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

Impaired IRE1α/XBP-1 pathway associated to DNA methylation might contribute to salivary gland... Abstract 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. Sjögren’s syndrome, glandular dysfunction, endoplasmic reticulum stress, unfolded protein response, IRE1α/XBP-1 pathway, gene promoter methylation, IFN-γ Rheumatology key messages The IRE1α/XBP-1 pathway of the unfolded protein response is attenuated in salivary glands of SS patients. In SS patients, reduced IRE1, XBP-1 and GRP78 mRNA levels is associated with the hypermethylation of their promoters. IFN-γ decreases the expression of the IRE1/XBP-1 pathway components in Sjögren's syndrome. Introduction Primary SS is a systemic chronic autoimmune disorder characterized by lymphocytic infiltration and functional changes of salivary and lachrymal glands [1]. Oral and ocular dryness has been associated with alterations in quantity and quality of mucins [2]. These are high-molecular-mass glycoproteins, containing oligosaccharide chains and intra-/intermolecular disulfide linkages, synthesized in the endoplasmic reticulum (ER) of salivary gland (SG) acinar cells [3]. One role of the ER is to ensure correct folding of proteins and glycoproteins destined for the secretory pathway [4]. Impaired ER function leads to the accumulation of unfolded proteins in the ER lumen, a condition termed ER stress, initiating an adaptive mechanism known as the unfolded protein response (UPR), seeking to restore ER homeostasis [5]. ER stress and the UPR have recently been linked to inflammation in a variety of human pathologies including autoimmune, metabolic, neurodegenerative and infectious disorders [6, 7]. In some circumstances, ER stress can promote the inflammation [8], but in others, the inflammation can be an important adjuvant of ER stress and can prompt the onset of disease in a genetically susceptible individual [9]. Pro-inflammatory cytokines deplete intracellular Ca2+ stores by interfering with the activity of the Ca2+-ATPase pump, thereby affecting calcium signalling in human salivary ductal and pancreatic cell lines [10, 11], a function also altered in the SGs of SS patients [12]. Moreover, calcium is required as a co-factor for Ca2+-dependent chaperones, including glucose-regulated protein 78 (GRP78), calnexin and calreticulin [4]. Studies have revealed that SGs from SS patients contain high levels of TNF-α, IL-1β, IL-6 and IFN-γ, among other cytokines [13], which could activate or exacerbate protein misfolding, thus inducing ER stress. Consequently, we postulate that SGs from SS patients are subject to chronic ER stress that would exacerbate SG inflammation. Additionally, SG acinar cells from SS patients show altered post-translational processing of mucin (MUC) 5B [14], intracellular accumulation of MUC1/SEC [15] and MUC7 [16], and dilatation of ER cisternae [17], all of them possibly related to ER stress. In our laboratory, we recently witnessed an increase of activating transcription factor 6α (ATF6α) signalling pathway activity and expression of ER-associated protein degradation (ERAD) machinery components in the SGs of SS patients, correlating with levels of pro-inflammatory cytokines [18]. Lastly, TNF-α or IFN-γ-treated 3D acini exhibited an increased expression and activation of ATF6α sensor and ERAD components [18]. Changes in the expression of components of the UPR pathways may also occur as a consequence of mutations, altered immune responses and epigenetic modifications [4, 19]. Interestingly, autoimmune diseases have been linked to modifications of epigenetic control [20, 21], and in particular, differential DNA methylation patterns have been observed in salivary epithelial and inflammatory cells of SS patients [22, 23]. Furthermore, specific genes have shown promoter hypo- (STAT1, IFI44L, LTA) or hypermethylation (RUNX1, FBXL16, BP230) [24, 25] and epigenome-wide association studies suggest a role of DNA methylation in the pathophysiology of primary SS [23, 26]. However, evidence linking epigenetic changes in specific genes of salivary epithelial cells to the pathogenesis of SS is scarce [23, 26] and no current information is available on the expression regulation of UPR pathway components by promoter methylation. Our study focused on the inositol-requiring enzyme 1α (IRE1α)/X box-binding protein 1 (XBP-1) pathway controlled by IRE1α (an ER resident serine/threonine-protein kinase/endoribonuclease) [5]. Under ER stress, IRE1α dimerizes/oligomerizes and then catalyses the splicing of the XBP-1 mRNA, resulting in the expression of transcription factor XBP-1s [5]. Consequently, XBP-1s induces UPR-related genes linked to the regulation of vesicle trafficking involved in secretion, protein folding, quality control, ERAD and ER/Golgi biogenesis [27, 28]. With this knowledge we analysed the expression, promoter methylation and localization of the IRE1α/XBP-1 pathway components, and then correlated these findings with clinical parameters of SS patients. The effect of inflammation in the induction of these components was also evaluated. Unexpectedly, our results showed a decreased activation of the IRE1α/XBP-1 pathway that could be explained by epigenetic control. Methods Patients with primary SS and control subjects The SS patients (n = 47) were diagnosed according to American–European Consensus Group Criteria [29], and control subjects (n = 37) were individuals who did not fulfil these criteria. In lip biopsy specimens from control subjects, only mild non-focal chronic sialadenitis was detected (supplementary Fig. S1, available at Rheumatology online). Scintigraphic evaluation of the SGs was performed according to Schall et al. [30]. Table 1 summarizes demographic, serological and histological characteristics of the samples. Patients gave written consent according to the Declaration of Helsinki and the study was approved by the Ethical Committee of the Faculty of Medicine, Universidad de Chile. Table 1 Demographic and serological characteristics of the SS patient and control groups Parameters Control subjects Patients with primary SS Number of individuals 37 47 Sex, n     Female 32 44     Male 5 3 Age, mean (range), years 42.8 (20–71) 45.7 (22–71) Xerophtalmia, n (%) 15 (40.5) 39 (83) Xerostomia, n (%) 10 (27) 39 (83) Focus scorea, n     1 0 21     2 0 17     3 0 9 USWSF, mean (s.d.), ml/15 min 3.24 (1.29) 1.38 (0.86) Scintigraphic data scoreb, n     1 7 3     2 19 18     3 11 12     4 0 12 Ro antibodies, n 0 35 Ro/La antibodies, n 0 21 ANA, n 0 41 RF, n 0 21 ESSDAI, median (range) — 3 (0–8) Parameters Control subjects Patients with primary SS Number of individuals 37 47 Sex, n     Female 32 44     Male 5 3 Age, mean (range), years 42.8 (20–71) 45.7 (22–71) Xerophtalmia, n (%) 15 (40.5) 39 (83) Xerostomia, n (%) 10 (27) 39 (83) Focus scorea, n     1 0 21     2 0 17     3 0 9 USWSF, mean (s.d.), ml/15 min 3.24 (1.29) 1.38 (0.86) Scintigraphic data scoreb, n     1 7 3     2 19 18     3 11 12     4 0 12 Ro antibodies, n 0 35 Ro/La antibodies, n 0 21 ANA, n 0 41 RF, n 0 21 ESSDAI, median (range) — 3 (0–8) a Number of foci/4 mm2 of tissue. b 1 = normal salivary gland function; 2 = mild impairment of salivary gland function; 3 = moderate impairment of salivary gland function; and 4 = severe impairment of salivary gland function. ESSDAI: EULAR SS disease activity index; USWSF: unstimulated whole salivary flow. Table 1 Demographic and serological characteristics of the SS patient and control groups Parameters Control subjects Patients with primary SS Number of individuals 37 47 Sex, n     Female 32 44     Male 5 3 Age, mean (range), years 42.8 (20–71) 45.7 (22–71) Xerophtalmia, n (%) 15 (40.5) 39 (83) Xerostomia, n (%) 10 (27) 39 (83) Focus scorea, n     1 0 21     2 0 17     3 0 9 USWSF, mean (s.d.), ml/15 min 3.24 (1.29) 1.38 (0.86) Scintigraphic data scoreb, n     1 7 3     2 19 18     3 11 12     4 0 12 Ro antibodies, n 0 35 Ro/La antibodies, n 0 21 ANA, n 0 41 RF, n 0 21 ESSDAI, median (range) — 3 (0–8) Parameters Control subjects Patients with primary SS Number of individuals 37 47 Sex, n     Female 32 44     Male 5 3 Age, mean (range), years 42.8 (20–71) 45.7 (22–71) Xerophtalmia, n (%) 15 (40.5) 39 (83) Xerostomia, n (%) 10 (27) 39 (83) Focus scorea, n     1 0 21     2 0 17     3 0 9 USWSF, mean (s.d.), ml/15 min 3.24 (1.29) 1.38 (0.86) Scintigraphic data scoreb, n     1 7 3     2 19 18     3 11 12     4 0 12 Ro antibodies, n 0 35 Ro/La antibodies, n 0 21 ANA, n 0 41 RF, n 0 21 ESSDAI, median (range) — 3 (0–8) a Number of foci/4 mm2 of tissue. b 1 = normal salivary gland function; 2 = mild impairment of salivary gland function; 3 = moderate impairment of salivary gland function; and 4 = severe impairment of salivary gland function. ESSDAI: EULAR SS disease activity index; USWSF: unstimulated whole salivary flow. Biopsies Labial SG (LSG) samples were obtained from SS patients and control subjects as described [31]. Following surgery, some samples were snap-frozen in liquid nitrogen and stored at −80°C until processed and the other samples were processed for morphological and immunofluorescence studies. Culture cell Human SG (HSG) cells were cultured as previously described [32] and 3D cell cultures were developed as previously documented [18]. These cultures were serum deprived over a period of 24 h and later incubated with recombinant human IFN-γ (Biolegend Inc., San Diego, CA, USA) (1 and 10 ng/ml) for a further 24 h. DNA, RNA and proteins were extracted from cells as described below. Gene expression by real-time PCR LSGs from SS patients (n = 23) and control subjects (n = 17) were analysed for detection of the