Background/Purpose: The impact of renal injury in lupus nephritis (LN) 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. While ongoing studies are exploiting single cell RNA sequencing to link phenotype to “biotype” and identify cell specific pathways in the kidney, this study was initiated to address the hypothesis that these pathways may be reflected in uninvolved skin which is more likely to be serially biopsied.

Methods: Single cell RNAseq was performed on cell suspensions prepared from ~2 mm punch biopsies of non-lesional, non-sun-exposed skin from the buttocks of 5 healthy controls, 4 SLE patients without LN and 18 SLE patients with proteinuria (with skin  biopsies obtained within 24 hrs of the kidney biopsy).  Histology revealed Class III (n=6), Class III/V or IV/V mixed (n=11), Class V (n=1), and nephrosclerosis (n=1).  Dissociation of cryostored skin biopsies with collagenase and trypsin enzymes was followed by scRNA-seq using the 10x Genomics platform using V2 and V3 reagents.

Results: We obtained 8,019 and 17,655 high-quality scRNA-seq profiles from single cell suspensions of control and SLE non-lesional, non-sun-exposed skin, respectively. A graph-based clustering method was applied and identified major clusters of cells as visualized by t-distributed stochastic neighbor embedding (tSNE). Differential gene expression analysis guided by established markers revealed these cell clusters as keratinocyte (KC), one smooth muscle cell cluster (SMC), fibroblast (FB), melanocyte (MEL), vascular endothelial cells (VEC), lymphatic endothelial cells (LEC), macrophages-dendritic cells (MAC-DC), T cells (TC) and sweat gland cells (SGC) (Figure 1A). Ranking cells by abundance, the result of the SLE skin cells was KC >FB >VEC >LEC, SMC, MAC-DC, TC, MEL and SGC. Overall, samples processed using the recent V3 single cell reagent kit showed higher genes and transcript captures compared to V2. However, these samples also captured more mitochondrial transcripts (Figure 1B). An analysis of gene expression changes in KC, SMC, and VSC from the LN patients versus controls demonstrated overexpression of interferon stimulated genes. However, the degree of interferon response varied in these cell types with KCs (basal KC, p=0.00312 and hair follicle KC, p=0.000012) showing the highest response followed by VECs (p=0.0043) and SMCs (p=0.0068). In addition to the interferon response signature, VECs from the LN patients also showed upregulation of MHC-II genes such as HLA-DRB5 and HLA-DRB1, suggesting increased antigen presentation capacity (Figure 1C).

Conclusion: scRNA-seq identifies major skin cell types and further clustering identifies rarer cell populations. KCs, SMCs, and VECs from the skin of LN patients reveal diverse IFN response states and additionally VECs also show higher antigen presentation potential. The V3 upgrade of 10x Genomics single cell reagents capture more genes and UMIs per cell, but also higher mitochondrial content compared to the V2 version.

Figure 1

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/evaluation-of-the-transcriptome-of-non-lesional-non-sun-exposed-skin-in-patients-with-lupus-nephritis/