Adipoclast: a multinucleated fat-eating macrophage

Cell membrane fusion and multinucleation in macrophages are associated with physiologic homeostasis as well as disease. Osteoclasts are multinucleated macrophages that resorb bone through increased metabolic activity resulting from cell fusion. Fusion of macrophages also generates multinucleated giant cells (MGCs) in white adipose tissue (WAT) of obese individuals. For years, our knowledge of MGCs in WAT has been limited to their description as part of crown-like structures (CLS) surrounding damaged adipocytes. However, recent evidence indicates that these cells can phagocytose oversized lipid remnants, suggesting that, as in osteoclasts, cell fusion and multinucleation are required for specialized catabolic functions. We thus reason that WAT MGCs can be viewed as functionally analogous to osteoclasts and refer to them in this article as adipoclasts. We first review current knowledge on adipoclasts and their described functions. In view of recent advances in single cell genomics, we describe WAT macrophages from a ‘fusion perspective’ and speculate on the ontogeny of adipoclasts. Specifically, we highlight the role of CD9 and TREM2, two plasma membrane markers of lipid-associated macrophages in WAT, which have been previously described as regulators of fusion and multinucleation in osteoclasts and MGCs. Finally, we consider whether strategies aiming to target WAT macrophages can be more selectively directed against adipoclasts

Immunolipidomics reveals a globoside network during the resolution of pro-inflammatory response in human macrophages

Toll-like receptor 4 (TLR4)-mediated changes in macrophages reshape intracellular lipid pools to coordinate an effective innate immune response. Although this has been previously well-studied in different model systems, it remains incompletely understood in primary human macrophages. Here we report time-dependent lipidomic and transcriptomic responses to lipopolysaccharide (LPS) in primary human macrophages from healthy donors. We grouped the variation of ~200 individual lipid species measured by LC-MS/MS into eight temporal clusters. Among all other lipids, glycosphingolipids (glycoSP) and cholesteryl esters (CE) showed a sharp increase during the resolution phase (between 8h or 16h post LPS). GlycoSP, belonging to the globoside family (Gb3 and Gb4), showed the greatest inter-individual variability among all lipids quantified. Integrative network analysis between GlycoSP/CEs levels and genome-wide transcripts, identified Gb4 d18:1/16:0 and CE 20:4 association with subnetworks enriched for T cell receptor signaling (PDCD1, CD86, PTPRC, CD247, IFNG) and DC-SIGN signaling (RAF1, CD209), respectively. Our findings reveal Gb3 and Gb4 globosides as sphingolipids associated with the resolution phase of inflammatory response in human macrophages

Multinucleation resets human macrophages for specialized functions at the expense of their identity

Macrophages undergo plasma membrane fusion and cell multinucleation to form multinucleated giant cells (MGCs) such as osteoclasts in bone, Langhans giant cells (LGCs) as part of granulomas or foreign-body giant cells (FBGCs) in reaction to exogenous material. How multinucleation per se contributes to functional specialization of mature mononuclear macrophages remains poorly understood in humans. Here, we integrate comparative transcriptomics with functional assays in purified mature mononuclear and multinucleated human osteoclasts, LGCs and FBGCs. Strikingly, in all three types of MGCs, multinucleation causes a pronounced downregulation of macrophage identity. We show enhanced lysosome-mediated intracellular iron homeostasis promoting MGC formation. The transition from mononuclear to multinuclear state is accompanied by cell specialization specific to each polykaryon. Enhanced phagocytic and mitochondrial function associate with FBGCs and osteoclasts, respectively. Moreover, human LGCs preferentially express B7-H3 (CD276) and can form granuloma-like clusters in vitro, suggesting that their multinucleation potentiates T cell activation. These findings demonstrate how cell–cell fusion and multinucleation reset human macrophage identity as part of an advanced maturation step that confers MGC-specific functionality

Unique Regulatory Properties of Mesangial Cells Are Genetically Determined in the Rat

Mesangial cells are glomerular cells of stromal origin. During immune complex mediated crescentic glomerulonephritis (Crgn), infiltrating and proliferating pro-inflammatory macrophages lead to crescent formation. Here we have hypothesised that mesangial cells, given their mesenchymal stromal origin, show similar immunomodulatory properties as mesenchymal stem cells (MSCs), by regulating macrophage function associated with glomerular crescent formation. We show that rat mesangial cells suppress conA-stimulated splenocyte proliferation in vitro, as previously shown for MSCs. We then investigated mesangial cell-macrophage interaction by using mesangial cells isolated from nephrotoxic nephritis (NTN)-susceptible Wistar Kyoto (WKY) and NTN-resistant Lewis (LEW) rats. We first determined the mesangial cell transcriptome in WKY and LEW rats and showed that this is under marked genetic control. Supernatant transfer results show that WKY mesangial cells shift bone marrow derived macrophage (BMDM) phenotype to M1 or M2 according to the genetic background (WKY or LEW) of the BMDMs. Interestingly, these effects were different when compared to those of MSCs suggesting that mesangial cells can have unique immunomodulatory effects in the kidney. These results demonstrate the importance of the genetic background in the immunosuppressive effects of cells of stromal origin and specifically of mesangial cell-macrophage interactions in the pathophysiology of crescentic glomerulonephritis

