Susanna Brauner

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 Sjögrens 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.

The Sjögren’s Syndrome-Associated Autoantigen Ro52 Is an E3 Ligase That Regulates Proliferation and Cell Death

Patients affected by Sjögren’s syndrome and systemic lupus erythematosus (SLE) carry autoantibodies to an intracellular protein denoted Ro52. Although the serologic presence of Ro52 autoantibodies is used clinically for diagnostic purposes, the function of the protein or why it is targeted as an autoantigen in several rheumatic conditions has not been elucidated. In this study, we show that the expression of Ro52 is significantly increased in PBMC of patients with Sjögren’s syndrome and SLE, and demonstrate that Ro52 is a RING-dependent E3 ligase involved in ubiquitination. Overexpression of Ro52, but not of Ro52 lacking the RING domain, in a mouse B cell line lead to decreased growth in steady state and increased cell death after activation via the CD40 pathway. The role of Ro52 in activation-mediated cell death was further confirmed as a reduction in Ro52 expression restored cell viability. These findings suggest that the increased expression of the Ro52 autoantigen in patients may be directly involved in the reduced cellular proliferation and increased apoptotic cell death observed in Sjögren’s syndrome and SLE, and may thus contribute to the autoantigenic load and induction of autoimmune B and T cell responses observed in rheumatic patients.

Expression of the Immune Regulator Tripartite-Motif 21 Is Controlled by IFN Regulatory Factors

Tripartite-motif 21 (TRIM21) is an E3 ubiquitin ligase that regulates innate immune responses by ubiquitinating IFN regulatory factors (IRFs). TRIM21 is mainly found in hematopoietic cells in which its expression is induced by IFNs during viral. infections and in systemic autoimmune diseases such as systemic lupus erythematosus and Sjögren’s syndrome. However, the exact molecular mechanism by which the expression of the Trim21 gene is regulated is unknown. In this study, we demonstrate that IFNs induce Trim21 expression in immune cells via IRFs and that IFN-α and IFN-β are the most potent inducers of Trim21. A functional IFN-stimulated response element but no conserved IFN-γ–activated site was detected in the promoter of Trim21. IRF1 and IRF2 strongly induced Trim21 expression in an IFN-stimulated response element–dependent manner, whereas IRF4 and IRF8 strongly repressed the IRF1-mediated induction of Trim21. Consistent with this observation, baseline expression of Trim21 was elevated in Irf4−/− cells. TRIM21, IRF1, and IRF2 expression was increased in PBMCs from patients with Sjögren’s syndrome compared with healthy controls. In contrast, IRF4 and IRF8 expression was not increased in PBMCs from patients. The IFN-γ–mediated induction of Trim21 was completely abolished by inhibiting protein synthesis with cycloheximide, and Trim21 expression could not be induced by IFN-γ in Irf1−/− cells, demonstrating that IFN-γ induces Trim21 indirectly via IRF1 and not directly via STAT1 activation. Our data demonstrate that multiple IRFs tightly regulate expression of Trim21 in immune cells, suggesting that a well-controlled expression of the E3 ligase TRIM21 is important for regulation of immune responses.

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.

Reduced expression of TRIM21/Ro52 predicts poor prognosis in diffuse large B-cell lymphoma patients with and without rheumatic disease.

TRIM21 (also known as Ro52) is an autoantigen in rheumatic disease and is predominantly expressed in leucocytes. Overexpression is associated with decreased proliferation, and the TRIM21 gene maps to a tumour suppressor locus. We therefore investigated the expression of TRIM21 in patients with diffuse large B‐cell lymphoma (DLBCL) and its potential usefulness as a prognostic biomarker.

Interferon beta treatment of multiple sclerosis increases serum interleukin-7.

Background: Interleukin-7 (IL-7) is a non-redundant cytokine for T-cell development and survival. The IL-7 signaling pathway has been genetically and functionally associated with several autoimmune diseases including multiple sclerosis (MS). Objective: The objective of this paper is to elucidate the effect of the widely used immunomodulatory MS therapy interferon beta (IFNβ) on IL-7 homeostasis. Methods: Swedish MS patients were screened for IL-7 concentration in serum and blood cell counts. IL-7 receptor alpha chain (IL-7Rα) expression was determined by semi-quantitative real-time polymerase chain reaction (PCR) and flow cytometry. Results: IFNβ treatment led to significantly increased serum IL-7 levels (mean: 17 pg/ml) compared with healthy controls (mean: 7.6 pg/ml) and natalizumab-treated patients (mean: 5.3 pg/ml). In vitro and in vivo, peripheral blood leukocytes showed decreased IL-7Rα expression and IL-7 consumption upon IFNβ exposure, suggesting that their IL-7 responsiveness is impaired during treatment. Conclusions: MS patients undergoing IFNβ treatment have increased serum IL-7 levels and decreased IL-7 consumption. Given IL-7’s important role in T-cell immunity, this relationship may be highly relevant for IFNβ’s treatment efficacy.

