Salivary gland epithelial cells from patients with Sjögren's syndrome induce B-lymphocyte survival and activation.

OBJECTIVE: Primary Sjögren's syndrome (pSS) is characterised by chronic hyperactivation of B lymphocytes. Salivary gland epithelial cells (SGECs) could play a role in promoting B-lymphocyte activation within the target tissue. We aimed to study the interactions between SGECs from patients with pSS or controls and B lymphocytes. METHODS: Patients had pSS according to 2016 European League Against Rheumatism/American College of Rheumatology criteria. Gene expression analysis of SGECs and B lymphocytes from pSS and controls isolated from salivary gland biopsies and blood was performed by RNA-seq. SGECs from pSS and controls were cocultured with B-lymphocytes sorted from healthy donor blood and were stimulated. Transwell and inhibition experiments were performed. RESULTS: Gene expression analysis of SGECs identified an upregulation of interferon signalling pathway and genes involved in immune responses (HLA-DRA, IL-7 and B-cell activating factor receptor) in pSS. Activation genes CD40 and CD48 were upregulated in salivary gland sorted B lymphocytes from patients with pSS. SGECs induced an increase in B-lymphocyte survival, which was higher for SGECs from patients with pSS than controls. Moreover, when stimulated with poly(I:C), SGECs from patients with pSS induced higher activation of B-lymphocytes than those from controls. This effect depended on soluble factors. Inhibition with anti-B-cell activating factor, anti-A proliferation-inducing ligand, anti-interleukin-6-R antibodies, JAK1/3 inhibitor or hydroxychloroquine had no effect, conversely to leflunomide, Bruton's tyrosine kinase (BTK) or phosphatidyl-inositol 3-kinase (PI3K) inhibitors. CONCLUSIONS: SGECs from patients with pSS had better ability than those from controls to induce survival and activation of B lymphocytes. Targeting a single cytokine did not inhibit this effect, whereas leflunomide, BTK or PI3K inhibitors partially decreased B-lymphocyte viability in this model. This gives indications for future therapeutic options in pSS.

Role of the IL-12/IL-35 balance in patients with Sjögren syndrome.

BACKGROUND: An interferon signature is involved in the pathogenesis of primary Sjögren syndrome (pSS), but whether the signature is type 1 or type 2 remains controversial. Mouse models and genetic studies suggest the involvement of TH1 and type 2 interferon pathways. Likewise, polymorphisms of the IL-12A gene (IL12A), which encodes for IL-12p35, have been associated with pSS. The IL-12p35 subunit is shared by 2 heterodimers: IL-12 and IL-35. OBJECTIVE: We sought to confirm genetic association of the IL12A polymorphism and pSS and elucidate involvement of the IL-12/IL-35 balance in patients with pSS by using functional studies. METHODS: The genetic study involved 673 patients with pSS from 2 French pSS cohorts and 585 healthy French control subjects. Functional studies were performed on sorted monocytes, irrespective of whether they were stimulated. IL12A mRNA expression and IL-12 and IL-35 protein levels were assessed by using quantitative RT-PCR and ELISA and a multiplex kit for IL-35 and IL-12, respectively. RESULTS: We confirmed association of the IL12A rs485497 polymorphism and pSS and found an increased serum protein level of IL-12p70 in patients with pSS carrying the risk allele (P = .016). Serum levels of IL-12p70 were greater in patients than control subjects (P = .0001), especially in patients with more active disease (P = .05); conversely, IL-35 levels were decreased in patients (P = .0001), especially in patients with more active disease (P = .05). In blood cellular subsets both IL12p35 and EBV-induced gene protein 3 (EBI3) mRNAs were detected only in B cells, with a trend toward a lower level among patients with pSS. CONCLUSION: Our findings emphasize involvement of the IL-12/IL-35 balance in the pathogenesis of pSS. Serum IL-35 levels were associated with low disease activity, in contrast with serum IL-12p70 levels, which were associated with more active disease.

Deregulation of microRNA expression in purified T and B lymphocytes from patients with primary Sjögren's syndrome.

