Faculty Bibliography

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.

OBJECTIVES/OBJECTIVE:There is a paucity of data regarding healthcare costs associated with damage accrual in systemic lupus erythematosus (SLE). We describe costs associated with damage states across the disease course using multi-state modeling. METHODS:Patients from 33 centres in 11 countries were enrolled in the Systemic Lupus International Collaborating Clinics (SLICC) inception cohort within 15 months of diagnosis. Annual data on demographics, disease activity, damage (SLICC/American College of Rheumatology (ACR) Damage Index [SDI]), hospitalizations, medications, dialysis, and selected procedures were collected. Ten-year cumulative costs (Canadian dollars) were estimated by multiplying annual costs associated with each SDI state by the expected state duration using a multi-state model. RESULTS:1687 patients participated, 88.7% female, 49.0% of Caucasian race/ethnicity, mean age at diagnosis 34.6 years (SD 13.3), and mean follow up 8.9 years (range 0.6-18.5). Annual costs were higher in those with higher SDIs (SDI ≥ 5: $22 006 2019 CDN, 95% CI $16 662, $27 350 versus SDI=0: $1833, 95% CI $1134, $2532). Similarly, 10-year cumulative costs were higher in those with higher SDIs at the beginning of the 10-year interval (SDI ≥ 5: $189 073, 95% CI $142 318, $235 827 versus SDI=0: $21 713, 95% CI $13 639, $29 788). CONCLUSION/CONCLUSIONS:Patients with the highest SDIs incur 10-year cumulative costs that are almost 9-fold higher than those with the lowest SDIs. By estimating the damage trajectory and incorporating annual costs, damage can be used to estimate future costs, critical knowledge for evaluating the cost-effectiveness of novel therapies.

AIMS/OBJECTIVE:Investigating human heart development and applying this to deviations resulting in disease is incomplete without molecular characterization of the cell types required for its normal functioning. We investigated fetal human heart single-cell transcriptomes from midgestational healthy and anti-Ro associated congenital heart block (CHB) samples, respectively. METHODS AND RESULTS/RESULTS:Three healthy fetal human hearts (19th-22nd week of gestation) and one fetal heart affected by autoimmune-associated CHB (21st week of gestation) were subjected to enzymatic dissociation using the Langendorff preparation to obtain single cell suspensions followed by 10x Genomics- and Illumina-based single cell RNA-sequencing (scRNA-seq). In addition to the myocytes, fibroblasts, immune cells, and other minor cell types, previously uncharacterized diverse subpopulations of endothelial cells were identified in the human heart. Differential gene expression analysis revealed increased and heterogeneous interferon responses in varied cell types the CHB heart compared to the healthy controls. In addition, we also identified matrisome transcripts enriched in CHB stromal cells that potentially contributing to extracellular matrix deposition and subsequent fibrosis. CONCLUSION/CONCLUSIONS:These data provide an information-rich resource to further understanding of human heart development, which, as illustrated by comparison to a heart exposed to a maternal autoimmune environment, can be leveraged to provide insight into the pathogenesis of disease. TRANSLATIONAL PERSPECTIVE/UNASSIGNED:This study provides a single cell transcriptomic atlas of cells obtained from healthy second trimester fetal hearts to further understand human heart development and impart insight into autoimmune associated congenital heart block. In addition to myocytes and fibroblasts, previously uncharacterized subpopulations of endothelial cells were identified. Leveraging an unprecedented opportunity, healthy heart transcriptomes were compared to an age matched anti-SSA/Ro exposed fetal heart with third degree block in which no maternal medications were taken. Differential gene expression analysis revealed a remarkable interferon response in many cell types of the diseased heart. In addition, matrisome transcripts were enriched in the stromal cells likely contributing to the extracellular matrix deposition and thereby fibrosis, a signature lesion of heart block. Thus, targeting the interferon pathway merits therapeutic consideration.

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.

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.

