A lipid-associated macrophage lineage rewires the spatial landscape of adipose tissue in early obesity.

Adipose tissue macrophage (ATM) infiltration is associated with adipose tissue dysfunction and insulin resistance in mice and humans. Recent single-cell data highlight increased ATM heterogeneity in obesity but do not provide a spatial context for ATM phenotype dynamics. We integrated single-cell RNA-Seq, spatial transcriptomics, and imaging of murine adipose tissue in a time course study of diet-induced obesity. Overall, proinflammatory immune cells were predominant in early obesity, whereas nonresident antiinflammatory ATMs predominated in chronic obesity. A subset of these antiinflammatory ATMs were transcriptomically intermediate between monocytes and mature lipid-associated macrophages (LAMs) and were consistent with a LAM precursor (pre-LAM). Pre-LAMs were spatially associated with early obesity crown-like structures (CLSs), which indicate adipose tissue dysfunction. Spatial data showed colocalization of ligand-receptor transcripts related to lipid signaling among monocytes, pre-LAMs, and LAMs, including Apoe, Lrp1, Lpl, and App. Pre-LAM expression of these ligands in early obesity suggested signaling to LAMs in the CLS microenvironment. Our results refine understanding of ATM diversity and provide insight into the dynamics of the LAM lineage during development of metabolic disease.

Rearrangement of T Cell genome architecture regulates GVHD.

WAPL, cohesin's DNA release factor, regulates three-dimensional (3D) chromatin architecture. The 3D chromatin structure and its relevance to mature T cell functions is not well understood. We show that in vivo lymphopenic expansion, and alloantigen-driven proliferation, alters the 3D structure and function of the genome in mature T cells. Conditional deletion of WAPL, cohesin's DNA release factor, in T cells reduced long-range genomic interactions and altered chromatin A/B compartments and interactions within topologically associating domains (TADs) of the chromatin in T cells at baseline. WAPL deficiency in T cells reduced loop extensions, changed expression of cell cycling genes and reduced proliferation following in vitro and in vivo stimulation, and reduced severity of graft-versus-host disease (GVHD) following experimental allogeneic hematopoietic stem cell transplantation. These data collectively characterize 3D genomic architecture of T cells in vivo and demonstrate biological and clinical implications for its disruption by cohesin release factor WAPL.

Human CD206+ macrophages associate with diabetes and adipose tissue lymphoid clusters.

Increased adipose tissue macrophages (ATMs) correlate with metabolic dysfunction in humans and are causal in development of insulin resistance in mice. Recent bulk and single-cell transcriptomics studies reveal a wide spectrum of gene expression signatures possible for macrophages that depends on context, but the signatures of human ATM subtypes are not well defined in obesity and diabetes. We profiled 3 prominent ATM subtypes from human adipose tissue in obesity and determined their relationship to type 2 diabetes. Visceral adipose tissue (VAT) and s.c. adipose tissue (SAT) samples were collected from diabetic and nondiabetic obese participants to evaluate cellular content and gene expression. VAT CD206+CD11c- ATMs were increased in diabetic participants, were scavenger receptor-rich with low intracellular lipids, secreted proinflammatory cytokines, and diverged significantly from 2 CD11c+ ATM subtypes, which were lipid-laden, were 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 show ATM subtype-specific profiles that uniquely contribute to the phenotypic variation in obesity.

Myeloid interleukin-4 receptor α is essential in postmyocardial infarction healing by regulating inflammation and fibrotic remodeling.

