Periodic Reporting for period 2 - MacinNASH (Revealing the contribution of liver macrophage populations to NASH in insulin resistance)
Berichtszeitraum: 2021-09-01 bis 2023-02-28
Non-alcoholic fatty liver disease (NAFLD) is a serious condition in which patients develop liver disease similar to that observe in alcoholism, but without excessive alcohol consumption. Obesity and its associated metabolic complication, insulin resistance, are high risk factors for NAFLD, due to the excessive and inappropriate accumulation of fat in the liver, also called steatosis. Through an onslaught of multiple factors the first stage of NAFLD, steatosis, can progress into Non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis and ultimately hepatocarcinoma. Today NASH has become the leading cause of liver-related malignancy and mortality. Despite its high prevalence (6% of the general population), no pharmacological treatment is currently available.
Macrophages are cells of the immune system whose main role is to defend the organism against pathogens. While macrophages are well known for their role in inflammation, we have demonstrated that liver macrophages could contribute to the development of NAFLD independently of their inflammatory phenotype. This work has been the steppingstone for the current study, which aims to unravel the roles and diversity of liver macrophages in the context of liver disease associated with obesity.
Using the power of gene sequencing we aim to first characterize in an unbiased manner the diversity of macrophage populations in the liver in healthy individuals and in obese patients with NAFLD, ranging from steatosis to NASH and severe fibrosis. Taking advantage of unique tools to manipulate gene expression in a macrophage-targeted manner and our pre-clinical models of human livers in a dish, we will functionally validate the relevance of macrophage populations in liver disease.
The overall objective of our project is to improve our understanding of the mechanisms driving the development and progression of NAFLD in order to pave the way for novel therapeutic strategies for NASH.
Macrophages are cells of the immune system whose main role is to defend the organism against pathogens. While macrophages are well known for their role in inflammation, we have demonstrated that liver macrophages could contribute to the development of NAFLD independently of their inflammatory phenotype. This work has been the steppingstone for the current study, which aims to unravel the roles and diversity of liver macrophages in the context of liver disease associated with obesity.
Using the power of gene sequencing we aim to first characterize in an unbiased manner the diversity of macrophage populations in the liver in healthy individuals and in obese patients with NAFLD, ranging from steatosis to NASH and severe fibrosis. Taking advantage of unique tools to manipulate gene expression in a macrophage-targeted manner and our pre-clinical models of human livers in a dish, we will functionally validate the relevance of macrophage populations in liver disease.
The overall objective of our project is to improve our understanding of the mechanisms driving the development and progression of NAFLD in order to pave the way for novel therapeutic strategies for NASH.
According to the timeline presented in the original proposal, patient recruitment should have been completed for the analyses of macrophage diversity in the liver of obese individuals with insulin resistance and different stages of fatty liver disease, from steatosis to NASH and fibrosis (Aim 1).
We have completed the part of the study in which samples had been collected prior to 2020. This study refers to the investigation of macrophage functional diversity in the liver of healthy individuals or obese patients with insulin resistance. We have demonstrated that multiple populations of macrophages with distinct function are present in the liver and can either contribute or protect from the development of metabolic stress associated with steatosis. This work has been recently submitted for publication in a peer-reviewed journal.
In parallel, we have investigated the functional diversity of liver macrophages in obesity in animal models. We demonstrated that a population of resident liver macrophages contribute to the oxidative stress associated with obesity and steatosis via the lipid transporter CD36. This collaborative study was published in Immunity in September 2021 (Bleriot et al, Immunity, 2021).
Due to the COVID19 pandemic patient recruitment was halted and the collection of samples from patients with NASH was delayed. While fresh biopsies are needed to perform single cell RNA sequencing (scRNAseq) using the SmartSeq2 protocol that allows deep sequencing and that we had proposed to preferentially use, we adapted to the pandemic situation and developed a new protocol to sequence single nuclei using cryopreserved material. We have already validated this technique provides data of the high quality we need. These samples have been sequenced and the analyses will provide valuable information on the diversity of macrophages in NASH livers.
While the goal of Aim 1 was to characterize the landscape of liver macrophages in liver disease associated with obesity, Aim 2 focused on studying the function of these macrophages. We have used pre-clinical models (mouse and liver spheroids) to validate the function of a resident population of liver macrophages and have recorded clear differences between species. Both the turnover and function of macrophages were different between mice and humans in health and fatty liver disease.
