Isabel Castro

Disruption of tight junction structure in salivary glands from Sjögren's syndrome patients is linked to proinflammatory cytokine exposure

OBJECTIVE Disorganization of acinar cell apical microvilli and the presence of stromal collagen in the acinar lumen suggest that the labial salivary gland (LSG) barrier function is impaired in patients with Sjögrens syndrome. Tight junctions define cell polarity and regulate the paracellular flow of ions and water, crucial functions of acinar cells. This study was undertaken to evaluate the expression and localization of tight junction proteins in LSGs from patients with SS and to determine in vitro the effects of tumor necrosis factor alpha (TNFalpha) and interferon-gamma (IFNgamma) on tight junction integrity of isolated acini from control subjects. METHODS Twenty-two patients and 15 controls were studied. The messenger RNA and protein levels of tight junction components (claudin-1, claudin-3, claudin-4, occludin, and ZO-1) were determined by semiquantitative reverse transcriptase-polymerase chain reaction and Western blotting. Tight junction protein localization was determined by immunohistochemistry. Tight junction ultrastructure was examined by transmission electron microscopy. Isolated acini from control subjects were treated with TNFalpha and IFNgamma. RESULTS Significant differences in tight junction protein levels were detected in patients with SS. ZO-1 and occludin were strongly down-regulated, while claudin-1 and claudin-4 were overexpressed. Tight junction proteins localized exclusively to apical domains in acini and ducts of LSGs from controls. In SS patients, the ZO-1 and occludin the apical domain presence of decreased, while claudin-3 and claudin-4 was redistributed to the basolateral plasma membrane. Exposure of isolated control acini to TNFalpha and IFNgamma reproduced these alterations in vitro. Ultrastructural analysis associated tight junction disorganization with the presence of endocytic vesicles containing electron-dense material that may represent tight junction components. CONCLUSION Our findings indicate that local cytokine production in LSGs from SS patients may contribute to the secretory gland dysfunction observed in SS patients by altering tight junction integrity of epithelial cells, thereby decreasing the quality and quantity of saliva.

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.

Changes in Rab3D Expression and Distribution in the Acini of Sjogren's Syndrome Patients Are Associated With Loss of Cell Polarity and Secretory Dysfunction

OBJECTIVE Oral and ocular dryness are frequent and serious symptoms of Sjögrens syndrome (SS) that reflect problems in secretion due to glandular dysfunction. Exocytosis, an important process in the secretory pathway, requires the participation of Rab family GTPases. This study was undertaken to analyze the expression and localization of Rab3D and Rab8A and to examine their correlation with acinar cell polarity and glandular secretory function. METHODS Nineteen patients with SS and 17 controls were evaluated. Levels of Rab3D and Rab8A messenger RNA (mRNA) and protein were determined by real-time polymerase chain reaction and Western blotting. Subcellular localization of proteins was determined by indirect immunofluorescence analysis. RESULTS In patients with SS, total Rab3D protein levels decreased significantly, while mRNA levels remained unchanged. For Rab8A, no changes in either mRNA or protein levels were detected. In serous acini of labial salivary glands from patients with SS, the following 4 patterns of Rab3D staining were distinguishable: severely decreased, distribution throughout the cytoplasm, distribution throughout the cytoplasm combined with loss of nuclear polarity, and normal apical localization. Basal localization of Rab8A was not modified. Rab3D changes were accompanied by apicobasolateral redistribution of ezrin, loss of nuclear polarity, thicker Golgi stacks, and mucin 7 accumulation in the cytoplasm. Finally, low Rab3D protein levels correlated with alterations in scintigraphy measurements. CONCLUSION Our findings indicate that Rab3D regulates the exocytosis of many components critical for the maintenance of oral physiology. Hence, the changes observed in Rab3D expression and distribution are likely to contribute to the decrease in or loss of saliva components (i.e., mucins), which may explain the variety of oral and ocular symptoms associated with SS.

Aberrant localization of fusion receptors involved in regulated exocytosis in salivary glands of Sjögren's syndrome patients is linked to ectopic mucin secretion.

Sjögrens syndrome (SS) is a chronic inflammatory autoimmune disease that mainly affects tear and salivary glands, whereby SS-patients frequently complain of eye and mouth dryness. Salivary acinar cells of SS-patients display alterations in their cell polarity; which may affect the correct localization and function of proteins involved in regulated exocytosis. Here we determined whether the expression and localization of SNARE proteins (membrane fusion receptors) involved in regulated secretion, such as VAMP8, syntaxin 3 (STX3), STX4 and SNAP-23 were altered in salivary glands (SG) from SS-patients. Additionally, we investigated SNARE proteins function, by evaluating their ability to form SNARE complexes under basal conditions. In SG from SS-patients and control subjects mRNA and proteins levels of SNARE complex components were determined by real-time PCR and Western blotting, respectively. SNARE protein distribution and mucin exocytosis were determined by indirect immunofluorescence. In SS-patients, the expression levels of mRNA and protein for VAMP8, STX4 and STX3 were altered. STX4, STX3, SNAP-23 and VAMP8 relocated from the apical to the basal region of acinar cells. Increased formation of SNARE complexes in a manner independent of external stimuli for secretion was detected. Mucins were detected in the extracellular matrix (ECM). Presence of mucins in the ECM, together with the observed alterations in SNARE protein localization is indicative of ectopic exocytosis. In the context of SS, such aberrantly localized mucins are likely to favor a pro-inflammatory response, which may represent an important initial step in the pathogenesis of this disease.

