Metabolomics studies hold promise for discovery of pathways linked to disease processes. Cardiovascular disease (CVD) represents the leading cause of death and morbidity worldwide. A metabolomics approach was used to generate unbiased small molecule metabolic profiles in plasma that predict risk for CVD. Three metabolites of the dietary lipid phosphatidylcholine, namely choline, trimethylamine N-oxide (TMAO), and betaine, were identified and then shown to predict risk for CVD in an independent large clinical cohort. Dietary supplementation of mice with choline, TMAO or betaine promoted up-regulation of multiple macrophage scavenger receptors linked to atherosclerosis, and supplementation with choline or TMAO promoted atherosclerosis. Studies using germ-free mice confirmed a critical role for dietary choline and gut flora in TMAO production, augmented macrophage cholesterol accumulation and foam cell formation. Suppression of intestinal microflora in atherosclerosis-prone mice inhibited dietary choline-enhanced atherosclerosis. Genetic variations controlling expression of flavin monooxygenases (FMOs), an enzymatic source of TMAO, segregated with atherosclerosis in hyperlipidemic mice. Discovery of a relationship between gut flora-dependent metabolism of dietary phosphatidylcholine and CVD pathogenesis provides opportunities for development of both novel diagnostic tests and therapeutic approaches for atherosclerotic heart disease.

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Inflammasome activation has been recently recognized to play a central role in the development of drug-induced and obesity-associated liver disease. However, the sources and mechanisms of inflammasome mediated liver damage remain poorly understood. Our aim was to investigate the effect of NLRP3 inflammasome activation on the liver using novel mouse models. We generated global and myeloid cell specific conditional mutant Nlrp3 knock-in mice expressing the D301N Nlrp3 mutation (ortholog of D303N in human NLRP3) resulting in a constitutively activated NLRP3. To study the presence and significance of NLRP3 initiated pyroptotic cell death, we separated hepatocytes from non-parenchymal cells and developed a novel flow cytometry-based (FACS) strategy to detect and quantify pyroptosis in vivo based on detection of active caspase1 and propidium iodide (PI) positive cells. Liver inflammation was quantified histologically, by FACS and via gene expression analysis. Liver fibrosis was assessed by Sirius-Red-staining and qPCR for markers of hepatic stellate cell-(HSC)-activation. NLRP3 activation resulted in shortened survival, poor growth, and severe liver inflammation; characterized by neutrophilic infiltration and HSC-activation with collagen deposition in the liver. These changes were partially attenuated by treatment with anakinra, an interleukin-1 receptor antagonist. Notably, hepatocytes from global Nlrp3 mutant mice showed marked hepatocyte pyroptotic cell death with more than a fivefold increase in active caspase1-PI double positive cells. Myeloid cell restricted mutant NLRP3 activation resulted in a less severe liver phenotype in the absence of detectable pyroptotic hepatocyte cell death. Conclusions Our data demonstrates that global and to a lesser extent myeloid-specific NLRP3 inflammasome activation results in severe liver inflammation and fibrosis, while identifying hepatocyte pyroptotic cell death as a novel mechanism of NLRP3 mediated liver damage.