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: Both plasmacytoid dendritic cells (pDCs) and switched memory B cells (SMBCs) are considered to be key effector cells in systemic lupus erythematosus. It seems likely that within these classical cell lineages, additional diversity of function will exist that will contribute to disease pathogenesis. To explore this question, we performed single-cell RNA sequencing in pDCs and SMBCs from SLE patients and controls to assess gene expression patterns and cellular sub-groupings within these lineages. Methods : pDCs and SMBCs from SLE patients (n=10) and Healthy controls (n=5) were purified by magnetic separation. For deep sequencing, we used the Fluidigm C1 HT system with 800 capture site chips to capture single cells. Single cell capture was verified by direct visualization using the Array Scan system, allowing us to remove empty wells and wells with multiple cells. After quality control and adaptor trimming, the data was analyzed using SeqGeq software. pDCs and SMBCs were clustered using UMAP and pseudo-time analysis was performed using the Monocle program. Type I IFN activity in SLE plasma was measured using reporter cell assay. Results : A total of 2774 pDCs and 2578 SMBCs from SLE and healthy controls passed the quality control and were used for further analysis. In pDCs, we observed unique clusters for patients with high interferon, low interferon, and controls, indicating that the IFN response is a major determinant of overall gene expression patterns in SLE patient pDCs. IFN signature in pDCs correlated with circulating type I IFN activity in the SLE patients measured at the same time. Other genes upregulated in pDCs included the type I interferon regulator AXL and MACC1. The SMBCs were heterogeneous in patients and controls, and in contrast to the pDCs, the overall clustering pattern was independent of the IFN score. SMBC clusters were predominantly defined by genes indicating cellular activation or proliferation such as HLA-DRs and CREB1, or genes associated with nucleic acid processing such as DNASE1 and SNORD3B-1. Conclusion : We find distinct clusters of cells defined transcriptionally within the pDC and SMBC lineages, and the transcripts which define these subgroups differ between cell lineages. Type I IFN induced transcripts are important to pDC diversity, while in SMBCs transcripts related to cellular activation and nucleic acid processing are critical markers of transcriptional heterogeneity

Background/Purpose : Pregnancy counseling of all anti-Ro positive women includes advice regarding the development of congenital heart block (CHB), albeit the risk is only 2% for primigravida women or those with previously unaffected offspring. Despite this low risk, the prevailing surveillance recommendation is weekly echocardiography. While evidence from basic research laboratories support that high titers of antibodies confer clinically meaningful risk, unfortunately the majority of commercial laboratories use the BioPlex assay, which provides positive and negative values with limited information on actual levels because the sera or plasma are not diluted past a specified cutoffgiven cost (e.g. values of anti-Ro inclusive of Ro52 or Ro60 by laboratories such as Quest or LabCorp provide positive as 1-8 or > 8 units with no further information). The present study was initiated to assess whether the Bio-Plex assay used by many commercial laboratories provides adequate stratification of risk for counseling regarding management. Methods : The study group comprised healthy non-pregnant donors (N = 9), healthy pregnant donors (N = 62), women testing positive for anti-Ro by commercial BioPlex but without CHB children (N = 60 SLE and 2 SS), and women with CHB children (N = 83). Anti-Ro60 reactivity was assessed using native antigen and anti-Ro52 using recombinant protein. Sera were applied to coated microtiter plates at serial dilutions ranging from 1:1000 -1:50,000 for 1h at RT and run in duplicate. Tested samples were multiplied by the dilution factor which gives an OD in the range of 0.3-0.8. Results were considered positive at 123 ELISA units (EU) for Ro60 and 215 EU for Ro52 as this represented the mean +3 SD of the values obtained for healthy control sera. Results : Of the 83 CHB mothers tested, 74 had titers of Ro60 and Ro52 > 1000 EU, in 1 anti-Ro60 was > 1000 EU and anti-52 Ro between 215 -1000, in 3 anti-Ro52 was > 1000 EU and anti-Ro60 between 300 -1000, and 1 mother had anti-Ro60 > 1000 EU and was negative for anti-Ro52. Albeit all positive, the sera from 4 CHB mothers obtained 15 years after the birth of the affected child were < 1000 EU for both anti-Ro60 and Ro52. With these results setting thresholds ( > 1000 EU in either Ro60 or Ro52 for CHB risk), we assessed patients testing positive for anti-Ro based on the BioPlex assay. Of 42 patients with values of > 8 on BioPlex testing, 14 had titers > 1000 EU for both anti-Ro60 and Ro52, 7 had anti-Ro60 > 1000 EU, and 8 had anti-Ro52 > 1000 EU. Thus, 13 of 42 (25%) with commercial Ro > 8 did not meet the threshold EU for CHB risk. Of 20 patients considered positive for anti-Ro by BioPlex with values between 1-8, none had levels of either anti-Ro60 or Ro52 at 1000 EU. No patient or healthy control testing negative by the BioPlex assay was positive for CHB risk in our ELISA. Conclusion : These data suggest that commercial testing using the BioPlex assay may fall short of stratifying risk for CHB. Women with positive values < 8 are not likely at risk, obviating the cost and burden of weekly fetal echo surveillance. Moreover, even those considered high titer on commercial testing may be at low risk supporting the need for more quantitative commercial testing than is currently available. (Figure Presented)

