Effect of SS-associated cytokines on salivary gland dysfunction

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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.