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.

The corepressors GPS2 and SMRT control enhancer and silencer remodeling via eRNA transcription during inflammatory activation of macrophages.

While the role of transcription factors and coactivators in controlling enhancer activity and chromatin structure linked to gene expression is well established, the involvement of corepressors is not. Using inflammatory macrophage activation as a model, we investigate here a corepressor complex containing GPS2 and SMRT both genome-wide and at the Ccl2 locus, encoding the chemokine CCL2 (MCP-1). We report that corepressors co-occupy candidate enhancers along with the coactivators CBP (H3K27 acetylase) and MED1 (mediator) but act antagonistically by repressing eRNA transcription-coupled H3K27 acetylation. Genome editing, transcriptional interference, and cistrome analysis reveals that apparently related enhancer and silencer elements control Ccl2 transcription in opposite ways. 4C-seq indicates that corepressor depletion or inflammatory signaling functions mechanistically similarly to trigger enhancer activation. In ob/ob mice, adipose tissue macrophage-selective depletion of the Ccl2 enhancer-transcribed eRNA reduces metaflammation. Thus, the identified corepressor-eRNA-chemokine pathway operates in vivo and suggests therapeutic opportunities by targeting eRNAs in immuno-metabolic diseases.

Blood Monocyte Phenotype Is A Marker of Cardiovascular Risk in Type 2 Diabetes.

BACKGROUND: Diabetes is a major risk factor for atherosclerotic cardiovascular diseases with a 2-fold higher risk of cardiovascular events in people with diabetes compared with those without. Circulating monocytes are inflammatory effector cells involved in both type 2 diabetes (T2D) and atherogenesis. METHODS: We investigated the relationship between circulating monocytes and cardiovascular risk progression in people with T2D, using phenotypic, transcriptomic, and metabolomic analyses. cardiovascular risk progression was estimated with coronary artery calcium score in a cohort of 672 people with T2D. RESULTS: Coronary artery calcium score was positively correlated with blood monocyte count and frequency of the classical monocyte subtype. Unsupervised k-means clustering based on monocyte subtype profiles revealed 3 main endotypes of people with T2D at varying risk of cardiovascular events. These observations were confirmed in a validation cohort of 279 T2D participants. The predictive association between monocyte count and major adverse cardiovascular events was validated through an independent prospective cohort of 757 patients with T2D. Integration of monocyte transcriptome analyses and plasma metabolomes showed a disruption of mitochondrial pathways (tricarboxylic acid cycle, oxidative phosphorylation pathway) that underlined a proatherogenic phenotype. CONCLUSIONS: In this study, we provide evidence that frequency and monocyte phenotypic profile are closely linked to cardiovascular risk in patients with T2D. The assessment of monocyte frequency and count is a valuable predictive marker for risk of cardiovascular events in patients with T2D. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT04353869.

Adrenocortical scavenger receptor class B type I deficiency exacerbates endotoxic shock and precipitates sepsis-induced mortality in mice.

Scavenger receptor class B type I (SR-BI)-deficient mice display reduced survival to endotoxic shock and sepsis. The understanding of the mechanisms underlying SR-BI protection has been hampered by the large spectrum of SR-BI functions and ligands. It notably plays an important role in the liver in high-density lipoprotein metabolism, but it is also thought to participate in innate immunity as a pattern recognition receptor for bacterial endotoxins, such as LPS. In this study, we sought to determine the tissue-specific contribution of SR-BI in the hyperinflammatory response and high mortality rates observed in SR-BI(-/-) mice in endotoxicosis or sepsis. Restoring plasma levels of high-density lipoprotein, which are critical lipoproteins for LPS neutralization, did not improve acute outcomes of LPS injection in SR-BI(-/-) mice. Mice deficient for SR-BI in hepatocytes, endothelial cells, or myeloid cells were not more susceptible to LPS-induced death. However, if SR-BI ablation in hepatocytes led to a moderate increase in systemic inflammatory markers, SR-BI deficiency in myeloid cells was associated with an anti-inflammatory effect. Finally, mice deficient for SR-BI in the adrenal cortex, where the receptor provides lipoprotein-derived cholesterol, had impaired secretion of glucocorticoids in response to stress. When exposed to an endotoxin challenge, these mice exhibited an exacerbated systemic and local inflammatory response, reduced activation of atrophy genes in muscle, and high lethality rate. Furthermore, polymicrobial sepsis induced by cecal ligature and puncture resulted in early death of these animals. Our study clearly demonstrates that corticoadrenal SR-BI is a critical element of the hypothalamic-pituitary-adrenal axis to provide effective glucocorticoid-dependent host defense after an endotoxic shock or bacterial infection.

