Faculty Bibliography

Background/Purpose: Histologic correlates of anti-Ro associated congenital heart block (CHB) are apoptosis and calcification of cardiomyocytes with fibrosis of the AV node surrounded by infiltrating macrophages and giant cells. This study leveraged an unprecedented opportunity to interrogate the transcriptome of different cell types in a fetal heart dying with CHB. Methods: Aortas from a 19 wk CHB fetal heart and a healthy 22 wk heart were cannulated using a Langendorff preparation with proteolytic enzymes to yield a single cell suspension. DAPI negative cells were isolated by flow using antibodies to CD14/CD45, CD31, and podoplanin, to yield leukocytes, endothelial cells, and fibroblasts, respectively. After RNA isolation and cDNA library preparation, RNA-Seq and transcriptome analysis were performed. Expression of lineage markers was consistent with the isolates based on flow markers. Data are expressed as log2 transcripts per million. Results: Transcriptomes of the two hearts for each isolated fraction were compared. For leukocytes, in CHB vs healthy there were 5000 genes greater than a threshold ratio of 0.78 (expressed as log2 difference). Based on the DAVID annotation, data were organized into clusters of closely related genes. Within the term inflammatory response (p=1.66E-4) for the ratio of CHB/healthy, the genes were IL8 (4.13), IL6 (6.72), TNFa (0.78), and EDN1 (5.17). In addition, leukocyte gene expression of FCGR3A, TLR7 and IRF5 was higher in CHB vs control (2.6,1.37 and 0.91, respectively), a result supporting that the requisite machinery is upregulated to effect anti-RohYRNA ligation and activate macrophages via TLR signaling. For endothelial cells, 7000 genes exceeded a ratio of 0.78 for CHB/healthy. Within the term inflammatory response (p=1E-5), FOS, FOSB, NFKB1A, and NFKBIZ were expressed with ratios of 1.47, 2.46, 1.03, and 2.75. Within the categories IMMUNE (p=4E-2) and cell adhesion (p=1.42E-06) higher expressed genes included CTGF (2.06), PTGS2 (2.53), SOCS3 (1.56), and OAS3 (2.52) along with ICAM1 (2.12), CCL2 (2.75), IL32 (2.81), VCAM1 (3.85), and SELE (2.4). Likewise within the death category (2.62E- 08), PPP1R15A (1.39), XAF1 (3.77), GADD45B (1.94), and TNFAIP3 (3.06) were increased in CHB. These data support endothelial cells in leukocyte recruitment. For the fibroblasts, 4500 genes were above the CHB/healthy threshold (Table 1). CHB fibroblasts showed increased expression of pro-fibrotic genes while attenuating anti-fibrotic genes. The cardiomyopathy marker, XIRP2 (3.15), as well as genes resisting and promoting vascular stiffness (ELN [-2.23] and TTN [2.12], respectively) were also identified. Conclusion: These data support that autoimmune CHB is a complex disease with contributions from death pathways, inflammation and fibrosis. Scarring likely results from a multi pronged pro-fibrotic environment whereby fibrosis promoting genes undermine genes that forestall fibrosis. (Table Presented)

Background/Purpose: Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by heterogeneity of presentation, an undulating course, and elevated risk for premature cardiovascular disease. Platelets have been understudied as a relevant contributor. Yet, these cells, which contain transcripts and the necessary molecular machinery to conduct translation, are intercellular regulators of inflammation and immune activation and play a key role in atherothrombosis. Platelets express low affinity type 2 receptors (FcgammaRIIA) whose ligand is the Fc portion of IgG. A single amino acid substitution, H131R, in the extracellular ligand binding domain increases the affinity for IgG and may account for individual variation in platelet activation, specifically an increase of function. Accordingly, this study addressed the hypothesis that FcyRIIA genotype associates with preclinical atherosclerosis and platelet hyperreactivity. Methods: Genotyping at rs1801274 (allelic discrimination, HWE P=NS) was performed in 71 SLE patients and 30 healthy controls. In 49 of the SLE patients and 30 healthy controls, carotid ultrasound for plaque (>50% increase over background IMT in any arterial segment); levels of soluble E-selectin as a proxy of endothelial cell activation; and C3, C4 to reflect complement activation were assessed. In 22 SLE patients, monocyteplatelet (MPA) and leukocyte-platelet aggregates (LPA), and light transmission aggregometry (LTA) in response to submaximal concentrations of collagen and arachidonic acid were evaluated. Results: Overall genotyping for FcgammaRIIA revealed 43 SLE patients carrying at least one copy of the variant allele and 28 patients who were homozygous for the ancestral allele. For the 49 with IMT, carotid plaque was reported in 22. A significant enrichment of carotid plaque was identified in patients with a variant compared to those who were homozygous ancestral (58% vs 25%, p=0.039). In contrast, among 30 healthy controls, the presence of carotid plaque was not associated with the variant or ancestral genotype (15% vs 15%). Soluble Eselectin (mean + 2SD, shown as dichotomous being above normal controls) was significantly increased in those patients with the variant vs ancestral (64% vs 23%, p=0.013). Complement levels, a proxy of circulating immune complexes, were lower in patients with the variant vs ancestral (64% vs 40%). With regard to platelet reactivity, among 22 SLE subjects evaluated, there was a significant increase in MPA and LPA (above controls, mean + 2SD) in those carrying at least one variant compared to the ancestral group (86% vs 37%, p=0.02 and 46% vs 12%, p=0.05, respectively). Platelet aggregation was more robust for those patients with the variant vs ancestral in response to 160 uM arachidonic acid and 1 ug/mL collagen (47% vs 19% and 47% vs 14%, respectively). Conclusion: These data suggest a model in which an FcgammaRIIA polymorphism associates with preclinical atherosclerosis and confers increased platelet activity in the setting of SLE, a disease characterized by circulating immune complexes

