Generation of functionally active resident macrophages from adipose tissue by 3D cultures

IntroductionWithin adipose tissue (AT), different macrophage subsets have been described, which played pivotal and specific roles in upholding tissue homeostasis under both physiological and pathological conditions. Nonetheless, studying resident macrophages in-vitro poses challenges, as the isolation process and the culture for extended periods can alter their inherent properties.MethodsStroma-vascular cells isolated from murine subcutaneous AT were seeded on ultra-low adherent plates in the presence of macrophage colony-stimulating factor. After 4 days of culture, the cells spontaneously aggregate to form spheroids. A week later, macrophages begin to spread out of the spheroid and adhere to the culture plate.ResultsThis innovative three-dimensional (3D) culture method enables the generation of functional mature macrophages that present distinct genic and phenotypic characteristics compared to bone marrow–derived macrophages. They also show specific metabolic activity and polarization in response to stimulation, but similar phagocytic capacity. Additionally, based on single-cell analysis, AT-macrophages generated in 3D culture mirror the phenotypic and functional traits of in-vivo AT resident macrophages.DiscussionOur study describes a 3D in-vitro system for generating and culturing functional AT-resident macrophages, without the need for cell sorting. This system thus stands as a valuable resource for exploring the differentiation and function of AT-macrophages in vitro in diverse physiological and pathological contexts

Etude phénotypique et fonctionnelle des macrophages issus de l'hématopoïèse du tissu adipeux dans un contexte de réparation tissulaire

Macrophages are key cells of innate immunity that play a major role in the inflammatory response and tissue repair. Indeed, a tissue injury induces an acute inflammation followed by a resolution phase finely orchestrated by macrophages in order to prevent the development of a chronic inflammation, and to promote tissue repair. They constitute a very heterogeneous cell population. They belong to distinct developmental origins that give them different characteristics and present a multitude of phenotypes in response to their environment. In adult mammal, two distinct origins have been described: embryonic hematopoiesis that gives rise to resident macrophages involved in the control of tissue homeostasis, and adult medullar hematopoiesis that generates pro-inflammatory macrophages involved in acute inflammatory response. In addition, a specific and quantitatively significant hematopoietic process has been identified in our lab in adipose tissue (AT), based on the presence of hematopoietic stem/progenitor cells in adult mice. This hematopoiesis generates mainly myeloid cells, including macrophages that are localized in the AT under physiological conditions where they contribute to the resident macrophage population and are involved in the control of tissue homeostasis. We hypothesized that these macrophages play a different role than medullary macrophages in tissue repair, and that a deregulation of this process is involved in the tissue alterations observed with aging. These different functions would be explained by intrinsic characteristics specific to each type of macrophages. Using hematopoietic chimeras, we investigated the role of macrophages derived from AT hematopoiesis in tissue repair in two injury models, partial AT ablation and myocardial infarction. We showed that macrophages derived from AT hematopoiesis are essential for AT regeneration because of a higher efferocytosis capacity than macrophages derived from circulating monocytes. On the other hand, although they are present in the heart after myocardial infarction, they play a deleterious role in post-injury cardiac remodeling and function. Using a 3D culture model, we compared the intrinsic properties of these macrophages with those of macrophages derived from circulating monocytes. Our results show that macrophages derived from AT hematopoiesis have an increased reactivity to signals from their environment compared to macrophages derived from medullar hematopoiesis. In conclusion, our work shows that macrophages derived from AT hematopoiesis have specific phenotypic and functional properties compared to macrophages derived from the bone marrow. Thanks to these properties, they play crucial positive or negative roles in tissue repair, in the AT and in other tissues.Les macrophages sont les cellules clés de l'immunité innée qui jouent un rôle prépondérant dans la réponse inflammatoire et la réparation tissulaire. En effet, une lésion tissulaire induit une inflammation aigüe suivie d'une phase de résolution finement orchestrée par les macrophages afin d'empêcher la mise en place d'une inflammation chronique, et favoriser la réparation du tissu. Ces macrophages constituent une population cellulaire très hétérogène. Ils possèdent des origines développementales distinctes qui leur confèrent des caractéristiques différentes, et présentent une multitude de phénotypes en réponse à leur environnement. Chez le mammifère adulte, on distingue classiquement les macrophages issus de l'hématopoïèse embryonnaire décrits comme macrophages résidents permettant le maintien de l'homéostasie tissulaire, et les macrophages issus de l'hématopoïèse médullaire adulte décrits comme pro-inflammatoires et intervenant dans la réponse inflammatoire aiguë. Les travaux du laboratoire ont cependant permis d'identifier un processus hématopoïétique spécifique et quantitativement significatif dans le tissu adipeux (TA), reposant sur la présence de cellules souches/progénitrices hématopoïétiques chez la souris adulte. Cette hématopoïèse génère principalement des cellules myéloïdes, dont des macrophages qui sont localisés dans le TA en conditions physiologiques où ils participent à la population de macrophages résidents, et sont impliqués dans le contrôle de l'homéostasie du tissu. Nous avons émis l'hypothèse selon laquelle ces macrophages jouent un rôle différent des macrophages médullaires dans la réparation tissulaire endogène et à distance, et qu'une dérégulation de ce processus est impliquée dans les altérations tissulaires observées avec le vieillissement. Ces fonctions différentes seraient expliquées par des caractéristiques intrinsèques spécifiques à chaque type de macrophages. Grâce à la construction de chimères hématopoïétiques, nous avons étudié le rôle des macrophages issus de l'hématopoïèse du TA dans la réparation tissulaire dans deux modèles de lésion, une ablation partielle du TA et l'infarctus du myocarde. Nous avons montré que ces macrophages sont indispensables à la régénération du TA grâce à une capacité d'efférocytose supérieure à celle des macrophages dérivés des monocytes circulants. En revanche, bien qu'ils soient présents dans le cœur après un infarctus du myocarde, ils jouent un rôle délétère dans le remodelage et la fonction cardiaque post lésion. Ces résultats nous ont conduits à étudier les caractéristiques intrinsèques des macrophages du TA et leur réponse à différents stimuli. Grâce à un modèle de culture 3D nous avons comparé les propriétés intrinsèques de ces macrophages avec celles des macrophages dérivés des monocytes circulants. Nos résultats montrent que les macrophages dérivés de l'hématopoïèse du TA possèdent une réactivité accrue aux signaux de leur environnement par rapport aux macrophages issus de l'hématopoïèse médullaire. En conclusion, nos travaux montrent que les macrophages issus de l'hématopoïèse du TA ont des propriétés phénotypiques et fonctionnelles différentes de celles des macrophages provenant de l'hématopoïèse médullaire. Ces propriétés en font des acteurs ayant un rôle clé dans la réparation tissulaire, qu'il soit bénéfique ou délétère, non seulement au sein du TA mais aussi dans d'autres tissus