Interleukin-4 receptor α (IL4Rα) signaling plays an important role in cardiac remodeling during myocardial infarction (MI). However, the target cell type(s) of IL4Rα signaling during this remodeling remains unclear. Here, we investigated the contribution of endogenous myeloid-specific IL4Rα signaling in cardiac remodeling post-MI. We established a murine myeloid-specific IL4Rα knockout (MyIL4RαKO) model with LysM promoter-driven Cre recombination. Macrophages from MyIL4RαKO mice showed significant downregulation of alternatively activated macrophage markers but an upregulation of classical activated macrophage markers both in vitro and in vivo, indicating the successful inactivation of IL4Rα signaling in macrophages. To examine the role of myeloid IL4Rα during MI, we subjected MyIL4RαKO and littermate floxed control (FC) mice to MI. We found that cardiac function was significantly impaired as a result of myeloid-specific IL4Rα deficiency. This deficiency resulted in a dysregulated inflammatory response consisting of decreased production of anti-inflammatory cytokines. Myeloid IL4Rα deficiency also led to reduced collagen 1 deposition and an imbalance of matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs), with upregulated MMPs and downregulated TIMPs, which resulted in insufficient fibrotic remodeling. In conclusion, this study identifies that myeloid-specific IL4Rα signaling regulates inflammation and fibrotic remodeling during MI. Therefore, myeloid-specific activation of IL4Rα signaling could offer protective benefits after MI.NEW & NOTEWORTHY This study showed, for the first time, the role of endogenous IL4Rα signaling in myeloid cells during cardiac remodeling and the underlying mechanisms. We identified myeloid cells are the critical target cell types of IL4Rα signaling during cardiac remodeling post-MI. Deficiency of myeloid IL4Rα signaling causes deteriorated cardiac function post-MI, due to dysregulated inflammation and insufficient fibrotic remodeling. This study sheds light on the potential of activating myeloid-specific IL4Rα signaling to modify remodeling post-MI. This brings hope to patients with MI and diminishes side effects by cell type-specific instead of whole body treatment.

Frontline Science: Rapid adipose tissue expansion triggers unique proliferation and lipid accumulation profiles in adipose tissue macrophages.

Obesity-related changes in adipose tissue leukocytes, in particular adipose tissue macrophages (ATMs) and dendritic cells (ATDCs), are implicated in metabolic inflammation, insulin resistance, and altered regulation of adipocyte function. We evaluated stromal cell and white adipose tissue (WAT) expansion dynamics with high fat diet (HFD) feeding for 3-56 days, quantifying ATMs, ATDCs, endothelial cells (ECs), and preadipocytes (PAs) in visceral epididymal WAT and subcutaneous inguinal WAT. To better understand mechanisms of the early response to obesity, we evaluated ATM proliferation and lipid accumulation. ATMs, ATDCs, and ECs increased with rapid WAT expansion, with ATMs derived primarily from a CCR2-independent resident population. WAT expansion stimulated proliferation in resident ATMs and ECs, but not CD11c+ ATMs or ATDCs. ATM proliferation was unperturbed in Csf2- and Rag1-deficient mice with WAT expansion. Additionally, ATM apoptosis decreased with WAT expansion, and proliferation and apoptosis reverted to baseline with weight loss. Adipocytes reached maximal hypertrophy at 28 days of HFD, coinciding with a plateau in resident ATM accumulation and the appearance of lipid-laden CD11c+ ATMs in visceral epididymal WAT. ATM increases were proportional to tissue expansion and adipocyte hypertrophy, supporting adipocyte-mediated regulation of resident ATMs. The appearance of lipid-laden CD11c+ ATMs at peak adipocyte size supports a role in responding to ectopic lipid accumulation within adipose tissue. In contrast, ATDCs increase independently of proliferation and may be derived from circulating precursors. These changes precede and establish the setting in which large-scale adipose tissue infiltration of CD11c+ ATMs, inflammation, and adipose tissue dysfunction contributes to insulin resistance.

Adipocyte hypertrophy-hyperplasia balance contributes to weight loss after bariatric surgery.

Predictors of weight loss responses are not well-defined. We hypothesized that adipose tissue phenotypic features related to remodeling would be associated with bariatric surgery weight loss responses. Visceral and subcutaneous adipose tissues collected from patients during bariatric surgery were studied with flow cytometry, immunohistochemistry, and QRTPCR, and results correlated with weight loss outcomes. Age, male sex, and a diagnosis of type 2 diabetes were associated with less weight loss. Adipocyte size was increased and preadipocyte frequency was decreased in visceral adipose tissue from diabetic subjects. Decreased adipose tissue preadipocyte frequency was associated with less weight loss in women but not men. These data suggest that phenotypic features of adipose tissue remodeling may predict responses to weight loss interventions.