IRE1α, IRE1β, XBP-1u, XBP-1s, XBP-1, GRP78 and h18S transcripts using specific primers designed with AmplifiX 1.4 software (supplementary Table S1, available at Rheumatology online). RNA extraction, yields and purity evaluation were performed as previously described [33]. Real-time PCR reactions were carried out using Brilliant II SYBR Green PCR Master Mix Kit (Stratagene) and MxPro-MX 3000P termocycler (Agilent Technologies, Santa Clara, CA, USA). Template cDNA obtained by reverse transcription of total RNA (1 μg) was subsequently amplified by real time-PCR as previously detailed [34] and each specific transcript tested was expressed as the ratio to h18S, using the efficiency-calibrated model [35]. Methylation-sensitive high resolution melting analysis Genomic DNA was extracted from LSGs from SS patients (n = 23) and control subjects (n = 17) using All Prep kit (Qiagen, Germantown, MD, USA) and treated with MethylCode Bisulfite Conversion kit (Thermo Fisher Scientific, Waltham, MA, USA). Methylation-sensitive high resolution melting primers for IRE1α, GRP78 and XBP-1 gene promoter analysis were designed using Methyl Primer Express Software v1.0 (Thermo Fisher Scientific), as recommended [36] (supplementary Table S1 and supplementary Fig. S2, available at Rheumatology online). Next, PCR amplification of bisulphite-modified templates was performed in quadruplicate and standard curves with dilutions of a representative DNA (with a recognized methylation percentage) were included in each assay. The melting curves were normalized by calculating the line that best fits the expected values, demonstrating the melting state of the template in a temperature range. This normalization enables the direct comparison of samples with differing levels of initial fluorescence. Standard curves were plotted and the methylation percentage was obtained from the linear regression analysis. Immunofluorescence detection of XBP-1 and GRP78 LSGs from SS patients (n = 6) and control subjects (n = 5) were fixed in 1% p-formaldehyde and embedded in paraffin, and tissue sections were incubated in an antigen recovery solution (0.01 M citrate, pH 6.0, at 92°C). These sections were then blocked with 0.25% casein/PBS (1 h at room temperature), and subsequently incubated with rabbit polyclonal anti-XBP-1 or anti-GRP78 primary antibodies (supplementary Table S2 and supplementary Fig. S3, available at Rheumatology online). Next, slides were incubated with an Alexa Fluor 488-conjugated secondary antibody/Hoechst 33342 (for nuclear staining) and afterwards visualized with a confocal laser scanning microscope (Olympus FluoView FV10i, Olympus, Center Valley, PA, USA). Finally, quantitative analysis of epithelial and/or inflammatory cell fluorescence intensity was performed using ImageJ software (for details see supplementary data section ‘Quantitative analysis of the fluorescence staining intensity’ and supplementary Fig. S6, available at Rheumatology online). Protein extraction and western blot analysis LSGs from SS patients (n = 18) and control subjects (n = 15) were homogenized as previously described [33]. Protein content was quantified by the Bradford method [37] and separated using SDS–PAGE (8% gel under reducing and denaturing conditions). The separated proteins were then transferred to nitrocellulose membranes and blocked (5% skimmed milk/protease-free prepared in Tris-buffered saline-Tween 20 (TBS-T)). Subsequently, blots were incubated with primary antibodies raised against XBP-1, IRE1α, GRP78 and β-actin (see supplementary Table S2 and supplementary Fig. S3, available at Rheumatology online). Later after washing in TBS-T, blots were incubated with secondary antibodies (horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit). Finally, protein bands were visualized by enhanced chemiluminescence kit (Thermo Fisher Scientific), quantified by densitometry and the protein levels were normalized to β-actin. Statistical analysis Mean values for measurements of variables in patient and control subject groups were compared using the Mann–Whitney test, and Spearman’s rank correlation analysis was also performed. P < 0.05 was considered significant. Results Decreased expression and promoter hypermethylation of IRE1α and XBP-1 in LSGs from SS patients In mammals, two IRE1 isoforms with similar kinase and endoribonuclease enzymatic activities have been described [38]. Here, relative mRNA levels of IRE1α and IRE1β variants were detected: IRE1α mRNA content was significantly decreased (P = 0.006) (Fig. 1A), while IRE1β mRNA remained unchanged (P = 0.72) in LSGs from SS patients (Fig. 1A). Additionally, relative IRE1α protein levels were decreased in LSGs from SS patients (P = 0.027) (Figs 1B and D). As mentioned previously, XBP-1s is produced by an IRE1α-dependent unconventional splicing of XBP-1 mRNA [5]. We found that relative mRNA levels of unspliced XBP-1 (XBP-1u) and XBP-1s were decreased in SS patients (P = 0.002 and P = 0.0003, respectively) (Fig. 1A), and mRNA levels for total XBP-1 (both variants) were also decreased (P = 0.015). For XBP-1 protein, two bands with differing electrophoretic mobility of 54 and 33 kDa were detected corresponding to XBP-1s and XBP-1u, respectively (Fig. 1C). Relative protein levels of XBP-1s were decreased in LSGs from SS patients (P = 0.002), while XBP-1u was similar between groups (P = 0.355) (Fig. 1D). Taking all this into account, these results validate the decreased expression of XBP-1 previously observed in a microarray analysis performed with enriched epithelial cell fractions obtained from SS patient LSGs (Fig. 1E). Fig. 1 View largeDownload slide Decreased expression and promoter hypermethylation of IRE1α and XBP-1 in LSGs from SS patients (A) Relative mRNA levels of IRE1α, IRE1β, XBP-1s, XBP-1u and total XBP-1 in LSG extracts from control subjects (C) and SS patients (P). (B and C) Representative western blots of IRE1α (130 kDa), XBP-1s (54 kDa) and XBP-1u (33 kDa) in LSG extracts from control subjects (C) and SS patients (P). β-Actin (47 kDa) was used as a loading control. (D) Relative protein levels are shown as box plots. (E) XBP-1 relative expression obtained by microarray. (F and G) Methylation percentage for IRE1α and XBP-1 gene promoters. *P < 0.05 was considered significant. Fig. 1 View largeDownload slide Decreased expression and promoter hypermethylation of IRE1α and XBP-1 in LSGs from SS patients (A) Relative mRNA levels of IRE1α, IRE1β, XBP-1s, XBP-1u and total XBP-1 in LSG extracts from control subjects (C) and SS patients (P). (B and C) Representative western blots of IRE1α (130 kDa), XBP-1s (54 kDa) and XBP-1u (33 kDa) in LSG extracts from control subjects (C) and SS patients (P). β-Actin (47 kDa) was used as a loading control. (D) Relative protein levels are shown as box plots. (E) XBP-1 relative expression obtained by microarray. (F and G) Methylation percentage for IRE1α and XBP-1 gene promoters. *P < 0.05 was considered significant. Additionally, a higher DNA methylation percentage of the IRE1α and XBP-1 promoters was observed in LSGs from SS patients compared with control subjects (P = 0.046 and P = 0.033, respectively) (Figs 1F and G). Moreover, mRNA levels showed a negative correlation with methylation percentage of their related promoters (supplementary Table S3, available at Rheumatology online). Subcellular XBP-1 distribution and content in LSGs from SS patients Using an XBP-1 antibody (see supplementary Fig. S3, available at Rheumatology online) that recognizes both XBP-1s and XBP-1u isoforms, we observed nuclear and cytoplasmic staining in acini with lower intensity in ducts from control LSG sections (Fig. 2A–C). Negative and positive staining for total XBP-1 was observed in nuclei of the same acinus of LSGs from SS patients (Fig. 2D and E, arrowheads). In ducts, cytoplasmic staining of XBP-1 in patients was similar to controls, but nuclear staining was weak in both groups (Fig. 2F). Additionally, inflammatory cell staining was found to be positive for the total XBP-1 (Fig. 2F, arrow). When quantifying with ImageJ software (see supplementary data section ‘Quantitative analysis of the fluorescence staining intensity’ and supplementary Fig. S6, available at Rheumatology online), a decrease of XBP-1 staining intensity was seen in epithelial cells of SS patients, and ∼40% of the total glandular XBP-1 staining intensity was contributed by the inflammatory cells (Fig. 2H). Fig. 2 View largeDownload slide XBP-1 localization in LSGs of controls (A−C) and SS patients (D−F) (A and D) Representative panoramic images showing XBP-1 staining in acini and ducts. (B and E) Higher magnification of acini selected in (A) and (D). (E) Nuclei with positive and negative (arrowheads) XBP-1 staining. (C and F) Representative ducts showing weak cytoplasmic staining. (F) Inflammatory cells with cytoplasmic staining (arrow). a: acini; d: duct; IC: inflammatory cells. Bars: 10 μm. (G) XBP-1 staining intensity was significantly lower in epithelial cells of LSGs from SS patients. (H) XBP-1 of inflammatory cells was ∼40% of the total glandular staining intensity. *P < 0.05 was considered significant. AU: arbitrary units. Fig. 2 View largeDownload slide XBP-1 localization in LSGs of controls (A−C) and SS patients (D−F) (A and D) Representative panoramic images showing XBP-1 staining in acini and ducts. (B and E) Higher magnification of acini selected in (A) and (D). (E) Nuclei with positive and negative (arrowheads) XBP-1 staining. (C and F) Representative ducts showing weak cytoplasmic staining. (F) Inflammatory cells with cytoplasmic staining (arrow). a: acini; d: duct; IC: inflammatory cells. Bars: 10 μm. (G) XBP-1 staining