OBJECTIVE: MicroRNAs (miRNAs) play an important role in the pathogenesis of autoimmune diseases such as primary Sjögren's syndrome (pSS). This study is the first to investigate miRNA expression patterns in purified T and B lymphocytes from patients with pSS using a high-throughput quantitative PCR (qPCR) approach. METHODS: Two independent cohorts of both patients with pSS and controls, one for discovery and one for replication, were included in this study. CD4+ T cells and CD19+ B cells were isolated from peripheral blood mononuclear cells by magnetic microbeads and expression of miRNAs was profiled using the Exiqon Human miRNome panel I analysing 372 miRNAs. A selection of differentially expressed miRNAs was replicated in the second cohort using specific qPCR assays. RESULTS: A major difference in miRNA expression patterns was observed between the lymphocyte populations from patients with pSS and controls. In CD4 T lymphocytes, hsa-let-7d-3p, hsa-miR-155-5 p, hsa-miR-222-3 p, hsa-miR-30c-5p, hsa-miR-146a-5p, hsa-miR-378a-3p and hsa-miR-28-5 p were significantly differentially expressed in both the discovery and the replication cohort. In B lymphocytes, hsa-miR-378a-3p, hsa-miR-222-3 p, hsa-miR-26a-5p, hsa-miR-30b-5p and hsa-miR-19b-3p were significantly differentially expressed. Potential target mRNAs were enriched in disease relevant pathways. Expression of B-cell activating factor (BAFF) mRNA was inversely correlated with the expression of hsa-miR-30b-5p in B lymphocytes from patients with pSS and functional experiments showed increased expression of BAFF after inhibiting hsa-miR-30b-5p. CONCLUSIONS: This study demonstrates major miRNAs deregulation in T and B cells from patients with pSS in two independent cohorts, which might target genes known to be involved in the pathogenesis of pSS.

Impact of anti-TNF therapy on NK cells function and on immunosurveillance against B-cell lymphomas.

OBJECTIVES: Rheumatoid arthritis (RA) is associated with an increased risk of lymphoma linked to activity of the disease. Immunosuppressive drugs have been suspected to induce an additional risk. Since, NK cells have been recently shown to participate to anti-lymphoma immunosurveillance, we aimed to assess if anti-TNF might impact their anti-lymphoma activity. METHODS: NK cells have been assessed ex vivo in patients with RA treated with methotrexate (MTX) with or without anti-TNF. Phenotype has been studied by flow cytometry and function has been assessed after NKp30-cross linking. NK have been cultured 6 days in presence of anti-TNF, TNF-R inhibitors or controls and phenotype has been studied. Then cytotoxicity against 2 B non-Hodgkin lymphoma cell lines [Farage (EBV+) and SU-DHL4 (EBV-)] was assessed. RESULTS: Exposure to anti-TNF was associated with a decreased activation of NK cells. NK cells exhibited an impaired function in patients treated with anti-TNF compared to patients treated with MTX alone as assessed by the percentage of degranulation (20.9% [18.5-32.9] vs 31.3% [21.5-49.1], p = 0.04) and a decreased IFN-γ secretion ((17.4% [8.9-25.9] vs to 29.7% [22.5-43.1], p = 0.007). In vitro, exposure to anti-TNF impaired NK cells function and impacted negatively anti-lymphoma activity. These effects may be the consequence of inhibition of TNFR1 signaling. CONCLUSIONS: Thus, even if meta-analysis of randomized controlled trials and of registries have not demonstrated to date an increased risk of lymphoma with anti-TNF, cautious must be pursued concerning this possible side effect in patients with long-term anti-TNF exposure.

Clinical relevance of RORγ positive and negative subsets of CD161+CD4+ T cells in primary Sjögren's syndrome.

OBJECTIVE: The relevance of the Th17 pathway in primary SS (pSS) is unclear. Published studies have relied on restimulating circulating CD161+ T cells in vitro for quantitation of IL-17-producing cells. While CD161 marks all IL-17+ T cells, it is also expressed by other Th subsets. The aim of this study was to directly analyse retinoic acid receptor-related orphan nuclear receptor (ROR)-γ expressing and non-expressing subsets of CD161+ T cells to determine the relevance of the Th17 pathway in pSS. METHODS: We quantitated the frequencies of both CD161- and RORγ-expressing T cells by comparative flow cytometry in peripheral blood mononuclear cells from a well-stratified cohort of pSS patients and control subjects. We also analysed the expression of antigen D-related HLA (HLA-DR) and CD161 in labial salivary glands from nine subjects undergoing a diagnostic biopsy. RESULTS: While the frequencies of both RORγ+ and RORγ- subsets of CD161+ CD4+ T cells were increased in peripheral blood from pSS patients, the increase in the RORγ+ subset positively correlated with humoral manifestations of the disease (anti-SSA/SSB autoantibodies and hypergammaglobulinaemia), but not with disease activity, and vice versa for the RORγ- subset. An increased frequency of HLA-DR+ CD161+CD4+ T cells was observed in labial salivary gland biopsies from pSS patients, suggesting chronic activation of CD161+CD4+ T cells in the target tissue of the disease. CONCLUSION: In addition to pointing to CD161 as a marker of a pathogenic subset of CD4+ T cells in pSS patients, our data indicate that even though the RORγ+ (Th17) CD161+ subset might contribute to humoral manifestations of the disease, the RORγ- (non-Th17) CD161+ subset is the one associated with disease activity in pSS patients.