Salivary mucins induce a Toll-like receptor 4-mediated pro-inflammatory response in human submandibular salivary cells: are mucins involved in Sjögren’s syndrome?

OBJECTIVES A hallmark characteristic of SS patients is the ectopic presence of the mucins MUC5B and MUC7 in the extracellular matrix of salivary glands that have lost apical-basolateral acinar-cell polarity. This study aims to determine whether exogenous salivary mucins induce gene expression of pro-inflammatory cytokines, as well as to evaluate whether the Toll-like receptor-4 (TLR4) pathway is involved in this response. METHODS Differentiated human submandibular gland (HSG) cells were stimulated with mucins or oligosaccharide residues at different concentrations and for different periods of time. The expression of pro-inflammatory cytokines and their receptors was determined by semi-quantitative real time PCR (sqPCR). TLR4-mediated responses induced by mucin were evaluated with the Toll-IL-1 receptor domain containing adaptor protein (TIRAP) inhibitory peptide or using anti-hTLR4 blocking antibody. TLR4-receptor expression was also determined in SS patients, controls and HSG cells. RESULTS Mucins induced a significant increase in CXCL8, TNF-α, IFN-α, IFN-β, IL-6 and IL-1β, but not B cell activating factor (BAFF). Cytokine induction was mediated by TLR4, as shown using TIRAP or using anti-hTLR4 antibody. Sugar residues present in MUC5B, such as sulpho-Lewis (SO3-3Galβ1-3GlcNAc), also induced cytokines. Unexpectedly, mucins induced MUC5B, but not MUC7 expression. CONCLUSION Salivary mucins were recognized by TLR4 in epithelial cells initiating a pro-inflammatory response that could attract inflammatory cells to amplify and perpetuate inflammation and thereby contribute to the development of a chronic state characteristic of SS. The ectopic localization of MUC5B and MUC7 in the salivary gland extracellular matrix from SS patients and the current results reveal the importance of salivary epithelial cells in innate immunity, as well as in SS pathogenesis.

Impaired IRE1α/XBP-1 pathway associated to DNA methylation might contribute to salivary gland dysfunction in Sjögren’s syndrome patients

Objectives Labial salivary glands (LSGs) of SS patients show alterations related to endoplasmic reticulum stress. Glandular dysfunction could be partly the consequence of an altered inositol-requiring enzyme 1α (IRE1α)/X box-binding protein 1 (XBP-1) signalling pathway of the unfolded protein response, which then regulates genes involved in biogenesis of the secretory machinery. This study aimed to determine the expression, promoter methylation and localization of the IRE1α/XBP-1 pathway components in LSGs of SS patients and also their expression induced by IFN-γ in vitro. Methods IRE1α, XBP-1 and glucose-regulated protein 78 (GRP78) mRNA and protein levels were measured by qPCR and western blot, respectively, in LSGs of SS patients (n = 47) and control subjects (n = 37). Methylation of promoters was evaluated by methylation-sensitive high resolution melting, localization was analysed by immunofluorescence and induction of the IRE1α/XBP-1 pathway components by IFN-γ was evaluated in 3D acini. Results A significant decrease of IRE1α, XBP-1u, XBP-1s, total XBP-1 and GRP78 mRNAs was observed in LSGs of SS patients, which was correlated with increased methylation levels of their respective promoters, and consistently the protein levels for IRE1α, XBP-1s and GRP78 were observed to decrease. IFN-γ decreased the mRNA and protein levels of XBP-1s, IRE1α and GRP78, and increased methylation of their promoters. Significant correlations were also found between IRE1α/XBP-1 pathway components and clinical parameters. Conclusion Decreased mRNA levels for IRE1α, XBP-1 and GRP78 can be partially explained by hypermethylation of their promoters and is consistent with chronic endoplasmic reticulum stress, which may explain the glandular dysfunction observed in LSGs of SS patients. Additionally, glandular stress signals, including IFN-γ, could modulate the expression of the IRE1α/XBP-1 pathway components.

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

Association of high 5-hydroxymethylcytosine levels with Ten Eleven Translocation 2 overexpression and inflammation in Sjögren's syndrome patients

Here, we determined the 5-hydroxymethylcytosine (5hmC), 5-methylcytosine (5mC), Ten Eleven Translocation (TETs), and DNA methyltransferases (DNMTs) levels in epithelial and inflammatory cells of labial salivary glands (LSG) from Sjögrens syndrome (SS)-patients and the effect of cytokines on HSG cells. LSG from SS-patients, controls and HSG cells incubated with cytokines were analysed. Levels of 5mC, 5hmC, DNMTs, TET2 and MeCP2 were assessed by immunofluorescence. In epithelial cells from SS-patients, an increase in TET2, 5hmC and a decrease in 5mC and MeCP2 were observed, additionally, high levels of 5mC and DNMTs and low levels of 5hmC were detected in inflammatory cells. Cytokines increased TET2 and 5hmC and decreased 5mC levels. Considering that the TET2 gene.promoter contains response elements for transcription factors activated by cytokines, together to in vitro results suggest that changes in DNA hydroxymethylation, resulting from altered levels of TET2 are likely to be relevant in the Sjögrens syndrome etiopathogenesis.