Pathophysiological mechanisms and clinical manifestations in primary Sjögren’s syndrome and Systemic lupus erythematosus (SLE)

Primary Sjögren’s syndrome (pSS) is a systemic inflammatory autoimmune condition. The etiology of pSS is mainly unknown, but it has been suggested that environmental factors trigger an immune response in genetically susceptible individuals. The disease is characterized by chronic inflammation which results in progressive destruction of exocrine glands, primarily the salivary and lacrimal glands, leading to symptoms of dryness, mainly in the mouth and eyes. Patients may also experience symptoms of exocrine dysfunction and dryness in other parts of the body. Autoantibodies against the antigens Ro/SSA (Ro52 and Ro60) and La/SSB, are a well-known sign of dysregulation of the immune system. Extraglandular manifestations (EGM) are present in a subset of patients with pSS. In a population based prospective study we included all patients referred to the Department of Rheumatology for suspected pSS during a five-year period. The patients were evaluated using a structured procedure according to the 2002 Revised American-European Consensus Criteria for Sjögren’s syndrome. Of referred individuals, 199 of 781 patients were diagnosed with pSS. We found an annual incidence rate of pSS in the Karolinska University Hospital catchment area of 3.1 (95% CI 2.3-4.3) cases per 100,000 adult inhabitants. In this cohort, we noted lower figures for severe EGM such as lung and neurological involvement than previously reported for prevalent pSS. Also, the frequency of autoantibodies including ANA, anti-Ro/SSA and anti-La/SSB was lower compared to other cohorts. Subsets of these patients were recruited and included in genetic studies. We contributed with 79 pSS patients in a candidate-gene association case control study in 540 patients with pSS and 532 controls, from Sweden and Norway. Three novel gene loci, not previously associated with pSS, were identified: the early B-cell factor 1 (EBF1) gene, the family with sequence similarity 167 member A–B-lymphoid tyrosine kinase (FAM167A–BLK) locus, and the tumor necrosis factor superfamily (TNFSF4=Ox40L) gene. Variations in the gene loci for interferon regulation factor 5 (IRF5) and signal transducer and activator of transcription 4 (STAT4) were confirmed. The three novel gene variants are involved in B-cell development and activation. In an international multicenter case control genome-wide association study (GWAS)/ large-scale association study of pSS, we contributed with 99 patients. The results confirmed associations with variations in the gene loci of the human leukocyte antigen (HLA) region, IRF5, STAT4, FAM167A-BLK, chemokine C-X-C motif receptor 5 (DX6-CXCR), and TNFAIP3 interacting protein 1 (TNIP1) at a genome-wide significance level. In addition, 29 further suggested associations (p meta < 5 × 10−5) were observed. The major part of these genes is involved in both innate and adaptive immune responses. We observed that the autoantigen Ro52 was expressed and upregulated in salivary glands of patients with primary Sjögren’s syndrome. This was noted in biopsies from 28 pSS patients and 19 non-pSS controls from Sweden and Norway. The degree of expression was correlated with the level of inflammation in the tissue. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by multiple organ manifestations and immunological dysregulation including production of a large set of autoantibodies targeting different epitopes. We showed that antibodies to the RING domain of Ro52, which is the functionally active domain with E3 ligase activity, were significantly correlated with disease activity as measured by the SLAM score in a well-characterized cohort of SLE patients. In conclusion, the results of these studies have highlighted the role of the immune system in the pathogenesis in pSS and SLE

Difference in clinical presentation between women and men in incident primary Sjögren’s syndrome

