Increased CD8+ tissue resident memory T cells, regulatory T cells and activated natural killer cells in systemic sclerosis lungs.

OBJECTIVE: Multiple observations indicate a role for lymphocytes in driving autoimmunity in 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 analysed these cell types in SSc-interstitial lung disease (ILD) lung tissue. This research aimed to identify and analyse the lymphoid subpopulations in SSc-ILD lung explants. METHODS: Lymphoid populations from 13 SSc-ILD and 6 healthy control (HC) lung explants were analysed 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 Treg cells were proportionately higher in SSc-ILD compared with HC lungs. Activated CD16+ NK cells in SSc-ILD showed upregulated granzyme B, IFN-γ 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 the tissue resident memory phenotype.

Fibrin hydrogel as a scaffold for differentiation of induced pluripotent stem cells into oligodendrocytes.

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.

Perivascular Adventitial Fibroblast Specialization Accompanies T Cell Retention in the Inflamed Human Dermis.

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. Although 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 current 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.

Derivation of preoligodendrocytes from human-induced pluripotent stem cells through overexpression of microRNA 338.

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 iPSC-derived OPCs can promote their differentiation into preoligodendrocyte which can be used in cell therapy of myelin-related diseases.

Enzyme-Linked Immunosorbent Spot (ELISpot) monitoring of cytokine-producing cells for the prediction of acute rejection in renal transplant patients.

The purpose of this study was to evaluate T-cell immunity markers using serial post-transplantation monitoring of cytokine-producing cells during the first post-transplant months for the prediction of acute rejection and potentially chronic rejection of kidney allograft. We followed 57 kidney allograft recipients for meanly 3 years post-transplantation. Blood samples were collected pre-transplant, 2, 4 and 12 weeks post-transplant. The frequencies of IL-10-, IL-17- and IFN-γ-producing cells were determined in all time-points using ELISPOT assay. The results of ELISpot monitoring and levels of IL-23 and TGF-ß were compared between recipients with acute (n = 12) or chronic rejection episodes and patients with stable graft function (n = 45). In all post-transplant time-points, significantly high frequencies of IFN-γ- and IL-17-producing cells and low frequency of IL-10-producing cells were observed in rejection group versus patients with stable graft function (P < 0.0001). The ROC curve analysis for determining the reliability of cytokine-producing cells for the prediction of acute rejection revealed that AUC was 0.046 for IL-10 (P < 0.001), 0.927 for IL-17 (P < 0.001) and 0.929 for INF-γ-producing cells (P < 0.001). Our results indicate that analyzing the frequencies of INF-γ/IL-10/IL-17-producing cells may define a reliable panel for the prediction of acute rejection within the first post-transplant year which could also be applicable for the prediction of chronic rejection episodes.

Altered Dermal Fibroblasts in Systemic Sclerosis Display Podoplanin and CD90.

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(+) dermal fibroblasts present in healthy human skin disappear in the skin of systemic sclerosis patients, and CD34(-), podoplanin(+), and CD90(+) 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-1ß. 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.

Chronic Toll-like receptor 4 stimulation in skin induces inflammation, macrophage activation, transforming growth factor beta signature gene expression, and fibrosis.

INTRODUCTION: The 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-ß) signaling, providing a potential mechanism for TLR4/Myeloid differentiation factor 88 (MyD88)-dependent fibrosis. METHODS: The 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-ß signaling and cellular markers genes by nanostring. We also identified cellular subsets by immunohistochemistry and flow cytometry. RESULTS: We 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-ß signature genes. CONCLUSIONS: We 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.

Comparison of the Th1, IFN-γ secreting cells and FoxP3 expression between patients with stable graft function and acute rejection post kidney transplantation.

There are limited clinical investigations identifying the percentage of T helper 1 (Th1) and T regulatory (Treg) cells in stable as well as rejected kidney allografts, a concept which needs to be more studied. The aim of our study was to compare the percentage of CD4+ IFN-γ+ cells, the number of IFN-γ secreting cells and the amount of FoxP3 expression in patients with or without stable graft function, to determine the roles of these immunological factors in stable and rejected renal allografts. In this prospective study, 3 months after transplantation 30 patients who received renal transplants from unrelated living donors were enrolled and divided into two groups, 20 patients with stable graft function and 10 patients with biopsy proven acute rejection. The percentage of Th1 CD4+ IFN-γ+ cells was determined on PBMC by flow cytometry and the number of IFN-γ secreting cells by ELISPOT method. Furthermore, FoxP3 expression of PBMCs was measured by Real Time PCR method. The results of these assessments in both groups were statistically analyzed by SPSS 14.0. Our results showed that the percentage of Th1 CD4+ IFN-γ+ cells and the number of IFN-γ secreting cells were significantly higher in the patients with acute rejection in comparison to the stable graft function group (p<0.001). In addition, the level of FoxP3 gene expression was higher in the group with stable graft compared to the acute rejection group. The higher percentage of CD4+ IFN-γ+Th1 subset and number of IFN-γ secreting cells and also the lower expression of Foxp3 could prone the patients to acute rejection episode post transplantation. By these preliminary data, it is suggested that monitoring of Th1 cells post transplantation, as an immunologic marker could predict the possibility of rejection episodes.

Thymic stromal lymphopoietin is up-regulated in the skin of patients with systemic sclerosis and induces profibrotic genes and intracellular signaling that overlap with those induced by interleukin-13 and transforming growth factor ß.

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 ß (TGFß). 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 TGFß, IL-13, poly(I-C), or TSLP by osmotic pump. Human fibroblasts and peripheral blood mononuclear cells (PBMCs) were stimulated with TGFß, 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 TGFß-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 TGFß 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, TGFß and IL-13, strongly suggesting that it might promote SSc fibrosis directly or indirectly by synergistically stimulating profibrotic genes, or production of these cytokines.