pSS Salivary Glands Research Articles

CD8+ tissue-resident memory T cells are expanded in primary Sjögren’s disease and can be therapeutically targeted by CD103 blockade

ObjectiveTissue-resident memory cells (Trm) are a subset of T cells residing persistently and long-term within specific tissues that contribute to persistent inflammation and tissue damage. We characterised the phenotype and...

WGCNA and machine learning analysis identifi ed SAMD9 and IFIT3 as primary Sjögren's Syndrome key genes

BackgroundCurrent treatments for primary Sjögren's Syndrome (pSS) are with limited effect, partially due to the heterogeneity and uncleared mechanism. MethodsWe got GSE40568 (Japan) and GSE40611 (USA), and analyzed them with WGCNA to find key Differentially expressed genes (DEGs) between pSS and healthy salivary glands (SG). Key pSS genes (KPGs) were further selected through 3 machine-learning methods. The expression of KPGs was validated via two other GEO datasets (GSE127952 and GSE154926). Infiltrated immune cells, ceRNA network, and potential compounds were explored. ResultsOur study identified 376 DEGs from the pSS patients, with 186 genes located in the “plum2" module, showing the strongest correlation with clinical characteristics. SAMD9 and IFIT3 emerged as KPGs with excellent diagnostic potential. SAMD9 demonstrated close association with immune cell infiltration. We constructed a lncRNA-miRNA-mRNA network comprising 2 KPGs, 12 miRNAs, 124 lncRNAs, and potential therapeutic targets. ConclusionIn the investigation of pSS public datasets, our study revealed two potential critical mediators in the pathological process of pSS salivary glands, namely SAMD9 and IFIT3. Furthermore, we put forth a hypothesis regarding the ceRNA network and made predictions regarding potential therapeutic drugs targeting these two genes.

A Link Between Mitochondrial Dysfunction and the Immune Microenvironment of Salivary Glands in Primary Sjogren's Syndrome.

BackgroundPrimary Sjogren’s syndrome (pSS) is a slowly progressive, inflammatory autoimmune disease characterized by lymphocytic infiltration into salivary and lacrimal glands. It becomes more recognized that morphology alterations of epithelial mitochondria are involved in altered cellular bioenergetics in pSS patients. The integrated analysis of the mitochondrial role in the pathogenesis and aberrant immune microenvironment in pSS remains unknown.MethodsThe mitochondria-related genes and gene expression data were downloaded from the MitoMiner, MitoCarta, and NCBI GEO databases. We performed novel transcriptomic analysis and constructed a network between the mitochondrial function and immune microenvironment in pSS-salivary glands by computer-aided algorithms. Subsequently, real-time PCR was performed in clinical samples in order to validate the bioinformatics results. Histological staining and transmission electron microscopy (TEM) were further studied on labial salivary gland samples of non-pSS and pSS patients characterized for mitochondria-related phenotypic observation in the different stages of the disease.ResultsThe bioinformatic analysis revealed that the expression of several mitochondria-related genes was altered in pSS. Quantitative real-time PCR showed that four hub genes, CD38, CMPK2, TBC1D9, and PYCR1, were differentially expressed in the pSS clinical samples. These hub genes were associated with the degree of immune cell infiltration in salivary glands, the mitochondrial respiratory chain complexes, mitochondrial metabolic pathway in gluconeogenesis, TCA cycle, and pyruvate/ketone/lipid/amino acid metabolism in pSS. Clinical data revealed that the gene expression of fission (Fis1, DRP1, and MFF) and fusion (MFN1, MFN2, and OPA1) was downregulated in pSS samples, consistent with the results from the public validation database. As the disease progressed, cytochrome c and Bcl-2 proteins were regionally distributed in salivary glands from pSS patients. TEM revealed cytoplasmic lipid droplets and progressively swollen mitochondria in salivary epithelial cells.ConclusionOur study revealed cross talk between mitochondrial dysfunction and the immune microenvironment in salivary glands of pSS patients, which may provide important insights into SS clinical management based on modulation of mitochondrial function.

