Ethanol inhibits activation of NLRP3 and AIM2 inflammasomes in human macrophages--a novel anti-inflammatory action of alcohol

Abstract

Objective: In the pathogenesis of coronary atherosclerosis, local macrophage-driven inflammation and secretion of proinflammatory cytokines, interleukin-1β (IL-1β) in particular, are recognized as key factors. Moderate alcohol consumption is associated with a reduced risk of coronary artery disease mortality. Here we examined in cultured human macrophages whether ethanol modulates the intracellular processes involved in the secretion of IL-1β.

Results: Ethanol decreased dose-dependently the production of mature IL-1β induced by activators of the NLRP3 inflammasome, i.e. ATP, cholesterol crystals, serum amyloid A and nigericin. Ethanol had no significant effect on the expression of NLRP3 or IL1B mRNA in LPS-primed macrophages. Moreover, secretion of IL-1β was decreased in parallel with reduction of caspase-1 activation, demonstrating that ethanol inhibits inflammasome activation instead of synthesis of pro-IL-1β. Acetaldehyde, a highly reactive metabolite of ethanol, had no effect on the ATP-induced IL-1β secretion. Ethanol also attenuated the secretion of IL-1β triggered by synthetic double-stranded DNA, an activator of the AIM2 inflammasome. Ethanol conferred the inhibitory functions by attenuating the disruption of lysosomal integrity and ensuing leakage of the lysosomal protease cathepsin B and by reducing oligomerization of ASC.

Conclusion: Ethanol-induced inhibition of the NLRP3 inflammasome activation in macrophages may represent a biological pathway underlying the protective effect of moderate alcohol consumption on coronary heart disease.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Ethanol inhibits dose-dependently the secretion of IL-1β and IL-18.

LPS-primed human macrophages were preincubated in the presence of the indicated concentrations of ethanol (EtOH: 2.5‰, 5‰, 10‰, and 20‰ corresponding to 43 mM, 86 mM, 171 mM, and 343 mM, respectively) prior to the activation of the NLRP3 inflammasome with (A) cholesterol crystals (CHC), (B) ATP or (C) nigericin (Nig). For activation with (D) SAA no LPS priming was used. To measure the secretion of IL-18, THP-1 cells were preincubated in the presence of the indicated concentrations of ethanol and activated with (E) SAA or (F) nigericin. Cytokine secretion by human primary macrophages (A,B,D) and THP-1 cells (C,E,F) was analyzed by ELISA. The results are expressed as means ± s.e.m from at least 4 individual experiments.

Figure 2. Ethanol inhibits the secretion of mature IL-1β and caspase-1, but not the expression of pro-IL-1β.

For the measurement of mRNA expression, LPS-primed human primary macrophages were incubated in the presence of the indicated concentrations of ethanol and (A) IL1B and (B) NLRP3 mRNA expression was determined by quantitative real-time RT-PCR. The data are expressed as fold changes compared to the control cells. The results are means ± s.e.m. from 5 individual experiments. For the Western blot studies LPS-primed human THP-1 cells were preincubated in 10‰ (171 mM) ethanol prior to the activation of the NLRP3 inflammasome with (C,G) nigericin and (D) ATP. For activation with (E,F) SAA no LPS priming was used. In IL-1β Western blots of supernatants (SN) or cell lysates (C–E), the p31 band represents pro-IL-1β and the p17 band mature IL-1β. In caspase-1 blots (F,G), the p45 band represents pro-caspase and the p10 band activated caspase-1. Results shown are representative of 3–4 individual experiments.

Figure 3. Ethanol inhibits lysosomal disruption and the secretion of active cathepsin B.

(A) THP-1 cells were preincubated in 10‰ (171 mM) ethanol prior to the activation of the NLRP3 inflammasome with cholesterol crystals. Live cells were stained with acridine orange (AO), which changes color from green to orange/red in the acidic pH of lysosomes, or with fluorescently labeled cathepsin B substrate z-Arg-Arg-cresyl violet (catB), which emits red signal upon cleavage by cathepsin. The cells were imaged using epifluorescence microscopy. The images are representative of 3 experiments. (B) LPS-primed THP-1 cells were preincubated in the presence of indicated concentrations of ethanol prior to activation of the NLRP3 inflammasome with nigericin. Cathepsin B/L activity was determined from cell culture media using a fluorescently labeled substrate Z-Phe-Arg-AMC, and the fluorescence was measured after a 60 min incubation at +37°C. The results are expressed as means of ± s.e.m from 3 individual experiments performed in duplicate.

Figure 4. Potassium efflux and ROS are not involved in the ethanol-induced inhibition.

