TRIM21/ Ro52 in B cell pathology

Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease affecting 0.2% of the population. Several B cell aberrances have been linked to pSS, such as autoantibody production, hypergammaglobulinemia and B cell associated genetic polymorphisms. In addition, pSS patients display a 16 fold increased risk to develop B cell lymphomas. Autoantibodies to TRIM21/ Ro52 are detected in approximately 70% of patients with pSS. The cellular role of TRIM21 was largely unknown when this thesis was initiated. However, TRIM21 had been implicated to belong to the TRIpartite Motif (TRIM) family, of which many proteins are E3 ligases, mediating ubiqiutination. The aims of this thesis were to characterize the role of B cells in primary Sjögren’s syndrome pathogenesis and to elucidate the cellular role of the autoantigen TRIM21. By using vaccination as a tool to study immune responses pSS in vivo, we detected a vigorous B cell hyperreactivity, specifically in IgG producing cells. Further, in vitro induction of IgG class switch revealed an increased response to endosomal Toll-like receptor (TLR) stimulation in B cells from patients. This phenomenon may explain the hypergammaglobulinemia observed in pSS patients, and possibly also the high autoantibody titers. In both in vivo and in vitro ubiquitination assays, we could show that TRIM21 is an E3 ligase. To better understand its role in immunity, a TRIM21-/- IRES-GFP mouse was generated. Studies revealed a hyperresponsive immune system. Mild immune activation induced Th17-dependent dermatitis and subsequently systemic autoimmunity with hypergammaglobulinemia, anti-nuclear antibodies and glomerulonephritis. We observed that TRIM21 regulates several interferon regulatory factors (IRFs), central transcription factors of pro-inflammatory responses, by ubiquitination. The loss of TRIM21 expression therefore resulted in loss of negative regulation of the transcription factors, and thereby accentuated immune responses. By using GFP as a reporter in the TRIM21-deficient-IRES-GFP mice, we observed that TRIM21 protein is almost exclusively expressed in hematopoietic cells. Further, overexpression of TRIM21 in a B cell lymphoma cell line resulted in markedly reduced proliferation and increased apoptosis. These findings prompted us to study the role of TRIM21 in lymphomagenesis. In three independent cohorts of diffuse large B cell lymphomas (DLBCL), a strong correlation between low TRIM21 expression and short overall and progression-free survival was demonstrated. In conclusion, these studies show that endosomal TLR hyperreactivity underlie hypergammaglobulinemia in primary Sjögren’s syndrome. Further, the major autoantigen TRIM21 is an E3 ligase, negatively regulating interferon and TLR responses. Loss of TRIM21 expression is associated both to aggravated immune responses and poor outcome in lymphoma development, implicating a central role for TRIM21 in the development of both systemic autoimmune diseases and lymphomas

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

Loss of the Lupus Autoantigen Ro52/Trim21 Induces Tissue Inflammation and Systemic Autoimmunity by Disregulating the IL-23–Th17 Pathway

Ro52/Trim21 is targeted as an autoantigen in systemic lupus erythematosus and Sjögren's syndrome. Polymorphisms in the Ro52 gene have been linked to these autoimmune conditions, but the molecular mechanism by which Ro52 may promote development of systemic autoimmune diseases has not been explored. To address this issue, we generated Ro52-null mice ($Ro52^{−/−}$), which appear phenotypically normal if left unmanipulated. However, $Ro52^{−/−}$ mice develop severe dermatitis extending from the site of tissue injury induced by ear tags. The affected mice further develop several signs of systemic lupus with hypergammaglobulinemia, autoantibodies to DNA, proteinuria, and kidney pathology. Ro52, which was recently identified as an E3 ligase, mediates ubiquitination of several members of the interferon regulatory factor (IRF) family, and the Ro52-deficient mice have an enhanced production of proinflammatory cytokines that are regulated by the IRF transcription factors, including cytokines involved in the Th17 pathway (interleukin [IL] 6, IL-12/IL-23p40, and IL-17). Loss of IL-23/IL-17 by genetic deletion of IL-23/p19 in the $Ro52^{−/−}$ mice conferred protection from skin disease and systemic autoimmunity. These data reveal that the lupus-associated Ro52 protein is an important negative regulator of proinflammatory cytokine production, and they provide a mechanism by which a defective Ro52 function can lead to tissue inflammation and systemic autoimmunity through the IL-23–Th17 pathway

Loss of the lupus autoantigen Ro52/Trim21 induces tissue inflammation and systemic autoimmunity by disregulating the IL-23-Th17 pathway.

