The role of the P2X7 nucleotide receptor in salivary gland inflammation

Salivary gland inflammation is a hallmark of Sjogren's syndrome (SS), a common autoimmune disease characterized by lymphocytic infiltration and the consequent impairment of the salivary gland and loss of saliva secretion, predominantly in women. The current therapeutic management of SS is relatively ineffective and does not address the underlying inflammatory processes contributing to the pathology of SS. In this study, two novel therapeutic approaches were evaluated to limit salivary gland inflammation and improve secretory function, i.e., antagonism of the P2X7 nucleotide receptor (P2X7R), which prevents salivary gland inflammation and activation of the P2Y[subscript2] nucleotide receptor (P2Y[subscript2]R) which stimulates the regeneration of damaged salivary glands. The P2X7R is an ATP-gated non-selective cation channel that regulates inflammatory responses in cells and tissues, including salivary gland epithelium. The P2X7R contributes to the pathology of a variety of inflammatory diseases, including rheumatoid arthritis and inflammatory bowel disease. In immune cells, P2X7R activation induces the production of pro-inflammatory cytokines, including IL-1[beta] and IL-18, by inducing the oligomerization of the multiprotein complex NLRP3-type inflammasome. This study (Chapter II) sheds light on the role of the P2X7R in salivary gland inflammation and hyposalivation. Our results show that in primary mouse submandibular gland (SMG) epithelial cells, P2X7R activation induces the assembly of the NLRP3 inflammasome and the maturation and release of IL-1[beta], responses that are absent in SMG cells isolated from mice devoid of P2X7Rs (P2X7R[superscript-/-]). P2X7R-mediated IL-1[beta] release in SMG epithelial cells is dependent on downhill transmembrane Na[superscript+] and/or K[superscript+] fluxes, the activation of heat shock protein 90 (HSP90), a protein required for the activation and stabilization of the NLRP3 inflammasome, and the generation of mitochondrial ROS. In vivo administration of the P2X7R antagonist A438079 in the CD28[superscript-/-], IFN[superscript][superscript-/-], NOD.H-2[superscripth4] 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. The P2Y[subscript2]R, a G protein-coupled receptor equipotently activated by ATP and UTP, is upregulated in a variety of tissues, including salivary gland epithelium, in response to injury or stress and is proposed to play a role in tissue regeneration. The results indicated that P2Y2R activation with UTP enhances the migration, aggregation and self-organization of dispersed salivary epithelial cells forming spheres that display characteristics similar to differentiated acini in salivary glands. One of the consequences of the chronic inflammatory disease SS is the fibrosis of the salivary gland. The role of transforming growth factor- [beta] (TGF-[beta]) is well established in the fibrosis and regeneration of various organs, including the liver, lung and kidney. In this study, results with a submandibular gland (SMG) duct ligation-induced mouse model of fibrosis indicated that 7 days of SMG duct ligation resulted in upregulation of TGF-β signaling components which correlated with the upregulation of the fibrosis markers collagen 1 and fibronectin, responses that were inhibited by administration of the TGF-[beta] receptor 1 inhibitors. These results suggest that TGF-[beta] signaling contributes to duct ligation-induced changes in salivary epithelium that correlate with glandular fibrosis.Includes biblographical reference

Increased Expression of TGF-β Signaling Components in a Mouse Model of Fibrosis Induced by Submandibular Gland Duct Ligation.

Transforming growth factor-β (TGF-β) is a multi-functional cytokine with a well-described role in the regulation of tissue fibrosis and regeneration in the liver, kidney and lung. Submandibular gland (SMG) duct ligation and subsequent deligation in rodents is a classical model for studying salivary gland damage and regeneration. While previous studies suggest that TGF-β may contribute to salivary gland fibrosis, the expression of TGF-β signaling components has not been investigated in relation to mouse SMG duct ligation-induced fibrosis and regeneration following ductal deligation. Following a 7 day SMG duct ligation, TGF-β1 and TGF-β3 were significantly upregulated in the SMG, as were TGF-β receptor 1 and downstream Smad family transcription factors in salivary acinar cells, but not in ductal cells. In acinar cells, duct ligation also led to upregulation of snail, a Smad-activated E-cadherin repressor and regulator of epithelial-mesenchymal transition, whereas in ductal cells upregulation of E-cadherin was observed while snail expression was unchanged. Upregulation of these TGF-β signaling components correlated with upregulation of fibrosis markers collagen 1 and fibronectin, responses that were inhibited by administration of the TGF-β receptor 1 inhibitors SB431542 or GW788388. After SMG regeneration following a 28 day duct deligation, TGF-β signaling components and epithelial-mesenchymal transition markers returned to levels similar to non-ligated controls. The results from this study indicate that increased TGF-β signaling contributes to duct ligation-induced changes in salivary epithelium that correlate with glandular fibrosis. Furthermore, the reversibility of enhanced TGF-β signaling in acinar cells of duct-ligated mouse SMG after deligation indicates that this is an ideal model for studying TGF-β signaling mechanisms in salivary epithelium as well as mechanisms of fibrosis initiation and their resolution

