Faculty Bibliography

Lupus nephritis, one of the most serious manifestations of systemic lupus erythematosus (SLE), has both a heterogeneous clinical and pathological presentation. For example, proliferative nephritis identifies a more aggressive disease class that requires immunosuppression. However, the current classification system relies on the static appearance of histopathological morphology which does not capture differences in the inflammatory response. Therefore, a biomarker grounded in the disease biology is needed to understand the molecular heterogeneity of lupus nephritis and identify immunologic mechanism and pathways. Here, we analyzed the patterns of 1000 urine protein biomarkers in 30 patients with active lupus nephritis. We found that patients stratify over a chemokine gradient inducible by interferon-gamma. Higher values identified patients with proliferative lupus nephritis. After integrating the urine proteomics with the single-cell transcriptomics of kidney biopsies, it was observed that the urinary chemokines defining the gradient were predominantly produced by infiltrating CD8 T cells, along with natural killer and myeloid cells. The urine chemokine gradient significantly correlated with the number of kidney-infiltrating CD8 cells. These findings suggest that urine proteomics can capture the complex biology of the kidney in lupus nephritis. Patient-specific pathways may be noninvasively tracked in the urine in real time, enabling diagnosis and personalized treatment.

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.

OBJECTIVE:Autoimmune disease is an emerging condition among persons exposed to the September 11, 2001, attack on the World Trade Center (WTC). Components of the dust cloud resulting from the collapse of the WTC have been associated with systemic autoimmune diseases (SAID), as has posttraumatic stress disorder (PTSD). We sought to determine whether dust exposure and PTSD were associated with an increased risk of SAID in a 9/11-exposed cohort. METHODS:Among 43,133 WTC Health Registry enrollees, 2,786 self-reported a post-9/11 SAID. We obtained consent to review medical records to validate SAID diagnoses for 1,041. SAIDs were confirmed by classification criteria, rheumatologist diagnosis, or having been prescribed SAID medication. Controls were enrollees who denied an autoimmune disease diagnosis (n=37,017). We used multivariable log-binomial regression to examine the association between multiple 9/11 exposures and risk of post-9/11 SAID, stratifying by responders and community members. RESULTS:We identified 118 persons with SAID. Rheumatoid arthritis was most frequent (n=71), followed by SjÓ§gren's syndrome (n=22), systemic lupus erythematosus (n=20), myositis (n=9), mixed connective tissue disease (n=7), and scleroderma (n=4). Among 9/11 responders, those with intense dust cloud exposure had almost twice the risk of SAID (adjusted risk ratio =1.86, 95% CI=1.02-3.40). Community members with PTSD had a nearly three-fold increased risk of SAID. CONCLUSION/CONCLUSIONS:Intense dust cloud exposure among responders and PTSD among community members were associated with a statistically significant increased risk of new-onset SAID. Clinicians treating 9/11 survivors should be aware of the potential increased risk of SAID in this population.

Autoimmune-mediated congenital heart block (CHB) is a severe manifestation of neonatal lupus in which conduction tissues of the fetal heart are damaged. This occurs due to passive transference of maternal anti-SSA/Ro and anti-SSB/La autoantibodies and subsequent inflammation and fibrosis of the atrioventricular (AV) node. Notably, the disease manifests after the fetal heart has structurally developed, ruling out other anatomical abnormalities that could otherwise contribute to the block of conduction. Complete AV block is irreversible and the most common manifestation of CHB, although other cardiac complications such as endocardial fibroelastosis (EFE), dilated cardiomyopathy, and valvular insufficiency have been observed. In this review, we detail the classification, prevalence, pathogenesis, and clinical management recommendations for autoimmune CHB.

OBJECTIVES/OBJECTIVE:Cardiac manifestations of neonatal lupus (NL) have been associated with significant morbidity and mortality; however, there is minimal information on long-term outcomes of affected individuals. This study was initiated to evaluate the presence of and the risk factors associated with cardiac dysfunction in NL after birth in multiple age groups to improve counselling, to further understand pathogenesis and to provide potential preventative strategies. METHODS:Echocardiogram reports were evaluated in 239 individuals with cardiac NL: 143 from age 0-1 year, 176 from age >1-17 years and 64 from age >17 years. Logistic regression analyses evaluated associations of cardiac dysfunction at each age group with demographic, fetal and postnatal factors, using imputation to address missing data. RESULTS:Cardiac dysfunction was identified in 22.4% at age 0-1 year, 14.8% at age >1-17 years and 28.1% at age >17 years. Dysfunction in various age groups was significantly associated with male sex, black race, lower fetal heart rates, fetal extranodal cardiac disease and length of time paced. In 106 children with echocardiograms at ages 0-1 year and >1-17 years, 43.8% with dysfunction at age 0-1 year were also affected at age >1-17 years, while the others reverted to normal. Of children without dysfunction at age 0-1 year, 8.9% developed new dysfunction between ages >1 and 17 years. Among 34 with echocardiograms at ages >1-17 years and >17 years, 6.5% with normal function at age >1-17 years developed dysfunction in adulthood. CONCLUSIONS:Risk factors in fetal life can influence cardiac morbidity into adulthood.Although limited by a small number of cases, cardiac dysfunction in the first year often normalises by later childhood. New-onset dysfunction, although rare, can occur de novo after the first year.

