M.-J. Barrera

Sjögren's syndrome and the epithelial target: A comprehensive review

The most difficult component in our understanding of human autoimmunity remains a rigorous dissection of etiological events. Indeed, the vast literature on autoimmune diseases focuses on the inflammatory response, with the hope of developing drugs that reduce inflammation. However, there is increasing recognition that understanding the immunobiology of target tissues will also have direct relevance to disease natural history, including breach of tolerance. Sjögrens syndrome is essentially an epitheliitis and there are major changes to normal architectural salivary organization. We propose that loss of homeostasis is the initial event that precipitates inflammation and that such inflammatory response includes not only the adaptive response, but also an intense innate immune/bystander response. To understand these events this review focuses on the architecture, phenotype, function and epithelial cell organization. We further submit that there are several critical issues that must be defined to fully understand epithelial cell immunobiology in Sjögrens syndrome, including defining epithelial cell polarity, cell-cell and cell to extracellular matrix interactions and a variety of chemical and mechanical signals. We also argue that disruption of tight junctions induces disorganization of the apical pole of salivary acinar cells in Sjögrens syndrome. In addition, there will be a critical role of inflammatory cytokines in the apico-basal relocation of tight junction proteins. Further, the altered disorganization and relocation of proteins that participate in secretory granule formation are also dysregulated in Sjögrens syndrome and will contribute to abnormalities of mucins within the extracellular matrix. Our ability to understand Sjögrens syndrome and develop viable therapeutic options will depend on defining these events of epithelial cell biology.

Oral dryness in Sjögren's syndrome patients. Not just a question of water

Sjögrens syndrome (SS) is a chronic autoimmune disease of undefined etiology. Patients with this syndrome suffer from severe alterations in both the quality and quantity of saliva and tears, due to impaired function of the relevant exocrine glands. Prevalent symptoms experienced by SS-patients include a persistent dry mouth sensation (xerostomia) and dry eyes (keratoconjunctivitis sicca). Water content of saliva depends of acetylcholine levels, glandular innervation, M3R signaling, calcium tunneling and water release, among other factors. However, unstimulated salivary flow correlates only poorly with symptoms of mouth dryness, raising the question as to which other components of saliva may be involved in mouth dryness experienced by SS-patients? Salivary mucins are glycoproteins characterized by the presence of large oligosaccharide side chains attached to the protein backbone. These molecules are key saliva components that are required to sequester water and thereby moisturize, as well as lubricate the oral mucosa. In the labial salivary glands of SS patients, morphological and functional alterations are detectable that affect the maturation and trafficking of salivary mucins. In this review, we will focus the discussion on these aspects of reduced salivary flow and decreased quality of salivary mucins, since they are likely to be responsible for xerostomia in SS-patients.

MUC1/SEC and MUC1/Y overexpression is associated with inflammation in Sjögren's syndrome

OBJECTIVES To evaluate the expression and localization of MUC1/SEC and MUC1/Y isoforms in labial salivary glands (LSG) from Sjögrens syndrome patients (SS patients), as well as their in vitro expression induced by cytokines. SUBJECTS AND METHODS Labial salivary gland from 27 primary SS patients and 22 non-SS sicca subjects were studied. Relative MUC1/SEC and MUC1/Y mRNA levels were determined by qPCR and protein levels by Western blotting. Induction of mucin mRNAs was assayed in vitro. Immunohistochemistry was used for localization. RESULTS Relative MUC1/SEC and MUC1/Y mRNA and protein levels were significantly higher in LSG from SS patients. These mRNAs were induced by cytokines. MUC1/SEC and MUC1/Y were detected in acini apical region of control LSGs, and significant cytoplasmic accumulation was observed in acini of SS patients. MUC1/Y localized in acinar nuclei and cytoplasm of inflammatory cells of LSG from SS patients. A strong positive correlation was observed between cellular MUC1/SEC levels and glandular function determined by scintigraphy. CONCLUSIONS We show for the first time that MUC1/SEC and MUC1/Y are expressed in LSG of both SS patients and non-SS sicca subjects. The observed overexpression and aberrant localization of MUC1/SEC and MUC1/Y and their induction by pro-inflammatory cytokines may favor the perpetuation of the inflammatory environment that disrupts the salivary glandular homeostasis in SS patients.

