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Single-cell RNA sequencing of emigrating cells from human psoriasis skin and control normal skin

ABSTRACT: Single-cell RNA sequencing is transforming how we understand skin immunology, but previous human skin single-cell RNA sequencing data included only a small fraction of inflammatory cells among the overall cell population, such that functional subsets may be difficult to ascertain. We have overcome these obstacles by harvesting inflammatory cells emigrating from a half of 6 mm punch biopsy skin after 48-hour incubation in culture medium without any enzyme, and then analyzing the harvested cells with single-cell RNA sequencing. By this strategy, we obtained single-cell RNA sequencing data of 24,354 cells (leukocytes 46.0%, keratinocytes 49.6%, and melanocytes 2.4%) from 13 human psoriasis skin and 5 healthy volunteer skin. Unsupervised clustering identified NK cells, CD161+ T-cells, CD8+ T-cells, CD4+ T-cells, regulatory T-cells, mature & semimature dendritic cells, melanocytes, and keratinocytes in different layers - S. (Stratum) corneum, S. granulosum, S. spinosum, and S. basale. To understand psoriasis immunopathogenesis at single-cell levels, we compared gene expression between psoriasis cells vs. control cells within each inflammatory cell subtype clusters.

ORGANISM(S): Homo sapiens

PROVIDER: GSE151177 | GEO | 2021/05/03

REPOSITORIES: GEO

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Project description:Herein we demonstrate the efficacy of an unbiased proteomics screening approach for studying protein expression changes in the KC-Tie2 psoriasis mouse model, identifying multiple protein expression changes in the mouse and validating these changes in human psoriasis. KC-Tie2 mouse skin samples (n=3) were compared with littermate controls (n=3) using gel-based fractionation followed by label-free protein expression analysis. 5482 peptides mapping to 1281 proteins were identified and quantitated: 105 proteins exhibited fold-changes ≥2.0 including: stefin A1 (average fold change of 342.4 and an average P = 0.0082; cystatin A, human orthologue); slc25a5 (average fold change of 46.2 and an average P = 0.0318); serpinb3b (average fold change of 35.6 and an average P = 0.0345; serpinB1, human orthologue); and kallikrein related peptidase 6 (average fold change of 4.7 and an average P = 0.2474; KLK6). We independently confirmed mouse gene expression-based increases of selected genes including serpinb3b (17.4-fold, P < 0.0001), KLK6 (9.0-fold, P = 0.002), stefin A1 (7.3-fold; P < 0.001) and slc25A5 (1.5-fold; P = 0.05) using qRT-PCR on a second cohort of animals (n=8). Parallel LC/MS/MS analyses on these same samples verified protein-level increases of 1.3-fold (slc25a5; P < 0.05), 29,000-fold (stefinA1; P < 0.01), 322-fold (KLK6; P < 0.0001) between KC-Tie2 and control mice. To underscore the utility and translatability of our combined approach, we analyzed gene and protein expression levels in psoriasis patient skin and primary keratinocytes vs. healthy controls. Increases in gene expression for slc25a5 (1.8-fold), cystatin A (3.0-fold), KLK6 (5.8-fold) and serpinB1 (76-fold; all P < 0.05) were observed between healthy controls and involved lesional psoriasis skin and primary psoriasis keratinocytes. Moreover slc25a5, cystatin A, KLK6 and serpinB1 protein were all increased in lesional psoriasis skin compared to normal skin. These results highlight the usefulness of preclinical disease models using readily-available mouse skin and demonstrate the utility of proteomic approaches for identifying novel peptides/proteins that are differentially regulated in psoriasis that could serve as sources of auto-antigens or provide novel therapeutic targets for the development of new anti-psoriatic treatments.

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Single-cell RNA sequencing of emigrating cells from human psoriasis skin and control normal skin

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