TRIM genes are part of the interferon signature observed in patients with primary Sjögren's syndrome

Background and objectives Autoimmune disease can arise when the immune system is overactive in response to a trigger. This results in an overproduction of proinflammatory cytokines and type I interferons (IFNs). The latter is associated with the pathology of several systemic autoimmune diseases such as systemic lupus erythematosus (SLE) and primary Sjögrens syndrome (pSS). In this study the authors wanted to identify interferon signature genes that contribute to the pathogenesis of pSS. To do so, the authors did gene expression profiling on patients with pSS. The authors identified several tripartite-motif (TRIM) genes that were overexpressed in patients and that are candidates for IFN signature effector genes. The human TRIM family consists of approximately 70 genes, many of which are important infection-restricting factors or immune regulators. Interestingly, altered expression of TRIMs has previously been reported in several autoimmune diseases. Materials and methods PBMCs from 14 female SSA positive and untreated patients with pSS and 18 age and gender matched healthy controls without any immunological disease were collected. mRNA was prepared using the QIAGEN RNeasy Plus Mini Kit and a gene expression array was performed using the Human Exon 1.0 ST chip from Affymetrix. CEL files were preprocessed using the RMA algorithm as implemented in Affymetrix Power Tools-1.12.0. Results A strong upregulation of IFN induced genes in PBMCs from patients with pSS compared with PBMCs from healthy controls was observed. Data also revealed differential expression of a set of TRIM genes between pSS patients and healthy controls. TRIM5, TRIM6, TRIM14, TRIM19, TRIM22 and TRIM25 were upregulated in the patients while TRIM18 and TRIM68 were downregulated. Of certain interest is TRIM5, a well-characterised retroviral restriction factor that acts on the viral capsid to destabilise it. Single nucleotide polymorphisms (SNPs) in the TRIM5 gene have been correlated to multiple sclerosis, indicating its possible importance in autoimmune disease. TRIM22 has also been identified as a retroviral restriction factor acting on HIV-1. TRIM68, also known as SS56, is an autoantigen in SLE and pSS. TRIM25 acts on the pattern recognition receptor RIG-1 after viral infection leading to an increased production of IFNα. TRIM25 is overexpressed in breast and ovarian cancers. Conclusions Our findings show that several TRIM genes are differentially expressed in patients with pSS compared to controls. TRIM genes are important for the proper regulation of immune responses suggesting that the identified TRIMs might contribute to the pathology of pSS.

Response to Comment on “Gene Disruption Study Reveals a Nonredundant Role for TRIM21/Ro52 in NF-κB-Dependent Cytokine Expression in Fibroblasts”

We thank Ozato and colleagues for initiating the discussion on similarities and differences between the two recently reported Ro52/Trim21 deficient mice ([1][1], [2][2]). When generating knockout mice the resulting phenotype may differ depending on several factors, including the gene targeting

IL-22 binding protein regulates murine skin inflammation

IL22 is a cytokine expressed by haematopoietic cells of both the innate and adaptive arm of the immune system.[1] Shortly after it was discovered in 2000, the existence of IL22 binding protein (IL22BP) was reported, a soluble decoy receptor transcribed from the gene IL22RA2.[2] IL-22BP binds with very high affinity to IL22 in such a way that it no longer can dock with the IL22 receptor (IL22R).[3] It was soon established that IL22 does not directly influence immune cells but rather the epithelial cells of the skin, lungs, and gut as well as cells of the liver, joints, pancreas, and kidney.[4] These tissues express IL22RA1, one of the two subunits of the IL-22R, whereas the other subunit, IL10RB, is expressed throughout the body. The IL22 pathway is implicated in several chronic inflammatory diseases, such as psoriasis, inflammatory bowel disease, and multiple sclerosis (MS). Interestingly, IL22BP has a protective role in a mouse model of inlammation-induced colon cancer but a disease promoting net effect in a model of MS.[5,6] Its role in inflammatory skin diseases has so far not been addressed.

Vaccination of patients with primary Sjögren's syndrome reveals hyperreactive B cell compartment with a skewed maturation pattern

Background Multiple immune disturbances underlying disease development and progression have been implicated in primary Sjögrens syndrome (pSS), especially defects in the B cell compartment. In this study we used vaccination as a tool to analyse defects in immune regulation in vivo in individuals with pSS, and specifically to characterise B cell development and responses. Methods Fourteen SSA positive women with pSS and 18 gender and age matched healthy controls were vaccinated against H1N1 influenza with a priming dose and booster 3 weeks apart. Blood samples were obtained before injections and after 1 and 3 weeks. Total Ig-levels and vaccine specific responses were analysed by ELISPOT, ELISA and hemagglutination tests. Lymphocyte phenotypes were characterised by flow cytometry and clinical routine laboratory tests were performed. Serum cytokine levels and microarray-based RNA expression analysis was performed, as well as genotyping for selected markers. Clinical parameters and potential adverse reactions were monitored with a clinical questionnaire. Results All subjects developed protective immunity to H1N1 influenza upon vaccination with a similar adverse effect profile. However, when dissecting the vaccine specific response patients developed significantly higher titers of IgG vaccine specific antibodies and significantly lower IgM titers. Interestingly, the IgG antibodies were of higher avidity, indicating that this was a specific response. In addition, hypergammaglobulinemia was induced in the patients and anti-SSA/Ro52 titers were transiently elevated. No significant phenotypic differences were observed in the lymphocyte compartment at the start of the study, but already 1 week after the first vaccination pSS patients had significantly fewer CD19 class switched B cells and more immature B cells in circulation. This skewed pattern of B cell maturation remained throughout the study. Paradoxically, plasmablasts appeared at a significantly higher level in patients after immunisation. In accordance, significantly higher levels of several pro-inflammatory cytokines (IL-6, IL-10, IL-12p40 and TNFα), as well as the B cell promoting cytokines IL-7 and BAFF were induced upon vaccination in the patients. Microarray analysis revealed an interferon-signature in the patients. Conclusions Patients with Sjögrens syndrome respond to vaccination with protective immunity. However, the induced IgG vaccine-specific antibodies are of significantly higher titers and of stronger avidity than in healthy controls, occuring in parallel with a skewed B cell maturation and induction of a pro-inflammatory cytokine response. This may reflect the mechanisms allowing high titers of autoantibodies to develop in pSS patients.