Faculty Bibliography

OBJECTIVE:Extant epidemiologic data of primary Sjögren's Syndrome (pSS) remains limited, particularly for racial/ethnic populations in the United States (US). The Manhattan Lupus Surveillance Program (MLSP), a population-based retrospective registry of cases with Systemic Lupus Erythematosus and related diseases including pSS in Manhattan, was used to provide estimates of the incidence and prevalence of pSS across major racial/ethnic populations. METHODS:MLSP cases were identified from hospitals, rheumatologists, and population databases. Three case definitions were used for pSS: physician diagnosis, rheumatologist diagnosis, and modified pSS criteria. Rates among Manhattan residents were age-adjusted, and capture-recapture analyses were conducted to assess case under-ascertainment. RESULTS:By physician diagnosis, age-adjusted overall incidence and prevalence rates of pSS among adult Manhattan residents were 3.5 and 13.1 per 100,000 person-years. Capture-recapture adjustment increased incidence and prevalence rates (4.1 and 14.2). Based on physician diagnosis, incidence and prevalence rates were approximately 6 times higher among women than men (p<0.01). Incidence of pSS was statistically higher among non-Latina Asian (10.5) and non-Latina White women (6.2) compared with Latina women (3.2). Incidence was also higher among non-Latina Asian women compared with non-Latina Black women (3.3). Prevalence of pSS did not differ by race/ethnicity. Similar trends were observed when more restrictive case definitions were applied. CONCLUSION/CONCLUSIONS:Data from the MLSP revealed disparities in pSS incidence and prevalence by sex among Manhattan residents and differences in pSS incidence by race/ethnicity among women. These data also provided epidemiologic estimates for the major racial/ethnic populations in the US.

OBJECTIVES/OBJECTIVE:The spectrum of antinuclear antibodies (ANA) is changing to include both nuclear staining as well as cytoplasmic and mitotic cell patterns (CMPs) and accordingly a change in terminology to anti-cellular antibodies. This study examined the prevalence of indirect immunofluorescence (IIF) anti-cellular antibody staining using the Systemic Lupus International Collaborating Clinics inception cohort. METHODS:Anti-cellular antibodies were detected by IIF on HEp-2000 substrate utilizing the baseline serum. Three serological subsets were examined: 1) ANA-positive (presence of either nuclear or mixed nuclear/CMP staining), 2) anti-cellular antibody-negative (absence of any intracellular staining), and 3) isolated CMP staining. The odds of being anti-cellular antibody-negative versus ANA or isolated CMP-positive was assessed by multivariable analysis. RESULTS:1137 patients were included; 1049/1137 (92.3%) were ANA-positive, 71/1137 (6.2%) were anti-cellular antibody-negative, and 17/1137 (1.5%) had isolated CMP. The isolated CMP group did not differ from the ANA-positive or anti-cellular antibody-negative group in clinical, demographic or serologic features. Patients who were older (OR 1.02 [95% CI: 1.00, 1.04]), of Caucasian race/ethnicity (OR 3.53 [95% CI: 1.77, 7.03]), or on high dose glucocorticoids at or prior to enrolment (OR 2.39 [95% CI: 1.39, 4.12]) were more likely to be anti-cellular antibody-negative. Patients on immunosuppressants (OR 0.35 [95% CI: 0.19, 0.64]) or with anti-SSA/Ro60 (OR 0.41 [95% CI: 0.23, 0.74]) or anti-UI-RNP (OR 0.43 [95% CI: 0.20, 0.93]) were less likely to be anti-cellular antibody-negative. CONCLUSIONS:In newly diagnosed SLE, 6.2% of patients were anti-cellular antibody-negative and 1.5% had isolated CMP. The prevalence of anti-cellular antibody-negative SLE will likely decrease as emerging nomenclature guidelines recommend that non-nuclear patterns should also be reported as a positive ANA.

BACKGROUND/PURPOSE/OBJECTIVE:To search for a transmissible agent involved in lupus pathogenesis, we investigated the faecal microbiota of patients with systemic lupus erythematosus (SLE) for candidate pathobiont(s) and evaluated them for special relationships with host immunity. METHODS:In a cross-sectional discovery cohort, matched blood and faecal samples from 61 female patients with SLE were obtained. Faecal 16 S rRNA analyses were performed, and sera profiled for antibacterial and autoantibody responses, with findings validated in two independent lupus cohorts. RESULTS:strain-restricted antibodies were detected in those with active nephritis (including Class III and IV) in the discovery cohort, with findings validated in two independent cohorts. CONCLUSION/CONCLUSIONS:These findings suggest a novel paradigm in which specific strains of a gut commensal may contribute to the immune pathogenesis of lupus nephritis.

Linked Comment: Ultrasound Obstet Gynecol 2019; 54: 87-95.

Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.

