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Severe, irreversible salivary gland disease and oral dryness is experienced by sufferers of Sjögren’s syndrome and those treated with irradiation for head and neck cancer. Therefore, major efforts have been made in the last decade to unravel key molecular signals that can drive salivary gland (SG) regeneration and functional restoration. However, the earliest molecular determinants that accompany SG regeneration remain incompletely defined. The present study examined the initial mitogenic events marking the regenerative response of the murine submandibular gland (SMG), following innate immune-mediated injury. Local intraductal administration of the synthetic double stranded (ds) RNA polyinosinic-polycytidylic acid (poly (I:C)) widely, but transiently, depleted the acinar and progenitor cells, 24 hours post poly (I:C) introduction. While the progenitor and duct cells started to proliferate and expand at 72 hours, the Mist1-positve acinar cells did not re-appear until 96 hours post poly (I:C) injury. The cellular replenishment during regeneration involved significant upregulation of the cell cycle promoter Aurora kinase B (AURKB). AURKB, which is expressed in healthy proliferating and cancerous cells, is a serine/threonine protein kinase, well known to orchestrate key events in cell division and cytokinesis. However, the expression and role of AURKB in regeneration of post mitotic salivary gland cells has not been previously explored. In vivo inhibition of AURKB using the selective inhibitor Barasertib (AZD1152-HQPA) interfered with SMG recovery from the transient, but severe poly (I:C)-mediated injury and cellular depletion. AURKB deficiency during regeneration of the injured tissues: disrupted cell cycle progression, repressed renewal of Mist1-positive acinar cells and prevented recovery of salivary secretion. The knowledge gained in this study may be utilized in the development of therapeutic targets for irreversible salivary gland disease.

The autoimmune disease primary Sjögren’s syndrome (pSS) is characterized by hypofunction of the salivary glands (SGs), the cause of which is not correlated to lymphocytic SG infiltration, as prevailing dogma often states. We knocked out the NF-κB proinflammatory pathway inhibitor A20 in keratin14+ epithelial cells, to investigate if immune activated epithelial cells are capable of initiating pSS SG hallmarks. We show that immune activated epithelial cells can cause T cell dominated leukocytic infiltration and immune foci development of the SGs, reflecting the early clinical picture. Infiltrating leukocytes invaded striated ducts, similar to early stage lymphoepithelial lesions observed clinically. Expression of proinflammatory cyto-/chemokines IFN, TNFα, IL-6, CXCL10 and CXCL13 increased in A20-/- SGs, and functionally both volume and mucin 10 content of whole stimulated saliva from A20-/-mice was significantly reduced. Epithelial cells may therefore represent the initial trigger for pSS SG pathologies, as opposed to simple reactionaries to pre-existing stimuli.

BackgroundSalivary 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.MethodsC57BL/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.ResultsInnate 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.ConclusionsIn 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.Publication cover imageVolume47, Issue2February 2018Pages 211-219

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.