Methylthioadenosine reprograms macrophage activation through adenosine receptor stimulation

Abstract

Regulation of inflammation is necessary to balance sufficient pathogen clearance with excessive tissue damage. Central to regulating inflammation is the switch from a pro-inflammatory pathway to an anti-inflammatory pathway. Macrophages are well-positioned to initiate this switch, and as such are the target of multiple therapeutics. One such potential therapeutic is methylthioadenosine (MTA), which inhibits TNFα production following LPS stimulation. We found that MTA could block TNFα production by multiple TLR ligands. Further, it prevented surface expression of CD69 and CD86 and reduced NF-KB signaling. We then determined that the mechanism of this action by MTA is signaling through adenosine A2 receptors. A2 receptors and TLR receptors synergized to promote an anti-inflammatory phenotype, as MTA enhanced LPS tolerance. In contrast, IL-1β production and processing was not affected by MTA exposure. Taken together, these data demonstrate that MTA reprograms TLR activation pathways via adenosine receptors to promote resolution of inflammation.

Figure 1. MTA inhibits TLR responses.

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. MTA alters LPS tolerance.

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.