The gut microbiota is reported to modulate the immune response in hepatocellular carcinoma (HCC). Here, we employ metagenomic and metabolomic studies to characterise gut microbiota in patients with non-alcoholic fatty liver disease (NAFLD) related cirrhosis, with or without HCC, and evaluate its effect on the peripheral immune response in an ex vivo model. We find that dysbiosis characterises the microbiota of patients with NAFLD-cirrhosis, with compositional and functional shifts occurring with HCC development. Gene function of the microbiota in NAFLD-HCC supports short chain fatty acid production, and this is confirmed by metabolomic studies. Ex vivo studies show that bacterial extracts from the NAFLD-HCC microbiota, but not from the control groups, elicit a T cell immunosuppressive phenotype, characterised by expansion of regulatory T cells and attenuation of CD8 + T cells. Our study suggest that the gut microbiota in NAFLD-HCC is characterised by a distinctive microbiome/metabolomic profile, and can modulate the peripheral immune response.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is a common but poorly understood form of heart failure, characterized by impaired diastolic function. It is highly heterogeneous with multiple comorbidities, including obesity and diabetes, making human studies difficult. METHODS: Metabolomic analyses in a mouse model of HFpEF showed that levels of indole-3-propionic acid (IPA), a metabolite produced by gut bacteria from tryptophan, were reduced in the plasma and heart tissue of HFpEF mice as compared with controls. We then examined the role of IPA in mouse models of HFpEF as well as 2 human HFpEF cohorts. RESULTS: The protective role and therapeutic effects of IPA were confirmed in mouse models of HFpEF using IPA dietary supplementation. IPA attenuated diastolic dysfunction, metabolic remodeling, oxidative stress, inflammation, gut microbiota dysbiosis, and intestinal epithelial barrier damage. In the heart, IPA suppressed the expression of NNMT (nicotinamide N-methyl transferase), restored nicotinamide, NAD ⁺ /NADH, and SIRT3 (sirtuin 3) levels. IPA mediates the protective effects on diastolic dysfunction, at least in part, by promoting the expression of SIRT3. SIRT3 regulation was mediated by IPA binding to the aryl hydrocarbon receptor, as Sirt3 knockdown diminished the effects of IPA on diastolic dysfunction in vivo. The role of the nicotinamide adenine dinucleotide circuit in HFpEF was further confirmed by nicotinamide supplementation, Nnmt knockdown, and Nnmt overexpression in vivo. IPA levels were significantly reduced in patients with HFpEF in 2 independent human cohorts, consistent with a protective function in humans, as well as mice. CONCLUSIONS: Our findings reveal that IPA protects against diastolic dysfunction in HFpEF by enhancing the nicotinamide adenine dinucleotide salvage pathway, suggesting the possibility of therapeutic management by either altering the gut microbiome composition or supplementing the diet with IPA.

The interaction between gut microbiota and host plays a central role in health. Dysbiosis, detrimental changes in gut microbiota and inflammation have been reported in non-communicable diseases. While diet has a profound impact on gut microbiota composition and function, the role of food additives such as titanium dioxide (TiO₂), prevalent in processed food, is less established. In this project, we investigated the impact of food grade TiO₂ on gut microbiota of mice when orally administered via drinking water. While TiO₂ had minimal impact on the composition of the microbiota in the small intestine and colon, we found that TiO₂ treatment could alter the release of bacterial metabolites in vivo and affect the spatial distribution of commensal bacteria in vitro by promoting biofilm formation. We also found reduced expression of the colonic mucin 2 gene, a key component of the intestinal mucus layer, and increased expression of the beta defensin gene, indicating that TiO₂ significantly impacts gut homeostasis. These changes were associated with colonic inflammation, as shown by decreased crypt length, infiltration of CD8⁺ T cells, increased macrophages as well as increased expression of inflammatory cytokines. These findings collectively show that TiO₂ is not inert, but rather impairs gut homeostasis which may in turn prime the host for disease development.

The microbiome-derived metabolite trimethylamine-N-oxide (TMAO) has attracted major interest and controversy both as a diagnostic biomarker and therapeutic target in atherothrombosis. Plasma TMAO increased in mice on ‘unhealthy’ high-choline diets and notably also on ‘healthy’ high-fibre diets. Interestingly, TMAO was found to be generated by direct oxidation in the gut in addition to oxidation by hepatic flavin-monooxygenases. Unexpectedly, two well-accepted mouse models of atherosclerosis, ApoE^−/− and Ldlr^−/− mice, which reflect the development of stable atherosclerosis, showed no association of TMAO with the extent of atherosclerosis. This finding was validated in the Framingham Heart Study showing no correlation between plasma TMAO and coronary artery calcium score or carotid intima-media thickness (IMT), as measures of atherosclerosis in human subjects. However, in the tandem-stenosis mouse model, which reflects plaque instability as typically seen in patients, TMAO levels correlated with several characteristics of plaque instability, such as markers of inflammation, platelet activation, and intraplaque haemorrhage. Dietary-induced changes in the microbiome, of both ‘healthy’ and ‘unhealthy’ diets, can cause an increase in the plasma level of TMAO. The gut itself is a site of significant oxidative production of TMAO. Most importantly, our findings reconcile contradictory data on TMAO. There was no direct association of plasma TMAO and the extent of atherosclerosis, both in mice and humans. However, using a mouse model of plaque instability we demonstrated an association of TMAO plasma levels with atherosclerotic plaque instability. The latter confirms TMAO as being a marker of cardiovascular risk.

Poor access to human left ventricular myocardium is a significant limitation in the study of heart failure (HF). Here, we utilise a carefully procured large human heart biobank of cryopreserved left ventricular myocardium to obtain direct molecular insights into ischaemic cardiomyopathy (ICM) and dilated cardiomyopathy (DCM), the most common causes of HF worldwide. We perform unbiased, deep proteomic and metabolomic analyses of 51 left ventricular (LV) samples from 44 cryopreserved human ICM and DCM hearts, compared to age-, gender-, and BMI-matched, histopathologically normal, donor controls. We report a dramatic reduction in serum amyloid A1 protein in ICM hearts, perturbed thyroid hormone signalling pathways and significant reductions in oxidoreductase co-factor riboflavin-5-monophosphate and glycolytic intermediate fructose-6-phosphate in both; unveil gender-specific changes in HF, including nitric oxide-related arginine metabolism, mitochondrial substrates, and X chromosome-linked protein and metabolite changes; and provide an interactive online application as a publicly-available resource.