Figure 1.
BMDM were incubated overnight with DMSO or 200 µM MTA in the absence or presence of the following TLR ligands: 10 ng/mL LPS, 10 µg/mL polyI:C, 1 µg/mL Pam3CSK4, 6.67 µM CpG or 2 µg/mL Imiquimod. Supernatants were assayed for TNFα (A), IL-6 (B) or IL-10 (C) production by ELISA while cells were harvested, stained and analyzed for surface CD69 (D,E) or CD86 (D, F) expression by FACS. Graphs represent mean ±sem of 3 (A, E, F), 4 (C) or 5 (B) experiments. * p<0.05, ** p<0.01, *** p<0.001.
Figure 2.
MTA inhibits TLR-induced TNFα mRNA production and NF-KB induction.
(A) BMDM were incubated for 4 h with either DMSO or 200 µM MTA in the absence or presence of either 100 EU/mL LPS or 1 µg/mL Pam3CSK4. Total RNA was extracted from cells. TNFα expression relative to β-actin was determined by Δ(ΔCT) method using real-time PCR. (B) RAW NF-KB reporter cells were treated with DMSO, 200 µM MTA or 200 µM adenosine (Ado) in the absence or presence of the indicated TLR ligands for 4 h at 37°C. Supernatants were assayed for luciferase, which was normalized to DMSO-treated cells that received no TLR stimulation. The graphs represent mean ±sem of 4 (A) or 5 (B) experiments. ** p<0.01, *** p<0.001.
Figure 3.
MTA inhibition of TLR ligands acts via Adenosine Receptors.
BMDM were incubated overnight with DMSO, the indicated concentrations of MTA or adenosine (Ado), presence or absence of 10 µM SCH442416 and 10 µM PSB1115 and the presence or absence of 10 ng/mL LPS. Supernatants were analyzed for TNFα production by ELISA (A) while cells were harvested, stained and analyzed for surface CD86 (B) expression by FACS. Data represent mean ±sem of at least 3 experiments. * p<0.05, ** p<0.01, *** p<0.001.
Figure 4.
BMDM were treated overnight with either 0 ng/mL, 10 ng/mL, or 100 ng/mL (A–C) or 100 EU/mL (D) LPS in the presence or absence of 200 µM MTA, and 10 µM SCH442416 with 10 µM PSB1115, washed and rested for 6 h. BMDM were then restimulated with either 0, 10 ng/mL, or 100 EU/mL LPS overnight. Supernatants were collected and analyzed for TNFα (A) by ELISA while cells were harvested, stained and analyzed for surface CD69 (B) or CD86 (C, D) expression by FACS. Data represent mean ±sem of 3 (A) or 4 (B–D) experiments. * p<0.05, ** p<0.01, *** p<0.001.
Figure 5.
IL-1β secretion is independent of MTA.
BMDM were primed for 4 h with 10 ng/mL LPS in the absence or presence of 200 µM or 500 µM MTA, then washed and treated in the presence (MTA in Sig 2) or absence of 200 µM MTA, inflammasome inhibitors YVAD or KCl and NLRP3 agonists 3 mM ATP, 2000 U/mL SLO or 20 µM nigericin for 30 min. Supernatants were collected and analyzed by ELISA (A) or TCA-precipitated, resolved by SDS-PAGE along with cell lysates, and transferred to PVDF (B). Blots were sequentially probed with 3ZD anti-IL-1β mAb, EPR3057 anti-HMGB1 rabbit mAb and anti-actin mAb coupled with relevant HRP-conjugated secondary antibodies. The graph is the mean ± sem of 4 experiments while the blot is a representative blot from 3 independent experiments.