Sl Blokland

Increased CCL25 and T Helper Cells Expressing CCR9 in the Salivary Glands of Patients With Primary Sjögren's Syndrome : Potential New Axis in Lymphoid Neogenesis

Follicular helper T (Tfh) cells play a critical role in germinal center formation and B cell activation, both of which are hallmarks of primary Sjögrens syndrome (SS). CCR9‐expressing T helper cells have “Tfh‐like” characteristics and their numbers are increased at mucosa‐associated sites in several inflammatory conditions. Because the characteristics of these cells are unique and evaluation has been limited, this study was undertaken to investigate the local and systemic CCL25/CCR9 axis in patients with primary SS.Introduction T follicular helper (Tfh)-cells play a critical role in germinal center formation and B-cell activation, both hallmarks of primary Sjogrens syndrome (pSS). CCR9-expressing Th-cells have “Tfh-like” characteristics and are increased at mucosa-associated sites in several inflammatory conditions. Because of their unique characteristics and limited evaluation we investigated the local and systemic CCL25/CCR9-axis in pSS. Methods CCL25 protein and mRNA levels and CCR9+ Th-cells were assessed in labial salivary glands (LSG) of pSS and non-Sjogrens sicca (nSS) patients and their correlation with inflammatory and clinical parameters was evaluated. Circulating CCR9+ and CXCR5+ Th-cells were compared based on phenotypic and functional properties. Results CCL25 protein and mRNA levels were elevated in LSG from pSS versus nSS patients and associated with B-cell hyperactivity, autoimmunity and levels of IL-21 and soluble IL-7Rα. The frequency of CCR9-expressing cells was increased in the LSG of pSS patients. Circulating CCR9+ Th-cells expressing PD-1 and ICOS were elevated in pSS patients. CCR9+ Th-cells displayed higher expression of IL-7Rα and secreted higher levels of IFN-γ, IL-17, IL-4 and IL-21 as compared to CXCR5+ Th-cells, ex vivo and upon triggering with antigen or IL-7. Both CCR9+ and CXCR5+ Th-cells induced IgG production by B-cells more potently than CCR9-CXCR5- Th-cells. Conclusion Enhanced CCL25 expression in pSS LSG can facilitate attraction of CCR9+ Th-cells, secreting high levels of pro-inflammatory cytokines when triggered with antigen or IL-7. Associations with B-cell hyperactivity, autoimmunity and markers of lymphoid neogenesis indicate the CCL25/CCR9-axis plays a significant role in pSS immunopathology, representing a novel therapeutic target. This article is protected by copyright. All rights reserved.

Serum microRNA screening and functional studies reveal miR-483-5p as a potential driver of fibrosis in systemic sclerosis

OBJECTIVE MicroRNAs (miRNAs) are regulatory molecules, which have been addressed as potential biomarkers and therapeutic targets in rheumatic diseases. Here, we investigated the miRNA signature in the serum of systemic sclerosis (SSc) patients and we further assessed their expression in early stages of the disease. METHODS The levels of 758 miRNAs were evaluated in the serum of 26 SSc patients as compared to 9 healthy controls by using an Openarray platform. Three miRNAs were examined in an additional cohort of 107 SSc patients and 24 healthy donors by single qPCR. MiR-483-5p expression was further analysed in the serum of patients with localized scleroderma (LoS) (n = 22), systemic lupus erythematosus (SLE) (n = 33) and primary Sjögrens syndrome (pSS) (n = 23). The function of miR-483-5p was examined by transfecting miR-483-5p into primary human dermal fibroblasts and pulmonary endothelial cells. RESULTS 30 miRNAs were significantly increased in patients with SSc. Of these, miR-483-5p showed reproducibly higher levels in an independent SSc cohort and was also elevated in patients with preclinical-SSc symptoms (early SSc). Notably, miR-483-5p was not differentially expressed in patients with SLE or pSS, whereas it was up-regulated in LoS, indicating that this miRNA could be involved in the development of skin fibrosis. Consistently, miR-483-5p overexpression in fibroblasts and endothelial cells modulated the expression of fibrosis-related genes. CONCLUSIONS Our findings showed that miR-483-5p is up-regulated in the serum of SSc patients, from the early stages of the disease onwards, and indicated its potential function as a fine regulator of fibrosis in SSc.