Hyperresponsive cytosolic DNA-sensing pathway in monocytes from primary Sjögren's syndrome.

ObjectivesCytosolic DNA-sensing pathway stimulation prompts type I IFN (IFN-I) production, but its role in systemic IFN-I pathway activation in primary SS (pSS) is poorly studied. Here we investigate the responsiveness of pSS monocytes and plasmacytoid dendritic cells (pDCs) to stimulator of interferon genes (STING) activation in relation to systemic IFN-I pathway activation and compare this with SLE.MethodsExpression of DNA-sensing receptors cGAS, IFI16, ZBP-1 and DDX41, signalling molecules STING, TBK1 and IRF3, positive and negative STING regulators, and IFN-I-stimulated genes MxA, IFI44, IFI44L, IFIT1 and IFIT3 was analysed in whole blood, CD14+ monocytes, pDCs, and salivary glands by RT-PCR, monocyte RNA sequencing data, flow cytometry and immunohistochemical staining. Peripheral blood mononuclear cells (PBMCs) from pSS, SLE and healthy controls (HCs) were stimulated with STING agonist 2′3′-cGAMP. STING phosphorylation (pSTING) and intracellular IFNα were evaluated using flow cytometry.ResultsSTING activation induced a significantly higher proportion of IFNα-producing monocytes, but not pDCs, in both IFN-low and IFN-high pSS compared with HC PBMCs. Additionally, a trend towards more pSTING+ monocytes was observed in pSS and SLE, most pronounced in IFN-high patients. Positive STING regulators TRIM38, TRIM56, USP18 and SENP7 were significantly higher expression in pSS than HC monocytes, while the dual-function STING regulator RNF26 was downregulated in pSS monocytes. STING was expressed in mononuclear infiltrates and ductal epithelium in pSS salivary glands. STING stimulation induced pSTING and IFNα in pSS and SLE pDCs.ConclusionpSS monocytes and pDCs are hyperresponsive to stimulation of the STING pathway, which was not restricted to patients with IFN-I pathway activation.

Low Mutational Burden of Extra Nodal Marginal Zone Lymphoma of Mucosa-Associated Lymphoid Tissue in Patients with Primary Sjogren's Syndrome

Background: Primary Sjogren's syndrome (pSS) is an autoimmune disease characterized by chronic inflammation of the exocrine glands. Patients with pSS are at increased risk of developing extra nodal marginal zone lymphoma of the mucosa-associated lymphoid tissue (MALT) type, predominantly in the salivary glands. The presence of cryoglobulinemia, low C4 levels and salivary gland enlargement, are important risk factors for lymphoma development in pSS. Although lymphoma associated mutations have been described in circulating B-cells of patients with pSS associated cryoglobulinemia, it is unknown which somatic aberrations underlie the development of pSS-associated MALT lymphomas. The aim of the current study was to define the genomic landscape of pSS salivary gland MALT lymphomas. Identification of specific recurrent mutations or copy number aberrations and defining the affected pathways would help to understand the mechanisms by which intra-epithelial B-cells undergo malignant transformation.Methods: Whole exome sequencing was performed on 14 fresh frozen and 3 paraffin embedded MALT tissue samples of pSS patients at the University Medical Center Groningen (UMCG). Matched peripheral white blood cell samples were available for 12 patients and were used as germline controls. Fluorescence in situ hybridization was performed for the detection of MALT1 translocations.Results: Presence of a clonal B-cell population, indicative of a MALT lymphoma was confirmed by IgH PCR (BIOMED-2) for all patients. Whole exome sequencing resulted in a median target coverage of 130x, ranging from 84-163. More than 90% of the target regions had a coverage of >50x. In total we identified 232 somatic mutations in 184 genes. Three cases had a relatively high mutational load (>25 / case), while the median number of mutations in the remaining 14 cases was 7. Across the 17 cases there were 18 recurrently mutated genes. PRKD1, associated with malignant epithelial tumor of the salivary glands, was the most frequently mutated gene (six cases). Besides PRKD1, five additional recurrently mutated genes are also involved in epithelial surface and/or extracellular matrix (MAMDC4, COL14A1, CAMSAP3, TMEM2 and MUC4). Five genes, all with mutations in two cases, were shown to be associated with lymphomagenesis (ID3, TBL1XR1, PAX5, IGLL5, APC). A total of 18 copy number alterations (CNAs) were detected in 8 of the 14 evaluable cases, with gains of part of chromosomes 2 and 19, and loss of chromosome 6 each being detected in two cases. With respect to outcome, only 2 cases with high mutational load relapsed outside of the salivary glands, suggesting a more advanced stage of lymphoma.Conclusion: The low mutational load and lack of a clear lymphoma related gene signature suggests that localized pSS MALT lymphomas are genetically stable and most likely depend on the inflammatory state of the micro-environment. Only those cases characterized by higher mutational load showed a relapse-remitting disease, as is typical for indolent lymphoma. These observations could be translated to the clinical setting and potentially have value as biomarker for identification of patients with a more aggressive disease. Confirmation in larger cohorts are required to confirm this potential association. This study also paves way for treatment strategies targeting the micro-environment. DisclosuresNo relevant conflicts of interest to declare.

