Faculty Bibliography

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: 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/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: 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: 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: The relationship between cell-bound complement activation products (CB-CAPs: EC4d, EC3d), anti-C1q, soluble complement C3/C4 and disease activity in systemic lupus erythematosus (SLE) was evaluated. METHODS: Per protocol, at baseline all SLE subjects enrolled in this longitudinal study presented with active disease and elevated CB-CAPs. At each monthly visit, the non-serological (ns) Safety of Estrogens in Lupus Erythematosus: National Assessment (SELENA-SLEDAI) and the British Isles Lupus Assessment Group (BILAG)-2004 index scores were determined as was a random urinary protein to creatinine ratio (uPCR). Short-form 36 (SF-36) questionnaires were also collected. All soluble markers were determined using immunoassays, while EC4d and EC3d were determined using flow cytometry. Statistical analysis consisted of linear mixed models with random intercept and fixed slopes. RESULTS: A total of 36 SLE subjects (mean age 34 years; 94% female) were enrolled and evaluated monthly for an average 11 visits per subject. Clinical improvements were observed during the study, with significant decreases in ns-SELENA-SLEDAI scores, BILAG-2004 index scores and uPCR, and increases in all domains of SF-36 (p<0.01). The longitudinal decrease in ns-SELENA-SLEDAI and BILAG-2004 index scores was significantly associated with reduced EC4d and EC3d levels, reduced anti-C1q titres and increased serum complement C3/C4 (p<0.05). The changes in uPCR significantly correlated with C3, C4, anti-C1q and EC4d, with EC4d outperforming C3/C4 by a multivariate analysis. The reduced EC4d or EC3d was associated with improvements in at least six out of the eight domains of SF-36 and outperformed C3/C4. Anti-dsDNA titres did not correlate with changes in disease activity. CONCLUSIONS: These data indicate that CB-CAPs and anti-C1q are helpful in monitoring patients with SLE.

At birth, the human immune system already contains substantial levels of polymeric IgM, that include autoantibodies to neo-epitopes on apoptotic cells (ACs) that are proposed to play homeostatic and anti-inflammatory roles. Yet the biologic origins and developmental regulation of these naturally arising antibodies remain poorly understood. Herein, we report that levels of IgM-antibodies to malondialdehyde (MDA) protein adducts, a common type of in vivo generated oxidative stress-related neoepitope, directly correlate with the relative binding of neonatal-IgM to ACs. Levels of IgM to phosphorylcholine (PC), a natural antibody prevalent in adults, were relatively scant in cord blood, while there was significantly greater relative representation of IgM anti-MDA antibodies in newborns compared to adults. To investigate the potential interrelationships between neonatal IgM with pathogenic IgG-autoantibodies, we studied 103 newborns born to autoimmune mothers with IgG anti-Ro (i.e., 70 with neonatal lupus and 33 without neonatal lupus). In these subjects the mean levels of IgM anti-Ro60 were significantly higher than in the newborns from non-autoimmune mothers. In contrast, levels of IgM anti-MDA in IgG anti-Ro exposed neonates were significantly lower than in neonates from non-autoimmune mothers. The presence or absence of neonatal lupus did not appear to influence the total levels of IgM in the anti-Ro exposed newborns. Taken together, our studies provide evidence that the immune development of the natural IgM-repertoire may be affected, and become imprinted by, the transfer of maternal IgG into the fetus.

Antibodies to DNA and chromatin drive autoimmunity in systemic lupus erythematosus (SLE). Null mutations and hypomorphic variants of the secreted deoxyribonuclease DNASE1L3 are linked to familial and sporadic SLE, respectively. We report that DNASE1L3-deficient mice rapidly develop autoantibodies to DNA and chromatin, followed by an SLE-like disease. Circulating DNASE1L3 is produced by dendritic cells and macrophages, and its levels inversely correlate with anti-DNA antibody response. DNASE1L3 is uniquely capable of digesting chromatin in microparticles released from apoptotic cells. Accordingly, DNASE1L3-deficient mice and human patients have elevated DNA levels in plasma, particularly in circulating microparticles. Murine and human autoantibody clones and serum antibodies from human SLE patients bind to DNASE1L3-sensitive chromatin on the surface of microparticles. Thus, extracellular microparticle-associated chromatin is a potential self-antigen normally digested by circulating DNASE1L3. The loss of this tolerance mechanism can contribute to SLE, and its restoration may represent a therapeutic opportunity in the disease.