High soluble IL-7 receptor expression in Sjögren's syndrome identifies patients with increased immunopathology and dryness

Increased expression of interleukin (IL)-7 and its receptor is suggested to play a critical role in immunopathology of primary Sjogrens syndrome (pSS).1–3 Data from humans and mice demonstrate that IL-7 drives a range of processes involved in pSS immunopathology, including epithelial cell apoptosis, lymphocyte infiltration and reduction of salivary output.3 The IL-7/IL-7R axis is involved in the formation of (ectopic) lymphoid structures in salivary glands (SGs),3 ,4 which is a predictor for lymphoma development in pSS.5 ,6 IL-7 activity is potentiated by the soluble form of its receptor (sIL-7R), which is produced in inflamed tissues by activated stromal cells.7 As sIL-7R is a possible biomarker for IL-7-driven immune activation and lymphoid neogenesis, we studied the expression of sIL-7R in pSS in relation to markers of inflammation and saliva production. Ninety-five patients with pSS were diagnosed according to the 2002 criteria (table 1).8 sIL-7R was measured in serum of 68 patients with pSS using ELISA as previously described9 and compared with 51 healthy individuals (HC). Labial SG biopsy tissues were taken from 27 patients and, after thorough rinsing, were incubated in 200 µL of sterile saline for 1 hour at room temperature. In these tissue supernatants, sIL-7R was measured …

Autoantibodies to Cytosolic 5 '-Nucleotidase 1A in Primary Sjogren's Syndrome and Systemic Lupus Erythematosus

Introduction Autoantibodies to cytosolic 5′-nucleotidase 1A (cN-1A; NT5C1A) have a high specificity when differentiating sporadic inclusion body myositis from polymyositis and dermatomyositis. In primary Sjögren’s syndrome (pSS) and systemic lupus erythematosus (SLE) anti-cN-1A autoantibodies can be detected as well. However, various frequencies of anti-cN-1A reactivity have been reported in SLE and pSS, which may at least in part be explained by the different assays used. Here, we determined the occurrence of anti-cN-1A reactivity in a large number of patients with pSS and SLE using one standardized ELISA. Methods Sera from pSS (n = 193) and SLE patients (n = 252) were collected in five European centers. Anti-cN-1A, anti-Ro52, anti-nucleosome, and anti-dsDNA reactivities were tested by ELISA (Euroimmun AG) in a single laboratory. Correlations of anti-cN-1A reactivity with demographic data and clinical data (duration of disease at the moment of serum sampling, autoimmune comorbidity and presence of muscular symptoms) were analyzed using SPSS software. Results Anti-cN-1A autoantibodies were found on average in 12% of pSS patients, with varying frequencies among the different cohorts (range: 7–19%). In SLE patients, the anti-cN-1A positivity on average was 10% (range: 6–21%). No relationship was found between anti-cN-1A reactivity and the presence or absence of anti-Ro52, anti-nucleosome, and anti-dsDNA reactivity in both pSS and SLE. No relationship between anti-cN-1A reactivity and duration of disease at the moment of serum sampling and the duration of serum storage was observed. The frequency of muscular symptoms or viral infections did not differ between anti-cN-1A-positive and -negative patients. In both disease groups anti-cN-1A-positive patients suffered more often from other autoimmune diseases than the anti-cN-1A-negative patients (15 versus 5% (p = 0.05) in pSS and 50 versus 30% (p = 0.02) in SLE). Conclusion Our results confirm the relatively frequent occurrence of anti-cN-1A in pSS and SLE patients and the variation in anti-cN-1A reactivity between independent groups of these patients. The explanation for this variation remains elusive. The correlation between anti-cN-1A reactivity and polyautoimmunity should be evaluated in future studies. We conclude that anti-cN-1A should be classified as a myositis-associated-, not as a myositis-specific-autoantibody based on its frequent presence in SLE and pSS.