Background/Purpose: Little is known about the association of healthcare costs with damage accrual in SLE. We describe the costs associated with damage states across the disease course using multi-state modeling. Methods: Patients fulfilling the revised ACR classification criteria for SLE from 32 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, SLE disease activity (SLEDAI-2K), damage (SLICC/ACR Damage Index [SDI] if > 6 months from diagnosis), hospitalizations, medications, dialysis, and utilization of selected medical/surgical procedures were collected. Annual health resource utilization was costed using 2015 Canadian prices. Annual costs associated with SDI states were obtained from multiple regressions adjusting for age, race/ethnicity, and disease duration. As there were relatively few transitions to SDI states 5 - 11, these were merged into a single SDI state. Five and 10-year cumulative costs were estimated by multiplying annual costs associated with each SDI state by the expected duration in each state, which was forecasted using a multi-state Markov model (Bruce IN et al. Ann Rheum Dis 2015;74:1706-13). Results: 1641 patients participated, 89.2% female, 48.9% Caucasian, mean age at diagnosis 35.2 years (SD 13.4), mean disease duration at enrollment 0.5 years (SD 0.3), and mean follow up 6.1 years (range 0.1 - 13.7 years). Health resource utilization and annual costs (after adjustment using regression) were markedly higher in those with higher SDIs Conclusion: Patients with the highest baseline SDIs incur annual costs and 10-year cumulative costs that are approximately 20-fold higher than those with the lowest baseline SDI. By estimating the expected duration in each SDI state and incorporating annual costs, disease severity at presentation can be used to predict future healthcare costs, critical knowledge for cost-effectiveness evaluations of novel therapies. (Table Presented)

Background/Purpose: Previous studies have established the value of measuring complement activation products (C4d) bound to platelets (PC4d) for the diagnosis and monitoring of Systemic Lupus Erythematosus (SLE). Separately, Antiphospholipid (APL) antibodies have been associated with complement activation and PC4d expression. In this study, we sought to validate the performance characteristics of PC4d, stratified by the presence or absence of APL antibodies. Methods: This multi-centered validation cross sectional study (16 sites in the US) enrolled 402 SLE subjects fulfilling the 1982 American College of Rheumatology Criteria revised in 1997 (mean age 41 years; 91% female), 411 subjects with rheumatic and autoimmune diseases other than SLE (mean age 55, 86% female consisting of 181 rheumatoid arthritis, 90 primary fibromyalgia, 92 other rheumatic diseases, and 48 autoimmune thyroiditis or hepatitis) and 198 healthy volunteers (mean age 41 years; 66% female). PC4d densities were determined using flow cytometry (expressed as mean fluorescence intensity [MFI]). Positive PC4d consisted of PC4d levels greater than 20 net MFI. Anticardiolipin IgG, anti-Beta-2-glycoprotein 1 IgG, or anti-Phosphatidylserine/Prothrombin (PSPT) complex IgG antibodies were determined using ELISA (INOVA diagnostics, San Diego, CA). Presence of APL antibodies consisted of any of these antibodies above manufacturer cutoff. SLE Disease activity was assessed using the non-serological SLE Disease Activity Index SELENA modification (ns-SELENA-SLEDAI, without the complement and anti-dsDNA). Performance characteristics were established using sensitivity, specificity, and ROC Curve Area Under the Curve (AUC). Statistical evaluation was by t-test (for disease activity), by chi-squared test for equality of proportions (for sensitivities and specificities) and by the method of DeLong (for ROC Curve AUC). Results: PC4d was highly specific in distinguishing SLE from other rheumatic diseases (Table) and normals. Among SLE subjects, 47% (n=187) presented with at least one APL antibody as compared to 21% (n=86) of subjects with other diseases and 15% of normals. PC4d sensitivity for SLE was higher among APL positive subjects by comparison to APL negative subjects (p=0.003). Specificity was not significantly different between APL positive and negative subjects (p>0.372). ROC AUC was significantly higher among the APL positive compared to negative subjects (p=0.002). The incidence of APL antibodies among all PC4d positive subjects was 60% compared to 27% among PC4d negative subjects (p<0.001). SLE subjects presenting with positive PC4d had higher disease activity (4.1+/-0.5) than those presenting with negative PC4d (3.0+/-0.2) (p=0.03). Conclusion: We confirm that PC4d is highly specific for SLE, and is associated with disease activity. (Table Presented)

