Defective Efferocytosis in a Murine Model of Sjögren's Syndrome Is Mediated by Dysfunctional Mer Tyrosine Kinase Receptor.

Sjögren's syndrome (SjS) is a chronic autoimmune disease primarily involving the exocrine glands in which the involvement of the innate immune system is largely uncharacterized. Mer signaling has been found to be protective in several autoimmune diseases but remains unstudied in SjS. Here, we investigated the role of Mer signaling in SjS. Mer knockout (MerKO) mice were examined for SjS disease criteria. SjS-susceptible (SjSS) C57BL/6.NOD-Aec1Aec2 mice were assessed for defective Mer signaling outcomes, soluble Mer (sMer) levels, A disintegrin and metalloprotease 17 (ADAM17) activity, and Rac1 activation. In addition, SjS patient plasma samples were evaluated for sMer levels via ELISA, and sMer levels were correlated to disease manifestations. MerKO mice developed submandibular gland (SMG) lymphocytic infiltrates, SMG apoptotic cells, anti-nuclear autoantibodies (ANA), and reduced saliva flow. Mer signaling outcomes were observed to be diminished in SjSS mice, as evidenced by reduced Rac1 activation in SjSS mice macrophages in response to apoptotic cells and impaired efferocytosis. Increased sMer was also detected in SjSS mouse sera, coinciding with higher ADAM17 activity, the enzyme responsible for cleavage and inactivation of Mer. sMer levels were elevated in patient plasma and positively correlated with focus scores, ocular staining scores, rheumatoid factors, and anti-Ro60 levels. Our data indicate that Mer plays a protective role in SjS, similar to other autoimmune diseases. Furthermore, we suggest a series of events where enhanced ADAM17 activity increases Mer inactivation and depresses Mer signaling, thus removing protection against the loss of self-tolerance and the onset of autoimmune disease in SjSS mice.

Transcriptomic and Network Analysis of Minor Salivary Glands of Patients With Primary Sjögren's Syndrome.

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterized primarily by immune-mediated destruction of exocrine tissues, such as those of the salivary and lacrimal glands, resulting in the loss of saliva and tear production, respectively. This disease predominantly affects middle-aged women, often in an insidious manner with the accumulation of subtle changes in glandular function occurring over many years. Patients commonly suffer from pSS symptoms for years before receiving a diagnosis. Currently, there is no effective cure for pSS and treatment options and targeted therapy approaches are limited due to a lack of our overall understanding of the disease etiology and its underlying pathology. To better elucidate the underlying molecular nature of this disease, we have performed RNA-sequencing to generate a comprehensive global gene expression profile of minor salivary glands from an ethnically diverse cohort of patients with pSS. Gene expression analysis has identified a number of pathways and networks that are relevant in pSS pathogenesis. Moreover, our detailed integrative analysis has revealed a primary Sjögren's syndrome molecular signature that may represent important players acting as potential drivers of this disease. Finally, we have established that the global transcriptomic changes in pSS are likely to be attributed not only to various immune cell types within the salivary gland but also epithelial cells which are likely playing a contributing role. Overall, our comprehensive studies provide a database-enriched framework and resource for the identification and examination of key pathways, mediators, and new biomarkers important in the pathogenesis of this disease with the long-term goals of facilitating earlier diagnosis of pSS and to mitigate or abrogate the progression of this debilitating disease.

Unique Sjögren's syndrome patient subsets defined by molecular features.