NKp30 Receptor Upregulation in Salivary Glands of Sjögren's Syndrome Characterizes Ectopic Lymphoid Structures and Is Restricted by Rituximab Treatment.

Primary Sjögren’s syndrome (pSS) is a chronic autoimmune disease resulting from the inflammatory infiltration of exocrine glands, mainly salivary and lacrimal glands, leading to secretory dysfunction and serious complications including debilitating fatigue, systemic autoimmunity, and lymphoma. Like other autoimmune disorders, a strong interferon (IFN) signature is present among subsets of pSS patients, suggesting the involvement of innate immunity in pSS pathogenesis. NCR3/NKp30 is a natural killer (NK) cell-specific activating receptor regulating the cross talk between NK and dendritic cells including type II IFN secretion upon NK-cell activation. A genetic association between single-nucleotide polymorphisms (SNPs) in the NCR3/NKp30 promoter gene and a higher susceptibility for pSS has been previously described, with pSS patients most frequently carrying the major allele variant associated with a higher NKp30 transcript and IFN-γ release as a consequence of the receptor engagement. In the present study, we combined RNA-sequencing and histology from pSS salivary gland biopsies to better characterize NKp30 (NCR3) and its ligand B7/H6 (NCR3LG1) in pSS salivary gland tissues. Levels of NCR3/NKp30 were significantly increased both in salivary glands and in circulating NK cells of pSS patients compared with sicca controls, especially in salivary glands with organized ectopic lymphoid structures. In line with this observation, a strong correlation between NCR3/NKp30 levels and salivary gland infiltrating immune cells (CD3, CD20) was found. Furthermore, NCR3/NKp30 levels also correlated with higher IFN-γ, Perforin, and Granzyme-B expression in pSS SGs with organized ectopic lymphoid structures, suggesting an activation state of NK cells infiltrating SG tissue. Of note, NKp30+ NK cells accumulated at the border of the inflammatory foci, while the NKp30 ligand, B7/H6, is shown to be expressed mainly by ductal epithelial cells in pSS salivary glands. Finally, immunomodulatory treatment, such as the B-cell depleting agent rituximab, known to reduce the infiltration of immune cells in pSS SGs, prevented the upregulation of NCR3/NKp30 within the glands.