Targeted invalidation of SR-B1 in macrophages reduces macrophage apoptosis and accelerates atherosclerosis.

AIMS: SR-B1 is a cholesterol transporter that exerts anti-atherogenic properties in liver and peripheral tissues in mice. Bone marrow (BM) transfer studies suggested an atheroprotective role in cells of haematopoietic origin. Here, we addressed the specific contribution of SR-B1 in the monocyte/macrophage. METHODS AND RESULTS: We generated mice deficient for SR-B1 in monocytes/macrophages (Lysm-Cre × SR-B1f/f) and transplanted their BM into Ldlr-/- mice. Fed a cholesterol-rich diet, these mice displayed accelerated aortic atherosclerosis characterized by larger macrophage-rich areas and decreased macrophage apoptosis compared with SR-B1f/f transplanted controls. These findings were reproduced in BM transfer studies using another atherogenic mouse recipient (SR-B1 KOliver × Cholesteryl Ester Transfer Protein). Haematopoietic reconstitution with SR-B1-/- BM conducted in parallel generated similar results to those obtained with Lysm-Cre × SR-B1f/f BM; thus suggesting that among haematopoietic-derived cells, SR-B1 exerts its atheroprotective role primarily in monocytes/macrophages. Consistent with our in vivo data, free cholesterol (FC)-induced apoptosis of macrophages was diminished in the absence of SR-B1. This effect could not be attributed to differential cellular cholesterol loading. However, we observed that expression of apoptosis inhibitor of macrophage (AIM) was induced in SR-B1-deficient macrophages, and notably upon FC-loading. Furthermore, we demonstrated that macrophages were protected from FC-induced apoptosis by AIM. Finally, AIM protein was found more present within the macrophage-rich area of the atherosclerotic lesions of SR-B1-deficient macrophages than controls. CONCLUSION: Our findings suggest that macrophage SR-B1 plays a role in plaque growth by controlling macrophage apoptosis in an AIM-dependent manner.

Loss of G protein pathway suppressor 2 in human adipocytes triggers lipid remodeling by upregulating ATP binding cassette subfamily G member 1.

OBJECTIVE: Adipogenesis is critical for adipose tissue remodeling during the development of obesity. While the role of transcription factors in the orchestration of adipogenic pathways is already established, the involvement of coregulators that transduce regulatory signals into epigenome alterations and transcriptional responses remains poorly understood. The aim of our study was to investigate which pathways are controlled by G protein pathway suppressor 2 (GPS2) during the differentiation of human adipocytes. METHODS: We generated a unique loss-of-function model by RNAi depletion of GPS2 in human multipotent adipose-derived stem (hMADS) cells. We thoroughly characterized the coregulator depletion-dependent pathway alterations during adipocyte differentiation at the level of transcriptome (RNA-seq), epigenome (ChIP-seq H3K27ac), cistrome (ChIP-seq GPS2), and lipidome. We validated the in vivo relevance of the identified pathways in non-diabetic and diabetic obese patients. RESULTS: The loss of GPS2 triggers the reprogramming of cellular processes related to adipocyte differentiation by increasing the responses to the adipogenic cocktail. In particular, GPS2 depletion increases the expression of BMP4, an important trigger for the commitment of fibroblast-like progenitors toward the adipogenic lineage and increases the expression of inflammatory and metabolic genes. GPS2-depleted human adipocytes are characterized by hypertrophy, triglyceride and phospholipid accumulation, and sphingomyelin depletion. These changes are likely a consequence of the increased expression of ATP-binding cassette subfamily G member 1 (ABCG1) that mediates sphingomyelin efflux from adipocytes and modulates lipoprotein lipase (LPL) activity. We identify ABCG1 as a direct transcriptional target, as GPS2 depletion leads to coordinated changes of transcription and H3K27 acetylation at promoters and enhancers that are occupied by GPS2 in wild-type adipocytes. We find that in omental adipose tissue of obese humans, GPS2 levels correlate with ABCG1 levels, type 2 diabetic status, and lipid metabolic status, supporting the in vivo relevance of the hMADS cell-derived in vitro data. CONCLUSION: Our study reveals a dual regulatory role of GPS2 in epigenetically modulating the chromatin landscape and gene expression during human adipocyte differentiation and identifies a hitherto unknown GPS2-ABCG1 pathway potentially linked to adipocyte hypertrophy in humans.

