RESUMEN
Resident macrophages orchestrate homeostatic, inflammatory, and reparative activities. It is appreciated that different tissues instruct specialized macrophage functions. However, individual tissues contain heterogeneous subpopulations, and how these subpopulations are related is unclear. We asked whether common transcriptional and functional elements could reveal an underlying framework across tissues. Using single-cell RNA sequencing and random forest modeling, we observed that four genes could predict three macrophage subsets that were present in murine heart, liver, lung, kidney, and brain. Parabiotic and genetic fate mapping studies revealed that these core markers predicted three unique life cycles across 17 tissues. TLF+ (expressing TIMD4 and/or LYVE1 and/or FOLR2) macrophages were maintained through self-renewal with minimal monocyte input; CCR2+ (TIMD4−LYVE1−FOLR2−) macrophages were almost entirely replaced by monocytes, and MHC-IIhi macrophages (TIMD4−LYVE1−FOLR2−CCR2−), while receiving modest monocyte contribution, were not continually replaced. Rather, monocyte-derived macrophages contributed to the resident macrophage population until they reached a defined upper limit after which they did not outcompete pre-existing resident macrophages. Developmentally, TLF+ macrophages were first to emerge in the yolk sac and early fetal organs. Fate mapping studies in the mouse and human single-cell RNA sequencing indicated that TLF+ macrophages originated from both yolk sac and fetal monocyte precursors. Furthermore, TLF+ macrophages were the most transcriptionally conserved subset across mouse tissues and between mice and humans, despite organ- and species-specific transcriptional differences. Here, we define the existence of three murine macrophage subpopulations based on common life cycle properties and core gene signatures and provide a common starting point to understand tissue macrophage heterogeneity.
Asunto(s)
Receptor 2 de Folato/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Macrófagos/inmunología , Proteínas de la Membrana/inmunología , Receptores CCR2/inmunología , Proteínas de Transporte Vesicular/inmunología , Animales , Estadios del Ciclo de Vida/inmunología , Activación de Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores CCR2/deficienciaRESUMEN
Hypertension affects one-third of the world's population, leading to cardiac dysfunction that is modulated by resident and recruited immune cells. Cardiomyocyte growth and increased cardiac mass are essential to withstand hypertensive stress; however, whether immune cells are involved in this compensatory cardioprotective process is unclear. In normotensive animals, single-cell transcriptomics of fate-mapped self-renewing cardiac resident macrophages (RMs) revealed transcriptionally diverse cell states with a core repertoire of reparative gene programs, including high expression of insulin-like growth factor-1 (Igf1). Hypertension drove selective in situ proliferation and transcriptional activation of some cardiac RM states, directly correlating with increased cardiomyocyte growth. During hypertension, inducible ablation of RMs or selective deletion of RM-derived Igf1 prevented adaptive cardiomyocyte growth, and cardiac mass failed to increase, which led to cardiac dysfunction. Single-cell transcriptomics identified a conserved IGF1-expressing macrophage subpopulation in human cardiomyopathy. Here we defined the absolute requirement of RM-produced IGF-1 in cardiac adaptation to hypertension.
Asunto(s)
Adaptación Fisiológica/fisiología , Hipertensión/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Macrófagos/metabolismo , Remodelación Ventricular/fisiología , Animales , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Humanos , Hipertensión/complicaciones , Hipertensión/inmunología , Lactante , Masculino , Ratones , Persona de Mediana Edad , Miocardio/inmunología , Miocardio/metabolismo , Miocardio/patologíaRESUMEN
In the version of this article initially published, the equal contribution of the third author was omitted. The footnote links for that author should be "Sara Nejat1,11" and the correct statement is as follows: "11These authors contributed equally: Sarah A. Dick, Jillian A. Macklin, Sara Nejat." The error has been corrected in the HTML and PDF versions of the article.
RESUMEN
Macrophages promote both injury and repair after myocardial infarction, but discriminating functions within mixed populations remains challenging. Here we used fate mapping, parabiosis and single-cell transcriptomics to demonstrate that at steady state, TIMD4+LYVE1+MHC-IIloCCR2- resident cardiac macrophages self-renew with negligible blood monocyte input. Monocytes partially replaced resident TIMD4-LYVE1-MHC-IIhiCCR2- macrophages and fully replaced TIMD4-LYVE1-MHC-IIhiCCR2+ macrophages, revealing a hierarchy of monocyte contribution to functionally distinct macrophage subsets. Ischemic injury reduced TIMD4+ and TIMD4- resident macrophage abundance, whereas CCR2+ monocyte-derived macrophages adopted multiple cell fates within infarcted tissue, including those nearly indistinguishable from resident macrophages. Recruited macrophages did not express TIMD4, highlighting the ability of TIMD4 to track a subset of resident macrophages in the absence of fate mapping. Despite this similarity, inducible depletion of resident macrophages using a Cx3cr1-based system led to impaired cardiac function and promoted adverse remodeling primarily within the peri-infarct zone, revealing a nonredundant, cardioprotective role of resident cardiac macrophages.
