Marie Wahren-Herlenius

Immunopathogenic mechanisms of systemic autoimmune disease

Systemic lupus erythematosus, Sjögrens syndrome, and dermatomyositis are systemic autoimmune diseases that develop after environmental triggering of genetically susceptible individuals. The precise cellular and molecular mechanisms leading to autoimmune disease, and what factors determine which organs are involved, remain poorly understood. Recent insights into genetic susceptibility now make obvious that environmental triggers often act via cellular pathways containing disease-associated polymorphisms. In the breaking of tolerance, the initiating tissue--including dendritic cells--provides a decisive microenvironment that affects immune-cell differentiation, leading to activation of adaptive immunity. Type 1 interferon produced by innate immune cells has a central role in systemic autoimmunity and activates B cells and T cells. In turn, B-cell-derived autoantibodies stimulate dendritic cells to produce type 1 interferon; thus, a positive feedforward loop is formed that includes both the innate and adaptive systems. New treatments could simultaneously and specifically target several such vital pathways in autoimmunity.

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

Sjögrens syndrome is a common autoimmune disease (affecting ∼0.7% of European Americans) that typically presents as keratoconjunctivitis sicca and xerostomia. Here we report results of a large-scale association study of Sjögrens syndrome. In addition to strong association within the human leukocyte antigen (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). We also observed suggestive associations (Pmeta < 5 × 10−5) with variants in 29 other regions, including TNFAIP3, PTTG1, PRDM1, DGKQ, FCGR2A, IRAK1BP1, ITSN2 and PHIP, among others. These results highlight the importance of genes that are involved in both innate and adaptive immunity in Sjögrens syndrome.

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.

Expression of the B Cell‐Attracting Chemokine CXCL13 in the Target Organ and Autoantibody Production in Ectopic Lymphoid Tissue in the Chronic Inflammatory Disease Sjögren's Syndrome

Sjögrens syndrome is an autoimmune disease characterized by lymphocytic infiltrates resembling secondary lymphoid organs in salivary glands. In this study, we demonstrate the expression of the lymphoid tissue homing chemokine CXCL13 (BCA‐1/BLC), which has attracting properties for B cells and subsets of activated T cells, in salivary glands of patients with Sjögrens syndrome using immunohistochemistry and in situ hybridization. CXCL13 expression was primarily observed in epithelial cells in acini and ducts of inflamed glands while its receptor, CXCR5 (BLR‐1), was expressed on the infiltrating mononuclear cells. In addition, cells producing antibodies against one of the major autoantigens in Sjögrens syndrome, Ro 52, were identified at the periphery of the follicular infiltrates indicating that the ectopic lymphoid tissue is directly involved in the disease process. Identification of CXCL13 and CXCR5 in salivary glands suggests that the target organ plays an essential role in the inflammatory process by recruiting B and T cells. These results also provide a molecular mechanism by which lymphoid neogenesis and ectopic germinal centre formation might occur in the glands of these patients, which may be the key step in the development of the chronic inflammatory process in Sjögrens syndrome.

Detection of anti-Ro/SSA and anti-La/SSB autoantibody-producing cells in salivary glands from patients with Sjögren's syndrome

OBJECTIVE To investigate and identify the presence of cells producing anti-Ro/SSA and anti-La/SSB autoantibodies in salivary glands from patients with Sjögrens syndrome (SS). METHODS Submucosal salivary gland biopsy samples from 10 SS patients (8 with and 2 without circulating Ro and La autoantibodies) and 14 control subjects were evaluated. Frozen tissue sections were immunostained by an avidin-biotin complex technique, using biotinylated recombinant Ro and La proteins as detection reagents. Autoantibody levels in SS patient sera were analyzed by enzyme-linked immunosorbent assay. RESULTS Cells producing autoantibodies to the Ro 52-kd, Ro 60-kd, and La proteins were recorded in 8, 6, and 7 of the 10 SS patient biopsy samples, respectively. Samples from the 2 SS patients without circulating Ro and La autoantibodies were negative for these autoantibody-producing cells, as were all control biopsy samples. A strong positive correlation between the presence of autoantibodies in sera and the presence of autoantibody-producing cells in glandular biopsy tissues was evident. The number of autoantibody-producing cells and the serum autoantibody levels were also correlated (r(s)=0.94, P < 0.0001). CONCLUSION Using a novel technique, we have demonstrated the presence of Ro and La autoantibody-producing cells in salivary gland biopsy tissues from patients with SS. These findings indicate that anti-Ro/ SSA and anti-La/SSB autoantibodies are produced and are present at sites of inflammation and indicate their potential involvement in the autoimmune exocrinopathy of this disease.

