Introduction: An altered gut microbiota is implicated in Kawasaki disease (KD), but the specific microbial-derived metabolites that causally drive inflammation remain undefined. We hypothesized that microbiota-derived trimethylamine N-oxide (TMAO) promotes cardiovascular inflammation during KD by amplifying inflammatory responses and enhancing oxidative stress.

Methods: We used a well-established mouse model of KD vasculitis that reproduces the cardiovascular lesions observed in KD patients. Gut microbiota composition was characterized using 16S rRNA gene sequencing and circulating TMAO levels were measured in serum by liquid chromatography with tandem mass spectrometry (LC-MS-MS). Published datasets from patients with acute KD were reanalyzed to assess enrichment for TMA-producing bacteria. Mechanistic studies included in vitro stimulation of bone marrow-derived macrophages with TMAO, quantification of IL-1β production, and immunofluorescence of vascular tissue sections to quantify macrophages with caspase-1 activity and reactive oxygen species (ROS). The TMAO pathway was modulated in vivo in mice by genetic deletion of the TMAO-producing enzyme flavin-containing monooxygenase 3 (Fmo3) and by pharmacological inhibition with indole-3 carbinol (I3C; an FMO3 inhibitor) or with the trimethylamine (TMA)-lyase inhibitors, 3,3-dimethyl butanol (DMB) and fluoromethylcholine (FMC).

Results: Mice developing KD-like vascular inflammation showed enrichment for Proteobacteria capable of generating TMA, a TMAO precursor, and elevated circulating TMAO levels. Reanalysis of gut microbiota datasets from patients with acute KD similarly revealed increased abundance of TMA-generating bacteria. Mechanistically, TMAO stimulation of macrophages in vitro enhanced their NLRP3 inflammasome activation and interleukin (IL)-1β production. In vivo, genetic deletion of the TMAO-producing enzyme Fmo3 attenuated the severity of vascular inflammation. Furthermore, pharmacological blockade of the TMAO pathway in mice, targeting either microbial TMA generation with DMB or FMC, or FMO3 with I3C, reduced the development of cardiovascular lesions. Mechanistically, these interventions were associated with significant reductions in ROS production and decreased infiltration of IL-1β-producing macrophages into cardiovascular lesions.

Conclusions: Microbiota-derived TMAO amplifies vascular inflammation in mice. Blockade of the TMAO pathway mitigated macrophage inflammasome activation, oxidative stress, and the development of cardiovascular lesions in mice. These findings define a novel gut-metabolite-immune axis during KD and indicate that targeting the TMAO pathway may have therapeutic value.