Skewed escape from X-inactivation underlies female bias in Sjögren’s syndrome

Abstract Many autoimmune diseases feature strikingly increased prevalence in females, such as systemic lupus erythematosus (female-to-male ratio 9:1), systemic sclerosis (female-to-male ratio 11:1) and Sjögren’s syndrome (female-to-male ratio 14:1). However, the molecular basis underlying female-biased autoimmunity remains elusive. To address this knowledge gap, we performed transcriptomic profiling of minor salivary gland-derived mesenchymal stromal cells (MSCs) from primary Sjögren’s syndrome (pSS) patients and control subjects. While autosomal gene expression profiles were largely comparable between pSS and control MSCs, we detected major differences in the regulation of X-linked genes. In control female MSCs, X-linked genes that escaped inactivation were expressed from both parental and maternal X chromosomes with a median paternal ratio of ~0.5. However, in pSS female MSCs, escapees exhibited preferential expression from one of the two X chromosomes. Concomitantly, pSS MSCs showed decrease in XIST levels and reorganization of H3K27me3+ foci in the nucleus, suggesting a global dysregulation of X-inactivation maintenance. The skewing in X escape in pSS MSCs was accompanied by mislocation of protein products encoded by the escapees. Given the hallmark features of inflammation and fibrosis in pSS salivary glands and growing evidence supporting MSCs contributing to tissue homeostasis, our data suggests that dysregulation of X-inactivation maintenance, a female-specific process, contributes to the female-bias in pSS susceptibility.

SMADS-Mediate Molecular Mechanisms in Sjögren's Syndrome.

There is considerable interest in delineating the molecular mechanisms of action of transforming growth factor-β (TGF-β), considered as central player in a plethora of human conditions, including cancer, fibrosis and autoimmune disease. TGF-β elicits its biological effects through membrane bound serine/threonine kinase receptors which transmit their signals via downstream signalling molecules, SMADs, which regulate the transcription of target genes in collaboration with various co-activators and co-repressors. Until now, therapeutic strategy for primary Sjögren’s syndrome (pSS) has been focused on inflammation, but, recently, the involvement of TGF-β/SMADs signalling has been demonstrated in pSS salivary glands (SGs) as mediator of the epithelial-mesenchymal transition (EMT) activation. Although EMT seems to cause pSS SG fibrosis, TGF-β family members have ambiguous effects on the function of pSS SGs. Based on these premises, this review highlights recent advances in unravelling the molecular basis for the multi-faceted functions of TGF-β in pSS that are dictated by orchestrations of SMADs, and describe TGF-β/SMADs value as both disease markers and/or therapeutic target for pSS.

Regulation of STAT4 by ETS1 in Sjögren syndrome pathogenesis

<h3>Objectives</h3> Primary Sjögren syndrome (pSS) is a chronic autoimmune disease mainly affecting women over the age of 40, causing dry mouth and eyes. The etiology of pSS is complex and includes the overexpression of inflammatory cytokines from affected salivary gland epithelial cells (SGECs). Our group has previously identified candidate biomarkers overexpressed in salivary gland tissues of patients with pSS. We showed that ETS proto-oncogene 1 (ETS1) had higher mRNA and protein expression in pSS salivary glands that correlated with higher levels of lymphocytic infiltration. It has also been shown that the signal transducer and activator of transcription 4 (STAT4) gene contains pSS susceptibility polymorphisms and is a significant mediator of cytokine (ie, interferon, tumor necrosis factor-α) production. Additionally, in previous studies unrelated to pSS, ETS1 was shown to regulate STAT4 expression at the transcription level. Thus, we hypothesized that ETS1 plays a significant role in regulating STAT4 transcription within SGECs of patients with pSS. Our objectives were as follows: (1) to determine whether downregulation of ETS1 mRNA by siRNA knockdown results in decreased mRNA and protein expression of STAT4 within the salivary gland cell line A253 and (2) to demonstrate the regulation of STAT4 mRNA expression by ETS1 siRNA knockdown in cultured SGECs from control patients with pSS and non-pSS sicca. <h3>Methods</h3> The salivary gland cell line, A253, was transfected with siRNA targeting ETS1 or a scrambled siRNA control. After 48 hours, mRNA and protein were isolated. cDNA synthesis was performed using 1 µg of mRNA. Expression of ETS1 and STAT4 mRNA was quantified by quantitative reverse transcription–polymerase chain reaction using the delta cycle threshold method to determine the relative fold changes normalized to glyceraldehyde 3-phosphate dehydrogenase in triplicate experiments. In addition, downregulation of STAT4 protein by ETS1 siRNA was determined by Western blot analysis and semiquantified by densitometry using cofilin as the loading control. SGECs were cultured from labial salivary gland biopsies of 2 patients with pSS (focus score [FS] ≥1) and 4 patients with non-pSS sicca (FS = 0, n = 2; 0 < FS < 1; n = 2) and transfected with siRNA targeting ETS1 or a scrambled siRNA control. After 48 hours, mRNA was isolated for cDNA synthesis and used to determine the mRNA expression of ETS1 and STAT4 as described above. <h3>Results</h3> In A253 cells, STAT4 mRNA expression was changed by −2.15-fold, on average, when treated with siRNA targeting ETS1. STAT4 protein expression was measured by Western blot analysis 48 hours post-ETS1 siRNA knockdown showing an average −1.25-fold change in A253 total cell lysate. SGECs cultured from patients with lymphocytic infiltration (ie, FS >0) showed a significantly higher expression of STAT4 (<i>P</i> = .0485) than patients with non-pSS sicca with FS = 0. However, we did not find a significant change in STAT4 mRNA expression (average, +1.14-fold) in cultured SGECs treated with siRNA targeting ETS1. <h3>Conclusions</h3> STAT4 is overexpressed by cultured SGECs in patients with pSS and those without pSS with focal lymphocytic infiltrates. STAT4 overexpression in SGECs of patients with pSS and those without pSS could be a result of inflammatory cytokine exposure by neighboring cells or effects of infiltrating lymphocyte exposure. Thus, our results suggest a separate regulatory mechanism of STAT4 in pSS bypassing ETS1 regulation, which might be due to pathogenic pathways involving other transcriptional regulation.