Background: A more severe disease phenotype has been reported in men compared to women in several rheumatic diseases. However, studies have not conclusively established sex-related clinical features in primary Sjögren’s syndrome (pSS). In this study, we therefore investigated the clinical presentation of pSS in women and men at diagnosis. Methods: Incident, treatment naïve patients (n = 199) during a 5-year period in a specified area were prospectively included and examined for items of classification criteria for pSS as well as extraglandular manifestations (EGM). Serum was sampled at the time of diagnosis and anti-Ro52/SSA levels were measured by ELISA. Replication of significant findings was confirmed in an independent cohort of pSS patients (n = 377), and meta-analysis was performed. Results: An increased frequency of extraglandular manifestations in men was observed and replicated (p = 0.05, p = 0.0003, and p meta = 0.002). This related to pulmonary involvement, vasculitis, and lymphadenopathy being more common in men, for whom a lower age at diagnosis was observed in the exploratory cohort. Additionally, SSA-positive male patients had significantly higher levels of anti-Ro52 levels than their female counterparts in sera available for analysis (p = 0.02). Conclusions: Our analysis of two independent cohorts of incident pSS demonstrates that the presence and number of EGM are significantly more frequent among men with pSS than women at diagnosis. Importantly, around half of the male patients presented with more than one EGM at diagnosis, supporting the conclusion that pSS in men represents a more severe form of disease, regardless of the lower risk for men to develop pSS

Autoantigenic properties of the aminoacyl tRNA synthetase family in idiopathic inflammatory myopathies

Objectives: Autoantibodies are thought to play a key role in the pathogenesis of idiopathic inflammatory myopathies (IIM). However, up to 40% of IIM patients, even those with clinical manifestations of anti-synthetase syndrome (ASSD), test seronegative to known myositis-specific autoantibodies. We hypothesized the existence of new potential autoantigens among human cytoplasmic aminoacyl tRNA synthetases (aaRS) in patients with IIM. Methods: Plasma samples from 217 patients with IIM according to 2017 EULAR/ACR criteria, including 50 patients with ASSD, 165 without, and two with unknown ASSD status were identified retrospectively, as well as age and gender-matched sera from 156 population controls, and 219 disease controls. Patients with previously documented ASSD had to test positive for at least one of the five most common anti-aaRS autoantibodies (anti-Jo1, -PL7, -PL12, -EJ, and -OJ) and present with one or more of the following clinical manifestations: interstitial lung disease, myositis, arthritis, Raynaud's phenomenon, fever, or mechanic's hands. Demographics, laboratory, and clinical data of the IIM cohort (ASSD and non-ASSD) were compared. Samples were screened using a multiplex bead array assay for presence of autoantibodies against a panel of 117 recombinant protein variants, representing 33 myositis-related proteins, including all nineteen cytoplasmic aaRS. Prospectively collected clinical data for the IIM cohort were retrieved and compared between groups within the IIM cohort and correlated with the results of the autoantibody screening. Principal component analysis was used to analyze clinical manifestations between ASSD, non-ASSD groups, and individuals with novel anti-aaRS autoantibodies. Results: We identified reactivity towards 16 aaRS in 72 of the 217 IIM patients. Twelve patients displayed reactivity against nine novel aaRS. The novel autoantibody specificities were detected in four previously seronegative patients for myositis-specific autoantibodies and eight with previously detected myositis-specific autoantibodies. IIM individuals with novel anti-aaRS autoantibodies (n = 12) all had signs of myositis, and they had either muscle weakness and/or muscle enzyme elevation, 2/12 had mechanic's hands, 3/12 had interstitial lung disease, and 2/12 had arthritis. The individuals with novel anti-aaRS and a pathological muscle biopsy all presented widespread up-regulation of major histocompatibility complex class I. The reactivities against novel aaRS could be confirmed in ELISA and western blot. Using the multiplex bead array assay, we could confirm previously known reactivities to four of the most common aaRS (Jo1, PL12, PL7, and EJ (n = 45)) and identified patients positive for anti-Zo, -KS, and -HA (n = 10) that were not previously tested. A low frequency of anti-aaRS autoantibodies was also detected in controls. Conclusion: Our results suggest that most, if not all, cytoplasmic aaRS may become autoantigenic. Autoantibodies against new aaRS may be found in plasma of patients previously classified as seronegative with potential high clinical relevance.publishedVersio

In Situ Coupled Electrochemical-Goniometry as a Tool to Reveal Conformational Changes of Charged Peptides

