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

Objective: 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. Methods: 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). Results: 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. Conclusion: 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.

Blood immunophenotyping identifies distinct kidney histopathology and outcomes in patients with lupus nephritis.

Lupus nephritis (LN) is a frequent manifestation of systemic lupus erythematosus, and fewer than half of patients achieve complete renal response with standard immunosuppressants. Identifying non-invasive, blood-based pathologic immune alterations associated with renal injury could aid therapeutic decisions. Here, we used mass cytometry immunophenotyping of peripheral blood mononuclear cells in 145 patients with biopsy-proven LN and 40 healthy controls to evaluate the heterogeneity of immune activation in patients with LN and to identify correlates of renal parameters and treatment response. Unbiased analysis identified 3 immunologically distinct groups of patients with LN that were associated with different patterns of histopathology, renal cell infiltrates, urine proteomic profiles, and treatment response at one year. Patients with enriched circulating granzyme B+ T cells at baseline showed more severe disease and increased numbers of activated CD8 T cells in the kidney, yet they had the highest likelihood of treatment response. A second group characterized primarily by a high type I interferon signature had a lower likelihood of response to therapy, while a third group appeared immunologically inactive by immunophenotyping at enrollment but with chronic renal injuries. Main immune profiles could be distilled down to 5 simple cytometric parameters that recapitulate several of the associations, highlighting the potential for blood immune profiling to translate to clinically useful non-invasive metrics to assess immune-mediated disease in LN.

Modeling of clinical phenotypes in systemic lupus erythematosus based on the platelet transcriptome and FCGR2a genotype.

BACKGROUND: The clinical heterogeneity of SLE with its complex pathogenesis remains challenging as we strive to provide optimal management. The contribution of platelets to endovascular homeostasis, inflammation and immune regulation highlights their potential importance in SLE. Prior work from our group showed that the Fcγ receptor type IIa (FcγRIIa)-R/H131 biallelic polymorphism is associated with increased platelet activity and cardiovascular risk in SLE. The study was initiated to investigate the platelet transcriptome in patients with SLE and evaluate its association across FcγRIIa genotypes and distinct clinical features. METHODS: Fifty-one patients fulfilling established criteria for SLE (mean age = 41.1 ± 12.3, 100% female, 45% Hispanic, 24% black, 22% Asian, 51% white, mean SLEDAI = 4.4 ± 4.2 at baseline) were enrolled and compared with 18 demographically matched control samples. The FCGR2a receptor was genotyped for each sample, and RNA-seq was performed on isolated, leukocyte-depleted platelets. Transcriptomic data were used to create a modular landscape to explore the differences between SLE patients and controls and various clinical parameters in the context of FCGR2a genotypes. RESULTS: There were 2290 differentially expressed genes enriched for pathways involved in interferon signaling, immune activation, and coagulation when comparing SLE samples vs controls. When analyzing patients with proteinuria, modules associated with oxidative phosphorylation and platelet activity were unexpectedly decreased. Furthermore, genes that were increased in SLE and in patients with proteinuria were enriched for immune effector processes, while genes increased in SLE but decreased in proteinuria were enriched for coagulation and cell adhesion. A low-binding FCG2Ra allele (R131) was associated with decreases in FCR activation, which further correlated with increases in platelet and immune activation pathways. Finally, we were able to create a transcriptomic signature of clinically active disease that performed significantly well in discerning SLE patients with active clinical disease form those with inactive clinical disease. CONCLUSIONS: In aggregate, these data demonstrate the platelet transcriptome provides insight into lupus pathogenesis and disease activity, and shows potential use as means of assessing this complex disease using a liquid biopsy.

High incidence of proliferative and membranous nephritis in SLE patients with low proteinuria in the Accelerating Medicines Partnership.

