Autoantibodies In Systemic Lupus Erythematosus Research Articles

Nuclear-penetrating scleroderma autoantibody inhibits topoisomerase 1 cleavage complex formation

The contributions of anti-Topoisomerase 1 (Top1) autoantibodies to the pathophysiology of diffuse cutaneous systemic sclerosis (dcSSc), the most aggressive scleroderma subtype, are unknown. Top1 catalyzes DNA relaxation and unwinding in cell nuclei, a site previously considered inaccessible to antibodies. The discovery of autoantibodies in systemic lupus erythematosus that penetrate nuclei and inhibit DNA repair raised the possibility that nuclear-penetrating autoantibodies contribute to mechanisms of autoimmunity. Here we show that an anti-Top1 autoantibody produced by a single B cell clone from a patient with dcSSc penetrates live cells and localizes into nuclei. Functionally, the autoantibody inhibits formation of the Top1 cleavage complex necessary for DNA nicking, which distinguishes it from cytotoxic camptothecin Top1 inhibitors used in cancer therapy that trap the cleavage complex rather than preventing its formation. Discovery of a patient-derived cell-penetrating scleroderma anti-Top1 autoantibody that inhibits Top1 cleavage complex formation supports the hypothesis that anti-Top1 autoantibodies contribute to cellular dysfunction in dcSSc and offers a valuable antibody reagent resource for future studies on anti-Top1 autoantibody contributions to scleroderma pathophysiology.

Immunoglobulin G/immunoglobulin M autoantibody ratios in incomplete systemic lupus erythematosus

Objective Immunoglobulin G (IgG) autoantibodies in systemic lupus erythematosus (SLE) are considered pathogenic, whereas immunoglobulin M (IgM) autoantibodies may have protective effects. The aim of this study was to identify whether IgG/IgM autoantibody ratios differ between patients with incomplete systemic lupus erythematosus (iSLE), patients with SLE, and healthy controls (HCs), and whether IgG/IgM autoantibody ratios relate to progression from iSLE to SLE. Method This prospective cohort study included 34 iSLE patients, 41 SLE patients, and 11 HCs. IgG and IgM anti-dsDNA, anti-Ro52, and anti-Ro60 were measured by fluoro-enzyme immunoassay in serum samples obtained at baseline in all groups and in follow-up samples of up to 5 years for iSLE patients. Correlations between IgG/IgM autoantibody ratios, interferon signature, and clinical parameters were also assessed. Results At baseline, IgG anti-dsDNA, anti-Ro52, anti-Ro60, and IgM anti-dsDNA were elevated in iSLE and SLE patients. IgG/IgM anti-dsDNA and anti-Ro52 ratios were similar between groups, while IgG/IgM anti-Ro60 ratios were significantly elevated in iSLE and SLE patients compared to HCs. IgG/IgM autoantibody ratios were not correlated with interferon signature or clinical parameters. IgG/IgM ratios at baseline were similar and remained relatively stable during a median follow-up of 18 months in non-progressors and six iSLE patients who progressed to SLE. Conclusion IgG anti-dsDNA, anti-Ro52, anti-Ro60, and IgM anti-dsDNA were elevated in iSLE and SLE patients, which was not apparent from the respective IgG/IgM ratios only. IgG/IgM autoantibody ratios remained relatively stable over up to 5 years in iSLE non-progressors and six patients who progressed to SLE.

Prevalence of concomitant rheumatologic diseases and autoantibody specificities among racial and ethnic groups in SLE patients.

Leveraging the Manhattan Lupus Surveillance Program (MLSP), a population-based registry of cases of systemic lupus erythematosus (SLE) and related diseases, we investigated the proportion of SLE with concomitant rheumatic diseases, including Sjögren's disease (SjD), antiphospholipid syndrome (APLS), and fibromyalgia (FM), as well as the prevalence of autoantibodies in SLE by sex and race/ethnicity. Prevalent SLE cases fulfilled one of three sets of classification criteria. Additional rheumatic diseases were defined using modified criteria based on data available in the MLSP: SjD (anti-SSA/Ro positive and evidence of keratoconjunctivitis sicca and/or xerostomia), APLS (antiphospholipid antibody positive and evidence of a blood clot), and FM (diagnosis in the chart). 1,342 patients fulfilled SLE classification criteria. Of these, SjD was identified in 147 (11.0%, 95% CI 9.2-12.7%) patients with women and non-Latino Asian patients being the most highly represented. APLS was diagnosed in 119 (8.9%, 95% CI 7.3-10.5%) patients with the highest frequency in Latino patients. FM was present in 120 (8.9%, 95% CI 7.3-10.5) patients with non-Latino White and Latino patients having the highest frequency. Anti-dsDNA antibodies were most prevalent in non-Latino Asian, Black, and Latino patients while anti-Sm antibodies showed the highest proportion in non-Latino Black and Asian patients. Anti-SSA/Ro and anti-SSB/La antibodies were most prevalent in non-Latino Asian patients and least prevalent in non-Latino White patients. Men were more likely to be anti-Sm positive. Data from the MLSP revealed differences among patients classified as SLE in the prevalence of concomitant rheumatic diseases and autoantibody profiles by sex and race/ethnicity underscoring comorbidities associated with SLE.