Asunto(s)
Macrófagos/fisiología , Infarto del Miocardio/inmunología , Miocardio/patología , Animales , Receptor 1 de Quimiocinas CX3C/metabolismo , Diferenciación Celular , Linaje de la Célula , Autorrenovación de las Células , Perfilación de la Expresión Génica , Antígenos de Histocompatibilidad Clase II/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Parabiosis , Receptores CCR2/genética , Receptores CCR2/metabolismo , Análisis de la Célula Individual , Remodelación Ventricular , Proteínas de Transporte Vesicular/metabolismoRESUMEN
The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small - but key - populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2-3 h.
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Células Dendríticas/inmunología , Corazón/fisiología , Macrófagos/inmunología , Animales , Citometría de Flujo , Humanos , RatonesRESUMEN
BACKGROUND: von Hippel-Lindau (vHL) syndrome is a rare autosomal-dominant disorder that confers a lifelong risk for developing both benign and malignant tumours in multiple organs. Recent evidence suggests that vHL may exhibit genetic anticipation (GA). The aim of this study was to determine if GA occurs in vHL, and if telomere shortening may be a factor in GA. METHODS: A retrospective chart review of vHL families seen at The Hospital for Sick Children between 1984 and 2016 was performed. Age of onset (AOO, defined as the age of first physician-diagnosed vHL-related manifestation) was confirmed for 96 patients from 20 unrelated families (80 clinically affected and 16 unaffected carriers). Flow-FISH(flow cytometry sorting of cells whose telomeres are labeled by Fluorescence In Situ Hybridization) was used to measure mean telomere length of six white blood cell subtypes from 14 known VHL pathogenic variant carriers. RESULTS: The median AOO for generations I, II and III were 32.5, 22.5 and 12.0 years, respectively. The differences in the AOO between generations were highly significant using a Cox proportional hazards model (P=6.00×10-12). Telomere lengths were significantly different for granulocytes and natural killer lymphocytes of patients with vHL compared with age-matched controls. For six vHL parent-child pairs, median white blood cell telomere lengths between parent and child were not significantly different. CONCLUSIONS: Our results suggest that vHL telomere abnormalities may be primarily somatic in origin rather than a cause of GA. As tumour development exhibits GA in our cohort, vHL surveillance guidelines may need to account for a patient's generational position within a vHL pedigree.
Asunto(s)
Anticipación Genética , Predisposición Genética a la Enfermedad , Acortamiento del Telómero/genética , Enfermedad de von Hippel-Lindau/genética , Adolescente , Adulto , Niño , Preescolar , Femenino , Citometría de Flujo , Granulocitos/metabolismo , Granulocitos/patología , Heterocigoto , Humanos , Hibridación Fluorescente in Situ , Lactante , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/patología , Masculino , Linaje , Telómero/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Adulto Joven , Enfermedad de von Hippel-Lindau/patologíaRESUMEN
BACKGROUND: To expand the current knowledge of DICER1 syndrome and to propose criteria for genetic testing based on experience at a pediatric tertiary care center. PROCEDURE: This study involved a retrospective chart review of the 78 patients (47 probands and 31 family members) seen in the Cancer Genetics Program at The Hospital for Sick Children (SickKids) who were offered genetic testing for DICER1. RESULTS: Of 47 probands offered genetic testing for DICER1, 46 pursued testing: 11 (23.9%) carried a pathogenic variant and one proband (2.1%) carried a missense variant of uncertain significance with evidence for pathogenicity. Thirty-one family members of variant-positive probands were offered testing: eight of the 25 who agreed to testing carried their familial variant (32.0%). Overall, 20 patients were identified to have a variant in DICER1 (eight males, 12 females). Of these, 13 (65.0%) presented with clinical manifestations associated with the syndrome. The most common lesions were pleuropulmonary blastoma (PPB) (five of 20 patients, 25.0%) and pineoblastoma (three of 20 patients, 15.0%). The average age at which individuals were diagnosed with a primary neoplasm was 5.2 years (range 0.8-20 years, median 3.0). Surveillance at our institution, with a median follow-up time of 23 months, has identified PPB in two asymptomatic individuals. These lesions were identified at early stages, thus potentially reducing treatment-related morbidity and mortality. CONCLUSION: This study further delineates the DICER1 syndrome phenotype and demonstrates the feasibility of a DICER1 syndrome surveillance protocol for the early detection of tumors.