intensity was significantly lower in epithelial cells of LSGs from SS patients. (H) XBP-1 of inflammatory cells was ∼40% of the total glandular staining intensity. *P < 0.05 was considered significant. AU: arbitrary units. Decreased expression and promoter hypermethylation of GRP78 in LSGs from SS patients In this study, we found that GRP78 mRNA and protein levels were diminished (P = 0.028 and P = 0.014, respectively) in LSGs from SS patients (Fig. 3A, C and D) and the antibody raised against GRP78 also recognized a reduced content of glucose-regulated protein 94 (GRP94) (another ER-resident chaperone protein) (P = 0.047) in patients (Fig. 3C and D). Similar to IRE1α and XBP-1, methylation of GRP78 promoter was increased (P = 0.033) in patients (Fig. 3B) and GRP78 mRNA levels showed a negative correlation with methylation percentage of its promoter (see supplementary Table S3, available at Rheumatology online). Fig. 3 View largeDownload slide Decreased expression, promoter hypermethylation and localization of GRP78 in LSGs from SS patients (A, C and D) Relative mRNA and protein levels of GRP78. (B) Methylation percentage for GRP78 gene promoter. (E and H) Overview images showing GRP78/GRP94 staining. (F and I) GRP78/GRP94 was observed in the basolateral region of acinar cells, which showed lower staining than inflammatory cells (IC) (I, arrow). (G and J) Representative ducts with low staining and focal inflammatory cells without staining. Bars: 10 μm. (K) GRP78/GRP94 staining was significantly decreased in epithelial cells of LSGs from SS patients. (L) GRP78/GRP94 of inflammatory cells was ∼40% of the total glandular staining. *P < 0.05 was considered significant. Fig. 3 View largeDownload slide Decreased expression, promoter hypermethylation and localization of GRP78 in LSGs from SS patients (A, C and D) Relative mRNA and protein levels of GRP78. (B) Methylation percentage for GRP78 gene promoter. (E and H) Overview images showing GRP78/GRP94 staining. (F and I) GRP78/GRP94 was observed in the basolateral region of acinar cells, which showed lower staining than inflammatory cells (IC) (I, arrow). (G and J) Representative ducts with low staining and focal inflammatory cells without staining. Bars: 10 μm. (K) GRP78/GRP94 staining was significantly decreased in epithelial cells of LSGs from SS patients. (L) GRP78/GRP94 of inflammatory cells was ∼40% of the total glandular staining. *P < 0.05 was considered significant. Subcellular GRP78/GRP94 distribution and content in LSGs from SS patients In LSG sections of control subjects, GRP78/GRP94 was primarily detected in the basolateral region of acinar cells, where the ER is localized (Fig. 3E–G). In LSG sections of SS patients, the distribution of GRP78/GRP94 was maintained; nevertheless acinar staining intensity decreased (Fig. 3H–K). Common to both groups, ductal staining was virtually undetectable (Fig. 3G and J), whereas some inflammatory cells showed strong staining (Fig. 3H–J). In SS patients, inflammatory cells harbouring GRP78/GRP94 labelling was ∼40% of total glandular staining intensity (Fig. 3L). Although no staining was detected in inflammatory cells adjacent to the ducts, several peripheral focus cells were positive (Fig. 3J), displaying morphological characteristics of plasma cells. Advancing this hypothesis, comparison of the localization pattern of GRP78-positive cells with CD20- (B-lymphocyte antigen absent in plasma cells) and CD38- (highly expressed on plasma cells, activated T and B cells) positive cells demonstrated that CD20-positive cells localized at the centre of the focus, whereas cells positive for CD38 and GRP78 localized at the periphery (supplementary Fig. S4A–F, available at Rheumatology online). Therefore, cells expressing GRP78 and CD38 showed an abundant cytoplasm and lack of CD20 staining (supplementary Fig. S4A–F, available at Rheumatology online). Finally, these results imply that GRP78-positive immune cells may potentially be plasma cells. Observations in LSGs from SS patients are simulated in IFN-γ-stimulated 3D acini We observed that 10 ng/ml IFN-γ decreased mRNA of IRE1α, XBP-1s, XBP-1u and GRP78 in HSG cell-formed 3D acini cultured for 24 h (Fig. 4A) and XBP-1s, GRP78 and IRE1α protein levels (Fig. 4B–H). Under these conditions, we also observed a significant hypermethylation of their promoters (Fig. 4I–K). A decreased MUC1/SEC secretion was observed after stimulation of HSG cells with secretagogues previously incubated with IFN-γ or tunicamycin (ER stressor) compared with non-treated cells (supplementary Fig. S5, available at Rheumatology online). Fig. 4 View largeDownload slide 3 D acini exposed to IFN-γ behave as LSGs from SS patients (A) IRE1α, XBP-1s, XBP-1u and GRP78 mRNA levels in 3D acini incubated with or without IFN-γ (1 or 10 ng/ml) for 24 h. (B, D and G) Representative western blots of IRE1α, XBP-1s, XBP-1u and GRP78, in 3D acini incubated with or without IFN-γ. (C, E, F and H) Respectively, IRE1α, XBP-1s, XBP-1u and GRP78 protein levels relative to β-actin. (I−K) DNA methylation percentage of IRE1α, XBP-1 and GRP78 gene promoters of 3D acini incubated with or without IFN-γ. Results are representative of three independent experiments. *P < 0.05 was considered significant. Fig. 4 View largeDownload slide 3 D acini exposed to IFN-γ behave as LSGs from SS patients (A) IRE1α, XBP-1s, XBP-1u and GRP78 mRNA levels in 3D acini incubated with or without IFN-γ (1 or 10 ng/ml) for 24 h. (B, D and G) Representative western blots of IRE1α, XBP-1s, XBP-1u and GRP78, in 3D acini incubated with or without IFN-γ. (C, E, F and H) Respectively, IRE1α, XBP-1s, XBP-1u and GRP78 protein levels relative to β-actin. (I−K) DNA methylation percentage of IRE1α, XBP-1 and GRP78 gene promoters of 3D acini incubated with or without IFN-γ. Results are representative of three independent experiments. *P < 0.05 was considered significant. Relative levels of IRE1α/XBP-1s pathway components and downstream target proteins correlate with clinical parameters of SS patients Regarding IRE1α protein levels, negative correlation with eye (r = −0.68; P = 0.005) and mouth dryness (r = −0.76; P = 0.001) was witnessed and positive correlation with unstimulated whole salivary flow (r = 0.78; P = 0.007) was observed (Table 2). Additionally, XBP-1s protein levels were negatively correlated with eye (r = −0.54; P = 0.04) and mouth dryness (r = −0.57; P = 0.03). Also, an inverse correlation was detected between XBP-1s and Ro (r = −0.54; P = 0.04) as well as XBP-1s and ANA auto-antibodies (r = −0.58; P = 0.02). Furthermore, GRP78 protein levels were negatively correlated with eye (r = −0.59; P = 0.01) and mouth dryness (r = −0.59; P = 0.01). Lastly, serological parameters correlated with dryness symptoms and scintigraphy data (Table 2). Table 2 Spearman’s rank correlation coefficients between protein levels and clinical parameters of SS patients and control subjects Parameters Dryness Serology Eye Mouth USWSF Scintigraphy Ro La RF ANA Eye dryness 1.00 Mouth dryness 0.65* 1.00 USWSF –0.28 –0.64* 1.00 Scintigraphy 0.39* 0.49* –0.37* 1.00 Ro 0.46* 0.6* –0.43* 0.79* 1.00 La 0.35* 0.49* –0.35 0.67* 0.84* 1.00 RF 0.56* 0.59* –0.62* 0.57* 0.66* 0.57* 1.00 ANA 0.52* 0.57* –0.44* 0.62* 0.72* 0.69* 0.61* 1.00 IRE1α protein –0.68* –0.76* 0.78* –0.24 –0.25 0.36 –0.39 –0.46 XBP-1s protein –0.54* –0.57* 0.2 –0.4 –0.54* 0.62* –0.31 –0.58* GRP78 protein –0.59* –0.59* 0.26 –0.02 –0.19 0.16 –0.14 –0.32 Parameters Dryness Serology Eye Mouth USWSF Scintigraphy Ro La RF ANA Eye dryness 1.00 Mouth dryness 0.65* 1.00 USWSF –0.28 –0.64* 1.00 Scintigraphy 0.39* 0.49* –0.37* 1.00 Ro 0.46* 0.6* –0.43* 0.79* 1.00 La 0.35* 0.49* –0.35 0.67* 0.84* 1.00 RF 0.56* 0.59* –0.62* 0.57* 0.66* 0.57* 1.00 ANA 0.52* 0.57* –0.44* 0.62* 0.72* 0.69* 0.61* 1.00 IRE1α protein –0.68* –0.76* 0.78* –0.24 –0.25 0.36 –0.39 –0.46 XBP-1s protein –0.54* –0.57* 0.2 –0.4 –0.54* 0.62* –0.31 –0.58* GRP78 protein –0.59* –0.59* 0.26 –0.02 –0.19 0.16 –0.14 –0.32 For scintigraphic data, we used lower values to indicate better glandular function. * P < 0.05. USWSF: unstimulated whole salivary flow. Table 2 Spearman’s rank correlation coefficients between protein levels and clinical parameters of SS patients and control subjects Parameters Dryness Serology Eye Mouth USWSF Scintigraphy Ro La RF ANA Eye dryness 1.00 Mouth dryness 0.65* 1.00 USWSF –0.28 –0.64* 1.00 Scintigraphy 0.39* 0.49* –0.37* 1.00 Ro 0.46* 0.6* –0.43* 0.79* 1.00 La 0.35* 0.49* –0.35 0.67* 0.84* 1.00 RF 0.56* 0.59* –0.62* 0.57* 0.66* 0.57* 1.00 ANA 0.52* 0.57* –0.44* 0.62* 0.72* 0.69* 0.61* 1.00 IRE1α protein –0.68* –0.76* 0.78* –0.24 –0.25 0.36 –0.39 –0.46 XBP-1s protein –0.54* –0.57* 0.2 –0.4 –0.54* 0.62* –0.31 –0.58* GRP78 protein –0.59* –0.59* 0.26 –0.02 –0.19 0.16 –0.14 –0.32 Parameters Dryness Serology Eye Mouth USWSF Scintigraphy Ro La RF ANA Eye dryness 1.00 Mouth dryness 0.65* 1.00 USWSF –0.28 –0.64* 1.00 Scintigraphy 0.39* 0.49* –0.37* 1.00 Ro 0.46* 0.6* –0.43* 0.79* 1.00 La 0.35* 0.49* –0.35 0.67* 0.84* 1.00 RF 0.56* 0.59* –0.62* 0.57* 0.66* 0.57* 1.00 ANA 0.52* 0.57* –0.44* 0.62* 0.72* 0.69* 0.61* 1.00 IRE1α protein –0.68* –0.76* 0.78* –0.24 –0.25 0.36 –0.39 –0.46 XBP-1s protein –0.54* –0.57* 0.2 –0.4 –0.54* 0.62* –0.31 –0.58* GRP78 protein –0.59* –0.59* 0.26 –0.02 –0.19 0.16 –0.14 –0.32 For scintigraphic data, we used lower values to indicate better glandular function. * P < 0.05. USWSF: unstimulated whole salivary flow. Discussion Here, we report reduced expression of IRE1α, XBP-1 and GRP78, suggesting that activation of the IRE1α/XBP-1 signalling pathway was impaired due to promoter hypermethylation; however, alternative mechanisms contributing to this