Adipocyte Reprogramming by the Transcriptional Coregulator GPS2 Impacts Beta Cell Insulin Secretion.

Glucose homeostasis is maintained through organ crosstalk that regulates secretion of insulin to keep blood glucose levels within a physiological range. In type 2 diabetes, this coordinated response is altered, leading to a deregulation of beta cell function and inadequate insulin secretion. Reprogramming of white adipose tissue has a central role in this deregulation, but the critical regulatory components remain unclear. Here, we demonstrate that expression of the transcriptional coregulator GPS2 in white adipose tissue is correlated with insulin secretion rate in humans. The causality of this relationship is confirmed using adipocyte-specific GPS2 knockout mice, in which inappropriate secretion of insulin promotes glucose intolerance. This phenotype is driven by adipose-tissue-secreted factors, which cause increased pancreatic islet inflammation and impaired beta cell function. Thus, our study suggests that, in mice and in humans, GPS2 controls the reprogramming of white adipocytes to influence pancreatic islet function and insulin secretion.

Isolation and Analysis of Human Monocytes and Adipose Tissue Macrophages.

Monocytes and macrophages are cells of the innate immune system, existing in circulation and permeating every tissue of the human body. These cells represent the first responders to stress, and their inflammatory activation forms part of virtually every human disease. It is for these reasons that several approaches have been developed in order to phenotypically and functionally analyze these cells and their subpopulations. These inflammatory cells have been consistently demonstrated to undergo inflammatory polarization through the engagement of lipid-activated nuclear receptors, namely, through PPARs and LXRs, which regulate both lipid metabolism and inflammation. Quantitative and phenotypic analyses of monocytes and macrophages are largely dependent on cytometric tools and antibody-based labelling of membrane markers, while functional analyses apply a range of in vitro approaches coupled with secondary analyses of gene or cytokine expression. An important aspect of phenotypic and functional analyses is the purification, enrichment, or appropriate differentiation of biological materials, ensuring experimentation is carried out on monocytes and/or macrophages and not on other cell types occupying the same physical niches. This chapter will focus on the most common analytical approaches applied to the mononuclear phagocytic system, namely, circulating monocytes and adipose tissue macrophages.

GPS2 Deficiency Triggers Maladaptive White Adipose Tissue Expansion in Obesity via HIF1A Activation.

Hypertrophic white adipose tissue (WAT) represents a maladaptive mechanism linked to the risk for developing type 2 diabetes in humans. However, the molecular events that predispose WAT to hypertrophy are poorly defined. Here, we demonstrate that adipocyte hypertrophy is triggered by loss of the corepressor GPS2 during obesity. Adipocyte-specific GPS2 deficiency in mice (GPS2 AKO) causes adipocyte hypertrophy, inflammation, and mitochondrial dysfunction during surplus energy. This phenotype is driven by HIF1A activation that orchestrates inadequate WAT remodeling and disrupts mitochondrial activity, which can be reversed by pharmacological or genetic HIF1A inhibition. Correlation analysis of gene expression in human adipose tissue reveals a negative relationship between GPS2 and HIF1A, adipocyte hypertrophy, and insulin resistance. We propose therefore that the obesity-associated loss of GPS2 in adipocytes predisposes for a maladaptive WAT expansion and a pro-diabetic status in mice and humans.

Ceramide Transporter CERT Is Involved in Muscle Insulin Signaling Defects Under Lipotoxic Conditions.

One main mechanism of insulin resistance (IR), a key feature of type 2 diabetes, is the accumulation of saturated fatty acids (FAs) in the muscles of obese patients with type 2 diabetes. Understanding the mechanism that underlies lipid-induced IR is an important challenge. Saturated FAs are metabolized into lipid derivatives called ceramides, and their accumulation plays a central role in the development of muscle IR. Ceramides are produced in the endoplasmic reticulum (ER) and transported to the Golgi apparatus through a transporter called CERT, where they are converted into various sphingolipid species. We show that CERT protein expression is reduced in all IR models studied because of a caspase-dependent cleavage. Inhibiting CERT activity in vitro potentiates the deleterious action of lipotoxicity on insulin signaling, whereas overexpression of CERT in vitro or in vivo decreases muscle ceramide content and improves insulin signaling. In addition, inhibition of caspase activity prevents ceramide-induced insulin signaling defects in C2C12 muscle cells. Altogether, these results demonstrate the importance of physiological ER-to-Golgi ceramide traffic to preserve muscle cell insulin signaling and identify CERT as a major actor in this process.

