20 research outputs found

    Recombinant PGRN promotes the induction of inducible regulatory T cells in vitro.

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    <p>Naïve CD4+GFP- cells in lymphocytes of spleen from Foxp3-GFP reporter mice were purified by using Mitenyi reagents and a MACS apparatus. The purity of cells was evaluated by FACS method. CD4+GFP- cells conversion assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112110#s2" target="_blank">Methods</a>, and GFP expression in TGF-β-unstimulated CD4+GFP- cells was taken as a control. After 3-4 days, cells were washed and GFP expression was analyzed by FACS. Data represent three independent experiments are shown. (A) No TGF-β and no PGRN. (B) No TGF-β plus 200 ng/ml of PGRN. (C) No TGF-β plus 1 µg/ml of PGRN. (D) 0.1 ng/ml of TGF-β and no PGRN. (E) 0.1 ng/ml of TGF-βplus 200 ng/ml of PGRN. (F) 0.1 ng/ml of TGF-β plus 1 µg/ml of PGRN. Data represent as a means ±SE of a representative experiment. *<i>p</i><0.05; **<i>p</i><0.01.</p

    Wnt signaling components expression in wild type and PGRN-deficient CD4+CD25+ T cells measured by real-time PCR.

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    <p>All values are shown as a relative ratio to GAPDH measured by 2-ΔΔct method. Data represent as a means ±SE of a representative experiment. *<i>p</i><0.05; **<i>p</i><0.01.</p

    PGRN deficiency does not alter the generation of CD4+CD25+Foxp3+ T cells in vivo.

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    <p>Flow cytometric evaluation of CD4+CD25+Foxp3+ T cells in one-, three-, and six-week-old wild type (WT) and PGRN-deficient mice. (A) The percentage of CD4+ and CD8+ T cells in thymus from one-week-old C57BL/6 mice and PGRN-deficient mice. (B) The percentage of CD4+CD25+Foxp3+ cells in thymus from one-week-old C57BL/6 mice and PGRN-deficient mice. (C) The proportion of CD4+CD25+Foxp3+ cells in spleen from three-week-old C57BL/6 mice and PGRN-deficient mice. (D) CD4+CD25+Foxp3+ cells in lymph nodes from three-week-old C57BL/6 mice and PGRN-deficient mice. (E) The proportion of CD4+CD25+Foxp3+ cells in spleen from six-week-old C57BL/6 mice and PGRN-deficient mice. (F) CD4+CD25+Foxp3+ cells in lymph nodes from six-week-old C57BL/6 mice and PGRN-deficient mice. All data was representative of three mice per group and indicated as mean ± SEM.</p

    PGRN deficiency does not affect the conversion of naïve CD4+CD25- T cells into iTreg mediated by TGF-β.

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    <p>Naïve CD4+ CD25- T cells isolated from both wildtype (WT) and PGRN-deficient mice were stimulated with plate-bound CD3 Ab and solute CD28 Ab and cultured the cells for 3-4 days in the presence or absence of TGF-β, and the expression of GFP was measured by FACS. (A) The GFP levels in CD4+ T cells in the absence of TGF-β. (B) The GFP levels in CD4+ T cells in the presence of 1 ng/ml TGF-β. (C) The GFP levels in CD4+ T cells in the presence of 10 ng/ml TGF-β. All data were repeated three times with similar results.</p

    In vivo proliferation of CD4+CD25+ T cells from spleen and lymph nodes analyzed by BrdU corporation.

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    <p>Wild type and PGRN-deficient mice were injected intraperitoneally with BrdU labeling reagents, and the degrees of BrdU incorporation by CD4+CD25+ T cells from lymphocytes of spleen and lymph nodes were determined by FACS. (A) The BrdU incorporation of wild type CD4+CD25+ T cells from splenocytes. (B) The BrdU incorporation of PGRN-deficient CD4+CD25+ T cells from splenocytes. (C) The percentage of wild type CD4+CD25+ T cells from lymphocytes of lymph nodes that incorporated BrdU. (D) The percentage of PGRN-deficient CD4+CD25+ T cells from lymphocytes of lymph nodes that incorporated BrdU. All data was representative of three mice per group and indicated as mean ±SEM.</p

    PGRN treatment does not change the proportions of CD4+CD25+Foxp3+ cells in normal conditions.

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    <p>One week-old Foxp3-RFP reporter mice were divided into two groups, three mice per group. PGRN group mice were treated with 100 µg PGRN every two days for 1 week, and PBS group mice were injected with the same volume of PBS as a control. The lymphocytes of spleen, peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN), and Peyer's patches (PP) were isolated and analyzed by FACS. All data are representative of three independent experiments.</p

    PGRN-deficient CD4+CD25+ Treg decreased the suppressive capacity to Teff proliferation.

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    <p>Freshly isolated, CFSE-labeled CD4+CD25- T cells from Thy1.1 mice were used as Teff and co-cultured with Tregs at ratios of 1∶2, 1∶1, 2∶1, 4∶1 and 8∶1. CFSE-based Teff proliferation suppression assay in vitro in which 5×10<sup>5</sup> CFSE-labeled Teff cells were stimulated with CD3 antibody in the presence of mitomycin-treated APC cells and different ratios of FACS sorted wild type or PGRN-deficient Tregs. The CFSE proliferation was evaluated by FACS. All data are representative of three separate experiments. (A) Negative control. (B) Positive control. (C) The CFSE dilution when Teff co-cultured with Tregs at ratios of 1∶2. (D) The CFSE dilution when Teff co-cultured with Tregs at ratios of 1∶1. (E) The CFSE dilution when Teff co-cultured with Tregs at ratios of 2∶1. (F) The CFSE dilution when Teff co-cultured with Tregs at ratios of 4∶1. (G) The CFSE dilution when Teff co-cultured with Tregs at ratios of 8∶1. Data represent as a means ±SE of a representative experiment. *<i>p</i><0.05; **<i>p</i><0.01.</p

    Fewer CD4+CD25+Foxp3+ Treg cells seen in PGRN-deficient CIA model.

