RNA-seq of human adipose tissue macrophage subtypes in obesity
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ABSTRACT: We profiled three prominent ATM subtypes from human visceral omental adipose tissue in obesity by RNA-seq. In the related manuscript, we evaluated differences in their signatures and their relationship to type 2 diabetes: Visceral (VAT) and subcutaneous (SAT) adipose tissue samples were collected from diabetic and non-diabetic obese subjects to evaluate cellular content and gene expression. VAT CD206+CD11c− ATMs were increased in diabetic subjects, scavenger receptor-rich with low intracellular lipids, secreted proinflammatory cytokines, and diverged significantly from two CD11c+ ATM subtypes, which were lipid-laden, lipid antigen presenting, and overlapped with monocyte signatures. Furthermore, diabetic VAT was enriched for CD206+CD11c− ATM and inflammatory signatures, scavenger receptors, and MHC II antigen presentation genes. VAT immunostaining found CD206+CD11c⁻ ATMs concentrated in vascularized lymphoid clusters adjacent to CD206⁻CD11c+ ATMs, while CD206+CD11c+ were distributed between adipocytes. Our results suggest ATM subtype-specific profiles that uniquely contribute to the phenotypic variation in obesity.
Project description:Adipose tissue stromal cells contribute to the regulation of adipose tissue in lean and obese states. Myeloid cells such as adipose tissue macrophages (ATMs) and dendritic cells (ATDCs) undergo both quantitative and qualitative changes with obesity. Due to similarity in markers the identify of adipose tissue dendritic cells and macrophages has been elusive. We have refined prior protocols to unambiguously discern ATM and ATDC in mice. We used microarrays to compare the profiles of ATMs and ATDC from gonadal adipose tissue from lean, obese, and formerly obese mice. We also isolated preadipocytes (PA) from lean and obese mice for comparison. Male C57Bl/6 mice were fed normal diet (ND) or high fat diet (HFD) for 16 weeks. Weight loss (WL) mice were switched from the HFD to ND for 8 weeks. RNA was purified from FACS sorted cell populations (live cells only) obtained from gonadal/epididymal adipose tissue depots. ATMs were defined as CD11c+ (CD45+CD64+ CD11c+) or CD11c- (CD45+CD64+ CD11c-) ATMs. ATDC were defined as CD64- CD11c+. PA were defined as CD31- CD45- Sca1+ PDGFRA+.
Project description:Context: It is not known whether biological differences reported between subcutaneous (SAT) and visceral (VAT) adipose tissue depots underlie the pathogenicity of visceral fat. Objective: We compared SAT and VAT gene expression according to obesity, visceral fat accumulation, insulin resistance and presence of the metabolic syndrome. Design: Subjects were assigned into 4 groups (lean, overweight, obese and obese with metabolic syndrome). Setting: Subjects were recruited at a university hospital. Patients: 32 women were included. Main Outcome Measures: Anthropometric measurements, euglycemic hyperinsulinemic clamps, blood analyses and computed tomography scans were performed and paired samples of SAT and VAT were obtained for DNA microarray-based gene expression profiling.
Project description:Obesity-associated insulin resistance is characterized by a state of chronic, low-grade inflammation that is associated with the accumulation of M1 proinflammatory macrophages in adipose tissue. Although different evidence explains the mechanisms linking the expansion of adipose tissue and adipose tissue macrophage (ATM) polarization, in the current study we investigated the concept of lipid-induced toxicity as the pathogenic link that could explain the trigger of this response. We addressed this question using isolated ATMs and adipocytes from genetic and diet-induced murine models of obesity. Through transcriptomic and lipidomic analysis, we created a model integrating transcript and lipid species networks simultaneously occurring in adipocytes and ATMs and their reversibility by thiazolidinedione treatment. We show that polarization of ATMs is associated with lipid accumulation and the consequent formation of foam cell–like cells in adipose tissue. Our study reveals that early stages of adipose tissue expansion are characterized by M2-polarized ATMs and that progressive lipid accumulation within ATMs heralds the M1 polarization, a macrophage phenotype associated with severe obesity and insulin resistance. Furthermore, rosiglitazone treatment, which promotes redistribution of lipids toward adipocytes and extends the M2 ATM polarization state, prevents the lipid alterations associated with M1 ATM polarization. Our data indicate that the M1 ATM polarization in obesity might be a macrophage-specific manifestation of a more general lipotoxic pathogenic mechanism. This indicates that strategies to optimize fat deposition and repartitioning toward adipocytes might improve insulin sensitivity by preventing ATM lipotoxicity and M1 polarization. 15 samples; 2 genotypes and 2 time points
Project description:The molecular background of mitochondrial dysfunction in adipose tissue of morbidly obese individuals and bariatric surgery-induced changes in adipose mitochondrial function remain incompletely understood. To evaluate the mechanisms behind the surgery-induced changes and differences between morbidly obese subjects and nonobese controls, we performed a LC-MS/MS proteomics analysis of abdominal subcutaneous (SAT) and visceral adipose tissue samples (VAT) collected from the bariatric surgery, SAT samples collected 6 months after surgery, and control SAT and VAT samples collected from baseline.
