Mouse Submandibular Gland Research Articles

3D Organoid Formation from the Murine Salivary Gland Cell Line SIMS.

Salivary glands consist of multiple phenotypically and functionally unique cell populations, such as the acinar, ductal, and myoepithelial cells that help produce, modify, and secrete saliva (Lombaert et al., 2011). Identification of mechanisms and factors that regulate these populations has been of key interest, as salivary gland-related diseases have detrimental effects on these cell populations. A variety of approaches have been used to understand the roles different signaling mechanisms and transcription factors play in regulating salivary gland development and homeostasis. Differentiation assays have been performed with primary salivary cells in the past (Maimets et al., 2016), however this approach may sometimes be limiting due to tissue availability, labor intensity of processing the tissue samples, and/or inability to long-term passage the cells. Here we describe in detail a 3D differentiation assay to analyze the differentiation potential of a salivary gland cell line, SIMS, which was immortalized from an adult mouse submandibular salivary gland (Laoide et al., 1996). SIMS cells express cytokeratin 7 and 19, which is characteristic for a ductal cell type. Although adult acinar and myoepithelial cells were found in vivo to preserve their own cell population through self-duplication (Aure et al., 2015; Song et al. 2018), in some cases duct cells can differentiate into acinar cells in vivo, such as after radiation injury (Lombaert et al., 2008; Weng et al., 2018). Thus, utilization of SIMS cells allows us to target and analyze the self-renewal and differentiation effects of ductal cells under specific in vitro controlled conditions.

Cholesterol metabolism plays a crucial role in the regulation of autophagy for cell differentiation of granular convoluted tubules in male mouse submandibular glands

It has been long appreciated that sex hormone receptors are expressed in various non-gonadal organs. However, it remains unclear how sex hormones regulate the morphogenesis of these non-gonadal organs. To address this issue, we used a male mouse model of androgen-dependent salivary gland morphogenesis. Mice with excessive cholesterol synthesis in the salivary glands exhibited defects in the maturation of granular convoluted tubules (GCTs), which is regulated through sex hormone-dependent cascades. We found that excessive cholesterol synthesis resulted in autophagy failure specifically in the duct cells of salivary glands, followed by the accumulation of NRF2, a transcription factor known as one of the specific substrates for autophagy. The accumulated NRF2 suppressed the expression of Foxa1, which forms a transcriptional complex with the androgen receptor to regulate target genes. Taken together, our results indicate that cholesterol metabolism plays a crucial role in GCT differentiation through autophagy.

Tissue-Resident Memory CD8+ T Cells Acting as Mediatorsof Salivary Gland Damage in a Murine Model of Sjögren's Syndrome.

Although a role for CD4+ T cells in the pathogenesis of Sjögren's syndrome (SS) has been documented, the pathogenic significance of CD8+ T cells is unclear. The aim of this study was to investigate the role of CD8+ T cells in the development of SS. Flow cytometry and immunofluorescence analyses were utilized to detect T cell infiltration within the labial salivary glands of patients with primary SS. In parallel, p40-/- CD25-/- mice were used as a murine model of SS. In addition, mice with genetic knockout of CD4, CD8a, or interferon-γ (IFNγ) were crossed with p40-/- CD25-/- mice to study the pathogenic significance of specific lineage subpopulations, including functional salivary gland tests as well as histopathologic and serologic data. A CD8+ T cell-specific depletion antibody was used in this murine SS model to evaluate its potential as a therapeutic strategy. CD8+ T cells with a tissue-resident memory phenotype outnumbered CD4+ T cells in the labial salivary glands of patients with SS, and were primarily colocalized with salivary duct epithelial cells and acinar cells. Furthermore, infiltrating CD8+ T cells with a CD69+CD103+/- tissue-resident phenotype and with a significant elevation of IFNγ production were dominant in the submandibular glands of mice in this murine SS model. CD8a knockout abrogated the development of SS in these mice. Knockout of IFNγ decreased CD8+ T cell infiltration and gland destruction. More importantly, depletion of CD8+ T cells fully protected mice against the pathologic manifestations of SS, even after the onset of disease. These data reveal the pathogenic significance of CD8+ T cells in the development and progression of SS in the salivary glands. Treatment directed against CD8+ T cells may be a rational therapy for the management of SS in human subjects.

