Faculty Bibliography

OBJECTIVES/OBJECTIVE:Current treatments are effective only in 30% of lupus nephritis patients emphasizing the need for novel therapeutic strategies. To develop mechanistic hypotheses and explore novel biomarkers, we analyzed the longitudinal urinary proteomic profiles in patients with lupus nephritis undergoing treatment. METHODS:We quantified 1,000 urinary proteins in 30 patients with lupus nephritis at the time of the diagnostic renal biopsy and after 3, 6, and 12 months. The proteins and molecular pathways detected in the urine proteome were then analyzed with respect to baseline clinical features and longitudinal trajectories. The intrarenal expression of candidate biomarkers was evaluated using single cell transcriptomics of renal biopsies from lupus nephritis patients. RESULTS:Our analysis revealed multiple biological pathways including chemotaxis, neutrophil activation, platelet degranulation, and extracellular matrix organization that could be noninvasively quantified and monitored in the urine. We identified 237 urinary biomarkers associated with lupus nephritis as compared to controls without SLE. IL-16, CD163, and TGF-β mirrored intrarenal nephritis activity. Response to treatment was paralleled by a reduction of urinary IL-16, a CD4 ligand with proinflammatory and chemotactic properties. Single cell RNA sequencing independently demonstrated that IL16 is the second most expressed cytokine by most infiltrating immune cells in lupus nephritis kidneys. IL-16 producing cells were found at key sites of kidney injury. CONCLUSION/CONCLUSIONS:Urine proteomics may profoundly change the diagnosis and management of lupus nephritis by noninvasively monitor active intrarenal biological pathways. These findings implicate IL-16 in lupus nephritis pathogenesis designating it as a potentially treatable target and biomarker.

OBJECTIVE:Previous studies suggest a link between high serum type I interferon (IFN) and lupus nephritis (LN). We determined whether serum IFN activity is associated with subtypes of LN and studied renal tissues and cells to understand the impact of IFN in LN. METHODS:). Podocyte cell line gene expression was measured by real-time PCR. RESULTS:expression was not closely co-localized with pDCs. IFN directly activated podocyte cell lines to induce chemokines and proapoptotic molecules. CONCLUSION/CONCLUSIONS:Systemic high IFN is involved in the pathogenesis of severe LN. We do not find co-localization of pDCs with IFN signature in renal tissue, and instead observe the greatest intensity of IFN signature in glomerular areas, which could suggest a blood source of IFN.

OBJECTIVE:To evaluate seroreactivity and disease flares after COVID-19 vaccination in a multi-ethnic/racial cohort of patients with systemic lupus erythematosus (SLE). METHODS:90 SLE patients and 20 healthy controls receiving a complete COVID-19 vaccine regimen were included. IgG seroreactivity to the SARS-CoV-2 spike receptor-binding domain (RBD) and SARS-CoV-2 microneutralization were used to evaluate B cell responses; IFN-γ production to assess T cell responses was measured by ELISpot. Disease activity was measured by the hybrid SLE disease activity index (SLEDAI) and flares were assigned by the SELENA/SLEDAI flare index. RESULTS:Overall, fully vaccinated SLE patients produced significantly lower IgG antibodies against SARS-CoV-2 spike RBD than controls. Twenty-six SLE patients (28.8%) generated an IgG response below that of the lowest control (<100 units/ml). In logistic regression analyses, the use of any immunosuppressant or prednisone and a normal anti-dsDNA level prior to vaccination associated with decreased vaccine responses. IgG seroreactivity to the SARS-CoV-2 Spike RBD strongly correlated with the SARS-CoV-2 microneutralization titers and antigen-specific IFN-γ production determined by ELISpot. In a subset of patients with poor antibody responses, IFN-γ production was likewise diminished. Pre-/post-vaccination SLEDAI scores were similar. Only 11.4% of patients had a post-vaccination flare; 1.3% were severe. CONCLUSION/CONCLUSIONS:In a multi-ethnic/racial study of SLE patients 29% had a low response to the COVID-19 vaccine which was associated with being on immunosuppression. Reassuringly, disease flares were rare. While minimal protective levels remain unknown, these data suggest protocol development is needed to assess efficacy of booster vaccination.

