Faculty Bibliography

Background: A transmissible agent has long been suspected in the pathogenesis of SLE. We therefore investigated the potential contribution of the intestinal microbiome to LN.
Method(s): Blood and fecal samples from SLE patients were obtained, unless a patient had selective IgA deficiency, prior cytotoxic drugs, or antibiotics within four months. Fecal 16S rRNA NGS was performed. Sera samples were profiled for autoantibodies. Sera from two independent lupus cohorts were studied for validation.
Result(s): Compared to controls, the intestinal microbiome from SLE patients (N=61) showed decreased species richness diversity. The microbiomes of patients in clinical remission (based on SLEDAI) were most similar to healthy controls, while reductions in taxonomic complexity were most pronounced in those with high disease activity. Notably, SLE patients had an overall 5-fold greater representation of a particular species in the Blautia genus of the Lachnospiracaea family of obligate anaerobic Gram-positive cocci. Abundance of this species significantly correlated with serum IgG to a cell wall moiety from a strain of this species (P=0.002, N=61, Spearman) but not with 7 other strains. There was also a significant correlation between the distribution of SLEDAI scores and levels of these circulating anti-strain IgG antibodies (P=0.02, N=48). Using antigen treated with DNAse/proteinase K, levels of IgG anti-strain antibodies were significantly higher in those with active nephritis at time of sampling compared to SLE without renal activity (Cohort 1 P=0.01 N=48; Cohort 2 P=0.001, N=53, Mann-Whitney). Levels of anti-strain antibodies also significantly correlated with high-titer serum IgG to native DNA (P<0.0001, N=27), and inversely correlated with C3 and C4 (each P<0.01, N=61). High titers of these anti-bacterial antibodies were found in active Class III, IV and V LN.
Conclusion(s): These findings suggest a novel paradigm for the pathogenesis of LN: Specific strains of common intestinal commensal bacteria affect IgG-autoantibody responses in patients with LN. This is reminiscent of post-streptococcal GN, although the postulated intestinal bacterial bloom occurs without clinical infection

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 hY3. The top ten upregulated transcripts in the stimulated fibroblasts reflected a type I interferon (IFN) response (e.g. IFI44L, MX1, MX2, and Rsad2). Within the fibrotic pathway, transcript levels of EDN1, PDE4D, CXCL2 and CXCL3 were upregulated, while others, including ADM, RAPGEF3, TIMP1, TIMP3 and DUSP1, were downregulated. Agnostic DAVID analysis revealed a significant increase in inflammatory genes, including C3AR1, the complement C3A receptor, F2RL3, and NCF2. In addition, the stimulated fibroblasts expressed high levels of phospho-MEF2C (a substrate of 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 ten most highly expressed transcripts in the 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.

While the relationship between maternal connective tissue diseases and neonatal rashes was described in the 1960s and congenital heart block in the 1970s, the "culprit" antibody reactivity to the SSA/Ro-SSB/La ribonucleoprotein complex was not identified until the 1980s. However, studies have shown that approximately 10-15% of cases of congenital heart block are not exposed to anti-SSA/Ro-SSB/La. Whether those cases represent a different disease entity or whether another antibody is associated has yet to be determined. Moreover, the cutaneous manifestations of neonatal lupus have also been identified in infants exposed only to anti-U1RNP antibodies. In this review, we describe what we believe to be the first case of congenital heart block exposed to maternal anti-U1RNP antibodies absent anti-SSA/Ro-SSB/La. The clinical and pathologic characteristics of this fetus are compared to those typically seen associated with SSA/Ro and SSB/La. Current guidelines for fetal surveillance are reviewed and the potential impact conferred by this case is evaluated.