effect cannot be ruled out. Changes in the IRE1α/XBP-1 axis were studied in LSG sections from SS patients, where acinar and ductal cells coexist with inflammatory cells and immunoreactivity for each protein corresponded to its expected localization in acinar cells. Moreover, in situ staining intensity of IRE1α/XBP-1 signalling pathway components was separately quantified in epithelial and inflammatory cells to allow comparison with results obtained in glandular extracts using other molecular analyses. Our results demonstrate that the population of inflammatory cells, localized in the periphery of focus, was positive for XBP-1 and GRP78/GRP94. Combined staining of specific cell markers and morphology characteristics (abundant cytoplasm and typical nucleus described as cartwheel) strongly suggest that these correspond to plasma cells. Here, it should be noted that all SS patients studied had a focus score of between 1 and 3, and ∼90% of remnant parenchyma could be observed in their LSGs (supplementary Fig. S1 and Table S1, available at Rheumatology online). Negative and positive XBP-1 staining was observed in nuclei of the same acinus of LSGs from SS patients, and this suggests an unknown paracrine mechanism that would control IRE1α/XBP-1 signalling among adjacent acinar cells. The antibody used recognizes XBP-1s and XBP-1u isoforms, both containing a nuclear localization signal. XBP-1u is a negative regulator of XBP-1s, switching off the transcription of target genes during ER stress [39]. The low focus score of LSGs analysed coupled with elevated remnant parenchyma and uncertain discrimination of XBP-1 isoforms by immunofluorescence, leads us to the interpretation that the reduction of XBP-1 mRNA and protein levels (together with the presence of unstained nuclei) suggests the inactivation of the IRE1α/XBP-1 pathway in epithelial cells. Lastly, the strong immunostaining observed of XBP-1 in CD20−/CD38+ inflammatory cells (with abundant cytoplasm), is consistent with plasma cells (highly secretory phenotype) subjected to basal stress conditions [7]. It has been reported that IRE1α endoribonuclease activity is important for XBP-1 mRNA processing [28]. Thus, the reduced levels of IRE1α protein obtained in LSGs from SS patients are consistent with decreased mRNA levels of XBP-1s. Through XBP-1s, the IRE1α signalling pathway leads to increased expression of chaperone proteins (i.e. GRP78) as well as glycosylation, ERAD and traffic and synthesis of exocytic proteins [28]. XBP-1s is also a transcriptional activator that regulates the expansion of the secretory machinery controlling lipid biosynthesis and biogenesis of the most important compartments of the secretory pathway, such as the ER/Golgi [40]. Evidence demonstrates that XBP-1−/−; LivXBP-1 mice show dilatation and disorganization of the ER cisternae, altered expression of chaperone proteins and alterations in secretory granules of pancreatic and SG acinar cells [40]. Similar ultrastructural changes have been previously reported in LSGs of SS patients [17]. Furthermore, an inverse correlation between XBP-1s protein levels and the presence of Ro auto-antibodies was also observed in this study and it is also known that XBP-1s is degraded by the ubiquitin–proteasome system [27, 39]. Ro52 is an E3 ubiquitin ligase [41], which mediates ERAD and the ubiquitination of several proteins. This finding allows us to postulate that Ro auto-antibodies combined with low XBP-1s protein level might be caused by high levels of Ro autoantigen observed in a previous expression profiling study [42]. Decreased XBP-1s levels suggest the decrease of GRP78 expression, a contributing factor to impairment of the secretory pathway in LSGs of SS patients [39, 43]. However, it has also been described that the expression of GRP78 depends on ATF6f [27, 44]. Intriguingly, ATF6f is increased in the LSG from SS patients compared with control subjects [18], which would suggest that decreased GRP78 levels could be attributed to stress inducers, acute or chronic stress, cellular types and/or epigenetic regulation. Interestingly, here we demonstrate that hypermethylation of CpG islands in XBP-1 and GRP78 gene promoters would also modulate its expression and prevent binding of ATF6f and other transcriptional factors to response elements (e.g. type endoplasmic reticulum stress element) thus causing transcript levels of XBP-1 and GRP78 to remain low (supplementary Fig. S2, available at Rheumatology online). Decreased GRP78 could generate defects in protein folding and, as a consequence, lead to protein accumulation, as has been observed for MUC1 in LSGs of SS patients [15]. The GRP78-detecting antibody employed in this study also recognizes GRP94 (an ER resident chaperone protein found decreased in LSGs of SS patients), which plays an important role in the trafficking and secretion of ER components [45] and whose decrease may lead to defective folding of newly synthesized peptides. Remarkably, loss of GRP94 decreases the XBP-1s level [46], which concurs with our observations. Moreover, the decrease of ER protein-folding chaperones, such as GRP78 and GRP94, suggests that the mucins of acinar cells might be misfolded, leading to their accumulation in the cytoplasm [15, 16]. Incorrect mucin folding or glycosylation might then explain the alterations in post-translational processing, such as sulfation [14], which is required for mucins to bind large amounts of water molecules and moisturize oral mucosa surfaces [2]. On the other hand, low GRP78, IRE1α and XBP-1s protein levels correlate with eye and mouth dryness symptoms (Table 2). Mucosa dryness is directly related to the quantity/quality of secreted mucins, dependent on effective protein folding and the quality control machinery in the ER. These findings clarify previous data showing that the UPR pathway triggered by IRE1α/XBP-1s regulates the functionality of secretory cells [40]. Understanding the inflammatory environment arising in the LSG from SS patients, we simulated such conditions by incubating HSG cells with IFN-γ and the expression pattern of XBP-1s, IRE1α and GRP78 as well as the hypermethylation of these promoters was observed to resemble that of the LSG. Under similar culture conditions the cytoplasmic staining of MUC1/SEC increased while its secretion decreased upon stimulation with secretagogues, suggesting that an attenuated activity of the IRE1α pathway aggravates secretory functions (supplementary Fig. S5, available at Rheumatology online). These results suggest a role for IFN-γ in SS pathogenesis [47, 48] and, therefore, our in vitro data confirmed its importance in the modulation of UPR signalling [18]. This is the first study on the methylation of CpG island promoters for IRE1α pathway components, and here decreased mRNA levels for IRE1α, XBP-1 and GRP78 might be, in part, explained by epigenetic regulation through the hypermethylation process in LSGs of SS patients. Furthermore, recent studies have reported differential global DNA methylation along with specific methylation of promoters (hypo- or hypermethylation) in SS [23, 24, 49]. The epithelial cells of SGs from SS patients are subjected to high and diverse stress factors and in response, the cellular metabolic machinery develops homeostasis-preserving mechanisms (including those aimed at modulating the expression of IRE1α/XBP-1 pathway components). Our study showed such response was reflected in an increased DNA methylation of gene promoters that are essential for transcription of important proteins of the IRE1α/XBP-1 pathway. Preliminary, unpublished data of our laboratory suggest that an increased expression of the DNA methyltransferases DNMT1, DNMT3a and 3b takes place in LSGs of SS patients. In conclusion, morphological and functional alterations of the LSGs from SS patients are indicative of elevated ER stress, although it is unclear whether ER stress detected in the acinar cells represents a cause or rather reflects the consequences. Given that SS is a chronic disease and usually diagnosed late in progression, the onset and timing of UPR-related events remain unknown. Furthermore, it is important to mention that responses elicited by the IRE1α/XBP-1s pathway are rapidly attenuated despite stress persistence, enabling restoration of homeostasis in the UPR adaptation phase [50]. Attenuation of IRE1α/XBP-1s pathway activity and sustained activation of the ATF6 pathway [18] in acinar cells may be an indicator of chronic ER stress, where local inflammation may contribute to the alterations in LSGs from SS patients. Acknowledgements We thank all the patients who participated in this study and gratefully acknowledge Dr Claudio Hetz (Biomedical Neuroscience Institute, Universidad de Chile) for providing N2a cells transfected with shXBP-1, and for revising critically the manuscript. We thank Sandra Gendler and Cathy S. Madsen (Mayo Clinic Scottsdale, College of Medicine, Department of Biochemistry and Molecular Biology) for providing antibodies for MUC1/SEC. Funding: This work was supported by Fondecyt-Chile [1160015 and 1120062 to M.J.G., S.A., C.M., S.G., I.C., 1130250 to A.F.G.Q.]; Fondecyt-Postdoctorado [3170023 to M.J.B.]; CONICYT-FONDAP Chile [15130011 to A.F.G.Q.]; CONICYT/Programa de Investigación Asociativa [ACT 1111 to A.F.G.Q.] and PhD fellowship Conicyt-Chile to D.S. Disclosure statement: The authors have declared no conflicts of interest. Supplementary data Supplementary data are available at Rheumatology online. References 1 Tzioufas AG , Kapsogeorgou EK , Moutsopoulos HM. Pathogenesis of Sjogren's syndrome: what we know and what we should learn . J Autoimmun 2012 ; 39 : 4 – 8 . Google Scholar CrossRef Search ADS PubMed 2 Castro I , Sepulveda D , Cortes J et al. Oral dryness in Sjogren's syndrome patients. Not just a question of water . Autoimmun Rev 2013 ; 12 : 567 – 74 . Google Scholar CrossRef Search ADS PubMed 3 Hattrup CL , Gendler SJ. Structure and function of the cell surface (tethered) mucins . 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Impaired IRE1α/XBP-1 pathway associated to DNA methylation might contribute to salivary gland dysfunction in Sjögren’s syndrome patients