We have also demonstrated that a miRNA, miR-144, that is important for the regulation of oxidative stress, downregulates the antioxidant response in the liver of obese individuals by increasing the activity of the mitochondria, the powerhouse of cells (Azzimato et al, Gastroenterology, 2021). We will use this knowledge to study cellular metabolism at the single cell level, as we had proposed, but now focusing on mitochondrial biogenesis.
The discovery of the difference in liver macrophage function and dynamics between mice and humans (Aims 1 and 2) has been crucial in designing a new set of experiments for Aim 3. In the future, we will thus study the effect of the liver niche on the acquisition of the liver macrophage phenotype in fatty liver disease, but more effort will now be made in the human study as mice do not seem to be the perfect model for this investigation. We have already developed a method to study liver macrophage dynamics in patients, using tissues from individuals who undergo liver transplantation and liver spheroids formed with monocytes.
In summary, we have characterized the diversity of liver macrophages in mice and humans in early stages of fatty liver disease. Our future work is focussing on the drivers of macrophage diversity in the later stages of the disease: NASH and fibrosis. Our results to date provide the foundation that could lead to the discovery of the therapeutic potential of macrophages in liver diseases such as NASH.
We have completed the part of the study in which samples had been collected prior to 2020. This study refers to the investigation of macrophage functional diversity in the liver of healthy individuals or obese patients with insulin resistance. We have demonstrated that multiple populations of macrophages with distinct function are present in the liver and can either contribute or protect from the development of metabolic stress associated with steatosis. This work has been recently submitted for publication in a peer-reviewed journal.
In parallel, we have investigated the functional diversity of liver macrophages in obesity in animal models. We demonstrated that a population of resident liver macrophages contribute to the oxidative stress associated with obesity and steatosis via the lipid transporter CD36. This collaborative study was published in Immunity in September 2021 (Bleriot et al, Immunity, 2021).
Due to the COVID19 pandemic patient recruitment was halted and the collection of samples from patients with NASH was delayed. While fresh biopsies are needed to perform single cell RNA sequencing (scRNAseq) using the SmartSeq2 protocol that allows deep sequencing and that we had proposed to preferentially use, we adapted to the pandemic situation and developed a new protocol to sequence single nuclei using cryopreserved material. We have already validated this technique provides data of the high quality we need. These samples have been sequenced and the analyses will provide valuable information on the diversity of macrophages in NASH livers.
While the goal of Aim 1 was to characterize the landscape of liver macrophages in liver disease associated with obesity, Aim 2 focused on studying the function of these macrophages. We have used pre-clinical models (mouse and liver spheroids) to validate the function of a resident population of liver macrophages and have recorded clear differences between species. Both the turnover and function of macrophages were different between mice and humans in health and fatty liver disease.
We have also demonstrated that a miRNA, miR-144, that is important for the regulation of oxidative stress, downregulates the antioxidant response in the liver of obese individuals by increasing the activity of the mitochondria, the powerhouse of cells (Azzimato et al, Gastroenterology, 2021). We will use this knowledge to study cellular metabolism at the single cell level, as we had proposed, but now focusing on mitochondrial biogenesis.
The discovery of the difference in liver macrophage function and dynamics between mice and humans (Aims 1 and 2) has been crucial in designing a new set of experiments for Aim 3. In the future, we will thus study the effect of the liver niche on the acquisition of the liver macrophage phenotype in fatty liver disease, but more effort will now be made in the human study as mice do not seem to be the perfect model for this investigation. We have already developed a method to study liver macrophage dynamics in patients, using tissues from individuals who undergo liver transplantation and liver spheroids formed with monocytes.
In summary, we have characterized the diversity of liver macrophages in mice and humans in early stages of fatty liver disease. Our future work is focussing on the drivers of macrophage diversity in the later stages of the disease: NASH and fibrosis. Our results to date provide the foundation that could lead to the discovery of the therapeutic potential of macrophages in liver diseases such as NASH.
Our studies on macrophage diversity are unique in their focus on the earlier stages of NAFLD, which represent a therapeutic window, compared to the late stages of the disease in which transplantation is the only option. In our translational approach we have particularly focused on studying these processes in humans, a challenging endeavor considering the inherent difficulty in studying cellular dynamics. We have designed methods and have collected precious samples to investigate macrophage turnover in humans. Our results so far have first highlighted species differences between humans and mice, but have also clearly defined the functional diversity of macrophages in the development of NAFLD. In the future, we will investigate the therapeutic potential of these populations in NAFLD. We will also study the drivers of the functional diversity of macrophages in NASH. The overall results will constitute a comprehensive analysis of the role and therapeutic potential of macrophages of the life-threatening disease NASH, for which there is currently no available pharmacological treatment.