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Single-cell analysis reveals innate lymphoid cell lineage infidelity in atopic dermatitis

ABSTRACT: Background: Although ample knowledge exists about phenotype and function of cutaneous T lymphocytes, much less is known about the lymphocytic components of the skin’s innate immune system. Objective: To better understand the biologic role of cutaneous innate lymphoid cells (ILCs), we investigated their phenotypic and molecular features under physiologic (normal human skin [NHS]) and pathologic (lesional skin of patients with atopic dermatitis [AD]) conditions. Methods: Skin punch biopsies and reduction sheets as well as blood specimens were obtained from either patients with AD or healthy individuals. Cell and/or tissue samples were analyzed by flow cytometry, immunohistochemistry, and single-cell RNA sequencing or subjected to in vitro / ex vivo culture. Results: Notwithstanding substantial quantitative differences between NHS and AD skin, we found that the vast majority of cutaneous ILCs belong to the CRTH2+ subset and reside in the upper skin layers. Single-cell RNA sequencing of cutaneous ILC-enriched cell samples confirmed the predominance of biologically heterogeneous group 2 ILCs and, for the first time, demonstrated considerable ILC lineage infidelity (coexpression of genes typical of either type 2 [GATA3 and IL13] or type 3/17 [RORC, IL22, and IL26] immunity within individual cells) in lesional AD skin, and to a much lesser extent, in NHS. Similar events were demonstrated in ILCs from skin explant cultures and in vitro expanded ILCs from the peripheral blood. Conclusion: These findings support the concept that instead of being a stable entity with well-defined components, the skin immune system consists of a network of highly flexible cellular players that are capable of adjusting their function to the needs and challenges of the environment.

ORGANISM(S): Homo sapiens

PROVIDER: GSE180885 | GEO | 2021/08/20

REPOSITORIES: GEO

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Project description:Netherton syndrome (NS) is a rare recessive skin disorder caused by loss-of-function mutations in the gene SPINK5 encoding the protease inhibitor LEKTI. NS patients suffer from a severe skin barrier defect, display inflammatory skin lesions and superficial scaling with atopic manifestations. They can present with typical ichthyosis linearis circumflexa (NS-ILC) or scaly erythroderma (NS-SE). Here we employed a combination of several molecular profiling methods to comprehensively characterize the skin, immune cell and allergic phenotypes of NS-ILC and NS-SE patients. This integrated multi-omic approach revealed abnormal epidermal proliferation and differentiation and IL-17/IL-36 signatures in lesional skin and blood in both NS endotypes. While the molecular profiles of NS-ILC and NS-SE lesional skin were very similar, non-lesional skin of each disease endotype displayed distinctive molecular features. NS-SE non-lesional and lesional epidermis showed activation of the type I IFN signaling pathway, which is involved in skin homeostasis and inflammation. NS-ILC lesional skin differed from NS-ILC non-lesional skin by upregulated complement activation and neutrophil infiltration. Serum cytokine profiling and immunophenotyping of circulating lymphocytes showed a Th2-driven allergic response in NS-ILC, whereasNS-SE patients displayed mainly a Th9 axis with increased CCL22/MDC and CCL17/TARC serum levels. This study identifies IL-17/IL-36 as predominant signaling axes in both NS endotypes and unveils distinct molecular features characterizing NS-ILC and NS-SE. These results identify new therapeutic targets and could pave the way for precision medicine of NS. Proteins were extracted from frozen skin samples using RapiGest containing buffer followed by reduction and alkylation using 5 mM Dithiothreitol and 15 mM Iodoacetamide. Proteins were digested by first incubating with LysC followed by incubating with Trypsin. A masterpool containing 10 microgram of desalted peptides (PreOmics) from each sample was fractionated (Agilent 1100 HPLC system). Fractions were measured using data dependent acquisition (DDA) on a Q-Exactive Plus (Thermo Scientific, San Jose, CA) mass spectrometer. For the proteomic comparison 1 microgram of peptide from each patient was measured in data independent acquisition (DIA) mode using the same gradient and instrument as for the masterpool fractions. The resulting .raw files for both the fractionated masterpool (16 DDA files) as well as the patient cohort measurements (33 DIA files) are uploaded here. Additionally, the converted mzML files and annotation files are provided. The complete data analysis of the library generation and the DIA data analysis is uploaded as compressed galaxy history files which can be downloaded, extracted and imported on https://usegalaxy.eu.

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Single-cell analysis reveals innate lymphoid cell lineage infidelity in atopic dermatitis

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