Faculty Bibliography

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

Background/Purpose : Despite the strong association of maternal anti-Ro60 autoantibodies in the development of SS, Neonatal Lupus (NL) and scLE, understanding causality is challenging given that the intracellular location of the target RNP antigen only becomes accessible to extracellular autoantibodies during cell death. While binding Y RNA is a property of Ro60 in dying cells, Ro60 contributes to events essential to proliferation including the degradation of non-coding RNAs. To define potentially targetable molecular events associated with Ro60 accessibility and their effects on proliferation, we evaluated two candidate chaperone Ro60 binding proteins, ZBP1 (also known as IGF2BP1, which has two RNA recognition domains and plays a role in nuclear export of protein) and PTBP1 (which binds RNA in heterogeneous nuclear complexes). Methods : Short hairpin (sh)RNA knockdown (KD) of both ZPB1 and PTBP1 was accomplished by using MISSION pLKO.1 derived lentiviral vehicles for targeted shRNA to yield each of the specific depletions in human fetal fibroblasts. Affinity purified anti-Ro60 antibodies (AP60) isolated from the serum of 2 mothers of children with cardiac NL and control IgG isolated from a healthy donor were used to evaluate Ro60 surface translocation in intact and apoptotic cells by flow cytometry. Fibroblast proliferation was determined using 5-ethynyl-2'-deoxyuridine (EdU) incorporation. Results : KD of targeted transcripts were confirmed by qPCR and western blot. Permeabilized KD and wildtype fi-broblasts demonstrated equivalent intracellular expression of Ro60. As expected, flow cytometry with AP60 revealed no surface staining of non-permeabilized wildtype or KD fibroblasts. The next set of experiments addressed binding of AP60 to polyHEMA-apoptotic fibroblasts. Staining with annexin V (a proxy of apoptosis) was uniformly consistent between the wildtype and KD cells. As expected, compared to non-apoptotic fibroblasts, AP60 but not control donor IgG readily bound the surface of wildtype apoptotic fibroblasts, supporting that Ro60 was indeed translocated during apoptosis (MFI of 97 + 5 vs 211 + 14, respectively; P< 0.05; N=3). In contrast, binding of AP60 was significantly attenuated in the ZPB1 KD fibroblasts (81.5 + 7; N = 3) vs anti-Ro binding of apoptotic wildtype fibroblasts(P< 0.05). However, the binding of AP60 was equivalent in the apoptotic PTBP1 KD fibroblasts (MFI of 203 + 6, N= 3, P=NS) compared to apoptotic wildtype fibroblasts. With regard to cell proliferation, the percent positive EdU cells of wildtype fetal fibroblasts, ZBP1 KD fibroblasts, and PTBP1 KD fibroblasts were 56%, 2% and 45%, respectively, a result suggesting that the loss of ZBP1 restrains cell cycle progression. Conclusion : ZBP1 represents a novel and required chaperone for the translocation of Ro60 to the cell surface during apoptosis in addition to contributing to cell proliferation. Given that Ro60 antigen accessibility is essential, not only to the generation of anti-Ro60 responses but also to the formation of surface immune complexes and subsequent tissue injury, ZBP1 may represent a newly targetable candidate to forestall both the initiation and sequelae of autoimmunity

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 : 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 : Based on encouraging bench to bedside results including experimental evidence supporting Toll-like receptor signaling in the pathogenesis of CHB, a case control study demonstrating CHB risk reduction in hydroxychoroquine (HCQ) exposed fetuses of anti-Ro positive SLE women, and a historical cohort study supporting a reduction in recurrence rate, an open label single arm Phase 2 clinical trial was initiated to evaluate whether HCQ reduces the CHB recurrence rate (pi) below the historical recurrence rate of 18%. Methods : A two-stage trial design (N=19 first stage; N=54 second stage) using Simon's optimal approach was employed to allow for early stopping due to absence of treatment efficacy. The null hypothesis, H 0 :pi <= 18%, would be rejected and HCQ considered efficacious at the end of the trial if <= 5 of 54 mothers with anti-Ro and a previous CHB child had a subsequent child with 2 nd or 3 rd degree block (primary outcome). The protocol required HCQ initiation or maintenance at 400mg by 10 wks gestation. Mothers underwent serial echocardiograms, with bloods drawn each trimester and delivery for cord blood to measure antibody and HCQ levels. Results : Sixty five mothers (all with previous CHB child and anti-Ro52 or Ro60 > 1,000 EU; 47.9% with anti-La; 71.4% White; 47.6% SLE and/or SS; 42.9% started HCQ solely for CHB prevention; 41% prior CHB child died, 3.2% had > 1 CHB child) signed consent. Ten were considered screen failures (2 miscarriages < 12 wks, 7 wherein dating of conception placed HCQ initiation at > 10 wks, 1 given dexamethasone (dex) 1mg at 10 wks) and 1 was lost to follow up before delivery leaving 54 pregnancies evaluable with serial fetal echos and birth or one yr EKG or echo results known. In Stage I, 2/19 fetuses had CHB, and the study proceeded to Stage II. By intention to treat analysis, 4/54 pregnancies resulted in CHB (7.4%; p = 0.02 for H 0 ), all at 19-20 wks. Three presented with 2 nd degree block, one reverted to NSR at birth following dex and two progressed to 3 rd degree despite dex and IVIG (one electively terminated). One presenting with 1 st degree was treated with dex prophylactically (eliminating this case from evaluating HCQ exposure alone), progressed to 2 nd but reverted to NSR at birth. At 2 yrs, the 2 in NSR had intermittent 2 nd degree on Holter monitor. In 8 mothers potentially confounding medications, IVIG and/or dex, were prescribed after enrollment for lupus flare, cardiac concerns apart from advanced block (APCs, echo brightness, 1 st degree block), and/or physician decision to consider additional prophylaxis. To evaluate HCQ alone, 9 additional mothers were enrolled, one whose fetus developed 3 rd degree block at 19 wks. Including only pregnancies exposed to HCQ alone prior to confirmed 2 nd or 3 rd degree block, 4/54 developed CHB (7.4%; p = 0.02). In total 5/63 pregnancies (7.9%) resulted in advanced block. HCQ levels in the second trimester confirmed a 98% adherence rate. Anti-Ro levels remained > 1,000 EU (considered vulnerable for CHB) throughout pregnancy. No CHB developed in any of the 7 mothers screened out because of low dose or delayed start of HCQ. Conclusion : These prospective data from a single-arm clinical trial support that HCQ significantly reduces the recurrence of CHB below the historical rate