Maldevelopment of the submandibular gland in a mouse model of apert syndrome.

Apert syndrome is characterized by craniosynostosis and bony syndactyly of the hands and feet. The cause of Apert syndrome is a single nucleotide substitution mutation (S252W or P253R) in fibroblast growth factor receptor 2 (FGFR2). Clinical experience suggests increased production of saliva by Apert syndrome patients, but this has not been formally investigated. FGFR2 signaling is known to regulate branching morphogenesis of the submandibular glands (SMGs). With the Apert syndrome mouse model (Ap mouse), we investigated the role of FGFR2 in SMGs and analyzed the SMG pathology of Apert syndrome. Ap mice demonstrated significantly greater SMG and sublingual gland (SMG/SLG complex) mass/body weight and percentage of parenchyma per unit area of the SMG compared with control mice. Furthermore, gene expression of Fgf1, Fgf2, Fgf3, Pdgfra, Pdgfrb, Mmp2, Bmp4, Lama5, Etv5, and Dusp6 was significantly higher in the SMG/SLG complex of Ap mice. FGF3 and BMP4 exhibited altered detection patterns. The numbers of macrophages were significantly greater in SMGs of Ap mice than in controls. Regarding functional evaluations of the salivary glands, no significant differences were observed. These results suggest that the gain-of-function mutation in FGFR2 in the SMGs of Ap mice enhances branching morphogenesis. Developmental Dynamics 247:1175-1185, 2018. © 2018 Wiley Periodicals, Inc.

A sialo-oligosaccharide-rich mucin-like molecule specifically detected in the submandibular glands of aged mice

ObjectivesMucins are heavily glycosylated large glycoproteins produced in the salivary glands that contribute to salivary viscosity. This study aimed to characterize age-related changes in mucin production in mouse submandibular salivary gland (SMG). MethodsThe paraffin sections of the SMGs of the young and aged mice were stained with HE or Alcian blue (AB). SMGs mucins derived from the young and aged mice were separated using supported molecular matrix electrophoresis (SMME). After SMME, the membranes were stained with AB and subsequent glycan analysis or subjected to immunoblotting. The expression of 18 mucin genes and 4 sialyltransferase genes in the young and aged SMGs were determined by qPCR. The neuraminidase activity in the SMG homogenates was determined using Neuraminidase Assay Kit. ResultsThe mouse SMG is more strongly stained by AB with increasing age. On SMME, a characteristic band not found in the young SMG is detected in aged SMG. Based on migration position and the MALDI MS, the band that appeared specifically with aging was determined to be acidic mucin. Additionally, most glycans of this acidic mucin were sialo-oligosaccharides. Furthermore, there was an increase in the expression of sialyltransferase genes ST6GalNAc I and ST6GalNAc II, but not a decrease in neuraminidase activity, in the SMG of aged mice. ConclusionA sialomucin or sialylated mucin-like molecule not found in the SMGs of young WT mice is expressed in aged WT mice. The increase in the sialo-oligosaccharide content in this aging-associated molecule may be attributed to the increased expression of the sialyltransferase genes.

Genetic and scRNA-seq Analysis Reveals Distinct Cell Populations that Contribute to Salivary Gland Development and Maintenance

Stem and progenitor cells of the submandibular salivary gland (SMG) give rise to, maintain, and regenerate the multiple lineages of mature epithelial cells including those belonging to the ductal, acinar, basal and myoepithelial subtypes. Here we have exploited single cell RNA-sequencing and in vivo genetic lineage tracing technologies to generate a detailed map of the cell fate trajectories and branch points of the basal and myoepithelial cell populations of the mouse SMG during embryonic development and in adults. Our studies show that the transcription factor p63 and alpha-smooth muscle actin (SMA) serve as faithful markers of the basal and myoepithelial cell lineages, respectively and that both cell types are endowed with progenitor cell properties. However, p63+ basal and SMA+ myoepithelial cells exhibit distinct cell fates by virtue of maintaining different cellular lineages during morphogenesis and in adults. Collectively, our results reveal the dynamic and complex nature of the diverse SMG cell populations and highlight the distinct differentiation potential of the p63 and SMA expressing subtypes in the stem and progenitor cell hierarchy. Long term these findings have profound implications towards a better understanding of the molecular mechanisms that dictate lineage commitment and differentiation programs during development and adult gland maintenance.