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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Abstract

Abstract 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. Sjögren’s syndrome, glandular dysfunction, endoplasmic reticulum stress, unfolded protein response, IRE1α/XBP-1 pathway, gene promoter methylation, IFN-γ Rheumatology key messages The IRE1α/XBP-1 pathway of the unfolded protein response is attenuated in salivary glands of SS patients. In SS patients, reduced IRE1, XBP-1 and GRP78 mRNA levels is associated with the hypermethylation of their promoters. IFN-γ decreases the expression of the IRE1/XBP-1 pathway components in Sjögren's syndrome. Introduction Primary SS is a systemic chronic autoimmune disorder characterized by lymphocytic infiltration and functional changes of salivary and lachrymal glands [1]. Oral and ocular dryness has been associated with alterations in quantity and quality of mucins [2]. These are high-molecular-mass glycoproteins, containing oligosaccharide chains and intra-/intermolecular disulfide linkages, synthesized in the endoplasmic reticulum (ER) of salivary gland (SG) acinar cells [3]. One role of the ER is to ensure correct folding of proteins and glycoproteins destined for the secretory pathway [4]. Impaired ER function leads to the accumulation of unfolded proteins in the ER lumen, a condition termed ER stress, initiating an adaptive mechanism known as the unfolded protein response (UPR), seeking to restore ER homeostasis [5]. ER stress and the UPR have recently been linked to inflammation in a variety of human pathologies including autoimmune, metabolic, neurodegenerative and infectious disorders [6, 7]. In some circumstances, ER stress can promote the inflammation [8], but in others, the inflammation can be an important adjuvant of ER stress and can prompt the onset of disease in a genetically susceptible individual [9]. Pro-inflammatory cytokines deplete intracellular Ca2+ stores by interfering with the activity of the Ca2+-ATPase pump, thereby affecting calcium signalling in human salivary ductal and pancreatic cell lines [10, 11], a function also altered in the SGs of SS patients [12]. Moreover, calcium is required as a co-factor for Ca2+-dependent chaperones, including glucose-regulated protein 78 (GRP78), calnexin and calreticulin [4]. Studies have revealed that SGs from SS patients contain high levels of TNF-α, IL-1β, IL-6 and IFN-γ, among other cytokines [13], which could activate or exacerbate protein misfolding, thus inducing ER stress. Consequently, we postulate that SGs from SS patients are subject to chronic ER stress that would exacerbate SG inflammation. Additionally, SG acinar cells from SS patients show altered post-translational processing of mucin (MUC) 5B [14], intracellular accumulation of MUC1/SEC [15] and MUC7 [16], and dilatation of ER cisternae [17], all of them possibly related to ER stress. In our laboratory, we recently witnessed an increase of activating transcription factor 6α (ATF6α) signalling pathway activity and expression of ER-associated protein degradation (ERAD) machinery components in the SGs of SS patients, correlating with levels of pro-inflammatory cytokines [18]. Lastly, TNF-α or IFN-γ-treated 3D acini exhibited an increased expression and activation of ATF6α sensor and ERAD components [18]. Changes in the expression of components of the UPR pathways may also occur as a consequence of mutations, altered immune responses and epigenetic modifications [4, 19]. Interestingly, autoimmune diseases have been linked to modifications of epigenetic control [20, 21], and in particular, differential DNA methylation patterns have been observed in salivary epithelial and inflammatory cells of SS patients [22, 23]. Furthermore, specific genes have shown promoter hypo- (STAT1, IFI44L, LTA) or hypermethylation (RUNX1, FBXL16, BP230) [24, 25] and epigenome-wide association studies suggest a role of DNA methylation in the pathophysiology of primary SS [23, 26]. However, evidence linking epigenetic changes in specific genes of salivary epithelial cells to the pathogenesis of SS is scarce [23, 26] and no current information is available on the expression regulation of UPR pathway components by promoter methylation. Our study focused on the inositol-requiring enzyme 1α (IRE1α)/X box-binding protein 1 (XBP-1) pathway controlled by IRE1α (an ER resident serine/threonine-protein kinase/endoribonuclease) [5]. Under ER stress, IRE1α dimerizes/oligomerizes and then catalyses the splicing of the XBP-1 mRNA, resulting in the expression of transcription factor XBP-1s [5]. Consequently, XBP-1s induces UPR-related genes linked to the regulation of vesicle trafficking involved in secretion, protein folding, quality control, ERAD and ER/Golgi biogenesis [27, 28]. With this knowledge we analysed the expression, promoter methylation and localization of the IRE1α/XBP-1 pathway components, and then correlated these findings with clinical parameters of SS patients. The effect of inflammation in the induction of these components was also evaluated. Unexpectedly, our results showed a decreased activation of the IRE1α/XBP-1 pathway that could be explained by epigenetic control. Methods Patients with primary SS and control subjects The SS patients (n = 47) were diagnosed according to American–European Consensus Group Criteria [29], and control subjects (n = 37) were individuals who did not fulfil these criteria. In lip biopsy specimens from control subjects, only mild non-focal chronic sialadenitis was detected (supplementary Fig. S1, available at Rheumatology online). Scintigraphic evaluation of the SGs was performed according to Schall et al. [30]. Table 1 summarizes demographic, serological and histological characteristics of the samples. Patients gave written consent according to the Declaration of Helsinki and the study was approved by the Ethical Committee of the Faculty of Medicine, Universidad de Chile. Table 1 Demographic and serological characteristics of the SS patient and control groups Parameters Control subjects Patients with primary SS Number of individuals 37 47 Sex, n     Female 32 44     Male 5 3 Age, mean (range), years 42.8 (20–71) 45.7 (22–71) Xerophtalmia, n (%) 15 (40.5) 39 (83) Xerostomia, n (%) 10 (27) 39 (83) Focus scorea, n     1 0 21     2 0 17     3 0 9 USWSF, mean (s.d.), ml/15 min 3.24 (1.29) 1.38 (0.86) Scintigraphic data scoreb, n     1 7 3     2 19 18     3 11 12     4 0 12 Ro antibodies, n 0 35 Ro/La antibodies, n 0 21 ANA, n 0 41 RF, n 0 21 ESSDAI, median (range) — 3 (0–8) Parameters Control subjects Patients with primary SS Number of individuals 37 47 Sex, n     Female 32 44     Male 5 3 Age, mean (range), years 42.8 (20–71) 45.7 (22–71) Xerophtalmia, n (%) 15 (40.5) 39 (83) Xerostomia, n (%) 10 (27) 39 (83) Focus scorea, n     1 0 21     2 0 17     3 0 9 USWSF, mean (s.d.), ml/15 min 3.24 (1.29) 1.38 (0.86) Scintigraphic data scoreb, n     1 7 3     2 19 18     3 11 12     4 0 12 Ro antibodies, n 0 35 Ro/La antibodies, n 0 21 ANA, n 0 41 RF, n 0 21 ESSDAI, median (range) — 3 (0–8) a Number of foci/4 mm2 of tissue. b 1 = normal salivary gland function; 2 = mild impairment of salivary gland function; 3 = moderate impairment of salivary gland function; and 4 = severe impairment of salivary gland function. ESSDAI: EULAR SS disease activity index; USWSF: unstimulated whole salivary flow. Table 1 Demographic and serological characteristics of the SS patient and control groups Parameters Control subjects Patients with primary SS Number of individuals 37 47 Sex, n     Female 32 44     Male 5 3 Age, mean (range), years 42.8 (20–71) 45.7 (22–71) Xerophtalmia, n (%) 15 (40.5) 39 (83) Xerostomia, n (%) 10 (27) 39 (83) Focus scorea, n     1 0 21     2 0 17     3 0 9 USWSF, mean (s.d.), ml/15 min 3.24 (1.29) 1.38 (0.86) Scintigraphic data scoreb, n     1 7 3     2 19 18     3 11 12     4 0 12 Ro antibodies, n 0 35 Ro/La antibodies, n 0 21 ANA, n 0 41 RF, n 0 21 ESSDAI, median (range) — 3 (0–8) Parameters Control subjects Patients with primary SS Number of individuals 37 47 Sex, n     Female 32 44     Male 5 3 Age, mean (range), years 42.8 (20–71) 45.7 (22–71) Xerophtalmia, n (%) 15 (40.5) 39 (83) Xerostomia, n (%) 10 (27) 39 (83) Focus scorea, n     1 0 21     2 0 17     3 0 9 USWSF, mean (s.d.), ml/15 min 3.24 (1.29) 1.38 (0.86) Scintigraphic data scoreb, n     1 7 3     2 19 18     3 11 12     4 0 12 Ro antibodies, n 0 35 Ro/La antibodies, n 0 21 ANA, n 0 41 RF, n 0 21 ESSDAI, median (range) — 3 (0–8) a Number of foci/4 mm2 of tissue. b 1 = normal salivary gland function; 2 = mild impairment of salivary gland function; 3 = moderate impairment of salivary gland function; and 4 = severe impairment of salivary gland function. ESSDAI: EULAR SS disease activity index; USWSF: unstimulated whole salivary flow. Biopsies Labial SG (LSG) samples were obtained from SS patients and control subjects as described [31]. Following surgery, some samples were snap-frozen in liquid nitrogen and stored at −80°C until processed and the other samples were processed for morphological and immunofluorescence studies. Culture cell Human SG (HSG) cells were