M.-J. González

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.

Oral manifestations and their treatment in Sjögren′s syndrome

Sjögrens syndrome (SS) is a complex, chronic, systemic, autoimmune disease that mainly affects the exocrine glands, especially the salivary and lacrimal glands, leading to dryness of the oral and ocular mucosae. Several factors have been studied that could explain the glandular hypofunction primarily related to water transport. Recent reports have shown alterations in secretory route and trafficking in labial salivary glands, explaining alterations in the saliva quality. The decrease in salivary flow and qualitative alterations in saliva could explain many of the oral manifestations. The exocrine manifestations and systemic involvement significantly impact the patients perception of health-related quality of life. For this reason and given its systemic nature, the treatment of these patients should be multidisciplinary. This review addresses some particular oral health aspects of SS patients and focuses on relevant topics concerning the treatment and prevention of common oral disorders associated with this disease.

Involvement of MT1-MMP and TIMP-2 in human periodontal disease

OBJECTIVES Periodontal disease is characterized by an increased collagen metabolism. Although membrane type-1 matrix metalloproteinase (MT1-MMP) plays a critical role in collagen degradation, its involvement in human periodontitis remains to be determined. METHODS MT1-MMP and TIMP-2 expression and distribution were evaluated in gingival tissue samples derived from 10 healthy and 12 periodontitis-affected human subjects. MT1-MMP and TIMP-2 expression were assessed through Western-blot of tissue homogenates. The main cell types involved in MT1-MMP and TIMP-2 production were evaluated by means of immunohistochemistry. RESULTS Both MT1-MMP and TIMP-2 were significantly increased in periodontitis-affected gingival tissues when compared to healthy gingiva. Moreover, the balance between MT1-MMP and its inhibitor TIMP-2 was altered in periodontitis-affected tissues, suggesting an imbalance in this proteolytic axis. Immunohistochemistry demonstrated the expression of MT1-MMP in fibroblasts and macrophages in gingival tissues. MT1-MMP was detected in cells in close association with the gingival collagen matrix. TIMP-2 expression was identified in fibroblasts, macrophages and epithelial cells. CONCLUSIONS Our observations show an increased expression of MT1-MMP and TIMP-2 in periodontitis-affected gingival tissues. The altered balance between these two molecular mediators of collagen remodeling suggests their involvement in human periodontal disease.

Tumor necrosis factor‐α‐stimulated membrane type 1‐matrix metalloproteinase production is modulated by epidermal growth factor receptor signaling in human gingival fibroblasts

BACKGROUND AND OBJECTIVES Membrane type 1-matrix metalloproteinase (MT1-MMP) is a collagenolytic enzyme involved in connective tissue remodeling. In periodontal tissues, either cytokines or growth factors regulate the production of proteolytic enzymes. Mice deficient in epidermal growth factor receptor (EGFR) show a reduced expression of MT1-MMP, suggesting that this receptor may play an important role in MT1-MMP production. The present study evaluated the role of the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and EGFR in the production of MT1-MMP in gingival fibroblasts. MATERIAL AND METHODS Primary cultures of human gingival fibroblasts were cultured over plastic or a type I collagen matrix and stimulated with TNF-alpha and EGF. A selective EGFR inhibitor (AG1478) was used to interfere with this signaling pathway. Production of MT1-MMP and activation of proMMP-2 were studied using Western blot and gelatin zymography, respectively. Activation of EGFR signaling was assessed through immunoprecipitation and Western blot. Expression of EGFR ligands was determined through reverse transcriptase-polymerase chain reaction. RESULTS Treatment of gingival fibroblasts cultured over a collagen matrix with TNF-alpha stimulated proMMP-2 activation and MT1-MMP production. However, after using AG1478, both responses were inhibited. Tumor necrosis factor-alpha induced EGFR transactivation and stimulated the expression of the mRNA for the EGFR ligands heparin binding-epidermal growth factor (HB-EGF) and transforming growth factor-alpha (TGF-alpha). CONCLUSIONS The present study shows that TNF-alpha may stimulate MT1-MMP production through transactivation of EGFR. Tumor necrosis factor-alpha may also modulate the expression of the EGFR ligands TGF-alpha and HB-EGF. Production of MT1-MMP by TNF-alpha requires interaction with EGFR, suggesting that tissue remodeling is controlled by cross-communication between diverse signaling pathways in gingival fibroblasts.

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