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BACKGROUND: Trem2 (triggering receptor on myeloid cells 2), a surface lipid receptor, is expressed on foamy macrophages within atherosclerotic lesions and regulates cell survival, proliferation, and anti-inflammatory responses. Studies examining the role of Trem2 in atherosclerosis have shown that deletion of Trem2 leads to impaired foamy macrophage lipid uptake, proliferation, survival, and cholesterol efflux. Thus, we tested the hypothesis that administration of a Trem2 agonist antibody (AL002a) to atherogenic mice would enhance macrophage survival and decrease necrotic core formation to improve plaque stability. METHODS: To model a therapeutic intervention approach, atherosclerosis-prone mice (Ldlr [low-density lipoprotein receptor]-/-) were fed a high-fat diet for 8 weeks, then transitioned to treatment with AL002a or isotype control for an additional 8 weeks while continuing on a high-fat diet. RESULTS: AL002a-treated mice had increased lesion size in both the aortic root and whole mount aorta, which correlated with an expansion of plaque macrophage area. This expansion was due to increased macrophage survival and proliferation in plaques. Importantly, plaques from AL002a-treated mice showed improved features of plaque stability, including smaller necrotic cores, increased fibrous caps, and greater collagen deposition. Single-cell RNA sequencing of whole aorta suspensions from isotype- and AL002a-treated atherosclerotic mice revealed that Trem2 agonism dramatically altered foamy macrophage transcriptome. This included upregulation of oxidative phosphorylation and increased expression of collagen genes. In vitro studies validated that Trem2 agonism with AL002a promoted foamy macrophage oxidized low-density lipoprotein uptake, survival, and cholesterol efflux. CONCLUSIONS: Trem2 agonism expands atherosclerotic plaque macrophages by promoting cell survival and proliferation but improves features of plaque stability by rewiring foamy macrophage function to enhance cholesterol efflux and collagen deposition.
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Aterosclerosis , Modelos Animales de Enfermedad , Células Espumosas , Glicoproteínas de Membrana , Ratones Endogámicos C57BL , Ratones Noqueados , Placa Aterosclerótica , Receptores Inmunológicos , Animales , Receptores Inmunológicos/agonistas , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/genética , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Ratones , Aterosclerosis/patología , Aterosclerosis/metabolismo , Aterosclerosis/genética , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/prevención & control , Células Espumosas/metabolismo , Células Espumosas/patología , Células Espumosas/efectos de los fármacos , Masculino , Receptores de LDL/genética , Receptores de LDL/metabolismo , Receptores de LDL/deficiencia , Proliferación Celular/efectos de los fármacos , Dieta Alta en Grasa , Supervivencia Celular/efectos de los fármacos , Necrosis , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/prevención & controlRESUMEN
Glucocorticoid synthesis by adrenal glands (AGs) is regulated by the hypothalamic-pituitary-adrenal axis to facilitate stress responses when the host is exposed to stimuli. Recent studies implicate macrophages as potential steroidogenic regulators, but the molecular mechanisms by which AG macrophages exert such influence remain unclear. In this study, we investigated the role of AG macrophages in response to cold challenge or atherosclerotic inflammation as physiologic models of acute or chronic stress. Using single-cell RNA sequencing, we observed dynamic AG macrophage polarization toward classical activation and lipid-associated phenotypes following acute or chronic stimulation. Among transcriptional alterations induced in macrophages, triggering receptor expressed on myeloid cells 2 (Trem2) was highlighted because of its upregulation following stress. Conditional deletion of macrophage Trem2 revealed a protective role in stress responses. Mechanistically, Trem2 deletion led to increased AG macrophage death, abolished the TGF-ß-producing capacity of AG macrophages, and resulted in enhanced glucocorticoid production. In addition, enhanced glucocorticoid production was replicated by blockade of TGF-ß signaling. Together, these observations suggest that AG macrophages restrict steroidogenesis through Trem2 and TGF-ß, which opens potential avenues for immunotherapeutic interventions to resolve stress-related disorders.
