Trimethylamine-N-Oxide Instigates NLRP3 Inflammasome Activation and Endothelial Dysfunction

Abstract

Background/aim: Plasma trimethylamine-N-oxide (TMAO), a product of intestinal microbial metabolism of dietary phosphatidylcholine has been recently associated with atherosclerosis and increased risk of cardiovascular diseases (CVD) in rodents and humans. However, the molecular mechanisms of how TMAO induces atherosclerosis and CVD progression are still unclear. The present study tested whether TMAO induces NLRP3 inflammasome formation and activation and thereby contributes to endothelial injury initiating atherogenesis.

Methods: Inflammasome formation and activation was determined by confocal microscopy, caspase-1 activity was measured by colorimetric assay, IL-1β production was measured using ELISA, cell permeability was determined by microplate reader and ZO-1 expression was determined by western blot analysis and confocal microscopy. In in vivo experiments, TMAO was infused by osmotic pump implantation.

Results: TMAO treatment significantly increased the colocalization of NLRP3 with Asc or NLRP3 with caspase-1, caspase-1 activity, IL-1β production, cell permeability in carotid artery endothelial cells (CAECs) compared to control cells. Pretreatment with caspase-1 inhibitor, WEHD or Nlrp3 siRNA abolished the TMAO-induced inflammasome formation, activation and cell permeability in these cells. In addition, we explored the mechanisms by which TMAO activates NLRP3 inflammasomes. TMAO-induced the activation of NLRP3 inflammasomes was associated with both redox regulation and lysosomal dysfunction. In animal experiments, direct infusion of TMAO in mice with partially ligated carotid artery were found to have increased NLRP3 inflammasome formation and IL-1β production in the intima of wild type mice.

Conclusion: The formation and activation of NLRP3 inflammasomes by TMAO may be an important initiating mechanism to turn on the endothelial inflammatory response leading to endothelial dysfunction.

Keywords: Endothelial cell permeability; Inflammasome; TMAO; Tight junction protein.

Figure 1. TMAO-induced NLRP3 inflammasome formation and activation in CAECs

Representative confocal fluorescence images show the colocalization of NLRP3 with ASC (A) or NLRP3 with caspase-1 (C). Summarized data shows the fold changes of pearson coefficient correlation (PCC) for the colocalization of NLRP3 with ASC (B) and NLRP3 with caspase-1 (D) in CAECs of Nlrp3^+/+ mice. * Significant difference (P<0.05) compared to the values from control cells, ^# Significant difference (P<0.05) compared to the values from TMAO treated group. Nlrp3 si, Nlrp3 siRNA; cells were transfected with Nlrp3 siRNA or WEHD and then stimulated with TMAO. N=5–6.

Figure 2. Effects of TMAO on caspase-1 activity and IL-1β production in CAECs

Values are arithmetic means ± SEM (n=6 each group) of caspase-1 activity (A), IL-1β production (B) in CAECs of Nlrp3^+/+ mice with or without stimulation of TMAO and/or Nlrp3 siRNA transfection. * Significant difference (P<0.05) compared to the values from control cells, ^# Significant difference (P<0.05) compared to the values from TMAO treated group. Nlrp3 si, Nlrp3 siRNA; cells were transfected with Nlrp3 siRNA or WEHD and then stimulated with TMAO. N=6.

Figure 3. Effects of Nlrp3 gene silencing on TMAO-induced tight junction protein ZO-1 in CAECs

A: Representative fluorescence images shows the ZO-1 expression in CAECs with or without stimulation of TMAO and/or Nlrp3 siRNA transfection (n=5). B: Representative Western blot gel document showing the expression of ZO-1 (n=3–5). C: Summarized data showing the expression of ZO-1 (n=3–5). * Significant difference (P<0.05) compared to the values from control cells.

Figure 4. Inhibition of inflammasome abolishes TMAO-induced cell permeability in CAECs

Values are arithmetic means ± SEM (n=6 each group) of cell permeability in CAECs of Nlrp3^+/+ mice with or without stimulation of TMAO and/or Nlrp3 siRNA transfection. * Significant difference (P<0.05) compared to the values from control cells, ^# Significant difference (P<0.05) compared to the values from TMAO treated group. Nlrp3 si: Nlrp3 siRNA; cells were transfected with Nlrp3 siRNA and then stimulated with TMAO.

Figure 5. Effect of cathespsin B inhibition, potassium channel blockade or ROS scavenging on TMAO- induced NLRP3 inflammasomes activation in CAECs

Summarized data showing the, caspase-1 activity in CAECs with or without stimulation of TMAO. Ca-074: Ca-074Me, cathepsin B inhibitor, Gly: Glybeclamide, potassium channel blocker, NAC: N-acetyl-L-cysteine, ROS scavenger. * P<0.05 vs. Ctrl group; # P<0.05 vs. TMAO (n=6).

Figure 6. Nlrp3 inflammasome formation and activation in wild type mice with or without stimulation of TMAO and PLCA

A: Summarized data showing the co-localization coefficient (PCC) of Nlrp3 with Asc. B: IL-1β production in the intima of vehicle or TMAO treated wild type mice. ^* Significant difference (P<0.05) compared to the values from control mice. ^# Significant difference (P<0.05) compared to the values from mice on the TMAO.