. Our studies show that among the TJs, claudin-1 is selectively downregulated by TNF1 and/or IFN3. In cells treated with TNF1, claudin-1 downregulation returns to normal levels upon incubation of cells for 24 h in cytokine- free medium. Under these conditions, recovery of claudin-1 expression corresponds with increases in TER and agonist-induced short circuit current (Isc). We have obtained new preliminary data demonstrating that silencing of claudin-1 expression decreases TER in Par-C10 monolayers. Furthermore, the cellular distribution of the TJ proteins occludin and ZO-1 was altered by TNF1 and/or IFN3 treatment of Par- C10 three-dimensional (3D) acinar spheres. Based on these preliminary findings, we will examine the overall hypothesis that cytokine- induced changes in the expression and/or distribution of TJ proteins affects TJ integrity in Par-C10 cell monolayers, consistent with a loss in saliva secretion associated with cytokine generation in the intact parotid gland. In addition, we propose to identify the cellular mechanisms underlying cytokine-induced disruption of TJ integrity using in vitro and in vivo models of SS. Studies in Specific Aim 1 will characterize the mechanisms underlying cytokine-induced decreases in claudin-1 expression. Studies in Specific Aim 2 will identify the TNF1- and IFN3-mediated changes in TJ protein phosphorylation and removal from the TJ complex in Par-C10 cells. Studies in Specific Aim 3 will characterize TJ mRNA and protein expression, TJ morphology, and TJ cellular distribution in parotid glands of the C57BL/6.NOD-Aec1Aec2 mouse model of SS and mice overexpressing TNF1, as compared to wild type mice. These studies will provide a greater understanding the mechanisms whereby saliva secretion is decreased during the pro-inflammatory stages of salivary gland diseases, a significant initial step in defining the poorly-studied signaling pathways that regulate TJ structural integrity in parotid acini. PUBLIC HEALTH RELEVANCE: Sjogren's syndrome (SS) is an autoimmune disease characterized by salivary gland dysfunction leading to severe dryness of the oral cavity. Pro-inflammatory cytokines are up-regulated in plasma and in salivary glands from patients with SS, however, little is known of their role in salivary gland dysfunction. We propose to determine the role of pro-inflammatory cytokines in salivary gland tight junction integrity causing reduction of saliva secretion.