Flow Cytometric Analysis of Macrophages and Dendritic Cell Subsets in the Mouse Lung
01 Oct 2013-American Journal of Respiratory Cell and Molecular Biology (American Thoracic Society)-Vol. 49, Iss: 4, pp 503-510
TL;DR: A panel of surface markers and an analysis strategy that accurately identify all known populations of macrophages and DCs, and their precursors in the lung during steady-state conditions and bleomycin-induced injury is developed.
Abstract: The lung hosts multiple populations of macrophages and dendritic cells, which play a crucial role in lung pathology. The accurate identification and enumeration of these subsets are essential for understanding their role in lung pathology. Flow cytometry is a mainstream tool for studying the immune system. However, a systematic flow cytometric approach to identify subsets of macrophages and dendritic cells (DCs) accurately and consistently in the normal mouse lung has not been described. Here we developed a panel of surface markers and an analysis strategy that accurately identify all known populations of macrophages and DCs, and their precursors in the lung during steady-state conditions and bleomycin-induced injury. Using this panel, we assessed the polarization of lung macrophages during the course of bleomycin-induced lung injury. Alveolar macrophages expressed markers of alternatively activated macrophages during both acute and fibrotic phases of bleomycin-induced lung injury, whereas markers of clas...
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TL;DR: The unique tissue location and function of alveolarmacrophages distinguish them from other macrophage populations and suggest that it is important to classify macrophages according to the site that they occupy.
Abstract: Alveolar macrophages exist in a unique microenvironment and, despite historical evidence showing that they are in close contact with the respiratory epithelium, have until recently been investigated in isolation. The microenvironment of the airway lumen has a considerable influence on many aspects of alveolar macrophage phenotype, function and turnover. As the lungs adapt to environmental challenges, so too do alveolar macrophages adapt to accommodate the ever-changing needs of the tissue. In this Review, we discuss the unique characteristics of alveolar macrophages, the mechanisms that drive their adaptation and the direct and indirect influences of epithelial cells on them. We also highlight how airway luminal macrophages function as sentinels of a healthy state and how they do not respond in a pro-inflammatory manner to antigens that do not disrupt lung structure. The unique tissue location and function of alveolar macrophages distinguish them from other macrophage populations and suggest that it is important to classify macrophages according to the site that they occupy.
999 citations
TL;DR: Alveolar macrophages differentiate from fetal monocytes in a GM-CSF–dependent fashion and colonize the alveolar space within a few days after birth.
Abstract: Tissue-resident macrophages can develop from circulating adult monocytes or from primitive yolk sac–derived macrophages. The precise ontogeny of alveolar macrophages (AMFs) is unknown. By performing BrdU labeling and parabiosis experiments in adult mice, we found that circulating monocytes contributed minimally to the steady-state AMF pool. Mature AMFs were undetectable before birth and only fully colonized the alveolar space by 3 d after birth. Before birth, F4/80hiCD11blo primitive macrophages and Ly6ChiCD11bhi fetal monocytes sequentially colonized the developing lung around E12.5 and E16.5, respectively. The first signs of AMF differentiation appeared around the saccular stage of lung development (E18.5). Adoptive transfer identified fetal monocytes, and not primitive macrophages, as the main precursors of AMFs. Fetal monocytes transferred to the lung of neonatal mice acquired an AMF phenotype via defined developmental stages over the course of one week, and persisted for at least three months. Early AMF commitment from fetal monocytes was absent in GM-CSF–deficient mice, whereas short-term perinatal intrapulmonary GM-CSF therapy rescued AMF development for weeks, although the resulting AMFs displayed an immature phenotype. This demonstrates that tissue-resident macrophages can also develop from fetal monocytes that adopt a stable phenotype shortly after birth in response to instructive cytokines, and then self-maintain throughout life.
946 citations
Cites background from "Flow Cytometric Analysis of Macroph..."
...Among tissue resident MFs, AMFs have a peculiar phenotype in that they are highly autofluorescent, express low levels of the phagocytic receptor CD11b, yet high levels of the integrin CD11c, and high levels of the lectin SiglecF, allowing them to be easily recognized among other myeloid cells of the lung (Gautier et al., 2012b; Misharin et al., 2013)....
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...…in that they are highly autofluorescent, express low levels of the phagocytic receptor CD11b, yet high levels of the integrin CD11c, and high levels of the lectin SiglecF, allowing them to be easily recognized among other myeloid cells of the lung (Gautier et al., 2012b; Misharin et al., 2013)....
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TL;DR: These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community providing the theory and key practical aspects offlow cytometry enabling immunologists to avoid the common errors that often undermine immunological data.
Abstract: These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.
