Effect of SS-associated cytokines on salivary gland dysfunction

描述（申请人提供）：干燥综合征（Sjogren's syndrome， SS）是一种以唾液腺炎症和功能障碍为特征的自身免疫性疾病，导致分泌功能受损。SS患者唾液腺中促炎细胞因子肿瘤坏死因子-1 （TNF1）、干扰素-3 （IFN3）、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）水平升高，尽管人们对这些细胞因子对唾液上皮细胞紧密连接（TJ）完整性的影响知之甚少，而TJ完整性是建立驱动唾液分泌的经上皮离子梯度所必需的。我们已经证明，长期暴露于极化大鼠腮腺（Par-C10）上皮细胞单层的TNF1和/或IFN3会降低经皮上皮抵抗（TER）和经皮上皮阴离子分泌，这些阴离子分泌是由一种毒蕈碱胆碱能受体激动剂或一种P2Y2核苷酸受体激动剂碳醇（carbachol）诱导的。相比之下，TNF1和/或IFN3对激动剂诱导的Par-C10细胞内钙浓度[Ca2+]i的升高没有影响，这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TJs中，claudin-1被TNF1和/或IFN3选择性下调。在用TNF1处理的细胞中，claudin-1的下调在不含细胞因子的培养基中孵育24小时后恢复到正常水平。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，表明沉默claudin-1表达会降低Par-C10单层中的TER。此外，tnf -1和/或IFN3处理Par- C10三维（3D）腺泡球可改变TJ蛋白occludin和ZO-1的细胞分布。基于这些初步发现，我们将检验细胞因子诱导的TJ蛋白表达和/或分布的变化影响parc10细胞单层中TJ完整性的总体假设，这与完整腮腺中与细胞因子生成相关的唾液分泌减少相一致。此外，我们建议通过体外和体内SS模型来确定细胞因子诱导的TJ完整性破坏的细胞机制。Specific Aim 1的研究将表征细胞因子诱导的claudin-1表达降低的细胞机制。特异性目标2的研究将确定TNF1-和ifn3介导的Par-C10细胞中TJ蛋白磷酸化和TJ复合物去除的变化。Specific Aim 3的研究将表征C57BL/6腮腺中TJ mRNA和蛋白的表达、TJ形态和TJ细胞分布。NOD-Aec1Aec2小鼠模型和过表达TNF1的小鼠，与野生型小鼠比较。这些研究将使我们更好地理解唾液腺疾病促炎阶段唾液分泌减少的机制，这是定义腮腺腺泡中调节TJ结构完整性的信号通路的重要的第一步。

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.