OBJECTIVE: Delayed detection of LN associates with worse outcomes. There are conflicting recommendations regarding a threshold level of proteinuria at which biopsy will likely yield actionable management. This study addressed the association of urine protein:creatinine ratios (UPCR) with clinical characteristics and investigated the incidence of proliferative and membranous histology in patients with a UPCR between 0.5 and 1. METHODS: A total of 275 SLE patients (113 first biopsy, 162 repeat) were enrolled in the multicentre multi-ethnic/racial Accelerating Medicines Partnership across 15 US sites at the time of a clinically indicated renal biopsy. Patients were followed for 1 year. RESULTS: At biopsy, 54 patients had UPCR <1 and 221 had UPCR ≥1. Independent of UPCR or biopsy number, a majority (92%) of patients had class III, IV, V or mixed histology. Moreover, patients with UPCR <1 and class III, IV, V, or mixed had a median activity index of 4.5 and chronicity index of 3, yet 39% of these patients had an inactive sediment. Neither anti-dsDNA nor low complement distinguished class I or II from III, IV, V or mixed in patients with UPCR <1. Of 29 patients with baseline UPCR <1 and class III, IV, V or mixed, 23 (79%) had a UPCR <0.5 at 1 year. CONCLUSION: In this prospective study, three-quarters of patients with UPCR <1 had histology showing class III, IV, V or mixed with accompanying activity and chronicity despite an inactive sediment or normal serologies. These data support renal biopsy at thresholds lower than a UPCR of 1.

Safety of procuring research tissue during a clinically indicated kidney biopsy from patients with lupus: data from the Accelerating Medicines Partnership RA/SLE Network.

OBJECTIVES: In lupus nephritis the pathological diagnosis from tissue retrieved during kidney biopsy drives treatment and management. Despite recent approval of new drugs, complete remission rates remain well under aspirational levels, necessitating identification of new therapeutic targets by greater dissection of the pathways to tissue inflammation and injury. This study assessed the safety of kidney biopsies in patients with SLE enrolled in the Accelerating Medicines Partnership, a consortium formed to molecularly deconstruct nephritis. METHODS: 475 patients with SLE across 15 clinical sites in the USA consented to obtain tissue for research purposes during a clinically indicated kidney biopsy. Adverse events (AEs) were documented for 30 days following the procedure and were determined to be related or unrelated by all site investigators. Serious AEs were defined according to the National Institutes of Health reporting guidelines. RESULTS: 34 patients (7.2%) experienced a procedure-related AE: 30 with haematoma, 2 with jets, 1 with pain and 1 with an arteriovenous fistula. Eighteen (3.8%) experienced a serious AE requiring hospitalisation; four patients (0.8%) required a blood transfusion related to the kidney biopsy. At one site where the number of cores retrieved during the biopsy was recorded, the mean was 3.4 for those who experienced a related AE (n=9) and 3.07 for those who did not experience any AE (n=140). All related AEs resolved. CONCLUSIONS: Procurement of research tissue should be considered feasible, accompanied by a complication risk likely no greater than that incurred for standard clinical purposes. In the quest for targeted treatments personalised based on molecular findings, enhanced diagnostics beyond histology will likely be required.

Sex Differences in Systemic Lupus Erythematosus: Epidemiology, Clinical Considerations, and Disease Pathogenesis.

Systemic lupus erythematosus (SLE) is a chronic, multiorgan, systemic autoimmune disease that is more common in women than men and is typically diagnosed during reproductive age, necessitating sex-specific considerations in care. In women there is no substantive evidence to suggest that SLE reduces fertility, but subfertility may occur as a result of active disease, immunosuppressive drugs, and age-related declines in fertility related to delays in childbearing. Although pregnancy outcomes have improved, SLE still poses risks in pregnancy that contribute to poorer maternal and fetal outcomes. Cyclophosphamide, an important agent for the treatment of severe or life-threatening lupus, may adversely affect fertility, particularly with increases in dose and patient age. Fertility preservation techniques are therefore an important consideration for women and men before cytotoxic treatment. There is mixed evidence as to whether exogenous estrogen in the form of oral contraceptive pills or hormone replacement therapy may increase the risk for the development of SLE, but among women with SLE already diagnosed, combined oral contraceptive pills and hormone replacement therapy do not confer risk for severe flare and remain important in reproductive care. The higher incidence of SLE in women may nonetheless be attributable to effects of endogenous estrogen, as well as failures in X chromosome inactivation, increased Toll-like receptor gene products, and changes in microRNA function. A greater appreciation of the biological underpinnings and consequences of sex differences in SLE may lead to more targeted treatments and improved outcomes for patients with SLE.