Synthesis and anti-HIV activity of thiocholesteryl-coupled phosphodiester antisense oligonucleotides incorporated into immunoliposomes

Encapsulation of oligonucleotides in antibody-targeted liposomes (immunoliposomes) which bind to target cells permits intracellular delivery of the oligonucleotides. This approach circumvents problems of extracellular degradation by nucleases and poor membrane permeability which free phosphodiester oligonucleotides are subject to, but leaves unresolved the inefficiency of encapsulation of oligonucleotides in liposomes. We have coupled oligonucleotides to cholesterol via a reversible disulfide bond. This modification of oligonucleotides improved their association with immunoliposomes by a factor of about 10 in comparison to unmodified oligonucleotides. The presence of cholesteryl-modified oligonucleotides incorporated in the bilayer of liposomes did not interfere with the coupling of the targeting protein to the liposome surface. Free or cholesterol coupled oligonucleotides associated with liposomes and directed against the tat gene of HIV-1 were tested for inhibition of HIV-1 proliferation in acutely infected cells. We demonstrate that the cholesteryl-modified as well as unmodified oligonucleotides acquire the target specificity of the antibody on the liposome. Their antiviral activity when delivered into cells is sequence-specific. The activity of these modified or unmodified oligonucleotides to inhibit the replication of HIV was the same on an equimolar basis (EC50 around 0.1 μM). Cholesterol coupled oligonucleotides thus offer increased liposome association without loss of antiviral activity

Epoxygenase inactivation exacerbates diet and aging-associated metabolic dysfunction resulting from impaired adipogenesis

• Cyp2j4 deletion in rats offers a simplified genetic landscape as opposed to the mice locus, which is under significant allelic expansion. • Under different metabolic stresses (aging, cafeteria diet), Cyp2j4−/− rats show an accelerated adipogenesis, which progress towards adipocyte dysfunction. • Adipocyte dysfunction in Cyp2j4−/− rats under cafeteria diet (CAF) is characterised by down-regulation of white adipose tissue (WAT) PPARγ and C/EBPα, adipocyte hypertrophy and extracellular matrix remodeling. • Cyp2j4−/− rats treated with CAF display exacerbated weight gain, insulin resistance, hepatic lipid accumulation and dysregulated gluconeogenesis. • Cyp2j4−/− rats display alternative arachidonic acid pathway usage in their adipose tissue upon CAF and aging

WEBSAGE: a web tool for visual analysis of differentially expressed human SAGE tags

The serial analysis of gene expression (SAGE) is a powerful method to compare gene expression of mRNA populations. To provide quantitative expression levels on a genome-wide scale, the Cancer Genome Anatomy Project (CGAP) uses SAGE. Over 7 million SAGE tags, from 171 human cell types have been assembled. The growing number of laboratories involved in SAGE research necessitates the use of software that provides statistical analysis of raw data, allowing the rapid visualization and interpretation of results. We have created the first simple tool that performs statistical analysis on SAGE data, identifies the tags differentially expressed and shows the results in a scatter plot. It is freely available and accessible at

Internalisation cellulaire et activité biologique de PNA bloqueurs stériques de la traduction, conjugués au peptide (R/W)9

