Acylated Anthocyanins From Black Carrots and Their Related Phenolic Acids Diminish Priming and Activation of the NLRP3 Inflammasome in THP-1 Monocytes

Abstract

Scope: Excessive activation of the nucleotide-binding oligomerization domain-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome contributes to chronic inflammation. Thus, targeting NLRP3 inflammasome activation by anthocyanins may prevent inflammatory diseases. Therefore, the present study determines the influence of a black carrot extract (BCE) with high amounts of acylated anthocyanins and their related phenolic acids on the NLRP3 inflammasome.

Methods and results: THP-1 monocytes are pretreated with a BCE, cyanidin-3-glucoside (C3G), or hydroxycinnamic acids. NLRP3 inflammasome assembly is initiated by priming THP-1 monocytes with lipopolysaccharide and/or activating the NLRP3 inflammasome with nigericin. Flow cytometry is used to assess apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) speck formation, as well as ASC and NLRP3 protein expression. Caspase-1 activity is measured using a bioluminescent assay, and cytokine concentrations are determined by enzyme-linked immunosorbent assays (ELISA). C3G and phenolic acids diminish ASC and NLRP3 protein expression. In addition, C3G and phenolic acids attenuate ASC speck formation. Furthermore, the BCE and C3G decline caspase-1 activity. Consistently, IL-1β and IL-18 secretion are reduced upon NLRP3 inflammasome activation.

Conclusion: The present study shows that a BCE with high amounts of acylated anthocyanins and their related phenolic acids diminish priming and activation of the NLRP3 inflammasome in THP-1 monocytes.

Keywords: ASC specks; NLRP3 inflammasome; anthocyanin‐rich black carrot extract; phenolic acids.

Figure 1

Gating strategy to assess apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC) speck formation in THP‐1 monocytes. A) First, debris were excluded using the forward light scatter area (FSC‐A) and side scatter area (SSC‐A). B) Then, doublets were excluded using FSC‐A and FSC height (FSC‐H). C) Next, ASC positive cells (dark gray filled histogram) were selected compared to the matching isotype control (light gray filled histogram) and D) ASC speck formation was assessed via the obvious reduction in phycoerythrin width (PE‐W) due to ASC condensation. Cell density is indicated by pseudocolor plot ranging from low (blue) to high (red).

Figure 2

Representative HPLC‐DAD chromatograms of anthocyanins at 520 nm as well as chlorogenic and caffeic acid at 320 nm in the black carrot extract. Acylated anthocyanins also generated signals at 320 nm. Peak assignment as follows. 1) Chlorogenic acid, 2) Caffeic acid. 3) Cyanidin‐3‐xylosyl‐glucosyl‐galactoside, 4) cyanidin‐3‐xylosyl‐galactoside, 5) cyanidin‐3‐xylosyl‐(sinapoyl‐glucosyl)‐galactoside, 6) cyanidin‐3‐xylosyl‐(feruloyl‐glucosyl)‐galactoside, 7) cyanidin‐3‐xylosyl‐(p‐coumaroyl‐glucosyl)‐galactoside. Detailed analytical data for anthocyanin identification has been compiled in Table 1.

Figure 3

Influence of a black carrot extract (BCE) with high amounts of acylated anthocyanins and their related phenolic acids on ASC and NLRP3 protein expression in THP‐1 monocytes. THP‐1 monocytes were preincubated with the indicated concentrations of the BCE, C3G, or phenolic acids before cells were primed with LPS. A, C) ASC and B, D) NLRP3 protein expression were assessed as median fluorescence intensity (MFI) by intracellular flow cytometry. Significant differences to LPS primed cells were calculated using one‐way ANOVA with Dunnett's multiple comparisons test (**p < 0.01). ANOVA, analysis of variance; ASC, apoptosis‐associated speck‐like protein containing a caspase recruitment domain; cyanidin‐3‐glucoside (C3G); p‐coumaric acid (CA); ferulic acid (FA); LPS, lipopolysaccharide; NLRP3, nucleotide‐binding oligomerization domain‐like receptor pyrin domain‐containing protein 3; sinapinic acid (SA).

Figure 4

ASC speck formation in THP‐1 monocytes. A) ASC pulse width (ASC‐W) analysis of THP‐1 monocytes by flow cytometry. Cells were treated with LPS and/or nigericin as indicated. Unstimulated cells were used as a negative control. A representative data set (n = 1) with 14.000 cells per treatment is shown and cell density is indicated by a pseudocolor plot ranging from low (blue) to high (red). B) The percentage of ASC speck‐positive cells was quantified. Significant differences to untreated cells were calculated using one‐way ANOVA with Dunnett's multiple comparisons test (****p < 0.0001). ANOVA, analysis of variance; ASC, apoptosis‐associated speck‐like protein containing a caspase recruitment domain; lipopolysaccharide (LPS).

Figure 5

Influence of a black carrot extract (BCE) with high amounts of acylated anthocyanins and their related phenolic acids on ASC speck formation in THP‐1 monocytes. THP‐1 monocytes were preincubated with the indicated concentrations of the BCE, cyanidin‐3‐glucoside, or phenolic acids before the NLRP3 inflammasome was activated. Significant differences to A, C) LPS and nigericin stimulated cells or B, D) only nigericin treated cells were calculated using one‐way ANOVA with Dunnett's multiple comparisons test (*p < 0.05 and **p < 0.01). ANOVA, analysis of variance; ASC, apoptosis‐associated speck‐like protein containing a caspase recruitment domain; C3G; CA; FA; LPS, lipopolysaccharide; NLRP3, nucleotide‐binding oligomerization domain‐like receptor pyrin domain‐containing protein 3; SA.

Figure 6

Influence of a black carrot extract (BCE) with high amounts of acylated anthocyanins on caspase‐1 activity in THP‐1 monocytes. THP‐1 monocytes were preincubated with the indicated concentrations of the BCE or C3G before the NLRP3 inflammasome was activated. Caspase‐1 activity was measured by using a bioluminescent assay and luminescence was measured as relative light unit (RLU). Significant differences to (A) LPS and nigericin stimulated cells or (B) only nigericin treated cells were calculated using one‐way ANOVA with Dunnett's multiple comparisons test (**p < 0.01, ***p < 0.001, and ****p < 0.0001). ANOVA, analysis of variance; C3G, cyanidin‐3‐glucoside; LPS, lipopolysaccharide; NLRP3, nucleotide‐binding oligomerization domain‐like receptor pyrin domain‐containing protein 3.

Figure 7

Influence of a black carrot extract (BCE) with high amounts of acylated anthocyanins on proinflammatory cytokine release in THP‐1 monocytes. THP‐1 monocytes were preincubated with the indicated concentrations of BCE or C3G before the NLRP3 inflammasome was activated. Release of (A) IL‐1β and (B) IL‐18 into the cell culture supernatant was measured by ELISA. Significant differences to LPS and nigericin‐stimulated cells were calculated using one‐way ANOVA with Dunnett's multiple comparisons test (*p < 0.05 and **p < 0.01). ANOVA, analysis of variance; C3G, cyanidin‐3‐glucoside; CA, p‐coumaric acid; ELISA, enzyme‐linked immunosorbent assays; FA, ferulic acid; LPS, lipopolysaccharide; NLRP3, nucleotide‐binding oligomerization domain‐like receptor pyrin domain‐containing protein 3; SA, sinapinic acid.