PD-1hiCXCR5- T peripheral helper cells promote B cell responses in lupus via MAF and IL-21.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathologic T cell-B cell interactions and autoantibody production. Defining the T cell populations that drive B cell responses in SLE may enable design of therapies that specifically target pathologic cell subsets. Here, we evaluated the phenotypes of CD4+ T cells in the circulation of 52 SLE patients drawn from multiple cohorts and identified a highly expanded PD-1hiCXCR5-CD4+ T cell population. Cytometric, transcriptomic, and functional assays demonstrated that PD-1hiCXCR5-CD4+ T cells from SLE patients are T peripheral helper (Tph) cells, a CXCR5- T cell population that stimulates B cell responses via IL-21. The frequency of Tph cells, but not T follicular helper (Tfh) cells, correlated with both clinical disease activity and the frequency of CD11c+ B cells in SLE patients. PD-1hiCD4+ T cells were found within lupus nephritis kidneys and correlated with B cell numbers in the kidney. Both IL-21 neutralization and CRISPR-mediated deletion of MAF abrogated the ability of Tph cells to induce memory B cell differentiation into plasmablasts in vitro. These findings identify Tph cells as a highly expanded T cell population in SLE and suggest a key role for Tph cells in stimulating pathologic B cell responses.

The immune cell landscape in kidneys of patients with lupus nephritis.

Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.

Siglec-1 Macrophages and the Contribution of IFN to the Development of Autoimmune Congenital Heart Block.

Given that diseases associated with anti-SSA/Ro autoantibodies, such as systemic lupus erythematosus and Sjögren syndrome, are linked with an upregulation of IFN and type I IFN-stimulated genes, including sialic acid-binding Ig-like lectin 1 (Siglec-1), a receptor on monocytes/macrophages, recent attention has focused on a potential role for IFN and IFN-stimulated genes in the pathogenesis of congenital heart block (CHB). Accordingly, three approaches were leveraged to address the association of IFN, IFN-stimulated genes, and the phenotype of macrophages in affected fetal cardiac tissue: 1) cultured healthy human macrophages transfected with hY3, an anti-SSA/Ro-associated ssRNA, 2) RNA isolated from freshly sorted human leukocytes/macrophages after Langendorff perfusion of three fetal hearts dying with CHB and three healthy gestational age-matched hearts, and 3) autopsy tissue from three additional human CHB hearts and one healthy heart. TLR ligation of macrophages with hY3 led to the upregulation of a panel of IFN transcripts, including SIGLEC1, a result corroborated using quantitative PCR. Using independent and agnostic bioinformatics approaches, CD45+CD11c+ and CD45+CD11c- human leukocytes flow sorted from the CHB hearts highly expressed type I IFN response genes inclusive of SIGLEC1. Furthermore, Siglec-1 expression was identified in the septal region of several affected fetal hearts. These data now provide a link between IFN, IFN-stimulated genes, and the inflammatory and possibly fibrosing components of CHB, positioning Siglec-1-positive macrophages as integral to the process.

Autoimmune reactivity to malondialdehyde adducts in systemic lupus erythematosus is associated with disease activity and nephritis.