CAR-T Cells for Systemic Lupus Erythematosus

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by an abnormal inflammatory response against nuclear antigens with consequent tissue damage. Autoreactive B cells and auto-antibodies have a fundamental role in SLE pathogenesis. Lupus nephritis (LN) has a great impact on patients' survival and quality of life and still represents and unmet clinical need due to the lack of specific treatments and to the poor response to conventional immunosuppression. Regulatory T cells (Tregs) physiologically maintain the immune tolerance and are impaired in SLE. Polyclonal Treg transfer obtained unsatisfactory results due to the low number of disease-relevant antigen-specific cells. Chimeric Antigen Receptors (CARs) are molecules capable of redirecting T cell specificity. CAR-Tregs proved effective in pre-clinical mouse models of autoimmunity. We aimed at developing a CAR-Treg based product to be employed in SLE in particular in LN. We isolated Tregs from Healthy Donors Peripheral Blood Mononuclear Cells (PBMCs) and expanded them with IL-2 and rapamycin. We transduced Tregs with a Lentiviral Vector encoding for a second-generation anti-CD19 CAR, considering the relevant role of autoreactive B cells and autoantibodies in SLE. Engineered cells retained their immune suppressive capabilities upon polyclonal stimulation. Noticeably, they acquired new antigen-specific suppressive capacities, being able to block autologous B cell proliferation. We set up a humanized mouse model of SLE. In vivo, CAR-Tregs delayed the occurrence of B cell lymphopenia, producing immunomodulatory cytokines and without showing toxicity or reprogramming towards Th17 pro-inflammatory cells. In inflamed organs, CAR-Tregs restored the normal composition of the immune system. In conclusion, we efficiently generated anti-CD19 CAR-Tregs and proved their efficacy both in vitro and in an in vivo humanized mouse model of lupus.

Identification of Autoantibodies Associated with Connective Tissue Disorders in Idiopathic Multicentric Castleman Disease

Background: Idiopathic multicentric Castleman disease (iMCD) is a rare, atypical lymphoproliferative disorder with significant morbidity and mortality and unknown etiology. While anti-interleukin-6 (IL-6) therapy with siltuximab is effective in 34-50% of iMCD patients, disease pathophysiology remains largely unknown. A fundamental question is whether iMCD is most appropriately considered an infectious disease caused by an as-yet-unknown pathogen, an autoimmune disease caused by autoantibodies, an autoinflammatory disease caused by germline mutations, or a neoplastic disease caused by somatic mutations in a clonal cell population. While limited evidence exists for each of these hypotheses, the pathogen hypothesis has been the most systematically evaluated to date. In this study, we quantified the levels of autoantibodies that may be involved in iMCD pathology. Methods: We constructed two custom bead-based protein arrays to screen serum samples for IgG autoantibodies from iMCD patients (n = 101) and healthy controls (HC, n = 30). The Connective Tissue Disease (CTD) array consisted of 52 antigens associated with traditional CTDs including in Scleroderma, Myositis/Overlap Syndromes, Systemic Lupus Erythematosus (SLE)/Sjögren's, GI/Endocrine, DNA-Associated, and Inflammation/Stress. The Anti-Cytokine Autoantibody (ACA) array included 38 cytokines, chemokines, and cell surface proteins. Serum samples came from the University of Pennsylvania (n = 38), University of Arkansas for Medical Sciences (n = 45), and Osaka University (Japan, n = 18). A “positive” autoantibody result in any sample was defined as greater than five standard deviations (SD) above the average mean fluorescent intensity (MFI) of the HC and at least 3,000 MFI. Fisher's exact tests were used to determine associations between autoantibody positivity and disease status. Results: To evaluate assay precision, we investigated anti-IL-6 antibodies given that a large portion (38%) of iMCD patients were on anti-IL-6 therapy. As expected, 40 iMCD samples were positive for anti-IL-6 antibodies, the most common autoantibody detected in iMCD subjects. IL-6 was then removed from further analyses. Next, we evaluated the proportion of CTD autoantibody positive iMCD patients. Overall, 46 (46%) iMCD subjects were positive for a CTD autoantibody compared to five (17%) in HCs (OR 4.1, P = 0.005) (Figure 1A). When investigating autoantibody prevalence in CTD subcategories, Myositis/Overlap Syndromes (OR 7.5, P = 0.026) and SLE/Sjögren's (OR 6.2, P = 0.073) autoantibodies trended higher in iMCD patients, but these associations did not survive multiple testing correction. The most common Myositis/Overlap Syndrome autoantibodies were anti-Mi2 (iMCD: 7/101 vs HC: 0/30), and anti-SRP54 (7/101 vs 0/30). The most common SLE/Sjögren's autoantibodies were anti-La/SSB (9/101 vs 0/30) and anti-Ro60 (5/101 vs 0/30). For the custom ACA array, there were significantly more ACA positive iMCD subjects (n = 40, 40%) than HC (n = 3, 10%; OR 5.8, P = 0.002) (Figure 1B). The most common ACAs in iMCD were anti-OSM (9/101 vs 0/30), anti-TNF (6/101 vs 0/30), anti-ITM2B (6/101 vs 0/30), and IFNe (5/101 vs 1/30). Conclusions: IgG autoantibodies associated with CTDs, such as anti-Mi2, anti-SRP54, anti-La, anti-Ro, and anti-Histone 3; and ACAs, such as anti-OSM, anti-TNF, and anti-ITM2B were common in iMCD patients. While autoantibodies suggest autoimmune involvement, the presence of autoantibodies does not directly implicate autoimmunity as a pathological mechanism. For example, Hodgkin's disease and other post-infectious etiologies can present with elevated autoantibodies. Although not surviving multiple hypothesis correction, myositis and SLE autoantibodies trended higher in iMCD patients. While most of these autoantibodies have not been reported, anti-Ro and and anti-La antibodies have been reported previously in iMCD case reports. The presence of anti-OSM is interesting, as OSM is a member of the IL-6 family. Two hypothesized mechanisms include that autoantibodies may be non-neutralizing leading to increased half-life and cytokine activity or autoantibodies are neutralizing and lack of negative feedback from OSM may lead to increased signaling via other IL-6-family cytokines. More research is needed to understand the role of these autoantibodies and, if pathogenic, whether iMCD should be considered an autoimmune disease.

