Periodic Reporting for period 1 - MICFIB (MICROBIOME-DRIVERS OF LIVER FIBROSIS)
Okres sprawozdawczy: 2022-06-01 do 2024-05-31
Liver fibrosis is a major public health problem and its burden continues to grow worldwide due to an increase in its main etiological factors: harmful alcohol consumption and non-alcoholic fatty liver disease (NAFLD) (Pimpin L, et al. J. Hepatol, 2018). The liver has a remarkable capacity to regenerate, however, if the healing does not proceed normally, chronic liver inflammation leads to fibrosis, cirrhosis, and the development of liver cancer called hepatocellular carcinoma (Pimpin L, et al. J. Hepatol, 2018). There is no specific treatment for fibrosis other than removing the underlying etiological agent (e.g. alcohol, losing weight), but this is not easily achieved in all the patients. However, among patients exposed to a hepatotoxic injury such as alcohol or metabolic syndrome, as many as 20% will develop liver fibrosis, partly due to host, environmental and genetic factors (EASL, 2018).
The molecular mechanisms leading to liver fibrosis are complex and involve an interplay between multiple cells including immune, vessel (called endothelial), and supporting ( called mesenchymal) cells located within areas of scarring, termed the fibrotic niche. The use of techniques that study the genetic material inside the cells (like single-cell RNA sequencing techniques) has increased our understanding of hepatic homeostasis in fibrosis by identifying different specific cell subpopulations (endothelial cells, macrophages and collagen-producing mesenchymal cells) that interact spatiotemporally to drive fibrosis within the fibrotic niche (Ramachandran P, et al. Nature, 2019).
However, the drivers of these particular cellular phenotypes and why fibrosis develops only in a subset of patients are concepts that are not yet fully understood. In recent years, there has been an increasing body of data suggesting that the microbiome (which encompasses the microorganisms inside the body: bacteria, fungi, and viruses) can modify liver homeostasis. The microorganisms and microbial-derived compounds that reach the liver (as the liver is connected to the gut directly through the portal vein) are rapidly recognized by liver cells. Activation of these cells, leads to hepatic inflammation and hepatocyte damage and in the end to liver fibrosis. This connection is often referred as the gut-liver axis. While normal microbiota prevents liver fibrosis in mice (Mazagova M, et al. FASEB J. 2015), in different liver diseases that develop fibrosis, there are alterations in the composition and function of the gut microbiota and of microbial-derived metabolites, that drive the individual susceptibility to develop liver inflammation (Llopis M, et al. Gut, 2016). Moreover, in animal models, manipulation of the gut microbiota using antibiotics or prebiotics, decreases liver fibrosis, suggesting the existence of fibrosis-associated intestinal microbiota (Seki, E. et al. Nat. Med, 2007, Llorente, C. et al. Nat. Comm., 2017, Kisseleva T, et al. Nat Rev Gastroenterol Hepatol., 2021).
The key objective of my project is to gain a comprehensive understanding of how microbiota modifies liver homeostasis and contributes to liver fibrosis using analysis of the network perturbation in disease, coupled with microbiome experimental techniques.
The molecular mechanisms leading to liver fibrosis are complex and involve an interplay between multiple cells including immune, vessel (called endothelial), and supporting ( called mesenchymal) cells located within areas of scarring, termed the fibrotic niche. The use of techniques that study the genetic material inside the cells (like single-cell RNA sequencing techniques) has increased our understanding of hepatic homeostasis in fibrosis by identifying different specific cell subpopulations (endothelial cells, macrophages and collagen-producing mesenchymal cells) that interact spatiotemporally to drive fibrosis within the fibrotic niche (Ramachandran P, et al. Nature, 2019).
However, the drivers of these particular cellular phenotypes and why fibrosis develops only in a subset of patients are concepts that are not yet fully understood. In recent years, there has been an increasing body of data suggesting that the microbiome (which encompasses the microorganisms inside the body: bacteria, fungi, and viruses) can modify liver homeostasis. The microorganisms and microbial-derived compounds that reach the liver (as the liver is connected to the gut directly through the portal vein) are rapidly recognized by liver cells. Activation of these cells, leads to hepatic inflammation and hepatocyte damage and in the end to liver fibrosis. This connection is often referred as the gut-liver axis. While normal microbiota prevents liver fibrosis in mice (Mazagova M, et al. FASEB J. 2015), in different liver diseases that develop fibrosis, there are alterations in the composition and function of the gut microbiota and of microbial-derived metabolites, that drive the individual susceptibility to develop liver inflammation (Llopis M, et al. Gut, 2016). Moreover, in animal models, manipulation of the gut microbiota using antibiotics or prebiotics, decreases liver fibrosis, suggesting the existence of fibrosis-associated intestinal microbiota (Seki, E. et al. Nat. Med, 2007, Llorente, C. et al. Nat. Comm., 2017, Kisseleva T, et al. Nat Rev Gastroenterol Hepatol., 2021).
The key objective of my project is to gain a comprehensive understanding of how microbiota modifies liver homeostasis and contributes to liver fibrosis using analysis of the network perturbation in disease, coupled with microbiome experimental techniques.
