THU0220 AUTOPHAGY IN SJOGREN’S SYNDROME SALIVARY GLAND EPITHELIAL CELLS (SGECS) IS ASSOCIATED WITH THE SEVERITY OF INFLAMMATION AND EPITHELIAL CELLS ACTIVATION.

Abstract

Background:Sjögren’s Syndrome (SS) is characterized by chronic inflammation supported by intrinsic activation of salivary gland epithelial cells (SGECs). Eventually, apoptosis of SGECs ensues, which leads to salivary gland dysfunction and exposition of autoantigens. Autophagy is a stress coping mechanisms of cells implicated in both survival and exposition of autoantigens, and is thereby plausibly implicated in the pathogenesis of SS. At present, the exact relationship between apoptosis and autophagy in SS SGECs is unclear, as is the link between these mechanisms and SGECs activation.Objectives:To explore autophagy in SGECs from patients with SS and to evaluate its relationship with apoptosis and SGECs activation.Methods:Consecutive patients with suspected SS referring to our “Sjogren Clinic” were enrolled, and minor salivary gland (MSG) biopsies were collected for: (1) SGECs culture, (2) PCR analysis, (3) IFI analysis. In SGECs cultures, the expression of autophagy (LC3II), apoptosis (annexin V/PI) and adhesion molecules (ICAM) was investigated by flow cytometry (results expressed as mean % ± SD). The expression of the autophagy gene MAP1LC3II was evaluated by PCR (expressed as 2^deltaCT normalized to GADPH) on both MSG sections and MSG acinar and ductal epithelium samples obtained by laser capture microdissection. Tissue expression of LC3II was evaluated by IFI on SS MSG.Results:Primary SGECs cultures were established from 14 MSG obtained for diagnostic purposes (SS n=8, Sicca n=6). These cells exhibited an inverse correlation between apoptosis and autophagy (p=0.007, r=-0.784), with lower levels of apoptosis (19.7±6.5 vs 24.5±8.5, p=ns) and higher levels of autophagy (59.7±13.1 vs 54.19±19.4, p=ns) in SS compared to Sicca. In SS, MAP1LC3 was positively correlated with Focus Score (p=0.021 r=0.478); however, PCR studies did not reveal significant differences in MAP1LC3 expression between SS (n=26) and Sicca (n=15) (0.024±0.010 vs 0.022±0.008, p=ns). Ductal SGECs (n=4) isolated by laser microdissection of MSG revealed a higher expression of MAP1LC3 (0.005±0.0005 vs 0.003±0.0008; p=0.057) compared to normal acinar epithelium (n=5); a major expression of LC3II in ducts was confirmed by IFI (Image).In SS, a higher expression of ICAM compared to sicca was observed (11.1±3.8 vs 6.9±6.9, p=0.006) and autophagy and apoptosis showed a trend of positive and negative correlation with this molecule, respectively (p=0.683 r=0.118 and p=0.106 r=-0.446).Figure.LC3-II staining in SS MSG [LC3-II+ (green) and Hoechst stain (blue); 60x magnification].Conclusion:In SS, autophagy is upregulated in SGECs and inversely correlated with apoptosis, thus supporting a role of this process in cells’ death prevention during inflammatory process. Indeed, the degree of msg inflammation is correlated more with the activation of autophagy than apoptosis. Interesting, in SS, SGECs autophagy is mainly observed at ductal level and is correlated with higher expression of adhesion molecules suggesting a link between this pathway and changes in SGECs immune phenotype.Disclosure of Interests: :Serena Colafrancesco: None declared, cristiana barbati: None declared, Valentina Iannizzotto: None declared, Linda Mastromanno: None declared, Saba Nayar: None declared, Elena Pipi: None declared, angelica gattamelata: None declared, francesco ciccia Grant/research support from: pfizer, novartis, roche, Consultant of: pfizer, novartis, lilly, abbvie, Speakers bureau: pfizer, novartis, lilly, abbvie, cristiano alessandri Grant/research support from: Pfizer, Francesca Barone: None declared, fabrizio conti Speakers bureau: BMS, Lilly, Abbvie, Pfizer, Sanofi, Roberta Priori: None declared