IRF5 governs liver macrophage activation that promotes hepatic fibrosis in mice and humans.

Hepatic fibrosis arises from inflammation in the liver initiated by resident macrophage activation and massive leukocyte accumulation. Hepatic macrophages hold a central position in maintaining homeostasis in the liver and in the pathogenesis of acute and chronic liver injury linked to fibrogenesis. Interferon regulatory factor 5 (IRF5) has recently emerged as an important proinflammatory transcription factor involved in macrophage activation under acute and chronic inflammation. Here, we revealed that IRF5 is significantly induced in liver macrophages from human subjects developing liver fibrosis from nonalcoholic fatty liver disease or hepatitis C virus infection. Furthermore, IRF5 expression positively correlated with clinical markers of liver damage, such as plasma transaminase and bilirubin levels. Interestingly, mice lacking IRF5 in myeloid cells (MKO) were protected from hepatic fibrosis induced by metabolic or toxic stresses. Transcriptional reprogramming of macrophages lacking IRF5 was characterized by immunosuppressive and antiapoptotic properties. Consequently, IRF5 MKO mice respond to hepatocellular stress by promoting hepatocyte survival, leading to complete protection from hepatic fibrogenesis. Our findings reveal a regulatory network, governed by IRF5, that mediates hepatocyte death and liver fibrosis in mice and humans. Therefore, modulating IRF5 function may be an attractive approach to experimental therapeutics in fibroinflammatory liver disease.

Loss of the co-repressor GPS2 sensitizes macrophage activation upon metabolic stress induced by obesity and type 2 diabetes.

Humans with obesity differ in their susceptibility to developing insulin resistance and type 2 diabetes (T2D). This variation may relate to the extent of adipose tissue (AT) inflammation that develops as their obesity progresses. The state of macrophage activation has a central role in determining the degree of AT inflammation and thus its dysfunction, and these states are driven by epigenomic alterations linked to gene expression. The underlying mechanisms that regulate these alterations, however, are poorly defined. Here we demonstrate that a co-repressor complex containing G protein pathway suppressor 2 (GPS2) crucially controls the macrophage epigenome during activation by metabolic stress. The study of AT from humans with and without obesity revealed correlations between reduced GPS2 expression in macrophages, elevated systemic and AT inflammation, and diabetic status. The causality of this relationship was confirmed by using macrophage-specific Gps2-knockout (KO) mice, in which inappropriate co-repressor complex function caused enhancer activation, pro-inflammatory gene expression and hypersensitivity toward metabolic-stress signals. By contrast, transplantation of GPS2-overexpressing bone marrow into two mouse models of obesity (ob/ob and diet-induced obesity) reduced inflammation and improved insulin sensitivity. Thus, our data reveal a potentially reversible disease mechanism that links co-repressor-dependent epigenomic alterations in macrophages to AT inflammation and the development of T2D.

Identification of the neutralizing epitopes of Merkel cell polyomavirus major capsid protein within the BC and EF surface loops.

Merkel cell polyomavirus (MCPyV) is the first polyomavirus clearly associated with a human cancer, i.e. the Merkel cell carcinoma (MCC). Polyomaviruses are small naked DNA viruses that induce a robust polyclonal antibody response against the major capsid protein (VP1). However, the polyomavirus VP1 capsid protein epitopes have not been identified to date. The aim of this study was to identify the neutralizing epitopes of the MCPyV capsid. For this goal, four VP1 mutants were generated by insertional mutagenesis in the BC, DE, EF and HI loops between amino acids 88-89, 150-151, 189-190, and 296-297, respectively. The reactivity of these mutants and wild-type VLPs was then investigated with anti-VP1 monoclonal antibodies and anti-MCPyV positive human sera. The findings together suggest that immunodominant conformational neutralizing epitopes are present at the surface of the MCPyV VLPs and are clustered within BC and EF loops.