BACKGROUND: Immunoglobulin M (IgM) autoreactivity to malondialdehyde (MDA) protein modifications is part of the natural antibody repertoire in health and may have beneficial functions. In contrast, IgG anti-MDA are increased in chronic inflammation and autoimmunity and may instead have pathogenic properties. METHODS: Herein, we investigated serum IgG anti-MDA levels by enzyme-linked immunosorbent assay (ELISA) in 398 systemic lupus erythematosus (SLE) patients in the Swedish Karolinska SLE cohort and compared these to findings in 225 US SLE patients from New York University and Johns Hopkins University. RESULTS: In two independent cohorts, IgG anti-MDA levels correlated positively with disease activity by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI; p < 0.0001, Spearman R = 0.3). Meta-analysis found an odds ratio of 2.7 (confidence interval (CI) 1.9-3.9; p < 0.0001) for high anti-MDA IgG levels with active disease (SLEDAI ≥ 6). Furthermore, IgG anti-MDA correlated directly with erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), soluble tumor necrosis factor receptors (sTNFR-1, sTNFR-2), and vascular cell adhesion molecule 1 (VCAM-1) measurements, and inversely with complement factors (C1q, C2, C3, C4). Importantly, IgG anti-MDA levels were significantly elevated in SLE patients with active nephritis (p = 0.0005) and correlated with cystatin C estimated glomerular filtration rate and albuminuria. CONCLUSIONS: Elevated IgG anti-MDA in SLE patients was associated with high disease activity, with active lupus nephritis, and with biomarkers of systemic inflammation. This natural antibody reactivity may have potential prognostic utility, and may also actively contribute to pathogenesis.

Systemic lupus erythematosus and the risk of perioperative major adverse cardiovascular events.

Systemic lupus erythematosus (SLE) is a significant risk factor for cardiovascular disease. The relationship between SLE and perioperative cardiovascular risks following non-cardiac surgery is uncertain. We investigated associations between a diagnosis of SLE and outcomes following major non-cardiac surgery in a large national database from the United States. Patients age ≥ 18 years requiring major non-cardiac surgery were identified from Healthcare Cost and Utilization Project's National Inpatient Sample data from 2004 to 2014. Systemic lupus erythematosus and perioperative major adverse cardiovascular events (MACE; myocardial infarction, ischemic stroke or death) were defined by ICD-9 diagnosis codes. Perioperative MACE were reported for SLE patients stratified by age and sex. From 2004 to 2014, a total of 17,853,194 hospitalizations for major non-cardiac surgery met study inclusion criteria. SLE was identified in 70,578 (0.4%) hospitalizations. Overall, the frequency of perioperative MACE was higher in patients with vs. without SLE [2.4 vs. 2.0%, p < 0.001; adjusted OR (aOR) 1.25; 95% CI 1.18-1.31]. Perioperative MACE associated with SLE was largely driven by increased death (aOR 1.58 95% CI 1.40-1.77) and myocardial infarction (aOR 1.32; 95% CI 1.05-1.66) in younger patients with SLE. The increased risk of perioperative MACE associated with SLE in younger patients was attenuated with increasing age. A diagnosis of SLE is associated with increased risk of perioperative MACE, particularly among younger patients. Efforts to improve the perioperative management and outcomes of patients with SLE are needed.

Apolipoprotein L1 risk variants associate with prevalent atherosclerotic disease in African American systemic lupus erythematosus patients.

OBJECTIVE: Atherosclerosis is exaggerated in African American (AA) systemic lupus erythematosus (SLE) patients, with doubled cardiovascular disease (CVD) risk compared to White patients. The extent to which common Apolipoprotein L1 (APOL1) risk alleles (RA) contribute to this trend is unknown. This retrospective cohort study assessed prevalent atherosclerotic disease across APOL1 genotypes in AA SLE patients. METHODS: One hundred thirteen AA SLE subjects were APOL1-genotyped and stratified as having: zero risk alleles, one risk allele, or two risk alleles. Chart review assessed CVD manifestations including abdominal aortic aneurysm, angina, carotid artery disease, coronary artery disease, myocardial infarction, peripheral vascular disease, stroke, and vascular calcifications. Associations between the genotypes and a composite endpoint defined as one or more CVD manifestations were calculated using logistic regression. Symptomatic atherosclerotic disease, excluding incidental vascular calcifications, was also assessed. RESULTS: The 0-risk-allele, 1-risk-allele and 2-risk-allele groups, respectively, comprised 34%, 53%, and 13% of the cohort. Respectively, 13.2%, 41.7%, and 60.0% of the 0-risk allele, 1-risk-allele, and 2-risk-allele groups met the composite endpoint of atherosclerotic CVD (p = 0.001). Adjusting for risk factors-including smoking, ESRD, BMI >25 and hypertension-we observed an association between carrying one or more RA and atherosclerotic CVD (OR = 7.1; p = 0.002). For symptomatic disease, the OR was 3.5 (p = 0.02). In a time-to-event analysis, the proportion of subjects free from the composite primary endpoint, symptomatic atherosclerotic CVD, was higher in the 0-risk-allele group compared to the 1-risk-allele and 2-risk-allele groups (χ2 = 6.5; p = 0.04). CONCLUSIONS: Taken together, the APOL1 RAs associate with prevalent atherosclerotic CVD in this cohort of AA SLE patients, perhaps reflecting a potentiating effect of SLE on APOL1-related cardiovascular phenotypes.

