Anti-histone and anti-nucleosome rather than anti-dsDNA antibodies associate with IFN-induced biomarkers in Sudanese and Swedish Systemic Lupus Erythematosus patients.

OBJECTIVES: In SLE, anti-dsDNA can co-occur with autoantibodies against other chromatin components, like histones and nucleosomes. These antibodies induce type-1 interferon production, a hallmark of SLE. We measured antinuclear antibody (ANA) sub-specificities and investigated their associations to inflammatory biomarkers including interferon-regulated chemokines. METHODS: We included 93 Sudanese and 480 Swedish SLE patients and matched controls (N = 104 + 192). Autoantibodies targeting ANA-subspecificites: dsDNA, Sm, Sm/U1RNPcomplex, U1RNP, SSA/Ro52, SSA/Ro60, SSB/La, ribosomal P, PCNA and histones were quantified in all subjects, anti-nucleosome only in the Swedish patients, with a bead-based multiplex immunoassay. Levels of 72 plasma biomarkers were determined with Proximity Extension Assay technique or ELISA. RESULTS: Among Sudanese patients, the investigated antibodies significantly associated with 9/72 biomarkers. Anti-histone antibodies showed the strongest positive correlations with MCP-3 and S100A12 as well as with interferon I-inducible factors MCP-1 and CXCL10. Anti-dsDNA antibodies associated with CXCL10 and S100A12, but in multivariate analyses, unlike anti-histone, associations lost significance.Among Swedish patients, MCP-1, CXCL10, SA100A12 also demonstrated stronger associations to anti-histone and anti-nucleosome antibodies, compared with anti-dsDNA and other ANA sub-specificities. In multiple regression models, anti-histone/nucleosome retained the strongest associations. When excluding anti-histone or anti-nucleosome positive patients, the associations between MCP-1/CXCL10 and anti-dsDNA were lost. In contrast, when excluding anti-dsDNA positive patients, associations with anti-histone and anti-nucleosome remained significant. CONCLUSION: In two cohorts of different ethnical origin, autoantibodies targeting chromatin correlate stronger with IFN-induced inflammatory biomarkers than anti-dsDNA or other ANA sub-specificities. Our results suggest that anti-histone/nucleosome autoantibodies may be main drivers of type-1 interferon activity in SLE.

Interleukin (IL) 16: a candidate urinary biomarker for proliferative lupus nephritis.

OBJECTIVE: Lupus nephritis (LN) is a severe manifestation of systemic lupus erythematosus (SLE). The pathogenesis is incompletely understood and diagnostic biomarkers are scarce. We investigated interleukin (IL) 16 as a potential biomarker for LN in a well-characterised cohort of patients with SLE. METHODS: We measured urinary (u-) and plasma (p-) levels of IL-16 in predefined patient groups using ELISA: LN (n=84), active non-renal SLE (n=63), inactive non-renal SLE (n=73) and matched population controls (n=48). The LN group included patients with recent biopsy-confirmed proliferative (PLN, n=47), mesangioproliferative (MES, n=11) and membranous (MLN, n=26) LN. Renal expression of IL-16 was investigated by immunohistochemistry. Associations between IL-16 measurements and clinical parameters and the diagnostic value for LN were explored. RESULTS: p-IL-16 was detected in all investigated cases and high p-IL-16 levels were observed in patients with active SLE. u-IL-16 was detected (dt-u-IL-16) in 47.6% of patients with LN, while only up to 17.8% had dt-u-IL-16 in other groups. In the LN group, 68% of patients with PLN had dt-u-IL-16, while the proportions in the MLN and MES groups were lower (11.5% and 45.5%, respectively). The highest u-IL-16 levels were detected in the PLN group. In the regression model, u-IL-16 levels differentiated PLN from other LN patient subgroups (area under the curve 0.775-0.896, p<0.0001). dt-u-IL-16 had superior specificity but slightly lower sensitivity than elevated anti-double-stranded DNA and low complement C3 or C4 in diagnosing PLN. A high proportion of LN kidney infiltrating cells expressed IL-16. CONCLUSIONS: We demonstrate that detectable u-IL-16 can differentiate patients with PLN from those with less severe LN subtypes and active non-renal SLE. Our findings suggest that u-IL-16 could be used as a screening tool at suspicion of severe LN. Furthermore, the high IL-16 levels in plasma, urine and kidney tissue imply that IL-16 could be explored as a therapeutic target in SLE.

