Faculty Bibliography

Background/Purpose : Many studies of cancer risk in SLE are limited by small sample size or use of administrative data, which rely on billing code diagnoses instead of clinical data. No studies to date focused on incident SLE. We studied cancer risk in the largest-ever cohort of clinically confirmed incident SLE patients. Methods : Patients meeting ACR criteria for new-onset SLE (within 15 months of diagnosis) were enrolled into the SLICC Inception Cohort, across 32 centres. Patients are followed yearly using a standard protocol, with detailed data collection including SLE Disease Activity Index-2000 (SLEDAI-2K) and damage, and drugs in the past year. New cancer diagnoses are recorded by the examining physician at the annual study visit, and confirmed with chart review including pathology reports. Multivariate proportional hazard regression was performed, using baseline variables for demographics (age at SLE onset, sex, race/ethnicity), and time-dependent variables for drugs (corticosteroids, anti-malarial drugs, immunosup-pressive drugs), smoking, and SLEDAI-2K. As well as cancer over-all, we evaluated risk factors for the most common cancer types. Results : Of 1848 new-onset SLE patients enrolled between 1999-2011, 1668 had at least one follow-up; these were the sample for the current analysis. End date was the first of death, last visit, or end of study interval for this analysis (Aug. 2015). Baseline demographics are shown in Table 1. Over 14,215 years (mean 8.5 years) there were 60 cancers (incidence 4. 2 events per 1,000 patient-years). This included 12 breast cancers, 9 non-melanoma skin, 7 lung, 6 hematological, 5 melanoma, 5 prostate, 3 cervical, 3 renal, 2 gastric, 2 head and neck, 2 thyroid, and one each rectal, sarcoma, thymoma, and uterine. Almost half of the cancer cases (including all of the lung cancers) were associated with baseline smoking, versus only one-third of those patients who did not develop cancer. Univariate analyses of all cancer types suggested a higher risk of cancer among patients of white race/ethnicity and among those with the highest quartile of disease activity at cohort entry. However, the multivariate proportional hazard regression indicated that among SLE patients, the over-all cancer risk was related primarily to male sex and older age at SLE diagnosis. In those analyses, the effect of race/ethnicity was not clearly evident, and the point estimate for highest quartile of disease activity actually reversed to suggest a nonsignificant trend towards lower cancer risk. In the multivariate analyses specifically for breast cancer, age at SLE diagnoses remained a risk factor, and antimalarials were associated with a decreased risk. This effect of anti-malarials was not clearly seen for any other cancer type. For non-melanoma skin cancer, both age at SLE diagnosis and cyclophosphamide were strongly linked with risk. Conclusion : This is the first large, multicentre cohort study to clearly show how different cancer types in SLE are associated with specific risk factors. Additional follow-up may allow additional determination of the possible effects of disease activity and drugs on cancer subtypes