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Neoplasias Encefálicas/genética , Síndromes Neoplásicos Hereditarios/genética , Glándula Pineal , Pinealoma/genética , Blastoma Pulmonar/genética , Adolescente , Adulto , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Niño , Preescolar , Femenino , Estudios de Seguimiento , Humanos , Lactante , Recién Nacido , Masculino , Síndromes Neoplásicos Hereditarios/mortalidad , Síndromes Neoplásicos Hereditarios/patología , Pinealoma/mortalidad , Pinealoma/patología , Blastoma Pulmonar/mortalidad , Blastoma Pulmonar/patologíaRESUMEN
Innate and adaptive immune cells modulate heart failure pathogenesis during viral myocarditis, yet their identities and functions remain poorly defined. We utilized a combination of genetic fate mapping, parabiotic, transcriptional, and functional analyses and demonstrated that the heart contained two major conventional dendritic cell (cDC) subsets, CD103+ and CD11b+, which differentially relied on local proliferation and precursor recruitment to maintain their tissue residency. Following viral infection of the myocardium, cDCs accumulated in the heart coincident with monocyte infiltration and loss of resident reparative embryonic-derived cardiac macrophages. cDC depletion abrogated antigen-specific CD8+ T cell proliferative expansion, transforming subclinical cardiac injury to overt heart failure. These effects were mediated by CD103+ cDCs, which are dependent on the transcription factor BATF3 for their development. Collectively, our findings identified resident cardiac cDC subsets, defined their origins, and revealed an essential role for CD103+ cDCs in antigen-specific T cell responses during subclinical viral myocarditis.
Asunto(s)
Antígenos CD/análisis , Infecciones por Cardiovirus/complicaciones , Células Dendríticas/inmunología , Virus de la Encefalomiocarditis , Insuficiencia Cardíaca/prevención & control , Cadenas alfa de Integrinas/análisis , Miocarditis/complicaciones , Animales , Antígeno CD11b/análisis , Linfocitos T CD8-positivos/inmunología , Infecciones por Cardiovirus/inmunología , Movimiento Celular , Femenino , Hematopoyesis , Memoria Inmunológica , Masculino , Ratones , Ratones Endogámicos C57BL , Miocarditis/inmunología , Receptores CCR2/fisiologíaRESUMEN
BACKGROUND: DICER1 syndrome, arising from a mutation in the DICER1 gene mapped to chromosome 14q32, is associated with an increased risk of a range of benign and malignant neoplasms. OBJECTIVE: To determine the spectrum of abnormalities and imaging characteristics in patients with DICER1 syndrome at a tertiary pediatric hospital. MATERIALS AND METHODS: This retrospective analysis evaluated imaging in patients ≤18 years with DICER1 germline variants between January 2004 and July 2016. An imaging database search including keywords pleuropulmonary blastoma, cystic nephroma, pineoblastoma, embryonal rhabdomyosarcoma, ovarian sex cord-stromal tumor, ovarian Sertoli-Leydig cell tumor and DICER1 syndrome, was cross-referenced against the institutional Cancer Genetics Program database, excluding patients with negative/unknown DICER1 gene testing. RESULTS: Sixteen patients were included (12 females; mean age at presentation: 4.2 years, range: 14 days to 17 years), with surveillance imaging encompassing the following modalities: chest X-ray and CT; abdominal, pelvic and neck US; and brain and whole-body MRI. Malignant lesions (68.8% of patients) included pleuropulmonary blastoma (5), pineoblastoma (3), ovarian Sertoli-Leydig cell tumor (1), embryonal rhabdomyosarcoma (1) and renal sarcoma (1); benign lesions (37.5% of patients) included thyroid cysts (2), thyroid nodules (2), cystic nephroma (2), renal cysts (1) and pineal cyst (1). A common lesional appearance observed across modalities and organs was defined as the "cracked windshield" sign. CONCLUSION: The spectrum of DICER1-related tumors and the young age at presentation suggest early surveillance of at-risk patients is critical, while minimizing exposure to ionizing radiation.