SAT0380 Impaired Ire1Alpha/XBP-1 Pathway is Associated with Glandular Dysfunction in SjÖgren's Syndrome

Background Labial salivary glands (LSG) of Sjögrens syndrome (SS) patients show alterations indicative of endoplasmic reticulum (ER) stress, such as ER cistern dilatation (1) and mucin accumulation (2). Pro-inflammatory cytokines induce ER stress, but it is not well known if the ER stress is an initial event in the onset of a chronic disease or if is secondary to the inflammation. ER stress triggers a complementary adaptive mechanism known as the “Unfolded Protein Response” (UPR) that seeks to restore ER homeostasis. IRE1α/XBP-1 signaling pathway is a UPR branch involved in secretory process regulation. Therefore, glandular dysfunction in SS-patients may be, at least in part, attributable to an altered signaling via the IRE1α/XBP-1s pathway. Objectives To determine the expression and localization of IRE1α/XBP-1 pathway components, their expression In vitro induced by pro-inflammatory cytokines, as well as their relationship to clinical parameters of SS-patients. Methods LSG of 19 SS-Patients and 21 controls were studied. Proteins localization was analyzed by immunofluorescence and mRNA and protein levels by qPCR and Western-blot. To evaluate the effect of pro-inflammatory cytokines on expression of IRE1α/XBP-1 pathway components, human submandibular gland (HSG) cells were stimulated with TNF-α or IFN-γ and mRNA levels were determined by qPCR. Results XBP-1 showed nuclear and cytoplasmic staining in acinar cells of LSG of SS-patients and controls, but mRNA and protein levels were significant decreased in LSG of SS-patients. Cytoplasmic GRP78 staining as well as their mRNA and protein levels decreased in acinar cells of SS-patients. PDIA3 staining was higher in nuclei and cytoplasm of acinar cells in SS-patients, coincident with increased mRNA and protein levels. Plasma cells but not lymphocytes showed strong staining for XBP-1, GRP78 and PDIA3. TNF-α increased and IFN-γ decreased mRNA levels of XBP-1s, IRE1α and GRP78, while PDIA3 mRNA levels increased with both cytokines. IRE1α levels correlated positively with salivary flow. XBP-1s levels correlated inversely with Ro. PDIA3 levels correlated positively with the presence of auto-antibodies and glandular dysfunction assayed by scintigraphy. Conclusions Considering the important role of IRE1α/XBP-1 pathway in the secretory process control, the altered expression and localization of IRE1α/XBP-1 pathway components may contribute to the observed changes in the physiology of LSG in SS-patients. In these glands, the expression of these components might be modulated by the inflammatory environment. References Goicovich E, Molina C, Pérez P, Aguilera S, Fernández J, Olea N, Alliende C, Leyton C, Romo R, Leyton L, González MJ. Arthritis Rheum. 2003 Sep;48(9):2573-84. Verόnica Bahamondes, Amelina Albornoz, Sergio Aguilera, Cecilia Alliende, Claudio Molina, Isabel Castro, Ulises Urzúa, Andrew F.G. Quest, María-José Barrera, Sergio González, Marianela Sánchez, Steffen Härtel, Marcela Hermoso, Cecilia Leyton and María-Julieta González. Arthritis Rheum. 2011 Oct;63(10):3126-35. Acknowledgements Fondecyt 1120062 (MJG, SA, CM, SG) and PhD fellowship Conicyt-Chile (DS MJB, JC). Disclosure of Interest None declared

OP0271 Perk Pathway Characterization in Labial Salivary Glands of Sjögren Syndrome's Patients: Could It Be An Adaptive Response?