Project description:The peritoneal cavity is home to various immune cells. Previous studies have investigated the heterogenous nature of peritoneal myeloid mononuclear cells. However, up to this date, there are no clear criteria to distinguish peritoneal macrophages and dendritic cells (DCs). In the present study, we delineate the subsets of myeloid mononuclear cells in the mouse peritoneal cavity. Considering phenotypical, functional, and ontogenic features, peritoneal myeloid mononuclear cells are divided into 5 subsets: large peritoneal macrophages (LPMs), small peritoneal macrophages (SPMs), DCs, and 2 MHCII+CD11c+CD115+ subpopulations (i.e., MHCII+CD11c+CD115+CD14-CD206- and MHCII+CD11c+CD115+CD14+CD206+). Among them, 2 subsets of competent antigen presenting cells are demonstrated with distinct functional characteristics, one being DCs and the other being MHCII+CD11c+CD115+CD14-CD206- cells. DCs are able to promote fully activated T cells and superior in expanding cytokine producing inflammatory T cells, whereas MHCII+CD11c+CD115+CD14-CD206- cells generate partially activated T cells and possess a greater ability to induce regulatory T cells under TGF-β and retinoic acid conditions. While the development of DCs and MHCII+CD11c+CD115+CD14-CD206- cells are responsive to the treatment of FLT3L and GM-CSF, the numbers of LPMs, SPMs, and MHCII+CD11c+CD115+CD14+CD206+ cells are only influenced by the injection of GM-CSF. In addition, the analysis of transcriptomes reveals that the gene expression profile of MHCII+CD11c+CD115+CD14+CD206+ cells share high similarity with that of SPMs. Collectively, our study identifies 2 distinct subpopulations of MHCII+CD11c+CD115+ cells, (i) MHCII+CD11c+CD115+CD14-CD206- cells closely related to DCs and (ii) MHCII+CD11c+CD115+CD14+CD206+ cells to SPMs.
Project description:In mammals, expansion of adipose tissue mass induces accumulation of adipose tissue macrophages (ATMs). We isolated CD11c- (FB) and CD11c+ (FBC) perigonadal ATMs from SVCs of lean (C57BL/6J Lep +/+) and obese leptin-deficient (C57BL/6J Lep ob/ob) mice. We used expression microarrays to generate transcription profiles of perigonadal ATMs from lean (C57BL/6J Lep +/+) and obese (C57BL/6J Lep ob/ob) mice. Profiling purified FBs and FBCs, we identified 521 transcripts whose expression was differentially (nominal p-value < 0.01) expressed between FBs from lean and obese mice and 1509 genes whose expression was differentially (nominal p-value <0.01) expressed between FBC from lean and obese mice RNA was isolated from sorted FBC (F4/80+, CD11b+, CD11c+) cells and FB ( F4/80+, CD11b+, CD11c-) cells and using RNeasy micro-kits (Qiagen), using a PicoPure RNA isolation kit then amplified two-rounds. Labeled cRNA Mouse Genome 430 2.0 arrays (purified FB and FBC adipose tissue macrophages. There was a total of sixteen samples. FB and FBC populations were isolated from 4 lean and 4 obese mice.
Project description:To determine whether expression microRNAs (miRNAs) in visceral adipose tissue (VAT) is altered in human non-alcoholic fatty liver disease (NAFLD), we first performed miRNA profiling of VAT from subjects with (n=15, macroscopic liver fat >30%) or without (<5%, non-NAFLD, n=15) NAFLD. VAT miRNA expression profiles in the two groups were similar except for a single miRNA, miR-192*, which was 1.9-fold increased in NAFLD. We next overexpressed miR-192* in cultured human adipocytes, measured its effect on the cellular triglyceride content, and characterized these adipocytes by transcriptomic profiling followed by pathway analysis, qPCR and quantification of select cellular proteins.
Project description:Obesity-associated insulin resistance is characterized by a state of chronic, low-grade inflammation that is associated with the accumulation of M1 proinflammatory macrophages in adipose tissue. Although different evidence explains the mechanisms linking the expansion of adipose tissue and adipose tissue macrophage (ATM) polarization, in the current study we investigated the concept of lipid-induced toxicity as the pathogenic link that could explain the trigger of this response. We addressed this question using isolated ATMs and adipocytes from genetic and diet-induced murine models of obesity. Through transcriptomic and lipidomic analysis, we created a model integrating transcript and lipid species networks simultaneously occurring in adipocytes and ATMs and their reversibility by thiazolidinedione treatment. We show that polarization of ATMs is associated with lipid accumulation and the consequent formation of foam cell–like cells in adipose tissue. Our study reveals that early stages of adipose tissue expansion are characterized by M2-polarized ATMs and that progressive lipid accumulation within ATMs heralds the M1 polarization, a macrophage phenotype associated with severe obesity and insulin resistance. Furthermore, rosiglitazone treatment, which promotes redistribution of lipids toward adipocytes and extends the M2 ATM polarization state, prevents the lipid alterations associated with M1 ATM polarization. Our data indicate that the M1 ATM polarization in obesity might be a macrophage-specific manifestation of a more general lipotoxic pathogenic mechanism. This indicates that strategies to optimize fat deposition and repartitioning toward adipocytes might improve insulin sensitivity by preventing ATM lipotoxicity and M1 polarization.
Project description:We report that obese fat tissue of mice contain multiple distinct populations of adipose tissue macrophage (ATM) with unique transcriptomes and chromatin landscapes. Mouse Ly6c ATMs express genes that are adipogenic, while CD9 ATMs express pro-inflammatory genes under the control of activating transcription factors. Adoptive transfer of Ly6c ATMs into lean mice activates gene programs typical of normal adipocyte physiology. By contrast, adoptive transfer of CD9 ATMs drives gene expression that is characteristic of obesity.
Project description:We report that obese fat tissue of mice contain multiple distinct populations of adipose tissue macrophage (ATM) with unique transcriptomes and chromatin landscapes. Mouse Ly6c ATMs express genes that are adipogenic, while CD9 ATMs express pro-inflammatory genes under the control of activating transcription factors. Adoptive transfer of Ly6c ATMs into lean mice activates gene programs typical of normal adipocyte physiology. By contrast, adoptive transfer of CD9 ATMs drives gene expression that is characteristic of obesity.