Faculty Bibliography

Background Mitochondria are intracellular organelles derived from the endosymbiosis between an a-proteobacterium and a primitive eukaryotic cell. Mitochondria thus display proinflammatory and antigenic properties, when released into the extracellular milieu. Several cross-sectional studies reported increased levels of anti-mitochondrial antibodies (AMAs) in patients with systemic lupus erythematosus (SLE) and the antiphospholipid syndrome (APS). These autoantibodies also displayed correlations with the SLE disease activity index 2000 (SLEDAI-2K) and associations with various clinical manifestations (e.g. lupus nephritis, thromboses, carotid plaque). In the present study, we aim to detect AMAs against either whole organelles (AwMA), mitochondrial DNA (mtDNA) or RNA (mtRNA) through time in samples from patients included in the SLICC cohort. Methods Clinically relevant variables (e.g., sociodemographic variables, disease-specific outcomes including death and arterial vascular events (AVE)) were documented and biosamples were harvested upon patient enrolment in the SLICC cohort, as well as at each follow-up visit. AMA levels were measured by in-house direct ELISAs whereas SLE autoantibodies were detected by clinical laboratories. Healthy individuals, defined as having no known illnesses and infectious symptoms at the time of the blood draw, were recruited. 90% confidence intervals were calculated for both limits of the 95% nonparametric two-sided reference intervals for values measured in healthy donors. AMA values were segregated into 3 categories: Normal values were determined as within the inner limits of the range while values outside this range were characterized as abnormal, either lower or higher than the reference interval. (figure 1). Marginal Cox models with AMAs in 3 categories were adjusted for covariables and are presented as [hazard ratio (95% CI)]. Interactions with sex were tested in models with the AMAs as continuous variables. Results Sera from healthy individuals (n=126) or SLE patients included in the SLICC cohort, from their inclusion, up to 7 years of follow-up (n=1114 patients at baseline, 3577 samples in total). AwMA displayed lower correlations with antibodies to mitochondrial nucleic acids (versus AmtDNA: rs=0.37, and vs AmtRNA: rs=0.38), while antibodies to mitochondrial DNA or RNA shown higher correlations (rs=0.59). During our preliminary analyses on the distribution of the variables, We made intriguing observations regarding patients with AMA levels that were either lower or higher than those of healthy individuals. This information led us to categorize SLE patients as described in the methods and in figure 1. For each of the three antibodies assessed, SLE patients displayed more abnormal AMA levels at baseline than controls. The percentage of patients with higher levels of AwMA and AmtRNA increased at subsequent follow-up visits, while a slight decrease was observed for AmtDNA (figure 2). SLE patients with higher levels of AwMA showed higher risks of death [2.12 (1.18-3.83)]. It is of interest that an inverse relationship was found between AmtRNA and AVEs, with a small subset of patients with low levels of AmtRNA (n = 4), this autoantibody was associated with increased risks of this manifestation [4.46 (1.71-11.66)]. Additionally, patients with higher levels of AmtDNA and AmtRNA displayed increased risks of lupus nephritis [respectively: 3.05(2.05-4.54), and 1.56(1.12-2.18)]. Interestingly, there was an interaction with sex for AmtRNA levels effect on AVEs [males: 0.32 (0.11-0.99). Females: 1.56 (1.11-2.19)], and AmtDNA association with nephritis was only significant in female patients [4.00 (2.51-6.36)] (table 1). Conclusion These results show that AMAs display different associations with disease manifestations in various clusters of patients. These results prompt for further analyses by machine-learning in order to delineate clusters of clinical interests by adding AMAs in the routine serological assessment of SLE autoantibodies. Acknowledgements We acknowledge the contribution of the study participants, individual center support staff as well as investigators of the Systemic Lupus International Collaborating Clinics (SLICC) Inception Cohort project who for the past 20 years have made this study possible. LAY ABSTRACT The mitochondrion is a part of the cell that controls various biological mechanisms (e.g., energy supply, whether the cell should live or die, control, or produce various cellular components). They are derived, through evolution, from a microbe. Mitochondria may sometimes be jettisoned out of their host cell and subsequently elicit immune reactions - including the production of antibodies. Previous studies indicated that patients with autoimmune conditions such as systemic lupus erythematosus (SLE) and the antiphospholipid syndrome (APS) have antibodies against mitochondria in their blood stream. Presence of these antibodies was associated with increased disease activity and clinical manifestations of these diseases (e.g. kidney disease, arterial vessel disease). In this study, we studied blood samples harvested by an international group dedicated to the study of SLE [i.e., the SLE International Collaborating Clinics (SLICC) cohort] and observed that patients may be clustered into groups, upon their levels of antibodies and/or sex, allowing to have a better appreciation of their risks of death, vascular events, and kidney disease. These results might lead to improved diagnosis and/or prognosis in SLE and thus, in improved care and quality of life for the people living with lupus