Four Systemic Lupus Erythematosus Subgroups, Defined by Autoantibodies Status, Differ Regarding HLA-DRB1 Genotype Associations and Immunological and Clinical Manifestations.

OBJECTIVE: The heterogeneity of systemic lupus erythematosus (SLE) constitutes clinical and therapeutical challenges. We therefore studied whether unrecognized disease subgroups can be identified by using autoantibody profiling together with HLA-DRB1 alleles and immunological and clinical data. METHODS: An unsupervised cluster analysis was performed based on detection of 13 SLE-associated autoantibodies (double-stranded DNA, nucleosomes, ribosomal P, ribonucleoprotein [RNP] 68, RNPA, Smith [Sm], Sm/RNP, Sjögren's syndrome antigen A [SSA]/Ro52, SSA/Ro60, Sjögren's syndrome antigen B [SSB]/La, cardiolipin [CL]-Immunoglobulin G [IgG], CL-Immunoglobulin M [IgM], and ß2 glycoprotein I [ß2 GPI]-IgG) in 911 patients with SLE from two cohorts. We evaluated whether each SLE subgroup is associated with HLA-DRB1 alleles, clinical manifestations (n = 743), and cytokine levels in circulation (n = 446). RESULTS: Our analysis identified four subgroups among the patients with SLE. Subgroup 1 (29.3%) was dominated by anti-SSA/Ro60/Ro52/SSB autoantibodies and was strongly associated with HLA-DRB1*03 (odds ratio [OR] = 4.73; 95% confidence interval [CI] = 4.52-4.94). Discoid lesions were more common for this disease subgroup (OR = 1.71, 95% CI = 1.18-2.47). Subgroup 2 (28.7%) was dominated by anti-nucleosome/SmRNP/DNA/RNPA autoantibodies and associated with HLA-DRB1*15 (OR = 1.62, 95% CI = 1.41-1.84). Nephritis was most common in this subgroup (OR = 1.61, 95% CI = 1.14-2.26). Subgroup 3 (23.8%) was characterized by anti-ß2 GPI-IgG/anti-CL-IgG/IgM autoantibodies and a higher frequency of HLA-DRB1*04 compared with the other patients with SLE. Vascular events were more common in Subgroup 3 (OR = 1.74, 95% CI = 1.2-2.5). Subgroup 4 (18.2%) was negative for the investigated autoantibodies, and this subgroup was not associated with HLA-DRB1. Additionally, the levels of eight cytokines significantly differed among the disease subgroups. CONCLUSION: Our findings suggest that four fairly distinct subgroups can be identified on the basis of the autoantibody profile in SLE. These four SLE subgroups differ regarding associations with HLA-DRB1 alleles and immunological and clinical features, suggesting dissimilar disease pathways.

Cytokines as Biomarkers in Systemic Lupus Erythematosus: Value for Diagnosis and Drug Therapy.

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease. The disease is characterized by activation and dysregulation of both the innate and the adaptive immune systems. The autoimmune response targets self-molecules including cell nuclei, double stranded DNA and other intra and extracellular structures. Multiple susceptibility genes within the immune system have been identified, as well as disturbances in different immune pathways. SLE may affect different organs and organ systems, and organ involvement is diverse among individuals. A universal understanding of pathophysiological mechanism of the disease, as well as directed therapies, are still missing. Cytokines are immunomodulating molecules produced by cells of the immune system. Interferons (IFNs) are a broad group of cytokines, primarily produced by the innate immune system. The IFN system has been observed to be dysregulated in SLE, and therefore IFNs have been extensively studied with a hope to understand the disease mechanisms and identify novel targeted therapies. In several autoimmune diseases identification and subsequent blockade of specific cytokines has led to successful therapies, for example tumor necrosis factor-alpha (TNF-α) inhibition in rheumatoid arthritis. Authors of this review have sought corresponding developments in SLE. In the current review, we cover the actual knowledge on IFNs and other studied cytokines as biomarkers and treatment targets in SLE.