698 citations
TL;DR: Using transcriptomic profiling of flow-sorted cells, it is found that monocyte to alveolar macrophage differentiation unfolds continuously over the course of fibrosis and its resolution, and suggests that selectively targeting alveolars macrophages differentiation within the lung may ameliorate fibrosis without the adverse consequences associated with global monocyte or tissue-resident alve Polar Macrophage depletion.
Abstract: Little is known about the relative importance of monocyte and tissue-resident macrophages in the development of lung fibrosis. We show that specific genetic deletion of monocyte-derived alveolar macrophages after their recruitment to the lung ameliorated lung fibrosis, whereas tissue-resident alveolar macrophages did not contribute to fibrosis. Using transcriptomic profiling of flow-sorted cells, we found that monocyte to alveolar macrophage differentiation unfolds continuously over the course of fibrosis and its resolution. During the fibrotic phase, monocyte-derived alveolar macrophages differ significantly from tissue-resident alveolar macrophages in their expression of profibrotic genes. A population of monocyte-derived alveolar macrophages persisted in the lung for one year after the resolution of fibrosis, where they became increasingly similar to tissue-resident alveolar macrophages. Human homologues of profibrotic genes expressed by mouse monocyte-derived alveolar macrophages during fibrosis were up-regulated in human alveolar macrophages from fibrotic compared with normal lungs. Our findings suggest that selectively targeting alveolar macrophage differentiation within the lung may ameliorate fibrosis without the adverse consequences associated with global monocyte or tissue-resident alveolar macrophage depletion.
657 citations
Cites background or methods from "Flow Cytometric Analysis of Macroph..."
...Data were acquired on BD LSR II flow cytometer (for information regarding instrument configuration and antibody panels, see Misharin et al., 2013, 2014; Bharat et al., 2016)....
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...…were stained with eFluor 506 (eBioscience) viability dyes, incubated with FcBlock (BD), and stained with fluorochrome-conjugated antibodies (antibodies, clones, fluorochromes, and manufacturers were described in detail in our previous publications; Misharin et al., 2013, 2014; Bharat et al., 2016)....
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...Bleomycin-induced lung fibrosis, TGF-β–induced lung fibrosis, and influenza A virus infection (A/WSN/33) were performed and assessed as previously described (Budinger et al., 2006; Misharin et al., 2013; Morales-Nebreda et al., 2014, 2015; Sennello et al., 2017)....
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... 1, B, E, and F; Misharin et al., 2013), and we used this gating strategy (Fig....
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...Furthermore we found that differential expression of Siglec F reliably distinguished Mo-AMs and TR-AMs over the course of bleomycin-induced fibrosis (Fig. 1, B, E, and F; Misharin et al., 2013), and we used this gating strategy (Fig....
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TL;DR: It is shown that MSCs manage intracellular oxidative stress by targeting depolarized mitochondria to the plasma membrane via arrestin domain-containing protein 1-mediated microvesicles and simultaneously shed micro RNA-containing exosomes that inhibit macrophage activation by suppressing Toll-like receptor signalling.
Abstract: Mesenchymal stem cells (MSCs) and macrophages are fundamental components of the stem cell niche and function coordinately to regulate haematopoietic stem cell self-renewal and mobilization. Recent studies indicate that mitophagy and healthy mitochondrial function are critical to the survival of stem cells, but how these processes are regulated in MSCs is unknown. Here we show that MSCs manage intracellular oxidative stress by targeting depolarized mitochondria to the plasma membrane via arrestin domain-containing protein 1-mediated microvesicles. The vesicles are then engulfed and re-utilized via a process involving fusion by macrophages, resulting in enhanced bioenergetics. Furthermore, we show that MSCs simultaneously shed micro RNA-containing exosomes that inhibit macrophage activation by suppressing Toll-like receptor signalling, thereby de-sensitizing macrophages to the ingested mitochondria. Collectively, these studies mechanistically link mitophagy and MSC survival with macrophage function, thereby providing a physiologically relevant context for the innate immunomodulatory activity of MSCs.
655 citations
References
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TL;DR: The identification of mechanisms and molecules associated with macrophage plasticity and polarized activation provides a basis for Macrophage-centered diagnostic and therapeutic strategies.