Grhl2 regulation of SPINT1 expression controls salivary gland development

Development of the salivary gland is characterized by extensive branching morphogenesis and lumen formation, the latter of which is closely associated with differentiation into acinar and ductal cells. Although various molecules, including signaling and cell adhesion molecules, have been implicated in salivary gland development, transcription factors (TFs) regulating the expression of those molecules and morphological development of the gland are largely unknown. Here we show that knockdown of the epithelial TF, Grainyhead-like 2 (Grhl2), with siRNA in developing mouse submandibular salivary gland (SMG) cultured ex vivo resulted in retardation of epithelial development. This retardation was concomitant with suppression of gene expression for the cell adhesion molecules, such as E-cadherin and the extracellular protease inhibitor SPINT1, and with the disorganized deposition of the basal lamina protein laminin. ChIP-PCR demonstrated the binding of Grhl2 protein to the Spint1 gene in the SMG. Notably, addition of recombinant SPINT1 protein in cultured SMG overcame the suppressive effects of Grhl2 siRNA on epithelial development and laminin deposition. These findings show that Grhl2 regulation of SPINT1 expression controls salivary gland development.

Technique improvement and experience sharing of cannulation of the mouse submandibular salivary gland via the Wharton’s duct

Objective This paper described my technique improvement and experience sharing of Cannulation of the mouse submandibular salivary gland via the Wharton’s duct in order to accelerate the submandibular gland gene therapy research in China. Methods The operation process of the technology was introduced in detail, and the improvement of the original technology was made. The micro pump and microsurgical instruments were introduced to make the operation more precise and precise, which made the success rate higher. Results On the basis of a certain surgical operation experience, we trained three times per week, every time three to five mouse. Due to inappropriate operation technique and unclear understanding of the anatomy, initially intubation success rate was very low. The location of intubation always was incorrect, intubation site bleeding aometimes happened, poor control of anesthesia led to the death of mice. With the accumulation of operating experience, intubation success rate rapidly improveed about four weeks later, even achieve 100% success rate. Different drugs need different injection speed. If we injection of saline only, there were no significant damage between injection speed 10 μl/min and 25 μl/min, the volume of 25 μl and 50 μl. If we injected nanoparticle+ small interfering RNA (siRNA, specific target gene siRNA), the injection speed of 25 μl/min can cause more damage than that of 10 μl/min.25 μl/min resulted in heavier tissue damage in female mice. Conclusion Retrograde intubation through the mouse Wharton’s Duct required some training to master. Familiar with the anatomy was the key. If the anatomical location was accurate, we could gradually training intubation techniques, force and the angle of the cannulation to continuously improve the success rate of this operation. Key words: Salivary glands; Submandibular gland; Gene therapy; Cannulation; Technology

Age associated decrease of sialin in salivary glands

Aging salivary glands are characterized by a reduced volume of acini, increased duct volume, decline in the rate of synthesis of proteins and their mRNA, and decreased saliva flow. Sialin is a versatile anion transporter that is highly expressed in salivary glands and may also participate in maintenance of salivary gland function. We investigated age-associated sialin expression in salivary glands. Submandibular glands of mice and human parotid gland were collected at different ages. Western blot and real-time reverse transcription-polymerase chain reaction were used to analyze the protein and mRNA expression levels of sialin, respectively. Immunohistochemical and immunofluorescence staining were used to evaluate the histological pattern of sialin expression. We found that the protein and mRNA expression levels of sialin decreased with aging. The expression of sialin in the striated and excretory ducts of both mouse and human glands were reduced with age. In the mouse glands, the basal 2/3 of the cytoplasm was stained, while in the human glands the stain extended to the luminal surface. Sialin was expressed in the basal cytoplasmic membrane of acini and in some myoepithelial-like cells in young salivary glands, but had nearly disappeared in the aged salivary glands. Changes in sialin expression may be associated with changes in physiological function of salivary glands with aging.