Background Labial-salivary-glands (LSG) of Sjögrens syndrome (SS) patients show an altered exocytic route that can induce endoplasmic reticulum (ER)-stress and activate the unfolded-protein-response (UPR)1,2. UPR helps to recover and preserve cellular homeostasis, reducing the load of unfolded proteins in the ER, through various mechanisms of cell survival. UPR is mediated by the activation of IRE1α, PERK and ATF6 sensors.Chronic activation of ER-stress sensors exhibits a time course-kinetics with attenuation of IRE1α pathway and sustained signaling of PERK and ATF6 pathways3. We have observed in SS-patients a decrease of IRE1α pathway activity4 and an increase of ATF6 pathway activity5. Objectives Considering that PERK pathway reduces levels of unfolded proteins by increasing expression of pro-survival or pro-apoptotic components, here our purpose was to evaluate the expression of PERK pathway components and its relation with an adaptive response of LSG from SS-patients. Methods RNA and proteins were extracted from LSG of 11 SS-patients and 12 controls. Expression of PERK pathway components was determined by q-PCR and Western-blot. Localization was addressed by immunofluorescence. Results Compared to controls, LSG of SS-patients showed a significant increase of pPERK/PERK ratio but no changes of peIF2α/eIF2α ratio. Also, an increase of ATF4 and CHOP protein levels were observed, which correlated with clinical parameters including scintigraphy and serology. On the contrary, significantly decreased expression of ERO1α, PDIA1 and PDIA4 (proteins involved in protein folding), and NRF2 (transcription factor) protein levels, an additional PERK substrate involved in the response to oxidative stress, were detected. Interestingly, SS-patients showed a significant increase of expression of Xc- system subunits, which is involved in glutathione synthesis. PERK, ERO1α, PDIA1, PDIA4, eIF2α and peIF2α were located in the basal cytoplasm of acinar cells, while eIF2α and peIF2α were also observed in the nucleus. NRF2 localized in cytoplasm and lateral plasma-membrane, next to E-cadherin. Conclusions PERK pathway is activated in LSG of SS-patients and controls; indicating that both are developing UPR. Considering that ATF4 mediates an integrated response to cellular stress6, its increased expression in SS-patients and its correlation with clinical data and Xc– expression, suggest that ATF4 could be part of an adaptive response against cellular stress. In addition, expression of ERO1α, PDIA1 and PDIA4 suggests alterations of the protein folding process. Finally, NRF2 protein levels and its location suggest an altered oxidative stress response. References Barrera MJ, et al. 2013. J Autoimmun 42:7–18. Goicovich E, et al. 2003. Arthritis Rheum; 48(9): 2573–2584. Hetz C. 2012. Nat Rev Mol Cell Biol; 13(2):89–102. Sepúlveda D, et al. 2015. Manuscript under review in Arthritis & Rheumatology. Barrera MJ, et al. Manuscript in preparation. Harding HP et al. 2003. Mol Cell; 11(3):619–33 Acknowledgement Supported by Fondecyt-Chile [#1120062] (MJG, SA, CM, SG), PhD fellowship Conicyt-Chile (MJB and JC). Disclosure of Interest None declared

AB0158 Tauroursodeoxycholic acid decreases the expression of erad components and the accumulation of salivary mucins induced by pro-inflammatory cytokines

Background The salivary glands of Sjögrens syndrome patients show endoplasmic reticulum (ER) stress characterized by intracellular accumulation of secretory products such as MUC11, dilated ER cisternae2 and high levels of pro-inflammatory cytokines. Previous results from our laboratory revealed an increase of the ATF6α pathway of the UPR3 and activation of ER-associated protein degradation (ERAD)3. Increased expression of proteins involved in ERAD (SEL1L and EDEM1) has been reproduced in vitro in human submandibular gland (HSG) cells treated with TNF-α or IFN-γ2. Tauroursodeoxycholic acid (TUDCA) is a chemical chaperone utilized for alleviate ER stress by enhancing the folding of proteins3. Objectives The aim of this study was to evaluate if TUDCA decreases EDEM1, SEL1L, and MUC1 expression induced by pro-inflammatory cytokines in salivary gland epithelial cells. Methods HSG-cells were incubated with 10 ng/mL of IFN-γ or TNF-α for 24h. Alternatively, cells were incubated with cytokines for 6h and then co-incubated with TUDCA (150 and 250 μM) up to 24h. EDEM1, SEL1L and MUC1 protein and mRNA levels were determined by Western-blot and RT-qPCR, respectively. EDEM1 and SEL1L localization was determined by immunofluorescence. Results HSG cells stimulated with IFN-γ or TNF-α showed a significant increase of EDEM1 and SEL1L protein and mRNA levels. Importantly, TUDCA co-incubation caused a significant decreased expression of both molecules. Treatment with both cytokines induced a cytoplasmic increase of staining intensity of EDEM1 and SEL1L, which was suppressed by TUDCA. HSG cells stimulated with cytokines showed a significant increase of MUC1 protein and mRNA levels, which was also suppressed by TUDCA. Conclusions Decreased expression of MUC1, SEL1L and EDEM1 in the presence of TUDCA suggests that this chemical chaperone promotes folding of proteins in the ER, by decreasing ERAD activity and ER stress induced by pro-inflammatory cytokines in HSG cells. These results enable us to propose that TUDCA might alleviate the ER stress of salivary glands from Sjögrens syndrome patients. References Oral Dis. 2015;21(6):730–8. Arthritis Rheum. 2003 Sep;48(9):2573–84. J Autoimmun. 2016;75:68–81. Prion. 2014;8(2). pii: 28938. Acknowledgements Supported by Fondecyt-Chile [#1160015] (MJG, SA, CM, SG, IC). Disclosure of Interest None declared