The Accelerating Medicines Partnership (AMP) Lupus Network was established as a partnership between the NIH, pharmaceutical companies, non-profit stakeholders and lupus investigators across multiple academic centers to apply high throughput technologies to the analysis of renal tissue, urine and blood from patients with lupus nephritis (LN). The AMP network provides publicly accessible data to the community with the goal of generating new scientific hypotheses and improving diagnostic and therapeutic tools so as to improve disease outcomes. We present here a description of the structure of the AMP Lupus Network and a summary of the preliminary results from the Phase 1 studies. The successful completion of Phase 1 sets the stage for analysis of a large cohort of LN samples in Phase 2 and provides a model for establishing similar discovery cohorts.

Background/Purpose : Autoantibody production precedes SLE or SS by years, including anti-Ro. Anti-Ro + mothers of children with congenital heart block (CHB) are a unique population at risk for pathologic autoimmunity, as many are asymptomatic (Asym/UAS) and become aware of autoantibodies due to fetal disease and yet have a 10-year progression rate to SS/SLE of 20%-30%. We hypothesized that variation in the oral microbiome correlates with transition to SLE or SS and pathogenicity involves sequence homology between Ro60 and bacterial von Willebrand factor type A domain protein (vWFA).
Method(s): The oral microbiome of 25 anti-Ro + mothers of CHB children (Asym/UAS, N=9; SS/SLE, N=16) and 7 healthy controls (HC) were processed using 16S ribosomal RNA sequencing. Analysis of variance methods compared the centered log ratio transformed relative abundances for 1) HC vs. anti-Ro + mothers, and 2) assuming an ordering of severity from HC < Asym/UAS < SS/SLE. To adjust for multiple comparisons, a taxonomic stepdown method coupled with false discovery rate (FDR) was used. The Basic Local Alignment Search Tool evaluated homology of Ro60 at aa 371-381 and peptides of vWFA. Results : Sequencing 16S rRNA identified microorganisms from 2 kingdoms, 16 phyla, 25 classes, 41 orders, 70 families, 164 genera, and 166 species. The Shannon Index (H) revealed that for each taxonomic level except species, there were significant reductions in diversity in the anti-Ro + mothers relative to HC (P <= 0.05). There were global differences in the microbiota of these mothers relative to HC (perMANOVA P=0.00049). The phylum Actinobacteria was more abundant in the anti-Ro + mothers vs HC (P FDR =0.0231). Within Actinobacteria , the class Coriobacteriia and subsequent lower taxonomic levels down to Atopobium parvulum , all exhibited increases in relative abundance in the anti-Ro + mothers compared to HC. There was a significant reduction in the relative abundance as clinical severity increased within one of the most frequent phyla, Proteobacteria (P FDR =0.030; mean+/-SD; HC 0.24+/-0.07; Asym/UAS 0.19+/-0.12; SS/SLE 0.11+/-0.08). The difference in the relative abundances between Asym/UAS and SS/SLE within Proteobacteria was significant (P=0.042). Within Proteobacteria , the common class Betaproteobacteria also showed reduced relative abundance with increasing clinical severity (P FDR =0.0037; HC 0.11+/-0.04; Asym/UAS 0.072+/-0.07; SS/ SLE 0.031+/-0.04). These ordered differences were maintained down the taxonomic hierarchy to the genus ( Lautropia , P FDR =0.0072) and species within this genus ( L. mirabilis , P FDR =0.012). Next, sequences of vWFA secreted by these taxa were evaluated. For a comparison of Ro60 T cell epitope, FLLAVDVSASMNQ, the vWFA, VLVVFDNSSSMTA vWFA of A. parvulum was not a fit due to the aromatic and polar aa at positions 5 and 9, respectively. In contrast, the vWFA of L. mirabilis , LLLLLDVSGSMAG, was identical at 7 of the first 11 aa. Conclusion : These data provide evidence that the microbiome differs along a clinical spectrum of autoimmunity. In part, the data refiect a path involving depletion of L. mirabilis , which is secondary to a pathologic role of anti-Ro along with an expansion of A. parvulum , an opportunistic taxon