A rapid separation and characterization of mucins from mouse submandibular glands by supported molecular matrix electrophoresis

Mucins are heavily glycosylated proteins with high molecular mass, and are involved in various diseases including infection, inflammation, and cancer. As easy separation method, such as gel electrophoresis, however, does not exist for mucins, due to their large molecular sizes and heterogeneities. In 2009, we published a supported molecular matrix electrophoresis (SMME) method that can be used to characterize mucins. For SMME analysis, mucins have been enriched by ultrafiltration of trypsin digests using a 100 KDa cutoff filter. However, this enrichment results in a loss of protein identification capability using proteomic approaches. In this study, we describe a simple enrichment of mucins without trypsinization for SMME analysis. The enrichment was developed using a porcine submandibular gland and then was applied to study and compare mouse submandibular glands between young and aged mice. From mouse submandibular glands, hyaluronic acid and some mucins were observed by SMME. One of the mucins was identified as MUC10 by proteomic analysis of the band on the SMME membrane and immunostaining using anti-MUC10 antibody. A major O-glycan of MUC10 was determined to be NeuAcα2-3Galβ1-3GalNAc. Furthermore, our experiments revealed that the concentrations of these molecules were lower in aged mice than in young mice, and that an unknown mucin-like molecule was detected only from the aged mouse submandibular gland.

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy.

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

A detrimental effect of interleukin-22 on salivary gland tissue integrity and function

Abstract Interleukin-22 (IL-22) has well-documented effects in promoting epithelial wound healing and protecting tissue damage in the intestine and lung. However, in certain inflammatory diseases such as psoriasis, IL-22 can also exert tissue-detrimental effect. Although the up-regulation of IL-22 has been reported in patients with Sjögren’s syndrome (SS), the role of IL-22 in this disease is not well-characterized. Herein, we investigated the effects of IL-22 on SS-like salivary gland disorder using both in vivo and in vitro approaches. To assess the effects of exogenous IL-22 on salivary gland tissues in vivo, IL-22 was directly injected into submandibular glands (SMGs) of 7–12 weeks old female C57BL/6 and the non-obese diabetic (NOD) mice, a model of SS disease. The salivary flow rate was decreased and the caspase-3 level was increased in the SMGs of the IL-22-treated C57BL/6 mice. IL-22 treatment also decreased the salivary flow rate and increased the level of capase-3 in the SMGs in the NOD mice. We determined the effects of endogenous IL-22 using an anti-CD3 antibody-induced acute exocrinopathy model. Anti-CD3 antibody was intraperitoneally injected into 8-week-old female C57BL/6 mice every other day for 3 days, together with a neutralizing anti-IL-22 antibody or its isotype control IgG. Neutralization of endogenous IL-22 improved saliva production and reduced caspase-3 activation in the SMGs in anti-CD3-treated C57BL/6 mice. Moreover, in vitro IL-22 treatment of a human salivary gland epithelial cells induced STAT3 activation and inhibited cell expansion. Taken together, these results provided strong evidence that IL-22 can directly act on salivary gland epithelial cells and promote damage and dysfunction of salivary gland tissues.

The G-Protein–Coupled Receptor ALX/Fpr2 Regulates Adaptive Immune Responses in Mouse Submandibular Glands

Lipoxin receptor (ALX)/N-formyl peptide receptor (FPR)-2 is a G-protein-coupled receptor that has multiple binding partners, including the endogenous lipid mediators resolvin D1, lipoxin A4, and the Ca2+-dependent phospholipid-binding protein annexin A1. Previous studies have demonstrated that resolvin D1 activates ALX/Fpr2 to resolve salivary gland inflammation in the NOD/ShiLtJ mouse model of Sjögren syndrome. Moreover, mice lacking the ALX/Fpr2 display an exacerbated salivary gland inflammation in response to lipopolysaccharide. Additionally, activation of ALX/Fpr2 has been shown to be important for regulating antibody production in B cells. These previous studies indicate that ALX/Fpr2 promotes resolution of salivary gland inflammation while modulating adaptive immunity, suggesting the need for investigation of the role of ALX/Fpr2 in regulating antibody production and secretory function in mouse salivary glands. Our results indicate that aging female knockout mice lacking ALX/Fpr2 display a significant reduction in saliva flow rates and weight loss, an increased expression of autoimmune-associated genes, an up-regulation of autoantibody production, and increased CD20-positive B-cell population. Although not all effects were noted among the male knockout mice, the results nonetheless indicate that ALX/Fpr2 is clearly involved in the adaptive immunity and secretory function in salivary glands, with further investigation warranted to determine the cause(s) of these between-sex differences.

