Immuno-metabolism in Metabolic (dysfunction) associated fatty liver disease (MAFLD)

Abstract

The findings from chronic complex diseases modelled in animals are difficult to extrapolate to humans. In metabolic (dysfunction) associated fatty liver disease (MAFLD), dietary models are commonly used to study the mechanisms for disease progression. The current dogma is that diets rich in fat, simple carbohydrates, and cholesterol can lead to systemic alterations in metabolism that leads to the accumulation of lipids in the adipose and liver tissues. This leads to lipotoxicity and a vicious cycle of inflammation and liver injury which can drive disease progression. Hence, there has been a considerable interest to model and comprehend the role of metabolism and the immune response in this disease. However, our knowledge of the underlying mechanisms in MAFLD is still limited. A major caveat is that pre-clinical models do not represent the full spectrum of human diseases. This has been a major source of failure in clinical trials. Understanding the impact of dietary challenges in animal models and where they resemble or diverge from human disease can help to resolve the current dilemmas that have hampered progress in the field.

In this thesis, I used different dietary models in mice and characterised liver pathology, the liver immune profile, and the landscape of liver gene expression. Initially, we used multiple dietary models containing simple (sucrose) or complex carbohydrates, with/without cholesterol (2%), and with/without an added bile acid (cholic acid): A) normal chow (NC); B) high sucrose (HS); C) high sucrose and high cholesterol (HS_Chol2%); D) high sucrose, high cholesterol (2%) and cholic acid (HS_Chol2%_CA); E) high cholesterol (2%) and cholic acid (Chol2%_CA); F) cholic acid (CA). From a liver pathology perspective, diets containing cholesterol (Diets C to E) induced a dramatic change in liver pathology. Consistently, immune profiling of the liver of mice fed these diets induced infiltration of a broad range of immune cells including myeloid and lymphoid cells (diets C to E). Of note, the combination of cholesterol and cholate (diets D and E) had synergistic effects and dramatically enhanced liver immune cell infiltration. Subsequently, we performed RNA sequencing on the liver of mice fed these 6 diets. In agreement, we detected the highest differentially expressed genes in diets containing cholesterol and cholate (diets D and E). We conclude that this combination disturbs liver homeostatic functions the greatest.

To gain a systems perspective of perturbations in liver homeostatic function, we undertook a systems approach and applied weighted gene co-expression network analysis (WGCNA) on liver transcriptomes. We noticed several gene expression modules (networks) that were associated with diets. Most of these modules were enriched for metabolic pathways and immune responses. Of interest, there was a negative correlation between immune and metabolic-related modules. This was reminiscent of immunometabolism and a co-variance in gene regulatory networks between metabolic and immune modules. The up-regulation of immune responses and down-regulation of metabolic networks within the liver were prominent in mice exposed to cholesterol and cholate. A published report and our unpublished data (not the subject of this thesis) indicated that diets containing cholesterol and cholate induce a heightened immune response with anti-tumorigenic properties. Thus, from a phenotypic perspective, this immune response and the outcomes are divergent from human fatty liver that increases the risk of liver cancer.

One caveat in our dietary models was that they contained supra-physiological levels of cholesterol (2%). I detected a suppression in the expression of genes in cholesterol biosynthesis pathway in all diets that contained cholesterol (Diet C) or cholic acid (Diets D to F). This was in contrast with a study on clinical fatty liver disease (in humans) which reported up-regulation of genes in cholesterol biosynthesis pathways. I hypothesised that reduction in cholesterol biosynthesis could be related to a higher immune response. Hence, I omitted cholic acid and reduced the amount of cholesterol to 0.2% and investigated the liver pathology in mice exposed to a HS_Chol0.2% diet. This diet also induced minimal liver pathology similar to the HS diet. Despite a reduction in the cholesterol content, I detected a suppression in the expression of genes in cholesterol biosynthesis in mice exposed to the 0.2% cholesterol in the diet. This indicates a diverged response in cholesterol metabolism between mice and human liver. This diet did not induce pathological features resembling human MAFLD, however it resembled some characteristics of metabolic syndrome such as adiposity with systemic glucose intolerance.

One of the models that is often used to simulate MAFLD is the MCD (methionine choline deficient) diet. This diet induced pathological features resembling human MAFLD, however, our analysis of liver transcriptome data on mice fed with MCD diet indicated down-regulation in metabolic pathways and cholesterol biosynthesis. Indeed, the behaviour of these modules in mice on the MCD diet resembles those in mice exposed to high levels of cholesterol, which are divergent from human fatty liver disease. Overall, my results have shown that mice dietary models do not fully resemble clinical fatty liver either phenotypically or at the gene expression level. A possible strategy to overcome these limitations is to use multiple models in which each model could represent a specific aspect of the disease. At the molecular level, undertaking a module-based approach to understand the link between the behaviour of preserved modules (e.g., between mice and humans) to phenotypic outcome is an alternative strategy. One unmet need in human fatty liver is dissociating liver tissue inflammation from protective immune responses (immunosurveillance), which I would like to delve into in my future endeavours.