Higher Genetic Risk Loads Confer More Diverse Manifestations and Higher Risk of Lupus Nephritis in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a highly heritable complex disorder with heterogeneous clinical manifestations. In this study, we aimed to identify the genetic risk load using clinical and serological manifestations in SLE patients. We genotyped a total of 1,655 Korean patients with SLE (n=1,243 as a discovery set and n=412 as a replication set) using a customized genome-wide single-nucleotide polymorphism (SNP) array, KoreanChip. A weighted genetic risk score (wGRS) for an individual was calculated from 112 well-validated non-HLA SNPs and HLA haplotypes of SLE-risk loci. We analyzed associations between individual wGRS and clinical SLE subphenotypes and autoantibodies using multivariable linear or logistic regression adjusted by onset age, sex, and disease duration. Childhood-onset SLE (<16 years) conferred the highest genetic risk compared with adult-onset (16-50 years) or late-onset (>50 years) SLE (P = 6.8 × 10-6 ). High wGRS significantly increased associations with SLE manifestations, regardless of onset age, sex, and disease duration. Individual wGRS significantly correlated positively with more clinical American College of Rheumatology criteria (β=0.143, P = 1.8 × 10-6 ). Subphenotype analysis revealed significant associations between the highest and lowest wGRS quartile with risk of renal disorder (hazard ratio [HR] 1.74, P = 2.2 × 10-8 ) and anti-Sm antibody production (HR 1.85, P = 2.8 × 10-5 ). Higher wGRS markedly modulated the pathogenesis of proliferative and membranous lupus nephritis class III or IV (HR 1.98, P = 1.6 × 10-5 ) and class V (HR 2.79, P = 1.0 × 10-3 ), but especially lupus nephritis class V in anti-Sm-positive SLE (area under the curve 0.68, P = 1.8 × 10-4 ). Patients with SLE and high wGRS tended to have earlier age of SLE onset, higher anti-Sm antibody positivity, and more diverse clinical phenotypes. Genetic profiling may predict high risk for lupus nephritis and a diverse clinical course in SLE patients.

EZH2 inhibition dampens autoantibody production in lupus by restoring B cell immune tolerance.

The aim of this study was to elucidate the role of enhancer of zeste homolog 2 (EZH2) in the breakdown of B cell immune tolerance and production of autoantibodies in systemic lupus erythematosus (SLE), and to explore the therapeutic effects of EZH2 inhibition on lupus. Peripheral blood mononuclear cells (PBMCs) were collected from new-onset SLE patients for flow cytometric analysis. Pristane-induced lupus mice were constructed, and the EZH2 inhibitor was administrated by intraperitoneal injection to treat lupus mice. Blood and urine were collected from lupus mice to detect autoantibodies and proteinuria, and renal pathology scores were assessed. Mouse spleen B cells were sorted with magnetic beads and subjected to flow cytometric apoptosis detection, real time quantitative PCR (RT-qPCR), and western blotting (WB). EZH2 expression was elevated in diverse B-cell subsets in both SLE patients and pristane-induced lupus mice. The EZH2 inhibitor attenuated lupus-like symptoms and dampened autoantibody production in pristane-induced lupus mice. Inhibition of EZH2 also reduced autoantibody secretion by plasma cells from lupus patients. Mechanistically, EZH2 mediated the impaired apoptosis of autoreactive B cells and the differentiation of autoantibody producing plasma cells by inhibiting multiple cyclin-dependent kinase inhibitor (CKI) genes. EZH2 mediated the breakdown of B-cell peripheral immune tolerance by inhibiting CKI genes and participated in the generation of autoantibodies in SLE. EZH2 inhibition could serve as a promising drug intervention for the treatment of SLE.