Background/Purpose: SLE is a complex multifactorial systemic autoimmune disease, which has been attributed to poorly understood interactions between genetic and environmental factors. Recent reports have begun to elucidate how imbalances within intestinal communities of commensal bacteria may lead to triggering of inflammatory and autoimmune conditions. Our studies are designed to shed light on the interactions of the immune system and the gut microbiome that may contribute to lupus pathogenesis. Methods: We have assembled a cohort of 60 female SLE patients and matched 20 healthy controls, and biobanked blood and stool samples. DNA was then extracted from fecal bacterial samples, and from sorted endogenous IgA-coated and non-coated bacterial fractions. 16S bacterial rRNA gene sequencing was then performed by illumina NGS technology. Fecal and serum total Igs and autoantibodies were measured by ELISA and by multiplex-bead autoantigen assays. Results: Our analyses showed that SLE patients have significantly reduced diversity (i.e., number of different taxa) in their gut microbiomes compared to controls (p=0.038). This dysbiosis was treatment independent, with more marked contractions in patients with high disease activity, based on SLEDAI (p=0.002). The distribution of microbiome taxa was more heterogeneous among SLE patients than healthy individuals (p=0.002). Interestingly, patients with the most active disease commonly displayed expansions of genus and species with putative pathobiont properties, with reciprocal contractions of others associated with protective properties (e.g. R. gnavus (p= 0.001) vs. F. prausnitzii (p= 0.022) and B. uniformis (p=0.016). In immunologic surveys, we found that IgA (the most highly produced Ig isotype in the body), was significantly elevated in SLE patients compared to healthy subjects (p<0.002). Only a limited proportion of bacterial taxa are specifically coated by endogenous intestinal IgA. Yet, SLE patients with high disease activity displayed an increased abundance among IgA-coated taxa of Prevotella copri (p= 0.018), a species which has recently been linked to new-onset RA. In addition, intestinal IgA in SLE patients included high levels of antibodies to lupus autoantigens, with the same IgA autoantibody profiles in the matched sera of individual patients. Conclusion: Our studies document that SLE is associated with a dysbiosis in the gut microbiome with expansions of specific pathobiont bacteria and reciprocal contractions that may contribute to immune dysregulation. This imbalance was more significant in patients with high disease activity. Certain microbial taxa/species are preferentially recognized by the adaptive immune system of SLE patients and coated in vivo by intestinal IgA, and this correlated with elevated overall levels of fecal and serum IgA. Taken together, these data support the hypothesis that the pathogenesis of SLE may arise from imbalances in the gut microbiome and immune recognition of certain bacterial taxa