Les Peptide Nucleic Acids (PNA) sont des oligonucléotides antisens analogues de l ADN, dont le squelette phosphodiester a été remplacé par un squelette pseudo-peptidique d unités 2-aminoéthylglycine, sur lequel sont greffées des bases azotées. Des PNA dirigés contre les ARN messagers peuvent inhiber la traduction in vitro et dans les cellules humaines. Lorsqu ils sont dirigés contre la partie codante du transcrit, des PNA polypyrimidiques peuvent bloquer physiquement l élongation de la traduction en stoppant la machinerie ribosomale. Le transcrit n est pas dégradé et une protéine tronquée est générée in vitro. Dans le cas de protéines dont la surexpression conduit à des pathologies, des protéines tronquées inactives peuvent jouer un rôle de dominant négatif dans les cellules. Des protéines tronquées de l Insulin-like Growth Factor-1 (IGF1R), récepteur cellulaire surexprimé dans de nombreux cancers, inhibent la tumorigénèse et la résistance à l apoptose de cellules cancéreuses. La pénétration cellulaire des PNA est la principale limite à leur utilisation in vivo et il est nécessaire de développer des transporteurs efficaces pour ces oligonucléotides neutres. Les Cell Penetrating Peptides (CPP) sont des peptides naturels ou synthétiques, qui peuvent être conjugués à différentes molécules pour promouvoir leur internalisation cellulaire. Les objectifs de ce travail de thèse étaient de comprendre les critères requis pour l arrêt de l élongation de la traduction par les PNA et d étudier leur internalisation cellulaire médiée par le CPP (R/W)9. Nous avons montré qu un couplage covalent entre ce peptide et deux PNA 13-mer permet l internalisation des conjugués dans un système cellulaire rapporteur, conduisant à leur activité biologique en présence d un agent lysosomotropique. Les conjugués interagissent avec les glycosaminoglycanes membranaires et sont internalisés par endocytose en moins d une heure. De plus, les conjugués formés avec un peptide analogue comportant des lysines sont six fois moins internalisés, mettant en évidence l importance des résidus arginines du peptide (R/W)9 pour l interaction avec la membrane. Enfin, nous avons montré que le peptide (R/W)9 couplé à un PNA dirigé contre la séquence codante de l IGF1R permet son internalisation dans les cellules de cancer de la prostate et que le conjugué inhibe spécifiquement l expression de la chaîne b du récepteur.Peptide nucleic acids (PNAs) are nucleic acid analogues in which the sugar-phosphate backbone has been replaced by a synthetic peptide backbone, usually comprised of N-(2-aminoethyl)-glycan units. PNAs targeted against mRNA can inhibit translation both in vitro and in human cells. Pyrimidine rich PNAs can physically block translation elongation at targets in the coding region of messenger RNA, giving rise to a truncated protein. Truncated proteins that lack a functional domain and can at the same time inhibit the function of the wild type protein are referred to as dominant negative. Truncated form of Insulin-like Growth Factor-1 receptor (IGF1R), protein overexpressed in numerous cancers, inhibits tumorigenesis and resistance to apoptosis of cancerous cells. One of the biggest limitations to the use of PNAs in vivo is their poor internalization. It is therefore necessary to develop efficient transporters able to enhance the cellular uptake of PNAs. Cell-penetrating peptides (CPPs) are natural or synthetic peptides that can be conjugated to different molecules in order to facilitate their cellular uptake. The objectives of this thesis were to understand the conditions required for the translation elongation arrest by PNAs and to study their cellular internalization mediated by CPP (R/W)9. We have shown that covalent coupling of two 13-mer PNAs to (R/W)9 facilitates their internalization in a reporter cell line, leading to their biological activity in the presence of a lysosomotropic agent such as chloroquine. The conjugates interact with membrane glycosaminoglycans and are internalized by endocytosis in less than one hour. Moreover, conjugates formed with an analogue peptide containing lysines in the place of arginines of (R/W)9 showed to be six time less efficiently internalized, suggesting the importance of arginine residues for the interaction of the conjugate with the membrane. We have also showed that the PNA targeted to the coding region of IGF1R coupled to (R/W)9 is efficiently internalized to prostate cancer cells where it inhibits the expression of the beta chain of the receptor.PARIS5-Bibliotheque electronique (751069902) / SudocPARIS-BIUM-Bib. électronique (751069903) / SudocSudocFranceF

Systems genetics identifies Sestrin 3 as a regulator of a proconvulsant gene network in human epileptic hippocampus

Gene-regulatory network analysis is a powerful approach to elucidate the molecular processes and pathways underlying complex disease. Here we employ systems genetics approaches to characterize the genetic regulation of pathophysiological pathways in human temporal lobe epilepsy (TLE). Using surgically acquired hippocampi from 129 TLE patients, we identify a gene-regulatory network genetically associated with epilepsy that contains a specialized, highly expressed transcriptional module encoding proconvulsive cytokines and Toll-like receptor signalling genes. RNA sequencing analysis in a mouse model of TLE using 100 epileptic and 100 control hippocampi shows the proconvulsive module is preserved across-species, specific to the epileptic hippocampus and upregulated in chronic epilepsy. In the TLE patients, we map the trans-acting genetic control of this proconvulsive module to Sestrin 3 (SESN3), and demonstrate that SESN3 positively regulates the module in macrophages, microglia and neurons. Morpholino-mediated Sesn3 knockdown in zebrafish confirms the regulation of the transcriptional module, and attenuates chemically induced behavioural seizures in vivo