Proteome study of cutaneous lupus erythematosus (CLE) and dermatomyositis skin lesions reveals IL-16 is differentially upregulated in CLE.

BACKGROUND: The objective of the study was to explore the disease pathways activated in the inflammatory foci of skin lesions in cutaneous lupus erythematosus (CLE) and dermatomyositis (DM). METHODS: Skin biopsies acquired from active CLE and DM lesions, patient (PC), and also healthy controls (HC) were investigated. Biopsy sections were examined by a pathologist, inflammatory foci were laser micro-dissected and captured, and proteins within captured tissue were detected in an unbiased manner by mass spectrometry. Protein pathway analysis was performed by the string-db.org platform. Findings of interest were confirmed by immunohistochemistry (IHC). RESULTS: Proteome investigation identified abundant expression of interferon-regulated proteins (IRP) as a common feature of CLE and DM. Interleukin (IL)-16 was the only abundant cytokine differentially expressed in CLE compared to DM. Caspase-3, an enzyme that cleaves IL-16 into its active form, was detected in low levels. Significantly higher proportion of IL-16- and caspase-3-positive cells was identified in CLE lesions in comparison with DM, PC, and HC. Proteomic results indicate more abundant complement deposition in CLE skin lesions. CONCLUSIONS: Using unbiased mass spectrometry investigation of CLE and DM inflammatory infiltrates, we confirmed that high IRP expression is a common feature of both CLE and DM, while IL-16 is the only differentially expressed cytokine in CLE. IHC confirmed high expression of IL-16 and caspase-3 in CLE. Our novel molecular findings indicate that IL-16 detection could be useful in differential diagnostics between the two conditions that can display similar histopathological appearance. IL-16 could be of interest as a future therapeutic target for CLE.

Interferon activation status underlies higher antibody response to viral antigens in patients with systemic lupus erythematosus receiving no or light treatment.

OBJECTIVES: Infections have been proposed as an environmental risk factor for autoimmune disease. Responses to microbial antigens may be studied in vivo during vaccination. We therefore followed patients with SLE and controls during split-virion influenza vaccination to quantify antibody responses against viral antigens and associated cellular and proteome parameters. METHODS: Blood samples and clinical data were collected from female patients with SLE with no or HCQ and/or low-dose prednisolone treatment (n = 29) and age- and sex-matched healthy controls (n = 17). Vaccine-specific antibody titres were measured by ELISA and IFN-induced gene expression in monocytes by quantitative PCR. Serum proteins were measured by proximity extension assay and disease-associated symptoms were followed by questionnaires. RESULTS: The vaccine-specific antibody response was significantly higher in patients compared with controls and titres of IgG targeting the viral proteins were higher in patients than controls at both 1 and 3 months after immunization. Clinical disease symptoms and autoantibody titres remained unchanged throughout the study. Notably, a positive pre-vaccination mRNA-based IFN score was associated with a significantly higher vaccine-specific antibody response and with a broader profile of autoantibody specificities. Screening of serum protein biomarkers revealed higher levels of IFN-regulated proteins in patients compared with controls and that levels of such proteins correlated with the vaccine-specific IgG response, with C-C motif chemokine ligand 3 exhibiting the strongest association. CONCLUSION: Augmented antibody responses to viral antigens develop in patients with SLE on no or light treatment and associate with markers of type I IFN system activation at the RNA and protein levels.

Sjögren Syndrome in Systemic Lupus Erythematosus: A Subset Characterized by a Systemic Inflammatory State.

