AbstractA long association has been established between infectious inflammation and diseases of the salivary glands (SGs). However, the prompt responses of the exocrine tissues to these types of injuries remain unclear. Accordingly, an acute inflammation model was developed by the retrograde intraductal injection of a double-stranded (ds) RNA analogue; polyinosinic:polycytidylic acid (poly(I:C)), into the submandibular glands (SMGs), through Wharton’s duct. The main aim of this study was to investigate the early functional and immune events downstream of exposure to this viral-like inflammagen. The first part of the current study highlighted that the SMGs responded to the locally injected poly (I:C) by a rapid decline in saliva secretion, which stopped completely after 24 hours of infection. The early loss of function perceived, paralleled the upregulated expression of Toll-like receptor-3 (TLR3) and that incited blocking of the receptor in vivo to assess its role in the loss of function. The outcome of these experiments confirmed that dysfunction of the acutely inflamed glands resulted from ligation of TLR3 and initiation and propagation of downstream cytokines. The current study revealed for the first time the possibility of using a protease inhibitor to restrict TLR3 neo-synthesis, dampen inflammation, fine-tune anti-viral innate immune responses and retrieve TLR3-induced loss of SMG function.
Next, the role played by the acute inflammatory cells which infiltrated the SMGs following poly (I:C) infection was examined. Despite successful depletion of these cells, the glands’ secretory functions were not retrieved, verifying that TLR3 has induced dysfunction independent of the invasive acute cell infiltration.
In the last part of this study, it was demonstrated that the TLR3-inhibited, functionally-protected SMGs revealed loss of the pro-inflammatory cytokine, inducible nitric oxide synthase (iNOS), which displayed extensive acinar upregulation in response to poly (I:C) infection. Selective blocking of iNOS by aminoguanidine protected the secretory function of the poly (I:C) treated glands and specified iNOS as the earliest signal which disrupts the secretory machinery after innate immune activation.
To assess the injurious contributions of iNOS and its rapidly derived cytotoxic oxidant peroxynitrite, in the acute SG model, the gland homogenates were immunoblotted with the peroxynitrite marker, 3-nitrotyrosine, which revealed extensive nitration of a plethora of the gland proteins, including proteins at the electrophoretic mobility of the endoplasmic reticulum (ER) SERCA2 channel. Immunohistochemistry further revealed the physical co-localization of peroxynitrite and the critical regulator of calcium homeostasis, SERCA2. To comprehensively investigate the impact of this finding on the cellular calcium levels of the infected glands, a novel protocol which allowed assaying of [Ca2+]i changes ex vivo was developed, optimized and applied to the aminoguanidine treated and non-treated mice. Through this protocol, iNOS-mediated dysregulations in resting and stimulated calcium were recorded. The set of experiments which followed these findings revealed upregulation of the unfolded protein response, global transcriptional downregulation of key water driving molecules and altered subcellular localization of these critical membranous receptors, water channels and ion transporters. All these changes in response to a single poly (I:C) dose, were remarkably reversed when the SMGs were treated with the iNOS inhibitor; aminoguanidine.
|Date of Award
|1 Aug 2017
|Gordon Proctor (Supervisor) & Guy Carpenter (Supervisor)