Background/Purpose : Different autoimmune diseases can co-exist in an individual and share similar genetic associations, autoimmune signaling pathways, and clinical manifestations. However, autoimmune diseases present varied cellular heterogeneities and may be distinguished by their primary target organs or tissues. The immune mechanisms that are shared between similar autoimmune diseases remain poorly understood due to limited access to affected human tissues and computational scalability. Recently, high resolution single-cell RNA-seq profiles have provided the opportunity for study of the contribution of diverse cell populations to disease pathogenesis. This advance has enabled unbiased comparison of disease across affected tissues with the goal of understanding autoimmune similarities. Methods : We have analyzed and integrated ~80,000 cells from 176 donors from publicly available single-cell RNAseq datasets generated from rheumatoid arthritis (RA) and osteoarthritis (OA) synovium 1 , pulmonary fibrosis and healthy lung, systemic lupus erythematosus (SLE) and healthy kidney and skin 2,3 , inflammatory bowel disease (IBD) and healthy colon, and Crohn intestinal mucosal biopsies. We use a robust integrative strategy 4 to cluster and project all the cells into two dimensional-space by correcting technical batch effect across tissue, donor, and single-cell platform (10X Genomics, Celseq2, Dropseq, Fluidigm, and Smartseq). Results : We identify 21 diverse cell type populations across multiple tissue sources (Figure 1A). In the myeloid cell population, we observed four distinct subsets including VSIG4+ M2-like macrophages, S100A8+ macrophages, SPP1+ MMP9+ inflammatory macrophages, and dendritic cells (DC). The SPP1+ MMP9+ inflammatory macrophage population with high expression of matrix metalloproteinases genes is co-localized between macrophages in RA synovium, SLE kidney, and alveolar from pulmonary fibrosis lung, and is absent in healthy lung tissue (Figure 1B). For CD8 T cells and nature killer cells, we identified a shared transcriptional gradient of granzyme-expressing cytotoxic effectors between RA synovium, SLE kidney, and fibrotic lung. Interestingly, the GzmK+ CD8 T cell population is absent in the healthy lung (Figure 1C). The patterns of cytotoxic effector states may suggest similarities between theprimary sites of inflamed RA and inflamed fibrotic lung, including potential common active pathways. In the stromal cell compartment, we observed distinct populations across different diseases and tissues, including fibroblasts, pericytes, mesangial, and tubular cells. Conclusion : We demonstrate that integrative analyses between disease tissues by single-cell transcriptomics is capable to discovering shared and unique disease-specific gene expression modules and cell states, and may help predict potential therapeutic targets for inflammatory and fibrotic diseases

Background/Purpose : Lupus nephritis (LN) complicates up to 60% of patients with systemic lupus erythematosus (SLE) and carries a high morbidity and mortality. The definitive diagnosis is based on kidney biopsy. This is invasive and not always readily available, thus delaying treatment. Sometimes multiple biopsies are required over the course of the disease. Importantly, while renal pathology is accurate at describing the morphology of renal disease, the underlying biology and molecular pathways are not thoroughly assessed. Urine proteomics is a non-invasive strategy that may provide insights regarding ongoing renal disease. Methods : One thousand proteins were quantified (RayBiotech Kiloplex assay) on a total of 112 longitudinal urine samples from 32 SLE patients with active LN and 7 healthy controls (HC) enrolled in the Accelerating Medicines Partnership (AMP). All patients underwent treatment as directed by their own physicians. Differentially excreted proteins at baseline (SLE vs HC, proliferative vs membranous LN, responders vs non responders) were identified using a linearmodel with moderated t statistic. Response to treatment was defined based on proteinuria at 1 year as complete (< 0.5g/24h) or partial (50% reduction but >0.5/24h). In the longitudinal analysis, a mixed model was employed to identify markers associated with proteinuria. Pathway enrichment analysis was performed using the genes coding for the differentially excreted analytes using Ingenuity Pathway Analysis (IPA) and other publicly available pathway libraries. Results : There were 186 proteins increased in SLE patients (Fig. 1). The most enriched pathway was TNFa (p< 0.001). We found 74 differentially excreted proteins comparing proliferative and pure membranous LN. CD4, MCP-1, MIP-1a, RANTES, IL-16, and IL-7, markers involved in CD4 T cell and monocyte biology, were enriched in proliferative disease. A few targets were exclusively identified in either class (i.e. CD4 in proliferative nephritis). We used a longitudinal model to identify specific urine proteins associated with worse proteinuria as a marker of severity. Proteinu-ria was associated with 105 markers (FDR < 0.05), the strongest association being CD163 (p = 10-9), a phagocyte marker. IPA implicated several pathways involving fibrosis, acute phase response, LPS/IL1, RXR, ICOS signaling and macrophage/fibroblasts (Fig. 2). Next, we identified 27 differentially excreted proteins in non-responders. IPA revealed that tretinoin, GM-CSF, TNF, and IL1 were among the top upstream regulators (Fig. 3). Conclusion : There is an inflammatory signature in the urine of patients with LN implicating monocyte and TNFa pathways. These signatures are associated with proliferative disease, worse proteinuria, and non-response to treatment. Of note, TNFa is involved in LN and has therapeutic potential. In phase 1 of AMP, monocytes were the main urine cell type identified by singe cell RNA sequencing in patients with LN. These results suggest that urine proteomics might identify and infer active pathological mechanisms in LN, paving the way for a more personalized approach to treatment. Further work in Phase 2 of AMP is being pursued to validate and extend these findings