Elevated CCL25 and CCR9-Expressing T Helper Cells in Salivary Glands of Primary Sjögren's Syndrome Patients: Potential New Axis in Lymphoid Neogenesis

Follicular helper T (Tfh) cells play a critical role in germinal center formation and B cell activation, both of which are hallmarks of primary Sjögrens syndrome (SS). CCR9‐expressing T helper cells have “Tfh‐like” characteristics and their numbers are increased at mucosa‐associated sites in several inflammatory conditions. Because the characteristics of these cells are unique and evaluation has been limited, this study was undertaken to investigate the local and systemic CCL25/CCR9 axis in patients with primary SS.Introduction T follicular helper (Tfh)-cells play a critical role in germinal center formation and B-cell activation, both hallmarks of primary Sjogrens syndrome (pSS). CCR9-expressing Th-cells have “Tfh-like” characteristics and are increased at mucosa-associated sites in several inflammatory conditions. Because of their unique characteristics and limited evaluation we investigated the local and systemic CCL25/CCR9-axis in pSS. Methods CCL25 protein and mRNA levels and CCR9+ Th-cells were assessed in labial salivary glands (LSG) of pSS and non-Sjogrens sicca (nSS) patients and their correlation with inflammatory and clinical parameters was evaluated. Circulating CCR9+ and CXCR5+ Th-cells were compared based on phenotypic and functional properties. Results CCL25 protein and mRNA levels were elevated in LSG from pSS versus nSS patients and associated with B-cell hyperactivity, autoimmunity and levels of IL-21 and soluble IL-7Rα. The frequency of CCR9-expressing cells was increased in the LSG of pSS patients. Circulating CCR9+ Th-cells expressing PD-1 and ICOS were elevated in pSS patients. CCR9+ Th-cells displayed higher expression of IL-7Rα and secreted higher levels of IFN-γ, IL-17, IL-4 and IL-21 as compared to CXCR5+ Th-cells, ex vivo and upon triggering with antigen or IL-7. Both CCR9+ and CXCR5+ Th-cells induced IgG production by B-cells more potently than CCR9-CXCR5- Th-cells. Conclusion Enhanced CCL25 expression in pSS LSG can facilitate attraction of CCR9+ Th-cells, secreting high levels of pro-inflammatory cytokines when triggered with antigen or IL-7. Associations with B-cell hyperactivity, autoimmunity and markers of lymphoid neogenesis indicate the CCL25/CCR9-axis plays a significant role in pSS immunopathology, representing a novel therapeutic target. This article is protected by copyright. All rights reserved.

THU0268 Decreased Expression of MIR-130A and MIR-708 in Type-1 Classical Dendritic Cells of Sjögren's Syndrome Patients Indicates Their Dysregulation

Background Primary Sjögrens syndrome (pSS) is a systemic autoimmune disease characterized by lymphocytic infiltration of the salivary and lachrymal exocrine glands that leads to dryness of mouth and eyes. Type-1 classical dendritic cells (cDCs) are very potent antigen presenting cells known to induce strong T-cell proliferation and cytokine production. Despite the fact that especially cDC1s are candidate key players in the activation of local T and B-cells in pSS, they have rarely been studied in pSS. Objectives Considering the critical role of micro-RNAs (miRNAs) in regulation of gene expression, we investigated miRNA expression in isolated cDC1s of pSS patients. Methods Two independent cohorts (discovery and validation) were established including a total of 29 pSS patients. 17 healthy controls (HC) were included as control group. cD1c+CD19- cells were isolated from peripheral blood using MACS and we performed miRNA profiling of 758 miRNA targets using the OpenArray platform in the donors included in the discovery cohort. A selection of the miRNAs found to be differentially expressed in pSS versus HC was measured in the validation cohort. We performed pathway enrichment with the experimentally supported targets of the validated miRNAs. Results A total of 24 miRNAs were downregulated in pSS patients versus HC in the discovery cohort (all at least p<0.05 and 2log expression difference). Of these, 16 targets were selected to be validated in validation cohort. Two miRNAs, miR-130a and miR-708, were significantly downregulated in pSS patients in both cohorts (p<0.05). Pathway enrichment showed that the experimentally supported targets of these miRNAs are mainly involved in vesicle trafficking and a number of growth-factor signalling pathways (p<0.05, FDR corrected), including epidermal growth factor (EGF) signalling and endocytosis. We are currently linking the miRNA expression data to RNAseq data and performing functional experiments with primary DCs to dissect the effects of miRNA dysregulation in these cells. Conclusions We here for the first time show differentially expressed miRNAs in an isolated immune cell subset of pSS patients. In addition, this is the first evidence for dysregulation of primary peripheral blood type-1 cDCs in pSS. Considering threimportant role of miRNAs in cell function, this suggests that targeting aberrant miRNA expression may provide tools to modulate cDC activity. Disclosure of Interest None declared

Circulating small non-coding RNAs reflect IFN status and B cell hyperactivity in patients with primary Sjögren’s syndrome