Effect of the microbiome profiles on liver fibrosis: To understand the impact of microbiota on liver fibrosis I induced liver fibrosis using a toxic model of liver fibrosis using chronic administration of Thioacetamide (TAA) intraperitoneally. To study the role of intestinal microbiota in liver fibrosis I treated mice with a combination of antibiotics in drinking water for 4 weeks (ampicillin (A), vancomycin (V), neomycin (N), and metronidazole (M)) while inducing liver fibrosis using TAA. Liver fibrosis was evaluated using commonly accepted methods: biochemical, histological and gene expression.
Microbiota profiling: Bacterial composition profiling of the luminal small intestine content was performed using shotgun metagenomic sequencing. This helped us identify the composition of the bacterial microbiome at the species level. Next, we identified which bacteria are different between fibrosis and control mice.
Role of microbiota in liver fibrosis at cellular level: To understand the impact of microbiota on the fibrotic niche at the cellular level, I performed single-cell RNA sequencing in the two groups of mice (fibrosis vs. no fibrosis) and two distinct microbiome states (antibiotic-treated vs control). This helped us identify how cells are modified and which are the genetic factors that could drive the susceptibility to develop fibrosis.
Identifying bacterial derived metabolites that influence the progression of fibrosis: The microbiome produces a large panel of metabolites that could affect the cell fate in the fibrotic niche. However, which metabolites and how they could be involved in the fibrotic process and cell transition is still unknown. To find which bacterially derived components affect the fibrosis process I performed untargeted metabolomics (a technique that allows the identification of a large panel of products) of blood serum from systemic blood and the portal vein that contains all the compounds that reach the liver, in healthy and fibrotic mice. By comparing the profiles between the different microbiome-associated states I identified candidate molecules that potentially affect the HSCs physiology.
Microbiota profiling: Bacterial composition profiling of the luminal small intestine content was performed using shotgun metagenomic sequencing. This helped us identify the composition of the bacterial microbiome at the species level. Next, we identified which bacteria are different between fibrosis and control mice.
Role of microbiota in liver fibrosis at cellular level: To understand the impact of microbiota on the fibrotic niche at the cellular level, I performed single-cell RNA sequencing in the two groups of mice (fibrosis vs. no fibrosis) and two distinct microbiome states (antibiotic-treated vs control). This helped us identify how cells are modified and which are the genetic factors that could drive the susceptibility to develop fibrosis.
Identifying bacterial derived metabolites that influence the progression of fibrosis: The microbiome produces a large panel of metabolites that could affect the cell fate in the fibrotic niche. However, which metabolites and how they could be involved in the fibrotic process and cell transition is still unknown. To find which bacterially derived components affect the fibrosis process I performed untargeted metabolomics (a technique that allows the identification of a large panel of products) of blood serum from systemic blood and the portal vein that contains all the compounds that reach the liver, in healthy and fibrotic mice. By comparing the profiles between the different microbiome-associated states I identified candidate molecules that potentially affect the HSCs physiology.
To study the effect of the microbiome on liver fibrosis development and evolution we depleted the microbiome using antibiotics and observed that mice treated with antibiotics develop less fibrosis.
We next investigated the microbial changes and identified changes in the levels of Lactobacilus and Turcibacter, suggesting that these 2 microorganisms could play a crucial role in fibrosis development.
To better understand how the microbiome and microbiome-derived molecules could influence fibrosis development, we performed single-cell sequencing and we observed an increase in activated stellate cells after fibrosis induction, of certain macrophage populations and a swift in the hepatocyte zonation, with a decrease in the periportal serpine1 + hepatocytes and an increase in the pericentral hepatocytes Cyp2e1+.
As the macrophages are key players in fibrosis formation as they are involved in the inflammatory response and repair, we studied the role of the Trem2+ macrophages in fibrosis but depletion of these macrophages didn’t have any effect on fibrosis development.
We next investigated the role of the microbiome on liver fibrosis at the cellular level and identified several transcription factors. Their role in liver fibrosis is under investigation.
The impact of the microbiome on the liver and stellate cells could be mediated by bacterial metabolites and we identified a decrease in the levels of several aminoacids, tryptophan derivatives, and in the bile acid profile. We are pursuing the investigation of the metabolic changes in fibrosis development by testing a panel of metabolites in vitro and in vivo. As the project is still ongoing, we cannot disclose the names of the most promising metabolites.
We next investigated the microbial changes and identified changes in the levels of Lactobacilus and Turcibacter, suggesting that these 2 microorganisms could play a crucial role in fibrosis development.
To better understand how the microbiome and microbiome-derived molecules could influence fibrosis development, we performed single-cell sequencing and we observed an increase in activated stellate cells after fibrosis induction, of certain macrophage populations and a swift in the hepatocyte zonation, with a decrease in the periportal serpine1 + hepatocytes and an increase in the pericentral hepatocytes Cyp2e1+.
As the macrophages are key players in fibrosis formation as they are involved in the inflammatory response and repair, we studied the role of the Trem2+ macrophages in fibrosis but depletion of these macrophages didn’t have any effect on fibrosis development.
We next investigated the role of the microbiome on liver fibrosis at the cellular level and identified several transcription factors. Their role in liver fibrosis is under investigation.
The impact of the microbiome on the liver and stellate cells could be mediated by bacterial metabolites and we identified a decrease in the levels of several aminoacids, tryptophan derivatives, and in the bile acid profile. We are pursuing the investigation of the metabolic changes in fibrosis development by testing a panel of metabolites in vitro and in vivo. As the project is still ongoing, we cannot disclose the names of the most promising metabolites.