OBJECTIVE: To address heterogeneity complicating primary SS (pSS) clinical trials, research and care by characterizing and clustering patients by their molecular phenotypes. METHODS: pSS patients met American-European Consensus Group classification criteria and had at least one systemic manifestation and stimulated salivary flow of ⩾0.1 ml/min. Correlated transcriptional modules were derived from gene expression microarray data from blood (n = 47 with appropriate samples). Patients were clustered based on this molecular information using an unbiased random forest modelling approach. In addition, multiplex, bead-based assays and ELISAs were used to assess 30 serum cytokines, chemokines and soluble receptors. Eleven autoantibodies, including anti-Ro/SSA and anti-La/SSB, were measured by Bio-Rad Bioplex 2200. RESULTS: Transcriptional modules distinguished three clusters of pSS patients. Cluster 1 showed no significant elevation of IFN or inflammation modules. Cluster 2 showed strong IFN and inflammation modular network signatures, as well as high plasma protein levels of IP-10/CXCL10, MIG/CXCL9, BLyS (BAFF) and LIGHT. Cluster 3 samples exhibited moderately elevated IFN modules, but with suppressed inflammatory modules, increased IP-10/CXCL10 and B cell-attracting chemokine 1/CXCL13 and trends toward increased MIG/CXCL9, IL-1α, and IL-21. Anti-Ro/SSA and anti-La/SSB were present in all three clusters. CONCLUSION: Molecular profiles encompassing IFN, inflammation and other signatures can be used to separate patients with pSS into distinct clusters. In the future, such profiles may inform patient selection for clinical trials and guide treatment decisions.

American Indians Have a Higher Risk of Sjögren's Syndrome and More Disease Activity Than European Americans and African Americans.

OBJECTIVE: To describe the clinical and serologic manifestations of Sjögren's syndrome (SS) in ethnic groups of the US. METHODS: This was a cross-sectional study of 648 patients with primary SS: 20 African American (AA), 164 American Indian (AI), 426 European American (EA), and 38 patients of other races evaluated in a multidisciplinary Sjögren's International Collaborative Clinical Alliance research clinic. RESULTS: AA subjects comprised 3.1% of the SS cohort, much lower than the percentage of AA in the state of Oklahoma (P = 3.01 × E - 05), the US (P = 2.24E - 13), or a systemic lupus erythematosus (SLE) cohort at the same institution (P = 4.26 × 10E - 27). In contrast, the percentage of AI in the SS cohort (25.3%) was much higher than expected (P = 3.17E - 09 versus SLE cohort, P = 6.36 - 26 versus Oklahoma, and P = 8.14E - 96 versus US population). The SS classification criteria were similar between AA and EA, but subjects of AI ancestry had lower rates of abnormal tear and salivary flow, as well as anti-Ro/SSA and anti-La/SSB antibodies. Paradoxically, AI had higher levels of disease activity (mean ± SD European League Against Rheumatism Sjögren's Syndrome Disease Activity Index score 3.77 ± 4.78) in comparison to EA (2.90 ± 4.12; P = 0.011) and more extraglandular manifestations affecting mainly the articular and glandular domains. Meanwhile, AA patients were characterized by higher rates of hypergammaglobulinemia (odds ratio [OR] 1.39 [95% confidence interval (95% CI) 1.39-8.65]; P = 0.01), elevated erythrocyte sedimentation rate (ESR) (OR 3.95 [95% CI 1.46-9.95]; P = 0.009), and parotid enlargement (OR 4.40 [95% CI 1.49-13.07]; P = 0.02). CONCLUSION: AI are affected at high rates with SS but present with few classical features, potentially preventing timely diagnosis. In contrast to SLE, SS is infrequent and not more severe among AA, but the triad of hypergammaglobulinemia, increased ESR, and parotid enlargement warrants extra vigilance for lymphomagenesis.

Sjögren Syndrome without Focal Lymphocytic Infiltration of the Salivary Glands.

OBJECTIVE: Primary Sjögren syndrome (SS) is characterized by a focal lymphocytic infiltrate in exocrine glands. We describe patients who lacked this key feature. METHODS: We evaluated patients with sicca in a comprehensive clinic at which medical, dental, and ophthalmological examinations were performed. All subjects underwent a minor salivary gland biopsy with focus score calculation. Extraglandular manifestations were also determined. We categorized subjects as high, intermediate, or low in terms of expression of interferon (IFN)-regulated genes. RESULTS: About 20% (51 of 229, 22%) of those classified as having primary SS had a focus score of zero. Compared to those with anti-Ro positivity and a focus score > 1.0, the patients with focus score of zero (who by classification criteria must be anti-Ro-positive) were statistically less likely to have anti-La (or SSB) and elevated immunoglobulin, as well as less severe corneal staining. The focus score zero patients were less likely to have elevated expression of IFN-regulated genes in peripheral blood mononuclear cells than anti-Ro-positive SS patients with a focal salivary infiltrate. CONCLUSION: There are only a few clinical differences between patients with primary SS with focus score zero and those with both anti-Ro and a focus score > 1.0. The small subset of focus score zero patients tested did not have elevated expression of IFN-regulated genes, but did have systemic disease. Thus, extraglandular manifestations are perhaps more related to the presence of anti-Ro than increased IFN. This may have relevance to pathogenesis of SS.

