Faculty Bibliography

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Nonadherence to treatment is a major cause of lupus flares. Hydroxychloroquine (HCQ), a major medication in systemic lupus erythematosus, has a long half-life and can be quantified by high-performance liquid chromatography. This international study evaluated nonadherence in 305 lupus patients with flares using drug levels (HCQ < 200 ng/ml or undetectable desethylchloroquine), and self-administered questionnaires (MASRI < 80%). Drug levels defined 18.4% of the patients as severely nonadherent. In multivariate analyses, younger age, nonuse of steroids, higher body mass index, and unemployment were associated with nonadherence by drug level. Questionnaires classified 23.4% of patients as nonadherent. Correlations between adherence measured by questionnaires, drug level, and physician assessment were moderate. Both methods probably measured two different patterns of nonadherence: self-administered questionnaires mostly captured relatively infrequently missed tablets, while drug levels identified severe nonadherence (i.e., interruption or erratic tablet intake). The frequency with which physicians miss nonadherence, together with underreporting by patients, suggests that therapeutic drug monitoring is useful in this setting. (Trial registration: ClinicalTrials.gov: NCT01509989.).

OBJECTIVE:To assess the prevalence of atherosclerotic cardiovascular disease (ASCVD) and its individual phenotypes of coronary artery disease (CAD), peripheral artery disease (PAD), and cerebrovascular disease by age and sex in a large US cohort of hospitalized patients with systemic lupus erythematosus (SLE). METHODS:A nested case-control study of adults with and without SLE was conducted from the January 1, 2008, through December 31, 2014, National Inpatient Sample. Hospitalized patients with SLE were matched (1:3) by age, sex, race, and calendar year to hospitalized patients without SLE. The prevalences of CAD, PAD, and cerebrovascular disease were evaluated, and associations with SLE were determined after adjustment for common cardiovascular risk factors. RESULTS:Among the 252,676 patients with SLE and 758,034 matched patients without SLE, the mean age was 51 years, 89% were women, and 49% were white. Patients with SLE had a higher prevalence of ASCVD vs those without SLE (25.6% vs 19.2%; OR, 1.45; 95% CI, 1.44-1.47; P<.001). After multivariable adjustment, SLE was associated with a greater odds of ASCVD (adjusted odds ratio [aOR], 1.46; 95% CI, 1.41-1.51). The association between SLE and ASCVD was observed in women and men and was attenuated with increasing age. Also, SLE was associated with increased odds of CAD (aOR, 1.42; 95% CI, 1.40-1.44), PAD (aOR, 1.25; 95% CI, 1.22-1.28), and cerebrovascular disease (aOR, 1.68; 95% CI, 1.65-1.71). CONCLUSION/CONCLUSIONS:In hospitalized US patients, SLE was associated with increased ASCVD prevalence, which was observed in both sexes and was greatest in younger patients.

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

Single-cell RNA sequencing (scRNA-seq) has recently undergone rapid advances in the development of this technology, leading to high throughput and accelerating discovery in many biological systems and diseases. The single-cell resolution of the technique allows for the investigation of heterogeneity in cell populations, and the pinpointing of pathological populations contributing to disease. Here we review the development of scRNA-seq technology and the analysis that has evolved with the ever-increasing throughput. Finally, we highlight recent applications of scRNA-seq to understand the molecular pathogenesis of lupus and lupus nephritis.

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.

OBJECTIVES/OBJECTIVE:In this study we aimed to investigate foetal and maternal pregnancy outcomes from a large multicentre cohort of women diagnosed with MCTD and anti-U1RNP antibodies. METHODS:This multicentre retrospective cohort study describes the outcomes of 203 pregnancies in 94 consecutive women ever pregnant who fulfilled the established criteria for MCTD with confirmed U1RNP positivity. RESULTS:The foetal outcomes in 203 pregnancies were as follows: 146 (71.9%) live births, 38 (18.7%) miscarriages (first trimester pregnancy loss of <12 weeks gestation), 18 (8.9%) stillbirths (pregnancy loss after 20 weeks gestation) and 11 (5.4%) cases with intrauterine growth restriction. Maternal pregnancy outcomes were as follows: 8 (3.9%) developed pre-eclampsia, 2 (0.9%) developed eclampsia, 31 (15.3%) developed gestational hypertension and 3 (1.5%) developed gestational diabetes. Women with MCTD and aPL and pulmonary or muscular involvement had worse foetal outcomes compared with those without. Moreover, we report a case of complete congenital heart block (0.45%) and a case of cutaneous neonatal lupus, both born to a mother with positive isolated anti-U1RNP and negative anti-Ro/SSA antibodies. CONCLUSION/CONCLUSIONS:In our multicentre cohort, women with MCTD had a live birth rate of 72%. While the true frequency of heart block associated with anti-U1RNP remains to be determined, this study might raise the consideration of echocardiographic surveillance in this setting. Pregnancy counselling should be considered in women with MCTD.

The molecular and cellular processes that lead to renal damage and to the heterogeneity of lupus nephritis (LN) are not well understood. We applied single-cell RNA sequencing (scRNA-seq) to renal biopsies from patients with LN and evaluated skin biopsies as a potential source of diagnostic and prognostic markers of renal disease. Type I interferon (IFN)-response signatures in tubular cells and keratinocytes distinguished patients with LN from healthy control subjects. Moreover, a high IFN-response signature and fibrotic signature in tubular cells were each associated with failure to respond to treatment. Analysis of tubular cells from patients with proliferative, membranous and mixed LN indicated pathways relevant to inflammation and fibrosis, which offer insight into their histologic differences. In summary, we applied scRNA-seq to LN to deconstruct its heterogeneity and identify novel targets for personalized approaches to therapy.

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)

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