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Glándulas Suprarrenales , Glucocorticoides , Macrófagos , Glicoproteínas de Membrana , Receptores Inmunológicos , Factor de Crecimiento Transformador beta , Animales , Macrófagos/metabolismo , Macrófagos/inmunología , Ratones , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Glucocorticoides/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/genética , Glándulas Suprarrenales/metabolismo , Masculino , Ratones Noqueados , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
Alzheimer's disease (AD) is a progressive neurodegenerative disease, and it is the most common cause of dementia worldwide. Recent genome-wide association studies (GWAS) identified TREM2 (triggering receptor expressed on myeloid cells 2) as one of the major risk factors for AD. TREM2 is a surface receptor expressed on microglia and largely mediates microglial functions and immune homeostasis in the brain. The functions of TREM2 in AD pathogenesis, including in the formation of the key pathology parenchymal amyloid-ß (Aß) plaques, have been investigated by introducing Trem2 deficiency in AD mouse models. However, the role of TREM2 in cerebrovascular amyloidosis, in particular cerebral amyloid angiopathy (CAA) remains unexplored. CAA features Aß deposition along the cerebral vessels, signifying an intersection between AD and vascular dysfunction. Using a well-characterized CAA-prone, transgenic mouse model of AD, Tg-SwDI (SwDI), we found that loss of TREM2 led to a marked increase in overall Aß load in the brain, but a dramatic decrease in CAA in microvessel-rich regions, along with reduced microglial association with CAA. Transcriptomic analysis revealed that in the absence of Trem2 , microglia were activated but trapped in transition to the fully reactive state. Like microglia, perivascular macrophages were activated with upregulation of cell junction related pathways in Trem2 -deficient SwDI mice. In addition, vascular mural cells and astrocytes exhibited distinct responses to Trem2 deficiency, contributing to the pathological changes in the brain of Trem2 -null SwDI mice. Our study provides the first evidence that TREM2 differentially modulates parenchymal and vascular Aß pathologies, which may have significant implications for both TREM2- and Aß-targeting therapies for AD.
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Pancreatic ductal adenocarcinoma (PDA) orchestrates a suppressive tumor microenvironment that fosters immunotherapy resistance. Tumor-associated macrophages (TAMs) are the principal immune cell infiltrating PDA and are heterogeneous. Here, by employing macrophage fate-mapping approaches and single-cell RNA sequencing, we show that monocytes give rise to most macrophage subsets in PDA. Tumor-specific CD4, but not CD8, T cells promote monocyte differentiation into MHCIIhi anti-tumor macrophages. By conditional major histocompatibility complex (MHC) class II deletion on monocyte-derived macrophages, we show that tumor antigen presentation is required for instructing monocyte differentiation into anti-tumor macrophages, promoting Th1 cells, abrogating Treg cells, and mitigating CD8 T cell exhaustion. Non-redundant IFNγ and CD40 promote MHCIIhi anti-tumor macrophages. Intratumoral monocytes adopt a pro-tumor fate indistinguishable from that of tissue-resident macrophages following loss of macrophage MHC class II or tumor-specific CD4 T cells. Thus, tumor antigen presentation by macrophages to CD4 T cells dictates TAM fate and is a major determinant of macrophage heterogeneity in cancer.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Monocitos , Linfocitos T CD4-Positivos , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/genética , Antígenos de Neoplasias , Antígenos de Histocompatibilidad Clase II , Microambiente Tumoral , Neoplasias PancreáticasRESUMEN
Atherosclerosis is driven by the expansion of cholesterol-loaded 'foamy' macrophages in the arterial intima. Factors regulating foamy macrophage differentiation and survival in plaque remain poorly understood. Here we show, using trajectory analysis of integrated single-cell RNA sequencing data and a genome-wide CRISPR screen, that triggering receptor expressed on myeloid cells 2 (Trem2) is associated with foamy macrophage specification. Loss of Trem2 led to a reduced ability of foamy macrophages to take up oxidized low-density lipoprotein (oxLDL). Myeloid-specific deletion of Trem2 showed an attenuation of plaque progression, even when targeted in established atherosclerotic lesions, and was independent of changes in circulating cytokines, monocyte recruitment or cholesterol levels. Mechanistically, we link Trem2-deficient macrophages with a failure to upregulate cholesterol efflux molecules, resulting in impaired proliferation and survival. Overall, we identify Trem2 as a regulator of foamy macrophage differentiation and atherosclerotic plaque growth and as a putative therapeutic target for atherosclerosis.
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Tissue-resident macrophages are present in all tissues where they perform homeostatic and immune surveillance functions. In many tissues, resident macrophages develop from embryonic progenitors, which mature into a self-maintaining population through local proliferation. However, tissue-resident macrophages can be supported by recruited monocyte-derived macrophages during scenarios such as tissue growth, infection, or sterile inflammation. Circulating blood monocytes arise from hematopoietic stem cell progenitors and possess unique gene profiles that support additional functions within the tissue. Determining cell origins (ontogeny) and cellular turnover within tissues has become important to understanding monocyte and macrophage contributions to tissue homeostasis and disease. Fate mapping, or lineage tracing, is a promising approach to tracking cells based on unique gene expression driving reporter systems, often downstream of a Cre-recombinase-mediated excision event, to express a fluorescent protein. This approach is typically deployed temporally with developmental stage, disease onset, or in association with key stages of inflammation resolution. Importantly, myeloid fate mapping can be combined with many emerging technologies, including single-cell RNA-sequencing and spatial imaging. The application of myeloid cell fate mapping approaches has allowed for impactful discoveries regarding myeloid ontogeny, tissue residency, and monocyte fate within disease models. This protocol outline will discuss a variety of myeloid fate mapping approaches, including constitutive and inducible labeling approaches in adult and embryo tissues. This article outlines basic approaches and models used in mice for fate mapping macrophages. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Adult Fate Mapping Basic Protocol 2: Embryonic Fate Mapping.