How immunological profile drives clinical phenotype of primary Sjögren's syndrome at diagnosis: analysis of 10,500 patients (Sjögren Big Data Project).

OBJECTIVES: To evaluate the influence of the main immunological markers on the disease phenotype at diagnosis in a large international cohort of patients with primary Sjögren's syndrome (SjS). METHODS: The Big Data Sjögren Project Consortium is an international, multicentre registry created in 2014. As a first step, baseline clinical information from leading centres on clinical research in SjS of the 5 continents was collected. The centres shared a harmonised data architecture and conducted cooperative online efforts in order to refine collected data under the coordination of a big data statistical team. Inclusion criteria were the fulfillment of the 2002 classification criteria. Immunological tests were carried out using standard commercial assays. RESULTS: By January 2018, the participant centres had included 10,500 valid patients from 22 countries. The cohort included 9,806 (93%) women and 694 (7%) men, with a mean age at diagnosis of primary SjS of 53 years, mainly White (78%) and included from European countries (71%). The frequency of positive immunological markers at diagnosis was 79.3% for ANA, 73.2% for anti-Ro, 48.6% for RF, 45.1% for anti- La, 13.4% for low C3 levels, 14.5% for low C4 levels and 7.3% for cryoglobulins. Positive autoantibodies (ANA, Ro, La) correlated with a positive result in salivary gland biopsy, while hypocomplementaemia and especially cryoglo-bulinaemia correlated with systemic activity (mean ESSDAI score of 17.7 for cryoglobulins, 11.3 for low C3 and 9.2 for low C4, in comparison with 3.8 for negative markers). The immunological markers with a great number of statistically-significant associations (p<0.001) in the organ-by-organ ESS- DAI evaluation were cryoglobulins (9 domains), low C3 (8 domains), anti-La (7 domains) and low C4 (6 domains). CONCLUSIONS: We confirm the strong influence of immunological markers on the phenotype of primary SjS at diagnosis in the largest multi-ethnic international cohort ever analysed, with a greater influence for cryoglobulinaemic-related markers in comparison with Ro/La autoantibodies and ANA. Immunological patterns play a central role in the phenotypic expression of the disease already at the time of diagnosis, and may guide physicians to design a specific personalised management during the follow-up of patients with primary SjS.

Minor salivary gland fibrosis in Sjögren's syndrome is elevated, associated with focus score and not solely a consequence of aging.

OBJECTIVES: Evaluate the presence of minor salivary gland (SG) fibrosis in primary Sjögren's syndrome (pSS) as a function of disease pathology or a consequence of ageing. METHODS: Subjects with sicca symptoms attending a Sjögren's research clinic were classified by American European Consensus Group (AECG) criteria as either pSS or non-SS (nSS). Discovery (n=34 pSS, n=28 nSS) and replication (n=35 pSS, n=31 nSS) datasets were evaluated. Minor SG cross-sections from haematoxylin and eosin stained slides were imaged, digitally reconstructed and analysed for percent area fibrosis. Relationships between SG fibrosis, age, and clinical measures were evaluated using Spearman correlations. Association with SS was assessed by: ROC curve, Variable Selection Using Random Forests (VSURF) and uni- and bi-variate regression analyses. RESULTS: SS subjects had significantly more fibrotic tissue in their minor labial salivary glands (median 24.39%, range 5.12-51.67%) than nSS participants (median 16.7%, range 5.97-38.65%, p<0.0001); age did not differ between groups (average ± SD pSS 50.2 ±13.9 years, nSS 53.8±12.4 years). In both the discovery and replication data sets, multiple regression models showed that the area of minor salivary gland fibrosis predicted pSS significantly better than age alone. Age-corrected linear regression revealed that the area of minor salivary gland fibrosis positively associated with vanBijsterveld score (p=0.042) and biopsy focus score (p=0.002). ROC curve and VSURF analyses ranked fibrosis as a significantly more important variable for subject discrimination than age. CONCLUSIONS: SG fibrosis is an element of pSS pathology that is related to focus score and is not solely attributable to age.

