(T1130-04-22) Membrane Raft Redox Signalling Pathway Mediates Gut Microbial Metabolite TMAO-Induced NLRP3 Inflammasome Activation and Endothelial Dysfunction

Purpose: Recently we have shown that gut microbial metabolite Trimethylamine-N-oxide (TMAO)-induced NLRP3 inflammasome activation contributes to endothelial dysfunction. However, the molecular mechanisms of how TMAO-induces the Nlrp3 inflammasome activation and endothelial dysfunction are still unclear. The present study tested whether membrane raft (MR) redox signaling pathway plays a central role in TMAO-induced NLRP3 inflammasomes activation and contributes to endothelial dysfunction.

Methods: Inflammasome formation and immunofluorescence expressions of ceramide, Asm, ZO-1, ZO-2, VE-Cadherin expression were quantified by confocal microscopy. IL-1β production was measured using ELISA and cell permeability assay was determined using microplate reader.

Results: Gut microbial metabolite TMAO treatment increased the MRs clustering in the membrane of endothelial cells in a dose dependent manner. Upon TMAO stimulation an aggregation of NADPH oxidase subunits, gp91(phox) was observed in the MR clusters, forming a MR redox signalling platform. The formation of gp91(phox) with MR signalling platform was blocked by prior treatment with MR disruptor MCD or NOX inhibitor DPI. In addition, TMAO treatment increased the ceramide expression, Asm expression, IL-β production and cell permeability compared to control cells. However, prior treatment with MCD and DPI significantly attenuated the TMAO-induced ceramide expression, Asm expression, IL-β production and cell permeability. Furthermore, immunofluorescence analysis demonstrated that TMAO treatment significantly decreased the ZO-1, ZO-2 and VE-Cadherin expression and prior treatments with DPI and MCD attenuated this TMAO-induced ZO-1, ZO-2 and VE-Cadherin reduction.

Conclusion: Our results demonstrate that TMAO-induced the ceramide production via ASMase and thereby stimulates membrane raft clustering in the membrane of endothelial cells to form redox signaling platforms by aggregation and activation of NADPH oxidase subunits, enhancing O₂^.- production and leading to Nlrp3 inflammasome formation and activation in ECs and ultimate endothelial dysfunction.

Acknowledgements:

Funding: Supported by NIH grant, R01HL148711.