Background/Purpose:

Given the widespread vasculopathy present in SLE, endothelial cell activation in the renal tubulointerstitium in lupus nephritis (LN) may be accompanied by similar activation in other tissue beds, even non-lesional skin. Faithful reflection of a relevant pathway in renal tissue by a more readily accessible compartment would be an advance. 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. Accordingly, this study utilized single-cell RNAseq as a novel approach to identify whether non-lesional non-sun exposed skin reflects diffuse activation of the microvasculature in patients with LN.  

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 SLE patients with proteinuria and known ISN/RPS Class and 1 healthy control.  The libraries were prepared on the Fluidigm C1 platform followed by sequencing on an Illumina HiSeq 2500. We generated 192 single-cell data sets with on average 3500 genes/cell and 50% of reads mapping to the reference genome.  The profile of differential expression analysis was used to assign in vivo cell-type compositions through unsupervised sampling and modeling of transcriptional states in single cells. Data are expressed as transcripts per million. 

Results:

Based on expression of PROCR, F2R, VWF, CD34, SERPINE1, ESAM and MCAM (CD146), assignments yielded 36 endothelial cells from 5 patients. From the two Class II subjects there were 10 single-cell transcriptomes. The other three subjects (1- Class IV,V; 1- Class V, 1-Class IV) yielded 26 single cell transcriptomes. Further consideration used evaluations of broad signatures involving the NFkB pathway (reflected by expression of SELE, ICAM1 & VCAM1) and IFNa response/Stat genes (IFI27, IFI44L, IFIH1, IFIT1, IFIT3, CXCL14 & SOCS3). The endothelial cells from patients with Class II had a significantly lower expression of NFkB-related genes compared to those with more advanced disease (4 of 10, i.e. 4 cells expressed at least one transcript and 6 none, vs 20 of 26, p<0.001). Both groups strongly expressed at least one IFNa response gene (10 of 10 vs 26 of 26).  In addition, standard lineage markers identified the major skin resident and infiltrating cell types including keratinocytes, and lymphocytes.  In contrast to the endothelial cells, these cells all displayed low expression of NFkB transcripts, independent of biopsy Class. However, IFNa responsive genes were strongly represented in all 5 patients. The skin biopsy from the healthy control showed neither NFkB nor IFNa responsive genes. 

Conclusion:

Single-cell RNAseq is feasible and informative in cell specific transcriptome analysis of fresh non-lesional skin biopsies from SLE patients with a spectrum of active renal disease. Insidious expression of endothelial activation and genes reflective of cytokine exposure support application of this novel approach to study readily accessible tissue. Insight into disease progression should facilitate earlier identification and treatment, critical to tissue survival in organs such as the kidney.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/single-cell-sequencing-of-non-lesional-non-sun-exposed-skin-from-sle-patients-with-proteinuria-supports-widespread-endothelial-activation/