Accumulation of Antigen-Driven Lymphoproliferations in Complement Receptor 2/CD21-/low B Cells From Patients With Sjögren's Syndrome.

OBJECTIVE: Patients with Sjögren's syndrome (SS) are prone to develop malignant lymphomas, and a correlation has been established between the lymphoproliferations occurring in these disorders and the presence in patients' blood of an unusual B cell population that down-regulates complement receptor 2/CD21. This study was undertaken to identify the B cell compartment from which these lymphoproliferations emerge and determine the mechanisms that promote clonal B cell expansion in patients with SS. METHODS: The reactivity of antibodies expressed by CD19+CD10-CD27-IgM+CD21-/low cells isolated from the blood of patients with SS was tested using a polymerase chain reaction-based approach that allows us to clone and express, in vitro, recombinant antibodies produced by single B cells. RESULTS: Clonal expansions were identified in CD21-/low B cells isolated from the peripheral blood of 3 patients with SS. These lymphoproliferations expressed B cell receptors (BCRs) that displayed somatic hypermutation lineage trees characteristic of a strong selection by antigens; one of these antigens was identified as a ribosomal self antigen. When the mutated BCR sequences expressed by the expanded CD21-/low B cell clones from patients with SS were reverted in vitro to their germline counterparts, one clone remained autoreactive. CONCLUSION: Clonal lymphoproliferations in patients with SS preferentially accumulate in the autoreactive CD21-/low B cell compartment often expanded in these subjects, and recognition of self antigens may drive the clonal B cell expansion while further refining BCR self-reactivity.

Fatty infiltration of the minor salivary glands is a selective feature of aging but not Sjögren's syndrome.

OBJECTIVE: Determine the presence and assess the extent of fatty infiltration of the minor salivary glands (SG) of primary SS patients (pSS) as compared to those with non-SS sicca (nSS). METHODS: Minor SG biopsy samples from 134 subjects with pSS (n = 72) or nSS (n = 62) were imaged. Total area and fatty replacement area for each glandular cross-section (n = 4-6 cross-sections per subject) were measured using Image J (National Institutes of Health, Bethesda, MD). The observer was blinded to subject classification status. The average area of fatty infiltration calculated per subject was evaluated by logistic regression and general linearized models (GLM) to assess relationships between fatty infiltration and clinical exam results, extent of fibrosis and age. RESULTS: The average area of fatty infiltration for subjects with pSS (median% (range) 4.97 (0.05-30.2)) was not significantly different from that of those with nSS (3.75 (0.087-41.9). Infiltration severity varied widely, and subjects with fatty replacement greater than 6% were equivalently distributed between pSS and nSS participants (χ2 p = .50). Age accounted for all apparent relationships between fatty infiltration and fibrosis or reduced saliva flow. The all-inclusive GLM for prediction of pSS versus non-SS classification including fibrosis, age, fatty replacement, and focus score was not significantly different from any desaturated model. In no iteration of the model did fatty replacement exert a significant effect on the capacity to predict pSS classification. CONCLUSIONS: Fatty infiltration is an age-associated phenomenon and not a selective feature of Sjögren's syndrome. Sicca patients who do not fulfil pSS criteria have similar rates of fatty infiltration of the minor SG.

A new MHC-linked susceptibility locus for primary Sjögren's syndrome: MICA.