AB0154 Role of Pro-Inflammatory Cytokines in The Endoplasmic Reticulum Associated-Protein Degradation in Sjögren's Syndrome Patients

Background Due to its high secretory activity and the complexity of synthesized secretory products, salivary gland (SG) acinar cells are susceptible to endoplasmic reticulum (ER) stress under physiological conditions. ER stress triggers a mechanism known as the “Unfolded Protein Response” (UPR) aiming to restore ER homeostasis. Various ER stress-related disorders have been described in SG of Sjögrens syndrome (SS) patients, including intracellular accumulation of mucins (1) and dilated ER cisternae (2). Also, high levels of pro-inflammatory cytokines present in SG of SS-patients may act as local ER stressors and may play an important role in ER stress induction and perpetuation (chronicity). Previous results from our laboratory indicate a decreased activation of the IRE1α/XBP-1s pathway of the UPR (unpublished data) indicating a chronic ER stress condition. Despite this, the SG acinar cells from SS-patients have low levels of apoptosis (3), suggesting that alternative UPR pathways such as ATF6α could be activated. ATF6α pathway promotes survival mechanisms such as ER-associated protein degradation (ERAD), which has been found hyper-activated in arthritis rheumatoid (4). Objectives To determine the expression and localization of ATF6α pathway components and molecules participating in ERAD in labial SG (LSG) of SS-patients. The effect of pro-inflammatory cytokines on both ERAD and cell survival was also studied using LSG extracts of SS patients and a three-dimensional (3D) acini model. Methods Expression of ATF6α, ERAD-components, pro-inflammatory cytokines, cIAP2 and cleaved-caspase-3 were determined in SG of SS-patients and controls by Western-blot and qPCR. Localization of selected molecules was evaluated by immunofluorescence. Correlation between cytokines and ERAD-components was analyzed with the Pearsons test. ATF6α and ERAD activation, pro- and anti-apoptotic markers and cell viability were determined in a 3D HSG cell culture system treated with cytokines. Results LSG of SS-patients showed high cytokines levels and high activities of ATF6α and ERAD pathways. No significant differences in cleaved-caspase-3 and cIAP2 levels between both groups were observed. Significant positive correlations were observed between pro-inflammatory cytokines and ERAD-components expression levels. These correlations were demonstrated in in vitro 3D cell cultures treated with TNF-α and/or IFN-γ for prolonged times, without increased apoptosis. Conclusions In SS-patients, the permanent action of pro-inflammatory cytokines establishes a chronic ER stress condition with ATF6α pathway activation and increased ERAD mechanism. Higher ATF6α pathway levels would increase ERAD activity, which may help to control chronic ER stress and prevent death by apoptosis. References Arthritis Rheum. 2011;63:3126–35. Arthritis Rheum. 2003;48:2573–84. Lab Invest. 2001, 81:95–105. Arthritis Res Ther. 2005;7:181–6. Acknowledgement Fondecyt 1120062 (MJG, SA, CM, SG) and PhD fellowship Conicyt-Chile (MJB, JC). Disclosure of Interest None declared

SAT0372 Ectopically Secreted Mucins Might Perpetuate the Inflammation in Salivary Glands of SjÖgren's Syndrome Patients