OBJECTIVE: An often-neglected subset of patients with systemic lupus erythematosus (SLE) is those with secondary Sjögren syndrome (SLE-sSS). Further, primary SS overlaps and can be difficult to delineate from SLE. To shed light on the SLE-sSS subset, we investigated a large and well-characterized SLE cohort, comparing patients with SLE-sSS and SLE patients without SS (SLE-nonsSS) and controls. METHODS: We included 504 consecutive patients with SLE, fulfilling the 1982 revised American College of Rheumatology criteria, and 319 controls from the general population, matched for age and sex to the first 319 patients. SLE-sSS was defined according to the American-European Consensus Criteria (AECC). A thorough clinical examination, including subjective and objective quantifications of sicca symptoms, was performed in all participants. Autoantibodies and 20 selected cytokines were measured by luminex and multiplex analysis, respectively. RESULTS: SLE-sSS, as defined by AECC, occurred in 23% of the patients with SLE. In comparison to SLE-nonsSS, the SLE-sSS group was older and more frequently female. Leukopenia and peripheral neuropathy were more frequent and nephritis less frequent. Circulating levels of 6/20 investigated proinflammatory cytokines [tumor necrosis factor-α, interleukin (IL) 6, monocyte chemoattractant protein 4, macrophage inflammatory protein 1ß, IL-12/IL-23p40, and interferon γ-induced protein 10], total IgG, anti-SSA/Ro52, anti-SSA/Ro60, anti-SSB/La antibodies, and rheumatoid factor (IgM and IgA) were higher in the SLE-sSS group (p < 0.05 for all comparisons). CONCLUSION: The frequency of SLE-sSS increased with age and affected roughly one-quarter of all patients with SLE. Despite less internal organ involvement, a systemic inflammatory state with high levels of proinflammatory cytokines is present in the SLE-sSS subgroup. This is a novel observation that may affect future understanding and treatment of the SLE-sSS subset.

Circulating Levels of Interferon Regulatory Factor-5 Associates With Subgroups of Systemic Lupus Erythematosus Patients.

Systemic Lupus Erythematosus (SLE) is a heterogeneous autoimmune disease, which currently lacks specific diagnostic biomarkers. The diversity within the patients obstructs clinical trials but may also reflect differences in underlying pathogenesis. Our objective was to obtain protein profiles to identify potential general biomarkers of SLE and to determine molecular subgroups within SLE for patient stratification. Plasma samples from a cross-sectional study of well-characterized SLE patients (n = 379) and matched population controls (n = 316) were analyzed by antibody suspension bead array targeting 281 proteins. To investigate the differences between SLE and controls, Mann-Whitney U-test with Bonferroni correction, generalized linear modeling and receiver operating characteristics (ROC) analysis were performed. K-means clustering was used to identify molecular SLE subgroups. We identified Interferon regulating factor 5 (IRF5), solute carrier family 22 member 2 (SLC22A2) and S100 calcium binding protein A12 (S100A12) as the three proteins with the largest fold change between SLE patients and controls (SLE/Control = 1.4, 1.4, and 1.2 respectively). The lowest p-values comparing SLE patients and controls were obtained for S100A12, Matrix metalloproteinase-1 (MMP1) and SLC22A2 (padjusted = 3 × 10-9, 3 × 10-6, and 5 × 10-6 respectively). In a set of 15 potential biomarkers differentiating SLE patients and controls, two of the proteins were transcription factors, i.e., IRF5 and SAM pointed domain containing ETS transcription factor (SPDEF). IRF5 was up-regulated while SPDEF was found to be down-regulated in SLE patients. Unsupervised clustering of all investigated proteins identified three molecular subgroups among SLE patients, characterized by (1) high levels of rheumatoid factor-IgM, (2) low IRF5, and (3) high IRF5. IRF5 expressing microparticles were analyzed by flow cytometry in a subset of patients to confirm the presence of IRF5 in plasma and detection of extracellular IRF5 was further confirmed by immunoprecipitation-mass spectrometry (IP-MS). Interestingly IRF5, a known genetic risk factor for SLE, was detected extracellularly and suggested by unsupervised clustering analysis to differentiate between SLE subgroups. Our results imply a set of circulating molecules as markers of possible pathogenic importance in SLE. We believe that these findings could be of relevance for understanding the pathogenesis and diversity of SLE, as well as for selection of patients in clinical trials.