OBJECTIVE:To assess the prevalence of atherosclerotic cardiovascular disease (ASCVD) and its individual phenotypes of coronary artery disease (CAD), peripheral artery disease (PAD), and cerebrovascular disease by age and sex in a large US cohort of hospitalized patients with systemic lupus erythematosus (SLE). METHODS:A nested case-control study of adults with and without SLE was conducted from the January 1, 2008, through December 31, 2014, National Inpatient Sample. Hospitalized patients with SLE were matched (1:3) by age, sex, race, and calendar year to hospitalized patients without SLE. The prevalences of CAD, PAD, and cerebrovascular disease were evaluated, and associations with SLE were determined after adjustment for common cardiovascular risk factors. RESULTS:Among the 252,676 patients with SLE and 758,034 matched patients without SLE, the mean age was 51 years, 89% were women, and 49% were white. Patients with SLE had a higher prevalence of ASCVD vs those without SLE (25.6% vs 19.2%; OR, 1.45; 95% CI, 1.44-1.47; P<.001). After multivariable adjustment, SLE was associated with a greater odds of ASCVD (adjusted odds ratio [aOR], 1.46; 95% CI, 1.41-1.51). The association between SLE and ASCVD was observed in women and men and was attenuated with increasing age. Also, SLE was associated with increased odds of CAD (aOR, 1.42; 95% CI, 1.40-1.44), PAD (aOR, 1.25; 95% CI, 1.22-1.28), and cerebrovascular disease (aOR, 1.68; 95% CI, 1.65-1.71). CONCLUSION/CONCLUSIONS:In hospitalized US patients, SLE was associated with increased ASCVD prevalence, which was observed in both sexes and was greatest in younger patients.

Nonadherence to treatment is a major cause of lupus flares. Hydroxychloroquine (HCQ), a major medication in systemic lupus erythematosus, has a long half-life and can be quantified by high-performance liquid chromatography. This international study evaluated nonadherence in 305 lupus patients with flares using drug levels (HCQ < 200 ng/ml or undetectable desethylchloroquine), and self-administered questionnaires (MASRI < 80%). Drug levels defined 18.4% of the patients as severely nonadherent. In multivariate analyses, younger age, nonuse of steroids, higher body mass index, and unemployment were associated with nonadherence by drug level. Questionnaires classified 23.4% of patients as nonadherent. Correlations between adherence measured by questionnaires, drug level, and physician assessment were moderate. Both methods probably measured two different patterns of nonadherence: self-administered questionnaires mostly captured relatively infrequently missed tablets, while drug levels identified severe nonadherence (i.e., interruption or erratic tablet intake). The frequency with which physicians miss nonadherence, together with underreporting by patients, suggests that therapeutic drug monitoring is useful in this setting. (Trial registration: ClinicalTrials.gov: NCT01509989.).

OBJECTIVES:To assess the prevalence of combined hormonal contraceptives (CHCs) in reproductive-age women with SLE with and without possible contraindications and to determine factors associated with their use in the presence of possible contraindications. METHODS:This observational cohort study included premenopausal women ages 18-45 years enrolled in the SLICC Registry ⩽15 months after SLE onset, with annual assessments spanning 2000-2017. World Health Organization Category 3 or 4 contraindications to CHCs (e.g. hypertension, aPL) were assessed at each study visit. High disease activity (SLEDAI score >12 or use of >0.5 mg/kg/day of prednisone) was considered a relative contraindication. RESULTS:A total of 927 SLE women contributed 6315 visits, of which 3811 (60%) occurred in the presence of one or more possible contraindication to CHCs. Women used CHCs during 512 (8%) visits, of which 281 (55%) took place in the setting of one or more possible contraindication. The most frequently observed contraindications were aPL (52%), hypertension (34%) and migraine with aura (22%). Women with one or more contraindication were slightly less likely to be taking CHCs [7% of visits (95% CI 7, 8)] than women with no contraindications [9% (95% CI 8, 10)]. CONCLUSION:CHC use was low compared with general population estimates (>35%) and more than half of CHC users had at least one possible contraindication. Many yet unmeasured factors, including patient preferences, may have contributed to these observations. Further work should also aim to clarify outcomes associated with this exposure.

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.

Linked Comment: Ultrasound Obstet Gynecol 2019; 54: 87-95.