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Systemic sclerosis (SSc) is a complex autoimmune disease characterized by fibrosis, vascular abnormalities, and immune dysfunction, with no definitive cure. Patients with progressive pulmonary fibrosis face a high mortality risk, underscoring the urgent need for effective treatments. Although fibroblasts are recognized as key drivers of fibrosis, the precise molecular mechanisms remain poorly understood. In this study, we employ single-cell RNA sequencing to explore the role of fibroblasts in pulmonary fibrosis. Using a mouse model induced by subcutaneous bleomycin administration, we identify two distinct fibroblast subpopulations: nephronectin-positive (NPNT) and peptidase inhibitor 16-positive cells(PI16). NPNT-positive fibroblasts, located around the alveoli, exhibit increased extracellular matrix expression following bleomycin treatment. To further understand pulmonary fibrosis, subcutaneous and intratracheal bleomycin-induced mouse models are compared. A comparative gene expression analysis reveals shared and unique features between the models, highlighting the complexity of the fibrotic process. These findings offer valuable insights into the molecular mechanisms of SSc-associated pulmonary fibrosis and may inform the development of therapies targeting specific fibroblast subpopulations or pathways., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Katsuto Tamai is a scientific founder of and received research funding from StemRIM. Katsuto Tamai and Takashi Shimbo are StemRIM stockholders. Yuya Ouchi, Tomomi Kitayama, Sho Yamazaki and Kastuto Tamai are employees of StemRIM., (Copyright © 2024. Published by Elsevier Inc.)

Macrophages are versatile myeloid leukocytes with flexible cellular states to perform diverse tissue functions beyond immunity. This plasticity is however often hijacked by diseases to promote pathology. Scanning kinetics of macrophage states by single-cell transcriptomics and flow cytometry, we observed atopic dermatitis drastically exhausted a resident subtype S1. Characterized by FRβ/CD163 expression, S1 exhibited strong efferocytosis and chemoattracted monocytes and eosinophils. Here we have delineated mechanisms regulating monocyte decision to acquire S1 identity in skin. During M-CSF driven macrophage differentiation in healthy skin, FRβ was expressed via intrinsic control of STAT6 and ALK5 activities, and did not require heterotypic cellular crosstalk. In contrast, CD163 expression required exposure to fibroblastic secretion. This process depended on SHP1 activity and involved STAT5 inactivation. Suppressed STAT5 activity caused CD163 expression and rendered macrophage insensitive to further induction by fibroblasts. Parsing coculture experiments with in silico ligand expression, we identified laminin-α2 and type-V collagen secreted by hypodermal fibroblasts as CD163-driving factors. S1 identity loss in AD followed a stepwise cascade: reduced laminins availability first dampened CD163 expression, IL4 and TGFβ subsequently acted on CD163 lo/- cells to downregulate FRβ. In AD skin, we showed that imitating this fibroblast-macrophage crosstalk with exogenous laminin-211 encouraged monocyte differentiation to S1 macrophages, fostered homeostatic commitment of extravasated eosinophils, and alleviated dermatitis. Hence, we demonstrated that reinforcing a steady-state cue from hypodermal fibroblasts could override maladaptive pressure on macrophage and restored tissue homeostasis., Competing Interests: Declarations. Conflict of interest: KT is a scientific founder, stockholder of and received research funding from StemRIM. ET and YO are employees of StemRIM. All other authors declare they have no competing interests. Human and animal rights: All animal experiments were conducted under the guidelines of and approved by the Institutional Animal Care and Use Committee of Osaka University Graduate School of Medicine with reference numbers 02-075-003 and 02-075-005. The study did not involve human subjects., (© 2024. The Author(s).)

Macrophages are versatile myeloid leukocytes with flexible cellular states to perform diverse tissue functions beyond immunity. This plasticity is however often hijacked by diseases to promote pathology. Scanning kinetics of macrophage states by single-cell transcriptomics and flow cytometry, we observed atopic dermatitis drastically exhausted a resident subtype S1. Characterized by FRβ/CD163 expression, S1 exhibited strong efferocytosis and chemoattracted monocytes and eosinophils. Here we have delineated mechanisms regulating monocyte decision to acquire S1 identity in skin. During M-CSF driven macrophage differentiation in healthy skin, FRβ was expressed via intrinsic control of STAT6 and ALK5 activities, and did not require heterotypic cellular crosstalk. In contrast, CD163 expression required exposure to fibroblastic secretion. This process depended on SHP1 activity and involved STAT5 inactivation. Suppressed STAT5 activity caused CD163 expression and rendered macrophage insensitive to further induction by fibroblasts. Parsing coculture experiments with in silico ligand expression, we identified laminin-α2 and type-V collagen secreted by hypodermal fibroblasts as CD163-driving factors. S1 identity loss in AD followed a stepwise cascade: reduced laminins availability first dampened CD163 expression, IL4 and TGFβ subsequently acted on CD163lo/− cells to downregulate FRβ. In AD skin, we showed that imitating this fibroblast-macrophage crosstalk with exogenous laminin-211 encouraged monocyte differentiation to S1 macrophages, fostered homeostatic commitment of extravasated eosinophils, and alleviated dermatitis. Hence, we demonstrated that reinforcing a steady-state cue from hypodermal fibroblasts could override maladaptive pressure on macrophage and restored tissue homeostasis. [ABSTRACT FROM AUTHOR]