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: Malondialdehyde (MDA) is a naturally occurring reactive aldehyde that arises during apoptosis or as a consequence of elevated reactive oxygen species and lipid peroxidation. Free MDA can posttranslationally modify lysine residues in a protein carbonylation process that generates neo-epitopes that can be recognized by autoantibodies. Methods: Serum IgG anti-MDA levels were compared in 71 healthy controls, 30 OA, and 283 SLE and 162 RA patients, identified by ACR criteria. IgG anti-MDA was measured by sandwich ELISA using MDA-modified BSA. After flow cytometry sorting of RA synovial memory B cells, and single cell Ab-gene PCR, 114 mAbs were expressed and tested for MDA-reactivity. Analyses used the 2-sided Mann-Whitney test or Spearman correlation. Results: In sera, IgG anti-MDA was significantly increased in SLE (17+/-21 RU/ml, p<0.0001) and RA patients (13+/-11 RU/ml, p<0.0001), compared to controls (5+/-3 RU/ml). In SLE, IgG anti-MDA correlated with disease activity by SELENA-SLEDAI (p<0.0001, R=0.34, n=219). Levels were also significantly higher in SLE patients with active disease (SLEDAI>6, 18.9+/-17.3 RU/ml, p=0.001) than with low disease activity (SLEDAI<6, 11.5+/-16.6). In RA patients, IgG anti-MDA correlated with DAS28-ESR (p<0.0001, R=0.35, n=157). Compared to RA patients with low disease activity (DAS28<3.2, 6+/-3), levels were significantly increased in RA with moderate activity (DAS28 3.2-5.1, 12+/-11 RU/ml, p=0.005) and high activity (DAS28>5.1, 15+/-12 RU/ml, p=0.001). In DMARD naive RA (n=62), IgG anti-MDA also correlated with serum TNFalpha (p=0.002, R=0.39), IL-6 (p=0.03, R=0.27), and CRP levels (p=0.003, R=0.37). In RA synovial mAbs, we identified four clones (3.5%) that recognized MDA-modified epitopes. The most reactive clone, 1276:01F04, showed high specific binding to MDA but was non-reactive with carbamylated or 4-HNE-modifications. Specificity was confirmed in antigen-competition studies with MDA or MDA acetaldehyde (MAA) protein adducts. This mAb also bound MDA-modified human fibrinogen and albumin. No cross-reactivity with citrullinated epitopes was detected, and mAbs with ACPA or RF reactivity did not bind MDA. Notably, 1276:01F04 originated from an IgG1-bearing B cell that was encoded by near germline variable genes (VH4-39, 1 R-mutation in HCDR3; VL1-51, 2 S-mutations in FR1). Conclusion: IgM binding to MDA-adducts may be common in health from birth and are part of the natural antibody pool. Yet, IgG anti-MDA are associated with inflammatory conditions including increased disease activity in autoimmune patients. Importantly, MDA-autoreactive B cells could also be isolated from RA synovium, the site of disease. Further studies are merited to investigate the potential pathogenic properties of IgG anti-MDA B-cells/autoantibodies

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: 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/Objective: The spectrum of the vascular pathology affecting SLE patients with secondary APS includes vasculopathy with endothelial cell hyperplasia as in APS nephropathy. Previous studies established a role for endothelial cell activation via the mammalian target of rapamycin (mTORC) but were limited to primary APS patients and the IgG fractions. We extend this finding by studying SLE patients with secondary APS and demonstrate this activity resides in both IgG and IgA fractions. Methods: Endothelial cell phenotypes were assessed using an immunofluorescent assay to report the mTORC biomarker, phospho-S6 ribosomal protein (S6RP) using the human microvascular cell line (HMEC1) in the presence and absence of purified human IgG, IgGFab2, and IgA with or without LY294002, a mTORC inhibitor. We studied eight SLE patients with secondary APS (mean age 46.8+/-.3, 89 % female and 67% white), four disease controls that were either SLE without APS or asymptomatic anti-Ro positive, and three normal controls. Purified IgG and IgA fractions were used as follows: SLE patients four IgG and IgA, three IgG and I IgA, four disease controls two IgG and IgA and two IgG, and controls two IgG and IgA. HMEC1 were serum starved (2 hr), and preincubated with beta2 glycoprotein I (5 ug/mL, 1 hour) followed by a five min exposure to test conditions. Fixed cells were acetone permeabilized and stained using an anti-phospho - S6RP (anti-mouse IgG TRITC) and counterstained with Hoechst 33342. Immunofluorescence was reported using both intensity (1-3+) and a staining scale reflecting % positive cells (3 fields) with 1, <10%; 2, 10-30%; 3, 40-50%; and 4>50%. Results: The phenotype of the endothelial cells which were coincubated with the IgG fractions of 3 (of 8) patients were diffusely positive for phospho- S6RP which was substantially attenuated by co-incubation with LY294002 (1+, 1 % positive cells). In addition, treatment of endothelial cells with IgA fractions from the same three individuals resulted in an increase of phosphorylation of S6RP in microvascular endothelial cells, suggesting the importance of both IgG and IgA APS isotypes. For the total group of eight SLE patients with APS antibodies however there was no association with APS titers and the ability to elicit the biomarker in HMEC1. The expression of phospho- S6RP by HMEC1 were within the ranges that is reported for no treatment (1+, <3 to report % positive cells) for IgG fractions isolated from both disease and healthy controls, Fab2 from a disease control and a control IgA. Conclusions: We report the novel finding that both IgG and IgA fractions from SLE patients with secondary APS activate endothelial cells via the mTORC pathway as demonstrated by S6RP phosphorylation. Future studies will explore these findings using Western blot analysis and explore endothelial cell gene expression for ICAM-1, E-selectin, NOS-2 and tissue factor in an effort to determine potential role of this pathway in the full spectrum of vascular pathology that accompanies APS in 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.

T follicular helper (Tfh) cells promote affinity maturation of B cells in germinal centers (GCs), whereas T follicular regulatory (Tfr) cells limit the GC reaction. Store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channels mediated by STIM and ORAI proteins is a fundamental signaling pathway in T lymphocytes. Conditional deletion of Stim1 and Stim2 genes in T cells abolished SOCE and strongly reduced antibody-mediated immune responses following viral infection caused by impaired differentiation and function of Tfh cells. Conversely, aging Stim1Stim2-deficient mice developed humoral autoimmunity with spontaneous autoantibody production due to abolished Tfr cell differentiation in the presence of residual Tfh cells. Mechanistically, SOCE controlled Tfr and Tfh cell differentiation through NFAT-mediated IRF4, BATF, and Bcl-6 transcription-factor expression. SOCE had a dual role in controlling the GC reaction by regulating both Tfh and Tfr cell differentiation, thus enabling protective B cell responses and preventing humoral autoimmunity.