The association of primary Sjögren's syndrome (pSS) with Major Histocompatibility Complex (MHC) alleles is quintessential of MHC-disease associations. Indeed, although disease associations with classical HLA class I and II alleles/haplotypes are amply documented, further dissection is often prevented by the strong linkage disequilibrium across the entire MHC complex. Here we study the association of pSS, not with HLA genes, but with the non-conventional MHC encoded class I gene, MICA (MHC class I chain-related gene A). MICA is selectively expressed within epithelia, and is the major ligand for the activatory receptor, NKG2D, both attributes relevant to pSS' etiology. MICA-pSS association was studied in two independent (French and UK) cohorts representing a total of 959 cases and 1,043 controls. MICA*008 allele was shown to be significantly associated with pSS (pcor=2.61 × 10-35). A multivariate logistic regression showed that this association was independent of all major known MHC-linked risk loci/alleles, as well as other relevant candidate loci that are in linkage disequilibrium with MICA*008 i.e. HLA-B*08:01, rs3131619 (T), MICB*008, TNF308A, HLA-DRB1*03:01 and HLA-DRB1*15:01 (P = 1.84 × 10-04). Furthermore, independently of the MICA*008 allele, higher levels of soluble MICA proteins were detected in sera of pSS patients compared to healthy controls. This study hence defines MICA as a new, MHC-linked, yet HLA-independent, pSS risk locus and opens a new front in our understanding of the still enigmatic pathophysiology of this disease. The fact that the soluble MICA protein is further amplified in MICA*008 carrying individuals, might also be relevant in other auto-immune diseases and cancer.

Effect of Tobacco Smoking on The Clinical, Histopathological, and Serological Manifestations of Sjögren's Syndrome.

OBJECTIVES: To assess the association of smoking habits with the clinical, serological, and histopathological manifestations of Sjögren's syndrome (SS) and non-Sjögren's sicca (non-SS sicca). METHODS: Cross-sectional case-control study of 1288 patients with sicca symptoms (587 SS and 701 non-SS sicca) evaluated in a multi-disciplinary research clinic. Smoking patterns were obtained from questionnaire data and disease-related clinical and laboratory data were compared between current, past, ever, and never smokers. RESULTS: Current smoking rates were 4.6% for SS patients compared to 14.1% in non-SS sicca (p = 5.17x10E-09), 18% in a local lupus cohort (p = 1.13x10E-14) and 16.8% in the community (p = 4.12x10E-15). Current smoking was protective against SS classification (OR 0.35, 95%CI 0.22-0.56, FDR q = 1.9E10-05), focal lymphocytic sialadenitis (OR 0.26, 95%CI 0.15-0.44, FDR q = 1.52x10E-06), focus score ≥1 (OR 0.22, 95%CI 0.13-0.39, FDR q = 1.43x10E-07), and anti-Ro/SSA(+) (OR 0.36, 95%CI 0.2-0.64, FDR q = 0.0009); ever smoking was protective against the same features and against anti-La/SSB(+) (OR 0.52, 95%CI 0.39-0.70, FDR q = 5.82x10E-05). Duration of smoking was inversely correlated with SS even after controlling for socioeconomic status, BMI, alcohol and caffeine consumption. CONCLUSIONS: Current tobacco smoking is negatively and independently associated with SS, protecting against disease-associated humoral and cellular autoimmunity. The overall smoking rate amongst SS patients is significantly lower than in matched populations and the effects of smoking are proportional to exposure duration. In spite of the protective effects of tobacco on SS manifestations, it is associated with other serious comorbidities such as lung disease, cardiovascular risk and malignancy, and should thus be strongly discouraged in patients with sicca.

Clinical and Serologic Features in Patients With Incomplete Lupus Classification Versus Systemic Lupus Erythematosus Patients and Controls.

OBJECTIVE: Incomplete lupus erythematosus (ILE) involves clinical and/or serologic manifestations consistent with but insufficient for systemic lupus erythematosus (SLE) classification. Because the nature of ILE is poorly understood and no treatment recommendations exist, we examined the clinical manifestations, medication history, and immunologic features in a diverse collection of ILE and SLE patients. METHODS: Medical records of subjects enrolled in the Lupus Family Registry and Repository were reviewed for medication history and American College of Rheumatology (ACR) classification criteria to identify ILE patients (3 ACR criteria; n = 440) and SLE patients (≥4 ACR criteria; n = 3,397). Participants completed the Connective Tissue Disease Screening Questionnaire. Anticardiolipin and plasma B lymphocyte stimulator (BLyS) were measured by enzyme-linked immunosorbent assay, antinuclear antibodies (ANAs) by indirect immunofluorescence, and 13 autoantibodies by bead-based assays. RESULTS: On average, ILE patients were older than SLE patients (46.2 years versus 42.0 years; P < 0.0001), and fewer ILE patients were African American (23.9% versus 32.2%; P < 0.001). ILE patients exhibited fewer autoantibody specificities than SLE patients (1.3 versus 2.6; P < 0.0001) and were less likely to have ANA titers ≥1:1,080 (10.5% versus 19.5%; P < 0.0001). BLyS levels were intermediate in ILE patients (controls < ILE; P = 0.016; ILE < SLE; P = 0.008). Pericarditis, renal, or neurologic manifestations occurred in 12.5% of ILE patients and were associated with non-European American race/ethnicity (P = 0.012). Hydroxychloroquine use increased over time, but was less frequent in ILE than SLE patients (65.2% versus 83.1%; P < 0.0001). CONCLUSION: Although usually characterized by milder symptoms, ILE manifestations may require immunomodulatory treatments. Longitudinal studies are necessary to understand how ILE affects organ damage and future SLE risk, and to delineate molecular pathways unique to ILE.

