To be or not to be a hepatic niche macrophage.

While single-cell analyses have improved our understanding of liver macrophage heterogeneity, their localization and cellular interactions remain unclear. In a recent issue of Cell, Guilliams et al. provide strategies to localize liver macrophage populations and their communication with neighboring cells during health and disease.

A subset of Kupffer cells regulates metabolism through the expression of CD36.

Tissue macrophages are immune cells whose phenotypes and functions are dictated by origin and niches. However, tissues are complex environments, and macrophage heterogeneity within the same organ has been overlooked so far. Here, we used high-dimensional approaches to characterize macrophage populations in the murine liver. We identified two distinct populations among embryonically derived Kupffer cells (KCs) sharing a core signature while differentially expressing numerous genes and proteins: a major CD206loESAM- population (KC1) and a minor CD206hiESAM+ population (KC2). KC2 expressed genes involved in metabolic processes, including fatty acid metabolism both in steady-state and in diet-induced obesity and hepatic steatosis. Functional characterization by depletion of KC2 or targeted silencing of the fatty acid transporter Cd36 highlighted a crucial contribution of KC2 in the liver oxidative stress associated with obesity. In summary, our study reveals that KCs are more heterogeneous than anticipated, notably describing a subpopulation wired with metabolic functions.

Hepatic miR-144 Drives Fumarase Activity Preventing NRF2 Activation During Obesity.

BACKGROUND AND AIMS: Oxidative stress plays a key role in the development of metabolic complications associated with obesity, including insulin resistance and the most common chronic liver disease worldwide, nonalcoholic fatty liver disease. We have recently discovered that the microRNA miR-144 regulates protein levels of the master mediator of the antioxidant response, nuclear factor erythroid 2-related factor 2 (NRF2). On miR-144 silencing, the expression of NRF2 target genes was significantly upregulated, suggesting that miR-144 controls NRF2 at the level of both protein expression and activity. Here we explored a mechanism whereby hepatic miR-144 inhibited NRF2 activity upon obesity via the regulation of the tricarboxylic acid (TCA) metabolite, fumarate, a potent activator of NRF2. METHODS: We performed transcriptomic analysis in liver macrophages (LMs) of obese mice and identified the immuno-responsive gene 1 (Irg1) as a target of miR-144. IRG1 catalyzes the production of a TCA derivative, itaconate, an inhibitor of succinate dehydrogenase (SDH). TCA enzyme activities and kinetics were analyzed after miR-144 silencing in obese mice and human liver organoids using single-cell activity assays in situ and molecular dynamic simulations. RESULTS: Increased levels of miR-144 in obesity were associated with reduced expression of Irg1, which was restored on miR-144 silencing in vitro and in vivo. Furthermore, miR-144 overexpression reduces Irg1 expression and the production of itaconate in vitro. In alignment with the reduction in IRG1 levels and itaconate production, we observed an upregulation of SDH activity during obesity. Surprisingly, however, fumarate hydratase (FH) activity was also upregulated in obese livers, leading to the depletion of its substrate fumarate. miR-144 silencing selectively reduced the activities of both SDH and FH resulting in the accumulation of their related substrates succinate and fumarate. Moreover, molecular dynamics analyses revealed the potential role of itaconate as a competitive inhibitor of not only SDH but also FH. Combined, these results demonstrate that silencing of miR-144 inhibits the activity of NRF2 through decreased fumarate production in obesity. CONCLUSIONS: Herein we unravel a novel mechanism whereby miR-144 inhibits NRF2 activity through the consumption of fumarate by activation of FH. Our study demonstrates that hepatic miR-144 triggers a hyperactive FH in the TCA cycle leading to an impaired antioxidant response in obesity.

Human resident liver myeloid cells protect against metabolic stress in obesity.

Although multiple populations of macrophages have been described in the human liver, their function and turnover in patients with obesity at high risk of developing non-alcoholic fatty liver disease (NAFLD) and cirrhosis are currently unknown. Herein, we identify a specific human population of resident liver myeloid cells that protects against the metabolic impairment associated with obesity. By studying the turnover of liver myeloid cells in individuals undergoing liver transplantation, we find that liver myeloid cell turnover differs between humans and mice. Using single-cell techniques and flow cytometry, we determine that the proportion of the protective resident liver myeloid cells, denoted liver myeloid cells 2 (LM2), decreases during obesity. Functional validation approaches using human 2D and 3D cultures reveal that the presence of LM2 ameliorates the oxidative stress associated with obese conditions. Our study indicates that resident myeloid cells could be a therapeutic target to decrease the oxidative stress associated with NAFLD.

Macrophage functional diversity in NAFLD - more than inflammation.

Macrophages have diverse phenotypes and functions due to differences in their origin, location and pathophysiological context. Although their main role in the liver has been described as immunoregulatory and detoxifying, changes in macrophage phenotypes, diversity, dynamics and function have been reported during obesity-related complications such as non-alcoholic fatty liver disease (NAFLD). NAFLD encompasses multiple disease states from hepatic steatosis to non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocarcinoma. Obesity and insulin resistance are prominent risk factors for NASH, a disease with a high worldwide prevalence and no approved treatment. In this Review, we discuss the turnover and function of liver-resident macrophages (Kupffer cells) and monocyte-derived hepatic macrophages. We examine these populations in both steady state and during NAFLD, with an emphasis on NASH. The explosion in high-throughput gene expression analysis using single-cell RNA sequencing (scRNA-seq) within the last 5 years has revolutionized the study of macrophage heterogeneity, substantially increasing our understanding of the composition and diversity of tissue macrophages, including in the liver. Here, we highlight scRNA-seq findings from the last 5 years on the diversity of liver macrophages in homeostasis and metabolic disease, and reveal hepatic macrophage function beyond their classically described inflammatory role in the progression of NAFLD and NASH pathogenesis.