Background/Purpose : In SLE Apolipoprotein L1 (APOL1) risk variants associate with cardiovascular and kidney damage. APOL1 is both a secreted and tissue intrinsic protein; the latter role mediates organ injury. Expression is driven by inflammatory stimuli which initially promotes survival through autophagy. At higher expression, APOL1 contributes to cell death partially by compromising mitochondria. The RV protein structure favors toxicity at lower thresholds. Macrophages express APOL1 and use metabolic plasticity to perform effector functions. We hypothesized that in activated macrophages, APOL1 expression impairs mitochondrial Methods : Healthy controls were genotyped for APOL1 reference allele (G0) risk variants (RV) (n=15; G0/G0=8, RV/ G0=4, RV/RV=3). Monocytes were isolated from PBMCs using a Miltenyi Biotec Pan Monocyte Isolation kit and differentiated into macrophages using GM-CSF. Cells were treated with SLE-relevant agonists ssRNA hY3 or IFNgamma; and APOL1 expression was observed by qPCR. As in-vivo correlates, APOL1 expression relative to IFN response gene, Siglec 1, in SLE patient monocytes (n=17); and expression in coronary artery tissue macrophages with (n=3) or without (n=3) atherosclerotic plaque were assessed through RNA seq data and immunohistochemistry respectively. To test mitochondrial respiration, cultured HC macrophages were left untreated or IFNgamma treated, and bioenergetics were measured by the Seahorse assay. Confirmatory fluorescent microscopy was done by staining cells with Mitoprobe (polarized mitochondria) and Mitotracker (all mitochondria) and the ratio of Mitoprobe to Mitotracker staining was quantified using ImageJ software. Results : Across the genotypes,hY3 and IFNgamma increased APOL1 mRNA expression 29.1+/-18.4 and 31.6+/-14.9 fold compared to untreated (p< 0.001 in each). In SLE monocytes, APOL1 significantly correlated with Siglec1 expression (R=0.64; P=0.005). APOL1 stained 4% of non-plaque containing and 18% of plaque-containing coronary arteries (p=0.05). APOL1 was apparent in multiple cell types including invading tissue macrophages (fig1). On the seahorse assay, there were genotype-dependent differences in Basal OCR (BO pmol/min), Spare Capacity (SC pmol/min), and total ATP production (ATP pmol/min) (G0/G0: BO: 99.4+/-11.3, SC:150.9+/-16.6, ATP: 88.1+/-9.5; RV/G0: BO: 46.1+/-4.5, SC: 52.1+/-8.8, ATP: 40.7+/-3.4; RV/RV: BO: 46+/-17.4, SC: 14.9+/-11.6, ATP: 36.7+/-9.5 each p< 0.001). Across genotype, these values fell with IFNgamma (G0/G0: BO: 73.9+/-6, SC: 129.4+/-17.3, ATP: 67.6+/-5.4; RV/G0: BO: 46+/-5.1, SC: 27.9+/-8.1, ATP: 39.5+/-3.9; RV/RV: BO: 42.1+/-8.6, SC: 9.5+/-8.3, ATP: 35.8+/-6.9) (fig2). Fluorescent microscopy confirmed findings with the mean respective MitoProbe/Mitotracker ratios in G0/G0, RV/G0, and RV/RV macrophages at rest 1.5+/-0.5, 0.86+/-0.3, and 0.89+/-0.3 and with IFNgamma 1.3+/-0.4, 0.74+/-0.3, and 0.6+/-0.2 (fig3). Conclusion : Inflammation both in vitro and in vivo due to hY3, IFNgamma, and ischemia increase macrophage APOL1 expression across genotypes. In RV carrying macrophages, this results in diminished mitochondrial energy production-a potential underpin of variant-mediated toxicity. (Figure Presented)