Differentiation and Metabolic Interrogation of Human Adipocytes.

Adipocytes differentiated from preadipocytes provide a valuable model for the study of human adipocyte metabolism. We describe methods for isolation of human stromal vascular cells, expansion of preadipocytes, differentiation into mature adipocytes, and in vitro metabolic interrogation of adipocytes.

Diabetes-Specific Regulation of Adipocyte Metabolism by the Adipose Tissue Extracellular Matrix.

Context: The role of the extracellular matrix (ECM) in regulating adipocyte metabolism in the context of metabolic disease is poorly defined. Objective: The objective of this study was to define the metabolic phenotype of adipocytes associated with human diabetes (DM) and the role of the ECM in regulating adipocyte metabolism. Design: Adipose tissues from obese patients were studied in standard 2-dimensional (2D) cell culture and an in vitro model of decellularized adipose tissue ECM repopulated with human adipocytes, and results were correlated with DM status. Setting: This study was conducted at the Academic University Medical Center and Veteran's Administration Hospital. Patients: Seventy patients with morbid obesity undergoing bariatric surgery were included in the study. Interventions: Visceral and subcutaneous adipose tissues were collected at the time of bariatric surgery. Outcome measures: This study used metabolic assays for glucose uptake, lipolysis, and lipogenesis in adipocytes in 2D cell culture and 3-dimensional ECM culture. Results: Adipocytes from subjects with DM manifest decreased glucose uptake and decreased lipolysis in 2D culture. ECM supports differentiation of mature adipocytes and recapitulates DM-specific differences in adipocyte metabolism observed in 2D culture. ECM from subjects without DM partially rescues glucose uptake and lipolytic defects in adipocytes from subjects with DM, whereas ECM from subjects with DM impairs glucose uptake in adipocytes from subjects without DM. Conclusions: DM is associated with adipocyte metabolic dysfunction. The ECM regulates adipocyte metabolism. Nondiabetic ECM rescues metabolic dysfunction in DM adipocytes, whereas DM ECM imparts features of metabolic dysfunction to nondiabetic adipocytes. These findings suggest the ECM as a target for manipulating adipose tissue metabolism.

Macrophage Proliferation Sustains Adipose Tissue Inflammation in Formerly Obese Mice.

Obesity causes dramatic proinflammatory changes in the adipose tissue immune environment, but relatively little is known regarding how this inflammation responds to weight loss (WL). To understand the mechanisms by which meta-inflammation resolves during WL, we examined adipose tissue leukocytes in mice after withdrawal of a high-fat diet. After 8 weeks of WL, mice achieved similar weights and glucose tolerance values as age-matched lean controls but showed abnormal insulin tolerance. Despite fat mass normalization, total and CD11c+ adipose tissue macrophage (ATM) content remained elevated in WL mice for up to 6 months and was associated with persistent fibrosis in adipose tissue. ATMs in formerly obese mice demonstrated a proinflammatory profile, including elevated expression of interferon-γ, tumor necrosis factor-α, and interleukin-1ß. T-cell-deficient Rag1-/- mice showed a degree of ATM persistence similar to that in WT mice, but with reduced inflammatory gene expression. ATM proliferation was identified as the predominant mechanism by which ATMs are retained in adipose tissue with WL. Our study suggests that WL does not completely resolve obesity-induced ATM activation, which may contribute to the persistent adipose tissue damage and reduced insulin sensitivity observed in formerly obese mice.

Adipose Tissue Dendritic Cells Are Independent Contributors to Obesity-Induced Inflammation and Insulin Resistance.