Genetics in Sjögren Syndrome.

The genes associated with Sjögren syndrome (SS) can be assigned to the NF-kB pathway, the IFN signaling pathway, lymphocyte signaling, and antigen presentation. The frequencies of risk variants show they are common with modest genetic effects. The strongest genetic association outside the human leukocyte antigen region is in IRF5, a gene relevant in the IFN signaling pathway and for B cell differentiation. Although no association has been found with the NF-kB gene itself, associations in TNFAIP3 and TNIP1 (both genome-wide significant), VCAM1 and IRAK1BP (both suggestive), point to genetic explanations for dysregulation of the NF-kB pathway in SS.

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.

Identification of a Systemic Lupus Erythematosus Risk Locus Spanning ATG16L2, FCHSD2, and P2RY2 in Koreans.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder whose etiology is incompletely understood, but likely involves environmental triggers in genetically susceptible individuals. Using an unbiased genome-wide association (GWA) scan and replication analysis, we sought to identify the genetic loci associated with SLE in a Korean population. METHODS: A total of 1,174 SLE cases and 4,246 population controls from Korea were genotyped and analyzed with a GWA scan to identify single-nucleotide polymorphisms (SNPs) significantly associated with SLE, after strict quality control measures were applied. For select variants, replication of SLE risk loci was tested in an independent data set of 1,416 SLE cases and 1,145 population controls from Korea and China. RESULTS: Eleven regions outside the HLA exceeded the genome-wide significance level (P = 5 × 10(-8) ). A novel SNP-SLE association was identified between FCHSD2 and P2RY2, peaking at rs11235667 (P = 1.03 × 10(-8) , odds ratio [OR] 0.59) on a 33-kb haplotype upstream of ATG16L2. In the independent replication data set, the SNP rs11235667 continued to show a significant association with SLE (replication meta-analysis P = 0.001, overall meta-analysis P = 6.67 × 10(-11) ; OR 0.63). Within the HLA region, the SNP-SLE association peaked in the class II region at rs116727542, with multiple independent effects observed in this region. Classic HLA allele imputation analysis identified HLA-DRB1*1501 and HLA-DQB1*0602, each highly correlated with one another, as most strongly associated with SLE. Ten previously established SLE risk loci were replicated: STAT1-STAT4, TNFSF4, TNFAIP3, IKZF1, HIP1, IRF5, BLK, WDFY4, ETS1, and IRAK1-MECP2. Of these loci, previously unreported, independent second risk effects of SNPs in TNFAIP3 and TNFSF4, as well as differences in the association with a putative causal variant in the WDFY4 region, were identified. CONCLUSION: Further studies are needed to identify true SLE risk effects in other loci suggestive of a significant association, and to identify the causal variants in the regions of ATG16L2, FCHSD2, and P2RY2.

Brief Report: Patients With Primary Sjögren's Syndrome Who Are Positive for Autoantibodies to Tripartite Motif-Containing Protein 38 Show Greater Disease Severity.