Interleukin-17 and -22 synergy linking inflammation and EMT-dependent fibrosis in Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is a chronic inflammatory, autoimmune and systemic disorder commonly associated with dry eyes and a dry mouth. Recently, the hypothetical link between epithelial-mesenchymal transition (EMT)-dependent salivary gland (SG) fibrosis and chronic inflammatory conditions has been suggested. In this study, we present data demonstrating a negative correlation of the epithelial marker E-cadherin expression and a positive correlation of mesenchymal vimentin and collagen type I expression with increasing degrees of tissue inflammation in pSS SG specimens. In addition, as it is not clear whether dysregulated cytokines in pSS, interleukin (IL)-17 and IL-22 may also contribute to the EMT-dependent fibrosis process, the effect of IL-17 and IL-22 treatment on EMT-dependent SG fibrosis was evaluated in primary human salivary gland epithelial cells (SGEC) isolated from healthy subjects. Here we present data demonstrating that IL-17 and IL-22 can induce SGEC to undergo a morphological and phenotypical transition to a mesenchymal phenotype. In support of this, vimentin and collagen type I were up-regulated while a decreased expression of E-cadherin occurs after interleukin treatment, and co-operation between IL-17 and Il-22 was required to induce the EMT.

The TGF-β1 Signaling Pathway as an Attractive Target in the Fibrosis Pathogenesis of Sjögren's Syndrome.

