An optimized protocol to identify keratinocyte subpopulations in vitro by single-cell RNA sequencing analysis

Here, we describe a protocol for single-cell isolation from the primary culture of normal human epidermal keratinocytes derived from neonatal foreskin. The cell culture conditions have been optimized for inducing expression of keratinocyte differentiation markers. Cells are cultured in the absence or presence of a bioactive lipid lysophosphatidic acid (LPA). Single cells are isolated by Fluidigm C1 system. This is followed by cDNA library preparation using Takara SMART-Seq v4 Ultra and Illumina Nextera XT kit for RNA sequencing

T cell–intrinsic prostaglandin E₂-EP2/EP4 signaling is critical in pathogenic T_H17 cell–driven inflammation

PGE2経路による病因細胞Th17の増殖機構を解明 --乾癬の慢性的な皮膚炎症を改善する新しい治療薬開発に向けて--. 京都大学プレスリリース. 2018-06-20.Background: IL-23 is the key cytokine for generation of pathogenic IL-17–producing helper T (TH17) cells, which contribute critically to autoimmune diseases. However, how IL-23 generates pathogenic TH17 cells remains to be elucidated. Objectives: We sought to examine the involvement, molecular mechanisms, and clinical implications of prostaglandin (PG) E2–EP2/EP4 signaling in induction of IL-23–driven pathogenic TH17 cells. Methods: The role of PGE2 in induction of pathogenic TH17 cells was investigated in mouse TH17 cells in culture in vitro and in an IL-23–induced psoriasis mouse model in vivo. Clinical relevance of the findings in mice was examined by using gene expression profiling of IL-23 and PGE2-EP2/EP4 signaling in psoriatic skin from patients. Results: IL-23 induces Ptgs2, encoding COX2 in TH17 cells, and produces PGE2, which acts back on the PGE receptors EP2 and EP4 in these cells and enhances IL-23–induced expression of an IL-23 receptor subunit gene, Il23r, by activating signal transducer and activator of transcription (STAT) 3, cAMP-responsive element binding protein 1, and nuclear factor κ light chain enhancer of activated B cells (NF-κB) through cyclic AMP–protein kinase A signaling. This PGE2 signaling also induces expression of various inflammation-related genes, which possibly function in TH17 cell–mediated pathology. Combined deletion of EP2 and EP4 selectively in T cells suppressed accumulation of IL-17A+ and IL-17A+IFN-γ+ pathogenic Th17 cells and abolished skin inflammation in an IL-23–induced psoriasis mouse model. Analysis of human psoriatic skin biopsy specimens shows positive correlation between PGE2 signaling and the IL-23/TH17 pathway. Conclusions: T cell–intrinsic EP2/EP4 signaling is critical in IL-23–driven generation of pathogenic TH17 cells and consequent pathogenesis in the skin

Single-cell RNA sequencing identifies a migratory keratinocyte subpopulation expressing THBS1 in epidermal wound healing

Keratinocyte differentiation is an intricate process that is regulated by multiple mediators. Using cultured human keratinocytes, we found that lysophosphatidic acid (LPA) induced the differentiation of a previously unsuspected keratinocyte subpopulation expressing the extracellular matrix protein, thrombospondin-1 (THBS1). This action of LPA was mediated by the RHO/ROCK-SRF signaling downstream of LPA₁ and LPA₅ receptors and required ERK activity. Suppression of THBS1 in vitro suggested a migratory role of THBS1⁺ keratinocytes. Moreover, we analyzed publicly deposited single-cell RNA sequencing dataset and identified Thbs1-expressing keratinocytes in the mouse wound skin. Immunohistochemistry analysis revealed that Thbs1⁺ keratinocytes were apparently differentiated from basal keratinocytes upon wounding, subsequently polarized and migrated suprabasally toward the wound front, and eventually underwent terminal differentiation in the neo-epidermis. Importantly, inhibition of Erk activity suppressed Thbs1⁺ keratinocyte differentiation in wound healing. Based on these findings, we suggest that THBS1⁺ keratinocyte is a migratory keratinocyte subpopulation that facilitates epidermal wound healing

Drought Stress Responses in Context-Specific Genome-Scale Metabolic Models of Arabidopsis thaliana

Drought perturbs metabolism in plants and limits their growth. Because drought stress on crops affects their yields, understanding the complex adaptation mechanisms evolved by plants against drought will facilitate the development of drought-tolerant crops for agricultural use. In this study, we examined the metabolic pathways of Arabidopsis thaliana which respond to drought stress by omics-based in silico analyses. We proposed an analysis pipeline to understand metabolism under specific conditions based on a genome-scale metabolic model (GEM). Context-specific GEMs under drought and well-watered control conditions were reconstructed using transcriptome data and examined using metabolome data. The metabolic fluxes throughout the metabolic network were estimated by flux balance analysis using the context-specific GEMs. We used in silico methods to identify an important reaction contributing to biomass production and clarified metabolic reaction responses under drought stress by comparative analysis between drought and control conditions. This proposed pipeline can be applied in other studies to understand metabolic changes under specific conditions using Arabidopsis GEM or other available plant GEMs

LPA induces keratinocyte differentiation and promotes skin barrier function through the LPAR1/LPAR5-RHO-ROCK-SRF axis

ヒト表皮細胞の分化と皮膚バリア機能の調節機構を解明 --アトピー性皮膚炎の新たな治療戦略へ向けて--. 京都大学プレスリリース. 2018-11-16.The skin barrier protects our body from water loss, allergens and pathogens. Profilaggrin (proFLG) is produced by differentiated keratinocytes and is processed into FLG monomers. These monomers crosslink keratin filaments and are also decomposed to natural moisturizing factors in the stratum corneum for skin hydration and barrier function. Deficits in FLG expression impair skin barrier function and underlie skin diseases such as dry skin and atopic dermatitis (AD). However, intrinsic factors that regulate FLG expression and their mechanism of action remain unknown. Here, we show that lysophosphatidic acid (LPA) induces FLG expression in human keratinocytes via the LPAR1 and LPAR5 receptors and the downstream RHO-ROCK-SRF pathway. Comprehensive gene profiling analysis further revealed that LPA not only induces FLG expression but also facilitates keratinocyte differentiation. Moreover, LPA treatment significantly upregulated FLG production in a three-dimensional culture model of human skin, and promoted barrier function in mouse skin in vivo. Thus, our work demonstrates a previously unsuspected role for LPA and its downstream signaling in the maintenance of skin homeostasis, which may serve as a novel therapeutic target for skin barrier dysfunction

An optimized protocol to identify keratinocyte subpopulations invitro by single-cell RNA sequencing analysis

Here, we describe a protocol for single-cell isolation from the primary culture of normal human epidermal keratinocytes derived from neonatal foreskin. The cell culture conditions have been optimized for inducing expression of keratinocyte differentiation markers. Cells are cultured in the absence or presence of a bioactive lipid lysophosphatidic acid (LPA). Single cells are isolated by Fluidigm C1 system. This is followed by cDNA library preparation using Takara SMART-Seq v4 Ultra and Illumina Nextera XT kit for RNA sequencing