Background Loss of apico-basal polarity in acinar cells is a characteristic of labial salivary glands (LSG) of Sjögrens syndrome (SS) patients (1). In these glands, the ectopic presence of salivary mucins in the extracellular matrix has been described (1). The structure of mucins consists of a protein backbone with large number of O-linked oligosaccharides covalently attached. In SS-patients, ectopic mucin oligosaccharides might be recognized as not-self antigens by innate immunity receptors, inducing or increasing a pro-inflammatory response. Objectives To determine whether exogenous salivary mucins or mucin oligosaccharides induce the expression of pro-inflammatory cytokines, to evaluate if the Toll-like receptor-4 (TLR4) is involved in this response and if pro-inflammatory cytokines are able to induce the expression of salivary mucins. Methods Human submandibular gland (HSG) cells were stimulated with mucin from bovine submaxillary glands (BSM), Sulfo-Lewis (SO3-3Galβ1-3GlcNAc) oligosaccharide and human recombinant TNF-α or IFN-γ at different concentrations and for different periods of time. For TLR4 signaling assays, HSG cells were pre-treated with the TIRAP inhibitory peptide TBX2 or with an anti-TLR4 blocking antibody and then stimulated with BSM. The mRNA expression of mucins and cytokines was determined by real time PCR. Results Mucins and Sulfo-Lewis induced a significant increase in CXCL8, TNF-α, IFN-α, IFN-β, IL-6 and IL-1β mRNA levels. The induction of cytokines was mediated by TLR4 as shown by reversion of the effect using TBX2 or the anti-TLR4 antibody. TNF-α and IFN-γ induced in vitro the expression of salivary mucins such as MUC1Y/ and MUC1/SEC, which are overexpressed in LSG of SS-patients. Conclusions Salivary mucins and mucin oligosaccharides were recognized by TLR4 in epithelial cells initiating a pro-inflammatory response that could attract inflammatory cells. The overexpression and aberrant localization of mucins in LSG of SS-patients and their induction upon stimulation with pro-inflammatory cytokines support a self-perpetuating mucin-cytokine signaling loop that may facilitate the maintenance of the inflammatory environment leading to disruption of salivary gland homeostasis in SS-patients. References Barrera MJ, Bahamondes V, Sepúlveda D, Quest AFG, Castro I, Cortés J, Aguilera S, Urzúa U, Molina C, Pérez P, Ewert P, Alliende C, Hermoso MA, González S, Leyton C and González MJ. Sjögrens Syndrome: J Autoimmun. 2013 May;42:7-18. Acknowledgements Fondecyt 1120062 (MJG, SA, CM, SG) and PhD fellowship Conicyt-Chile (MJB and JC). Disclosure of Interest None declared

THU0053 Three Dimensional HSG Cells Culture as A Model to Study the Exocitic Process in Salivary Glands of SjÖGren's Syndrome Patients

Background Labial salivary glands (LSG) from Sjögrens syndrome (SS) patients show impaired cell-polarity, alterations of SNARE proteins localization and presence of salivary mucins in the extracellular matrix. Exocytosis and Ca+ signaling are polarized processes in acinar-cells. Synaptotagmin-I (Syt-I) is a Ca+-sensor protein localized in the membrane of secretory granules and is a linker between Ca+ stimulus and the exocytic machinery. A significant upregulation of SytI observed in a microarray assay and a Ca+ response less sensitive to acetylcholine stimulation have been observed in LSG from SS patients. These data suggest that impaired cell-polarity observed in SS-patients could affect the behavior of Ca+ signaling and alter the expression of Syt-I and the exocytic process. Objectives To determine the expression and localization of Syt-I in LSG from SS patients and controls. To evaluate Ca+ signaling and exocytic events in normal and polarity-altered three dimensional (3D) HSG acini. Methods LSG were obtained from 22 healthy controls and 28 SS patients selected according to the American/European classification criteria (2002). Syt-I mRNA and protein levels were determined in protein extracts of LSG by qPCR and Western blotting, respectively. Syt-I localization was addressed by immunofluorescence and confocal microscopy. To evaluate the formation of SDS-resistant SNARE complexes, some aliquots of proteins were maintained at 25°C and other aliquots were boiled for 3 min at 100°C to disassemble SNARE complexes. Ca+ signaling was measured in 3D HSG acini obtained by culturing HSG cells on basal lamina extract. 3D acini like structures were treated with the a6 integrin blocking antibody (GoH3) that alters cell polarity. Ca+ measurements were performed with fluorescent dyes sensitive to changes of Ca+ concentration upon carbachol and isoproterenol stimulation. Exocytosis events were measured by FM 1-43 fluorescence and carbon fiber amperometry. Results LSG of SS-patients showed a significant increase of mRNA and protein levels of Syt-I. An increased Syt-I staining intensity co-localizing with STX4 mainly in the basal pole was also observed in LSG of SS-patients. An increased amount of Syt-I was observed in pre-formed SNARE complexes of LSG extracts from SS-patients. Ca+ signaling and exocytic events were dependent on extracellular Ca+ and acinar cell-polarity in 3D HSG acini. Conclusions Altered distribution of SNARE complexes and increased Syt-I levels in LSG from SS-patients might indicate Ca+ signaling alterations. These alterations could explain altered polarized exocytosis and the presence of salivary mucins in the extracellular matrix of LSG from SS-patients. We demonstrate that 3D HSG acini are useful to evaluate the effects of disrupting Ca+ signaling and Syt-I over-expression on exocytosis, as a model to explain observations in LSG from SS-patients. Acknowledgements Fondecyt-Chile 1120062 (MJGB, SA, CM, SG), CONICYT-Chile PhD fellowship (MJB, JC and HU). Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.2393

