Faculty Bibliography

Background/Purpose: The Accelerating Medicines Project (AMP) has enabled significant increases in understanding of SLE nephritis pathology, providing a profile of dozens of leukocyte subsets within affected kidneys by single-cell RNA sequencing of nephritis biopsies. While these results suggest a complex network of interactions between cell populations during nephritis, the spatial positioning of these cells is lost during the sequencing process. Inferred interactions between the diverse identified cell types would be greatly strengthened by detailed spatial information, placing these cells in context with each other and with the surrounding structures of the kidney.
Method(s): In consultation with AMP, we have used CODEX, a multicycle imaging technology allowing for staining of up to 40 targets on a single tissue sample without tissue degradation, to capture preliminary images of the AMP tissue biopsies available at New York University. Extensive antibody screening, sample preparation, optimization of antigen retrieval, and imaging steps are required, which remain under active optimization to allow for imaging the entirety of the AMP biopsy cohort available at the Grossman School of Medicine, which has been fully processed for future staining.
Result(s): At present we are able to image sixteen targets capturing dense interstitial T and B cell infiltrates, intratubular and interstitial myeloid populations, and sparser glomerular infiltrating cells in our demonstration cohort, with clear imaging of the glomeruli, tubules, and interstitial spaces. Further targets will be added as they are optimized, further allowing subsetting of T, B, and myeloid populations, with the goal of capturing the populations identified previously in single-cell sequencing.
Conclusion(s): CODEX imaging of renal biopsy samples provides spatial context for prior observations across a range of SLE nephritis samples, with complex interstitial populations found around glomeruli and tubules in active disease. Deeper profiling with expanded antigen targets to enable further sub-population phenotyping and activation states and imaging of the full cohort of biopsies available at NYU will provide a spatial atlas to SLE nephritis and further reveal underlying mechanisms of disease

Background/Purpose: Anti-Ro autoantibody production often precedes the development of Systemic Lupus Erythematosus (SLE) or Sjogren's syndrome (SS) by years. For anti-Ro+ mothers enrolled in the Research Registry for Neonatal Lupus (RRNL), progression to SS or SLE occurs in about a quarter, while most remain asymptomatic or develop only minor rheumatic symptoms (Asym/UAS). Thus, RRNL mothers uniquely offer a promise to identify genotype-phenotype relationships that are important to preclinical autoimmunity. Since multiple SLE risk alleles from Class II HLA genes are present in anti-Ro+ mothers, we examined interactions of specific microbiome taxa with Class II HLA by independent analytic paths with the goal to identify HLA-related microbiome associations in Anti-Ro+ Mothers of Children with Neonatal Lupus.
Method(s): Subjects included 125 RRNL mothers and 23 healthy controls. Stool microbiomes of anti-Ro+ women in RRNL (Asym/UAS, SS/SLE), and healthy controls (HC) were processed using 16S ribosomal RNA sequencing. Sera/ plasma were evaluated for cytokines and autoantibody levels. Alleles from HLA Class II genes were genotyped using NextGen sequencing of HLA region or imputed (HIBAG) from GWAS data. Independent analytic paths sought to explore associations of specific taxa and class II HLA included: 1) use of a cumulative logit model to test interactions between FDR significant genera and HLA alleles and 2) assignment of SLE, SS, UAS patients and HC to molecular phenotype clusters by Random Forest (RF), an unsupervised machine learning tool using Z-score transformed cytokine soluble mediators and autoantibody values with settings and the gap statistic that were used to estimate the optimal number of patients and HC within clusters. The overlapping distribution of SS/SLE, HLA alleles and taxa at clusters were then examined.
Result(s): Findings related to DRB1*15:01 and an interaction with genera of the Ruminococcaceae family were tested. Oscillibacter, with FDR-adjusted significance was shown to exhibit evidence of an interaction (P=0.033 (OR=0.60 (0.37-0.96)). In order to authenticate that SLE HLA risk alleles modify the strength of the association, we examined the molecular phenotype clusters from RF clustering. Radar plots were used to visualize the distribution HLA alleles and the enrichment of microbiome taxa within these clinically relevant phenotypic clusters (Figure 1). DRB1*1501 shows enrichment at cluster 4. Interestingly, the distribution of Oscillibacter, but not Coprococcus 3 was nearly superimposable with the Class II HLA allele with enrichment at cluster 4. However, the distribution of DRB1*1501 was not enriched at cluster profiles representing evaluations of DRB1*0301 and SS/SLE disease classification (Figure 2) demonstrating a limitation of DRB1*1501 to predict risk for transition from benign to pathologic autoimmunity in anti-Ro+ mothers of children with neonatal lupus.
Conclusion(s): These data support the use of molecular phenotypes that are linked to genetic-environmental interactions to identify HLA-related microbiome associations

