Sergio 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.

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

OBJECTIVESnA 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.nnnMETHODSnDifferentiated 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.nnnRESULTSnMucins 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.nnnCONCLUSIONnSalivary 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.

Pro-inflammatory cytokines enhance ERAD and ATF6α pathway activity in salivary glands of Sjögren's syndrome patients

Salivary gland (SG) acinar-cells are susceptible to endoplasmic reticulum (ER) stress related to their secretory activity and the complexity of synthesized secretory products. SGs of Sjögrens syndrome patients (SS)-patients show signs of inflammation and altered proteostasis, associated with low IRE1α/XBP-1 pathway activity without avert increases in apoptosis. Acinar-cells may avoid apoptosis by activation of the ATF6α pathway and ER-associated protein degradation (ERAD). The aim of this study was to evaluate the role of pro-inflammatory cytokines in ATF6α pathway/ERAD activation and cell viability in labial salivary glands (LSG) of SS-patients. In biopsies from SS-patients increased ATF6α signaling pathway activity, as evidenced by generation of the ATF6f cleavage fragment, and increased expression of ERAD machinery components, such as EDEM1, p97, SEL1L, gp78, UBE2J1, UBE2G2, HERP and DERLIN1, were observed compared to controls. Alternatively, for pro- (active-caspase-3) and anti-apoptotic (cIAP2) markers no significant difference between the two experimental groups was detected. Increased presence of ATF6f and ERAD molecules correlated significantly with increased expression of pro-inflammatory cytokines. These observations were corroborated inxa0vitro in 3D-acini treated with TNF-α and/or IFN-γ, where an increase in the expression and activation of the ATF6α sensor and ERAD machinery components was detected under ER stress conditions, while changes in cell viability and caspase-3 activation were not observed. Cytokine stimulation protected cells from death when co-incubated with an ERAD machinery inhibitor. Alternatively, when cytokines were eliminated from the medium prior to ERAD inhibition, cell death increased, suggesting that the presence of pro-inflammatory cytokines in the medium is essential to maintain cell viability. In conclusion, the ATF6α pathway and the ERAD machinery are active in LSG of SS-patients. Both were also activated by TNF-α and IFN-γ inxa0vitro in 3D-acini and aided in preventing apoptosis. IFN-γ levels were elevated in SS-patients and UPR responses triggered inxa0vitro by this cytokine closely matched those observed in LSG from SS-patients, suggesting that cytokines may induce ER stress.

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

ObjectivesnLabial 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.nnnMethodsnIRE1α, 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.nnnResultsnA 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.nnnConclusionnDecreased 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.

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

Avances en el Estudio de la Diversidad Bacteriana Oral Asociada a Caries Dental Mediante el Estudio Genómico

La caries es una enfermedad infecciosa, transmisible y multifactorial, que conduce a la perdida de minerales reversible o irreversible de los tejidos duros susceptibles del diente, por accion de productos acidos provenientes de la fermentacion de los hidratos de carbono de la dieta por la actividad metabolica del biofilm adherido a la superficie dentaria. Aunque tradicionalmente se ha considerado al Streptococcus mutans como el responsable de la enfermedad, actualmente otras bacterias, denominadas no mutans, se han asociado con el inicio, progresion y actividad de la enfermedad en esmalte, dentina y cemento radicular. Para profundizar el estudio de la diversidad bacteriana oral asociada a caries dental se han aplicado diversas metodologias, dentro de las cuales destaca el estudio del metagenoma oral. Este nos permite estudiar comunidades bacterianas completas mediante el analisis del DNA, en un determinado ambiente sin necesidad de aislar y cultivar las especies, entregando informacion sobre la diversidad taxonomica y filogenetica de estas comunidades. Existen diferentes metodos de analisis de la diversidad bactariana, entre los que tenemos el analisis del ARNr 16S mediante electroforesis, PCR, microarreglos, secuenciamiento de ultima generacion, entre otros. El estudio del metagenoma oral ha permitido identificar especies que no han podido ser aisladas por metodos convencionales, ademas de identificar su presencia o ausencia en las distintas etapas del desarrollo de la enfermedad de caries dental, permitiendo un mejor conocimiento del desarrollo de esta patologia. El estudio basado en el metagenoma ha dado a conocer una diversidad microbiana oral inesperada, dando informacion relevante para la actualizacion de los conocimientos y asi identificar nuevos objetivos terapeuticos. El proposito de esta revision bibliografica es exponer los principales resultados que ha aportado el estudio del metagenoma sobre la diversidad microbiana, aplicado especificamente a la comunidad bacteriana oral.