Tissue injury triggers the activation and differentiation of multiple cell types to minimize damage and initiate repair processes. In systemic sclerosis, these repair processes appear to run unchecked, leading to aberrant remodeling and fibrosis of the skin and multiple internal organs, yet the fundamental pathological defect remains unknown. We describe herein a transition wherein the abundant CD34 þ dermal fibroblasts present in healthy human skin disappear in the skin of systemic sclerosis patients, and CD34 À , podoplanin þ , and CD90 þ fibroblasts appear. This transition is limited to the upper dermis in several inflammatory skin diseases, yet in systemic sclerosis, it can occur in all regions of the dermis. In vitro, primary dermal fibroblasts readily express podoplanin in response to the inflammatory stimuli tumor necrosis factor and IL-1b. Furthermore, we show that on acute skin injury in both human and murine settings, this transition occurs quickly, consistent with a response to inflammatory signaling. Transitioned fibroblasts partially resemble the cells that form the reticular networks in organized lymphoid tissues, potentially linking two areas of fibroblast research. These results allow for the visualization and quantification of a basic stage of fibroblast differentiation in inflammatory and fibrotic diseases in the skin. (Am J Pathol 2016, 186: 2650e2664; http://dx

IntroductionThe crucial role of innate immunity in the pathogenesis of systemic sclerosis (SSc) is well established, and in the past few years the hypothesis that Toll-like receptor 4 (TLR4) activation induced by endogenous ligands is involved in fibrogenesis has been supported by several studies on skin, liver, and kidney fibrosis. These findings suggest that TLR4 activation can enhance transforming growth factor beta (TGF-β) signaling, providing a potential mechanism for TLR4/Myeloid differentiation factor 88 (MyD88)-dependent fibrosis.MethodsThe expression of TLR4, CD14 and MD2 genes was analyzed by real-time polymerase chain reaction from skin biopsies of 24 patients with diffuse cutaneous SSc. In order to investigate the effects of the chronic skin exposure to endotoxin (Lipopolysaccharide (LPS)) in vivo we examined the expression of inflammation, TGF-β signaling and cellular markers genes by nanostring. We also identified cellular subsets by immunohistochemistry and flow cytometry.ResultsWe found that TLR4 and its co-receptors, MD2 and CD14, are over-expressed in lesional skin from patients with diffuse cutaneous SSc, and correlate significantly with progressive or regressive skin disease as assessed by the Delta Modified Rodnan Skin Score. In vivo, a model of chronic dermal LPS exposure showed overexpression of proinflammatory chemokines, recruitment and activation of macrophages, and upregulation of TGF-β signature genes.ConclusionsWe delineated the role of MyD88 as necessary for the induction not only for the early phase of inflammation, but also for pro-fibrotic gene expression via activation of macrophages. Chronic LPS exposure might be a model of early stage of SSc when inflammation and macrophage activation are important pathological features of the disease, supporting a role for innate immune activation in SSc skin fibrosis.

Objective To explore the expression of thymic stromal lymphopoietin (TSLP) in patients with diffuse cutaneous systemic sclerosis (dcSSc) and compare its effects in vivo and in vitro with those of interleukin-13 (IL-13) and transforming growth factor β (TGFβ). Methods Skin biopsy specimens from patients with dcSSc (n = 14) and healthy controls (n = 13) were analyzed by immunohistochemistry and immunofluorescence for TSLP, TSLP receptor, CD4, CD8, CD31, and CD163 markers. Wild-type, IL-4Rα1–, and TSLP-deficient mice were treated with TGFβ, IL-13, poly(I-C), or TSLP by osmotic pump. Human fibroblasts and peripheral blood mononuclear cells (PBMCs) were stimulated with TGFβ, IL-13, poly(I-C), or TSLP. Microarray analysis and quantitative polymerase chain reaction were performed to determine gene expression, and protein levels of phospho-Smad2 and macrophage marker CD163 were tested. Results TSLP was highly expressed in the skin of dcSSc patients, more strongly in perivascular areas and in immune cells, and was produced mainly by CD163+ cells. The skin of TSLP-treated mice showed up-regulated clusters of gene expression that overlapped strongly with those in IL-13– and TGFβ-treated mice. TSLP up-regulated specific genes, including CXCL9, proteasome, and interferon (IFN)–regulated genes. TSLP treatment in IL-4Rα1–deficient mice promoted similar cutaneous inflammation as in wild-type mice, though TSLP-induced arginase 1, CCL2, and matrix metalloproteinase 12 messenger RNA levels were blocked. In PBMCs, TSLP up-regulated tumor necrosis factor α, Mx-1, IFNγ, CXCL9, and mannose receptor 1 gene expression. TSLP-deficient mice treated with TGFβ showed less fibrosis and blocked expression of plasminogen activator inhibitor 1 and osteopontin 1. Poly(I-C)–treated mice showed high levels of cutaneous TSLP. Conclusion TSLP is highly expressed in the skin of dcSSc patients and interacts in a complex manner with 2 other profibrotic cytokines, TGFβ and IL-13, strongly suggesting that it might promote SSc fibrosis directly or indirectly by synergistically stimulating pro-fibrotic genes, or production of these cytokines.