Cardiac fibroblast transcriptome analyses support a role for interferogenic, profibrotic, and inflammatory genes in anti-SSA/Ro-associated congenital heart block.

The signature lesion of SSA/Ro autoantibody-associated congenital heart block (CHB) is fibrosis and a macrophage infiltrate, supporting an experimental focus on cues influencing the fibroblast component. The transcriptomes of human fetal cardiac fibroblasts were analyzed using two complementary approaches. Cardiac injury conditions were simulated in vitro by incubating human fetal cardiac fibroblasts with supernatants from macrophages transfected with the SSA/Ro-associated noncoding Y ssRNA. The top 10 upregulated transcripts in the stimulated fibroblasts reflected a type I interferon (IFN) response [e.g., IFN-induced protein 44-like (IFI44L), of MX dynamin-like GTPase (MX)1, MX2, and radical S-adenosyl methionine domain containing 2 (Rsad2)]. Within the fibrotic pathway, transcript levels of endothelin-1 (EDN1), phosphodiesterase (PDE)4D, chemokine (C-X-C motif) ligand (CXCL)2, and CXCL3 were upregulated, while others, including adenomedullin, RAP guanine nucleotide exchange factor 3 (RAPGEF3), tissue inhibitor of metalloproteinase (TIMP)1, TIMP3, and dual specificity phosphatase 1, were downregulated. Agnostic Database for Annotation, Visualization and Integrated Discovery analysis revealed a significant increase in inflammatory genes, including complement C3A receptor 1 (C3AR1), F2R-like thrombin/trypsin receptor 3, and neutrophil cytosolic factor 2. In addition, stimulated fibroblasts expressed high levels of phospho-MADS box transcription enhancer factor 2 [a substrate of MAPK5 (ERK5)], which was inhibited by BIX-02189, a specific inhibitor of ERK5. Translation to human disease leveraged an unprecedented opportunity to interrogate the transcriptome of fibroblasts freshly isolated and cell sorted without stimulation from a fetal heart with CHB and a matched healthy heart. Consistent with the in vitro data, five IFN response genes were among the top 10 most highly expressed transcripts in CHB fibroblasts. In addition, the expression of matrix-related genes reflected fibrosis. These data support the novel finding that cardiac injury in CHB may occur secondary to abnormal remodeling due in part to upregulation of type 1 IFN response genes.NEW & NOTEWORTHY Congenital heart block is a rare disease of the fetal heart associated with maternal anti-Ro autoantibodies which can result in death and for survivors, lifelong pacing. This study provides in vivo and in vitro transcriptome-support that injury may be mediated by an effect of Type I Interferon on fetal fibroblasts.

Single cell RNA sequencing to dissect the molecular heterogeneity in lupus nephritis.

Lupus nephritis is a leading cause of mortality among systemic lupus erythematosus (SLE) patients, and its heterogeneous nature poses a significant challenge to the development of effective diagnostics and treatments. Single cell RNA sequencing (scRNA-seq) offers a potential solution to dissect the heterogeneity of the disease and enables the study of similar cell types distant from the site of renal injury to identify novel biomarkers. We applied scRNA-seq to human renal and skin biopsy tissues and demonstrated that scRNA-seq can be performed on samples obtained during routine care. Chronicity index, IgG deposition, and quantity of proteinuria correlated with a transcriptomic-based score composed of IFN-inducible genes in renal tubular cells. Furthermore, analysis of cumulative expression profiles of single cell keratinocytes dissociated from nonlesional, non-sun-exposed skin of patients with lupus nephritis also revealed upregulation of IFN-inducible genes compared with keratinocytes isolated from healthy controls. This indicates the possible use of scRNA-seq analysis of skin biopsies as a biomarker of renal disease. These data support the potential utility of scRNA-seq to provide new insights into the pathogenesis of lupus nephritis and pave the way for exploiting a readily accessible tissue to reflect injury in the kidney.