High levels of circulating interferons type I, type II and type III associate with distinct clinical features of active systemic lupus erythematosus.

BACKGROUND AND AIM: Interferons (IFNs) are considered to be key molecules in the pathogenesis of systemic lupus erythematosus (SLE). We measured levels of type I, II and III IFNs in a large cohort of patients with systemic lupus erythematosus (SLE) and controls and explored associations among high levels of different IFN types and distinct SLE features. METHODS: Four hundred ninety-seven well-characterized SLE patients and 322 population controls were included. Disease activity was assessed by SLE Disease Activity Index (SLEDAI) and Systemic Lupus Activity Measure (SLAM). Functional type I IFN activity was estimated by a WISH reporter cell assay. Levels of IFN-γ were estimated by MSD 30-plex assay. IFN-α and IFN-λ1 were measured by ELISA. Values above the third quartile of patients' measurements were defined as high. Associations among high IFN results and SLE features were investigated by nominal regression analysis. RESULTS: All IFN measurements were higher in SLE patients than in controls. High type I IFN activity correlated with levels of IFN-γ and IFN-α and associated with active SLE in most domains: weight loss, fatigue, fever, rash, lymphadenopathy, arthritis, nephritis and haematological manifestations. Specific SLE subsets were linked to the upregulation of different subtypes of circulating IFNs: high IFN-γ to arthritis, nephritis and anti-Ro60 antibodies and high IFN-α to mucocutaneous engagement and anti-Ro52 and anti-La antibodies. Isolated high IFN-λ1 was coupled to anti-nucleosome antibodies and less severe SLE. CONCLUSIONS: High functional type I IFN activity captures active SLE in most domains, but more distinct patterns of organ involvement are associated with profiles of circulating IFNs. High IFN-γ as well as high functional type I IFN activity is a characteristic of severe SLE with nephritis and arthritis, while elevated levels of IFN-α associate with active mucocutaneous inflammation and a more benign cardiovascular profile. IFN-λ1 in isolation is associated with milder disease. Our findings suggest that IFNs contribute to the heterogeneity of clinical manifestations in SLE, and measuring circulating IFNs could assist in designing clinical trials with therapies targeting IFN pathways.

TNF-α and plasma albumin as biomarkers of disease activity in systemic lupus erythematosus.

OBJECTIVES: Composite criteria/indices are presently used to diagnose and monitor patients with systemic lupus erythematosus (SLE). Biomarkers for these purposes would be helpful in clinical practice. We therefore evaluated a large panel of cytokines and basic laboratory tests and investigated their performance as discriminators versus controls and as biomarkers of disease activity (DA). METHODS: We examined 437 patients with SLE, fulfilling American College of Rheumatology-82 criteria, and 322 matched controls. DA was assessed according to both SLE DA Index 2000 (SLEDAI-2K) and SLE Activity Measure (SLAM). British Isles Lupus Activity Group (BILAG) was used to assess renal DA. Additionally, 132 patients self-assessed their Global Disease Activity (PtGDA). Mesoscale Discovery 30-plex cytokine assay and routine blood chemistry was performed on fasting EDTA-plasma. RESULTS: Of 26 tested biomarkers, we identified TNF-α as the superior discriminator between patients with SLE and controls (median=4.5 pg/mL, IQR=3.1-6.2 vs median=2.3 pg/mL, IQR=2.0-2.8). The strongest correlations to SLEDAI-2K and SLAM were obtained with TNF-α (Spearman rho (ρ)=0.32 and ρ=0.34, respectively), partly driven by the nephritis subgroup, and with p-albumin (ρ=-0.33 and ρ=-0.31, respectively). P-albumin was decreased and TNF-α was increased in patients with kidney involvement (renal BILAG A/B vs C/D/E, p=4×10-16 and p=6×10-9 respectively). IP-10 was increased in patients with joint involvement (SLAM item 24≥2 vs ≤1, p=0.0005) but did not differ when comparing patients with active/inactive kidney involvement. The most powerful correlations to PtGDA was observed with p-albumin (ρ=-0.42), IL-6 (ρ=0.30) and TNF-α (ρ=0.29). CONCLUSION: TNF-α and p-albumin both performed well as discriminators between patients with SLE and controls and as proxies for DA according to both rheumatologists' and patients' assessments. In particular, renal DA was well reflected by TNF-α. We propose that the TNF-α and p-albumin merit further investigations as clinically useful biomarkers in SLE. We also observed that the pattern of activated cytokines varies with organ involvement.

