2-Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine is an anti-inflammatory TLR-2, -4 and -5 response mediator in human monocytes

Abstract

Objective and design: To elucidate the influence of 2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP), a canonical Wnt/β-catenin pathway activator, on the inflammatory response of TLR-engaged innate cells in vitro.

Material or subject: Primary human monocytes.

Treatment: AMPMB (0-10 μM), LPS (0-1.0 μg/ml), Pam3CSK4, FSL-1, or S. typhimurium flagellin (0-0.25 μg/ml).

Methods: TLR-induced cytokine release (TNF, IL-6, IL-12 p40) was monitored by ELISA while Wnt-related signals (GSK3β, p65, IκB, β-catenin) were assessed by Western blot, pharmaceutical inhibition and gene silencing.

Results: AMBMP induced the rapid phosphorylation of NFκB p65 at Ser(536) and abrogated total IκB, accompanied by a subsequent increase in pro-inflammatory cytokine production (TNF, IL-6, IL-12 p40) in otherwise naive monocytes. However, in TLR2, -4 and -5-engaged monocytes, AMBMP-suppressed cytokine production. In the context of LPS stimulation, this occurred concomitant with the phosphorylative inactivation of GSK3β at Ser(9), β-catenin accumulation and abrogation of NFκB p65 phosphorylation. AMBMP-mediated suppression of the TLR4 -induced inflammatory response was reversed by two pharmaceutical Wnt/β-catenin pathway inhibitors, IWP-2 and PNU-74654 and by Wnt3a silencing.

Conclusions: Herein, we show that AMBMP induces canonical Wnt signaling events and acts as a suppressor of inflammation in surface TLR-engaged primary human monocytes.

Keywords: 2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine; Cytokines; GSK3β; Inflammation; TLR; Wnt; β-catenin.

Fig. 1

AMBMP stimulation induces pro-inflammatory cytokines, augments phosphop65 and suppresses total IκB. AMBMP was added to human monocytes and a TNF, b IL-6, and c IL-12/23 p40 measured in 20 h cell-free supernatants by ELISA. Data represent the mean ± SD of three separate experiments. ***p < 0.001. AMBMP led to (d) a rapid increase in the p65 (Ser⁵³⁶) signal and (e) a reduction in total IκB, measured by Western blot in cell lysates (20 μg protein). The densitometric ratio of phospho-65 and IκB to β-actin is shown in e and f, respectively. Typical data are presented

Fig. 2

AMBMP suppresses TLR-4 induced pro-inflammatory cytokine production. AMBMP (10.0 μM) was added to LPS-stimulated (100 ng/ml) human monocytes and a TNF, b IL-6, and c IL-12/23 p40 measured in 20 h cell-free supernatants by ELISA. Data represent the mean ± SD of three separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001, compared to cells stimulated with LPS only

Fig. 3

Kinetics of AMBMP suppression of TLR-4 induced pro-inflammatory cytokine production. AMBMP (10.0 μM) was added to LPS-stimulated (100 ng/ml) human monocytes and cytokine release measured in cell-free supernatants at multiple time points (2, 4, 8, 12, 16, and 24 h) by ELISA. Data represent the mean ± SD of three separate experiments

Fig. 4

AMBMP suppression of TLR-2/1, -2/6 and -5-induced pro-inflammatory cytokine production. AMBMP (10.0 μM) was added to TLR agonist [Pam3CSK4; FSL-1; or S. typhimurium flagellin (all, 0.25 μg/ml)] stimulated human monocytes and cytokine release measured in cell-free supernatants at 20 h by ELISA. Data represent the mean ± SD of three separate experiments. *p < 0.05, **p < 0.01 compared to solvent controls. Other than the TNF signal under AMBMP and flagellin co-stimulation, all conditions exhibited significantly higher cytokine release compared to the unstimulated controls (p < 0.05). AMBMP also suppressed pro-inflammatory cytokine production in innate cells stimulated with TLR3 (Poly(I:C) and Poly(I:C) LMW, 1 μg/ml) and TLR7 (Imiquimod, 1 μg/ml) agonists (data not shown)

Fig. 5

AMBMP augments TLR-4-induced GSK3β inactivation and β-catenin accumulation but abrogates NFκB p65 phosphorylation. Purified human monocytes were treated with LPS (100 ng/ml) in the presence and absence of AMBMP (10.0 μM) or pre-treated with GSK3β inhibitor, SB216763 (25 μM) for 2 h and then stimulated with LPS (100 ng/ml). Levels of (a) phosphorylated GSK3β (Ser⁹), (c) total β-catenin and phosphorylated p65 (Ser⁵³⁶) (e) were determined in cell lysates (20 μg protein) by Western blot. Total levels of β-actin were also monitored to ensure equivalent protein loading of cell lysates. The densitometric ratios of phospho-GSK3β, total-β-catenin and phospho-p65 to β-actin are shown in b, d and f, respectively. g Monocytes pre-treated with SB216763 exhibited reduced phosphorylation of the GSK3 specific substrate, glycogen synthase (GS) (S640/641). Typical data are presented (a–g). h GSK3 inhibition significantly decreased the production of TNF, IL-12, IL-6 in LPS stimulated cells. Data represent the mean ± SD of three separate experiments. ***p < 0.001, compared to LPS-stimulated controls

Fig. 6

Blockade of the canonical Wnt pathway rescues the inflammatory response to LPS. Purified human monocytes were pre-treated with two different pharmacological inhibitors of the canonical Wnt pathway, IWP-2 (100 nM) and PNU-74654 (1 μM), for 2 h or transfected with Wnt3a siRNA and then stimulated with LPS (100 ng/ml). Cell-free supernatants were collected after 24 h and the production of a TNF, b IL-6 and c IL-12 p40 analyzed by ELISA. siRNA-mediated knockdown of Wnt3a protein levels (d) enhanced the production of IL-12, TNF, and IL-6 in LPS-stimulated cells (f). The densitometric ratios of Wnt3a to β-actin (e) were assessed to ensure the equivalent loading of cell lysates. Data represent the mean ± SD of three separate experiments. *p < 0.05, ***p < 0.001, compared to LPS stimulated cells