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)

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 : Since one of the strongest associations with antibodies (abs) to SSA/Ro (Ro60) is the development of congenital heart block (CHB), this model provides an exceptional opportunity to define novel insights that link maternal abs with an inflammatory cellular response which eventuates in fibrotic replacement of the AV node. We recently compiled risk genes based on an agnostic transcriptomic survey of macrophages isolated from hearts of fetuses dying with CHB and healthy aged matched fetuses electively terminated, noting that IFN related genes (IRGs), including IFN induced Protein with Tetratricopeptide Repeats 1(IFIT1) and Sialic Acid Binding Ig Like Lectin 1 (SIGLEC1), are highly upregulated in the CHB hearts. Accordingly, this study addressed the hypothesis that IRGs contribute to CHB pathogenesis. Methods : hY3 RNA, a noncoding ssRNA and TLR7/8 agonist, was used as a proxy of the Ro60 immune complex. Human derivatives included healthy peripheral blood macrophages and fibroblasts isolated from a healthy human fetal heart. Neutralizing IFNalpha and IFNbeta abs were used to assess the contribution of the respective cytokines to the model. Macrophage readouts included the expression of IFIT1 and SIGLEC1 transcripts (qPCR, units, fold change based on 2-DELTADELTACT, relative expression of transcript normalized to GAPDH) and myofibroblast phenotype (EdU imaging and SMAc by IF, respectively). Results : As expected, exposure of macrophages to IFNalpha resulted in a significant upregulation of IFIT1 and SIGLEC1 compared to untreated macrophages (70+/-25 vs 1, and 17+/-9, vs 1, respectively with both N=3, P< 0.05). Similarly, exposure to IFNbeta also resulted in the upregulation of these transcripts (254+/-237 vs 1, p=0.03, and 21+/-14 vs 1, respectively with both N=4, p< 0.03). The expression of these transcripts by IFNalpha-and IFNbeta-treated macrophages was completely attenuated by co-treatment using respective Type I IFN-specific neutralizing antibodies. In parallel, transfection of human macrophages with hY3 also resulted in upregulation of IFIT1 (112+/-30 vs 1, p=0.02, N=3) and SIGLEC (13+/-7 vs 1, N=3). To confirm TLR7/8 dependency of IRGs, the addition of TLR7/8 antagonist IRS661 to our in vitro model resulted in a significant decrease of IFIT1 expression to 14% (14+/-10, n=6) and SIGLEC1 to 54% (7+/-5, n=7, both P=0.03). Co-treatment with neutralizing antibody against IFNalpha reduced the expression of IFIT1 to 9% (10+/-9, n=3) and SIGLEC1 to 35% (5+/-3, n=3). Co-treatment with neutralizing antibody against IFNbeta also reduced the expression of IFIT1 to 24% (24+/-6, n=2) and SIGLEC1 to 59% (3+/-5, n=3). For a survey of direct effects of type I IFN, IFNalpha and IFNbeta were shown sharing the capacity to stimulate fibroblast proliferation (EdU, % positive) yielding a result of untreated (16%), IFNalpha (40%), and IFNbeta (48%). In addition, exposure of human fibroblasts to IFNalpha as well as IFNbeta induced expression of the myofibroblast marker, SMAc (IF) versus no expression by the untreated fibroblasts. Conclusion : These results suggest that type I IFN contributes to the inflammatory and profibrosing milieu associated with the development of CHB. Feed forward expression of IFN related genes in response to TLR signaling may provide new targets towards the prevention of disease