Abstract: Diversity and plasticity are hallmarks of cells of the monocyte-macrophage lineage. In response to IFNs, Toll-like receptor engagement, or IL-4/IL-13 signaling, macrophages undergo M1 (classical) or M2 (alternative) activation, which represent extremes of a continuum in a universe of activation states. Progress has now been made in defining the signaling pathways, transcriptional networks, and epigenetic mechanisms underlying M1-M2 or M2-like polarized activation. Functional skewing of mononuclear phagocytes occurs in vivo under physiological conditions (e.g., ontogenesis and pregnancy) and in pathology (allergic and chronic inflammation, tissue repair, infection, and cancer). However, in selected preclinical and clinical conditions, coexistence of cells in different activation states and unique or mixed phenotypes have been observed, a reflection of dynamic changes and complex tissue-derived signals. The identification of mechanisms and molecules associated with macrophage plasticity and polarized activation provides a basis for macrophage-centered diagnostic and therapeutic strategies.
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TL;DR: A fate-mapping study of the murine monocyte and macrophage compartment taking advantage of constitutive and conditional CX(3)CR1 promoter-driven Cre recombinase expression is reported, establishing that short-lived Ly6C(+) monocytes constitute obligatory steady-state precursors of blood-resident Ly 6C(-) cells and that the abundance of Ly6 C(+) blood monocytes dynamically controls the circulation lifespan of their progeny.
2,302 citations
TL;DR: This work identifies two distinct phases of monocyte participation after MI and proposes a model that reconciles the divergent properties of these cells in healing and identifies new therapeutic targets that can influence healing and ventricular remodeling after MI.
Abstract: Healing of myocardial infarction (MI) requires monocytes/macrophages These mononuclear phagocytes likely degrade released macromolecules and aid in scavenging of dead cardiomyocytes, while mediating aspects of granulation tissue formation and remodeling The mechanisms that orchestrate such divergent functions remain unknown In view of the heightened appreciation of the heterogeneity of circulating monocytes, we investigated whether distinct monocyte subsets contribute in specific ways to myocardial ischemic injury in mouse MI We identify two distinct phases of monocyte participation after MI and propose a model that reconciles the divergent properties of these cells in healing Infarcted hearts modulate their chemokine expression profile over time, and they sequentially and actively recruit Ly-6Chi and -6Clo monocytes via CCR2 and CX3CR1, respectively Ly-6Chi monocytes dominate early (phase I) and exhibit phagocytic, proteolytic, and inflammatory functions Ly-6Clo monocytes dominate later (phase II), have attenuated inflammatory properties, and express vascular–endothelial growth factor Consequently, Ly-6Chi monocytes digest damaged tissue, whereas Ly-6Clo monocytes promote healing via myofibroblast accumulation, angiogenesis, and deposition of collagen MI in atherosclerotic mice with chronic Ly-6Chi monocytosis results in impaired healing, underscoring the need for a balanced and coordinated response These observations provide novel mechanistic insights into the cellular and molecular events that regulate the response to ischemic injury and identify new therapeutic targets that can influence healing and ventricular remodeling after MI
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TL;DR: It is shown, by direct examination of blood monocyte functions in vivo, that a subset of monocytes patrols healthy tissues through long-range crawling on the resting endothelium, which initiated an early immune response and differentiated into macrophages.
Abstract: The cellular immune response to tissue damage and infection requires the recruitment of blood leukocytes. This process is mediated through a classical multistep mechanism, which involves transient rolling on the endothelium and recognition of inflammation followed by extravasation. We have shown, by direct examination of blood monocyte functions in vivo, that a subset of monocytes patrols healthy tissues through long-range crawling on the resting endothelium. This patrolling behavior depended on the integrin LFA-1 and the chemokine receptor CX(3)CR1 and was required for rapid tissue invasion at the site of an infection by this "resident" monocyte population, which initiated an early immune response and differentiated into macrophages.
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23 May 2013
TL;DR: In this paper, a fate-mapping study of the macrophage compartment is presented, taking advantage of constitutive and conditional CX(3)CR1 promoter-driven Cre recombinase expression.
Abstract: Mononuclear phagocytes, including monocytes, macrophages, and dendritic cells, contribute to tissue integrity as well as to innate and adaptive immune defense. Emerging evidence for labor division indicates that manipulation of these cells could bear therapeutic potential. However, specific ontogenies of individual populations and the overall functional organization of this cellular network are not well defined. Here we report a fate-mapping study of the murine monocyte and macrophage compartment taking advantage of constitutive and conditional CX(3)CR1 promoter-driven Cre recombinase expression. We have demonstrated that major tissue-resident macrophage populations, including liver Kupffer cells and lung alveolar, splenic, and peritoneal macrophages, are established prior to birth and maintain themselves subsequently during adulthood independent of replenishment by blood monocytes. Furthermore, we have established that short-lived Ly6C(+) monocytes constitute obligatory steady-state precursors of blood-resident Ly6C(-) cells and that the abundance of Ly6C(+) blood monocytes dynamically controls the circulation lifespan of their progeny.
1,691 citations