Autoantibodies to dense fine speckles 70 (DFS70) are purported to rule out the diagnosis of SLE when they occur in the absence of other SLE-related autoantibodies. This study is the first to report the prevalence of anti-DFS70 in an early, multinational inception SLE cohort and examine demographic, clinical, and autoantibody associations. Patients were enrolled in the Systemic Lupus International Collaborating Clinics (SLICC) inception cohort within 15 months of diagnosis. The association between anti-DFS70 and multiple parameters in 1137 patients was assessed using univariate and multivariate logistic regression. The frequency of anti-DFS70 was 7.1% (95% CI: 5.7-8.8%), while only 1.1% (95% CI: 0.6-1.9%) were monospecific for anti-DFS70. In multivariate analysis, patients with musculoskeletal activity (Odds Ratio (OR) 1.24 [95% CI: 1.10, 1.41]) or with anti-beta2 glycoprotein 1 (OR 2.17 [95% CI: 1.22, 3.87]) were more likely and patients with anti-dsDNA (OR 0.53 [95% CI: 0.31, 0.92]) or anti-SSB/La (OR 0.25 [95% CI: 0.08, 0.81]) were less likely to have anti-DFS70. In this study, the prevalence of anti-DFS70 was higher than the range previously published for adult SLE (7.1 versus 0-2.8%) and was associated with musculoskeletal activity and anti-beta2 glycoprotein 1 autoantibodies. However, 'monospecific' anti-DFS70 autoantibodies were rare (1.1%) and therefore may be helpful to discriminate between ANA-positive healthy individuals and SLE.

PURPOSE OF REVIEW: To provide new insights into pathogenesis, prevention and management of cardiac manifestations of neonatal lupus (cardiac neonatal lupus) and issues pertinent to all anti-SSA/Ro positive individuals of childbearing age. RECENT FINDINGS: Antibody specificity with high risk for cardiac neonatal lupus remains elusive, but high titers of Ro60, Ro52 or Ro52p200 antibodies appear to be required. Varying antibody specificities to the p200 region of Ro52 can induce first-degree block in a rodent model. In consideration of the contribution of macrophages to inflammation and fibrosis in cardiac neonatal lupus, hydroxychloroquine (HCQ) is being considered as preventive therapy. Cord blood biomarkers support the association of fetal reactive inflammatory and fibrotic components with the development and morbidity of cardiac neonatal lupus. Data from U.S. and French registries do not provide evidence that the prompt use of fluorinated steroids in cases of isolated block significantly alters fetal/neonatal morbidity or mortality. SUMMARY: The search for a high-risk cardiac neonatal lupus antibody profile remains, but high-titer antibodies to Ro60 and R052 are a consistent finding, and this may guide the need for fetal echocardiographic surveillance. The uniform use of fluorinated steroids to prevent progression of cardiac neonatal lupus or reduce mortality does not appear justified. HCQ, based on diminishing an inflammatory component of cardiac neonatal lupus, is under consideration as a potential preventive approach.

It is currently recommended that hydroxychloroquine (HCQ) be maintained during pregnancy in patients with systemic lupus erythematosus. Recent data suggest that this Toll-like receptor inhibitor may also reduce the recurrence rate of anti-SSA/Ro associated congenital heart block (CHB). This case report describes a unique situation in which a CHB-afflicted, HCQ-exposed pregnancy was electively terminated. The heart did not reveal any characteristic features of cardiotoxicity, providing further evidence supporting the safety of foetal exposure to HCQ.

BACKGROUND AND OBJECTIVES: Kidney disease is a critical concern in counseling patients with lupus considering pregnancy. This study sought to assess the risk of renal flares during pregnancy in women with previous lupus nephritis in partial or complete remission, particularly in those with antidouble-stranded DNA antibodies and low complement levels, and the risk of new-onset nephritis in patients with stable/mildly active SLE. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We assessed active nephritis (renal flares and de novo kidney disease) and associated predictors during pregnancy in patients with lupus with urine protein 500 mg and/or red blood cell casts. RESULTS: Of 118 patients with previous kidney disease, 13 renal flares (11%) occurred (seven of 89 in complete remission and six of 29 in partial remission) compared with four with de novo kidney involvement (2%) in 255 patients without past kidney disease (P<0.001). Active nephritis was not associated with ethnicity, race, age, creatinine, BP, or antihypertensive and other medications. In multivariable logistic regression analyses, patients with past kidney disease in complete or partial remission more often experienced active nephritis (adjusted odds ratio, 6.88; 95% confidence interval, 1.84 to 25.71; P=0.004 and adjusted odds ratio, 20.98; 95% confidence interval, 4.69 to 93.98; P<0.001, respectively) than those without past kidney disease. Low C4 was associated with renal flares/de novo disease (adjusted odds ratio, 5.59; 95% confidence interval, 1.64 to 19.13; P<0.01) but not low C3 or positive anti-dsDNA alone. CONCLUSIONS: De novo kidney involvement in SLE, even in ethnic/racial minorities, is uncommon during pregnancy. Past kidney disease and low C4 at baseline independently associate with higher risk of developing active nephritis. Antibodies to dsDNA alone should not raise concern, even in patients with past kidney disease, if in remission.

