Although the physiological control of salivary secretion has been well studied, the impact of disease on salivary gland function and how this changes the composition and function of saliva is less well understood and is considered in this review. Secretion of saliva is dependent upon nerve-mediated stimuli, which activate glandular fluid and protein secretory mechanisms. The volume of saliva secreted by salivary glands depends upon the frequency and intensity of nerve-mediated stimuli, which increase dramatically with food intake and are subject to facilitatory or inhibitory influences within the central nervous system. Longer-term changes in saliva secretion have been found to occur in response to dietary change and aging, and these physiological influences can alter the composition and function of saliva in the mouth. Salivary gland dysfunction is associated with different diseases, including Sjögren syndrome, sialadenitis, and iatrogenic disease, due to radiotherapy and medications and is usually reported as a loss of secretory volume, which can range in severity. Defining salivary gland dysfunction by measuring salivary flow rates can be difficult since these vary widely in the healthy population. However, saliva can be sampled noninvasively and repeatedly, which facilitates longitudinal studies of subjects, providing a clearer picture of altered function. The application of omics technologies has revealed changes in saliva composition in many systemic diseases, offering disease biomarkers, but these compositional changes may not be related to salivary gland dysfunction. In Sjögren syndrome, there appears to be a change in the rheology of saliva due to altered mucin glycosylation. Analysis of glandular saliva in diseases or therapeutic interventions causing salivary gland inflammation frequently shows increased electrolyte concentrations and increased presence of innate immune proteins, most notably lactoferrin. Altering nerve-mediated signaling of salivary gland secretion contributes to medication-induced dysfunction and may also contribute to altered saliva composition in neurodegenerative disease.

Little is known about the key molecules that regulate cell division during organogenesis. Here we determine the role of the cell cycle promoter aurora kinase B (AURKB) during development, using embryonic salivary glands (E-SGs) as a model. AURKB is a serine/threonine kinase that regulates key events in mitosis, which makes it an attractive target for tailored anticancer therapy. Many reports have elaborated on the role of AURKB in neoplasia and cancer; however, no previous study has shown its role during organ development. Our previous experiments have highlighted the essential requirement for AURKB during adult exocrine regeneration. To investigate if AURKB is similarly required for progression during embryonic development, we pharmacologically inhibited AURKB in developing submandibular glands (SMGs) at embryonic day (E)13.5 and E16.5, using the highly potent and selective drug Barasertib. Inhibition of AURKB interfered with the expansion of the embryonic buds. Interestingly, this effect on SMG development was also seen when the mature explants (E16.5) were incubated for 24 h with another cell cycle inhibitor Aphidicolin. Barasertib prompted apoptosis, DNA damage and senescence, the markers of which (cleaved caspase 3, γH2AX, SA-βgal and p21, respectively), were predominantly seen in the developing buds. In addition to a reduction in cell cycling and proliferation of the epithelial cells in response to AURKB inhibition, Barasertib treatment led to an excessive generation of reactive oxygen species (ROS) that resulted in downregulation of the acinar differentiation marker Mist1. Importantly, inhibition of ROS was able to rescue this loss of identity, with Mist1 expression maintained despite loss of AURKB. Together, these data identify AURKB as a key molecule in supporting embryonic development and differentiation, while inhibiting senescence-inducing signals during organogenesis.

Understanding the key regulators which impact the innate immune response during initial phases of tissue injury, can advance the use of therapeutic approaches which aim at attenuating inflammation and organ damage. Recognition of microbial components by TLRs, initiates the transcription of innate immune signal pathways, that induce the expression of key inflammatory mediators: cytokines, chemokines and adhesion molecules. Beside regulating apoptotic cell death, recent studies have revealed distinct roles for caspases in the optimal production of inflammatory cytokines and host defense against injurious infections. Whether caspases can play an immune regulatory role in vivo has not been sufficiently investigated. This study aims to explore whether the pan caspase inhibitor z-VAD-fmk can control inflammation and cytokine production subsequent to challenging the innate immunity of the exocrine secretory tissues in vivo. Submandibular glands (SMGs) of the C57BL/6 mice were challenged with the TLR3 stimulant: polyinosinic-polycytidylic acid (poly (I:C)). Results obtained from the current study provide evidence that caspases can control immune responses downstream of TLR3 ligation. The present work proposes a novel mechanism that can prevent overactivation of the innate immunity, which typically leads to fatal immune disorders.

