Faculty Bibliography

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 : 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 : 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)

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 : 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

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.

OBJECTIVES/OBJECTIVE:The spectrum of antinuclear antibodies (ANA) is changing to include both nuclear staining as well as cytoplasmic and mitotic cell patterns (CMPs) and accordingly a change in terminology to anti-cellular antibodies. This study examined the prevalence of indirect immunofluorescence (IIF) anti-cellular antibody staining using the Systemic Lupus International Collaborating Clinics inception cohort. METHODS:Anti-cellular antibodies were detected by IIF on HEp-2000 substrate utilizing the baseline serum. Three serological subsets were examined: 1) ANA-positive (presence of either nuclear or mixed nuclear/CMP staining), 2) anti-cellular antibody-negative (absence of any intracellular staining), and 3) isolated CMP staining. The odds of being anti-cellular antibody-negative versus ANA or isolated CMP-positive was assessed by multivariable analysis. RESULTS:1137 patients were included; 1049/1137 (92.3%) were ANA-positive, 71/1137 (6.2%) were anti-cellular antibody-negative, and 17/1137 (1.5%) had isolated CMP. The isolated CMP group did not differ from the ANA-positive or anti-cellular antibody-negative group in clinical, demographic or serologic features. Patients who were older (OR 1.02 [95% CI: 1.00, 1.04]), of Caucasian race/ethnicity (OR 3.53 [95% CI: 1.77, 7.03]), or on high dose glucocorticoids at or prior to enrolment (OR 2.39 [95% CI: 1.39, 4.12]) were more likely to be anti-cellular antibody-negative. Patients on immunosuppressants (OR 0.35 [95% CI: 0.19, 0.64]) or with anti-SSA/Ro60 (OR 0.41 [95% CI: 0.23, 0.74]) or anti-UI-RNP (OR 0.43 [95% CI: 0.20, 0.93]) were less likely to be anti-cellular antibody-negative. CONCLUSIONS:In newly diagnosed SLE, 6.2% of patients were anti-cellular antibody-negative and 1.5% had isolated CMP. The prevalence of anti-cellular antibody-negative SLE will likely decrease as emerging nomenclature guidelines recommend that non-nuclear patterns should also be reported as a positive ANA.