Cytometry by time-of-flight immunophenotyping identifies a blood Sjögren's signature correlating with disease activity and glandular inflammation.

BACKGROUND: Mass cytometry has recently emerged as a promising tool for clinical research. However, few studies have demonstrated its benefit for patient stratification and biomarker identification. Primary Sjögren's syndrome (pSS) is a prototype of chronic autoimmune disease, the pathogenesis of which remains unclear and for which treatment does not exist. OBJECTIVE: This observational case-control study was designed to discover new cellular biomarkers and therapeutic targets in patients with pSS. METHODS: Forty-nine patients with pSS and 45 control subjects were enrolled for clinical evaluation and mass cytometry quantification of 34 protein markers in blood. For a third of these subjects, matched labial salivary gland biopsy specimens were also analyzed by mass cytometry and immunohistochemistry. RESULTS: In salivary gland biopsy specimens from patients with pSS, we identified a high number of activated CD8(+) T cells, terminally differentiated plasma cells, and activated epithelial cells, pointing to new pathogenic mechanisms for future clinical intervention. In blood, we identified a 6-cell disease signature defined by decreased numbers of CD4 and memory B lymphocytes, decreased plasmacytoid dendritic cell numbers, and increased representation of activated CD4 and CD8 T cells and plasmablasts. These blood cellular components correlated with clinical parameters and, when taken together, clustered patients into subsets with distinct disease activity and glandular inflammation. CONCLUSION: This first application of mass cytometry to a well-stratified clinical cohort and small biopsy tissues establishes the benefits of such an approach for the discovery of new biomarkers and therapeutic targets. Similar high-dimensional immunophenotyping strategies could be implemented in longitudinal and interventional clinical settings in this and other disease areas.

Increased Dickkopf-1 in Recent-onset Rheumatoid Arthritis is a New Biomarker of Structural Severity. Data from the ESPOIR Cohort.

Rheumatoid arthritis (RA) is the most common chronic inflammatory rheumatic condition over the world. RA is potentially disabling because chronic inflammation of the joints leads to joint destruction. To date, the best predictor of radiographic progression for patients with early RA is the presence of radiographic erosions at baseline, but a limited number of predictive biomarkers of structural progression are currently used in daily practice. Here, we investigated Dickkopf-1 (DKK-1) and sclerostin (SOST) serum levels in patients with recent inflammatory arthritis from the ESPOIR cohort. This cohort is a prospective, multicenter French cohort of 813 patients with early arthritis. We observed that mean baseline DKK-1 level was higher among RA patients with than without radiological progression within the first 2 years of evolution. DKK-1 level was still associated with radiographic progression in a model including other main predictors of severity (erosions at baseline, and anti-CCP antibody positivity). This study demonstrates that increased DKK-1 level at baseline predicted structural progression after 2-year follow-up and suggests that DKK-1 might be a new structural biomarker for early RA.

Germline variation of TNFAIP3 in primary Sjögren's syndrome-associated lymphoma.

BACKGROUND AND OBJECTIVE: A germline and coding polymorphism (rs2230926) of TNFAIP3 (A20), a central gatekeeper of nuclear factor-kappa B (NF-kB) activation, was recently found associated with primary Sjögren's syndrome (pSS)-associated lymphoma in a French cohort. We aimed to replicate this association. PATIENTS AND METHODS: The rs2230926 polymorphism was genotyped in cases and controls of European ancestry from two independent cohorts from UK and France. Case control association tests were performed (Fisher's test) in the two cohorts, followed by a meta-analysis of the two cohorts. RESULTS: The UK cohort included 308 controls and 590 patients with pSS including 31 with a history of lymphoma. The French cohort consisted of 448 controls and 589 patients with pSS including 47 with lymphoma. In both cohorts, the rs2230926 missense polymorphism was not associated with pSS. However, in the UK cohort, the rs2230926G variant was significantly associated with pSS-associated lymphoma (OR=2.74, 95% CI (1.07 to 7.03), p=0.0423, compared with patients with pSS without lymphoma, and OR=3.12, 95% CI (1.16 to 8.41), p=0.0314, compared with healthy controls) as observed in the French cohort. The meta-analysis of the two cohorts confirmed these results (OR=2.48, 95% CI (1.87 to 3.28) p=0.0037 and OR=2.60, 95% CI (1.91 to 3.53) p=0.0031, respectively). CONCLUSIONS: This study confirms the role of A20 impairment in pSS-associated lymphoma. Subtle germline abnormalities of genes leading to impaired control of NF-kB activation in B cells continuously stimulated by autoimmunity enhance the risk of lymphoma.

Overlap between differentially methylated DNA regions in blood B lymphocytes and genetic at-risk loci in primary Sjögren's syndrome.