Impacts of the A20 protein polymorphism, rs2230926, in human B cells

Abstract Patients with autoimmune diseases, such as systemic lupus erythematosus (SLE), often have skewed B cell population ratios, accumulation of autoantibodies, and an increased amount of proinflammatory cytokines aiding in disease development. Genome-wide association studies (GWAS) have strongly linked polymorphisms in A20, encoded by TNFAIP3, to autoimmune diseases including SLE. A20 functions to inhibit the activation of nuclear factor kappa B (NF-κB) through ubiquitination and deubiquitylation of key signaling proteins and has been extensively studied in epithelial cells in response to TNF. Given the role of autoantibodies in SLE and the importance of NF-κB in regulating B cell fate, the role of SLE linked polymorphisms in A20 and their effects on both the canonical and noncanonical NF-κB pathways in human B cells is poorly understood. Our lab has identified an A20 polymorphism, rs2230926 (F127C), that is enriched in a cohort of African American females with SLE. Herein, we investigate the impact of rs2230926 on B cell function. We employed CRISPR/Cas9 genome-editing and generated a TNFAIP3knockout lymphoblastoid Raji human B cell line. To determine how the polymorphism rs2230926 affects the NF-kB pathway, we re-introduced WT A20, A20-rs2230926, and a truncated version of A20 into the TNFAIP3-KO cell line. Using a combination of qRT-PCR, western blotting, and flow cytometry, we determined how rs2230926 influences the expression of proinflammatory cytokines, response to various B cell stimuli, and ultimately NF-κB activity. Dissecting A20’s role in NF-kB signaling will help to understand the dysfunction and regulation of B cells in autoimmunity. Supported by grants from NIH to CDS (R01 AI148471)

Argonaute, Vault, and Ribosomal Proteins Targeted by Autoantibodies in Systemic Lupus Erythematosus.

To expand, in an unbiased manner, our knowledge of autoantigens and autoantibodies in patients with systemic lupus erythematosus (SLE) and evaluate their associations with serological and clinical variables. Human proteome arrays (> 21,000 proteins) were screened with serum from patients with SLE (n = 12) and healthy controls (n = 6) for IgG and IgA binding. Top hits were validated with 2 cohorts of patients with SLE (cohort 1, n = 49; cohort 2, n = 46) and other rheumatic diseases by ELISA. Clinical associations of the autoantibodies were tested. Ro60 was the top hit in the screen, and the 10 following proteins included 2 additional known SLE autoantigens plus 8 novel autoantigens involved in microRNA processing (Argonaute protein 1 [AGO1], AGO2, and AGO3), ribosomes (ribosomal protein lateral stalk subunit P2 and ovarian tumor deubiquitinase 5 [OTUD5]), RNA transport by the vault (major vault protein), and the immune proteasome (proteasome activator complex subunit 3). Patient serum contained IgG reactive with these proteins and IgA against the AGO proteins. Using the 95th percentile of healthy donor reactivity, 5-43% were positive for the novel antigens, with OTUD5 and AGO1 showing the highest percentages of positivity. Autoantibodies against AGO1 proteins were more prevalent in patients with oral ulcers in a statistically significant manner. IgG autoantibodies against AGO proteins were also seen in other rheumatic diseases. We discovered new autoantigens existing in cytosolic macromolecular protein assemblies containing RNA (except the proteasome) in cells. A more comprehensive list of autoantigens will allow for a better analysis of how proteins are targeted by the autoimmune response. Future research will also reveal whether specific autoantibodies have utility in the diagnosis or management of SLE.

Systemic Lupus Erythematosus Pathogenesis: Interferon and Beyond.