Background/Purpose: The clinical heterogeneity of SLE with its complex pathogenesis remains challenging as we strive to provide optimal management. The contribution of platelets to endovascular homeostasis, inflammation and immune regulation highlights their potential importance in SLE. Prior work from our group showed that the Fcgamma receptor type IIa (FcgammaRIIa)-R/H131 biallelic polymorphism is associated with increased platelet activity and cardiovascular risk in SLE. The study was initiated to investigate the platelet transcriptome in patients with SLE and evaluate its association across FcgammaRIIa genotypes and distinct clinical features.
Method(s): RNA-sequencing was done on platelets isolated from 51 patients fulfilling criteria for the classification of SLE based on recent EULAR/ACR definitions, and 18 healthy controls matched on age, sex, and race. Unsupervised clustering, differential gene expression, and gene set enrichment analysis (GSEA) were used to analyze differences between SLE patients and controls, and SLE subpopulations, based on SELENA SLEDAI Hybrid disease activity, specific organ manifestations, and FcgammaRIIa genotype. Weighted Gene Correlation Network Analysis (WGCNA) was performed to create a modular transcriptomic framework.
Result(s): Our cross-sectional SLE cohort (N=51, age = 41.1+/-12.3, 100% female, 45% Hispanic, 24% black, 22% Asian, 51% white, SLEDAI = 4.4+/-4.2) was comprised of patients consecutively enrolled excluding those on aspirin or anticoagulants. Compared to the 18 controls, there were 2290 (p.adj < 0.05) differentially expressed genes. ( Figure 1 A, B) GSEA revealed positive enrichment for pathways related to interferon response, TNFa signaling, and coagulation in SLE. ( Figure 1C) WGCNA was used to create a modular transcriptomic framework. ( Figure 2A ) Modules enriched for platelet activity, immune response, and WNT signaling were significantly increased in SLE versus controls. Moreover, modules enriched for interferon response and WNT signaling paralleled increases in disease activity. ( Figure 2B) When analyzing patients with proteinuria, modules associated with oxidative phosphorylation and platelet activity were unexpectedly decreased. (Figure 2C) Analyzing the ratio of fold changes between SLE/Control vs SLE Proteinuria/SLE No Proteinuria, genes increased in SLE and those with proteinuria were enriched for immune effector processes, while genes increased in SLE but decreased in proteinuria were enriched for coagulation and cell adhesion. (Figure 2D) The module enriched for FCR activation was decreased in SLE and was affected by the FcgammaRIIa genotype. (Figure 3A) FcgammaRIIa R131 and H131 patients showed significantly different platelet transcriptomes. (Figure 3B) The combination of SLE with an FcgammaRIIa R131 variant leads to a significant increase in the platelet activity module not seen in controls. (Figure 3C)
Conclusion(s): These analyses reveal that SLE patients have a significantly different platelet transcriptome from controls, different phenotypic presentations of SLE patients associate with distinct platelet transcriptomic signatures, and FCGR2a variants may differentially influence the role of platelets in the contribution to SLE disease activity