Transforming growth factor β1 (TGF-β1) plays a crucial role in the induction of fibrosis, often associated with chronic phases of inflammatory diseases contributing to marked fibrotic changes that compromise normal organ function. The TGF-β1 signal exerts its biological effects via the TGF-β/SMAD/Snail signaling pathway, playing an important pathogenic role in several fibrotic diseases. It has as yet been poorly investigated in the chronic autoimmune disease Sjögren's syndrome (SS). Here, we firstly tested, by immunohistochemistry, whether the TGF-β1/SMAD/Snail signaling pathway is triggered in human pSS salivary glands (SGs). Next, healthy salivary gland epithelial cell (SGEC) cultures derived from healthy donors were exposed to TGF-β1 treatment, and the relative gene and protein levels of SMAD2/3/4, Snail, E-cadherin, vimentin, and collagen type I were compared by semiquantitative RT-PCR, quantitative real-time PCR, and Western blot analysis. We observed, both at gene and protein levels, higher expression of SMAD2, 3, and 4 and Snail in the SGEC exposed by TGF-β1 compared to untreated healthy SGEC. Additionally, in TGF-β1-treated samples, we found a significant reduction in the epithelial phenotype marker E-cadherin and an increase in the mesenchymal phenotype markers vimentin and collagen type I compared to those in untreated SGEC, indicating that TGF-β1 induces the EMT via the TGF-β1/SMAD/Snail signaling pathway. Therefore, by using the specific TGF-β receptor 1 inhibitor SB-431542 in healthy SGEC treated with TGF-β1, we showed a significant reduction of the fibrosis markers vimentin and collagen type I while the epithelial marker E-cadherin returns to levels similar to untreated healthy SGEC. These data demonstrate that TGF-β1 is an important key factor in the transition phase from SG chronic inflammation to fibrotic disease. Characteristic changes in the morphology and function of TGF-β1-treated healthy SGEC further confirm that TGF-β1 plays a significant role in EMT-dependent fibrosis.

Phosphatidylinositol 3-kinase delta pathway: a novel therapeutic target for Sjögren’s syndrome

BackgroundThe phosphatidylinositol 3-kinase delta isoform (PI3Kδ) belongs to an intracellular lipid kinase family that regulate lymphocyte metabolism, survival, proliferation, apoptosis and migration and has been successfully targeted in B-cell malignancies....

Dysregulation of NF-kB in glandular epithelial cells results in Sjögren's-like features.

The autoimmune disease primary Sjögren’s syndrome (pSS) is characterized by hypofunction of the salivary glands (SGs), the cause of which is not correlated to lymphocytic SG infiltration, as prevailing dogma often states. We knocked out the NF-κB proinflammatory pathway inhibitor A20 in keratin14+ epithelial cells, to investigate if immune activated epithelial cells are capable of initiating pSS SG hallmarks. We show that immune activated epithelial cells can cause T cell dominated leukocytic infiltration and immune foci development of the SGs, reflecting the early clinical picture. Infiltrating leukocytes invaded striated ducts, similar to early stage lymphoepithelial lesions observed clinically. Expression of proinflammatory cyto-/chemokines IFNɣ, TNFα, IL-6, CXCL10 and CXCL13 increased in A20-/- SGs, and functionally both volume and mucin 10 content of whole stimulated saliva from A20-/- mice was significantly reduced. Epithelial cells may therefore represent the initial trigger for pSS SG pathologies, as opposed to simple reactionaries to pre-existing stimuli.

Abnormal distribution of AQP4 in minor salivary glands of primary Sjögren’s syndrome patients

A decreased saliva production occurs in primary Sjögren’s syndrome (pSS), an autoimmune disease characterized by oral and ocular dryness due to dysfunction of the lacrimal and salivary glands (SGs). Since water movement is involved in saliva secretion, the expression, localization, and function of the water channels aquaporins (AQPs) have been extensively studied in SGs. To date, the presence of AQP4 remains controversial and ambiguous in human SGs. We investigated by immunohistochemistry, high-resolution confocal microscopy and quantitative image analysis, Western blot and real-time RT-PCR, the presence of the AQP4 gene, and the distribution of AQP4 protein in healthy controls and pSS SG biopsies. Through the immunohistochemical analysis, we demonstrated that AQP4 presence is confined to the basal region of acini, to the lateral and apical membrane of intercalated and striated ducts in both control and pSS glands. The most striking observation was the discovery of AQP4 localization in myoepithelial cells (MECs) that surround acini lobules and intercalated ducts, and the demonstration of AQP4-downregulated immunoreactivity in pSS MECs. Our studies suggest that the capacity for water flow across the membrane of MECs may be altered in pSS, identifying AQP4 as a promising new therapeutic agent to treat xerostomia.