Ro52/Trim21 is targeted as an autoantigen in systemic lupus erythematosus and Sjögren\u27s syndrome. Polymorphisms in the Ro52 gene have been linked to these autoimmune conditions, but the molecular mechanism by which Ro52 may promote development of systemic autoimmune diseases has not been explored. To address this issue, we generated Ro52-null mice (Ro52(-/-)), which appear phenotypically normal if left unmanipulated. However, Ro52(-/-) mice develop severe dermatitis extending from the site of tissue injury induced by ear tags. The affected mice further develop several signs of systemic lupus with hypergammaglobulinemia, autoantibodies to DNA, proteinuria, and kidney pathology. Ro52, which was recently identified as an E3 ligase, mediates ubiquitination of several members of the interferon regulatory factor (IRF) family, and the Ro52-deficient mice have an enhanced production of proinflammatory cytokines that are regulated by the IRF transcription factors, including cytokines involved in the Th17 pathway (interleukin [IL] 6, IL-12/IL-23p40, and IL-17). Loss of IL-23/IL-17 by genetic deletion of IL-23/p19 in the Ro52(-/-) mice conferred protection from skin disease and systemic autoimmunity. These data reveal that the lupus-associated Ro52 protein is an important negative regulator of proinflammatory cytokine production, and they provide a mechanism by which a defective Ro52 function can lead to tissue inflammation and systemic autoimmunity through the IL-23-Th17 pathway

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

IFN-λ1 with Th17 axis cytokines and IFN-α define different subsets in systemic lupus erythematosus (SLE).

BACKGROUND: Interferon (IFN)-α is thought to have a pivotal role in systemic lupus erythematosus (SLE), and type III IFNs (IFN-λ) were recently also associated with SLE. In this study, we measured levels of IFN-α, IFN-λ1, and related cytokines, such as IL-17A, IL-23, and interferon-γ-induced protein 10 (IP-10), in a Karolinska University Hospital cohort of patients with SLE and control subjects. The objective of the study was to investigate if cytokine measurements could identify different subsets of patients with active SLE and higher disease damage. METHODS: We included 261 patients with SLE and 261 population control subjects. All participants underwent a standardized clinical examination. Medical files were reviewed. Patients with SLE were assessed for current organ manifestations, disease activity, and damage. Routine blood parameters, complement levels, and serology were analyzed at the time of inclusion. Levels of IFN-λ1, IFN-α, IL-17A, IL-23, and IP-10 were measured by enzyme-linked immunosorbent assay. RESULTS: IFN-λ1 and IFN-α were detected in 29% and 44% of patients, respectively, but their levels did not correlate. High serum levels of IFN-λ1 were positively associated with antinucleosome antibodies and lymphopenia but negatively with musculoskeletal damage. Positive correlations between levels of IFN-λ1, IL-17A, and IL-23 were observed. Patients with high levels of these three cytokines had more disease damage, especially renal impairment. High levels of IFN-α were associated with mucocutaneous disease; leukopenia; and low complement, Ro/SSA, and La/SSB. Vascular events and antiphospholipid antibodies were uncommon. We identified two subgroups with high disease activity: one with double-high IFN-λ1 and IFN-α and another with IP-10(high). The former had more neuropsychiatric manifestations, and the latter had more arthritis. Increased levels of both types I and III IFNs were found in a proportion of population control subjects. Therefore, high IFN levels do not seem to be SLE-specific biomarkers. CONCLUSIONS: Measurements of circulating IFN-λ1 and IFN-α define subsets of patients with SLE with different characteristics. Levels of IFN-λ1 correlate with T-helper type 17 cytokines and identify a subgroup with more damage. High disease activity is associated with either simultaneous upregulation of IFN-λ1 and IFN-α or independently with IP-10. Our findings could be of major importance when tailoring therapy for patients with SLE with agents targeting IFN pathways

Sex influences eQTL effects of SLE and Sjögren’s syndrome-associated genetic polymorphisms

Abstract Background Systemic lupus erythematosus (SLE) and primary Sjögren’s syndrome (pSS) are autoimmune disorders characterized by autoantibodies, dysregulated B cells, and notably high female-to-male incidence ratios. Genome-wide association studies have identified several susceptibility SNPs for both diseases. Many SNPs in the genome are expression quantitative trait loci (eQTLs), with context-dependent effects. Assuming that sex is a biological context, we investigated whether SLE/pSS SNPs act as eQTLs in B cells and used a disease-targeted approach to understand if they display sex-specific effects. Methods We used genome-wide genotype and gene expression data from primary B cells from 125 males and 162 females. The MatrixEQTL R package was used to identify eQTLs within a genomic window of 2 Mb centered on each of 22 established SLE and/or pSS susceptibility SNPs. To find sex-specific eQTLs, we used a linear model with a SNP * sex interaction term. Results We found ten SNPs affecting the expression of 16 different genes (FDR < 0.05). rs7574865-INPP1, rs7574865-MYO1B, rs4938573-CD3D, rs11755393-SNRPC, and rs4963128-PHRF1 were novel observations for the immune compartment and B cells. By analyzing the SNP * sex interaction terms, we identified six genes with differentially regulated expression in females compared to males, depending on the genotype of SLE/pSS-associated SNPs: SLC39A8 (BANK1 locus), CD74 (TNIP1 locus), PXK, CTSB (BLK/FAM167A locus), ARCN1 (CXCR5 locus), and DHX9 (NCF2 locus). Conclusions We identified several unknown sex-specific eQTL effects of SLE/pSS-associated genetic polymorphisms and provide novel insight into how gene-sex interactions may contribute to the sex bias in systemic autoimmune diseases