Faculty Bibliography

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 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)

BACKGROUND:Systemic lupus erythematosus (SLE) is a complex autoimmune disease associated with an elevated risk for premature cardiovascular disease. Platelets express receptors contributing to inflammation and immunity including FcγRIIA, the low affinity receptor of the Fc portion of IgG antibodies. The variation at a single amino acid substitution, H131R, in the extracellular binding domain alters the affinity for IgG, which may account for individual variation in platelet activity and platelet mediated disease. OBJECTIVES/OBJECTIVE:This study was performed to investigate the association between FcγRIIA genotype, preclinical atherosclerosis, platelet reactivity, and vascular health. METHODS:FcγRIIA was genotyped in 80 SLE patients and 30 healthy controls. Carotid ultrasound plaque, soluble E-selectin, and platelet aggregability were evaluated in SLE and matched controls. RESULTS:Carotid plaque was significantly more prevalent in SLE patients carrying a variant allele compared to those who were homozygous ancestral (58% vs. 25%, P=0.04). In contrast, prevalent carotid plaque was not associated with genotype in controls. Consistently, SLE variant FcγRIIA carriers vs. ancestral had a significant increase in the levels of soluble E-selectin, which was not observed in controls. Monocyte and leukocyte-platelet aggregation and platelet aggregation in response to submaximal agonist stimulation were significantly elevated in SLE patients with the variant vs. ancestral genotype. CONCLUSIONS:Carotid ultrasound plaque, soluble E-selectin levels and platelet activity were more frequently prevalent in SLE patients carrying variant FcγRIIA. The interplay between FcγRIIA-mediated platelet activation and endothelial cells might represent a mechanism underlying the pathogenesis of cardiovascular disease in SLE patients.

Given that diseases associated with anti-SSA/Ro autoantibodies, such as systemic lupus erythematosus and Sjögren syndrome, are linked with an upregulation of IFN and type I IFN-stimulated genes, including sialic acid-binding Ig-like lectin 1 (Siglec-1), a receptor on monocytes/macrophages, recent attention has focused on a potential role for IFN and IFN-stimulated genes in the pathogenesis of congenital heart block (CHB). Accordingly, three approaches were leveraged to address the association of IFN, IFN-stimulated genes, and the phenotype of macrophages in affected fetal cardiac tissue: 1) cultured healthy human macrophages transfected with hY3, an anti-SSA/Ro-associated ssRNA, 2) RNA isolated from freshly sorted human leukocytes/macrophages after Langendorff perfusion of three fetal hearts dying with CHB and three healthy gestational age-matched hearts, and 3) autopsy tissue from three additional human CHB hearts and one healthy heart. TLR ligation of macrophages with hY3 led to the upregulation of a panel of IFN transcripts, including SIGLEC1, a result corroborated using quantitative PCR. Using independent and agnostic bioinformatics approaches, CD45⁺CD11c⁺ and CD45⁺CD11c^- human leukocytes flow sorted from the CHB hearts highly expressed type I IFN response genes inclusive of SIGLEC1. Furthermore, Siglec-1 expression was identified in the septal region of several affected fetal hearts. These data now provide a link between IFN, IFN-stimulated genes, and the inflammatory and possibly fibrosing components of CHB, positioning Siglec-1-positive macrophages as integral to the process.

Photosensitivity, or skin sensitivity to ultraviolet radiation (UVR), is a feature of lupus erythematosus (LE) and other autoimmune and dermatologic conditions. Photosensitivity is associated with increased keratinocyte apoptosis, but mechanisms that modulate keratinocyte apoptosis that are dysfunctional in photosensitivity are poorly understood. Here, we identify a Langerhans cell (LC)-keratinocyte axis that limits UVR-induced keratinocyte apoptosis and skin injury via keratinocyte epidermal growth factor receptor (EGFR) stimulation. LCs express EGFR ligands and ADAM17, the metalloprotease that activates EGFR ligands. LC ADAM17 is required for skin protection and is activated by UVR, suggesting that LCs protect by providing activated EGFR ligands to keratinocytes. Photosensitive systemic LE (SLE) models have reduced LC ADAM17, and topical EGFR ligand reduces photosensitivity. Human SLE skin shows reduced EGFR activation. Together, our data identify a new tissue protective function for LCs, delineate a novel mechanism that limits skin injury, and suggest EGFR stimulation as a treatment for photosensitivity and possibly other dermatologic conditions.
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