Abstract
Salivary gland inflammation is a hallmark of Sjögren's syndrome (SS), a common autoimmune disease characterized by lymphocytic infiltration of the salivary gland and loss of saliva secretion, predominantly in women. The P2X7 receptor (P2X7R) is an ATP-gated nonselective cation channel that induces inflammatory responses in cells and tissues, including salivary gland epithelium. In immune cells, P2X7R activation induces the production of proinflammatory cytokines, including IL-1β and IL-18, by inducing the oligomerization of the multiprotein complex NLRP3-type inflammasome. Here, our results show that in primary mouse submandibular gland (SMG) epithelial cells, P2X7R activation also induces the assembly of the NLRP3 inflammasome and the maturation and release of IL-1β, a response that is absent in SMG cells isolated from mice deficient in P2X7Rs (P2X7R-/-). P2X7R-mediated IL-1β release in SMG epithelial cells is dependent on transmembrane Na+ and/or K+ flux and the activation of heat shock protein 90 (HSP90), a protein required for the activation and stabilization of the NLRP3 inflammasome. Also, using the reactive oxygen species (ROS) scavengers N-acetyl cysteine and Mito-TEMPO, we determined that mitochondrial reactive oxygen species are required for P2X7R-mediated IL-1β release. Lastly, in vivo administration of the P2X7R antagonist A438079 in the CD28-/-, IFNγ-/-, NOD.H-2h4 mouse model of salivary gland exocrinopathy ameliorated salivary gland inflammation and enhanced carbachol-induced saliva secretion. These findings demonstrate that P2X7R antagonism in vivo represents a promising therapeutic strategy to limit salivary gland inflammation and improve secretory function.
Highlights
Salivary gland inflammation is a hallmark of Sjögren’s syndrome (SS), a common autoimmune disease characterized by lymphocytic infiltration of the salivary gland and loss of saliva secretion, predominantly in women
We have previously shown that P2X7 receptor (P2X7R) activation in primary mouse submandibular gland (SMG) epithelial cell aggregates increases the activity of caspase-1, the protease responsible for IL-1 processing [19]; it is unclear how P2X7R activation in salivary glands regulates IL-1 maturation and release
ATP-induced IL-1 release was robust in the enriched SMG epithelial cells and the standard SMG cell preparations (Fig. 1A), suggesting that SMG epithelial cells are the primary source of ATP-induced IL-1 release in our in vitro experiments
Summary
P2X7 receptor antagonism prevents IL-1 release from salivary epithelial cells and reduces inflammation in a mouse model of autoimmune exocrinopathy. Our previous studies showed that P2X7R activation by ATP induces inflammatory responses, e.g. increases in caspase-1 activity and membrane blebbing, in freshly prepared mouse submandibular gland (SMG) cells from wild-type mice, but not from P2X7Rdeficient (P2X7RϪ/Ϫ) mice [19]. We demonstrate for the first time in salivary gland epithelial cells that P2X7R activation promotes NLRP3 inflammasome activation and assembly and IL-1 release through a mechanism involving transmembrane Naϩ and Kϩ flux, ROS production, and heat shock protein 90 (HSP90) function. Our in vivo results show that administration of the P2X7R antagonist, A438079, in the CD28Ϫ/Ϫ, IFN␥Ϫ/Ϫ, NOD.H-2h4 mouse model of autoimmune exocrinopathy [35], reduced salivary gland lymphocytic infiltrates and enhanced saliva secretion, suggesting that antagonism of the P2X7R is a promising approach to prevent salivary gland inflammation and associated hyposalivation in the autoimmune disease SS
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