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.

The detection of cardiac conduction defects in an 18-24 week old foetus in the absence of structural abnormalities predicts with near certainty the presence of autoantibodies against 60kD and 52kD SSA/Ro in the mother regardless of her health status. Previous studies have emphasized these autoantibodies as key mediators of tissue injury. The aim of this study was to focus on the anti-Ro52 response to determine whether these autoantibodies originate from progenitors that are inherently self-reactive or from B-cells that acquire self-reactivity during an immune response. We traced the evolution of two anti-Ro52 autoantibodies isolated from circulating IgG1-switched B-cells from an asymptomatic mother of a child with third degree congenital heart block. The autoantibodies were expressed as their immune form and as pre-immune ancestors by reverting somatic mutations to germline sequence. The reactivity of pre-immune and immune antibodies for Ro52, Ro60, La and DNA was measured. Both anti-Ro52 autoantibodies exhibited a low frequency of somatic mutations (3-4%) and utilised the same heavy and light chain genes but represented distinct clones based on differing complementarity determining region sequences. Pre- and post-immune antibodies showed specific binding to Ro52 with no measurable reactivity for other autoantigens. Ro52 binding was higher for immune antibodies compared to pre-immune counterparts demonstrating that autoreactivity was enhanced by affinity maturation. These data indicate that Ro52 reactivity is an intrinsic property of the germline antibody repertoire in a mother with a pathogenic antibody defined by cardiac injury in her offspring, and implies defects in both central and peripheral tolerance mechanisms.

OBJECTIVE: Systemic lupus erythematosus (SLE) is associated with the premature development of cardiovascular disease. The platelet-endothelium interaction is important in the pathogenesis of cardiovascular disease. In this study, we investigated the platelet phenotype from patients with SLE and matched controls, and their effect on endothelial cells. APPROACH AND RESULTS: Platelet aggregability was measured in 54 SLE subjects off antiplatelet therapy (mean age 40.1+/-12.8 years; 82% female; 37% white) with age- and sex-matched controls. Platelets were coincubated with human umbilical vein endothelial cells (HUVECs) and changes to gene expression assessed by an RNA array and quantitative reverse transcription polymerase chain reaction. SLE disease activity index ranged from 0 to 22 (mean 5.1+/-3.9). Compared with controls, patients with SLE had significantly increased monocyte and leukocyte-platelet aggregation and platelet aggregation in response to submaximal agonist stimulation. An agnostic microarray of HUVECs cocultured with SLE platelets found a platelet-mediated effect on endothelial gene pathways involved in cell activation. Sera from SLE versus control subjects significantly increased (1) activation of control platelets; (2) platelet adhesion to HUVECs; (3) platelet-induced HUVEC gene expression of interleukin-8, and ICAM-1; and (4) proinflammatory gene expression in HUVECs, mediated by interleukin-1beta-dependent pathway. Incubation of SLE-activated platelets with an interleukin-1beta-neutralizing antibody or HUVECs pretreated with interleukin-1 receptor antibodies attenuated the platelet-mediated activation of endothelial cells. CONCLUSIONS: Platelet activity measurements and subsequent interleukin-1beta-dependent activation of the endothelium are increased in subjects with SLE. Platelet-endothelial interactions may play a role in the pathogenesis of cardiovascular disease in patients with SLE.