Silymarin suppresses basal and stimulus-induced activation, exhaustion, differentiation, and inflammatory markers in primary human immune cells

<div><p>Silymarin (SM), and its flavonolignan components, alter cellular metabolism and inhibit inflammatory status in human liver and T cell lines. In this study, we hypothesized that SM suppresses both acute and chronic immune activation (CIA), including in the context of HIV infection. SM treatment suppressed the expression of T cell activation and exhaustion markers on CD4+ and CD8+ T cells from chronically-infected, HIV-positive subjects. SM also showed a trend towards modifying CD4+ T cell memory subsets from HIV+ subjects. In the HIV-negative setting, SM treatment showed trends towards suppressing pro-inflammatory cytokines from non-activated and pathogen-associated molecular pattern (PAMP)-activated primary human monocytes, and non-activated and cytokine- and T cell receptor (TCR)-activated mucosal-associated invariant T (MAIT) cells. The data suggest that SM elicits broad anti-inflammatory and immunoregulatory activity in primary human immune cells. By using novel compounds to alter cellular inflammatory status, it may be possible to regulate inflammation in both non-disease and disease states.</p></div

Flow Cytometry Panel Used to Determine Activation and Exhaustion in PBMC Samples.

<p>Flow Cytometry Panel Used to Determine Activation and Exhaustion in PBMC Samples.</p

SM inhibits basal and PAMP-induction of pro-inflammatory cytokines from primary human monocytes.

<p>Luminex analysis of MIP1α (A), MIP1β (B), TNF-α (C), IL1α (D), IL1β (E), and RANTES (F) from supernatants collected from CD14+ monocytes cultured in the absence or presence of SM (80 μM) for 24 hours in different activating conditions: rested (black), LPS-stimulated, a TLR4 agonist (dark gray), or ssRNA-stimulated, a TLR8 agonist (light gray). Data shown are from three different donors, and data are displayed as mean +/- SEM. Dotted line represents limit of detection (LOD).</p

SM suppresses inflammation in resting and cytokine- and TCR-activated MAIT cells.

<p>Expression of IFN-γ (A) and granzyme B (B) by CD8+ Va7.2+ CD161hi sorted MAIT cells cultured for 24 hours at rest, 100ng/ml IL-12, 15, and 18, or a combination of IL-12, 15, 18 and anti-CD3/CD28 beads, in the presence or absence of SM (80 μM). Data shown are from three different donors, and data are displayed as mean +/- SEM.</p

Inclusion and Exclusion Criteria for selecting PBMC samples.

<p>Inclusion and Exclusion Criteria for selecting PBMC samples.</p

Hepatitis C Virus Core Protein Inhibits Interferon Production by a Human Plasmacytoid Dendritic Cell Line and Dysregulates Interferon Regulatory Factor-7 and Signal Transducer and Activator of Transcription (STAT) 1 Protein Expression

<div><p>Plasmacytoid Dendritic Cells (pDCs) represent a key immune cell population in the defense against viruses. pDCs detect viral pathogen associated molecular patterns (PAMPs) through pattern recognition receptors (PRR). PRR/PAMP interactions trigger signaling events that induce interferon (IFN) production to initiate local and systemic responses. pDCs produce Type I and Type III (IFNL) IFNs in response to HCV RNA. Extracellular HCV core protein (Core) is found in the circulation in chronic infection. This study defined how Core modulates PRR signaling in pDCs. Type I and III IFN expression and production following exposure to recombinant Core or β-galactosiade was assessed in human GEN2.2 cells, a pDC cell line. Core suppressed type I and III IFN production in response to TLR agonists and the HCV PAMP agonist of RIG-I. Core suppression of IFN induction was linked with decreased IRF-7 protein levels and increased non-phosphorylated STAT1 protein. Circulating Core protein interferes with PRR signaling by pDCs to suppress IFN production. Strategies to define and target Core effects on pDCs may serve to enhance IFN production and antiviral actions against HCV.</p></div

Characteristics of Subjects Whose PBMCs Were Analyzed by Flow Cytometry.

<p>Characteristics of Subjects Whose PBMCs Were Analyzed by Flow Cytometry.</p

SM suppresses the expression of activation and exhaustion markers on CD4+ and CD8+ T cells from HIV+ subjects.

<p>Expression of activation markers, CD38 and HLA-DR on CD4+ (A) and CD8+ (B) T cells, and exhaustion markers, CTLA4 and PD1, on CD4+ (C) and CD8+ (D) T cells from PBMC cultures treated for 72 hours with SM (80 μM; empty symbols) or DMSO (vehicle control; solid symbols). Data are from 25 different PBMC samples. P values are derived from Wilcoxon signed rank tests.</p

Silibinin but not SIL inhibits innate inflammatory and antiviral signaling.

<p>A, B, effect of silibinin and SIL on NF-κB dependent transcription. Huh7 cells were transfected with an NF-κB responsive reporter plasmid (pRDII-luc) and twenty-four hours later, cells were pretreated with the indicated doses of silibinin (A) or SIL (B). Cells were then treated with 10 ng/ml TNF-α and luciferase activity measured by Britelite assay 3.5 hours later. C, D, effect of silibinin and SIL on IRF-3 driven transcription from the IFN-B promoter. Huh7.5.1 cells were co-transfected with a luciferase reporter plasmid under control of the IFN-B promoter and IRF-35D, a constitutively active mutant of IRF-3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016464#pone.0016464-Lin1" target="_blank">[28]</a>. Twenty-four hours later, cells were pretreated with the indicated doses of silibinin (C) or SIL (D). Luciferase activity measured by Britelite assay 24 hours later. Fluorescence is reported as relative light units (RLU). Error bars represent standard deviation from triplicate cultures.</p

Antiviral Activity of SIL and silibinin.

<p>A, effect of SIL on HCV replication in genotype 1b subgenomic replicon cells (BB7) and JFH-1 infection of Huh7.5.1 cells. The top panels show HCV NS5A protein expression detected by western blot, while the lower graph depicts HCV RNA levels determined by real time RT-PCR. Cells were treated with 0, 6.9, 27.6, 138, and 414 µM of SIL for 72 hours before protein and RNA isolations. B, effect of silibinin on HCV replication in genotype 1b subgenomic replicon cells (BB7) and JFH-1 infection of Huh7.5.1 cells. Cells were treated with DMSO, 15.5, 31.1, and 62.1 µM of silibinin for 72 hours before protein and RNA isolations. C, effects of SIL and silibinin on HCV replication in subgenomic JFH-1 replicon cells. Cells were treated with 0, 6.9, 27.6, 138, and 414 µM of SIL or DMSO, 20.7, 41.4, and 82.8 µM of silibinin for 72 hours before proteins were extracted and NS5A detected by western blot. D, effect of SIL and silibinin on progeny virus production. Huh7.5.1 cells were treated with 20 µg/ml silibinin, 300 µg/ml SIL or DMSO and PBS controls immediately after 5 hours of adsorption with JFH-1 at an m.o.i. of 0.05. Culture supernatants were harvested 72 hours later and carry over silibinin or SIL was removed by concentration through 10,000 molecular filters. Supernatants were diluted 1∶100 in Huh7 media and used to infect naïve Huh7.5.1 cells in triplicate and immunofluorescent detection of HCV core protein was performed. Foci were counted manually and used to calculate infectious virus yields expressed as focus forming units per milliliter (FFU/ml). Error bars represent standard deviations of triplicate cultures.</p