The opportunity to manipulate cell functions by regulating bioactive surfaces is a potentially promising approach for organic bioelectronics. Here, the tuning of the orientation of charged peptides by means of an electrical input observed via optical tensiometry is reported. A stimuli-responsive self-assembled monolayer (SAM) with specially designed charged peptides is used as a model system to switch between two separate hydrophilic states. The underwater contact angle (UCA) technique is used to measure changes in the wetting property of a dichloromethane droplet under electrical stimuli. The observed changes in the UCA of the bio-interface can be understood in terms of a change in the surface energy between the ON and OFF states. Molecular dynamics simulations in an electric field have been performed to verify the hypothesis of the orientational change of the charged peptides upon electrical stimulation. In addition, X-ray photoelectron spectroscopy (XPS) is performed to clarify the stability of the functionalized electrodes. Finally, the possibility of using such a novel switching system as a tool to characterize bioactive surfaces is discussed

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

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.Fil: Brito Zerón, Pilar. Hospital Sanitas CIMA; España. Universidad de Barcelona; EspañaFil: Acar Denizli, Nihan. Mimar Sinan Fine Arts University; TurquíaFil: Ng, Wan Fai. University of Newcastle; Reino UnidoFil: Zeher, Margit. University of Debrecen; HungríaFil: Rasmussen, Astrid. Oklahoma Medical Research Foundation; Estados UnidosFil: Mandl, Thomas. Lund University; SueciaFil: Seror, Raphaele. Université Paris Sud; FranciaFil: Xiaolin, Li. Anhui Provincial Hospital; ChinaFil: Baldini, Chiara. Università degli Studi di Pisa; ItaliaFil: Gottenberg, Jaques. Université de Strasbourg; Francia. Centre National de la Recherche Scientifique; FranciaFil: Danda, Debashish. Christian Medical College & Hospital; IndiaFil: Quartuccio, Luca. University Hospital “Santa María della Misericordia”; ItaliaFil: Priori, Roberta. Università degli Studi di Roma "La Sapienza"; ItaliaFil: Hernandez Molina, Gabriela. Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán; MéxicoFil: Armagan, Berkan. Hacettepe University. Faculty of Medicine.Department of Internal Medicine; TurquíaFil: Kruize, Aike. University Medical Center Utrecht; Países BajosFil: Kwok, Seung Ki. The Catholic University of Korea; Corea del SurFil: Kvarnström, Marika. Karolinska University Hospital.Department of Medicine.Unit of Rheumatology. Karolinska Institutet ; SueciaFil: Praprotnik, Sonja. University Medical Centre; EsloveniaFil: Sene, Damien. Université Paris Diderot - Paris 7; FranciaFil: Bartoloni, Elena. Università di Perugia; ItaliaFil: Solans, R.. Hospital Vall d’Hebron; ItaliaFil: Rischmueller, M.. University of Western Australia; AustraliaFil: Suzuki, Y.. Kanazawa University Hospital; JapónFil: Isenberg, D. A.. University College London; Estados UnidosFil: Valim, V.. Federal University of Espírito Santo; BrasilFil: Wiland, P.. Wroclaw Medical Hospital; PoloniaFil: Nordmark, G.. Uppsala Universitet; SueciaFil: Fraile, G.. Hospital Ramón y Cajal; EspañaFil: Retamozo, Maria Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; Argentina. Hospital Privado Centro Medico de Córdoba; Argentina; Argentina. Instituto Universitario de Ciencias Biomédicas de Córdoba; Argentin

H1N1 vaccination in Sjögren’s syndrome triggers polyclonal B cell activation and promotes autoantibody production