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    <p>Wild type (n = 6) and PGRN-deficient mice (n = 6) were intradermally immunized with 100 µl of chicken type II collagen emulsified with an equal volume of complete Freund's adjuvant (CFA). 21 days post immunization, draining lymph nodes were extracted and CD4+CD25+Foxp3+ T cells were analyzed by FACS. Data represent as a means ±SE of a representative experiment. **<i>p</i><0.01.</p

    Image1_The interleukin-1 receptor type-1 in disturbed flow-induced endothelial mesenchymal activation.jpeg

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    IntroductionAtherosclerosis is a progressive disease that develops in areas of disturbed flow (d-flow). Progressive atherosclerosis is characterized by bulky plaques rich in mesenchymal cells and high-grade inflammation that can rupture leading to sudden cardiac death or acute myocardial infarction. In response to d-flow, endothelial cells acquire a mesenchymal phenotype through endothelial-to-mesenchymal transition (EndMT). However, the signaling intermediaries that link d-flow to EndMT are incompletely understood.Methods and ResultsIn this study we found that in human atherosclerosis, cells expressing SNAI1 (Snail 1, EndMT transcription factor) were highly expressed within the endothelial cell (EC) layer and in the pre-necrotic areas in unstable lesions, whereas stable lesions did not show any SNAI1 positive cells, suggesting a role for EndMT in lesion instability. The interleukin-1 (IL-1), which signals through the type-I IL-1 receptor (IL-1R1), has been implicated in plaque instability and linked to EndMT formation in vitro. Interestingly, we observed an association between SNAI1 and IL-1R1 within ECs in the unstable lesions. To establish the causal relationship between EndMT and IL-1R1 expression, we next examined IL-1R1 levels in our Cre-lox endothelial-specific lineage tracing mice. IL-1R1 and Snail1 were highly expressed in ECs under atheroprone compared to athero-protective areas, and oscillatory shear stress (OSS) increased IL-1R1 protein and mRNA levels in vitro. Exposure of ECs to OSS resulted in loss of their EC markers and higher induction of EndMT markers. By contrast, genetic silencing of IL-1R1 significantly reduced the expression of EndMT markers and Snail1 nuclear translocation, suggesting a direct role for IL-1R1 in d-flow-induced EndMT. In vivo, re-analysis of scRNA-seq datasets in carotid artery exposed to d-flow confirmed the IL-1R1 upregulation among EndMT population, and in our partial carotid ligation model of d-flow, endothelial cell specific IL-1R1 KO significantly reduced SNAI1 expression.DiscussionGlobal inhibition of IL-1 signaling in atherosclerosis as a therapeutic target has recently been tested in the completed CANTOS trial, with promising results. However, the data on IL-1R1 signaling in different vascular cell-types are inconsistent. Herein, we show endothelial IL-1R1 as a novel mechanosensitive receptor that couples d-flow to IL-1 signaling in EndMT.</p

    Image2_The interleukin-1 receptor type-1 in disturbed flow-induced endothelial mesenchymal activation.jpeg

    No full text
    IntroductionAtherosclerosis is a progressive disease that develops in areas of disturbed flow (d-flow). Progressive atherosclerosis is characterized by bulky plaques rich in mesenchymal cells and high-grade inflammation that can rupture leading to sudden cardiac death or acute myocardial infarction. In response to d-flow, endothelial cells acquire a mesenchymal phenotype through endothelial-to-mesenchymal transition (EndMT). However, the signaling intermediaries that link d-flow to EndMT are incompletely understood.Methods and ResultsIn this study we found that in human atherosclerosis, cells expressing SNAI1 (Snail 1, EndMT transcription factor) were highly expressed within the endothelial cell (EC) layer and in the pre-necrotic areas in unstable lesions, whereas stable lesions did not show any SNAI1 positive cells, suggesting a role for EndMT in lesion instability. The interleukin-1 (IL-1), which signals through the type-I IL-1 receptor (IL-1R1), has been implicated in plaque instability and linked to EndMT formation in vitro. Interestingly, we observed an association between SNAI1 and IL-1R1 within ECs in the unstable lesions. To establish the causal relationship between EndMT and IL-1R1 expression, we next examined IL-1R1 levels in our Cre-lox endothelial-specific lineage tracing mice. IL-1R1 and Snail1 were highly expressed in ECs under atheroprone compared to athero-protective areas, and oscillatory shear stress (OSS) increased IL-1R1 protein and mRNA levels in vitro. Exposure of ECs to OSS resulted in loss of their EC markers and higher induction of EndMT markers. By contrast, genetic silencing of IL-1R1 significantly reduced the expression of EndMT markers and Snail1 nuclear translocation, suggesting a direct role for IL-1R1 in d-flow-induced EndMT. In vivo, re-analysis of scRNA-seq datasets in carotid artery exposed to d-flow confirmed the IL-1R1 upregulation among EndMT population, and in our partial carotid ligation model of d-flow, endothelial cell specific IL-1R1 KO significantly reduced SNAI1 expression.DiscussionGlobal inhibition of IL-1 signaling in atherosclerosis as a therapeutic target has recently been tested in the completed CANTOS trial, with promising results. However, the data on IL-1R1 signaling in different vascular cell-types are inconsistent. Herein, we show endothelial IL-1R1 as a novel mechanosensitive receptor that couples d-flow to IL-1 signaling in EndMT.</p
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