Background/Purpose: Few studies have analyzed the risk factors for thrombosis in Systemic Lupus Erythematosus (SLE) patients with antiphospholipid antibodies (aPL) and most had small sample sizes and homogenous patient populations. We examined whether a history of nephritis is an additional risk factor for thrombosis on a large multi-ethnic SLE cohort database. Methods: The NYU SLE SAMPLE biorepository and registry was initiated in September 2013 and includes 612 patients fulfilling ACR and/or SLICC criteria for SLE. Within SAMPLE, we identified patients with a positive aPL test (lupus anticoagulant, IgG or IgM anti-beta2- glycoprotein-I antibodies and/or IgG or IgM anticardiolipin antibodies) and determined if these patients had also ever fulfilled nephritis criteria. We reviewed each patient's medical record for presence/absence of venous and arterial thrombosis, and obstetric events as well as the non-criteria manifestations such as thrombocytopenia or valvulitis. We compared the prevalence of antiphospholipid syndrome (APS) manifestations in SLE patients with and without a history of lupus nephritis. Results: Of the initial 612 SLE patients (90% female; mean age 43.0+/-0.9 years), 54% were Caucasian, 31% African American, 15% Asian; 30% Hispanic white, and 5% Hispanic Black. Of the 612 patients, 105 had aPL, including 93 females and 12 males (mean age 43.0+/-0.2 years), 56% Caucasian, 33% African American, 11% Asian, 24% Hispanic white and 4% Hispanic Black. The total number of patients with thrombotic events was 45/105 (43%), including 26/43 (60%) in the nephritis subset and 19/62 (30%) (p=0.04) among patients without prior renal involvement. The most common thrombotic event was deep vein thrombosis (DVT) followed by stroke. Conclusion: The prevalence of APS criteria in the NYU SLE SAMPLE was 17%. Within this group, adverse events were more common among the patients with, versus without, a prior history of renal involvement (60% vs 30%). It remains uncertain if this association can be explained by, for example, by the presence of other accompanying findings that distinguish nephritis from non-nephritis SLE (e.g. anti-dsDNA, complement consumption) or drug treatment. This observation provides further support for the universal use of hydroxychloroquine in SLE, especially in those with current or previous nephritis and suggests that event-free aPL positive patients with a prior history of nephritis may be at increased risk for future thrombosis. Further studies are needed to determine whether such patients could benefit from prophylaxis with low-dose aspirin, statins or even other mild anti-thrombotic agents

Background/Purpose: Maternal autoantibodies (Ab) reactive with the Ro/La ribonucleoprotein complex are associated with the development of cardiac injury in a fetus passively exposed to these Ab. This study evaluated the irreversible scarring phenotype characteristic of heart block involving the mitogen activated protein kinase (MAPK) pathway and its regulation by cAMP. Methods: The aorta from a healthy 2nd trimester fetal heart was cannulated using a Langendorff preparation with the addition of proteolytic enzymes to yield a single cell suspension of primary human fetal cardiac fibroblasts. Cultured cells were treated with secreted products generated from activated macrophages with or without BIX 02189 (10uM, a specific MEK5/ERK5 inhibitor) and forskolin (10uM to raise cAMP). After RNA isolation and cDNA library preparation, RNA-Seq, transcriptome analysis (Data as log2 transcripts per million) and qPCR were performed. Results: Incubation of fibroblasts with supernatants from macrophages transfected with hY3 (ssRNA associated with Ro60), shown to induce a pro-fibrotic phenotype, resulted in the increased expression of 3836 genes. Based on DAVID functional annotation, the top clusters represented were Actin Binding, Cytoskeletal Protein Binding, Cell Adhesion, Signal Peptide, and Contractile Fiber, all processes considered typical of the myofibroblast phenotype. In addition, RAPGEF3, an endogenous ERK5 inhibitor, and Adrenomedullin, which increases cAMP, were downregulated while PDE4D, an inhibitor of cAMP generation, was upregulated (Table 1). These data are consistent with previous literature supporting the association of lowered intracellular cAMP and upregulation of pro-fibrotic genes. Given that cAMP attenuates the activity of ERK5, BIX 02189 was used to evaluate the transcriptome. Of the 3836 genes upregulated by hY3 macrophage supernatants, 617 were reversed by the subsequent addition of BIX. Among the upregulated genes were pro-fibrosing genes such as EDN1 and TGFbeta2 and among those downregulated were genes that resist fibrosis including CLU, RAPGEF3, and ADM. The latter two are associated with a cAMP dependent inhibition of ERK5 and increased cAMP, respectively. The pro-fibrosing EDN1 result was confirmed by qPCR and as expected was attenuated by forskolin. Conclusion: These data support that the link of anti-Ro immune complex activated macrophages and the pathogenic fibroblast phenotype may relate to a decrease in cAMP levels. These results highlight potential novel targets for therapy and solidify the role of ERK5 in the transdifferentiation of fetal fibroblasts in the context of congenital heart block. (Table Presented)