Angiogenic factor imbalance early in pregnancy predicts adverse outcomes in patients with lupus and antiphospholipid antibodies: results of the PROMISSE study.

BACKGROUND: Over 20% of pregnancies in patients with systemic lupus erythematosus (SLE) and/or antiphospholipid antibodies (APL) result in an adverse pregnancy outcome (APO) related to abnormal placentation. The ability to identify, early in pregnancy, patients who are destined for poor outcomes would significantly impact care of this high-risk population. In nonautoimmune patients, circulating angiogenic factors are dysregulated in disorders of placentation, such as preeclampsia (PE) and fetal growth restriction. OBJECTIVE: We sought to determine whether early dysregulation of circulating angiogenic factors can predict APO in high-risk SLE and/or APL pregnancies. STUDY DESIGN: We used data and samples from the Predictors of Pregnancy Outcome: Biomarkers in APL Syndrome and SLE (PROMISSE), a multicenter prospective study that enrolled 492 pregnant women with SLE and/or APL from September 2003 through August 2013. Patients were followed through pregnancy from <12 weeks gestation. Circulating levels of soluble fms-like tyrosine kinase-1 (sFlt1), placental growth factor (PlGF), and soluble endoglin were measured monthly and subjects followed up for APO, classified as severe (PE <34 weeks, fetal/neonatal death, indicated preterm delivery <30 weeks) or moderate (PE ≥34 weeks, indicated preterm delivery 30-36 weeks, growth restriction without PE). RESULTS: Severe APOs occurred in 12% and moderate APOs in 10% of patients. By 12-15 weeks, sFlt1, PlGF, and soluble endoglin levels were markedly altered in women who developed severe APO. After adjusting for clinical risk factors, sFlt1 was the strongest predictor of severe APO among 12-15 week measures (odds ratio, 17.3 comparing highest and lowest quartiles; 95% confidence interval [CI], 3.5-84.8; positive predictive value [PPV], 61%; negative predictive value [NPV], 93%). At 16-19 weeks, the combination of sFlt1 and PlGF was most predictive of severe APO, with risk greatest for subjects with both PlGF in lowest quartile (<70.3 pg/mL) and sFlt1 in highest quartile (>1872 pg/mL; odds ratio, 31.1; 95% CI, 8.0-121.9; PPV, 58%; NPV, 95%). Severe APO rate in this high-risk subgroup was 94% (95% CI, 70-99.8%), if lupus anticoagulant or history of high blood pressure was additionally present. In contrast, among patients with both sFlt1 <1872 pg/mL and PlGF >70.3 pg/mL, rate of severe APO was only 4.6% (95% CI, 2.1-8.6%). CONCLUSION: Circulating angiogenic factors measured during early gestation have a high NPV in ruling out the development of severe adverse outcomes among patients with SLE and/or APL syndrome. Timely risk stratification of patients is important for effective clinical care and optimal allocation of health care resources.

Targeting downstream transcription factors and epigenetic modifications following Toll-like receptor 7/8 ligation to forestall tissue injury in anti-Ro60 associated heart block.

