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Systemic sclerosis (SSc) is an autoimmune fibrotic disease of unknown aetiology that is characterized by vascular changes in the skin and visceral organs. Autologous haematopoietic stem cell transplantation can improve skin and organ fibrosis in patients with progressive disease and a high risk of organ failure, indicating that cells originating in the bone marrow are important contributors to the pathogenesis of SSc. Animal studies also indicate a pivotal function of myeloid cells in the development of fibrosis leading to changes in the tissue architecture and dysfunction in multiple organs such as the heart, lungs, liver and kidney. In this Review, we summarize current knowledge about the function of myeloid cells in fibrogenesis that occurs in patients with SSc. Targeted therapies currently in clinical studies for SSc might affect myeloid cell-related pathways. Therefore, myeloid cells might be used as cellular biomarkers of disease through the application of high-dimensional techniques such as mass cytometry and single-cell RNA sequencing.

Background: Activation of melanocortin 1 receptor (MC1R) is known to exert broad anti-inflammatory and anti-fibrotic effects. The purpose of this study is to investigate the potential of dersimelagon, a novel oral MC1R agonist, as a therapeutic agent for systemic sclerosis (SSc). Methods: The effects of dersimelagon phosphoric acid (MT-7117) on skin fibrosis and lung inflammation were evaluated in bleomycin (BLM)-induced SSc murine models that were optimized for prophylactic and therapeutic evaluation. Microarray-based gene expression analysis and serum protein profiling were performed in the BLM-induced SSc models. The effect of MT-7117 on transforming growth factor-β (TGF-β)-induced activation of human dermal fibroblasts was evaluated in vitro. Immunohistochemical analyses of MC1R expression in the skin of SSc patients were performed. Results: Prophylactic treatment with MT-7117 (≥ 0.3 mg/kg/day p.o.) significantly inhibited skin fibrosis and lung inflammation, and therapeutic treatment with MT-7117 (≥ 3 mg/kg/day p.o.) significantly suppressed the development of skin fibrosis in the BLM-induced SSc models. Gene array analysis demonstrated that MT-7117 exerts an anti-inflammatory effect via suppression of the activation of inflammatory cells and inflammation-related signals; additionally, vascular dysfunction was extracted as the pathology targeted by MT-7117. Serum protein profiling revealed that multiple SSc-related biomarkers including P-selectin, osteoprotegerin, cystatin C, growth and differentiation factor-15, and S100A9 were suppressed by MT-7117. MT-7117 inhibited the activation of human dermal fibroblasts by suppressing TGF-β-induced ACTA2(encoding α-smooth muscle actin) mRNA elevation. MC1R was expressed by monocytes/macrophages, neutrophils, blood vessels (endothelial cells), fibroblasts, and epidermis (keratinocytes) in the skin of SSc patients, suggesting that these MC1R-positive cells could be targets for MT-7117. Conclusions: MT-7117 demonstrates disease-modifying effects in preclinical models of SSc. Investigations of its mechanism of action and target expression analyses indicate that MT-7117 exerts its positive effect by affecting inflammation, vascular dysfunction, and fibrosis, which are all key pathologies of SSc. The results of the present study suggest that MT-7117 is a potential therapeutic agent for SSc. A phase 2 clinical trial investigating the efficacy and tolerability of MT-7117 in patients with early, progressive diffuse cutaneous SSc is currently in progress.

The goal of this study was to characterize the performance of fluorine-19 (19F) cardiac magnetic resonance (CMR) for the specific detection of inflammatory cells in a mouse model of myocarditis. Intravenously administered perfluorocarbons are taken up by infiltrating inflammatory cells and can be detected by 19F-CMR. 19F-labeled cells should, therefore, generate an exclusive signal at the inflamed regions within the myocardium.