Anti-SSA/Ro antibodies, while strongly linked to fetal cardiac injury and neonatal rash, can associate with a spectrum of disease in the mother, ranging from completely asymptomatic to overt Systemic Lupus Erythematosus (SLE) or Sjögren's Syndrome (SS). This study was initiated to test the hypothesis that the microbiome, influenced in part by genetics, contributes to disease state. The stool microbiome of healthy controls (HC) was compared to that of anti-SSA/Ro positive women whose children had neonatal lupus. At the time of sampling, these women were either asymptomatic (Asym), had minor rheumatic symptoms or signs considered as an undifferentiated autoimmune syndrome (UAS), or were diagnosed with SLE or SS. Differences in microbial relative abundances among these three groups were tested assuming an ordering in clinical severity (HC<Asym/UAS<SS/SLE) and then again without the ordinal assumption. Those taxa that showed differential relative abundances were then tested for whether the effect size differed depending on the women's HLA SLE-risk allele genotype (DRB1*03:01, DRB1*15:01, DQB1*02:01 and DQB1*06:02) or anti-SSA/Ro autoantibody levels. Multiple genera within the families Ruminococcaceae and Lachnospiraceae showed evidence of an HLA-by-genus interaction (P < .05). Four genera exhibited evidence of an interaction with anti-Ro52 IgA: Lachnoclostridium, Romboutsia, Bacteroides and Actinomyces (P < .01). In addition to documenting differences in microbial relative abundances across clinical severity of disease, these data provide a first-time demonstration that microbial differences are correlated with HLA SLE-risk alleles. Taken together, these data suggest that the clinical spectrum from benign to overt clinical autoimmunity may partially result from or trigger a complex interplay among specific microbial profiles, anti-Ro autoantibodies, and genetics.

Introduction Fetal cardiac disease is strongly associated with maternal anti-SSA/Ro antibodies, but gaps in our knowledge include the influence of antibody specificity and titer, maternal diagnosis, overall non-cardiac adverse pregnancy outcomes (APOs), optimal surveillance protocols, and efficacy of rapid treatment. Methods The multi-center Surveillance and Treatment To Prevent Fetal AV Block Likely to Occur Quickly (STOP BLOQ) study recruited pregnant women with commercially positive anti-Ro antibodies and stratified them into high and low titers of anti-Ro60 and Ro52 based on a research ELISA, using a cutoff defined by that obtained for 50 mothers with previous AVB offspring. Mothers with anti-Ro60 and/or 52 antibodies at or above 1,000 I.U. were trained to perform FHRM. From 17-25 weeks of gestation, FHRM was completed 3x/day in addition to weekly or biweekly fetal echocardiograms (echo). Mothers texted all audio sounds to the coordinating center. Texts deemed abnormal by mothers 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. Results 250 anti-Ro pregnant women (22% Hispanic, 50% white, 12% Black, 12% Asian, 4% other) have been consented, including 28 whose previous child had AVB. Of mothers tested to date, 153 were provided home monitors given high titer anti-Ro60 and/or 52 antibodies (26 high titer anti-Ro60 alone, 21 high titer anti-Ro52 alone,105 high titer antibodies to both antigens). The 83 patients with low titers were surveilled with echos per local standard of care. Regarding maternal diagnosis, of 161 assessed to date, 39% were asym/UAS, 11% RA, 31% SS, 19% SLE. Antibody titers did not significantly differ by ethnicity, race or diagnosis (table 1). Non-AVB APOs occurred in 18% and were not predicted by Ro60 or 52 titers but rather SLE diagnosis (table 2). In total, 24,759 FHRM audiotexts were received from 131 patients (90 of whom have delivered) during the monitoring period. Of these, 22 were evaluated by the on-call pediatric cardiologist, who prompted an emergency echo (all completed in < 6 hrs). In 11 cases, the emergency echo was normal. In 9, there were premature atrial contractions, confirming the mother's perception. In 2 with 2degree block on urgent echo (both treated per protocol with IVIG and dexamethasone), 1 reverted to normal sinus rhythm and the other progressed to 3degree block. In 2 others, the mother did not perceive abnormal FHRM for > 24 hrs, echo identified 3degree block, and retrospective cardiology review of FHRM audio captures identified an abnormality prior to obtaining the echo. All 4 AVB developed in fetuses of mothers with high titer antibodies to both Ro60 and 52 (mean 32,451 and 34,991 respectively). Of the 18 mothers with a previous AVB child who followed the 400mg hydroxychloroquine PATCH protocol, 1 developed AVB in accord with the results of Step 1 in that study. Conclusion These data support that APOs in this clinically diverse group of mothers are not influenced by anti-Ro titer or specificity, but rather SLE diagnosis. All conduction defects were initially identified by FHRM and in mothers with high titer anti-Ro60 and 52. Hydroxychloroquine continues to show efficacy in reducing the AVB recurrence rate with rapid intervention of emergent block being promising