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-10high. 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.

Interferon (IFN)-λ is a potential mediator in lupus nephritis.

OBJECTIVES: Interferon (IFN)-α is thought to be central in the pathogenesis for lupus nephritis (LN) and recent studies also indicate a role for IFNλ. Little is known about these cytokines in the context of treatment response. We studied levels of IFNα and IFNλ in patients with LN in association with clinical and histological response (HR) to treatment. METHODS: Fifty-six patients with active LN were included. Renal biopsies were performed at baseline and after immunosuppressive therapy. Serum levels of IFNα and IFNλ were analysed at both biopsy occasions and in 163 controls. The biopsies were evaluated according to the International Society of Nephrology/Renal Pathology Society classification. Clinical response was defined according to recent definitions. HR was defined as class I, II or III/IV-C on repeat biopsies. The expression of IFNλ in renal tissue was assessed by immunohistochemistry. RESULTS: At baseline, serum levels of both IFNα and IFNλ were higher in patients versus controls (p=0.01 and p=0.03, respectively). There was no correlation between IFNα and IFNλ. Overall, IFNα decreased after treatment (p=0.003) but IFNλ remained unchanged. However in patients with HR, IFNλ decreased (p=0.01). The highest levels of IFNλ were seen in patients with poor HR. Immunostaining of renal tissue revealed expression of IFNλ, particularly in crescent formations, inflammatory infiltrates and tubular cells. CONCLUSIONS: The study supports a role for IFNλ in LN, both in circulation and at a tissue level. Levels of IFNα and IFNλ did not correlate and were affected differently by immunosuppression, indicating that they are differently involved in subgroups of LN. Persistent increased levels of IFNλ were associated to an unfavourable HR to treatment.

The immunobiology of Ro52 (TRIM21) in autoimmunity: a critical review.

Ro52 is a common target of circulating autoantibodies in autoimmune disease. Data indicate that anti-Ro52 antibodies are associated with distinct clinical manifestations. It is therefore of major interest to understand how it becomes an antigenic target and what cells express this protein under what conditions and what cellular function it has. Ro52 contains a RING and a B-box motif, followed by a coiled-coil domain and a B30.2 (or PRYSPRY) region in the C-terminal end. This molecular structure places Ro52 within the family of tripartite motif proteins (TRIM), and it is also denoted TRIM21. Like several other TRIM proteins, Ro52 has E3 ligase activity and functions in the process of ubiquitination. Ro52 is expressed in the immune system as a predominantly cytoplasmic protein that can be upregulated and translocate to the nucleus in a proinflammatory environment. Reported substrates for Ro52-mediated ubiquitination include IRF3, IRF5, IRF7 and IRF8, and via these transcription factors Ro52 regulates type 1 interferon and cytokine production. Ro52 is upregulated at the site of autoimmune inflammation, such as cutaneous lupus lesions. This implies that Ro52 may have an important role in the pathogenesis of autoimmunity, and this paper will review the available data on the role of Ro52 in immune responses and autoimmune pathogenesis.

Photoprovocation in cutaneous lupus erythematosus: a multicenter study evaluating a standardized protocol.