Kupffer Cell mRNA Sequencing.

The liver is an important organ for the regulation of whole-body metabolism, as well as for immunity. Kupffer cells (KCs) are specialized liver-resident macrophages and the major population of immune cells in the liver. These cells have been shown to play an important role for the regulation of liver homeostasis, and many studies have thus linked these cells to the development of various liver diseases. However, the complexity of macrophage populations and the lack of specific and exclusive markers have so far made it difficult to interpret results from many of these studies. Today, new technologies have emerged including next-generation sequencing allowing for more in depth investigation of multifaceted cell populations such as KCs. Here, we describe a protocol to isolate and prepare cDNA libraries for mRNA sequencing of murine liver macrophages. Using mRNA sequencing to study the gene expression of macrophages in the liver provides a great tool to study the various functions of these cells in the regulation of homeostasis and immunity.

Liver macrophages inhibit the endogenous antioxidant response in obesity-associated insulin resistance.

Obesity and insulin resistance are risk factors for nonalcoholic fatty liver disease (NAFLD), the most common chronic liver disease worldwide. Because no approved medication nor an accurate and noninvasive diagnosis is currently available for NAFLD, there is a clear need to better understand the link between obesity and NAFLD. Lipid accumulation during obesity is known to be associated with oxidative stress and inflammatory activation of liver macrophages (LMs). However, we show that although LMs do not become proinflammatory during obesity, they display signs of oxidative stress. In livers of both humans and mice, antioxidant nuclear factor erythroid 2-related factor 2 (NRF2) was down-regulated with obesity and insulin resistance, yielding an impaired response to lipid accumulation. At the molecular level, a microRNA-targeting NRF2 protein, miR-144, was elevated in the livers of obese insulin-resistant humans and mice, and specific silencing of miR-144 in murine and human LMs was sufficient to restore NRF2 protein expression and the antioxidant response. These results highlight the pathological role of LMs and their therapeutic potential to restore the impaired endogenous antioxidant response in obesity-associated NAFLD.

Glucan-Encapsulated siRNA Particles (GeRPs) for Specific Gene Silencing in Adipose Tissue Macrophages.

Macrophages are cells of the immune system that have been suggested as important regulators of whole-body metabolism in mammals. In obesity, adipose tissue macrophages (ATMs) are thought to play both a detrimental and a beneficial role in the regulation of insulin sensitivity. Here, we describe a protocol to prepare and administer glucan-encapsulated RNAi particles (GeRPs), for specific delivery of siRNA and subsequent gene silencing in ATMs in obese mice. Using the GeRP technology to silence genes provides a unique method to study the function of factors expressed by ATMs in the regulation of metabolism.

Liver macrophages regulate systemic metabolism through non-inflammatory factors.

Liver macrophages (LMs) have been proposed to contribute to metabolic disease through secretion of inflammatory cytokines. However, anti-inflammatory drugs lead to only modest improvements in systemic metabolism. Here we show that LMs do not undergo a proinflammatory phenotypic switch in obesity-induced insulin resistance in flies, mice and humans. Instead, we find that LMs produce non-inflammatory factors, such as insulin-like growth factor-binding protein 7 (IGFBP7), that directly regulate liver metabolism. IGFBP7 binds to the insulin receptor and induces lipogenesis and gluconeogenesis via activation of extracellular-signal-regulated kinase (ERK) signalling. We further show that IGFBP7 is subject to RNA editing at a higher frequency in insulin-resistant than in insulin-sensitive obese patients (90% versus 30%, respectively), resulting in an IGFBP7 isoform with potentially higher capacity to bind to the insulin receptor. Our study demonstrates that LMs can contribute to insulin resistance independently of their inflammatory status and indicates that non-inflammatory factors produced by macrophages might represent new drug targets for the treatment of metabolic diseases.

Author Correction: Liver macrophages regulate systemic metabolism through non-inflammatory factors.

In the version of this article initially published, author Volker M. Lauschke had affiliation number 13; the correct affiliation number is 12. The error has been corrected in the HTML and PDF versions of the article.

Isolation of Kupffer Cells and Hepatocytes from a Single Mouse Liver.

Liver perfusion is a common technique used to isolate parenchymal and non-parenchymal liver cells for in vitro experiments. This method allows hepatic cells to be separated based on their size and weight, by centrifugation using a density gradient. To date, other methods allow the isolation of only one viable hepatic cellular fraction from a single mouse; either parenchymal (hepatocytes) or non-parenchymal cells (i.e., Kupffer cells or hepatic stellate cells). Here, we describe a method to isolate both hepatocytes and Kupffer cells from a single mouse liver, thereby providing the unique advantage of studying different liver cell types that have been isolated from the same organism.