41 research outputs found

    Fringe Controls Naïve CD4<sup>+</sup>T Cells Differentiation through Modulating Notch Signaling in Asthmatic Rat Models

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    <div><p>The ability of Notch signaling to regulate T helper cell development and differentiation has been widely accepted. Fringe, <em>O-fucose-β1,3-N</em>-acetylglucosaminyltransferases modulate Notch receptor expression and promote the Notch signaling pathway through receptor-ligand binding. In this study, we assayed the expression levels of three Fringe homologs in naive CD4<sup>+</sup>T cells in asthmatic rats. We found that Radical Fringe (Rfng) was highly expressed, whereas both Lunatic Fringe (Lfng) and Manic Fringe (Mfng) were expressed at low levels. Down-regulation of Rfng using siRNA, and overexpression of Lfng or Mfng enhanced Th1 subset lineages and diminished Th2 subset lineages. Notch signaling was more activated in asthmatic naïve CD4<sup>+</sup>T cells than in control cells, and Lfng, but not Mfng or Rfng, partly inhibited Notch signaling in asthmatic naïve CD4<sup>+</sup>T lymphocytes. Lfng overexpression resulted in significantly decreased Th2 cytokine production in asthma, which was the same effect as the GSI (γ-secretase inhibitor) treatment alone, but had an increased effect on Th1 cytokines than GSI treatment. Collectively, these data identify the essential role of Fringe modulating naïve CD4<sup>+</sup>T cells differentiation through Notch signaling. Lfng regulated Th2 cells differentiation via a Notch-dependent manner and Th1 cells differentiation via a Notch-independent manner. Fringe could be a therapeutic strategy for the management and prevention of allergic asthma.</p> </div

    Asthmatic naïve CD4<sup>+</sup>T cells are efficiently transfected with SiRNA.

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    <p>CD4<sup>+</sup>T (5×10<sup>6</sup>) cells were transfected by Amaxa Nucleofection, protocol U-14, with 40 nM, 60 nM, 80 nM, 100 nM FAM-tagged SiRNA. The transfection conditions optimized by flow cytometry analysis. Q-PCR and Western blot were performed to determined the mRNA levels and protein levels after SiRNA-Rfng transfection. <b>A</b>, The optimal final concentration of SiRNA was 100nM (the MOI is 58.97%). <b>B</b>, The optimal time of transfection is more than 6 hr (64.27%). <b>C</b>, Real-time PCR analysis of Rfng levels in transfected naïve CD4<sup>+</sup>T cells. Blank-treated results were taken as 1. Results are from three independent experiments. The data for each group are expressed as means±SEM. *** <i>p</i><0.05, significant differences between siRNA-Rfng and blank-treated, mock-treated or SiRNA-scrambled CD4<sup>+</sup>T cells. <b>D</b>, Rfng protein levels in transfected CD4<sup>+</sup>T cells. CD4<sup>+</sup>T cells were unmanipulated (blank, lane 1), transfected with reagent alone (mock, lane 2), siRNA scrambled (lane 3), or siRNA-Rfng (Lane 4). Transfected CD4<sup>+</sup>T cells were lysates and collected to assess the expression of Rfng by Western blot. Gapdh was used as a loading control. Representative of one of three similar experiments.</p