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.