BACKGROUND: Beyond genetics, epigenetics alterations and especially those related to DNA methylation, play key roles in the pathogenesis of autoimmune diseases such as primary Sjögren's syndrome (pSS) and systemic lupus erythematosus. This study aimed to assess the role of methylation deregulation in pSS pathogeny through a genome-wide methylation approach. PATIENTS AND METHODS: 26 female patients with pSS and 22 age-matched controls were included in this study. CD4+ T cells and CD19+ B cells were isolated from peripheral blood mononuclear cells by magnetic microbeads and their genome-wide DNA methylation profiles were analysed using Infinium Human Methylation 450 K BeadChips. Probes with a median DNA methylation difference of at least 7% and p<0.01 between patients and controls were considered significantly differentially methylated. RESULTS: Methylation alterations were mainly present in B cells compared with T cells. In B cells, an enrichment of genes with differentially methylated probes in genetic at-risk loci was observed, suggesting involvement of both genetic and epigenetic abnormalities in the same genes. Methylation alterations in B cells were more frequent in some specific pathways including Interferon Regulated Genes, mainly among patients who were autoantibody positive. Moreover, genes with differentially methylated probes were over-represented in B cells from patients with active disease. CONCLUSIONS: This study demonstrated more important deregulation of DNA methylation patterns in B cells compared with T cells, emphasising the importance of B cells in the pathogenesis of the disease. Overlap between genes with differentially methylated probes in B lymphocytes and genetic at-risk loci is a new finding highlighting their importance in pSS.

Increase in Dickkopf-1 Serum Level in Recent Spondyloarthritis. Data from the DESIR Cohort.

OBJECTIVES: To investigate DKK-1 and SOST serum levels among patients with recent inflammatory back pain (IBP) fulfilling ASAS criteria for SpA and associated factors. METHODS: The DESIR cohort is a prospective, multicenter French cohort of 708 patients with early IBP (duration >3 months and <3 years) suggestive of AxSpA. DKK-1 and SOST serum levels were assessed at baseline and were compared between the subgroup of patients fulfilling ASAS criteria for SpA (n = 486; 68.6%) and 80 healthy controls. RESULTS: Mean SOST serum levels were lower in ASAS+ patients than healthy controls (49.21 ± 25.9 vs. 87.8 ± 26 pmol/L; p<0.0001). In multivariate analysis, age (p = 5.4 10-9), CRP level (p<0.0001) and serum DKK-1 level (p = 0.001) were associated with SOST level. Mean DKK-1 serum levels were higher in axial SpA patients than controls (30.03 ± 15.5 vs. 11.6 ± 4.2 pmol/L; p<0.0001). In multivariate analysis, DKK-1 serum levels were associated with male gender (p = 0.03), CRP level (p = 0.006), SOST serum level (p = 0.002) and presence of sacroiliitis on radiography (p = 0.05). Genetic association testing of 10 SNPs encompassing the DKK-1 locus failed to demonstrate a significant contribution of genetics to control of DKK-1 serum levels. CONCLUSIONS: DKK-1 serum levels were increased and SOST levels were decreased among a large cohort of patients with early axial SpA compared to healthy controls. DKK-1 serum levels were mostly associated with biological inflammation and SOST serum levels.

Germline and somatic genetic variations of TNFAIP3 in lymphoma complicating primary Sjogren's syndrome.

Several autoimmune diseases, including primary Sjögren's syndrome (pSS), are associated with an increased risk for lymphoma. Polymorphisms of TNFAIP3, which encodes the A20 protein that plays a key role in controlling nuclear factor κB activation, have been associated with several autoimmune diseases. Somatic mutations of TNFAIP3 have been observed in the mucosa-associated lymphoid tissue lymphoma subtype frequently associated with pSS. We studied germline and somatic abnormalities of TNFAIP3 in 574 patients with pSS, including 25 with lymphoma. Nineteen additional patients with pSS and lymphoma were available for exome sequence analysis. Functional abnormalities of A20 were assessed by gene reporter assays. The rs2230926 exonic variant was associated with an increased risk for pSS complicated by lymphoma (odds ratio, 3.36 [95% confidence interval, 1.34-8.42], and odds ratio, 3.26 [95% confidence interval, 1.31-8.12], vs controls and pSS patients without lymphoma, respectively; P = .011). Twelve (60%) of the 20 patients with paired germline and lymphoma TNFAIP3 sequence data had functional abnormalities of A20: 6 in germline DNA, 5 in lymphoma DNA, and 1 in both. The frequency was even higher (77%) among pSS patients with mucosa-associated lymphoid tissue lymphoma. Some of these variants showed impaired control of nuclear factor κB activation. These results support a key role for germline and somatic variations of A20 in the transformation between autoimmunity and lymphoma.