Based on the consistent demonstration of fibrosis of the atrioventricular node surrounded by macrophages and multinucleated giant cells in anti-Ro antibody exposed fetuses dying with heart block, this study focuses on macrophage signaling stimulated by ssRNA associated with the Ro60 protein and the impact of antagonizing innate cell drivers such as TLR7/8. Transcriptome and epigenetic modifications which affect transcription factors, NF-κB and STAT1, were selected to evaluate the phenotype of macrophages in which TLR7/8 was ligated following treatment with either anti-Ro60/Ro60/hY3 RNA immune complexes or transfection with hY3. Based on microarray, TNF and IL6 were among the most highly upregulated genes in both stimulated conditions, each of which was significantly inhibited by preincubation with hydroxychloroquine (HCQ). In contrast, following stimulation of macrophages with either TNF-α or IFN-α, which do not signal through TLR, the resultant gene expression was refractory to HCQ. Ligation of TLR7/8 resulted in increased histone methylation as measured by increased H3K4me2, a requirement for binding of NF-κB at certain promoters, specifically the kB1 region in the TNF promoter (ChIP-qPCR), which was significantly decreased by HCQ. In summary, these results support that the HCQ-sensitive phenotype of hY3 stimulated macrophages reflects the bifurcation of TLR downstream signals involving NF-κB and STAT 1 pathways and for the former dimethylation of H3K4. Accordingly, HCQ may act more as a preventive measure in downregulating the initial production of IFN-α or TNF-α and not affect the resultant autocoid stimulation reflected in TNF-α and IFN-α responsive genes. The beneficial scope of antimalarials in the prevention of organ damage, inclusive of heart block in an anti-Ro offspring or more broadly SLE, may include in part, a mechanism targeting TLR-dependent epigenetic modification.

A central role of plasmin in cardiac injury initiated by fetal exposure to maternal anti-Ro autoantibodies.

OBJECTIVE: Cardiac neonatal lupus (cardiac-NL), initiated by surface binding of anti-Ro60 autoantibodies to apoptotic cardiocytes during development, activates the urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) system. Subsequent accumulation of apoptotic cells and plasmin generation facilitates increased binding of anti-Ro60 by disrupting and cleaving circulating ß2-glycoprotein I (ß2GPI) thereby eliminating its protective effect. The association of soluble levels of components of the uPA/uPAR system with cardiac-NL was examined. METHODS: Levels of the uPA/uPAR system were assessed by ELISA in cord blood and immunohistological evaluation of autopsies. RESULTS: uPA, uPAR and plasminogen levels were each significantly higher in cord blood from cardiac-NL (n = 35) compared with non-cardiac-NL (n = 26) anti-Ro-exposed neonates: 3.3 ± 0.1 vs 1.9 ± 0.05 ng/ml (P < 0.0001), 6.6 ± 0.3 vs 2.1 ± 0.2 ng/ml (P < 0.0001) and 435 ± 34 vs 220 ± 19 ng/ml (P < 0.0001), respectively. In three twin pairs discordant for cardiac-NL, the twin with cardiac-NL had higher levels of uPA, uPAR and plasminogen than the unaffected twin (3.1 ± 0.1 vs 1.9 ± 0.05 ng/ml; P = 0.0086, 6.2 ± 1.4 vs 2.2 ± 0.7 ng/ml; P = 0.147 and 412 ± 61 vs 260 ± 27 ng/ml; P = 0.152, respectively). Immunohistological evaluation of three hearts from fetuses dying with cardiac-NL revealed macrophages and giant cells expressing uPA and plasminogen in the septal region. CONCLUSION: Increased soluble uPA, uPAR and plasminogen in cord blood and expression in affected tissue of fetuses with cardiac-NL supports the hypothesis that fetal cardiac injury is in part mediated by plasmin generation initiated by anti-Ro binding to the apoptotic cardiocyte.

Complement receptor 3 influences toll-like receptor 7/8-dependent inflammation: implications for autoimmune diseases characterized by antibody reactivity to ribonucleoproteins.