Background Considering the important role of miRNAs in the regulation of post–transcriptional expression of target genes, we investigated circulating small non-coding RNAs (snc)RNA levels in patients with primary Sjögren’s syndrome (pSS). In addition we assessed if serum sncRNA levels can be used to differentiate patients with specific disease features. Methods Serum RNA was isolated from 37 pSS patients as well as 21 patients with incomplete Sjögren’s Syndrome (iSS) and 17 healthy controls (HC) allocated to two independent cohorts: discovery and validation. OpenArray profiling of 758 sncRNAs was performed in the discovery cohort. Selected sncRNAs were measured in the validation cohort using single-assay RT-qPCR. In addition, unsupervised hierarchical clustering was performed within the pSS group. Results Ten sncRNAs were differentially expressed between the groups in the array. In the validation cohort, we confirmed the increased expression of U6-snRNA and miR-661 in the iSS group as compared to HC. We were unable to validate differential expression of any miRNAs in the pSS group. However, within this group several miRNAs correlated with laboratory parameters. Unsupervised clustering distinguished three clusters of pSS patients. Patients in one cluster showed significantly higher serum IgG, prevalence of anti-SSB autoantibodies, IFN-score, and decreased leukocyte counts compared to the two other clusters. Conclusion We were unable to identify any serum sncRNAs with differential expression in pSS patients. However, we show that circulating miRNA levels are associated with disease parameters in pSS patients and can be used to distinguish pSS patients with more severe B cell hyperactivity. As several of these miRNAs are implicated in the regulation of B cells, they may play a role in the perpetuation of the disease.

FRI0281 Transcriptomic profiling of pdcs from patients with pss identifies consistently altered gene networks that indicate an activated phenotype and enhanced anti-viral state

Background Primary Sjögren’s syndrome is an autoimmune disease characterised by lymphocytic infiltration of the exocrine glands and dryness of mouth and eyes. Type-I interferons (IFN) are thought to play an important role in pSS pathogenesis and plasmacytoid dendritic cells (pDCs) are capable of producing high levels of IFN. These cells infiltrate the salivary glands of pSS patients and their numbers correlate with local IFN-production. Objectives To understand the molecular mechanisms behind systemic dysregulation of pDCs, we performed RNA sequencing on pDCs isolated from peripheral blood of patients with pSS, non-Sjögren’s sicca (nSS) and healthy controls. Methods We established two independent cohorts (each n=31), of patients and controls. pSS patients (n=25) were classified according to the 2002 AECG criteria. nSS patients (n=20) presented with dryness complaints without a known cause, did not have any generalised autoimmune disease, and did not fulfil the classification criteria for pSS. Healthy donors (n=17) were included as control group. Peripheral blood pDCs were isolated using MACS and RNA sequencing was performed for both cohorts.±20 million paired-end sequencing reads per sample were obtained using using Illumina HiSeq 2500 platform. Results 8556 genes were differentially expressed (p-value<0.05) between all three groups in the discovery cohort. Of these, 3144 genes were also differential in the replication cohort. We generated gene modules from both cohorts and found 5 gene clusters comprising 1259 genes that were consistently dysregulated in both analyses. Pathway analysis showed that the 5 modules contain genes associated with cellular activation, including a group of genes involved in IFN-signalling and viral sensing, as well as regulation of intracellular transport. Generally, pDCs from patients with nSS displayed an intermediate phenotype. Conclusions We mapped transcriptomic differences in circulating pDCs from patients with nSS and pSS and identified gene clusters that are robustly replicated in two independent cohorts. We found 5 gene clusters that are dysregulated in patients with pSS and indicate enhanced cellular activation, including IFN-signalling and viral sensing which are key pathways in pSS pathogenesis. nSS patients showed similar transcriptomic dysregulation at an intermediate level. These data can help us better understand the role of pDCs in pSS. Disclosure of Interest None declared

AB0149 Decreased microrna-130a expression drives activation of classical dendritic cells from patients with primary sjögren’s syndrome