(A) THP-1 cells were first preincubated with or without the ROS scavenger N-acetyl-L-cysteine (NAC), then in the presence of 10‰ (171 mM) ethanol, and the NLRP3 inflammasome was activated with SAA. Secretion of IL-1β into the culture medium was analyzed by ELISA. The results are expressed as the means ± s.e.m. from 4 individual experiments, performed in duplicate. (B) THP-1 cells were preincubated in 10‰ (171 mM) ethanol prior to activation of the NLRP3 inflammasome with nigericin. Intracellular potassium was determined by ICP-MS measurement of cell lysates. The results are expressed as means ± s.e.m. from 3 individual experiments.

Figure 5. Ethanol inhibits release and oligomerization of ASC.

(A) LPS-primed THP-1 cells were preincubated in 10‰ (171 mM) ethanol prior to the activation of the NLRP3 inflammasome with nigericin. ASC (22 kDa) was blotted from the supernatants (SN) and cell lysates. Western blots shown are representative of 4 experiments. (B) The intensities of the ASC bands of the cell culture supernatants were analyzed from 4 individual experiments, and are expressed as fold changes compared to the LPS-primed nigericin-activated cells. The results are expressed as means of ± s.e.m. (C) THP-1 cells were preincubated in 10‰ (171 mM) ethanol prior to the activation of the NLRP3 inflammasome with nigericin. Cells were fixed and analyzed for ASC expression using epifluorescence microscopy. (D) To quantify the extent of speck formation, the number of specks was divided by the number of cells per field and expressed as a percentage. The results are expressed as means ± s.e.m. from 4 individual experiments.

Figure 6. Ethanol inhibits AIM2 inflammasome-induced secretion of IL-1β, but not activation of caspase-8.

(A) For the measurement of AIM2 mRNA expression THP-1 cells were preincubated in the presence of the indicated concentrations of ethanol prior to the activation of the AIM2 inflammasome by transfecting the cells with poly (dA:dT). The results are expressed as fold changes compared to unstimulated cells. The data are expressed as means ± s.e.m. from 4 individual experiments. (B) To study the activation of the AIM2 inflammasome, THP-1 cells were preincubated in the presence of the indicated concentrations of ethanol and thereafter the AIM2 inflammasome was activated by transfecting the cells with poly (dA:dT), and the secretion of IL-1β into the culture medium was determined by ELISA. The results are expressed as means ± s.e.m. from 5 individual experiments. (C) THP-1 cells were preincubated in the presence of 10‰ (171 mM) ethanol prior to the activation of the cells with SAA. The activated caspase-8 (p18) was detected from the supernatants and cell lysates. Beta-actin is shown as a loading control. (D) The intensities of the activated caspase-8 bands of the cell culture supernatants were analyzed from 4 individual experiments, and are expressed as fold changes compared to the activated cells. The results are expressed as means of ± s.e.m.

Figure 7. The effect of ethanol on the NLRP3 inflammasome-activating danger signals.

1. Priming (left). Binding of lipopolysaccharide (LPS) or serum amyloid A to a toll-like receptor (TLR) activates the receptor which then initiates the synthesis of NLRP3 (nucleotide-binding domain and leucine-rich repeat containing family, pyrin domain containing 3) and pro-interleukin-1β (pro-IL-1β). 2. Activation (right). The pathways leading to activation of NLRP3 inflammasome by the 4 agents (ATP, nigericin, serum amyloid A, and cholesterol crystals) used in this study are shown. Extracellular ATP binds to the P2X purinergic receptor 7 (P2X7), so inducing potassium efflux and reciprocal lowering of the intracellular potassium concentration. Likewise, nigericin induces K+ efflux through its H+/K+ antiporter function and ensuing lowering of the intracellular potassium concentration. Finally, also cholesterol crystals lower the concentration of intracellular potassium. Both ATP and nigericin also trigger the formation of reactive oxygen species (ROS). Nigericin and serum amyloid A, the latter by activating the P2X7 receptor, induce the release of cathepsin B from intact lysosomes into the cytoplasm (intact lysosomes not shown for clarity). Phagocytosed cholesterol crystals cause lysosomal disruption and the leakage of cathepsin B into the cytoplasm. The cytoplasmic elevation of ROS, lowering of potassium, and the elevation of active cathepsin B, each can activate the NLRP3 inflammasome. The activated NLRP3 inflammasome contains active enzyme caspase-1, which cleaves pro-IL-1β and so converts it into the active secretable form. Ethanol inhibits the activation of the NLRP3 inflammasome, thereby reducing the activation of caspase-1 and the secretion of mature IL-1β at two levels: 1) by inhibiting the release of cathepsin B from both the intact and disrupted lysosomes, and 2) by diminishing the assembly of the NLRP3 inflammasome complexes.