Objectives: Vaccination of patients with rheumatic disease has been reported to result in lower antibody titres than in healthy individuals. However, studies primarily include patients on immunosuppressive therapy. Here, we investigated the immune response of treatment-naïve patients diagnosed with primary Sjögren’s syndrome (pSS) to an H1N1 influenza vaccine. Methods: Patients with Sjögren’s syndrome without immunomodulatory treatment and age-matched and gender-matched healthy controls were immunised with an H1N1 influenza vaccine and monitored for serological and cellular immune responses. Clinical symptoms were monitored with a standardised form. IgG class switch and plasma cell differentiation were induced in vitro in purified naïve B cells of untreated and hydroxychloroquine-treated patients and healthy controls. Gene expression was assessed by NanoString technology. Results: Surprisingly, treatment-naïve patients with Sjögren’s syndrome developed higher H1N1 IgG titres of greater avidity than healthy controls on vaccination. Notably, off-target B cells were also triggered resulting in increased anti-EBV and autoantibody titres. Endosomal toll-like receptor activation of naïve B cells in vitro revealed a greater propensity of patient-derived cells to differentiate into plasmablasts and higher production of class switched IgG. The amplified plasma cell differentiation and class switch could be induced in cells from healthy donors by preincubation with type 1 interferon, but was abolished in hydroxychloroquine-treated patients and after in vitro exposure of naïve B cells to chloroquine. Conclusions: This comprehensive analysis of the immune response in autoimmune patients to exogenous stimulation identifies a mechanistic basis for the B cell hyperactivity in Sjögren’s syndrome, and suggests that caution is warranted when considering vaccination in non-treated autoimmune patients

Genetic and clinical basis for two distinct subtypes of primary Sjögren's syndrome

Objectives Clinical presentation of primary Sjögren’s syndrome (pSS) varies considerably. A shortage of evidence-based objective markers hinders efficient drug development and most clinical trials have failed to reach primary endpoints. Methods We performed a multicentre study to identify patient subgroups based on clinical, immunological and genetic features. Targeted DNA sequencing of 1853 autoimmune-related loci was performed. After quality control, 918 patients with pSS, 1264 controls and 107 045 single nucleotide variants remained for analysis. Replication was performed in 177 patients with pSS and 7672 controls. Results We found strong signals of association with pSS in the HLA region. Principal component analysis of clinical data distinguished two patient subgroups defined by the presence of SSA/SSB antibodies. We observed an unprecedented high risk of pSS for an association in the HLA-DQA1 locus of odds ratio 6.10 (95% CI: 4.93, 7.54, P=2.2×10−62) in the SSA/SSB-positive subgroup, while absent in the antibody negative group. Three independent signals within the MHC were observed. The two most significant variants in MHC class I and II respectively, identified patients with a higher risk of hypergammaglobulinaemia, leukopenia, anaemia, purpura, major salivary gland swelling and lymphadenopathy. Replication confirmed the association with both MHC class I and II signals confined to SSA/SSB antibody positive pSS. Conclusion Two subgroups of patients with pSS with distinct clinical manifestations can be defined by the presence or absence of SSA/SSB antibodies and genetic markers in the HLA locus. These subgroups should be considered in clinical follow-up, drug development and trial outcomes, for the benefit of both subgroups.publishedVersio

Complement C4 Copy Number Variation is Linked to SSA/Ro and SSB/La Autoantibodies in Systemic Inflammatory Autoimmune Diseases

Objective Copy number variation of the C4 complement components, C4A and C4B, has been associated with systemic inflammatory autoimmune diseases. This study was undertaken to investigate whether C4 copy number variation is connected to the autoimmune repertoire in systemic lupus erythematosus (SLE), primary Sjögren's syndrome (SS), or myositis. Methods Using targeted DNA sequencing, we determined the copy number and genetic variants of C4 in 2,290 well-characterized Scandinavian patients with SLE, primary SS, or myositis and 1,251 healthy controls. Results A prominent relationship was observed between C4A copy number and the presence of SSA/SSB autoantibodies, which was shared between the 3 diseases. The strongest association was detected in patients with autoantibodies against both SSA and SSB and 0 C4A copies when compared to healthy controls (odds ratio [OR] 18.0 [95% confidence interval (95% CI) 10.2–33.3]), whereas a weaker association was seen in patients without SSA/SSB autoantibodies (OR 3.1 [95% CI 1.7–5.5]). The copy number of C4 correlated positively with C4 plasma levels. Further, a common loss-of-function variant in C4A leading to reduced plasma C4 was more prevalent in SLE patients with a low copy number of C4A. Functionally, we showed that absence of C4A reduced the individuals’ capacity to deposit C4b on immune complexes. Conclusion We show that a low C4A copy number is more strongly associated with the autoantibody repertoire than with the clinically defined disease entities. These findings may have implications for understanding the etiopathogenetic mechanisms of systemic inflammatory autoimmune diseases and for patient stratification when taking the genetic profile into account.publishedVersio

