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

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 HIV-1 infection in primary cells and a T cell line.

<p><b>A</b>, SIL inhibits LAI and BAL infection of PBMCs derived from 5 different donors. PBMC were activated with PHA and 3 days later, cells were washed to remove PHA and resuspended in media containing IL-2. The cells were plated in 96 well plates in the presence or absence of 243 µM SIL. Cells were then infected with 3-fold serial dilutions of LAI and BAL virus stocks until the end point dilution was reached. Cultures were incubated 24 hours, input virus removed, and cultures were fed with media containing IL-2 and SIL. Supernatants were harvested 6 days later and assayed for p24 levels using HIV p24 Antigen Capture ELISA. <b>B</b>, Dose response of LAI inhibition by SIL. The five PBMC cultures were treated as described above and infected with LAI in the presence of the indicated concentrations of SIL. p24 ELISA was performed at 7 days post-infection. <b>C</b>, SIL inhibits HIV-1 infection of PBMCs and CEM cells. PBMCs were treated as described above. CEM cells were infected with LAI (MOI = 0.001) in the presence of the indicated amounts of SIL and p24 antigen was measured in culture supernatants at 4–7 days post-infection. The data represent pooled data from individual experiments of PBMCs infected with LAI (N = 10) or BAL (N = 6) and CEM infected with LAI (N = 8). <b>D</b>, SIL's anti-HIV effects are durable. PBMCs were activated and infected with LAI in the presence of 243 µM of SIL and p24 antigen was measured at the indicated times (days post-infection). Cells were fed every 3–4 days with medium containing fresh SIL. Virus control refers to cells that only received virus and no SIL.</p

SIL suppresses HIV-1 Infection of TZM-bl cells.

<p><b>A</b>, Cytotoxicity profile of SIL in TZM-bl cells. Cells were infected with LAI, a CXCR4-using virus, or BAL, a CCR5-using virus, at an MOI of 0.05 in the presence of the indicated concentrations of SIL and ATP was measured using the ATPlite kit 48 hours later. The data are representative of 2 (BAL) and 3 (LAI) independent technical repeats. <b>B</b>, Antiviral profile of SIL in TZM-bl cells. Serial dilutions of SIL were tested for inhibition of infection in TZM cells. Following addition of compounds and virus, cells were incubated for 48 hours before luciferase activity was measured. Percent inhibition refers to percent reduction in luciferase activity of SIL versus untreated cultures. Error bars represent standard deviation of 3 independent technical repeats. <b>C</b>, SIL inhibits pseudovirus replication in TZM-bl cells. TZM-bl cells were infected with the indicated viruses in the presence of the indicated concentrations of SIL and luciferase activity was measured 48 hours post-infection. The D013M12 psuedovirus contains a subtype D envelope sequence, while the D769 psuedovirus contains a subtype A envelope sequence. Error bars represent standard deviations of triplicate wells per condition.</p