Dynamic changes of adipose tissue leukocytes, including adipose tissue macrophage (ATM) and adipose tissue dendritic cells (ATDCs), contribute to obesity-induced inflammation and metabolic disease. However, clear discrimination between ATDC and ATM in adipose tissue has limited progress in the field of immunometabolism. In this study, we use CD64 to distinguish ATM and ATDC, and investigated the temporal and functional changes in these myeloid populations during obesity. Flow cytometry and immunostaining demonstrated that the definition of ATM as F4/80+CD11b+ cells overlaps with other leukocytes and that CD45+CD64+ is specific for ATM. The expression of core dendritic cell genes was enriched in CD11c+CD64- cells (ATDC), whereas core macrophage genes were enriched in CD45+CD64+ cells (ATM). CD11c+CD64- ATDCs expressed MHC class II and costimulatory receptors, and had similar capacity to stimulate CD4+ T cell proliferation as ATMs. ATDCs were predominantly CD11b+ conventional dendritic cells and made up the bulk of CD11c+ cells in adipose tissue with moderate high-fat diet exposure. Mixed chimeric experiments with Ccr2-/- mice demonstrated that high-fat diet-induced ATM accumulation from monocytes was dependent on CCR2, whereas ATDC accumulation was less CCR2 dependent. ATDC accumulation during obesity was attenuated in Ccr7-/- mice and was associated with decreased adipose tissue inflammation and insulin resistance. CD45+CD64+ ATM and CD45+CD64-CD11c+ ATDCs were identified in human obese adipose tissue and ATDCs were increased in s.c. adipose tissue compared with omental adipose tissue. These results support a revised strategy for unambiguous delineation of ATM and ATDC, and suggest that ATDCs are independent contributors to adipose tissue inflammation during obesity.

Prosurvival Factors Improve Functional Engraftment of Myogenically Converted Dermal Cells into Dystrophic Skeletal Muscle.

In Duchenne muscular dystrophy (DMD) and other muscle wasting disorders, cell therapies are a promising route for promoting muscle regeneration by supplying a functional copy of the missing dystrophin gene and contributing new muscle fibers. The clinical application of cell-based therapies is resource intensive, and it will therefore be necessary to address key limitations that reduce cell engraftment into muscle tissue. A pressing issue is poor donor cell survival following transplantation, which in preclinical studies limits the ability to effectively test the impact of cell-based therapy on whole muscle function. We, therefore, sought to improve engraftment and the functional impact of in vivo myogenically converted dermal fibroblasts (dFbs) using a prosurvival cocktail (PSC) that includes heat shock followed by treatment with insulin-like growth factor-1, a caspase inhibitor, a Bcl-XL peptide, a KATP channel opener, basic fibroblast growth factor, Matrigel, and cyclosporine A. Advantages of dFbs include compatibility with the autologous setting, ease of isolation, and greater proliferative potential than DMD satellite cells. dFbs expressed tamoxifen-inducible MyoD and carried a mini-dystrophin gene driven by a muscle-specific promoter. After transplantation into muscles of mdx mice, a 70% reduction in donor cells was observed by day 5, and a 94% reduction by day 28. However, treatment with PSC gave a nearly three-fold increase in donor cells in early engraftment, and greatly increased the number of donor-contributed muscle fibers and total engrafted area in transplanted muscles. Furthermore, dystrophic muscles that received dFbs with PSC displayed reduced injury with eccentric contractions and an increase in maximum isometric force. Thus, enhancing survival of myogenic cells increases engraftment and improves structure and function of dystrophic muscle.

Adipose tissue fibrosis, hypertrophy, and hyperplasia: Correlations with diabetes in human obesity.