OBJECTIVE: Autoantibodies reactive with Ro52 (tripartite motif-containing protein 21 [TRIM21]) are detected in 70% of patients with primary Sjögren's syndrome (SS). TRIM21 belongs to a 34-member C-IV family of TRIM proteins. Although autoantibodies against other TRIM proteins within the C-IV family have been detected in the sera of patients with primary SS, their clinical relevance remains unclear. This study was undertaken to investigate the frequency of anti-TRIM38 in patients with primary SS and evaluate its association with various clinical measures of the disease. METHODS: Serum samples from patients with primary SS (n = 235) and controls (n = 50) were analyzed for reactivity with in vitro-transcribed and -translated (35) S-methionine-labeled TRIM38 protein. The associations of anti-TRIM38 with various laboratory and clinical measures of primary SS were evaluated. Reactivity of anti-TRIM38 with different structural domains of TRIM38 was analyzed. Affinity-purified anti-TRIM38 antibodies were used to immunoprecipitate TRIM21. RESULTS: TRIM38-reactive autoantibodies were detected in the sera of 24 of the 235 patients with primary SS and 2 of the 50 controls. Anti-TRIM38 positivity was significantly associated with the presence of anti-Ro60, anti-Ro52, anti-La, rheumatoid factor, and hypergammaglobulinemia. Clinically, anti-TRIM38 was associated with significantly higher ocular surface staining scores, lower Schirmer's test scores, and minor labial salivary gland biopsy focus scores of ≥3.0. Anti-TRIM38 antibodies mainly recognized the cortactin-binding protein 2 (CortBP-2; amino acids 128-238) and the B30.2/SPRY (amino acids 268-465) domains on TRIM38. Affinity-purified antibodies to TRIM38-CortBP-2 and TRIM38-B30.2/SPRY domains reacted with TRIM21. CONCLUSION: Our data demonstrate that anti-TRIM38 specificity arising in a subset of patients with primary SS is associated with increased severity of the disease.

Lupus Risk Variant Increases pSTAT1 Binding and Decreases ETS1 Expression.

Genetic variants at chromosomal region 11q23.3, near the gene ETS1, have been associated with systemic lupus erythematosus (SLE), or lupus, in independent cohorts of Asian ancestry. Several recent studies have implicated ETS1 as a critical driver of immune cell function and differentiation, and mice deficient in ETS1 develop an SLE-like autoimmunity. We performed a fine-mapping study of 14,551 subjects from multi-ancestral cohorts by starting with genotyped variants and imputing to all common variants spanning ETS1. By constructing genetic models via frequentist and Bayesian association methods, we identified 16 variants that are statistically likely to be causal. We functionally assessed each of these variants on the basis of their likelihood of affecting transcription factor binding, miRNA binding, or chromatin state. Of the four variants that we experimentally examined, only rs6590330 differentially binds lysate from B cells. Using mass spectrometry, we found more binding of the transcription factor signal transducer and activator of transcription 1 (STAT1) to DNA near the risk allele of rs6590330 than near the non-risk allele. Immunoblot analysis and chromatin immunoprecipitation of pSTAT1 in B cells heterozygous for rs6590330 confirmed that the risk allele increased binding to the active form of STAT1. Analysis with expression quantitative trait loci indicated that the risk allele of rs6590330 is associated with decreased ETS1 expression in Han Chinese, but not other ancestral cohorts. We propose a model in which the risk allele of rs6590330 is associated with decreased ETS1 expression and increases SLE risk by enhancing the binding of pSTAT1.

Antibody-secreting cell specificity in labial salivary glands reflects the clinical presentation and serology in patients with Sjögren's syndrome.

OBJECTIVE: The serologic hallmark of primary Sjögren's syndrome (SS) is the presence of IgG antibodies specific for Ro (SSA) and La (SSB). The molecular characteristics of gland-derived B cells at the site of primary SS inflammation have been described previously; however, parallels between glandular antibody-secreting cells (ASCs) and serologic antibody specificities have not been evaluated. We used recombinant monoclonal antibody (mAb) technology to study the specificities of salivary gland (SG)-derived ASCs, evaluate their molecular characteristics, and identify IgG antibody specificity. METHODS: Human antibodies were generated from glandular IgG ASCs. Heavy chain and light chain use and immunoglobulin subclass were analyzed by sequencing. Enzyme-linked immunosorbent assay, indirect immunofluorescence, enzyme immunoassay, and (35) S-labeled protein immunoprecipitation analysis were used to determine antibody specificity. RESULTS: Evaluation of single ASCs in SG biopsy specimens from a patient with primary SS and a patient with SS and overlapping systemic lupus erythematosus revealed significant concordance between serum autoantibody and glandular ASC specificities. Gland-derived ASC heavy chains and light chains were extensively somatically hypermutated, which is indicative of antigen-driven responses. Specifically, we produced the first fully human mAb derived from SGs. CONCLUSION: In patients with SS, the SGs are a site for the production of antibodies that extend beyond the canonical Ro and/or La SS specificities. Glandular antibody production strongly reflected the serologic humoral response in the 2 patients whom we studied.