Toll-like receptor (TLR) signaling is an important component in the inflammatory response generated in diseases characterized by autoantibody reactivity to proteins such as SSA/Ro in complex with endogenous nucleic acids. Complement receptor 3 (CR3), a genetic variant of which has been identified as a risk factor in systemic lupus erythematosus, has been shown to induce tolerogenic responses in dendritic cells and suppress TLR4 responses in a murine sepsis model. Accordingly, this study addressed the hypothesis that activation of CR3, influenced by genotype of CD11b, negatively regulates TLR7/8-dependent effector function. Allosteric activation of CD11b via pretreatment with the small molecule, leukadhedrin 1 (LA1), significantly attenuated TLR7/8-induced (hY3 RNA, R848) secretion of TNFα in THP-1 cells and human macrophages isolated from donors homozygous for the ancestral common ITGAM allele at rs1143679. This inhibition was accompanied by profound degradation of the adaptor protein MyD88, an effect not observed with direct inhibition of TLR ligation by an antagonist oligonucleotide. In contrast, the addition of LA1 after incubation with the TLR agonists did not result in MyD88 degradation and subsequent attenuation of TNFα secretion. In TLR7/8-stimulated macrophages isolated from donors heterozygous for the CD11b variant, pretreatment with LA1 did not down-regulate TNFα release. These novel findings support a negative cross-talk between CR3 and TLR pathways likely to be induced by antibodies reactive with ribonucleoproteins and point to the development of CR3-specific agonists as potential therapeutics for diseases such as neonatal lupus.

Derivation and validation of the Systemic Lupus International Collaborating Clinics classification criteria for systemic lupus erythematosus.

OBJECTIVE: The Systemic Lupus International Collaborating Clinics (SLICC) group revised and validated the American College of Rheumatology (ACR) systemic lupus erythematosus (SLE) classification criteria in order to improve clinical relevance, meet stringent methodology requirements, and incorporate new knowledge regarding the immunology of SLE. METHODS: The classification criteria were derived from a set of 702 expert-rated patient scenarios. Recursive partitioning was used to derive an initial rule that was simplified and refined based on SLICC physician consensus. The SLICC group validated the classification criteria in a new validation sample of 690 new expert-rated patient scenarios. RESULTS: Seventeen criteria were identified. In the derivation set, the SLICC classification criteria resulted in fewer misclassifications compared with the current ACR classification criteria (49 versus 70; P = 0.0082) and had greater sensitivity (94% versus 86%; P < 0.0001) and equal specificity (92% versus 93%; P = 0.39). In the validation set, the SLICC classification criteria resulted in fewer misclassifications compared with the current ACR classification criteria (62 versus 74; P = 0.24) and had greater sensitivity (97% versus 83%; P < 0.0001) but lower specificity (84% versus 96%; P < 0.0001). CONCLUSION: The new SLICC classification criteria performed well in a large set of patient scenarios rated by experts. According to the SLICC rule for the classification of SLE, the patient must satisfy at least 4 criteria, including at least one clinical criterion and one immunologic criterion OR the patient must have biopsy-proven lupus nephritis in the presence of antinuclear antibodies or anti-double-stranded DNA antibodies.

Dysregulation of the microvasculature in nonlesional non-sun-exposed skin of patients with lupus nephritis.

OBJECTIVE: Membrane endothelial protein C receptor (mEPCR) is highly expressed in peritubular capillaries of kidneys from patients with active and poorly responsive lupus nephritis (LN). We investigated the hypothesis that changes in the microvasculature are widespread with extension to the dermal vasculature. METHODS: Skin biopsies from uninvolved skin (buttocks) were performed in 27 patients with LN and 5 healthy controls. Sections were stained with specific antibodies reactive with mEPCR, adiponectin, intercellular adhesion molecule-1 (ICAM-1), and CD31; then assessed by enumeration of stained blood vessels (percentage positive blood vessels) blinded to knowledge of clinical information. RESULTS: There was a significant increase in the prevalence of blood vessels that stained for mEPCR and ICAM-1 in patients compared to controls [94% vs 59% (p = 0.045) and 81% vs 67% (p = 0.037), respectively]. Adiponectin staining and CD31 staining were similar between the groups (45% vs 43% and 98% vs 92%). Dermal staining for mEPCR was greater in patients with proliferative glomerulonephritis than in those with membranous disease (96% vs 60%; p = 0.029). A composite of poor prognostic renal markers and death was significantly associated with greater expression of mEPCR staining. CONCLUSION: These data are consistent with the notion that in patients with LN, activation of the microvasculature extends beyond the clinically targeted organ. The insidious expression of this widespread vasculopathy may be a contributor to longterm comorbidities.