Background Lupus nephritis (LN) is characterized by considerable variability in its clinical manifestations and histopathological findings. Understanding the cellular and molecular mechanisms underlying this heterogeneity is key for the development of personalized treatments for LN. Methods Droplet-based single-cell RNA-sequencing was applied to the analysis of dissociated kidney samples, collected from 155 LN patients with active kidney disease and 30 living donor controls as part of the Accelerating Medicines Partnership (AMP) in SLE consortium -a large-scale, multi-center study. 73,440 immune cells passing quality control were identified, spanning 134 cell subsets, representing various populations of tissue-resident and infiltrating leukocytes, as well as the activation states these cells assume as part of their diseaserelated activation and differentiation (figure 1). Principal component analysis (PCA) was used to characterize the variability in cell subset frequencies across the LN patients. Relationships between the resulting principal components (PCs) and the demographic, clinical and histopathological features of the patients were then assessed. Results The main source of variability in immune cell subset frequencies, as represented by the first PC (PC1), reflected the balance between lymphocytes and monocytes/macrophages. Subsequent PCs represented the balance between B cells and T cells (PC2); the levels of cytotoxic T lymphocytes and NK cells, as compared to plasma cells (PC3); and the degree of macrophage differentiation to an alternatively activated phagocytic profile (PC4). PC1 was significantly correlated with the Chronicity index, such that patients with a higher percentage of lymphocytes compared to monocytes/macrophages had a higher Chronicity score (rho = -0.422, p-value < 0.001; figure 2A). A high degree of macrophage differentiation, as represented by PC4, was associated with a high Activity score (rho = 0.387, p-value < 0.001; figure 2B), and, in addition, with proliferative or mixed histology class, compared to pure membranous nephritis (p-value = 0.001, Kruskal-Wallis test). The ratio of B cells to T cells, as represented by PC2, demonstrated a positive correlation with the Activity index (rho = 0.311, p-value < 0.001). We further identified a significant correlation of PC1 with age; specifically, older patients had a higher relative frequency of lymphocytes compared to monocytes/macrophages (rho = -0.239, p-value = 0.003). Our analysis indicated that these relations are not driven by demographic, clinical and technical sources of variation in our data, including race, ethnicity, the mixture of different nephritic classes, and the inclusion of both first and later biopsies. Conclusion Our work identifies distinct leukocyte populations active in different LN patients and, possibly, different stages of disease, and points to potential therapeutic targets, that must be validated in mechanistic studies. This approach may pave the way to personalized treatment of LN

Background Lupus nephritis can occur as an isolated component of disease activity or be accompanied by diverse extrarenal symptoms that can adversely affect a patient's quality of life (QOL). Whether renal disease absent other activity is sufficient to decrease QOL is unknown. A lack of reported QOL impairment may place patients at risk for delayed diagnosis of nephritis or medication noncompliance yet nephritis trials have largely neglected QOL. As such, this study leveraged the multi-center multi-racial Accelerating Medicines Partnership (AMP) lupus nephritis cohort to assess QOL measured by PROMIS-29. Methods Patients (n=182) fulfilling ACR or SLICC criteria for SLE with a uPCR >= .5 and biopsy Class III, IV, V, or mixed were consecutively enrolled in AMP at the time of renal biopsy and clinical history, PROMIS-29, and disease activity as assessed by the hybrid SELENA-SLEDAI were recorded. Patients were determined to have extrarenal clinical activity if, after excluding all laboratory parameters from the SLEDAI, the score remained >= 1. Raw PROMIS-29 scores were transformed to t-scores with the mean of 50 +/- 10 representing the US population and a difference of 5 points considered clinically meaningful. PROMIS-29 physical and mental health summary scores were calculated according to published formulas. Results Forty-three percent of patients (n=78) had extrarenal clinical manifestations including vasculitis (4%), arthritis (39%), rash (45%), alopecia (42%), mucosal ulcers (13%), pleurisy (12%), pericarditis (8%), and fever (4%). Patients with isolated renal disease (n=104, 57%) did not have PROMIS-29 scores that differed clinically from the US population whereas patients with extrarenal disease reported deficits in physical functioning, fatigue, social functioning, and pain (table 1). Patients with extrarenal disease had significantly lower physical health summary scores compared to patients with isolated disease (median [IQR]: 40.31 [35.79, 47.02] p<0.001 vs. 48.6 [40.14, 57.08]) and significantly lower mental health summary scores (44.12 [38.63, 51.39], p=0.024 vs. 48.67 [40.51, 55.07]). Female and African American patients and those with nephrotic range proteinuria or undergoing first biopsy had significantly lower physical health summary scores, but mental health summary scores did not differ by these variables. Patients on greater than 20 mg of prednisone had both significantly lower physical and mental health summary scores compared to those on lower doses. PROMIS-29 scores did not differ by low complements, anti-dsDNA, or anti-Ro antibodies. Stepwise multivariable linear regression analysis demonstrated that the association between extrarenal disease and lower PROMIS-29 summary scores was primarily driven by arthritis and independent of potential confounders (tables 2 and 3). Conclusion The majority of patients had isolated renal disease and report a QOL similar to that of the general population. In contrast, those with extrarenal manifestations report significantly worse QOL outcomes. These results reinforce the critical importance of routine laboratory surveillance and medication compliance for nephritis even in patients with seemingly quiescent clinical disease since lupus nephritis is often asymptomatic