Background Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease characterized by lymphocytic infiltration of the exocrine glands and dryness of mouth and eyes. Classical dendritic cells are very potent antigen presenting cells known to induce strong T-cell proliferation and cytokine production. Objectives Considering the critical role of microRNAs (miRNAs) in regulation of gene expression, we investigated miRNA expression in circulating CD1c+ dendritic cells (cDCs) of patients with pSS. Methods Two independent cohorts consisting of pSS patients and healthy controls were established: a discovery cohort (15 pSS, 6 HC) was used to screen the expression of a large panel of 758 miRNAs. An independent validation cohort (14 pSS, 11 HC) was used to test the reproducibility of the results. cDCs were isolated from peripheral blood using MACS and miRNA profiling of 758 targets was performed using the OpenArray platform in the discovery cohort. A selection of 16 differentially expressed miRNAs was measured in the validation cohort using a custom-made array. Isolated cDCs from HC were stimulated with a panel of Toll-like receptor (TLR) ligands and the expression of miR-130a and miR-708 was measured by qPCR. The effect of transfection with miR-130a on protein synthesis was analysed by using the pulsed stable isotope labelling by amino acids in cell culture (pSILAC) method (quantitative mass spectrometry-based technique) in a HEK-293T cell-line. Results A total of 24 miRNAs was downregulated in pSS patients versus HC in the discovery cohort (p<0.05, with a difference between the groups of >log2). Of the 16 miRNAs that were selected for replication, the decreased expression of miR-130a and miR-708 in pSS was validated. Activation of cDCs via TLR3 and TLR7/8 induced downregulation of both miRNA-130a and miRNA-708. Transfection with a miR-130a mimic resulted in downregulation of several proteins with a seed match for the miRNA. These proteins are known to be involved in membrane trafficking and cell activation trough CREB/NF-kB signalling. Conclusions miR-130a and miR-708 are significantly downregulated in cDCs of patients with pSS. We show that the expression of these miRNAs is decreased upon cDC activation and that upregulation of miR-130a decreases the expression of proteins involved in the CREB/NF-kB pathway. As such, these miRNAs seem to be involved in cDC activation and reflect enhanced activation of circulating cDCs from pSS patients. Disclosure of Interest None declared

AB0170 Ccr9+ pathogenic thelper cells from primary sjÖgren’s syndrome patients are characterised by deregulated pathways associated with effector t cell function

Background CCR9 +Th cells produce large amounts of IFN-γ and IL-10, lack CXCR5 expression but have features similar to Tfh cells and the recently described pathogenic PD1 +CXCR5 cells, including expression of ICOS, PD-1, IL-21 and Bcl6, but no CXCR5 expression. CCR9 Th cells and their ligand CCL25 are elevated in salivary glands of primary Sjögren’s syndrome (pSS) patients. Since CCR9 Th cells strongly induce antibody production and robustly respond to IL-7, which is indicated to play an essential role in pSS pathogenesis and in formation of ectopic lymphoid structures, these cells may play an important role in pSS immunopathology. Objectives The goal of this study was to identify the molecular dysregulation of CCR9 Th cells in pSS patients. Methods CCR9+, CXCR5 +and CCR9-CXCR5- Th cells were sorted from peripheral blood of 7 healthy individuals and 7 pSS patients and RNA sequencing of these sorted cell subsets was performed. Computational analysis identified differentially expressed genes (DEG) and gene expression networks. Pathway enrichment analysis was performed in order to assess differentially regulated pathways. Target genes are technically validated and knockdown experiments will assess the functional role of identified targets. Results The sorted Th subsets could robustly be distinguished based on their transcriptomes. In the CCR9 +Th cell subset 2777 DEGs (1249 up and 1528 down) were identified between healthy controls and pSS patients, and 1416 and 1077 in the CXCR5 +and CCR9-CXCR5- subsets, respectively. Using network analysis 15 modules were constructed, consisting of genes showing coherent expression patterns. Four modules of interest were selected based on pathway enrichment analysis, revealing pathways involved in e.g. cytokine and chemokine production, proliferation and migration. DEGs of interest within these networks were selected, including upregulated expression of integrin αE, integrin α1 and downregulation of regulatory T cell associated genes FoxP3 and IL2RA. Expression of these markers is being validated using flow cytometry. In addition, knockdown of predicted key transcription factors is studied to reveal their role in the pathogenic potential of CCR9 +Th cells. Conclusions Transcriptomic analysis of CCR9 +Th cells from pSS patients revealed multiple dysregulated pathways previously shown to be involved in increased effector T cell function. Upregulation of genes associated with pathogenicity and downregulation of regulatory T cell associated genes were found in pSS patients. Targeting predicted key molecules might reveal (novel) therapeutic targets to halt pathogenic processes induced by CCR9 +Th cells. Disclosure of Interest None declared