cultured as previously described [32] and 3D cell cultures were developed as previously documented [18]. These cultures were serum deprived over a period of 24 h and later incubated with recombinant human IFN-γ (Biolegend Inc., San Diego, CA, USA) (1 and 10 ng/ml) for a further 24 h. DNA, RNA and proteins were extracted from cells as described below. Gene expression by real-time PCR LSGs from SS patients (n = 23) and control subjects (n = 17) were analysed for detection of the IRE1α, IRE1β, XBP-1u, XBP-1s, XBP-1, GRP78 and h18S transcripts using specific primers designed with AmplifiX 1.4 software (supplementary Table S1, available at Rheumatology online). RNA extraction, yields and purity evaluation were performed as previously described [33]. Real-time PCR reactions were carried out using Brilliant II SYBR Green PCR Master Mix Kit (Stratagene) and MxPro-MX 3000P termocycler (Agilent Technologies, Santa Clara, CA, USA). Template cDNA obtained by reverse transcription of total RNA (1 μg) was subsequently amplified by real time-PCR as previously detailed [34] and each specific transcript tested was expressed as the ratio to h18S, using the efficiency-calibrated model [35]. Methylation-sensitive high resolution melting analysis Genomic DNA was extracted from LSGs from SS patients (n = 23) and control subjects (n = 17) using All Prep kit (Qiagen, Germantown, MD, USA) and treated with MethylCode Bisulfite Conversion kit (Thermo Fisher Scientific, Waltham, MA, USA). Methylation-sensitive high resolution melting primers for IRE1α, GRP78 and XBP-1 gene promoter analysis were designed using Methyl Primer Express Software v1.0 (Thermo Fisher Scientific), as recommended [36] (supplementary Table S1 and supplementary Fig. S2, available at Rheumatology online). Next, PCR amplification of bisulphite-modified templates was performed in quadruplicate and standard curves with dilutions of a representative DNA (with a recognized methylation percentage) were included in each assay. The melting curves were normalized by calculating the line that best fits the expected values, demonstrating the melting state of the template in a temperature range. This normalization enables the direct comparison of samples with differing levels of initial fluorescence. Standard curves were plotted and the methylation percentage was obtained from the linear regression analysis. Immunofluorescence detection of XBP-1 and GRP78 LSGs from SS patients (n = 6) and control subjects (n = 5) were fixed in 1% p-formaldehyde and embedded in paraffin, and tissue sections were incubated in an antigen recovery solution (0.01 M citrate, pH 6.0, at 92°C). These sections were then blocked with 0.25% casein/PBS (1 h at room temperature), and subsequently incubated with rabbit polyclonal anti-XBP-1 or anti-GRP78 primary antibodies (supplementary Table S2 and supplementary Fig. S3, available at Rheumatology online). Next, slides were incubated with an Alexa Fluor 488-conjugated secondary antibody/Hoechst 33342 (for nuclear staining) and afterwards visualized with a confocal laser scanning microscope (Olympus FluoView FV10i, Olympus, Center Valley, PA, USA). Finally, quantitative analysis of epithelial and/or inflammatory cell fluorescence intensity was performed using ImageJ software (for details see supplementary data section ‘Quantitative analysis of the fluorescence staining intensity’ and supplementary Fig. S6, available at Rheumatology online). Protein extraction and western blot analysis LSGs from SS patients (n = 18) and control subjects (n = 15) were homogenized as previously described [33]. Protein content was quantified by the Bradford method [37] and separated using SDS–PAGE (8% gel under reducing and denaturing conditions). The separated proteins were then transferred to nitrocellulose membranes and blocked (5% skimmed milk/protease-free prepared in Tris-buffered saline-Tween 20 (TBS-T)). Subsequently, blots were incubated with primary antibodies raised against XBP-1, IRE1α, GRP78 and β-actin (see supplementary Table S2 and supplementary Fig. S3, available at Rheumatology online). Later after washing in TBS-T, blots were incubated with secondary antibodies (horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit). Finally, protein bands were visualized by enhanced chemiluminescence kit (Thermo Fisher Scientific), quantified by densitometry and the protein levels were normalized to β-actin. Statistical analysis Mean values for measurements of variables in patient and control subject groups were compared using the Mann–Whitney test, and Spearman’s rank correlation analysis was also performed. P < 0.05 was considered significant. Results Decreased expression and promoter hypermethylation of IRE1α and XBP-1 in LSGs from SS patients In mammals, two IRE1 isoforms with similar kinase and endoribonuclease enzymatic activities have been described [38]. Here, relative mRNA levels of IRE1α and IRE1β variants were detected: IRE1α mRNA content was significantly decreased (P = 0.006) (Fig. 1A), while IRE1β mRNA remained unchanged (P = 0.72) in LSGs from SS patients (Fig. 1A). Additionally, relative IRE1α protein levels were decreased in LSGs from SS patients (P = 0.027) (Figs 1B and D). As mentioned previously, XBP-1s is produced by an IRE1α-dependent unconventional splicing of XBP-1 mRNA [5]. We found that relative mRNA levels of unspliced XBP-1 (XBP-1u) and XBP-1s were decreased in SS patients (P = 0.002 and P = 0.0003, respectively) (Fig. 1A), and mRNA levels for total XBP-1 (both variants) were also decreased (P = 0.015). For XBP-1 protein, two bands with differing electrophoretic mobility of 54 and 33 kDa were detected corresponding to XBP-1s and XBP-1u, respectively (Fig. 1C). Relative protein levels of XBP-1s were decreased in LSGs from SS patients (P = 0.002), while XBP-1u was similar between groups (P = 0.355) (Fig. 1D). Taking all this into account, these results validate the decreased expression of XBP-1 previously observed in a microarray analysis performed with enriched epithelial cell fractions obtained from SS patient LSGs (Fig. 1E). Fig. 1 View largeDownload slide Decreased expression and promoter hypermethylation of IRE1α and XBP-1 in LSGs from SS patients (A) Relative mRNA levels of IRE1α, IRE1β, XBP-1s, XBP-1u and total XBP-1 in LSG extracts from control subjects (C) and SS patients (P). (B and C) Representative western blots of IRE1α (130 kDa), XBP-1s (54 kDa) and XBP-1u (33 kDa) in LSG extracts from control subjects (C) and SS patients (P). β-Actin (47 kDa) was used as a loading control. (D) Relative protein levels are shown as box plots. (E) XBP-1 relative expression obtained by microarray. (F and G) Methylation percentage for IRE1α and XBP-1 gene promoters. *P < 0.05 was considered significant. Fig. 1 View largeDownload slide Decreased expression and promoter hypermethylation of IRE1α and XBP-1 in LSGs from SS patients (A) Relative mRNA levels of IRE1α, IRE1β, XBP-1s, XBP-1u and total XBP-1 in LSG extracts from control subjects (C) and SS patients (P). (B and C) Representative western blots of IRE1α (130 kDa), XBP-1s (54 kDa) and XBP-1u (33 kDa) in LSG extracts from control subjects (C) and SS patients (P). β-Actin (47 kDa) was used as a loading control. (D) Relative protein levels are shown as box plots. (E) XBP-1 relative expression obtained by microarray. (F and G) Methylation percentage for IRE1α and XBP-1 gene promoters. *P < 0.05 was considered significant. Additionally, a higher DNA methylation percentage of the IRE1α and XBP-1 promoters was observed in LSGs from SS patients compared with control subjects (P = 0.046 and P = 0.033, respectively) (Figs 1F and G). Moreover, mRNA levels showed a negative correlation with methylation percentage of their related promoters (supplementary Table S3, available at Rheumatology online). Subcellular XBP-1 distribution and content in LSGs from SS patients Using an XBP-1 antibody (see supplementary Fig. S3, available at Rheumatology online) that recognizes both XBP-1s and XBP-1u isoforms, we observed nuclear and cytoplasmic staining in acini with lower intensity in ducts from control LSG sections (Fig. 2A–C). Negative and positive staining for total XBP-1 was observed in nuclei of the same acinus of LSGs from SS patients (Fig. 2D and E, arrowheads). In ducts, cytoplasmic staining of XBP-1 in patients was similar to controls, but nuclear staining was weak in both groups (Fig. 2F). Additionally, inflammatory cell staining was found to be positive for the total XBP-1 (Fig. 2F, arrow). When quantifying with ImageJ software (see supplementary data section ‘Quantitative analysis of the fluorescence staining intensity’ and supplementary Fig. S6, available at Rheumatology online), a decrease of XBP-1 staining intensity was seen in epithelial cells of SS patients, and ∼40% of the total glandular XBP-1 staining intensity was contributed by the inflammatory cells (Fig. 2H). Fig. 2 View largeDownload slide XBP-1 localization in LSGs of controls (A−C) and SS patients (D−F) (A and D) Representative panoramic images showing XBP-1 staining in acini and ducts. (B and E) Higher magnification of acini selected in (A) and (D). (E) Nuclei with positive and negative (arrowheads) XBP-1 staining. (C and F) Representative ducts showing weak cytoplasmic staining. (F) Inflammatory cells with cytoplasmic staining (arrow). a: acini; d: duct; IC: inflammatory cells. Bars: 10 μm. (G) XBP-1 staining intensity was significantly lower in epithelial cells of LSGs from SS patients. (H) XBP-1 of inflammatory cells was ∼40% of the total glandular staining intensity. *P < 0.05 was considered significant. AU: arbitrary units. Fig. 2 View largeDownload slide