Groups for TGF-β1 signaling pathway experiments.

<p>Groups for TGF-β1 signaling pathway experiments.</p

The involvement of TGF-β1 in the modulatory effect of MSCs on IDO induction.

<p>(<b>A</b>) Untreated HCE-T, HCE-T monocultures exposed to IFN-γ/TNF for 24 h and 48 h with or without 10 ng/ml TGF-β1 pre-treatment. The cytokines-induced IDO expression was significantly inhibited by TGF-β1 at 24 h and 48 h (p&lt;0.05 at both time points). (<b>B</b>) HCE-T/MSC co-cultures without and with TGF-β1 10 ng/ml 24 h treatment express a similar low level of IDO as untreated HCE-T monocultures (less than 2 folds). The IDO expression is significant lower in the HCE-T/MSC co-cultures with 48 h TGF-β1 treatment compared to the untreated HCE-T monolayers (p&lt;0.01). While IDO expression from HCE-Ts stimulated with IFN-γ/TNF for 24 h was more than 180 folds higher than untreated HCE-Ts. (<b>C</b>) Untreated HCE-T, HCE-T monocultures stimulated with IFN-γ/TNF for 24 h and 48 h, or HCE-T/MSC co-cultures stimulated by IFN-γ/TNF with or without combined SB-431542 (1 µM) and anti-TGF-β1 antibody (1 µg/ml) treatment. Blockade of the TGF-β1 signaling pathway in IFN-γ/TNF-treated MSC/HCE-T co-cultures using combined neutralizing anti-TGF-β1 antibody and SB-431542, a TGF-β receptor I blocker, significantly reversed the down-regulatory effect of MSC on IDO expression by 48 h (p&lt;0.05). The upper panels are representative images of individual western blots for IDO (42 kDa) and GAPDH (37 kDa) expression in HCE-T cells. Each bar summarizes the data from 3 individual experiments (n = 3) ±SEM. *: p&lt;0.05, **: p&lt;0.01. The “+” or “−“ symbol denotes the presence or absence of the denoted treatment or cell type at the left.</p

Modulatory effects of MSC on IgSF molecule expression.

<p>Bar graphs showing the relative expressions of ICAM-1 (<b>A</b>), HLA-ABC (<b>B</b>), HLA-DR (<b>C</b>) and HLA-G (<b>D</b>) on HCE-T cells, as determined by FCM. HCE-Ts were either untreated or treated for 24 h and 48 h with IFN-γ (100 U/ml) and TNF (100 U/ml) on monocultures and HCE-T/MSC co-cultures. The inserts are representative FCM histograms, and the abscissa represents log10 fluorescent intensity in arbitrary units (AU) [a. isotype control Alexa 488 conjugated mouse IgG; b. untreated HCE-T; c. 24 h IFN-γ/TNF treatment on HCE-T monoculture; d. 24 h IFN-γ/TNF treatment on HCE-T/MSC co-culture; e. 48 h combination cytokines stimulation on HCE-T monoculture; f. 48 h combination cytokine stimulation on HCE-T/MSC co-culture]. Data represents mean ± SEM of four separate experiments (n = 4). *: p&lt;0.05, **: p&lt;0.01. The “+” or “-“ symbol denotes the presence or absence of the denoted treatment or cell type at the left.</p

Groups for exogenous TGF-β1 treatment.

<p>Groups for exogenous TGF-β1 treatment.</p

Antibodies, conjugates and immunoreagents.

<p>FCM: flow cytometry; WB: Western blotting; IF: immunofluorescence staining.</p

Blocked NF-κB activation inhibits cytokine-upregulated IgSF molecules expression, and MSC modulates cytokine-induced NF-κB nuclear translocation.

<p>A to C shows FCM analysis of HCE-T cells treated with BMS-345541 (5 µM), a NF-κB pathway inhibitor, significantly inhibited IFN-γ (100 U/ml) and TNF (100 U/ml) induced ICAM-1 (A), HLA-ABC (B), and HLA-DR (C) expression. Data represents mean ± SEM of four separate experiments (n = 4). *: p&lt;0.05, **: p&lt;0.01. D and E (upper panel) show representative bands of western blots for NF-κB p65 (65 kDa) in nuclear extracts (NE, D) and cytoplasmic extracts (CE, E). Histone H2B (15 kDa) and GAPDH (37 kDa) were used as the loading controls for nuclear and cytoplasmic extracts, respectively. Protein was prepared from HCE-T cells with or without IFN-γ/TNF stimulation, or HCE-T/MSC co-cultured with 24 h and 48 h IFN-γ/TNF treatment. The lower panel shows the fold changes of NF-κB within nuclear extracts and cytoplasmic extracts compared with the untreated group, using Histone H2B and GAPDH as loading controls. Data represents mean ± SEM of four separate experiments (n = 4). *: p&lt;0.05. F to I show representative immunocytochemistry images. Untreated HCE-T cells displayed weak NF-κB nuclear and cytoplasmic staining (F); NF-κB expression showed marked nuclear and perinuclear translocation after 24 h IFN-γ/TNF stimulation of HCE-T monocultures (G); NF-κB perinuclear and nuclear translocation decreased at 24 h in IFN-γ/TNF in stimulated HCECs/MSC co-cultures (H); HCE-T monolayer showed no obvious staining with rabbit IgG (I). The “+” or “−“ symbol denotes the presence or absence of the denoted treatment or cell type at the left.</p

Immunophenotype of rat MSCs.

<p>Rat MSCs display spindle shape and reach confluent at 14 days post-isolation (<b>A</b>). More than 98% of the population are positive to the mesenchymal markers CD29 (<b>B, D</b>) and CD44 (<b>E</b>), and negative to the hematopoietic markers CD34 (<b>C, F</b>) and CD45 (<b>G</b>). Green shaded profile, antibodies staining; blue open profile, isotype control.</p

The effect of MSC on TGF-β1 secretion.

<p>The concentration of (<b>A</b>) total and (<b>B</b>) active TGF-β1 secretion in conditioned media collected from untreated HCE-T monocultures, MSC monocultures and HCE-T/MSC co-cultures, or HCE-T monocultures, MSC monocultures and HCE-T/MSC co-cultured with IFN-γ/TNF stimulation for 24 h and 48 h was measured by ELISA. (<b>A</b>) A similar level of TGF-β1 was detected from untreated HCE-T monocultures and HCE-T/MSC co-cultures. IFN-γ/TNF-stimulated HCE-T/MSC co-culture showed increased total TGF-β1 from HCE-Ts at 24 h (p = 0.052), and significantly increased at 48 h (P&lt;0.01). (<b>B</b>) IFN-γ/TNF treatment significantly increased active TGF-β1 secretion from HCE-T (p&lt;0.01) and MSC (p&lt;0.05) monocultures at 48 h. Compared to untreated monocultures, IFN-γ/TNF-stimulated HCE-T/MSC co-culture showed a significant increase in active TGF-β1 after 48 h, (p&lt;0.05). Data represents the results from three individual experiments (n = 3) ±SEM. *: p&lt;0.05, **: p&lt;0.01. The “+” or “−“ symbol denotes the presence or absence of the denoted treatment or cell type at the left.</p

Groups for NF-κB pathway.

<p>DMSO - Dimethyl sulfoxide (Research Organics, OH USA).</p