Autoreactive B cells and interferons are central players in systemic lupus erythematosus (SLE) pathogenesis. The partial success of drugs targeting these pathways, however, supports heterogeneity in upstream mechanisms contributing to disease pathogenesis. In this review, we focus on recent insights from genetic and immune monitoring studies of patients that are refining our understanding of these basic mechanisms. Among them, novel mutations in genes affecting intrinsic B cell activation or clearance of interferogenic nucleic acids have been described. Mitochondria have emerged as relevant inducers and/or amplifiers of SLE pathogenesis through a variety of mechanisms that include disruption of organelle integrity or compartmentalization, defective metabolism, and failure of quality control measures. These result in extra- or intracellular release of interferogenic nucleic acids as well as in innate and/or adaptive immune cell activation. A variety of classic and novel SLE autoantibody specificities have been found to recapitulate genetic alterations associated with monogenic lupus or to trigger interferogenic amplification loops. Finally, atypical B cells and novel extrafollicular T helper cell subsets have been proposed to contribute to the generation of SLE autoantibodies. Overall, these novel insights provide opportunities to deepen the immunophenotypic surveillance of patients and open the door to patient stratification and personalized, rational approaches to therapy. Expected final online publication date for the Annual Review of Immunology, Volume 41 is April 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Oxidized mitochondrial DNA induces gasdermin D oligomerization in systemic lupus erythematosus

Although extracellular DNA is known to form immune complexes (ICs) with autoantibodies in systemic lupus erythematosus (SLE), the mechanisms leading to the release of DNA from cells remain poorly characterized. Here, we show that the pore-forming protein, gasdermin D (GSDMD), is required for nuclear DNA and mitochondrial DNA (mtDNA) release from neutrophils and lytic cell death following ex vivo stimulation with serum from patients with SLE and IFN-γ. Mechanistically, the activation of FcγR downregulated Serpinb1 following ex vivo stimulation with serum from patients with SLE, leading to spontaneous activation of both caspase-1/caspase-11 and cleavage of GSDMD into GSDMD-N. Furthermore, mtDNA oxidization promoted GSDMD-N oligomerization and cell death. In addition, GSDMD, but not peptidyl arginine deiminase 4 is necessary for extracellular mtDNA release from low-density granulocytes from SLE patients or healthy human neutrophils following incubation with ICs. Using the pristane-induced lupus model, we show that disease severity is significantly reduced in mice with neutrophil-specific Gsdmd deficiency or following treatment with the GSDMD inhibitor, disulfiram. Altogether, our study highlights an important role for oxidized mtDNA in inducing GSDMD oligomerization and pore formation. These findings also suggest that GSDMD might represent a possible therapeutic target in SLE.

Interrelation of T cell cytokines and autoantibodies in systemic lupus erythematosus: A cross-sectional study

T-helper cytokines interferon gamma (IFNɣ), interleukin 17 (IL-17) and IL-10 impact systemic lupus erythematosus (SLE) directly and indirectly via modulation of autoAb production. We determined the separate and combined effects on clinical manifestations of SLE (N = 62). IFNɣ, IL-17 but not IL-10 were significantly elevated in patients with SLE. IFNɣ positively correlated with anti-DNA and anti-SSA. IL-17 positively correlated with anti-SSA and was significantly higher in patients with discoid rash and class V LN. IL-10 did not correlate with circulating autoantibodies but was significantly elevated in patients with LN. Patients with LN had elevated plasma levels of anti-DNA and anti-Sm/ribonuclear protein (RNP). Anti-Sm/RNP levels were decreased in patients with acute mucocutaneous manifestations, including photosensitivity and/or malar rash. The study provides critical insights into pathological mechanisms of LN, which could help guide future diagnoses and therapies.

Systemic Lupus Erythematosus Patients With Related Organic Damage Are at High Risk of Hypothyroidism.

PurposeThe aim of this study included determining the prevalence of hypothyroidism in patients with systemic lupus erythematosus (SLE), clarifying the clinical characteristics of SLE patients with hypothyroidism, and identifying the relationship between hypothyroidism and SLE-related organic damage. Another purpose was to analyze the relationship between SLE and thyroid autoantibody. We also intended to discuss the pathogenesis of hypothyroidism in SLE patients, which would provide clues for further investigation.MethodsThis study recruited 856 SLE patients and 856 age- and sex-matched healthy population and compared the prevalence of hypothyroidism between the cases and controls. Univariate and multivariate logistic analyses were applied to identify risk factors for hypothyroidism in SLE patients.ResultsSLE patients had higher prevalence of clinical hypothyroidism (9.10%) and TgAb+TPOAb- (10.40%) than controls. The prevalence of hypothyroidism was the highest in SLE patients aged 16-26 years (18.9%) and decreased with age. The prevalence of autoimmune hypothyroidism in SLE group was higher than that in the control group (64.4% vs. 51.5%, P=0.042), which was mainly due to TgAb; the prevalence of non-autoimmune hypothyroidism in SLE group was also significantly higher than that in the control group (67.3% vs. 47.8%, P<0.001). Based on multivariate analysis, the use of glucocorticoids/immunosuppressants, liver abnormality, lupus nephritis (LN), and cardiac insufficiency were independently associated with hypothyroidism in SLE patients.ConclusionThe prevalence of hypothyroidism in SLE patients was higher than that in controls and decreased with age. The results suggested that young SLE patients combined with LN, liver abnormality and cardiac insufficiency were at higher risk of hypothyroidism. According to the results of this study, we speculated that SLE might have impact on thyroid, and SLE might be one of the causes of hypothyroidism.