Inducible nitric oxide synthase (iNOS) is a key regulator of the innate immune system. The aim of the current study was to explore whether innate immune-mediated iNOS and reactive nitrogen species acutely perturb acinar cell physiology and calcium homeostasis of exocrine salivary tissues. Innate immunity in the submandibular gland of C57BL/6 mice was locally activated via intraductal retrograde infusion of polyinosinic:polycytidylic acid (poly (I:C). Expressions of iNOS and the activity of the reactive nitrogen species peroxynitrite, were evaluated by immunohistochemistry. Mice were pre-treated with the selective iNOS inhibitor aminoguanidine in order to substantiate the injurious effect of the nitrosative signal on the key calcium regulator sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2b) and calcium signalling. Challenging salivary gland innate immunity with poly (I:C) prompted upregulated expression of iNOS and the generation of peroxynitrite. Inhibition of iNOS/peroxynitrite revealed the role played by upregulated nitrosative signalling in: dysregulated expression of SERCA2b, perturbed calcium homeostasis and loss of saliva secretion. iNOS mediates disruption of exocrine calcium signalling causing secretory dysfunction following activation of innate immunity in a novel salivary gland injury model.

Background: Salivary gland (SG) injurious agents are all translated into loss of salivation (xerostomia). An association has been established between activation of innate immunity and SG injury and dysfunction. However, it remains unclear how the secretory epithelia respond by halting saliva production.Methods: C57BL/6 submandibular glands (SMGs) were acutely challenged using a single dose of the innate immune stimulant: polyinosinic‐polycytidylic acid (poly (I:C)). Secretory capacity of the infected SMGs was substantiated by assessing the flow rate in response to pilocarpine stimulation. Depletion of the acute inflammatory cells was achieved by pre‐treating mice with RB6‐8C5 depletion antibody. Flow cytometry, histology and immunohistochemistry were conducted to verify the immune cell depletion. Epithelial expression of saliva‐driving molecules: muscarinic 3 receptor (M3R), aquaporin 5 water channel (AQP5), Na‐K‐CL‐Cotransporter 1 (NKCC1) and transmembrane member 16A (TMEM16A), was characterized using RT‐qPCR and immunohistochemistry. Tight junction (TJ) protein; zonula occludens (ZO‐1) and basement membrane (BM) protein; and laminin were assessed by immunohistochemistry.Results: Innate immune challenge prompted dysfunction in the exocrine SGs. Dysregulated gene and protein expression of molecules that drive saliva secretion was substantiated. Aberrant expression of TJ and BM proteins followed innate immune activation. Hyposalivation in the current model was independent of myeloperoxidase (MPO)‐positive, acute inflammatory cells.Conclusions: In this study, we developed a novel injury model of the SGs, featuring acute secretory dysfunction and immediate structural disruptions. Our results ruled out the injurious role of aggressively infiltrating inflammatory cells.

Calcium ions (Ca2+) impact nearly every aspect of cellular life and intracellular calcium [Ca2+]i is a critical factor in the regulation of a plethora of physiological functions, including: muscle contraction, saliva secretion, metabolism, gene expression, cell survival and death. By measuring the changes of [Ca2+]i levels, critical physiologic functions can be characterized and aberrant pathologic conditions or drug responses can be efficiently monitored. We developed a protocol for assessment of Ca2+ signaling in the acinar units of submandibular glands isolated from C57BL/6 mice, using benchtop, multi-mode, high throughput plate reader (FlexStation 3). This method represents a powerful tool for unlimited in vitro studies to monitor changes in receptor-mediated Ca2+ responses while retaining functional and morphological features of a native setting.