Targeting the Ca2 + Sensor STIM1 by Exosomal Transfer of Ebv-miR-BART13-3p is Associated with Sjögren's Syndrome

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease that is associated with inflammation and dysfunction of salivary and lacrimal glands. The molecular mechanism(s) underlying this exocrinopathy is not known, although the syndrome has been associated with viruses, such as the Epstein Barr Virus (EBV). We report herein that an EBV-specific microRNA (ebv-miR-BART13-3p) is significantly elevated in salivary glands (SGs) of pSS patients and we show that it targets stromal interacting molecule 1 (STIM1), a primary regulator of the store-operated Ca2+ entry (SOCE) pathway that is essential for SG function, leading to loss of SOCE and Ca2+-dependent activation of NFAT. Although EBV typically infects B cells and not salivary epithelial cells, ebv-miR-BART13-3p is present in both cell types in pSS SGs. Importantly, we further demonstrate that ebv-miR-BART13-3p can be transferred from B cells to salivary epithelial cells through exosomes and it recapitulates its functional effects on calcium signaling in a model system.

Co-culture system of human salivary gland epithelial cells and immune cells from primary Sjögren’s syndrome patients: an in vitro approach to study the effects of Rituximab on the activation of the Raf-1/ERK1/2 pathway

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disorder of the exocrine glands with associated lymphocytic infiltrates in the affected glands. Dryness of the mouth and eyes results from involvement of the salivary and lacrimal glands. The efficacy of Rituximab (RTX) in pSS is still open to debate. This study delineates the signaling pathway involved in RTX-mediated down-regulation of pro-inflammatory factors in a co-culture system of pSS salivary gland epithelial cells (SGEC) with syngeneic pSS B-lymphocytes. In addition, the effects of RTX on the activation of the Raf-1/ERK1/2 pathway in pSS SGEC co-cultured with syngeneic pSS T-lymphocytes were also investigated. This study demonstrated that RTX may interfere with the ERK1/2 pathway in a syngeneic co-culture of pSS SGEC with pSS B-lymphocytes, leading to decreased cytokine production by SGEC. These novel findings reveal that syngeneic co-culture of pSS SGEC with pSS B-lymphocytes leads to a down-regulation of Raf-1 in epithelial cells that adversely regulates the activity of the ERK1/2 pathway and determines a subsequent reduction of the release of pro-inflammatory factors.

Rituximab‐mediated Raf kinase inhibitor protein induction modulates NF‐κB in Sjögren syndrome

Primary Sjögren syndrome (pSS) is an autoimmune disorder characterized by an epithelial injury surrounded by dense lymphocytic infiltrates. The conditions for the long-term maintenance of human salivary gland epithelial cells from pSS patients and a co-culture system with pSS lymphocytes were used to assess the effect of Rituximab (RTX) on the inflammatory condition and progression in pSS. Quantitative real-time PCR, genes and protein array analysis, Western blot, flow cytometry, small interfering RNA transfection and nuclear factor-κB (NF-κB) DNA binding assays were used as methods. Supporting the benefits of RTX, this study demonstrates that RTX decreases NF-κB activity and interrupts the NF-κB signalling pathway through the up-regulation of the Raf-1 kinase inhibitor protein (RKIP). Over-expression of RKIP down-regulates interleukins, their receptors and the expression of genes encodes proteins that attracted lymphocytes. Silencing of the RKIP gene leads to significantly increased expression and release of pro-inflammatory mediators supporting that RKIP expression could be involved in the suppression of NF-κB activation in pSS salivary gland epithelial cells.