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

Objectives We previously reported in a single centre prevalent SLE cohort that antibodies against the cytokinesis-associated protein M-Phase Phosphoprotein 1 (anti-MPP-1) were associated with SLE-related cranial neuropathy (CN), a rare manifestation of neuropsychiatric SLE (NPSLE). The purpose of this study was to assess whether anti-MPP-1 is a biomarker for CN or other NPSLE manifestations using an international SLE inception cohort. Methods SLE patients fulfilling the updated 1997 ACR classification criteria for SLE were included. Anti-MPP-1 antibody testing was performed on baseline samples (within 15 months of diagnosis) or first annual assessment using an addressable laser bead immunoassay (ALBIA) with purified recombinant human protein with results expressed as median florescence units (MFU). Based on healthy controls, a dilution of >=1:500 MFU was considered positive. NPSLE manifestations occurring over the first 5 years of follow up were documented annually based on ACR case definitions using published NPSLE attribution rules1). The frequency of anti-MPP-1 positivity between patients with versus without each of the 19 NPSLE manifestations was compared using univariate logistic regression. For any NPSLE manifestations where anti-MPP-1 positivity differed between patients with versus without the manifestation, baseline demographic and clinical characteristics were compared using t-tests and twosample tests of proportions. For NPSLE manifestations associated with anti-MPP-1 positivity in the univariate analysis, multivariable logistic regression analysis using penalized maximum likelihood estimates was then performed to assess the relationship between anti-MPP-1 and the NPSLE manifestation, adjusting for age at anti-MPP-1 testing, female, White race/ethnicity, and significantly different baseline clinical characteristics. Results Seven hundred and ninety-five SLE patients were assessed; 29.8% were anti-MPP-1 positive, 88.7% female, and 52.1% White. The frequency of anti-MPP-1 positivity differed only for those with versus without CN (70.0% vs. 29.3%; odds ratio [OR] 5.16, 95%CI 1.44, 18.54) (table 1). Compared to patients without CN (n=785), patients with CN (n=10) were more likely to fulfill the ACR hematologic (difference: 23.9%, 95%CI 5.0%, 42.8%) and antinuclear antibody criteria (difference: 4.3%, 95%CI 2.9%, 5.8%) (table 2). (Table Presanted)In the multivariate analysis, anti-MPP-1 remained associated with CN (OR 5.24, 95%CI 1.44, 19.09) after adjusting for age at anti-MPP-1 testing, female, White race/ethnicity, hematologic disorder, and antinuclear antibody (table 3). Conclusion Anti-MPP-1 is a potential biomarker for CN. Although anti-MPP-1 is differentially expressed in a variety of neurological cells and tissues, the link to a pathogenic role requires further study

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 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

OBJECTIVE:In systemic lupus erythematosus (SLE), disease activity and glucocorticoid (GC) exposure are known to contribute to irreversible organ damage. We aimed to examine the association between GC exposure and organ damage occurrence. METHODS:We conducted a literature search (PubMed (Medline), Embase and Cochrane January 1966-October 2021). We identified original longitudinal observational studies reporting GC exposure as the proportion of users and/or GC use with dose information as well as the occurrence of new major organ damage as defined in the Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index. Meta-regression analyses were performed. Reviews, case-reports and studies with <5 years of follow-up, <50 patients, different outcomes and special populations were excluded. RESULTS:We selected 49 articles including 16 224 patients, 14 755 (90.9%) female with a mean age and disease duration of 35.1 years and of 37.1 months. The mean follow-up time was 104.9 months. For individual damage items, the average daily GC dose was associated with the occurrence of overall cardiovascular events and with osteoporosis with fractures. A higher average cumulative dose adjusted (or not)/number of follow-up years and a higher proportion of patients on GC were associated with the occurrence of osteonecrosis. CONCLUSIONS:We confirm associations of GC use with three specific damage items. In treating patients with SLE, our aim should be to maximise the efficacy of GC and to minimise their harms.