Suppression mediated by dsRNA is not TLR3-dependent and can be blocked by anti-miR to miR-150.

<p><u><b>a</b></u>. Treatment of CS-effector cells from either wild type (WT) or TLR3^-/- mice with OX Ts Sup of WT mice results in equal suppression of the CS immune response (Group B and D). <u><b>b</b></u><b>.</b> Among different miRNA antagonists (groups C-G), only anti-miR to miR-150 is able to reverse the suppression of CS mediated by TNP Ts Sup QRNA (group C).</p

Possible Ag-specific suppression by Ts Sup exRNA PCE.

<p><u><b>a</b></u><b>.</b> OX specific CS-effector cells were suppressed by OX Ts Sup PCE, but not TNP Ts Sup PCE (Group D vs C). <u><b>b</b></u>. In a dual reciprocal criss cross Ag-specificity experiment, TNP vs OX-specific CS-effector cells were suppressed by PCE from Ts Sup induced by tolerization with their respective homologous TNP or OX haptens (Groups B and F), but not from mice tolerized with the heterologous hapten (Groups C and E).</p

Enzymatic treatment of nucleic acids and associated proteins from PCE and QRNA of TNP Ts Sup eliminates suppressive activity.

<p><u><b>a</b></u>. TNP Ts Sup-derived PCE suppression is sensitive to RNase A (Sigma 4375), but not DNase treatment (Group D vs C). <u><b>b</b></u>. Purer RNase A (Sigma 5250, Group E) and RNase III (Group H), as well as proteinase K with and without SDS (Groups F and G) treatment of QRNA from Sup of TNP Ts, eliminates its suppressive activity.</p

Dose-dependent suppression mediated by PCE and QRNA fractions of TNP Ts Sup.

<p><u><b>a</b></u>. Three-fold decreasing doses of TNP Ts Sup-derived PCE regressively suppresses adoptive CS responses down to a dose of 15μg per eventual recipient (Groups B and C). <u><b>b</b></u>. Five-fold decreasing dose-response of QRNA from TNP Ts Sup PCE results in suppression of adoptive CS responses down to a dose of 0.6μg per recipient (Group C).</p

RNA from Phenol-Chloroform Extract (PCE) and Qiagen column fraction (QRNA) from CD3+ CD8+ suppressor T lymphocyte (Ts) culture supernatant (Sup) is suppressive.

<p><u><b>a</b></u><b>.</b> PCE from TNP Ts Sup suppresses the adoptive transfer of CS-effector cells (Groups B and D). <u><b>b</b></u>. Treatment of Ts cells with anti-CD3 mAb plus complement (C’) eliminates production of suppressive Sup (Group D). <u><b>c</b></u><b>.</b> Similar treatment of Ts cells with anti-CD8 mAb plus C’ also eliminates generation of suppressive Sup (Group D), while anti-CD4 mAb plus C’ treatment does not (Group C). <u><b>d</b></u><b>.</b> QRNA from TNP Ts Sup PCE separated on a Qiagen column suppresses adoptively transferred CS, while the QDNA fraction is inactive (Group C vs D). <u><b>e</b></u><b>.</b> TNP- and OX-specific Ts Sup-derived PCE (Groups B and C) and QRNA (Groups E and F) inhibit HT-2 cell in vitro responsiveness to IL-2, compared to Nl Cell Sup PCE and QRNA (Groups A and D).</p

Suppression by extracellular QRNA from TNP Ts Sup free of exosomes, or miR-150 alone, associating with B-cell derived Ag-specific exosomes from the assayed CS-effector cell mixture.