The biphasic effect of extracellular glucose concentration on carbachol-induced fluid secretion from mouse submandibular glands.

Cholinergic agonists evoke elevations of the cytoplasmic free-calcium concentration ([Ca2+ ]i ) to stimulate fluid secretion in salivary glands. Salivary flow rates are significantly reduced in diabetic patients. However, it remains elusive how salivary secretion is impaired in diabetes. Here, we used an exvivo submandibular gland perfusion technique to characterize the dependency of salivary flow rates on extracellular glucose concentration and activities of glucose transporters expressed in the glands. The cholinergic agonist carbachol (CCh) induced sustained fluid secretion, the rates of which were modulated by the extracellular glucose concentration in a biphasic manner. Both lowering the extracellular glucose concentration to less than 2.5mM and elevating it to higher than 5mM resulted in decreased CCh-induced fluid secretion. The CCh-induced salivary flow was suppressed by phlorizin, an inhibitor of the sodium-glucose cotransporter 1 (SGLT1) located basolaterally in submandibular acinar cells, which is altered at the protein expression level in diabetic animal models. Our data suggest that SGLT1-mediated glucose uptake in acinar cells is required to maintain the fluid secretion by sustaining Cl- secretion in real-time. High extracellular glucose levels may suppress the CCh-induced secretion of salivary fluid by altering the activities of ion channels and transporters downstream of [Ca2+ ]i signals.

The apical anion exchanger Slc26a6 promotes oxalate secretion by murine submandibular gland acinar cells

The solute carrier family 26 (SLC26) gene family encodes at least 10 different anion exchangers. SLC26 member 6 (SLC26A6 or CFEX/PAT-1) and the cystic fibrosis transmembrane conductance regulator (CFTR) co-localize to the apical membrane of pancreatic duct cells, where they act in concert to drive HCO3- and fluid secretion. In contrast, in the small intestine, SLC26A6 serves as the major pathway for oxalate secretion. However, little is known about the function of Slc26a6 in murine salivary glands. Here, RNA sequencing-based transcriptional profiling and Western blots revealed that Slc26a6 is highly expressed in mouse submandibular and sublingual salivary glands. Slc26a6 localized to the apical membrane of salivary gland acinar cells with no detectable immunostaining in the ducts. CHO-K1 cells transfected with mouse Slc26a6 exchanged Cl- for oxalate and HCO3-, whereas two other anion exchangers known to be expressed in salivary gland acinar cells, Slc4a4 and Slc4a9, mediated little, if any, Cl-/oxalate exchange. Of note, both Cl-/oxalate exchange and Cl-/HCO3- exchange were significantly reduced in acinar cells isolated from the submandibular glands of Slc26a6-/- mice. Oxalate secretion in submandibular saliva also decreased significantly in Slc26a6-/- mice, but HCO3- secretion was unaffected. Taken together, our findings indicate that Slc26a6 is located at the apical membrane of salivary gland acinar cells, where it mediates Cl-/oxalate exchange and plays a critical role in the secretion of oxalate into saliva.

Expression and localization of phosphodiesterase 2A in the submandibular gland of mice

