Grape/Blueberry Anthocyanins and Their Gut-Derived Metabolites Attenuate LPS/Nigericin-Induced Inflammasome Activation by Inhibiting ASC Speck Formation in THP-1 Monocytes

Abstract

Inflammasomes are multi-protein complexes, which are formed in response to tissue injury, infections, and metabolic stress. However, aberrant inflammasome activation has been linked to several inflammatory diseases. Anthocyanins have been reported to attenuate NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation, but the influence of grape/blueberry anthocyanins and especially their gut-derived metabolites on NLRP3 inflammasome activation in human monocytes remains unclear. Therefore, human leukemic monocytes (THP-1 cells, Tohoku Hospital Pediatrics-1 cells) were preincubated with different concentrations of grape/blueberry anthocyanins, homovanillyl alcohol, or 2,4,6-trihydroxybenzaldehyde (THBA) before the NLRP3 inflammasome was activated by lipopolysaccharide and/or nigericin. Apoptosis-associated speck-like protein containing a CARD (ASC) speck formation, as well as ASC and NLRP3 protein expression, were determined using flow cytometry. Caspase-1 activity was measured in cultured cells, and pro-inflammatory cytokine secretion was determined using enzyme-linked immunosorbent assays. Anthocyanins and their metabolites had no effect on ASC or NLRP3 protein expression. However, THBA significantly inhibited ASC speck formation in primed and unprimed THP-1 monocytes, while caspase-1 activity was significantly declined by grape/blueberry anthocyanins. Furthermore, reduced inflammasome activation resulted in lower pro-inflammatory cytokine secretion. In conclusion, our results show for the first time that grape/blueberry anthocyanins and their gut-derived metabolites exert anti-inflammatory effects by attenuating NLRP3 inflammasome activation in THP-1 monocytes.

Keywords: ASC specks; NLRP3 inflammasome; anthocyanins; blueberries; grapes; gut-derived metabolites.

Figure 1

Effect of grape/blueberry anthocyanins and their gut-derived metabolites on THP-1 cell viability. THP-1 monocytes were incubated with the indicated concentrations of (a) the GBE, (b) HVA, or (c) THBA for 24 h before cell viability was measured by flow cytometry. Data are presented as mean ± SD (n = 3). Significant differences compared to the untreated control were calculated using one-way ANOVA followed by Dunnett’s multiple comparisons test. ** p < 0.01.

Figure 2

Detection of ASC speck formation in THP-1 cells using flow cytometry. THP-1 monocytes were either left untreated or primed with LPS followed by activation of the NLRP3 inflammasome with nigericin as mentioned in the methods section. Flow analysis was performed and the percentage of ASC speck-positive cells was quantified. Data are presented as (a) representative dot plots and (b) column bars with mean ± SD (n = 3). Significant differences compared to the untreated control were calculated using one-way ANOVA followed by Dunnett’s multiple comparisons test. **** p < 0.0001.

Figure 3

Effect of grape/blueberry anthocyanins and their gut-derived metabolites on ASC speck formation in THP-1 cells. THP-1 monocytes were preincubated with the indicated concentrations of grape/blueberry anthocyanins and their gut-derived metabolites before the NLRP3 inflammasome was activated as mentioned in the methods section. Cells were flow cytometrically analyzed and the percentage of ASC speck-positive cells was quantified. Data are presented as mean ± SD (n = 3). Significant differences compared to (a,c) LPS- and nigericin-stimulated cells or (b,d) cells treated only with nigericin were calculated using one-way ANOVA followed by Dunnett’s multiple comparisons test. * p < 0.05 and ** p < 0.01.

Figure 4

Effect of grape/blueberry anthocyanins and their gut-derived metabolites on caspase-1 activity in THP-cells. THP-1 monocytes were preincubated with the indicated concentrations of grape/blueberry anthocyanins and their gut-derived metabolites before the NLRP3 inflammasome was activated as mentioned in the methods section. Caspase-1 activity was measured by using the Caspase-Glo^® 1 Inflammasome Assay and luminescence was measured. Data are presented as mean ± SD (n = 3). Significant differences compared to (a,c) LPS- and nigericin-stimulated cells or (b,d) cells treated only with nigericin were calculated using one-way ANOVA followed by Dunnett’s multiple comparisons test. * p < 0.05, **** p < 0.0001. RLU, relative light unit.

Figure 5

Effect of grape/blueberry anthocyanins and their gut-derived metabolites on inflammatory cytokine secretion in THP-1 cells. THP-1 monocytes were preincubated with the indicated concentrations of grape/blueberry anthocyanins and their gut-derived metabolites before the NLRP3 inflammasome was activated as mentioned in the methods section. The release of (a,c) IL-1β and (b,d) IL-18 into the cell-culture supernatant was measured using ELISA. Data are presented as mean ± SD of at least three replicated experiments. Significant differences compared to LPS- and nigericin-stimulated cells were calculated using one-way ANOVA followed by Dunnett’s multiple comparisons test. ** p < 0.01.