Background/Purpose: Congenital Heart Block (CHB) complicates 2% of anti-Ro/ SSA antibody positive pregnancies and carries substantial perinatal morbidity and mortality. Almost all survivors require lifelong pacing. Data suggests the potential of anti-inflammatory treatment of 1degree and 2degree CHB in preventing progression to immutable complete block. However, the optimal surveillance strategy to detect rapidly transitioning and potentially reversible conduction disease is unknown. This study addresses the feasibility, acceptance and accuracy of the fetal heart rate and rhythm technique (FHRM) in high risk mothers.
Method(s): Prospective data from the Surveillance To Prevent AV Block Likely to Occur Quickly (STOP BLOQ) study were leveraged. Mothers referred to the study all had commercially positive anti-Ro/ SSA antibodies and were stratified into high and low titers of anti-Ro60 and Ro52 based on a research ELISA which used a threshold cutoff defined as the titer above or below that obtained for 50 mothers with a previous CHB offspring. Mothers with anti-Ro60 or 52 antibodies at or above 1,000 I.U or with a previous CHB offspring, were trained to perform FHRM with an educational video and personal instruction from a pediatric cardiologist. From 17-25 weeks of gestation, FHRM was completed 3x/day in addition to weekly or biweekly fetal echocardiograms (echo). Mothers texted all FHRM sounds to the study's data coordinating center. For those FHRM deemed abnormal by the mothers, texts were immediately sent to an on call pediatric cardiologist who either reassured if FHRM was normal or referred for emergency fetal echo in < 6 hours if abnormal. Postnatal electrocardiograms were evaluated for CHB.
Result(s): Fifty-six mothers with commercial anti-Ro/ SSA positivity were consented to the study. Of these, 37 (inclusive of 6 with previous CHB) performed FRHM since they had high titer anti-Ro60 (n=8) or 52 antibodies (n=7) or both (n=21), albeit one mother had unexpectedly low titer antibodies to both Ro60 and 52 and a child with incomplete CHB 4 yrs prior to enrollment. In total 3,360 FHRM audiotexts were received during the monitoring period. Of these, 39 recordings from 5 concerned mothers prompted an immediate call with the cardiologist. All but 2 recordings were deemed to be normal based on review of the audiotext alone; the cardiologist requested that the patient send repeat recordings after review as part of re-training and to provide additional reassurance. In the 2 cases an emergency echo was completed in < 6 hrs. In both there were premature atrial contractions which confirmed the mother's perception of the FHRM abnormality. However, there was no evidence of conduction disease. All surveillance echoes were normal. Thus, the overall rate of false positive recordings for the concern of a conduction defect perceived by the mothers was 1.1% (38/3360). There were no cases of CHB at birth.
Conclusion(s): These data support that FHRM is feasible and accurate. Mothers can be empowered to detect rhythm abnormalities with very few false perceptions thus supporting this technique to substantially enhance the management of anti-Ro/ SSA pregnancies

Background/Purpose: Systemic Lupus Erythematosus (SLE) is a complex chronic heterogeneous autoimmune disease, which increases the risk of atherothrombosis. In addition to their well described role in thrombosis and hemostasis, platelets are key mediators of inflammation and have immune effector cell properties. This study was initiated to investigate the role of platelet associated Lectin Galactoside-binding Soluble 3 Binding Protein (LGALS3BP), which binds to macrophage-associated lectin Mac-2, as a mediator of inflammation in SLE and potential biomarker associated with clinical phenotypes.
Method(s): RNA transcriptome analysis was performed on platelets isolated from 51 patients with SLE (not taking aspirin or anticoagulants) and 18 age, sex and race/ethnicity matched controls. LGALS3BP protein expression was determined in platelet releasates by ELISA and western blot analysis. Gene and protein expression of LGALS3BP in Megakaryocyte cell line (MEG-01) was investigated upon stimulation with IFN-alpha. Correlations between circulating serum LGALS3BP and LGALS3BP platelet mRNA and releasates were assessed. Subsequently, correlation analysis between clinical features of SLE and circulating serum LGLAS3BP was performed. Finally, the effects of platelets and LGALS3BP on macrophage inflammatory response were studied in vitro.
Result(s): Platelet transcriptome analysis revealed that LGALS3BP was one of the most differentially expressed transcripts in SLE versus matched-healthy controls (Fold change, 3.9, adjusted P-value = 2.5 x 10^-11) (Figure1A). Consistently, LGALS3BP in platelet releasates was significantly higher in 40 patients with SLE than 20 controls (p = 0.002) (Figure1B). Platelet LGALS3BP gene and protein expression were highly correlated with circulating LGALRS3BP in serum (r² = 0.370, p = 0.003 and r² = 0.689, p < 0.0001 respectively) (Figure1E and F). LGALS3BP measured in serum of 115 patients with SLE correlated with the SELENA SLEDAI hybrid disease activity index (r²= 0.322, p = 0.0005) (Figure1G). In particular, higher serum LGALS3BP levels were observed in SLE patients with lupus nephritis compared to those with SLE and inactive disease (P=0.0001) (Figure1H). In longitudinal analysis of 22 patients without proteinuria at baseline who went on to develop proteinuria over time, circulating plasma LGALS3BP tracked with flares of nephritis (p=0.06). In vitro, IFN-alpha induced the expression and production of LGALS3BP in MEG-01 cells in a dose dependent manner (Figure2A, B and C), which was completely inhibited by IFN-alpha neutralizing antibody (Figure2D, E and F). Recombinant LGALS3BP (Figure 3A and B) and Platelet releasates from SLE (Figure 3C) induced the production of pro-inflammatory cytokines such as IL-8 (p=0.04) and IL-6 (p=0.073) by macrophages.
Conclusion(s): These data show that platelets isolated from patients with SLE highly express and secrete LGALS3BP which induces a proinflammatory macrophage and is associated with SLE disease clinical phenotype. LGALS3BP may contribute to pathogenesis and serve as a novel biomarker of SLE disease activity

