1) Lipid-orchestrated paracrine circuit coordinates mast cell maturation and anaphylaxis through functional interaction with fibroblasts & 2) Epidermal ABHD12B-driven atypical acylceramide safeguards against scleroderma [skin scRNA-seq]

1) Interaction of mast cells (MCs) with fibroblasts is essential for MC maturation within tissue microenvironments, although the underlying mechanism is incompletely understood. Through a phenotypic screening of >30 mouse lines deficient in lipid-related genes, we found that deletion of the lysophosphatidic acid (LPA) receptor LPA1, like that of the phospholipase PLA2G3, the prostaglandin D2 (PGD2) synthase L-PGDS, or the PGD2 receptor DP1, impairs MC maturation and thereby anaphylaxis. Mechanistically, MC-secreted PLA2G3 acts on extracellular vesicles (EVs) to supply lysophospholipids, which are converted by fibroblast-derived autotaxin (ATX) to LPA. Fibroblast LPA1 then integrates multiple pathways required for MC maturation by facilitating integrin-mediated MC-fibroblast adhesion, IL-33-ST2 signaling, L-PGDS-driven PGD2 generation, and feedforward ATX-LPA1 amplification. Defective MC maturation resulting from PLA2G3 deficiency is restored by supplementation with LPA1 agonists or PLA2G3-modified EVs. Thus, the lipid-orchestrated paracrine circuit involving PLA2G3-driven lysophospholipid, eicosanoid, integrin, and cytokine signaling fine-tunes MC-fibroblast communication, ensuring MC maturation. 2) Systemic scleroderma (SSc) is a devastating fibrotic disease with poor prognosis, yet its underlying mechanisms remain poorly understood. Given that lipid dysregulation is often associated with skin disorders, we screened cutaneous expression of various lipid hydrolases in SSc patients and found that ABHD12B, a putative hydrolase expressed in terminally differentiated epidermal keratinocytes, was most robustly downregulated in the affected skin. ABHD12B deficiency disturbed skin barrier and exaggerated skin fibrosis in mouse scleroderma models. Single-cell RNA-sequencing and untargeted lipidomic analyses of the skin revealed that ABHD12B deficiency facilitated epidermal- and endothelial-to-mesenchymal transitions and selectively reduced the levels of 1-O-acylceramides with very-long-chain fatty acids. Topical application of 1-O-acylceramide to ABHD12B-deficient mice reversed skin barrier abnormality and fibrosis. Thus, our results reveal a functional connection of ABHD12B with 1-O-acylceramide biosynthesis, highlight an unexplored anti-fibrotic role of this unique lipid, and point to this lipid-driven pathway as a potential drug target for SSc. Overall design: Whole cells and CD326-negative cells isolated by AutoMACS from ear skin of Abhd12b deficient (Abhd12b-/-) and wild-type (Abhd12b+/+) developing control or hypochrorite (HClO)-induced SSc model and analyzed using scRNA-seq. Preparation of the scRNA-seq library was performed as described previously (Shichino et al. Commun Biol 2022). In brief, cells prepared from mouse skin were stained with BD Ms Single-Cell Multiplexing Kit (anti-mouse CD45, 30-F11) and Custom BD Single-Cell Multiplexing Set (anti-mouse MHC-H2 class I, M1/42) (BD Biosciences). Obtained single-cell suspensions were subjected to a BD Rhapsody system with Targeted mRNA and AbSeq Amplification Kit (BD Biosciences) following the manufacture's instructions. Resultant beads were reverse transcribed, treated with exonuclease I, and subjected to TAS-Seq workflow for cDNA and hashtag library amplification. Illumina cDNA libraries were constructed from amplified cDNA libraries by using NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs). Ligand products were purified and performed barcording PCR. Size distribution of amplified products was analyzed by a MultiNA system (MCE-202; Shimadzu). Resultant libraries and barcorded hastag libraries were quantified using KAPA Library Quantification Kit (Roche). Pooled libraries were sequenced on an Illumina NovaSeq 6000 sequencer with an Illumina NovaSeq 6000 S4 Reagent Kit v1.5 (both from Illumina). Two billion clusters, equivalent to 400 Gb/analysis, were obtained by pair-ended analysis of 100 bp of sequencing data. Fastq data preprocessing and generation of the single-cell gene-expression matrix was performed as described previously (Shichino et al. Commun Biol 2022). The resultant dataset was analyzed using R software package Seurat v4.0.4120 in R 4.1.3.