POS0455 TOFACITINIB DECREASES INFLAMMATORY MARKERS AND MITOCHONDRIAL MORPHOLOGICAL DAMAGE IN SALIVARY GLANDS OF A MURINE MODEL OF SJÖGREN’S SYNDROME

Abstract

BackgroundAltered homeostasis of salivary gland (SG) epithelial cells in Sjögren’s syndrome (SS) patients could be the initiating factor that leads to inflammation, as well as secretory dysfunction. Mitochondria are important organelles involved in cellular metabolism and their dysfunction can induce a loss of homeostasis and inflammation. Altered mitochondrion can release mitochondrial components that can act as damage-associated molecular patterns (DAMPs) and induce an inflammatory response via pattern recognition receptors (PRRs) such as the NLRP3 inflammasome, TLR9, cGAS/STING, and ZBP1 (1). Previously we determined that SG from SS patients showed and altered autophagy, which is associated to an increased pro-inflammatory cytokines expression. Interestingly, increased expression of pro-inflammatory markers such as IL-6, was reversed by JAK inhibitor tofacitinib in three-dimensional (3D)-acini deficient in autophagy (2). It is not clear whether the alterations in autophagy found in SG patients include alterations in mitochondrial clearance (mitophagy) that may lead to the accumulation of damaged mitochondria and enhanced inflammation. In this context, recent results of our laboratory showed, for the first time, severe ultrastructural alterations of mitochondria in SG cells from SS patients (1). However, it remains to be determined if these alterations are related to inflammation and if an anti-inflammatory agent could regulate these processes.ObjectivesTo analyze the effect of tofacitinib on the mitochondrial ultrastructure in submandibular glands of a murine model of SS. In addition, to evaluate the effect of tofacitinib on the expression and activation of some PRRs involved in the recognition of mitochondrial DAMPs in the same murine model.MethodsSix-month-old female NOD.B10Sn-H2b/J mice (Jackson Laboratories, USA) were used with 4-5 mice per group. Procedures were approved by the Universidad de Chile Animal Care and Use Committee. 30 mg/kg/day tofacitinib citrate was administered by oral gavage. After 28 days of tofacitinib or vehicle administration, their submandibular glands were obtained, which were processed to evaluate the mitochondrial ultrastructure by electron microscopy or lysed in RIPA buffer to obtain proteins. The protein levels of PRRs: NLRP3, TLR9, ZBP-1, and cGAs, as well as molecules activated downstream of cGAS and ZBP-1 such as TBK1, pTBK1, pSTING, and STING were determined by Western blotting.ResultsThe results show that the mitochondria of the glandular epithelial cells of NOD.B10Sn-H2b/J mice treated with vehicle (control) present alterations such as swelling, disruption of membranes and crest disorganization that previously were reported in patients with SS (1). Interestingly, tofacitinib treatment improves the architecture of mitochondria. On the other hand, the protein levels of PRRs such as NLRP3 and cGAS decreased in mice treated with tofacitinib, as well as pTBK1.ConclusionThe altered morphology of mitochondria together with the increased protein levels of PRRs and downstream markers of these PRRs suggests release of mitochondrial DAMPs in submandibular glands of NOD.B10Sn-H2b/J mice. The improvement in mitochondrial morphology as well as the decrease in PRRs activation under tofacitinib treatment suggest a potential use of this anti-inflammatory agent in mitochondrial alterations associated with inflammation. Many questions remain to be addressed, such as determining which mitochondrial DAMP might be being released and whether this is associated with impaired mitochondrial function in SS.