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.