Maltol, a Natural Flavor Enhancer, Inhibits NLRP3 and Non-Canonical Inflammasome Activation

Abstract

Maltol (3-hydroxy-2-methyl-4-pyrone) is used widely as a food and cosmetic supplement, and it has antioxidant and anti-inflammatory activities. Inflammasome causes the maturation and secretion of interleukin (IL)-1β and -18 through the activation of caspase-1 (Casp1), which contributes to various inflammatory diseases. This study examined the effects of maltol on the inflammasome activation in macrophages and mice. Lipopolysaccharide (LPS)-primed macrophages were treated with a trigger of NLRP3, NLRC4, AIM2, or non-canonical (NC) inflammasomes in the presence of maltol. The secretion of IL-1β and IL-18 and the cleavage of Casp1 were analyzed as indices of inflammasome activation. Mice were injected with LPS and an NLRP3 trigger with or without maltol, and the peritoneal IL-1β secretions were observed. The effects of maltol on reactive oxygen species (ROS) production and Casp1 activity were analyzed to determine the mechanism. Maltol inhibited the activation of NLRP3 and NC inflammasomes, but it did not alter the other inflammasomes. Maltol also attenuated IL-1β secretion resulting from the inflammasome activation in mice. The anti-inflammatory mechanism of maltol was revealed by the inhibition of ROS production and Casp1 activity. Maltol is suggested to be promising as a anti-inflammasome molecule.

Keywords: inflammasome; interleukin-1beta; macrophages; maltol.

Figure 1

Effects of maltol on NLRP3 and AIM2 inflammasome activation. (A), Chemical structure of maltol (C₆H₆O₃). (B), Schematic diagram describing the mode of inflammasome activation. Macrophages were primed with LPS for 3 h and treated with an inflammasome trigger with maltol. The indices of inflammasome activation were analyzed by ELISA and Western blotting. (C), LPS-primed BMDMs were induced to activate NLRP3 or AIM2 inflammasome with ATP or dsDNA in the presence of maltol, as indicated. The maturation of secretion of Casp1 (p20), IL-1β, IL-18, and LDH was analyzed by immunoblotting or ELISA. All immunoblot data shown are representative of at least two independent experiments. The bar graph presents the mean ± SD.

Figure 2

Effect of maltol on various inflammasome activation in murine and human macrophages (A), LPS-primed BMDMs were activated NLRP3 (NG and MSU), NC (LPS), or NLRC4 (flagellin) inflammasomes by a specific trigger and co-treated with maltol as indicated. IL-1β secretions were analyzed by ELISA. (B), PMA-treated THP-1, human macrophage-like cells, were treated with NLRP3 (ATP, NG, MSU), NC (LPS), AIM2 (dsDNA), or NLRC4 (flagellin) inflammasome triggers to activate the inflammasome in the presence of maltol. IL-1β secretion was measured by ELISA. The bar graph presents the mean ± SD.

Figure 3

Effect of maltol on the inflammasome activation in mice. (A), Schematic diagram describes the mode of the maltol injecting experiment. (B), The peritoneal IL-1β production of the mice treated similar to panel A was measured. (C), The procedure for oral maltol administration is shown. (D), The secretion of peritoneal IL-1β of the maltol-fed animals was analyzed. IL-1β releases were measured by ELISA. The bar graph presents the mean, and each gray circle indicates a value from each mouse.

Figure 4

Effect of maltol on the ROS production and Casp1 activity. (A), LPS-primed BMDMs were treated with rotenone (Rot) in the presence of maltol or DPI (a ROS scavenger). IL-1β secretion was analyzed by ELISA. (B), LPS-primed BMDMs were treated with DHR123 to measure ROS production in the presence of Rot with/without Maltol. Relative fluorescence unit (RFU) as the level of ROS was measured. (C), Recombinant human (rh) Casp1 was incubated with maltol or Z-VAD-FMK (Z-VAD, a pan-caspase inhibitor), and the Casp1 activity was analyzed using an assay kit. The bar graph presents the mean ± SD.