Antibodies to double-stranded DNA (dsDNA) are prevalent in systemic lupus erythematosus (SLE), particularly in patients with lupus nephritis, yet the nature and regulation of antigenic cell-free DNA (cfDNA) are poorly understood. Null mutations in the secreted DNase DNASE1L3 cause human monogenic SLE with anti-dsDNA autoreactivity. We report that >50% of sporadic SLE patients with nephritis manifested reduced DNASE1L3 activity in circulation, which was associated with neutralizing autoantibodies to DNASE1L3. These patients had normal total plasma cfDNA levels but showed accumulation of cfDNA in circulating microparticles. Microparticle-associated cfDNA contained a higher fraction of longer polynucleosomal cfDNA fragments, which bound autoantibodies with higher affinity than mononucleosomal fragments. Autoantibodies to DNASE1L3-sensitive antigens on microparticles were prevalent in SLE nephritis patients and correlated with the accumulation of cfDNA in microparticles and with disease severity. DNASE1L3-sensitive antigens included DNA-associated proteins such as HMGB1. Our results reveal autoantibody-mediated impairment of DNASE1L3 activity as a common nongenetic mechanism facilitating anti-dsDNA autoreactivity in patients with severe sporadic SLE.

OBJECTIVE:Hydroxychloroquine (HCQ) is a mainstay of therapy in the treatment of SLE. The effect of HCQ on platelets and vascular health is uncertain. We investigated the relationship between HCQ use and dose with platelet activity, platelet transcriptomics and vascular health in patients with SLE. METHODS:Platelet aggregation, platelet mRNA expression and vascular health (sublingual capillary perfused boundary region (PBR), red blood cell filling (RBCF) and brachial artery reactivity testing) were analysed by HCQ use and dose. RESULTS:Among 132 subjects with SLE (age: 39.7±12.9 years, 97% female), 108 were on HCQ. SLE disease activity was similar between subjects on and off HCQ. Platelet aggregation in response to multiple agonists was significantly lower in patients on HCQ. There were inverse relationships between HCQ dose and gene expression pathways of platelet activity. Gene expression of P-selectin (SELP) was inversely correlated with HCQ dose (r=-0.41, p=0.003), which was validated at the protein level. Subjects on HCQ had improved vascular function correlating with HCQ dose as measured by lower PBR (r=-0.52, p=0.007), higher RBCF (r=0.55, p=0.004) and greater brachial artery reactivity (r=0.43, p=0.056). CONCLUSION/CONCLUSIONS:HCQ use was associated with decreased platelet activation and activation-related transcripts and improved vascular health in SLE.