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

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 : Osteoporosis and bone fractures are a frequent cause of morbidity in systemic lupus erythematosus (SLE), and are felt to be related both to disease activity and glucocorticoid (GC) exposure. Dual energy X-ray absorptiometry (DEXA) is the standard tool to assess bone density, but it does not measure bone quality or strength and is not a robust predictor of fractures in SLE. Clinical 3T MRI scans have been shown to assess information about bone not captured by DEXA. This study aims to evaluate factors associated with bone structure measured by DEXA and MRI in an SLE cohort. Methods : DEXAs were performed on 31 women with SLE and 3T MRI of the non-dominant hip were performed on 29 of these cases. Results were associated with multiple demographic, clinical and laboratory measures. MRI parameters measured included trabecular plate width (PW), trabecular plate to rod ratio (PRR), plate volume fraction (PVF), rod volume fraction (RVF), trabecular bone thickness (Tb.Th), trabecular spacing (Tb.Sp) and trabecular network area (TNA). DEXA BMD was measured, and osteoporosis (OP) was defined as hip, spine or femoral neck Z score < -2.0 in premenopausal women, and T score < -2.5 in others, and low bone density (LBD) as Z score < -2.0 in premenopausal women and T score < -1.0 in others. Results : By DEXA, 8/31 (25.8%) had OP and 12 (38.7%) had LBD. History of lymphopenia (75.0% vs. 31.8%, p=0.049) and lower concurrent HCQ dose (340 vs. 400 mg, p=0.006) associated with DEXA OP, while older age (48.3 vs. 36.3 y, p=0.024) associated with LBD. Higher ESR was inversely correlated with favorable bone structure (PW r(22) = -.49, p=0.025, PRR rs = -.51, p=0.018, PVF rs = -.51, p=0.018, RVF rs = .51, p=0.018, Tb.Th rs = -.58, p=0.005, Tb.Sp rs = .44, p=0.046, TNA rs = -.50, p=0.022). Higher CRP was likewise inversely correlated with favorable bone structure (PW r(20) = -.61, p=0.004, PRR rs = -.57, p=0.009, PVF rs = -.57, p=0.009, RVF rs =.57, p=0.009, Tb.Th rs = -.56, p=.011, Tb.Sp rs =.67, p=0.001, TNA rs = -.64, p=0.002). A history of lupus nephritis was associated with unfavorable bone structure (PW 705.3 vs. 833.3 mum, p=0.048, PRR 6.6 vs. 8.1, p=0.024, PVF 0.83 vs. 0.89, p=0.024, RVF 0.17 vs. 0.11, p=0.024, Tb.Th 178.1 vs. 193.4 mm, p=0.012, Tb.Sp 358.6 vs. 296.5 mm, p=0.056, TNA 0.41 vs. 0.54 (1/mm), p=0.009). ESR, CRP and history of lupus nephritis were not significantly associated with DEXA hip BMD, OP or LBD. MRI parameters for favorable bone structure were inversely correlated with DEXA hip BMD (PW r(28) = -.47, p=0.011, Tb.Th rs = -.53, p=0.003) and BMI (PW r(28) = -.54, p=0.003, TbTh rs = -.72, p< 0.001, TNA rs = -.44, p=0.017). Conclusion : Higher ESR and CRP and a history of lupus nephritis associated with MRI parameters of unfavorable bone structure, but did not associate with DEXA abnormalities in SLE patients. MRI may be a more sensitive tool than DEXA to measure inflammatory effects on bone and potentially cumulative dose of steroid exposure. There were inverse correlations of MRI parameters with traditional osteoporosis risk factors and BMD measures on DEXA, and it is possible that each tool evaluates different aspects of bone health. Further evaluation of MRI screening for fracture risk in SLE and GC exposed individuals is warranted to better quantify risk and guide treatment