P2X7 receptor antagonism prevents IL-1β release from salivary epithelial cells and reduces inflammation in a mouse model of autoimmune exocrinopathy

Salivary gland inflammation is a hallmark of Sjogren'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-1 beta 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-1 beta, a response that is absent in SMG cells isolated from mice deficient in P2X7Rs (P2X7R(-/-)). P2X7R-mediated IL-1 beta 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-1 beta release. Lastly, in vivo administration of the P2X7R antagonist A438079 in the CD28(-/-), IFN gamma(-/-), NOD.H-2(h4) 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.National Institute of Dental and Craniofacial Research, National Institutes of Health [DE023342]12 month embargo; Published online: 10 Aug 17.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

P2 receptors as therapeutic targets in the salivary gland: From physiology to dysfunction

Although often overlooked in our daily lives, saliva performs a host of necessary physiological functions, including lubricating and protecting the oral cavity, facilitating taste sensation and digestion and maintaining tooth enamel. Therefore, salivary gland dysfunction and hyposalivation, often resulting from pathogenesis of the autoimmune disease Sjögren’s syndrome or from radiotherapy of the head and neck region during cancer treatment, severely reduce the quality of life of afflicted patients and can lead to dental caries, periodontitis, digestive disorders, loss of taste and difficulty speaking. Since their initial discovery in the 1970s, P2 purinergic receptors for extracellular nucleotides, including ATP-gated ion channel P2X and G protein-coupled P2Y receptors, have been shown to mediate physiological processes in numerous tissues, including the salivary glands where P2 receptors represent a link between canonical and non-canonical saliva secretion. Additionally, extracellular nucleotides released during periods of cellular stress and inflammation act as a tissue alarmin to coordinate immunological and tissue repair responses through P2 receptor activation. Accordingly, P2 receptors have gained widespread clinical interest with agonists and antagonists either currently undergoing clinical trials or already approved for human use. Here, we review the contributions of P2 receptors to salivary gland function and describe their role in salivary gland dysfunction. We further consider their potential as therapeutic targets to promote physiological saliva flow, prevent salivary gland inflammation and enhance tissue regeneration

Submandibular gland ductal ligation followed by deligation results in reversible acinar cell atrophy, immune cell infiltration and glandular fibrosis.

<p>(A, D, G) Control SMG, (B, E, H) 7 day duct-ligated SMG and (C, F, I) 7 day duct-ligated SMG followed by deligation and recovery for 28 days were subjected to immunofluorescence staining with (A-C) antibodies to the acinar cell marker AQP5 (green) and Hoechst nuclear stain (blue), (D-F) antibodies to the pan-immune cell marker CD45 (red) and Hoechst nuclear stain (blue) and (G-I) hematoxylin and eosin. Results indicate that ligation of the main SMG excretory duct induces (B) loss of acinar cells, (E) substantial immune cell infiltration and (H) atrophy of acinar cells (white arrow) and loss of secretory granules (pink) within ductal cells (black arrow) that is reversed by subsequent deligation (C, F, I). Images are representative of results from at least 3 independent experiments and scale bar = 20 μm.</p

Upregulation of Snail and Slug in response to SMG duct ligation is reversible upon duct deligation.