Variants at multiple loci implicated in both innate and adaptive immune responses are associated with Sjögren’s syndrome

Sjögren’s syndrome is a common autoimmune disease (~0.7% of European Americans) typically presenting as keratoconjunctivitis sicca and xerostomia. In addition to strong association within the HLA region at 6p21 (Pmeta=7.65×10−114), we establish associations with IRF5-TNPO3 (Pmeta=2.73×10−19), STAT4 (Pmeta=6.80×10−15), IL12A (Pmeta =1.17×10−10), FAM167A-BLK (Pmeta=4.97×10−10), DDX6-CXCR5 (Pmeta=1.10×10−8), and TNIP1 (Pmeta=3.30×10−8). Suggestive associations with Pmeta<5×10−5 were observed with 29 regions including TNFAIP3, PTTG1, PRDM1, DGKQ, FCGR2A, IRAK1BP1, ITSN2, and PHIP amongst others. These results highlight the importance of genes involved in both innate and adaptive immunity in Sjögren’s syndrome

Genome-wide association study identifies Sjögren’s risk loci with functional implications in immune and glandular cells

Sjögren’s disease is a complex autoimmune disease with twelve established susceptibility loci. This genome-wide association study (GWAS) identifies ten novel genome-wide significant (GWS) regions in Sjögren’s cases of European ancestry: CD247, NAB1, PTTG1-MIR146A, PRDM1-ATG5, TNFAIP3, XKR6, MAPT-CRHR1, RPTOR-CHMP6-BAIAP6, TYK2, SYNGR1. Polygenic risk scores yield predictability (AUROC = 0.71) and relative risk of 12.08. Interrogation of bioinformatics databases refine the associations, define local regulatory networks of GWS SNPs from the 95% credible set, and expand the implicated gene list to >40. Many GWS SNPs are eQTLs for genes within topologically associated domains in immune cells and/or eQTLs in the main target tissue, salivary glands.Research reported in this publication was supported by the National Institutes of Health (NIH): R01AR073855 (C.J.L.), R01AR065953 (C.J.L.), R01AR074310 (A.D.F.), P50AR060804 (K.L.S.), R01AR050782 (K.L.S), R01DE018209 (K.L.S.), R33AR076803 (I.A.), R21AR079089 (I.A.); NIDCR Sjögren’s Syndrome Clinic and Salivary Disorders Unit were supported by NIDCR Division of Intramural Research at the National Institutes of Health funds - Z01-DE000704 (B.W.); Birmingham NIHR Biomedical Research Centre (S.J.B.); Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy – EXC 2155 – Projektnummer 390874280 (T.W.); Research Council of Norway (Oslo, Norway) – Grant 240421 (TR.R.), 316120 (M.W-H.); Western Norway Regional Health Authority (Helse Vest) – 911807, 912043 (R.O.); Swedish Research Council for Medicine and Health (L.R., G.N., M.W-H.); Swedish Rheumatism Association (L.R., G.N., M.W-H.); King Gustav V’s 80-year Foundation (G.N.); Swedish Society of Medicine (L.R., G.N., M.W-H.); Swedish Cancer Society (E.B.); Sjögren’s Syndrome Foundation (K.L.S.); Phileona Foundation (K.L.S.). The Stockholm County Council (M.W-H.); The Swedish Twin Registry is managed through the Swedish Research Council - Grant 2017-000641. The French ASSESS (Atteinte Systémique et Evolution des patients atteints de Syndrome de Sjögren primitive) was sponsored by Assistance Publique-Hôpitaux de Paris (Ministry of Health, PHRC 2006 P060228) and the French society of Rheumatology (X.M.).publishedVersio