Genome-wide DNA methylation patterns in naive CD4+ T cells from patients with primary Sjögren's syndrome.

OBJECTIVE: Primary Sjögren's syndrome (SS) is a systemic autoimmune disease with incompletely understood etiology. This study was undertaken to investigate the role of epigenetic dysregulation in the pathogenesis of primary SS. METHODS: A genome-wide DNA methylation study was performed in naive CD4+ T cells from 11 patients with primary SS compared to age-, sex-, and ethnicity-matched healthy controls. Cytosine methylation was quantified using the Illumina Infinium HumanMethylation450 BeadChip array, and the data were validated using bisulfite sequencing. RESULTS: Genome-wide analyses identified 553 hypomethylated CpG sites and 200 hypermethylated CpG sites in naive CD4+ T cells from patients with primary SS as compared to healthy controls, representing 311 hypomethylated and 115 hypermethylated gene regions. The hypomethylated genes in patients with primary SS included LTA (encoding lymphotoxin α). Other relevant genes, such as CD247, TNFRSF25, PTPRC, GSTM1, and PDCD1, were also hypomethylated. The interferon signature pathway was represented by hypomethylation of STAT1, IFI44L, USP18, and IFITM1. A group of genes encoding members of the solute carrier proteins were differentially methylated. In addition, the transcription factor gene RUNX1 was hypermethylated in patients with primary SS, suggesting a possible connection to lymphoma predisposition. Gene ontology (GO) analysis of hypomethylated genes demonstrated enrichment of genes involved in lymphocyte activation and immune response. GO terms for hypermethylated genes included antigen processing and presentation. CONCLUSION: This is the first epigenome-wide DNA methylation study in patients with primary SS. These findings highlight a role for DNA methylation in primary SS and identify disease-associated DNA methylation changes in several genes and pathways in naive CD4+ T cells from patients with primary SS that may be involved in the pathogenesis of this disease.

Comparison of the American-European Consensus Group Sjogren's syndrome classification criteria to newly proposed American College of Rheumatology criteria in a large, carefully characterised sicca cohort.

OBJECTIVE: To compare the performance of the American-European Consensus Group (AECG) and the newly proposed American College of Rheumatology (ACR) classification criteria for Sjögren's Syndrome (SS) in a well-characterised sicca cohort, given ongoing efforts to resolve discrepancies and weaknesses in the systems. METHODS: In a multidisciplinary clinic for the evaluation of sicca, we assessed features of salivary and lacrimal gland dysfunction and autoimmunity as defined by tests of both AECG and ACR criteria in 646 participants. Global gene expression profiles were compared in a subset of 180 participants. RESULTS: Application of the AECG and ACR criteria resulted in classification of 279 and 268 participants with SS, respectively. Both criteria were met by 244 participants (81%). In 26 of the 35 AECG+/ACR participants, the minor salivary gland biopsy focal score was ≥1 (74%), while nine had positive anti-Ro/La (26%). There were 24 AECG-/ACR+ who met ACR criteria mainly due to differences in the scoring of corneal staining. All patients with SS, regardless of classification, had similar gene expression profiles, which were distinct from the healthy controls. CONCLUSIONS: The two sets of classification criteria yield concordant results in the majority of cases and gene expression profiling suggests that patients meeting either set of criteria are more similar to other SS participants than to healthy controls. Thus, there is no clear evidence for increased value of the new ACR criteria over the old AECG criteria from the clinical or biological perspective. It is our contention, supported by this report, that improvements in diagnostic acumen will require a more fundamental understanding of the pathogenic mechanisms than is at present available.