<p>RT-PCR analysis of cDNA prepared from whole SMGs shows significant upregulation of (A) Snail and (B) Slug mRNA after a 7 day duct ligation, which is reversed to control levels after deligation and a 28 day recovery. Data represent means ± S.E.M. (n = 4 for control, n = 8 for 7 day ligation, n = 5 for 7 day ligation, deligation and a 28 day recovery), where ***<i>P</i><0.001 and *<i>P</i><0.05 indicate significant differences in mRNA expression, as compared to control SMG. (C) Immunofluorescence analysis reveals that Snail (red) expression is primarily upregulated in acinar cells after a 7 day duct ligation, and returns to control levels after deligation and recovery. (D) Dual-immunofluorescence staining confirmed the colocalization of Snail (red) with the acinar marker AQP5 (green) following a 7 day duct ligation. Hoechst nuclear stain in blue and scale bar = 20 μm. Images are representative of results from at least 3 independent experiments.</p

Upregulation of TGF-β1, TGF-β3 and TGF-β R1 in response to SMG duct ligation is reversible upon deligation.

<p>(A) Western analysis of whole gland lysates shows upregulation of pro-TGF-β1/2/3 expression (top) in response to 7 day SMG duct ligation that is reversible following deligation and recovery for 28 days. Ponceau S staining (bottom) shows equal amounts of total protein in each well. (B) RT-PCR analysis of cDNA prepared from whole SMGs shows significant upregulation of TGF-β1 and TGF-β3, but not TGF-β2, mRNA expression after 7 days of SMG excretory duct ligation (grey bars), as compared to contralateral control glands (white bars). When ducts were ligated for 7 days then deligated for 28 days (black bars), expression levels of TGF-β1 and TGF-β3 mRNA return to control levels, whereas TGF-β2 mRNA levels remain unchanged. Data represent means ± S.E.M. (n = 7 for control and 7 day ligation, n = 5 for 7 day ligation, deligation and a 28 day recovery), where *<i>P</i><0.05 and **<i>P</i><0.01 indicate significant differences in mRNA expression, as compared to control. (C) Dual-immunofluorescence analysis of 8 μm frozen SMG sections for control, 7 day ligation and 7 day ligation followed by deligation and recovery for 28 days reveals that TGF-β R1 (red) expression is upregulated primarily in acinar cells (marked by residual AQP5 expression; green) after a 7 day SMG duct ligation, whereas little staining is visible in ductal cells. After a 7 day duct ligation followed by deligation and a 28 day recovery, TGF-β R1 expression levels are similar to control. Hoechst nuclear stain in blue and scale bar = 20 μm. Images are representative of results from at least 3 independent experiments.</p

TGF-β R1 inhibitors SB431542 and GW788388 attenuate duct ligation-induced upregulation of fibrosis markers.

<p>RT-PCR analysis of 7 day ligated and contralateral unligated control SMGs shows significant attenuation of 7 day duct ligation-induced collagen 1 and fibronectin mRNA upregulation in mice treated with either SB431542 (20 mg/kg mouse weight) or GW788388 (2 mg/kg mouse weight), as compared to DMSO-treated controls. Data represent means ± S.E.M. (n = 6 for DMSO, n = 6 for SB431542, n = 5 for GW788388), where *<i>P</i><0.05 and ***<i>P</i><0.001 indicate significant differences in mRNA expression, as compared to DMSO-treated controls.</p

Upregulation of Smad2/3, TAK1 and TAB1 in response to SMG duct ligation is reversible upon duct deligation.

<p>(A) Western blot analysis of whole gland lysates from unligated control SMG, SMG after a 7 day duct ligation or a 7 day ligation followed by deligation and a 28 day recovery. Duct ligation increases Smad2/3 expression (top) and phospho-Smad2/3 levels (middle) that return to control levels after deligation and recovery. Ponceau S staining (bottom) shows equal amounts of total protein in each well. Immunofluorescence analysis revealed that increases in (B) Smad2/3 (red) expression and (C) p-Smad2/3 (red) levels after a 7 day duct ligation are restricted to acinar cells (marked by residual AQP5 expression; green), where p-Smad2/3 is localized to the nucleus, as determined by colocalization with Hoechst nuclear stain (blue). Smad2/3 and p-Smad2/3 returned to control levels after deligation of SMG ducts and recovery. Images are representative of results from at least 3 independent experiments and scale bar = 20 μm. (D) RT-PCR analysis of whole gland lysates shows increased TAK1 and TAB1 mRNA expression following a 7 day duct ligation, which was reversed to control levels following deligation and a 28 day recovery. Data represent means ± S.E.M. (n = 6 for control, n = 8 for 7 day ligation, n = 5 for 7 day ligation, deligation and a 28 day recovery), where *<i>P</i><0.05 and **<i>P</i><0.01 indicate significant differences in mRNA expression, as compared to control SMG.</p