ObjectivesPhosphodiesterases comprise a superfamily of enzymes that hydrolyze and inactivate cyclic AMP (cAMP) and/or cyclic GMP (cGMP), thereby regulating cellular signaling mechanisms. We herein investigated the production of phosphodiesterase 2A (PDE2A) in the mouse submandibular gland. DesignThe expression and localization of the mRNA and protein of PDE2A were examined in the submandibular gland of male and female mice using the reverse transcription-polymerase chain reaction, in situ hybridization, Western blotting, and immunohistochemistry. ResultsAmong the different species of phosphodiesterases examined in the mouse submandibular gland, PDE2A, which hydrolyzes cAMP and cGMP, exhibited a marked sexual difference; it was more abundantly expressed in females. The mRNA and protein signals for PDE2A were intense in all acinar and duct portions, including the striated duct, in females, whereas in males, these signals were markedly weaker in the granular convoluted duct, the counterpart of the female striated duct, than in acini and other duct portions. Furthermore, the signals for protein kinases A and G1, which are intracellular effectors of cAMP and cGMP, respectively, were markedly weaker in the male granular convoluted duct. ConclusionsThese results suggest that cyclic nucleotide-dependent signaling mechanisms function poorly in granular convoluted duct cells in the mouse submandibular gland.

FGF2-dependent mesenchyme and laminin-111 are niche factors in salivary gland organoids.

Epithelial progenitor cells are dependent upon a complex 3D niche to promote their proliferation and differentiation during development, which can be recapitulated in organoids. The specific requirements of the niche remain unclear for many cell types, including the proacinar cells that give rise to secretory acinar epithelial cells that produce saliva. Here, using ex vivo cultures of E16 primary mouse submandibular salivary gland epithelial cell clusters, we investigated the requirement for mesenchymal cells and other factors in producing salivary organoids in culture. Native E16 salivary mesenchyme, but not NIH3T3 cells or mesenchymal cell conditioned medium, supported robust protein expression of the progenitor marker Kit and the acinar/proacinar marker AQP5, with a requirement for FGF2 expression by the mesenchyme. Enriched salivary epithelial clusters that were grown in laminin-enriched basement membrane extract or laminin-111 together with exogenous FGF2, but not with EGF, underwent morphogenesis to form organoids that displayed robust expression of AQP5 in terminal buds. Knockdown of FGF2 in the mesenchyme or depletion of mesenchyme cells from the organoids significantly reduced AQP5 levels even in the presence of FGF2, suggesting a requirement for autocrine FGF2 signaling in the mesenchyme cells for AQP5 expression. We conclude that basement membrane proteins and mesenchyme cells function as niche factors in salivary organoids.

Regulatory mechanisms of branching morphogenesis in mouse submandibular gland rudiments.

SummaryBranching morphogenesis is an important developmental process for many organs, including the salivary glands. Whereas epithelial–mesenchymal interactions, which are cell-to-cell communications, are known to drive branching morphogenesis, the molecular mechanisms responsible for those inductive interactions are still largely unknown. Cell growth factors and integrins are known to be regulators of branching morphogenesis of salivary glands. In addition, functional microRNAs (miRNAs) have recently been reported to be present in the developing submandibular gland. In this review, the authors describe the roles of various cell growth factors, integrins and miRNAs in branching morphogenesis of developmental mouse submandibular glands.

Distinct hormonal regulation of two types of sexual dimorphism in submandibular gland of mice.

The submandibular gland (SMG) of mice exhibits prominent sexual dimorphism in two aspects: the preferential development of granular convoluted tubule (GCT) cells and the earlier disappearance of granular intercalated duct (GID) cells in males after puberty. The former is dependent on androgens and thyroid hormones, whereas the hormonal dependence of the latter remains obscure. In the present study, we examined the effects of the postnatal administration of androgens and thyroid hormones to wild-type (WT) and androgen-receptor-knockout (ARKO) mice on these two types of sexual dimorphism by counting the numbers of GCT and GID cells labeled with nerve growth factor and submandibular gland protein C, respectively, as immunohistochemical markers. WT females and ARKO males and females exhibited a lower number of GCT cells and higher number of GID cells at 5 and 11weeks postpartum than WT males. The administration of dihydrotestosterone for 1-2weeks prior to these ages caused an increase in GCT cells and decrease in GID cells in WT females to similar levels as those in WT males, whereas it had no effects in ARKO, indicating that both types of sexual dimorphism are androgen-dependent. In contrast, the administration of thyroxine caused an increase in GCT cells but did not cause a decrease in GID cells in WT females or ARKO, indicating that the former is dependent on thyroid hormones, whereas the latter is not. The present results suggest that the two types of sexual dimorphism in the mouse SMG undergo distinct forms of hormonal regulation and, therefore, have different mechanisms.

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 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.