<p><u><b>a.</b></u> Treatment of TNP-CS-effector cells with two day immune B-1 B cell-derived TNP-specific exosomes that are supplemented with QRNA from TNP Ts Sup (Group D) results in significant suppression of the adoptive transfer. <u><b>b.</b></u> Similarly, two day immune B-1 B cell-derived exosomes, supplemented with decreasing doses of miR-150 alone, mediated suppression of TNP-CS-effector cell adoptive transfer (Groups E-G), down to a dose of 750pg per eventual recipient, which is 50 femtomoles per eventual recipient (Group G). <u><b>c.</b></u> The in vitro HT-2 cell responsiveness to IL-2 was suppressed by QRNA from Ts Sup and this was inhibited by pre-incubation of the QRNA with anti-miRNA-150, but not by anti-miRNA-150* (Groups B and C). Further, pure miR-150 alone was inhibitory compared to mimic control (Groups F vs G).</p

Undifferentiated Bronchial Fibroblasts Derived from Asthmatic Patients Display Higher Elastic Modulus than Their Non-Asthmatic Counterparts

<div><p>During asthma development, differentiation of epithelial cells and fibroblasts towards the contractile phenotype is associated with bronchial wall remodeling and airway constriction. Pathological fibroblast-to-myofibroblast transition (FMT) can be triggered by local inflammation of bronchial walls. Recently, we have demonstrated that human bronchial fibroblasts (HBFs) derived from asthmatic patients display some inherent features which facilitate their FMT <i>in vitro</i>. In spite of intensive research efforts, these properties remain unknown. Importantly, the role of undifferentiated HBFs in the asthmatic process was systematically omitted. Specifically, biomechanical properties of undifferentiated HBFs have not been considered in either FMT or airway remodeling <i>in vivo</i>. Here, we combine atomic force spectroscopy with fluorescence microscopy to compare mechanical properties and actin cytoskeleton architecture of HBFs derived from asthmatic patients and non-asthmatic donors. Our results demonstrate that asthmatic HBFs form thick and aligned ‘ventral’ stress fibers accompanied by enlarged focal adhesions. The differences in cytoskeleton architecture between asthmatic and non-asthmatic cells correlate with higher elastic modulus of asthmatic HBFs and their increased predilection to TGF-β-induced FMT. Due to the obvious links between cytoskeleton architecture and mechanical equilibrium, our observations indicate that HBFs derived from asthmatic bronchi can develop considerably higher static tension than non-asthmatic HBFs. This previously unexplored property of asthmatic HBFs may be potentially important for their myofibroblastic differentiation and bronchial wall remodeling during asthma development.</p></div

Additional file 1: Figure S1. of miR-200b downregulates CFTR during hypoxia in human lung epithelial cells

Endogenous levels of miR-200b in NHBEC, Calu3 and 16HBE14o- cells. (A) Comparison of CFTR mRNA (white) and miR-200b (grey) relative levels between NHBEC (primary cells), Calu3 and 16HBE14o- cells during normoxic conditions. CFTR mRNA levels from 2 independent experiments (n = 8) are plotted normalized to 18S rRNA levels and expressed as a fold change over the NHBEC levels. miR-200b levels from 2 independent experiments (n = 8) are plotted normalized to RNU48 levels and expressed as a fold change over the NHBEC levels. (B) NHBEC, Calu3 and 16HBE14o- cells were transfected with miR-200b antagomir (left) or mimic (right) and the miRNA levels were monitored in qRT-PCR experiments. miR-200b levels from 2 independent experiments (n = 8) are plotted normalized to RNU48 levels and expressed as a fold change over the transfection control. Error bars represent standard deviations (SD). Significant changes (P < 0.05) are marked with an asterisk. (PDF 348 kb

Characteristics of study participants.

<p>^aData presented as means with standard deviation</p><p>Characteristics of study participants.</p

Gene expression profile and motility of AS and NA HBFs.

<p>A lack of α-SMA (myofibroblast marker) and desmin (muscle cell marker) expression in HBF samples from AS and NA groups (A) was accompanied by a lack of significant differences in the total expression of actin and vinculin between AS and NA samples, as shown by densitometric analysis (B). Human cardiac mesenchymal stromal cells (hcMSC) and TGF-β₁-stimulated AS HBFs were used as positive controls for desmin and α-SMA, respectively. Relative optical density (ROD) values represent vinculin and actin levels (mean ± s.d.) calculated for each group by compilation of 4 AS and 4 NA samples, respectively, normalized against GAPDH levels. Time-lapse analyses of cell locomotion did not reveal any significant differences in the motile activity (the total length of displacement and the total length of trajectory) between AS and NA cells (C). Values are means ± s.d. for each group compiled from 4 AS and 4 NA samples, respectively. (*) P < 0.05.</p