Glycation end-products specific auto-antibodies in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease, which is highly inflammatory. Compared to a healthy control group, SLE patients exhibit a higher concentration of advanced glycation end products (AGEs) and a lower concentration of receptors for AGEs (RAGE) in serum, however, the exact aetiology is still unclear. In the present study, non-enzymatic glycation induced modification of human serum albumin (HSA) has been studied by biophysical techniques. Glycated HSA (G-HSA) was used as an antigen, and serum autoantibody levels were estimated in SLE and normal humans (NH) against it, using direct binding ELISA and competitive inhibition ELISA. Compared to N-HSA, remarkable structural modifications were observed in G-HSA. Modified HSA also showed increased pentosidine fluorescence (213.7 ± 13.4 AU). Glycation of HSA induced a conversion of α-helix and random coil to β-sheet and β-turns. Serum immuno assays results exhibited significantly (p < 0.001) higher binding of G-HSA with serum autoantibodies from SLE patients when compared with native HSA (N-HSA). Furthermore, competitive ELISA results showed significantly (p < 0.001) high percent inhibition of serum IgG from SLE patients with modified antigen. Chronic inflammation with excessive oxidative stress in SLE patients could be a possible reason for structural alterations in blood proteins, generating highly immunogenic unique new-epitopes. These in turn induce the generation of specific autoantibodies against G-HSA that may serve as a potential biomarker for SLE pathogenesis.

A High Prevalence of Anti-EBNA1 Heteroantibodies in Systemic Lupus Erythematosus (SLE) Supports Anti-EBNA1 as an Origin for SLE Autoantibodies.

BackgroundThat Epstein–Barr virus (EBV) infection is associated with systemic lupus erythematosus (SLE) is established. The challenge is to explain mechanistic roles EBV has in SLE pathogenesis. Previous studies identify four examples of autoantibody cross-reactions between SLE autoantigens and Epstein–Barr nuclear antigen 1 (EBNA1). For two of these examples, the earliest detected autoantibody specifically cross-reacts with EBNA1; thereby, defined EBNA1 epitopes induce a robust autoantibody response in animals. These results suggest that the autoantibodies initiating the process leading to SLE may emerge from the anti-EBNA1 heteroimmune response. If this hypothesis is true, then anti-EBNA1 responses would be more frequent in EBV-infected SLE patients than in EBV-infected controls. We tested this prediction.MethodsWe evaluated published East Asian data by selecting those with a positive anti-viral capsid antigen (VCA) antibody immunoglobulin G (IgG) test and determining whether anti-EBNA1 was more common among the EBV-infected SLE cases than among matched EBV-infected controls with conditional logistic regression analysis.ResultsAll the qualifying SLE patients (100%) in this dataset were EBV-infected compared to age- and sex-matched controls (92.2%) [odds ratio (OR) = 28.6, 95% CI 6.4–∞, p = 8.83 × 10-8], confirming the known close association of EBV infection with SLE. Furthermore, virtually all the SLE cases have both anti-VCA IgG and anti-EBNA1 IgG antibodies [124 of 125 (99.2%)], which are more frequently present than in age- and sex-matched EBV-infected controls [232 of 250 (93.2%)] (OR = 9.7, 95% CI 1.5–414, p = 0.0078) for an 89.7% SLE attributable risk from anti-EBNA1, which is in addition to the 100% SLE risk attributable to EBV infection in these data.ConclusionsThe association of EBV infection with SLE is reconfirmed. The prediction that anti-EBNA1 is more frequent in these SLE cases than in EBV-infected controls is true, consistent with the hypothesis that anti-EBNA1 contributes to SLE. This second EBV-dependent risk factor is consistent with a molecular mimicry model for the generation of SLE, starting with EBV infection, progressing to anti-EBNA1 response; then molecular mimicry leads to anti-EBNA1 antibodies cross-reacting with an SLE autoantigen, causing autoantibody epitope spreading, and culminating in clinical SLE. These results support the anti-EBNA1 heteroimmune response being a foundation from which pathogenic SLE autoimmunity emerges.

Up-regulation of proBDNF/p75NTR signaling in antibody-secreting cells drives systemic lupus erythematosus.

Inappropriate expansion of antibody-secreting cells (ASCs) is typical of systemic lupus erythematosus (SLE), but the regulatory signaling of pathogenic ASCs is unclear. The present study shows that brain-derived neurotrophic factor precursor (proBDNF) and its high-affinity pan-75 neurotrophin receptor (p75NTR) are highly expressed in CD19+CD27hiCD38hi ASCs in patients with SLE and in CD19+CD44hiCD138+ ASCs in lupus-like mice. The increased proBDNF+ ASCs were positively correlated with clinical symptoms and higher titers of autoantibodies in SLE. Administration of monoclonal antibodies against proBDNF or specific knockout of p75NTR in CD19+ B cells exerted a therapeutic effect on lupus mice by limiting the proportion of ASCs, reducing the production of autoantibodies and attenuating kidney injury. Blocking the biological function of proBDNF or p75NTR also inhibits ASC differentiation and antibody production in vitro. Together, these findings suggest that proBDNF-p75NTR signaling plays a critical pathogenic role in SLE through promoting ASC dysfunction.

