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

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

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

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

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

Silibinin Inhibits HIV-1 Infection by Reducing Cellular Activation and Proliferation

<div><p>Purified silymarin-derived natural products from the milk thistle plant (<em>Silybum marianum</em>) block hepatitis C virus (HCV) infection and inhibit T cell proliferation in vitro. An intravenous formulation of silibinin (SIL), a major component of silymarin, displays anti-HCV effects in humans and also inhibits T-cell proliferation in vitro. We show that SIL inhibited replication of HIV-1 in TZM-bl cells, PBMCs, and CEM cells in vitro. SIL suppression of HIV-1 coincided with dose-dependent reductions in actively proliferating CD19+, CD4+, and CD8+ cells, resulting in fewer CD4+ T cells expressing the HIV-1 co-receptors CXCR4 and CCR5. SIL inhibition of T-cell growth was not due to cytotoxicity measured by cell cycle arrest, apoptosis, or necrosis. SIL also blocked induction of the activation markers CD38, HLA-DR, Ki67, and CCR5 on CD4+ T cells. The data suggest that SIL attenuated cellular functions involved in T-cell activation, proliferation, and HIV-1 infection. Silymarin-derived compounds provide cytoprotection by suppressing virus infection, immune activation, and inflammation, and as such may be relevant for both HIV mono-infected and HIV/HCV co-infected subjects.</p> </div

SIL inhibits activation marker expression on CD4+ T cells.

<p>PBMCs were activated with either SEB (0.5 µg/ml) or PHA (2 µg/ml) for 24 hours, and treated with the indicated concentrations of SIL for 12 hours. Representative flow cytometry dot plots showing expression of HLA-DR (A), CD38 (B), Ki67 (C), and CCR5 (D). Data are representative of 3 HIV-seronegative individuals tested for each marker.</p

SIL inhibits stimulus-induced expansion of CD4+ T cells expressing the HIV co-receptors CXCR4 and CCR5 (panel A) but does not affect the relative frequency of CD4+ T cells expressing co-receptors (panel B).

<p>PBMCs were stimulated with PHA for 3 days prior to exposure to IL-2 and the indicated concentrations of SIL. Twenty-four hours later, cells were stained for CD4, CXCR4, and CCR5 and analyzed by flow cytometry. A, Y-axis represents the concentration of the indicated cell type. The cell concentration is expressed per µl of cell suspension and was determined using counting beads. Panel B shows the percentage of total CD4+ T cells that express one, both, or neither co-receptor.</p