Perivascular accumulation of lymphocytes can be a prominent histopathologic feature of various human inflammatory skin diseases. Select examples include systemic sclerosis, spongiotic dermatitis and cutaneous lupus. While a large body of work has described various aspects of the endothelial and vascular smooth muscle layers in these diseases, the outer adventitial compartment is poorly explored. The goal of the present study was to characterize perivascular adventitial fibroblast states in inflammatory human skin diseases and relate these states to perivascular lymphocyte accumulation. In normal skin, adventitial fibroblasts are distinguished by CD90 expression and dense perivascular lymphocytic infiltrates are uncommon. In systemic sclerosis, this compartment expands, but lymphocyte infiltrates remain sparse. In contrast, perivascular adventitial fibroblast expression of VCAM1 is upregulated in spongiotic dermatitis and lupus and is associated with a dense perivascular T cell infiltrate. VCAM1 expression marks transitioned fibroblasts that show some resemblance to the reticular stromal cells in secondary lymphoid organs. Expanded adventitial compartments with perivascular infiltrates similar to the human settings were not seen in the inflamed murine dermis. This species difference may hinder the dissection of aspects of perivascular adventitial pathology. The altered perivascular adventitial compartment and its associated reticular network form a niche for lymphocytes and appear to be fundamental in the development of an inflammatory pattern.

The importance of tissue engineering has been established as a promising approach in treating neurodegenerative diseases. The purpose of the current study is to determine the effect of fibrin hydrogel on the differentiation of iPSC into oligodendrocyte. For this purpose, iPSCs transduced by miR‐338 expressing lentiviruses. They were treated with basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and platelet‐derived growth factor (PDGF)‐AA. The process was traced by a 6‐day treatment in a mitogen‐free medium. At the end of the process, multipolar preoligodendrocytes appeared. In comparison to tissue culture plate (TCP), MTT assay demonstrated a significant increase in the viability of cells cultured in fibrin hydrogel. SEM analysis showed cells with elongated morphology and intertwined intercellular interactions. An immunofluorescent assay confirmed the expression of oligodendrocyte markers Olig2 and O4. In comparison to TCP, real‐time PCR data indicated a significant increase in the expression of some markers such as Olig2, MBP, Sox10, and PDGFRα on cells encapsulated in fibrin hydrogel. Overall, the results suggest that fibrin hydrogel improves viability of cells and promotes the differentiation of iPSCs into preoligodendrocytes. Hence, it can be used as an appropriate option in the tissue engineering in order to treat neurodegenerative diseases. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:192–200, 2020.

MicroRNAs (miRNAs) control gene expression at the posttranscriptional level and have a critical role in many biological processes such as oligodendrocyte differentiation. Recent studies have shown that microRNA 338 (miR-338) is overexpressed during the oligodendrocyte development process in the central nervous system; this finding indicates a potentially important role for miR-338 in oligodendrocyte development. To evaluate this assumption, we studied the effect of miR-338 overexpression on promoting the differentiation of oligodendrocyte progenitor cells (OPCs), derived from human-induced pluripotent stem cells (hiPSC), into preoligodendrocyte. hiPSCs were differentiated into OPCs after treating for 16 days with basic fibroblast growth factor (BFGF), epidermal growth factor (FGF), and platelet-derived growth factor (PDGF)-AA.Bipolar OPCs appeared and the expression of OPC-related markers, including Nestin, Olig2, Sox10, PDGFRα, and A2B5 was confirmed by real-time polymerase chain reaction (PCR) and immunofluorescence. Then, OPCs were transduced by miR-338 expressing lentivirus or were treated with triiodothyronine (T3) for 6 days. Data obtained from real-time PCR and immunofluorescence experiment indicated that preoligodendrocyte markers such as Sox10, O4, and MBP were expressed at higher levels in transduced cells with miR-338 in comparison with the T3 group. So, the overexpression of miR-338 in iPSCderived OPCs can promote their differentiation into preoligodendrocyte which can be used in cell therapy of myelin-related diseases. K E Y W O R D S differentiation, induced pluripotent stem cells, microRNA 338, oligodendrocyte J Cell Biochem. 2019;120:9700-9708. wileyonlinelibrary.com/journal/jcb | 9707

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Objective Multiple observations indicate a role for lymphocytes in driving autoimmunity in systemic sclerosis (SSc). While T and NK cells have been studied in SSc whole blood and bronchoalveolar lavage fluid, their role remains unclear, partly because no studies have analyzed these cell types in SSc-ILD lung tissue. This research aimed to identify and analyze the lymphoid subpopulations in SSc-ILD lung explants. Methods Lymphoid populations from 13 SSc-ILD and 6 healthy control (HC) lung explants were analyzed using Seurat following single cell RNA sequencing. Lymphoid clusters were identified by their differential gene expression. Absolute cell numbers and cell proportions in each cluster were compared between cohorts. Additional analyses were performed using pathway analysis, pseudotime, and cell ligand-receptor interactions. Results Activated CD16+ NK cells, CD8+ tissue resident memory T cells, and regulatory T cells (Tregs) were proportionately higher in SSc-ILD compared with HC lungs. Activated CD16+ NK in SSc-ILD showed upregulated granzyme B, interferon-gamma, and CD226. Amphiregulin, highly upregulated by NK cells, was predicted to interact with epidermal growth factor receptor on several bronchial epithelial cell populations. Shifts in CD8+ T cell populations indicated a transition from resting to effector to tissue resident phenotypes in SSc-ILD. Conclusions SSc-ILD lungs show activated lymphoid populations. Activated cytotoxic NK cells suggest they may kill alveolar epithelial cells, while their expression of amphiregulin suggests they may also induce bronchial epithelial cell hyperplasia. CD8+ T cells in SSc-ILD appear to transition from resting to tissue resident memory phenotype.