Identification of Adipose Tissue as a Reservoir of Macrophages after Acute Myocardial Infarction

International audienceMedullary and extra-medullary hematopoiesis has been shown to govern inflammatory cell infiltration and subsequently cardiac remodeling and function after acute myocardial infarction (MI). Emerging evidence positions adipose tissue (AT) as an alternative source of immune cell production. We, therefore, hypothesized that AT could act as a reservoir of inflammatory cells that participate in cardiac homeostasis after MI. To reveal the distinct role of inflammatory cells derived from AT or bone marrow (BM), chimeric mice were generated using standard repopulation assays. We showed that AMI increased the number of AT-derived macrophages in the cardiac tissue. These macrophages exhibit pro-inflammatory characteristics and their specific depletion improved cardiac function as well as decreased infarct size and interstitial fibrosis. We then reasoned that the alteration of AT-immune compartment in type 2 diabetes could, thus, contribute to defects in cardiac remodeling. However, in these conditions, myeloid cells recruited in the infarcted heart mainly originate from the BM, and AT was no longer used as a myeloid cell reservoir. Altogether, we showed here that a subpopulation of cardiac inflammatory macrophages emerges from myeloid cells of AT origin and plays a detrimental role in cardiac remodeling and function after MI. Diabetes abrogates the ability of AT-derived myeloid cells to populate the infarcted heart

Driving regeneration, instead of healing, in adult mammals: the decisive role of resident macrophages through efferocytosis

International audienceTissue repair after lesion usually leads to scar healing and thus loss of function in adult mammals. In contrast, other adult vertebrates such as amphibians have the ability to regenerate and restore tissue homeostasis after lesion. Understanding the control of the repair outcome is thus a concerning challenge for regenerative medicine. We recently developed a model of induced tissue regeneration in adult mice allowing the comparison of the early steps of regenerative and scar healing processes. By using studies of gain and loss of function, specific cell depletion approaches, and hematopoietic chimeras we demonstrate here that tissue regeneration in adult mammals depends on an early and transient peak of granulocyte producing reactive oxygen species and an efficient efferocytosis specifically by tissue-resident macrophages. These findings highlight key and early cellular pathways able to drive tissue repair towards regeneration in adult mammals

Driving regeneration, instead of healing, in adult mammals: the decisive role of resident macrophages through efferocytosis

AbstractTissue repair after lesion usually leads to scar healing and thus loss of function in adult mammals. In contrast, other adult vertebrates such as amphibians have the ability to regenerate and restore tissue homeostasis after lesion. Understanding the control of the repair outcome is thus a concerning challenge for regenerative medicine. We recently developed a model of induced tissue regeneration in adult mice allowing the comparison of the early steps of regenerative and scar healing processes. By using studies of gain and loss of function, specific cell depletion approaches, and hematopoietic chimeras we demonstrate here that tissue regeneration in adult mammals depends on an early and transient peak of granulocyte producing reactive oxygen species and an efficient efferocytosis specifically by tissue-resident macrophages. These findings highlight key and early cellular pathways able to drive tissue repair towards regeneration in adult mammals.</jats:p