Systemic lupus erythematosus carries an increased risk of pregnancy complications, including preeclampsia and fetal adverse outcomes. To identify the underlying molecular mechanisms, we longitudinally profiled the blood transcriptome of 92 lupus patients and 43 healthy women during pregnancy and postpartum and performed multicolor flow cytometry in a subset of them. We also profiled 25 healthy women undergoing assisted reproductive technology to monitor transcriptional changes around embryo implantation. Sustained down-regulation of multiple immune signatures, including interferon and plasma cells, was observed during healthy pregnancy. These changes appeared early after embryo implantation and were mirrored in uncomplicated lupus pregnancies. Patients with preeclampsia displayed early up-regulation of neutrophil signatures that correlated with expansion of immature neutrophils. Lupus pregnancies with fetal complications carried the highest interferon and plasma cell signatures as well as activated CD4⁺ T cell counts. Thus, blood immunomonitoring reveals that both healthy and uncomplicated lupus pregnancies exhibit early and sustained transcriptional modulation of lupus-related signatures, and a lack thereof associates with adverse outcomes.

Background Classification and treatment decisions in lupus nephritis (LN) are largely based on renal histology. Single-cell RNA sequencing (scRNAseq) analysis may accurately differentiate types of renal involvement at the transcriptomic level, and better inform treatment decisions and prognosis. Methods scRNAseq was performed on kidney and non-lesional skin tissue collected from 20 SLE patients undergoing a clinically indicated renal biopsy. Cell types were determined using principal component analysis and t-distributed stochastic neighbor embedding (tSNE) plotting, resulting in the definitive identification of keratinocytes, tubular cells, mesangial cells, fibroblasts, endothelial cells, and leukocytes. Results LN patients expressed upregulated IFN response genes in both their tubular cells and keratinocytes. This IFN response signature in tubular cells predicted poor response to therapy 6 months post-biopsy. Tubular cells of non-responder patients also expressed upregulated extracellular matrix proteins and fibrotic markers (figure 1A and 1B). Using logistic regression analysis, a 4-gene tubular fibrosis score was created and able to predict response to treatment with an area under curve of 0.9 (figure 1C). Keratinocytes of non-responders also upregulated certain extracellular matrix genes and this response was not observed in peripheral blood mononuclear cells. Differential expression analysis between histology classes indicated an upregulation of IFN and TNF signaling in the tubular cells of patients with proliferative LN compared with membranous. Conclusions scRNAseq from 2-10 mm of renal biopsy tissue in SLE can differentiate between the different classes of LN, and provide important insights into potential pathogenic mechanisms. Further, changes in the skin of LN patients can provide a useful source of biomarkers and may reflect important information concerning concurrent kidney pathological events

Background The impact of renal injury in lupus nephritis is widespread with consequences to resident cells in other tissue beds, even non-lesional non-sun exposed skin. Faithful reflection of a relevant renal tissue pathway in a more readily accessible compartment would allow for less invasive diagnostic alternatives. Single-cell transcriptional states as performed in this study may provide a framework for understanding how in vivo biological function emerges from complex cell ensembles, thus allowing for a clearer understanding of potential mutual pathways. Methods Patients with proteinuria and known ISN/RPS Class and controls were recruited to discovery 1 and 2 cohorts. Single cell RNAseq was performed on cell suspensions prepared from ~2 mm punch biopsies of non-lesional non sun-exposed skin from the buttocks. The libraries were prepared on the Fluidigm C1 platform (discovery 1) and 10X Genomics platform (discovery 2) along with Illumina HiSeq 2500 sequencing. Results Sorting based on COL1A1, COL1A2, COL3A1, MFAP5 and MFAP4 expression yielded 12 fibroblasts from 3 patients. The 1 Class II subject yielded 5 single-cell transcriptomes. The other 2 subjects (1 Class IV,V; 1 Class III,V) yielded 7 single-cell transcriptomes. 22 transcriptomes were derived from 3 controls. The aggregate data were used to determine the top upregulated genes in cases versus controls, most of which belonged to the interferon-stimulated gene category and the extracellular matrix category (DAVID databases). Fewer cells were obtained using Fluidigm C1 (36 single-cell) than 10X Genomics (7280 single-cell). For the latter, the major biopsy classes were represented (Class III, III/IV, III/V, V and no LN). We applied graph-based clustering and identified 12 major clusters of cells from the patient skin as visualized by t-distributed stochastic neighbor embedding (t-SNE; figure 1). Differential gene expression analysis guided by established lineage markers revealed three keratinocyte clusters (KC1- KC3), two fibroblast clusters (FB1, FB2), smooth muscle cells (SMC), two endothelial cell clusters (VEC, LEC), melanocytes (MEL), sweat gland cells (SG), macrophages/dendritic cells (MAC-DC) and T cells (TC). Ranked by abundance, patient skin exhibited KC>FB>EC>MAC-DC>SMC>TC>SG> MEL. Conclusions Single-cell RNAseq is both feasible and informative in cell-specific transcriptome analysis of fresh non-lesional non-sun exposed LN skin biopsies. 10X genomics significantly increases cell numbers and facilitates identification of major cell clusters compared to Fluidigm C1. The expression of fibroblasts and genes reflective of fibrotic and interferonrelated pathways support the application of this approach to study readily accessible tissue for biomarkers related to disease progression. (Figure Presented)