T Cell Homeostatic Proliferation Promotes a Redox State That Drives Metabolic and Epigenetic Upregulation of Inflammatory Pathways in Lupus.

Significance: Numerous abnormalities in T cells have been described in patients with systemic lupus erythematosus (SLE), including lymphopenia, DNA demethylation, expression of endogenous retroviruses (ERVs), increased cell death, enlarged mitochondria, production of reactive oxygen species (ROS), and the appearance of unusual CD4-CD8- T cells. Our studies propose a model in which accelerated homeostatic proliferation of T cells promotes an epigenetic and metabolic program, leading to this cluster of abnormalities. Recent Advances: Growing knowledge of the innate immune disorders in SLE has included increased mitochondrial size and ROS production that induces oligomerization of the mitochondrial antiviral signaling (MAVS) protein and type I interferon production, as well as DNA demethylation, upregulation of inflammatory genes, and expression of certain ERVs in SLE peripheral blood mononuclear cells. All these events are part of the cellular program that occurs during homeostatic proliferation of T cells. Evidence from a murine model of SLE as well as in human SLE reveals that increased T cell homeostatic proliferation may be a driving factor in these processes. Critical Issues: Despite extensive knowledge of the myriad autoantibodies in SLE and other immune abnormalities, a cogent model has been lacking to link the numerous and seemingly disparate immune aberrations. This may partly explain the general lack of new drugs specifically for SLE in over 50 years. A more coherent model of SLE would not only unify the variety of immune abnormalities is SLE but would also suggest new therapies. Future Directions: The model of augmented homeostatic proliferation leading to increased mitochondrial mass, ROS, DNA demethylation, and upregulation of inflammatory genes suggests strategic new targets for SLE, including antioxidants and certain inhibitors of metabolism. Antioxid. Redox Signal. 36, 410-422.

Shared Pathogenetic Features Between Common Variable Immunodeficiency and Sjögren's Syndrome: Clues for a Personalized Medicine.

Common variable immunodeficiency disorders (CVID) are a group of rare diseases of the immune system and the most common symptomatic primary antibody deficiency in adults. The “variable” aspect of CVID refers to the approximately half of the patients who develop non-infective complications, mainly autoimmune features, in particular organ specific autoimmune diseases including thyroiditis, and cytopenias. Among these associated conditions, the incidence of lymphoma, including mucosal associated lymphoid tissue (MALT) type, is increased. Although these associated autoimmune disorders in CVID are generally attributed to Systemic Lupus Erythematosus (SLE), we propose that Sjogren’s syndrome (SS) is perhaps a better candidate for the associated disease. SS is an autoimmune disorder characterized by the lymphocytic infiltrates of lacrimal and salivary glands, leading to dryness of the eyes and mouth. Thus, it is a lymphocyte aggressive disorder, in contrast to SLE where pathology is generally attributed to auto-antibody and complement activation. Although systemic lupus erythematosus (SLE) shares these features with SS, a much higher frequency of MALT lymphoma distinguishes SS from SLE. Also, the higher frequency of germ line encoded paraproteins such as the monoclonal rheumatoid factor found in SS patients would be more consistent with the failure of B-cell VDJ switching found in CVID; and in contrast to the hypermutation that characterizes SLE autoantibodies. Thus, we suggest that SS may fit as a better “autoimmune” association with CVID. Examining the common underlying biologic mechanisms that promote lymphoid infiltration by dysregulated lymphocytes and lymphoma in CVID may provide new avenues for treatment in both the diseases. Since the diagnosis of SLE or rheumatoid arthritis is usually based on specific autoantibodies, the associated autoimmune features of CVID patients may not be recognized in the absence of autoantibodies.

Ultrastructural Alterations of Platelets and Monocytes Associated with Autoantibodies in Systemic Lupus Erythematosus