Differential effects on BAFF and APRIL levels in rituximab-treated patients with systemic lupus erythematosus and rheumatoid arthritis

The objective of this study was to investigate the interaction between levels of BAFF (B-cell activation factor of the tumour necrosis factor [TNF] family) and APRIL (a proliferation-inducing ligand) and B-cell frequencies in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) treated with the B-cell-depleting agent rituximab. Ten patients with SLE were treated with rituximab in combination with cyclophosphamide and corticosteroids. They were followed longitudinally up to 6 months after B-cell repopulation. Nine patients with RA, resistant or intolerant to anti-TNF therapy, treated with rituximab plus methotrexate were investigated up to 6 months after treatment. The B-cell frequency was determined by flow cytometry, and serum levels of BAFF and APRIL were measured by enzyme-linked immunosorbent assays. BAFF levels rose significantly during B-cell depletion in both patient groups, and in patients with SLE the BAFF levels declined close to pre-treatment levels upon B-cell repopulation. Patients with SLE had normal levels of APRIL at baseline, and during depletion there was a significant decrease. In contrast, patients with RA had APRIL levels 10-fold higher than normal, which did not change during depletion. At baseline, correlations between levels of B cells and APRIL, and DAS28 (disease activity score using 28 joint counts) and BAFF were observed in patients with RA. In summary, increased BAFF levels were observed during absence of circulating B cells in our SLE and RA patient cohorts. In spite of the limited number of patients, our data suggest that BAFF and APRIL are differentially regulated in different autoimmune diseases and, in addition, differently affected by rituximab treatment.

Ro/SSA autoantibodies directly bind cardiomyocytes, disturb calcium homeostasis, and mediate congenital heart block

Congenital heart block develops in fetuses after placental transfer of Ro/SSA autoantibodies from rheumatic mothers. The condition is often fatal and the majority of live-born children require a pacemaker at an early age. The specific antibody that induces the heart block and the mechanism by which it mediates the pathogenic effect have not been elucidated. In this study, we define the cellular mechanism leading to the disease and show that maternal autoantibodies directed to a specific epitope within the leucine zipper amino acid sequence 200–239 (p200) of the Ro52 protein correlate with prolongation of fetal atrioventricular (AV) time and heart block. This finding was further confirmed experimentally in that pups born to rats immunized with p200 peptide developed AV block. p200-specific autoantibodies cloned from patients bound cultured cardiomyocytes and severely affected Ca2+ oscillations, leading to accumulating levels and overload of intracellular Ca2+ levels with subsequent loss of contractility and ultimately apoptosis. These findings suggest that passive transfer of maternal p200 autoantibodies causes congenital heart block by dysregulating Ca2+ homeostasis and inducing death in affected cells.

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.

Mutations in the gene encoding fibroblast growth factor 10 are associated with aplasia of lacrimal and salivary glands

Autosomal dominant aplasia of lacrimal and salivary glands (ALSG; OMIM 180920 and OMIM 103420) is a rare condition characterized by irritable eyes and dryness of the mouth. We mapped ALSG to 5p13.2–5q13.1, which coincides with the gene fibroblast growth factor 10 (FGF10). In two extended pedigrees, we identified heterozygous mutations in FGF10 in all individuals with ALSG. Fgf10+/− mice have a phenotype similar to ALSG, providing a model for this disorder. We suggest that haploinsufficiency for FGF10 during a crucial stage of development results in ALSG.

The complexity of Sjögren's syndrome: Novel aspects on pathogenesis

In Sjögrens syndrome, like in most other autoimmune diseases, the enigma leading to a pathogenic attack against self has not yet been solved. By definition, the disease must be mediated by specific immune reactions against endogenous tissues to qualify as an autoimmune disease. In Sjögrens syndrome the autoimmune response is directed against the exocrine glands, which, as histopathological hallmark of the disease, display persistent and progressive focal mononuclear cell infiltrates. Clinically, the disease in most patients is manifested by two severe symptoms: dryness of the mouth (xerostomia) and the eyes (keratoconjunctivitis sicca). A number of systemic features have also been described and the presence of autoantibodies against the ubiquitously expressed ribonucleoprotein particles Ro (Sjögrens-syndrome-related antigen A - SSA) and La (SSB) further underline the systemic nature of Sjögrens syndrome. The original explanatory concept for the pathogenesis of Sjögrens syndrome proposed a specific, self-perpetuating, immune mediated loss of acinar and ductal cells as the principal cause of salivary gland hypofunction. Although straightforward and plausible, the hypothesis, however, falls short of accommodating several Sjögrens syndrome-related phenomena and experimental findings. Consequently, researchers considered immune-mediated salivary gland dysfunction prior to glandular destruction and atrophy as potential molecular mechanisms underlying the symptoms of dryness in Sjögrens syndrome. Accordingly, apoptosis, fibrosis and atrophy of the salivary glands would represent consequences of salivary gland hypofunction. The emergence of advanced bio-analytical platforms further enabled the identification of potential biomarkers with the intent to improve Sjögrens syndrome diagnosis, promote the development of prognostic tools for Sjögrens syndrome and the long-term goal to identify possible processes for therapeutic treatment interventions. In addition, such approaches allowed us to glimpse at the apparent complexity of Sjögrens syndrome.