XBP-1 localization in LSGs of controls (A−C) and SS patients (D−F) (A and D) Representative panoramic images showing XBP-1 staining in acini and ducts. (B and E) Higher magnification of acini selected in (A) and (D). (E) Nuclei with positive and negative (arrowheads) XBP-1 staining. (C and F) Representative ducts showing weak cytoplasmic staining. (F) Inflammatory cells with cytoplasmic staining (arrow). a: acini; d: duct; IC: inflammatory cells. Bars: 10 μm. (G) XBP-1 staining intensity was significantly lower in epithelial cells of LSGs from SS patients. (H) XBP-1 of inflammatory cells was ∼40% of the total glandular staining intensity. *P < 0.05 was considered significant. AU: arbitrary units. Decreased expression and promoter hypermethylation of GRP78 in LSGs from SS patients In this study, we found that GRP78 mRNA and protein levels were diminished (P = 0.028 and P = 0.014, respectively) in LSGs from SS patients (Fig. 3A, C and D) and the antibody raised against GRP78 also recognized a reduced content of glucose-regulated protein 94 (GRP94) (another ER-resident chaperone protein) (P = 0.047) in patients (Fig. 3C and D). Similar to IRE1α and XBP-1, methylation of GRP78 promoter was increased (P = 0.033) in patients (Fig. 3B) and GRP78 mRNA levels showed a negative correlation with methylation percentage of its promoter (see supplementary Table S3, available at Rheumatology online). Fig. 3 View largeDownload slide Decreased expression, promoter hypermethylation and localization of GRP78 in LSGs from SS patients (A, C and D) Relative mRNA and protein levels of GRP78. (B) Methylation percentage for GRP78 gene promoter. (E and H) Overview images showing GRP78/GRP94 staining. (F and I) GRP78/GRP94 was observed in the basolateral region of acinar cells, which showed lower staining than inflammatory cells (IC) (I, arrow). (G and J) Representative ducts with low staining and focal inflammatory cells without staining. Bars: 10 μm. (K) GRP78/GRP94 staining was significantly decreased in epithelial cells of LSGs from SS patients. (L) GRP78/GRP94 of inflammatory cells was ∼40% of the total glandular staining. *P < 0.05 was considered significant. Fig. 3 View largeDownload slide Decreased expression, promoter hypermethylation and localization of GRP78 in LSGs from SS patients (A, C and D) Relative mRNA and protein levels of GRP78. (B) Methylation percentage for GRP78 gene promoter. (E and H) Overview images showing GRP78/GRP94 staining. (F and I) GRP78/GRP94 was observed in the basolateral region of acinar cells, which showed lower staining than inflammatory cells (IC) (I, arrow). (G and J) Representative ducts with low staining and focal inflammatory cells without staining. Bars: 10 μm. (K) GRP78/GRP94 staining was significantly decreased in epithelial cells of LSGs from SS patients. (L) GRP78/GRP94 of inflammatory cells was ∼40% of the total glandular staining. *P < 0.05 was considered significant. Subcellular GRP78/GRP94 distribution and content in LSGs from SS patients In LSG sections of control subjects, GRP78/GRP94 was primarily detected in the basolateral region of acinar cells, where the ER is localized (Fig. 3E–G). In LSG sections of SS patients, the distribution of GRP78/GRP94 was maintained; nevertheless acinar staining intensity decreased (Fig. 3H–K). Common to both groups, ductal staining was virtually undetectable (Fig. 3G and J), whereas some inflammatory cells showed strong staining (Fig. 3H–J). In SS patients, inflammatory cells harbouring GRP78/GRP94 labelling was ∼40% of total glandular staining intensity (Fig. 3L). Although no staining was detected in inflammatory cells adjacent to the ducts, several peripheral focus cells were positive (Fig. 3J), displaying morphological characteristics of plasma cells. Advancing this hypothesis, comparison of the localization pattern of GRP78-positive cells with CD20- (B-lymphocyte antigen absent in plasma cells) and CD38- (highly expressed on plasma cells, activated T and B cells) positive cells demonstrated that CD20-positive cells localized at the centre of the focus, whereas cells positive for CD38 and GRP78 localized at the periphery (supplementary Fig. S4A–F, available at Rheumatology online). Therefore, cells expressing GRP78 and CD38 showed an abundant cytoplasm and lack of CD20 staining (supplementary Fig. S4A–F, available at Rheumatology online). Finally, these results imply that GRP78-positive immune cells may potentially be plasma cells. Observations in LSGs from SS patients are simulated in IFN-γ-stimulated 3D acini We observed that 10 ng/ml IFN-γ decreased mRNA of IRE1α, XBP-1s, XBP-1u and GRP78 in HSG cell-formed 3D acini cultured for 24 h (Fig. 4A) and XBP-1s, GRP78 and IRE1α protein levels (Fig. 4B–H). Under these conditions, we also observed a significant hypermethylation of their promoters (Fig. 4I–K). A decreased MUC1/SEC secretion was observed after stimulation of HSG cells with secretagogues previously incubated with IFN-γ or tunicamycin (ER stressor) compared with non-treated cells (supplementary Fig. S5, available at Rheumatology online). Fig. 4 View largeDownload slide 3 D acini exposed to IFN-γ behave as LSGs from SS patients (A) IRE1α, XBP-1s, XBP-1u and GRP78 mRNA levels in 3D acini incubated with or without IFN-γ (1 or 10 ng/ml) for 24 h. (B, D and G) Representative western blots of IRE1α, XBP-1s, XBP-1u and GRP78, in 3D acini incubated with or without IFN-γ. (C, E, F and H) Respectively, IRE1α, XBP-1s, XBP-1u and GRP78 protein levels relative to β-actin. (I−K) DNA methylation percentage of IRE1α, XBP-1 and GRP78 gene promoters of 3D acini incubated with or without IFN-γ. Results are representative of three independent experiments. *P < 0.05 was considered significant. Fig. 4 View largeDownload slide 3 D acini exposed to IFN-γ behave as LSGs from SS patients (A) IRE1α, XBP-1s, XBP-1u and GRP78 mRNA levels in 3D acini incubated with or without IFN-γ (1 or 10 ng/ml) for 24 h. (B, D and G) Representative western blots of IRE1α, XBP-1s, XBP-1u and GRP78, in 3D acini incubated with or without IFN-γ. (C, E, F and H) Respectively, IRE1α, XBP-1s, XBP-1u and GRP78 protein levels relative to β-actin. (I−K) DNA methylation percentage of IRE1α, XBP-1 and GRP78 gene promoters of 3D acini incubated with or without IFN-γ. Results are representative of three independent experiments. *P < 0.05 was considered significant. Relative levels of IRE1α/XBP-1s pathway components and downstream target proteins correlate with clinical parameters of SS patients Regarding IRE1α protein levels, negative correlation with eye (r = −0.68; P = 0.005) and mouth dryness (r = −0.76; P = 0.001) was witnessed and positive correlation with unstimulated whole salivary flow (r = 0.78; P = 0.007) was observed (Table 2). Additionally, XBP-1s protein levels were negatively correlated with eye (r = −0.54; P = 0.04) and mouth dryness (r = −0.57; P = 0.03). Also, an inverse correlation was detected between XBP-1s and Ro (r = −0.54; P = 0.04) as well as XBP-1s and ANA auto-antibodies (r = −0.58; P = 0.02). Furthermore, GRP78 protein levels were negatively correlated with eye (r = −0.59; P = 0.01) and mouth dryness (r = −0.59; P = 0.01). Lastly, serological parameters correlated with dryness symptoms and scintigraphy data (Table 2). Table 2 Spearman’s rank correlation coefficients between protein levels and clinical parameters of SS patients and control subjects Parameters Dryness Serology Eye Mouth USWSF Scintigraphy Ro La RF ANA Eye dryness 1.00 Mouth dryness 0.65* 1.00 USWSF –0.28 –0.64* 1.00 Scintigraphy 0.39* 0.49* –0.37* 1.00 Ro 0.46* 0.6* –0.43* 0.79* 1.00 La 0.35* 0.49* –0.35 0.67* 0.84* 1.00 RF 0.56* 0.59* –0.62* 0.57* 0.66* 0.57* 1.00 ANA 0.52* 0.57* –0.44* 0.62* 0.72* 0.69* 0.61* 1.00 IRE1α protein –0.68* –0.76* 0.78* –0.24 –0.25 0.36 –0.39 –0.46 XBP-1s protein –0.54* –0.57* 0.2 –0.4 –0.54* 0.62* –0.31 –0.58* GRP78 protein –0.59* –0.59* 0.26 –0.02 –0.19 0.16 –0.14 –0.32 Parameters Dryness Serology Eye Mouth USWSF Scintigraphy Ro La RF ANA Eye dryness 1.00 Mouth dryness 0.65* 1.00 USWSF –0.28 –0.64* 1.00 Scintigraphy 0.39* 0.49* –0.37* 1.00 Ro 0.46* 0.6* –0.43* 0.79* 1.00 La 0.35* 0.49* –0.35 0.67* 0.84* 1.00 RF 0.56* 0.59* –0.62* 0.57* 0.66* 0.57* 1.00 ANA 0.52* 0.57* –0.44* 0.62* 0.72* 0.69* 0.61* 1.00 IRE1α protein –0.68* –0.76* 0.78* –0.24 –0.25 0.36 –0.39 –0.46 XBP-1s protein –0.54* –0.57* 0.2 –0.4 –0.54* 0.62* –0.31 –0.58* GRP78 protein –0.59* –0.59* 0.26 –0.02 –0.19 0.16 –0.14 –0.32 For scintigraphic data, we used lower values to indicate better glandular function. * P < 0.05. USWSF: unstimulated whole salivary flow. Table 2 Spearman’s rank correlation coefficients between protein levels and clinical parameters of SS patients and control subjects Parameters Dryness Serology Eye Mouth USWSF Scintigraphy Ro La RF ANA Eye dryness 1.00 Mouth dryness 0.65* 1.00 USWSF –0.28 –0.64* 1.00 Scintigraphy 0.39* 0.49* –0.37* 1.00 Ro 0.46* 0.6* –0.43* 0.79* 1.00 La 0.35* 0.49* –0.35 0.67* 0.84* 1.00 RF 0.56* 0.59* –0.62* 0.57* 0.66* 0.57* 1.00 ANA 0.52* 0.57* –0.44* 0.62* 0.72* 0.69* 0.61* 1.00 IRE1α protein –0.68* –0.76* 0.78* –0.24 –0.25 0.36 –0.39 –0.46 XBP-1s protein –0.54* –0.57* 0.2 –0.4 –0.54* 0.62* –0.31 –0.58* GRP78 protein –0.59* –0.59* 0.26 –0.02 –0.19 0.16 –0.14 –0.32 Parameters Dryness Serology Eye Mouth USWSF Scintigraphy Ro La RF ANA Eye dryness 1.00 Mouth dryness 0.65* 1.00 USWSF –0.28 –0.64* 1.00 Scintigraphy 0.39* 0.49* –0.37* 1.00 Ro 0.46* 0.6* –0.43* 0.79* 1.00 La 0.35* 0.49* –0.35 0.67* 0.84* 1.00 RF 0.56* 0.59* –0.62* 0.57* 0.66* 0.57* 1.00 ANA 0.52* 0.57* –0.44* 0.62* 0.72* 0.69* 0.61* 1.00 IRE1α protein –0.68* –0.76* 0.78* –0.24 –0.25 0.36 –0.39 –0.46 XBP-1s protein –0.54* –0.57* 0.2 –0.4 –0.54* 0.62* –0.31 –0.58* GRP78 protein –0.59* –0.59* 0.26 –0.02 –0.19 0.16 –0.14 –0.32 For scintigraphic data, we used lower values to indicate better glandular function. * P < 0.05. USWSF: unstimulated whole salivary flow. Discussion Here, we report reduced expression of IRE1α, XBP-1 and GRP78, suggesting that activation of the IRE1α/XBP-1 signalling pathway was impaired due to promoter hypermethylation; however, alternative mechanisms contributing to this effect cannot be ruled out. Changes in the IRE1α/XBP-1 axis were studied in LSG sections from SS patients, where acinar and ductal cells coexist with inflammatory cells and immunoreactivity for each protein corresponded to its expected localization in acinar cells. Moreover, in situ staining intensity of IRE1α/XBP-1 signalling pathway components was separately quantified in epithelial and inflammatory cells to allow comparison with results obtained in glandular extracts using other molecular analyses. Our results demonstrate that the population of inflammatory cells, localized in the periphery of focus, was positive for XBP-1 and GRP78/GRP94. Combined staining of specific cell markers and morphology characteristics (abundant cytoplasm and typical nucleus described as cartwheel) strongly suggest that these correspond to plasma cells. Here, it should be noted that all SS patients studied had a focus score of between 1 and 3, and ∼90% of remnant parenchyma could be observed in their LSGs (supplementary Fig. S1 and Table S1, available at Rheumatology online). Negative and positive XBP-1 staining was observed in nuclei of the same acinus of LSGs from SS patients, and this suggests an unknown paracrine mechanism that would control IRE1α/XBP-1 signalling among adjacent acinar cells. The antibody used recognizes XBP-1s and XBP-1u isoforms, both containing a nuclear localization signal. XBP-1u is a negative regulator of XBP-1s, switching off the transcription of target genes during ER stress [39]. The low focus score of LSGs analysed coupled with elevated remnant parenchyma and uncertain discrimination of XBP-1 isoforms by immunofluorescence, leads us to the interpretation that the reduction of XBP-1 mRNA and protein levels (together with the presence of unstained nuclei) suggests the inactivation of the IRE1α/XBP-1 pathway in epithelial cells. Lastly, the strong immunostaining observed of XBP-1 in CD20−/CD38+ inflammatory cells (with abundant cytoplasm), is consistent with plasma cells (highly secretory phenotype) subjected to basal stress conditions [7]. It has been reported that IRE1α endoribonuclease activity is important for XBP-1 mRNA processing [28]. Thus, the reduced levels of IRE1α protein obtained in LSGs from SS patients are consistent with decreased mRNA levels of XBP-1s. Through XBP-1s, the IRE1α signalling pathway leads to increased expression of chaperone proteins (i.e. GRP78) as well as glycosylation, ERAD and traffic and synthesis of exocytic proteins [28]. XBP-1s is also a transcriptional activator that regulates the expansion of the secretory machinery controlling lipid biosynthesis and biogenesis of the most important compartments of the secretory pathway, such as the ER/Golgi [40]. Evidence demonstrates that XBP-1−/−; LivXBP-1 mice show dilatation and disorganization of the ER cisternae, altered expression of chaperone proteins and alterations in secretory granules of pancreatic and SG acinar cells [40]. Similar ultrastructural changes have been previously reported in LSGs of SS patients [17]. Furthermore, an inverse correlation between XBP-1s protein levels and the presence of Ro auto-antibodies was also observed in this study and it is also known that XBP-1s is degraded by the ubiquitin–proteasome system [27, 39]. Ro52 is an E3 ubiquitin ligase [41], which mediates ERAD and the ubiquitination of several proteins. This finding allows us to postulate that Ro auto-antibodies combined with low XBP-1s protein level might be caused by high levels of Ro autoantigen observed in a previous expression profiling study [42]. Decreased XBP-1s levels suggest the decrease of GRP78 expression, a contributing factor to impairment of the secretory pathway in LSGs of SS patients [39, 43]. However, it has also been described that the expression of GRP78 depends on ATF6f [27, 44]. Intriguingly, ATF6f is increased in the LSG from SS patients compared with control subjects [18], which would suggest that decreased GRP78 levels could be attributed to stress inducers, acute or chronic stress, cellular types and/or epigenetic regulation. Interestingly, here we demonstrate that hypermethylation of CpG islands in XBP-1 and GRP78 gene promoters would also modulate its expression and prevent binding of ATF6f and other transcriptional factors to response elements (e.g. type endoplasmic reticulum stress element) thus causing transcript levels of XBP-1 and GRP78 to remain low (supplementary Fig. S2, available at Rheumatology online). Decreased GRP78 could generate defects in protein folding and, as a consequence, lead to protein accumulation, as has been observed for MUC1 in LSGs of SS patients [15]. The GRP78-detecting antibody employed in this study also recognizes GRP94 (an ER resident chaperone protein found decreased in LSGs of SS patients), which plays an important role in the trafficking and secretion of ER components [45] and whose decrease may lead to defective folding of newly synthesized peptides. Remarkably, loss of GRP94 decreases the XBP-1s level [46], which concurs with our observations. Moreover, the decrease of ER protein-folding chaperones, such as GRP78 and GRP94, suggests that the mucins of acinar cells might be misfolded, leading to their accumulation in the cytoplasm [15, 16]. Incorrect mucin folding or glycosylation might then explain the alterations in post-translational processing, such as sulfation [14], which is required for mucins to bind large amounts of water molecules and moisturize oral mucosa surfaces [2]. On the other hand, low GRP78, IRE1α and XBP-1s protein levels correlate with eye and mouth dryness symptoms (Table 2). Mucosa dryness is directly related to the quantity/quality of secreted mucins, dependent on effective protein folding and the quality control machinery in the ER. These findings clarify previous data showing that the UPR pathway triggered by IRE1α/XBP-1s regulates the functionality of secretory cells [40]. Understanding the inflammatory environment arising in the LSG from SS patients, we simulated such conditions by incubating HSG cells with IFN-γ and the expression pattern of XBP-1s, IRE1α and GRP78 as well as the hypermethylation of these promoters was observed to resemble that of the LSG. Under similar culture conditions the cytoplasmic staining of MUC1/SEC increased while its secretion decreased upon stimulation with secretagogues, suggesting that an attenuated activity of the IRE1α pathway aggravates secretory functions (supplementary Fig. S5, available at Rheumatology online). These results suggest a role for IFN-γ in SS pathogenesis [47, 48] and, therefore, our in vitro data confirmed its importance in the modulation of UPR signalling [18]. This is the first study on the methylation of CpG island promoters for IRE1α pathway components, and here decreased mRNA levels for IRE1α, XBP-1 and GRP78 might be, in part, explained by epigenetic regulation through the hypermethylation process in LSGs of SS patients. Furthermore, recent studies have reported differential global DNA methylation along with specific methylation of promoters (hypo- or hypermethylation) in SS [23, 24, 49]. The epithelial cells of SGs from SS patients are subjected to high and diverse stress factors and in response, the cellular metabolic machinery develops homeostasis-preserving mechanisms (including those aimed at modulating the expression of IRE1α/XBP-1 pathway components). Our study showed such response was reflected in an increased DNA methylation of gene promoters that are essential for transcription of important proteins of the IRE1α/XBP-1 pathway. Preliminary, unpublished data of our laboratory suggest that an increased expression of the DNA methyltransferases DNMT1, DNMT3a and 3b takes place in LSGs of SS patients. In conclusion, morphological and functional alterations of the LSGs from SS patients are indicative of elevated ER stress, although it is unclear whether ER stress detected in the acinar cells represents a cause or rather reflects the consequences. Given that SS is a chronic disease and usually diagnosed late in progression, the onset and timing of UPR-related events remain unknown. Furthermore, it is important to mention that responses elicited by the IRE1α/XBP-1s pathway are rapidly attenuated despite stress persistence, enabling restoration of homeostasis in the UPR adaptation phase [50]. Attenuation of IRE1α/XBP-1s pathway activity and sustained activation of the ATF6 pathway [18] in acinar cells may be an indicator of chronic ER stress, where local inflammation may contribute to the alterations in LSGs from SS patients. Acknowledgements We thank all the patients who participated in this study and gratefully acknowledge Dr Claudio Hetz (Biomedical Neuroscience Institute, Universidad de Chile) for providing N2a cells transfected with shXBP-1, and for revising critically the manuscript. We thank Sandra Gendler and Cathy S. Madsen (Mayo Clinic Scottsdale, College of Medicine, Department of Biochemistry and Molecular Biology) for providing antibodies for MUC1/SEC. 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RheumatologyOxford University Press

Published: Mar 9, 2018

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