Introduction. Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with a high titer of autoantibodies, and SLE patients are predisposed to thrombosis. Monocytes and platelets are potentially involved in the pathogenesis of SLE. Monocytes are a source of immune modulators for T and B lymphocytes and they increase the risk of thrombosis through exposing tissue factor on their cell surface. Platelets are activated in SLE and may promote thrombotic events by expressing the procoagulant phosphatidylserine. The aim of this work was to investigate the ultrastructural changes of monocytes and platelets in SLE patients in association with titers of autoantibodies.Methods. SLE patients were included in the study based on the diagnostic criteria of the American College of Rheumatology. Platelets from 5 SLE patients and 3 healthy donors were isolated by gel-filtration on Sepharose 2B from citrated platelet-rich plasma. Monocytes of 6 SLE patients and 3 healthy donors were isolated using density gradient centrifugation of whole citrated blood followed by negative immune-separation. The >96% purity of the isolated monocytes was confirmed using flow cytometry and the viability of the isolated monocytes assessed by exclusion of trypan blue was 95-99%. The ultrastructure of platelets and monocytes from SLE patients and healthy donors was studied with transmission electron microscopy. A total 192 individual platelets and 154 monocytes from SLE patients were analyzed and compared with 60 individual platelets and 22 monocytes from healthy donors.Results . Most platelets from SLE patients and healthy donors had a normal discoid shape without or with 1-2 short filopodia. Intracellular components (α-granules, dense granules, open canalicular system, lysosomes and mitochondria) were well visualized, albeit somewhat less clear in the SLE platelets. The biggest morphological difference between platelets from healthy subjects and SLE patients was revealed in the platelet plasma membrane. All platelets from healthy donors had normal ultrastructure of the plasma membrane, while the membrane of SLE platelets was very often (in 70% of the cells) shaggy and rough and not as smooth as in normal platelets. About 38% of the SLE platelets were fully bordered with a shaggy plasma membrane, while 32% of the platelets had only partially abnormal plasma membrane. Cells from a patient with anti-cardiolipin and anti-beta-2-glypoprotein antibodies had the most striking membrane abnormalities that were found in 100% of platelets analyzed.Monocytes with normal ultrastructure (ruffled cell surface, formation of filopodia, an indented nucleus, and uniform chromatin distribution) comprised 64% of the total in the SLE patients and 95% in the healthy donors. Unlike monocytes from healthy subjects, 19% of monocytes from the SLE patients had ultrastructural signs of apoptosis with a smooth cell surface, electron-dense cytoplasm, mitochondrial swelling, and a spherical nucleus with perinuclear accumulation of condensed chromatin or condensation of chromatin into dense spherical particles. About 17% of monocytes from the SLE patients and 5% from the healthy donors had necrotic features, such as smooth cell surface without or with quite a few filopodia, low electron-density cytoplasm, small electron-dense mitochondria, and spherical pyknotic nucleus with more diffuse euchromatin. The SLE patients with a high titer of anti-dsDNA antibodies (>200 IU/ml) had significantly more apoptotic (25%, p<0.01, χ2-test) and necrotic (20%, p<0.05) monocytes than those of the SLE patients with a normal titer of anti-dsDNA antibodies (<25 IU/ml) (6% and 10%, respectively).Conclusions . In SLE patients, most platelets have abnormal ultrastructure of the plasma membrane (loss of smoothness, shagginess), likely due to deposition of pathological immune complexes. A substantial fraction of monocytes in SLE undergo morphological alternations of apoptotic or necrotic types. Such alterations were more pronounced in patients with an increased level of anti-cardiolipin, anti-beta-2-glypoprotein, and anti-dsDNA autoantibodies. The results confirm the involvement of both platelets and monocytes in the pathogenesis of SLE, including functional and structural alterations associated with increased production of various autoantibodies.The work was supported by the Program for Competitive Growth at Kazan Federal University. DisclosuresNo relevant conflicts of interest to declare.

Autoantibodies targeting LINE-1-encoded ORF1p are associated with systemic lupus erythematosus diagnosis but not disease activity.

Long Interspersed Element 1 (LINE-1) is an endogenous retroelement that constitutes a significant portion of the human genome and has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). The LINE-1 RNA chaperone protein ORF1p was recently identified as an SLE autoantigen. Here we analyse ORF1p for qualities underlying SLE autoantigen status, compared anti-ORF1p antibodies to markers of SLE disease activity, and performed screening for antibodies against LINE-1 reverse transcriptase ORF2p. ORF1p was examined in epithelial cell lines treated with cytotoxic lymphocyte granules and UV irradiation. Anti-ORF1p and anti-ORF2p antibodies were assayed by ELISA and analysed in two SLE cohorts. We found that ORF1p localises to cytoplasmic RNA-containing blebs in apoptotic cells, and is a substrate of the cytotoxic protease granzyme B (GrB). Anti-ORF1p antibodies were present in 4.2% of healthy controls, compared to 15.8% (p=0.0157) and 15.5% (p=0.036) of subjects in the two SLE cohorts. Anti-ORF1p antibodies were not associated with SLE disease activity nor peripheral blood markers of interferon (IFN) activation. Anti-ORF1p titres demonstrated stability over serial time points. Anti-ORF1p antibodies were not associated with anti-DNA, anti-RNP, or other SLE autoantibodies. There was no difference in anti-ORF2p ELISA results in controls versus SLE patients. LINE-1 ORF1p is a component of apoptotic blebs and a substrate for GrB. Anti-ORF1p antibodies are enriched in SLE subjects but are not associated with dynamic markers of disease activity. These data support a potential role for LINE-1 dysregulation in SLE pathogenesis.