Background/Purpose: Thrombocytopenia is a common feature of both systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) and in the former most frequently results from antiplatelet antibodies (i.e., AITP) or aPL antibodies. Patients with AITP paradoxically have an increased risk of thrombosis and it has been speculated that this can result from co-presence of aPL. Primary APS is associated with both thrombosis and thrombocytopenia. We assessed whether there was an association between a history of thrombocytopenia and the prevalence of a thrombotic event in a large, multiethnic cohort of SLE patients. Methods: We analyzed the NYU SLE SAMPLE registry, consisting of patients fulfilling ACR and/or SLICC criteria for SLE. We identified 105 patients whose SLE criteria included the presence of one or more of the following aPL antibodies: lupus anticoagulant, IgG or IgM anti-beta2-glycoprotein-I, and/or IgG or IgM anticardiolipin antibodies; and determined whether these patients had thrombocytopenia (<100,000/muL) recorded among their SLE classification criteria. We reviewed each patient's medical record to identify the prevalence of arterial or venous thrombosis (defined as DVT, PE, stroke, arterial occlusion with gangrene or amputation), and/or obstetric events. We compared the prevalence of thrombotic events in SLE patients with and without history of thrombocytopenia. Results: The NYU SLE SAMPLE currently includes 612 patients (90% female, mean age 43.0+/-0.9 years, and mean age of 41.0+/-0.3 years in the males); 54% of the subjects were Caucasian, 31% African American, 15% Asian, 30% Hispanic White, and 5% Hispanic Black: 17% had aPL antibodies, of whom 89% were female and 11% male (mean age 43.0+/-0.2 years, 56% Caucasian, 33% African American, and 11% Asian, 24% Hispanic white and 4% Hispanic Black). The total numbers of patients with thrombotic events were 45 (43%), with 5/21 (23%) in the SLE patients with aPL and prior history of thrombocytopenia and 40/84 (47%; p=0.042) in the patients without thrombocytopenia. The most common thrombotic event was DVT followed by stroke. Conclusion: The prevalence of aPL in the NYU SLE registry was 17%, and adverse thrombotic events were less common in the patients with prior history of thrombocytopenia (5/21, 23%) as compared to those without (40/84, 47%). This unexpected finding could be explained by protective benefit of anti-platelet antibodies when co-occur with aPL in SLE, or less thrombogenic aPL in SLE when there is concomitant thrombocytopenia. Additionally, our data suggest that there might be different consequences of aPL between primary APS patients and SLE patients with aPL

Background/Objective: Few studies have analyzed the risk factors for thrombosis in SLE patients with antiphospholipid antibodies and most had small sample sizes and homogenous patient populations. We examined whether a history of nephritis is a risk factor for thrombosis using a large multi-ethnic SLE cohort. Methods: The NYU SLE SAMPLE Biorepository and Registry was initiated in September 2013 and includes 612 patients fulfilling ACR and/or SLICC criteria for SLE. Within SAMPLE, we identified patients with a positive test for antiphospholipid antibodies (aPL) (lupus anticoagulant, IgG or IgM anti-beta2-glycoprotein-I antibodies, IgG or IgM anticardiolipin antibodies) and determined if these patients had also ever fulfilled nephritis criteria. We reviewed each patient's medical record for presence/absence of thrombosis (DVT, PE, CVA, arterial occlusion with gangrene or amputation), and obstetric events as well as the noncriteria manifestations thrombocytopenia or valvulitis. We compared the prevalence of APLS manifestations in SLE patients with and without history of lupus nephritis. Results: Of the initial 612 SLE patients, 90% were female (mean age 43.0+/-0.9 years), and 10% were male (mean age 41.0+/-0.3 years). 54% were white, 31% African American, and 15% Asian; 30% were Hispanic white, and 5% Hispanic Black. 105 of the 612 patients had aPL, including 93 females and 12 males (mean age 43.0+/-0.2 years, 56% Caucasian, 33% African American, and 11% Asian). 24% were Hispanic white and 4% Hispanic Black. The total number of patients with thrombotic events was 45/105 (43%), including 26/43 (60%) in the nephritis subset and 19/62 (30%) (p: 0.04) among patients without prior renal involvement. The most common thrombotic event was DVT followed by CVA. Conclusion: The prevalence of aPL criteria in NYU SLE SAMPLE was 17%. Within this group, adverse events were more common among the patients with, versus without, prior history of renal involvement (60% vs 30%). It remains uncertain if association is explained by different accompanying antibodies that distinguish nephritis from non-nephritis SLE, e.g., dsDNA, complement, or drug treatment. This observation provides further support for universal use of hydroxychloroquine in SLE especially in setting of nephritis and suggests that event-free aPL positive patients with prior history of nephritis may be at increased risk for future thrombosis. Further studies are needed to determine whether such patients could benefit from prophylaxis with aspirin or other mild antithrombotic agents