Objective: The risk of fetal atrioventricular block (AVB) in anti-Ro exposed pregnancies approximates 2% with no prior affected pregnancies. Antibody titer may be contributory to AVB, but there are no data on the negative predictive value (NPV) of antibody titer identifying fetuses unlikely to develop AVB, thus averting costly serial echocardiographs currently recommended for all anti-Ro pregnancies. Using a commercial core laboratory specifying anti-Ro52 and anti-Ro60 antibodies, we sought to develop a threshold level identifying fetuses unlikely to develop AVB, thus averting costly echo surveillance currently recommend for all anti Ro pregnancies.
Study Design: We performed a retrospective multicenter (Children's Hospital Colorado fetal database and NYU Research Registry for Neonatal Lupus) review of women screened for anti-Ro antibodies by several local commercial laboratories because of rheumatic disease or a previous child with AVB. Serum from anti-Ro positive women was sent to a 2nd commercial core laboratory at Associated Regional and University Pathologists laboratories (Salt Lake City, UT) to determine anti-Ro52 and anti-Ro60 antibody (TheraDiag, France) levels. We calculated the NPV individually of anti-Ro52 and anti-Ro60 and the two combined (anti-Ro) using a logistic regression model and a parallel testing strategy.
Result(s): Of 332 women, 142 had a child with AVB and 190 never had a child with AVB. An anti-Ro60 threshold of <101 AU/mL achieved 90% NPV for AVB with 79 titers falling below the threshold (Figure). The logistic regression model with both anti-Ro52 and anti-Ro60 using a threshold of a predicted probability <22% achieved a 91% NPV with a larger sample size below the cutoff (n=87) than any other method (Table).
Conclusion(s): While we could not determine a cutoff for no AVB, the likelihood of AVB developing if the maternal anti-Ro60 titer <101 AU/mL is 10%. Our logistic regression model gives a likelihood of 9% to develop fetal AVB below the threshold. These data may help to guide surveillance of future anti-Ro positive pregnancies. [Formula presented] [Formula presented]
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Background/Purpose: Current management of lupus nephritis (LN) is guided by histopathological features on kidney biopsy and measurement of proteinuria. Urine proteomics is a non-invasive source of novel biomarkers which may better reflect the complex dynamic immunobiology of LN in real time. Two composite measures include CKD273, which can predict the risk of progression of chronic kidney disease in the general population, and LN120, which was designed to diagnose LN. Both are multidimensional urine proteomic classifiers consisting of 273 or 120 peptides, respectively, with major components including collagen fragments, abundant blood-derived proteins, and proteins involved in inflammation. We investigated the ability of these classifiers to predict traditional biopsy features and disease response in LN.
Method(s): A total of 31 adults with biopsy-proven LN were included in this study. All participants met the SLICC and 2019 EULAR/ACR Classification Criteria for SLE based on a spot urine protein-to-creatinine ratio of >0.5 and class III, IV, and/or V LN on renal biopsy. Urine samples were collected at week 0 (at the time of renal biopsy) and week 12 and then subjected to peptidome analysis using a capillary electrophoresis-mass spectrometry (CE-MS) platform. This peptidome data was used to calculate CKD273 and LN120 classifiers at each time point. LN response status was determined at week 52 based on proteinuria, creatinine, and prednisone dosage (no more than 10 mg daily). Spearman's rank correlation and t-tests were used to compare proteomic classifiers with renal biopsy characteristics and response.
Result(s): At week 0, both CKD273 and LN120, but not proteinuria, exhibited a moderate to strong correlation with histological activity index on renal biopsy (Figure 1; rho = 0.65 with p = 0.00024 for CKD273; rho = 0.47 with p = 0.013 for LN120). CKD273 also correlated with chronicity index (rho = 0.54, p = 0.0037). Neither classifier significantly correlated with lupus nephritis ISN class. With respect to response, CKD273 and LN120 were not significantly different between groups at week 0. However, a reduction in LN120 was observed in 100% of complete responders, 60% of partial responders, and 50% of non-responders at week 12 (Figure 2). The magnitude of this change in LN120 in complete responders versus non-responders did not reach statistical significance (p = 0.13), though this is potentially because of the small number of responders with CE-MS data available at both time points (n = 4). CKD273 did not significantly change with time in any response group (Figure 3).
Conclusion(s): This work provides proof of concept that urine proteomic classifiers can noninvasively predict histological activity and chronicity in LN. Complete responders, but not partial responders or non-responders, exhibited an impressive numerical decrease in LN120 by week 12, suggesting that proteomic scores may track with and predict a durable treatment response. Larger studies are needed to validate these findings

Background - Based on inhibition of viral replication and limited reports on clinical efficacy, hydroxychloroquine (HCQ) is being considered as prophylaxis and treatment of COVID-19. Although HCQ is generally considered safe during pregnancy based on studies in patients with systemic lupus erythematous and other rheumatic conditions, there may still be reluctance to institute this antimalarial during pregnancy for the sole purpose of antiviral therapy. Methods - To provide data regarding any potential fetal/neonatal cardiotoxicity, we leveraged a unique opportunity in which neonatal electrocardiograms (ECGs) and HCQ blood levels were available in a recently completed study evaluating the efficacy of HCQ 400mg daily to prevent the recurrence of congenital heart block associated with anti-SSA/Ro antibodies. Results - Forty-five ECGs were available for QTc measurement, and levels of HCQ were assessed during each trimester of pregnancy and in the cord blood, providing unambiguous assurance of drug exposure. Overall, there was no correlation between cord blood levels of HCQ and the neonatal QTc (R = 0.02, P = 0.86) or the mean of HCQ values obtained throughout each individual pregnancy and the QTc (R = 0.04, P = 0.80). In total 5 (11%; 95% CI: 4% - 24%) neonates had prolongation of the QTc > 2SD above historical healthy controls (2 markedly and 3 marginally) but ECGs were otherwise normal. Conclusions - In aggregate, these data provide reassurances that the maternal use of HCQ is associated with a low incidence of infant QTc prolongation. However, if included in clinical COVID-19 studies, early postnatal ECGs should be considered.