Here, we describe a basic protocol for the in vitro differentiation of mouse embryonic stem (ES) cells into insulin-producing cells. The three-step protocol comprises (i) the formation of embryoid bodies, (ii) the spontaneous differentiation of embryoid bodies into progenitor cells of ecto-, meso- and endodermal lineages, and (iii) the induction of differentiation of early progenitors into the pancreatic lineage. Differentiated cells can be obtained within approximately 33 d. Differentiation induction by growth and extracellular-matrix factors, including laminin, nicotinamide and insulin, leads to the formation of ES-derived progeny that resembles cells committed to the pancreatic lineage. During differentiation, transcript levels of genes expressed in early pancreatic cells are upregulated. Continued differentiation results in the development of C-peptide/insulin-positive islet-like clusters that release insulin upon glucose stimulation. Differentiated ES cells that overexpress the pancreatic developmental control gene Pax4 develop insulin-secretory granules and reveal functional properties with respect to the pancreas-specific ATP-modulated K+ channel and the normalization of glycemia of streptozotocin-treated diabetic mice.

Ubiquitination is a universal protein degradation pathway in which the molecules of 8.5-kDa proteolytic peptide ubiquitin are covalently attached to the ε-amino group of the substrate's lysine residues. Little is known about the importance of this highly conserved mechanism for protein recycling in mammalian gametogenesis and fertilization. The data obtained by the students and faculty of the international training course Window to the Zygote 2000 demonstrate the accumulation of ubiquitin-cross-reactive structures in the trophoblast, but not in the inner cell mass of the expanding bovine and mouse blastocysts. This observation suggests that a major burst of ubiquitin-dependent proteolysis occurs in the trophoblast of mammalian peri-implantation embryos. This event may be important for the success of blastocyst hatching, differentiation of embryonic stem cells into soma and germ line, and/or implantation in both naturally conceived and reconstructed mammalian embryos.

Regulatory T cells (Tregs) represent a major population in the control of immune homeostasis and autoimmunity. Here we show that Fos-like 2 (Fosl2), a TCR-induced AP1 transcription factor, represses Treg development and controls autoimmunity. Mice overexpressing Fosl2 (Fosl2tg) indeed show a systemic inflammatory phenotype, with immune infiltrates in multiple organs. This phenotype is absent in Fosl2tg× Rag2−/−mice lacking T and B cells, and Fosl2 induces T cell-intrinsic reduction of Treg development that is responsible for the inflammatory phenotype. Fosl2tgT cells can transfer inflammation, which is suppressed by the co-delivery of Tregs, while Fosl2 deficiency in T cells reduces the severity of autoimmunity in the EAE model. We find that Fosl2 could affect expression of FoxP3 and other Treg development genes. Our data highlight the importance of AP1 transcription factors, in particular Fosl2, during T cell development to determine Treg differentiation and control autoimmunity.

Systemic sclerosis (SSc) refers to an autoimmune disease, which is manifested by inflammation, vasculopathy, and fibrosis of the skin and internal organs. There are a number of different animal models recapitulating specific aspects of SSc. The experimental mouse model of bleomycin‐induced skin fibrosis is commonly used to study the pathogenesis observed in SSc. In this model, repetitive intradermal injections of the cytotoxic agent bleomycin trigger progressive skin thickening, associated with excessive accumulation of collagen, infiltration of immune cells, and formation of α‐smooth muscle actin (α‐SMA)‐positive myofibroblasts. In this article, we provide a detailed protocol for the induction of skin fibrosis in experimental mice by bleomycin. Moreover, we describe procedures for processing and analyzing affected skin tissue, provide troubleshooting, highlight advantages and limitations of the presented model, and critically discuss representative results. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Intradermal bleomycin injections to induce skin fibrosis in mice Support Protocol: Mouse tissue collection for fibrosis evaluation and for other molecular assays Basic Protocol 2: Evaluation of mouse skin thickness using Masson's trichrome staining Basic Protocol 3: Measurement of hydroxyproline content in skin tissue using a colorimetric assay Basic Protocol 4: Evaluation of myofibroblasts in mouse skin by immunohistochemistry