SAT0175 Alterations of VAMP2 and SINTAXIN-2 in salivary acinar cells modify the secretion process in sjögren’s syndrome patients

Background Sjögren’s syndrome patients (SS) have oral and ocular dryness attributed to alterations in the quantity and quality of saliva and tears (1). We have demonstrated that disruption of cell-cell and cell-extracellular matrix (ECM) interactions that could modify the secretory pathway (2). Salivary acinar cells of SS-patients display alterations in their cell-polarity, affecting the correct localization and function of proteins composing the secretory machinery. SNARE-complexes are responsible for the secretion of proteins which are essential components of saliva. VAMP-2 and STX2 are SNARE-proteins that participate in the constitutive and regulated secretion. VAMP-2 is located in secretory granules next to the basolateral plasma membrane and it is involved in vesicular traffic towards the apical and basolateral poles, while STX2 is exclusively located in the apical plasma membrane (3). Objectives To determine the expression and localization of VAMP-2 and STX2, as well as their ability to form SNARE complexes under basal conditions in salivary acinar cells of SS-patients. Methods In labial salivary glands from SS-patients (n=27) and control subjects (n=17) mRNA and proteins levels of SNARE complex components were determined by real-time PCR and Western blotting, respectively. Protein localization was evaluated by immunofluorescence and confocal microscopy. Results In controls and SS-patients, STX2 and VAMP-2 mRNA levels remained unchanged. STX2 protein levels were found significantly augmented in SS-patients, while VAMP-2 protein levels did not change. In SS-patients, VAMP-2 showed a significant increase in apical staining, while STX2 showed decreased apical staining and apico-basal redistribution. Interestingly, increased formation of SNARE complexes containing VAMP-2 or STX2 in a manner independent of external secretory stimuli was detected in SS-patients. Conclusions In SS-patients, VAMP-2 was relocated to the apical cytoplasm; a change that might compensate the significant decrease of VAMP-8 observed in the apical region, as reported previously (4). Apical relocalization of VAMP-2 has been reported in VAMP-8 knockout mice, associated to a significant increase of VAMP-2 protein levels. Regardless of VAMP-2 relocalization, these mice show a decreased the secretory response to secretagogues (5). Conversely, STX2 protein levels increased significantly and showed a strong relocation from apical to basal plasma membrane. These findings suggest a probable loss of exocytic ability by the apical pole of acinar cells that may affect the destination of secretion proteins, thus impairing physiological production of saliva. Furthermore, a higher quantity of fusion complexes containing the studied proteins has been detected in SS-patients. The ectopic formation of SNARE-complexes in the basal domain of acinar cells is probably enhanced. FONDECYT #1120062, 1080006 and CONICYT PhD fellowship (BMJ). References Fox RI. Lancet. 2005;366:321-31 Ewert P. et al. Arthritis Rheum 2010;62:1280–9. Gaisano HY et al. Gastroenterology. 1996;111:1661-9 Barrera MJ et al. J. Autoimmunity, 2012 in press Wang CC et al. Dev Cell. 2004;7:359-71. Disclosure of Interest None Declared