Photosensitivity is an important and distinguishing sign in various subtypes of cutaneous lupus erythematosus (CLE); however, it remains poorly defined. The purpose of this study was to evaluate whether standardized photoprovocation is a reproducible method to assess photosensitivity in subjects with CLE. A total of 47 subjects with CLE (subacute cutaneous lupus erythematosus (SCLE), n=14; discoid lupus erythematosus (DLE), n=20; lupus erythematosus tumidus (LET), n=13) and 13 healthy volunteers underwent photoprovocation at seven European sites. Of these, 22 (47%) subjects (57% SCLE, 35% DLE, and 54% LET) and none of the healthy volunteers developed photoprovoked lesions according to clinical analysis. Of these 22 subjects, 19 (86%) developed lesions that were histopathologically confirmed as specific for lupus erythematosus (LE). In CLE subjects who developed UV-induced lesions, 86% had Fitzpatrick's phototypes I or II, and the mean minimal erythema dose (MED) was significantly lower compared with subjects without UV-induced lesions (P=0.004). No significant differences in photoprovocation results were observed between study sites. Safety parameters showed no clinically meaningful differences between CLE subjects and healthy volunteers after photoprovocation. In conclusion, a standardized, safe, and reproducible protocol for photoprovocation using UVA and UVB radiation induced skin lesions in approximately half of all CLE subjects and showed comparable results across multiple sites. This method may therefore be used for future diagnostic testing and clinical trials.

High Ro52 expression in spontaneous and UV-induced cutaneous inflammation.

Ro52 is an E3 ubiquitin ligase with a recently identified regulatory role in inflammation. The protein is targeted by autoantibodies in rheumatic diseases, and Ro52 autoantibodies are specifically associated with cutaneous lupus erythematosus (CLE) and photosensitivity. The aim of this study was to investigate cutaneous Ro52 expression in CLE patients and to examine whether UVR might modulate Ro52. Ro52 expression was assessed by immunohistochemistry in biopsies derived from CLE lesions (n=25), nonlesional (n=7), and healthy control skin using four anti-Ro52 mAbs generated by us. Ro52 expression was also analyzed in psoriatic, lichenoid, and eczematous lesions. It was increased in the epidermis of spontaneous CLE lesions as compared with unaffected skin of patients and healthy controls. High epidermal Ro52 expression was also observed in other inflammatory dermatoses investigated. Ro52 was upregulated in experimentally photoprovoked CLE lesions as observed by immunohistochemistry in sequential biopsies, which was confirmed in vitro both at the mRNA and protein levels by exposing cultured patient-derived primary keratinocytes to UVR. In conclusion, Ro52 expression is upregulated in keratinocytes in inflammatory skin conditions and in response to UVR. High Ro52 expression might lead to the breaking of tolerance and the generation of Ro52 autoantibodies in genetically susceptible subjects. Further, the upregulation of Ro52 in keratinocytes after sun exposure might also be a triggering factor for skin lesions in patients with Ro52 antibodies.

The autoantigen Ro52 is an E3 ligase resident in the cytoplasm but enters the nucleus upon cellular exposure to nitric oxide.

Patients with the systemic autoimmune diseases Sjögrens's syndrome and systemic lupus erythematosus often have autoantibodies against the intracellular protein Ro52. Ro52 is an E3 ligase dependent on the ubiquitin conjugation enzymes UBE2D1 and UBE2E1. While Ro52 and UBE2D1 are cytoplasmic proteins, UBE2E1 is localized to the nucleus. Here, we investigate how domains of human Ro52 regulate its intracellular localization. By expressing fluorescently labeled Ro52 and Ro52 mutants in HeLa cells, an intact coiled-coil domain was found to be necessary for the cytoplasmic localization of Ro52. The amino acids 381-470 of the B30.2 region were essential for translocation into the nucleus. Furthermore, after exposure of HeLa cells to the inflammatory mediator nitric oxide (NO), Ro52 translocated to the nucleus. A nuclear localization of Ro52 in inflamed tissue expressing inducible NO synthetase (iNOS) from cutaneous lupus patients was observed by immunohistochemistry and verified in NO-treated cultures of patient-derived primary keratinocytes. Our results show that the localization of Ro52 is regulated by endogenous sequences, and that nuclear translocation is induced by an inflammatory mediator. This suggests that Ro52 has both cytoplasmic and nuclear substrates, and that Ro52 mediates ubiquitination through UBE2D1 in the cytoplasm and through UBE2E1 in the nucleus.