OBJECTIVE: The relationship between adipose tissue fibrosis, adipocyte hypertrophy, and preadipocyte hyperplasia in the context of obesity and the correlation of these tissue-based phenomena with systemic metabolic disease are poorly defined. The goal of this study was to clarify the relationship between adipose tissue fibrosis, adipocyte hypertrophy, and preadipocyte hyperplasia in human obesity and determine the correlation of these adipose-tissue based phenomena with diabetes. METHODS: Visceral and subcutaneous adipose tissues from humans with obesity collected during bariatric surgery were studied with QRTPCR, immunohistochemistry, and flow cytometry for expression of collagens and fibrosis-related proteins, adipocyte size, and preadipocyte frequency. Results were correlated with clinical characteristics including diabetes status. RESULTS: Fibrosis was decreased, hypertrophy was increased, and preadipocyte frequency and fibrotic gene expression were decreased in adipose tissues from diabetic subjects compared to non-diabetic subjects. These differences were greater in visceral compared to subcutaneous adipose tissue. CONCLUSIONS: These data are consistent with the hypothesis that adipose tissue fibrosis in the context of human obesity limits adipocyte hypertrophy and is associated with a reciprocal increase in adipocyte hyperplasia, with beneficial effects on systemic metabolism. These findings suggest adipose tissue fibrosis as a potential target for manipulation of adipocyte metabolism.

Extraocular muscle satellite cells are high performance myo-engines retaining efficient regenerative capacity in dystrophin deficiency.

Extraocular muscles (EOMs) are highly specialized skeletal muscles that originate from the head mesoderm and control eye movements. EOMs are uniquely spared in Duchenne muscular dystrophy and animal models of dystrophin deficiency. Specific traits of myogenic progenitors may be determinants of this preferential sparing, but very little is known about the myogenic cells in this muscle group. While satellite cells (SCs) have long been recognized as the main source of myogenic cells in adult muscle, most of the knowledge about these cells comes from the prototypic limb muscles. In this study, we show that EOMs, regardless of their distinctive Pax3-negative lineage origin, harbor SCs that share a common signature (Pax7(+), Ki67(-), Nestin-GFP(+), Myf5(nLacZ+), MyoD-positive lineage origin) with their limb and diaphragm somite-derived counterparts, but are remarkably endowed with a high proliferative potential as revealed in cell culture assays. Specifically, we demonstrate that in adult as well as in aging mice, EOM SCs possess a superior expansion capacity, contributing significantly more proliferating, differentiating and renewal progeny than their limb and diaphragm counterparts. These robust growth and renewal properties are maintained by EOM SCs isolated from dystrophin-null (mdx) mice, while SCs from muscles affected by dystrophin deficiency (i.e., limb and diaphragm) expand poorly in vitro. EOM SCs also retain higher performance in cell transplantation assays in which donor cells were engrafted into host mdx limb muscle. Collectively, our study provides a comprehensive picture of EOM myogenic progenitors, showing that while these cells share common hallmarks with the prototypic SCs in somite-derived muscles, they distinctively feature robust growth and renewal capacities that warrant the title of high performance myo-engines and promote consideration of their properties for developing new approaches in cell-based therapy to combat skeletal muscle wasting.

Emerging strategies for cell and gene therapy of the muscular dystrophies.

The muscular dystrophies are a heterogeneous group of over 40 disorders that are characterised by muscle weakness and wasting. The most common are Duchenne muscular dystrophy and Becker muscular dystrophy, which result from mutations within the gene encoding dystrophin; myotonic dystrophy type 1, which results from an expanded trinucleotide repeat in the myotonic dystrophy protein kinase gene; and facioscapulohumeral dystrophy, which is associated with contractions in the subtelomeric region of human chromosome 1. Currently the only treatments involve clinical management of symptoms, although several promising experimental strategies are emerging. These include gene therapy using adeno-associated viral, lentiviral and adenoviral vectors and nonviral vectors, such as plasmid DNA. Exon-skipping and cell-based therapies have also shown promise in the effective treatment and regeneration of dystrophic muscle. The availability of numerous animal models for Duchenne muscular dystrophy has enabled extensive testing of a wide range of therapeutic approaches for this type of disorder. Consequently, we focus here on the therapeutic developments for Duchenne muscular dystrophy as a model of the types of approaches being considered for various types of dystrophy. We discuss the advantages and limitations of each therapeutic strategy, as well as prospects and recent successes in the context of future clinical applications.