Background/Purpose : Null mutations in DNASE1L3 cause severe familial SLE with prominent anti-DNA antibodies (Abs), suggesting that DNASE1L3 is a key driver of tolerance to DNA. Indeed, DNASE1L3-deficient mice rapidly develop anti-chromatin and anti-DNA Abs followed by renal involvement. DNASE1L3 is a secreted DNase that has the preferential capacity to digest DNA within nucleosomes and/or encapsulated by membranes. Our previous studies suggest that chromatin carried by circulating apoptotic microparticles (MP) is an important physiological substrate of DNASE1L3. Accordingly, the present study was initiated to address the hypothesis that dysfunction of DNASE1L3 and its downstream consequences contribute to the development of sporadic human SLE. Methods : Reactivity to DNASE1L3-sensitive antigens on MP was measured by incubating plasma samples with control or DNASE1L3-treated MP and analyzing IgG binding to MP by flow cytometry. DNASE1L3 activity was measured based on its preferential capacity to digest complex DNA substrates with the readout expressed as the fraction of activity measured in healthy control subjects. The DNA load of MP was measured by qPCR of genomic DNA purified from the MP and MP-depleted fractions of plasma. In considering a spectrum from benign to clinical autoimmunity, subjects included those with active biopsy proven lupus nephritis as well as those with anti-Ro antibodies absent SLE. Results : IgG-binding to MP was assessed in 116 SLE patients, 45 anti-Ro Ab-positive mothers whose children have neonatal lupus (17 asymptomatic or having an undifferentiated autoimmune syndrome, 2 SLE, 12 SS, 14 SLE/SS) and 16 healthy controls. IgG-reactivity to MP was not detected in any healthy controls. In contrast, 36% of SLE patients showed IgG binding to MP reversed by DNASE1L3 pretreatment (DNASE1L3-senstive reactivity). Only two of these patients were heterozygous for the known hypomorphic DNASE1L3 (R206C) variant. DNASE1L3-sensitive reactivity correlated with anti-dsDNA Abs (p< .0001) but did not overlap, indicating that reactivity is directed to more complex DNA. DNASE1L3-sensitive reactivity correlated with active proteinuria (p=.0003), low complement levels (p=.01) and overall SLEDAI (p< .0001). Among 45 anti-Ro-positive mothers, all were unreactive except the only 2 SLE mothers that developed lupus nephritis. The DNA load of MP and DNASE1L3 activity were assessed in a subset of SLE patients (n=40). Of these, patients with proteinuria showed lower plasma DNASE1L3 activity (p=.034) than those without proteinuria (Fig. 1). Moreover, increased partitioning of plasma DNA into MP correlated with reduced DNASE1L3 activity, and both parameters strongly correlated with DNASE1L3-sensitive IgG binding to MPs (p< 0.0001) (Fig. 2). Conclusion : The activity of plasma DNASE1L3 is reduced in a significant fraction of SLE patients with renal disorder unrelated to a genetic explanation and is associated with DNA accumulation in MP targeted by Abs. Collectively, these data suggest that digestion of circulating chromatin by DNASE1L3 is a fundamental mechanism of tolerance to DNA which when disrupted may lead to sporadic SLE, particularly lupus nephritis

Background/Purpose : Compared to Caucasian, African-American ethnicity is associated with a higher risk of developing systemic lupus erythematosus, lupus nephritis, high-risk histological features, resistance to treatment, and mortality. In phase 1 of the Accelerating Medicines Partnership (AMP), we used single-cell genomics to identify ethnicity associated features. Methods : Single cell RNA sequencing was performed on renal biopsies obtained for clinical purpose; one pipeline applying CEL-Seq2 in a leukocyte enriched sample and the other Fluidigm C1 800 in an agnostic approach to dissociated renal cells. Differential abundance of cell populations was determined using a logistic mixed model. Then, the differential expression profile was determined for each cell cluster and interpreted using pathway enrichment analysis. Results : Samples from 19 African-American and 20 Caucasian patients were obtained. We identified 30 cell clusters. Type I and II interferon inducible genes were upregulated in most cell populations. A cluster of T cells with exceptionally high interferon signature was found to be increased in African-Americans (OR 4.8). Macrophages and DC4-like dendritic cells were instead less abundant (OR 0.3). In African-Americans, type I and II interferon response pathways were enriched in several cell types including T cells, B cells, plasma cells, and activated monocytes. The majority of the differentially expressed genes was specifically inducible by type II interferon. In addition, while there was no local expression of type I interferons, interferon gamma was abundantly expressed by infiltrating NK and CD8 T cells. Conclusion : African-American patients with lupus nephritis have a stronger interferon response pathway activation, especially type II. Our findings suggest an intrinsic biological factor underlying the outcome gap and highlight the role of interferon gamma in lupus nephritis, implicating this pathway as a potential therapeutic target in SLE. Further work in Phase 2 of AMP is being pursued to validate and extend these findings