The metalloproteinase ADAM17 and the epidermal growth factor receptor (EGFR) signaling drive the inflammatory epithelial response in Sjögren’s syndrome

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disorder that particularly compromises the function of exocrine glands. The pathogenetic mechanisms of this autoimmune exocrinopathy have not been fully elucidated. Since increasing evidence actually suggests that the epidermal growth factor receptor (EGFR) pathway has a major impact on the inflammatory/immune reactions of the epithelial cells, in the apparent effort of enhancing innate immune defense while opposing overactivation of pro-inflammatory functions, the focus of the work presented here is clarify whether the EGFR-extracellular-signal-regulated kinase (ERK) pathway plays a role in the pro-inflammatory responses mounted by pSS salivary gland epithelial cells (SGEC). Investigations revealed that the EGFR-mediated activation of the downstream effectors ERK1/2 in pSS SGEC appeared to require ADAM17-dependent release of the endogenous EGFR ligand amphiregulin and transactivation of the EGFR. Moreover, blockade of amphiregulin bioactivity using a neutralizing Ab significantly reduced EGFR transactivation and ERK1/2 phosphorylation. In addition, pSS SGEC treated with the specific ADAM17 inhibitor TAPI-1 and with the EGFR inhibitor AG1478 exhibited deactivated AREG/EGFR/ERK signaling pathway and reduced pro-inflammatory cytokines released.

Salivary glands of primary Sjögren's syndrome patients express factors vital for plasma cell survival

IntroductionThe presence of circulating Ro/SSA and La/SSB autoantibodies has become an important marker in the classification criteria for primary Sjögren's syndrome (pSS). Plasma cells producing these autoantibodies are mainly high affinity plasma cells originating from germinal centre reactions. When exposed to the right microenvironment these autoimmune plasma cells become long-lived and resistant to immunosuppressive treatment. Since autoimmune plasma cells have been detected in the salivary glands of SS patients, we wanted to investigate if the glandular microenvironment is suitable for plasma cell survival and if glandular residing plasma cells are the long-lived plasma cell subset.MethodsSingle, double and triple immunohistochemistry as well as immunofluorescence staining was performed on minor salivary gland tissue retrieved from pSS, chronically inflamed and normal subjects.ResultsWe detected significant numbers of CD138+, non-proliferating, Bcl-2 expressing plasma cells in the salivary glands of pSS patients with high focus score (FS). Furthermore, we demonstrated that CXCL12 and interleukin (IL)-6 survival factors were highly expressed in pSS salivary gland epithelium and by focal mononuclear infiltrating cells. Notably, adipocytes when present in the salivary gland tissue were an important source of CXCL12. We clearly demonstrate that plasma cells are localised in close proximity to CXCL12 and IL-6 expressing cells and thus that the environment of salivary glands with high FS provide factors vital for plasma cell survival.ConclusionsPlasma cells residing in the salivary glands of pSS patients with high FS showed phenotypic characteristics of the long-lived plasma cell subtype. Furthermore, the pSS salivary gland microenvironment provided niches rich in factors vital for plasma cell survival.

Autoimmune response and target autoantigens in Sjogren’s syndrome

Primary Sjogren's syndrome (pSS) is characterized by the presence of autoantibodies targeting mainly the Ro/La ribonucleoprotein complex. It is now appreciated that the production of autoantibodies is an antigen-driven immune response. In this review, candidate mechanisms for autoantigen presentation and perpetuation of the autoimmune response within the autoimmune tissue lesion of pSS are discussed. Several studies have shown that the epithelial cell in labial salivary glands of patients with Sjogren's syndrome is activated, bearing characteristics of an antigen-presenting cell, as suggested by inappropriate expression of class II HLA and co-stimulatory molecules. Other studies have confirmed that in salivary glands, there is an increased autoantigen presentation via apoptotic blebs and bodies, exosomes and heat shock protein-mediated cross-priming. There is also an increased expression of interferon (IFN)-induced genes, such as the autoantigen Ro52, which provide negative feedback regulation in inflammation. Ro60 and La autoantigens also appear to play a major role in the local autoimmune response in Sjogren's syndrome. In this regard, La and Ro60 the messenger RNA (mRNA) expression is upregulated in the affected salivary glands with different isoforms of La autoantigen mRNA to be expressed in patients with pSS. At the protein level, La/SSB in pSS salivary glands is found to be post-translationally modified. Autoantigen alterations in a microenvironment of local inflammation with increased in situ apoptosis, Toll-like receptor (TLR) signalling and antigen presentation may drive the autoimmune response and local autoantibody production in pSS.