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Macrófagos , Monocitos , Animales , Diferenciación Celular/fisiología , Células Madre Hematopoyéticas , Inflamación/metabolismo , Ratones , Monocitos/metabolismoRESUMEN
Despite the ubiquitous function of macrophages across the body, the diversity, origin, and function of adrenal gland macrophages remain largely unknown. We define the heterogeneity of adrenal gland immune cells using single-cell RNA sequencing and use genetic models to explore the developmental mechanisms yielding macrophage diversity. We define populations of monocyte-derived and embryonically seeded adrenal gland macrophages and identify a female-specific subset with low major histocompatibility complex (MHC) class II expression. In adulthood, monocyte recruitment dominates adrenal gland macrophage maintenance in female mice. Adrenal gland macrophage sub-tissular distribution follows a sex-dimorphic pattern, with MHC class IIlow macrophages located at the cortico-medullary junction. Macrophage sex dimorphism depends on the presence of the cortical X-zone. Adrenal gland macrophage depletion results in altered tissue homeostasis, modulated lipid metabolism, and decreased local aldosterone production during stress exposure. Overall, these data reveal the heterogeneity of adrenal gland macrophages and point toward sex-restricted distribution and functions of these cells.
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Glándulas Suprarrenales , Macrófagos , Monocitos , Caracteres Sexuales , Glándulas Suprarrenales/metabolismo , Animales , Femenino , Antígenos de Histocompatibilidad Clase II/genética , Recuento de Leucocitos , Macrófagos/metabolismo , Masculino , RatonesRESUMEN
Arterial stiffness is an important biomarker for many cardiovascular diseases. Shear wave elastography is a recent technique aimed at estimating local arterial stiffness using guided wave inversion (GWI), i.e. matching the computed and measured wave dispersion. This paper develops and validates a new GWI approach by synthesizing various recent observations and algorithms: (a) refinements to signal processing to obtain more accurate experimental dispersion curves; (b) an efficient forward model to compute theoretical dispersion curves for immersed, incompressible cylindrical waveguides; (c) an optimization framework based on the recent observation that the measured dispersion curve is multimodal, i.e. it matches for not one but two different wave modes in two different frequency ranges. The resulting inversion approach is validated using extensive experimental data from rubber tube phantoms, not only for modulus estimation but also to simultaneously estimate modulus and wall thickness. The observations indicate that the modulus estimates are best performed with the information on wall thickness. The approach, which takes less than half a minute to run, is shown to be accurate, with the modulus estimated with less than 4% error for 70% of the experiments.
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Diagnóstico por Imagen de Elasticidad , Rigidez Vascular , Algoritmos , Módulo de Elasticidad , Fantasmas de ImagenRESUMEN
While macrophages are among the most abundant immune cell type found within primary and metastatic mammary tumors, how their complexity and heterogeneity change with metastatic progression remains unknown. Here, macrophages were isolated from the lungs of mice bearing orthotopic mammary tumors for single-cell RNA sequencing (scRNA-seq). Seven distinct macrophage clusters were identified, including populations exhibiting enhanced differential expression of genes related to antigen presentation (H2-Aa, Cd74), cell cycle (Stmn1, Cdk1), and interferon signaling (Isg15, Ifitm3). Interestingly, one cluster demonstrated a profile concordant with lipid-associated macrophages (Lgals3, Trem2). Compared with nontumor-bearing controls, the number of these cells per gram of tissue was significantly increased in lungs from tumor-bearing mice, with the vast majority costaining positively with the alveolar macrophage marker Siglec-F. Enrichment of genes implicated in pathways related to lipid metabolism as well extracellular matrix remodeling and immunosuppression was observed. In addition, these cells displayed reduced capacity for phagocytosis. Collectively, these findings highlight the diversity of macrophages present within metastatic lesions and characterize a lipid-associated macrophage subset previously unidentified in lung metastases. SIGNIFICANCE: scRNA-seq of macrophages isolated from lung metastases reveals extensive macrophage heterogeneity and identifies a novel subpopulation enriched for genes involved in lipid metabolism, extracellular matrix remodeling, and immunosuppression.