POS0997 INFLAMMATION IN SJӦGREN DISEASE INDUCES SALIVARY GLAND EPITHELIAL CELLS (SGECS) METABOLIC REPROGRAMMING: FOCUS ON GLYCOLYSIS AND ITS REGULATION OF EPITHELIAL CELL ACTIVATION
Authors: S. Colafrancesco, E. Simoncelli, E. Pontarini, G. Cavallaro, D. Lucchesi, A. Marino, G. Buoncuore, C. Barbati, F. Conti, R. Priori, C. Pitzalis, M. Bombardieri
Journal: Annals of the Rheumatic Diseases (2024)

<h3>Background:</h3> In Sjögren Disease (SD) salivary gland epithelial cells (SGEC) sustain inflammation due to their acquired capacity to express/secrete adhesion/pro-inflammatory molecules. However, the mechanisms responsible for SGECs activation remain undetermined. The link between immune cells' function and cells' metabolic reprogramming is known under the term "immunometabolism" and has become one of the most exciting areas of investigation in the field of immuno-rheumatology, despite its relevance in SD has not been investigated as yet. We hypothesize that the altered cell energy metabolism of SGECs is a central driver of SG inflammation in SD and one that is targetable for the development of innovative therapies. <h3>Objectives:</h3> <i>I–</i>To determine (<i>in vivo</i>) in a viral-induced model of experimental sialoadenitis the relationship between SG inflammation and cell metabolic reprogramming; <i>II–</i>To identify <i>ex vivo</i> the main metabolic pathways differing between SD and sicca (control) SGECs; <i>III–</i>To assess <i>ex vivo</i> and <i>in vitro</i> the expression of glycolysis in SGECs and to investigate its contribution to SGECs activation. <h3>Methods:</h3> Aim I- SD mouse models were produced. After culling at different time points [day_0 (non-cannulated), day_5-12-19 (cannulated)], SG bulk RNA-seq analysis focused on metabolic pathways was conducted. Aim II- The metabolic profile of cultured SGECs from SD and sicca patients was analysed (mass spectrometry). Aim III- The expression of the glycolysis marker GLUT-1, along with lymphocytes (CD45+) and epithelial cells (PanCK+, CK7+, CK14+) markers, was determined (IFI) on paraffin-embedded sections of SD and sicca minor SG. SGECs cultures were treated as follows: 1) fluorescent glucose (6-NBDG), to assess glucose up-take; 2) 2-deoxy-glucose (2DG), to evaluate the effect of glycolysis inhibition on SGECs activation [ICAM-1 expression (IFI) and IL-6/type-I IFN production (Elisa and IFNα/β reporter cells)]; treatment conditions are in Figure 1d. <h3>Results:</h3> <i>Aim-I</i>: Following the inflammatory insult, bulk RNA-seq analysis in AdV-induced sialoadenitis, demonstrated changes in the SG metabolic profile. Down-regulation of specific pathways (i.e. oxidative phosphorylation and pyruvate metabolism), occurred up to day 12 post-cannulation (Figure 1a). <i>Aim-II</i>: A metabolic difference between SGECs from SD and sicca patients was revealed (i.e. glycolysis and TCA cycles) (Figure 1b). <i>Aim III</i>: As glycolysis turned out to be one of the most expressed pathways in SD, we further investigated its expression in SGECs. The expression of the glucose transporter GLUT-1 was highly detected in SD SG tissue by SGECs wile no expression was detected in sicca. Such GLUT-1 expression was mainly localized in ducts, especialy in ductal luminal cytokeratin-7 positive (CK7+) cells (Figure 1c). <i>In vitro</i> treatment of SGECs with fluorescent glucose demonstrated an higher glucose uptake in SD SGECs (representative example-Figure 1c). In primary SGECs culture, following stimulation with Poly(I:C), which mimics viral sensing by SGEC, an increase in IL-6, type-I IFN and the adhesion molecule ICAM-1 was observed. SGECs treatment with the glycolisis-inhibitor 2DG prevented [if Poly(I:C) and 2DG were administered concomitantly] or reduced [if 2DG was added following 12h stimulation with Poly(I:C)] the expression of adhesion/pro-inflammatory molecules (Figure 1d). <h3>Conclusion:</h3> <i>In vivo</i> experiments confirm that inflammation is intimately linked with metabolic changes occurring in SD SG. After induction of inflammation, we observed a down-regulation of oxidative phosphorylation and changes in glucose metabolism. Mass spectometry analysis confirmed the higher expression of specific metabolic pathways in SD SGECs, such as glycolysis. The increased expression of GLUT-1 in SD CK7+ ductal epithelial cells and the higher glucose up-take in SD cultured SGECs confirm such a shift towards glycolysis. Remarkably, glycolysis inhibition affects SGECs activation, indicating that metabolic reprogramming is likely responsible for their pathogenic behaviour. <i>In vivo</i> investigations on the potential utility of drugs targeting glycolysis in SD are currently ongoing. <h3>REFERENCES:</h3> <b>NIL.</b> <h3>Acknowledgements:</h3> The presenting author of this abstract (SC) is recipient of a "Career Research Grant" funded by FOREUM – Foundation for Research in Rheumatology. <h3>Disclosure of Interests:</h3> <b>None declared.</b>