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Cited by 949Immune cells in cancerImmune Response and InflammationImmune responses and vaccinations

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The fate and lifespan of human monocyte subsets in steady state and systemic inflammation

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Patel, Amit A., Zhang, Yan, Fullerton, James N., Boelen, Lies, Rongvaux, Anthony, Maini, Alexander A., Bigley, Venetia, Flavell, Richard A., Gilroy, Derek W., Asquith, Becca, Macallan, Derek, Yona, Simon·2017·Journal of Experimental Medicine
DOI 10.1084/jem.20170355PMC PMC5502436
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Abstract

In humans, the monocyte pool comprises three subsets (classical, intermediate, and nonclassical) that circulate in dynamic equilibrium. The kinetics underlying their generation, differentiation, and disappearance are critical to understanding both steady-state homeostasis and inflammatory responses. Here, using human in vivo deuterium labeling, we demonstrate that classical monocytes emerge first from marrow, after a postmitotic interval of 1.6 d, and circulate for a day. Subsequent labeling of intermediate and nonclassical monocytes is consistent with a model of sequential transition. Intermediate and nonclassical monocytes have longer circulating lifespans (∼4 and ∼7 d, respectively). In a human experimental endotoxemia model, a transient but profound monocytopenia was observed; restoration of circulating monocytes was achieved by the early release of classical monocytes from bone marrow. The sequence of repopulation recapitulated the order of maturation in healthy homeostasis. This developmental relationship between monocyte subsets was verified by fate mapping grafted human classical monocytes into humanized mice, which were able to differentiate sequentially into intermediate and nonclassical cells.

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Figure 1. In vivo labeling and a methodological approach of modeling human monocyte subset kinetics at steady state. (a) Polychromatic flow cytometry gating strategy for blood monocyte subsets. Peripheral blood mononuclear phagocyte cells were identified as Lin− (CD3, CD19, CD20, CD56, CD66b) HLA-DR+ cells. This population comprises classical monocytes (CD14+ CD16−: black gated population), intermediate monocytes (CD14+ CD16+: gray gated population), and nonclassical monocytes (CD14lo CD16+: red gated population) representative of >10 subjects. Representative cytospin images from 10 healthy volunteers stained with hematoxylin and eosin (bottom). Bar, 25 µm. (b) Flow cytometry viSNE analysis of monocyte subsets illustrating membrane expression for CCR2, CD62L, CD36, CD64, CD11b, CD11c, HLA-DR, SLAN, and CX3CR1, representative of eight healthy volunteers. (c) Schematic of protocol for labeling newly divided cells. Healthy volunteers received 20 g deuterium-labeled glucose over 3 h. Monocytes subsets were then sorted from whole blood over a 30-d period, DNA was extracted to quantify the deuterium enrichment in each monocyte subset by gas chromatography mass spectrometry. (d) Percentage of deuterium label in peripheral blood classical (black), intermediate (gray), and nonclassical (red) monocytes following oral admission of deuterated glucose in four healthy volunteers; values shown are mean ± SEM. (e) Model of circulating monocyte kinetics. Cartoon depicts the sequential model for the fate of circulating monocyte subsets. Monocytes mature in the bone marrow, where their precursors proliferate at a rate p. Classical monocytes leave the bone marrow at rate r1, after a delay of Δ1 days between the last proliferation and release into the circulation. In the blood, classical monocytes either mature into intermediate monocytes at rate α2 r2, where α = proportion of the subset, or they disappear from the blood (either by death or by moving to other organs) at rate (1–α2)r2. The total disappearance rate is thus r2. Likewise, a proportion α3 of the intermediate monocyte subset develop into nonclassical monocytes, the remainder disappearing from blood. A parameter Δ3 has been included to allow for a potential delay in the differentiation of intermediate monocyte to nonclassical monocytes. (f) Polychromatic flow cytometry comparing BM with circulating monocyte subsets. Human BM was initially gated as Lin−HLA-DR+. Human BM obtained as either an aspirate or femoral head excavated biopsy was examined by flow cytometry to identify resident monocyte subsets. Only classical monocytes could be detected in the biopsy specimen. These data are representative of three donors for each procedure. (g) Summary of the steady-state kinetics for monocyte subsets. Figures in black bold text denote lifespans in each compartment figures in italics denote the relative probability of each cell undergoing the respective fate (death/disappearance versus phenotype transition). Progenitor cells in the bone marrow proliferate at rate of 0.42/d (blue), where the postmitotic cells remain within the bone marrow for 1.6 d before being released into the circulation as classical monocytes. Classical monocytes contribute 87% to the total monocyte pool, whereas intermediate and nonclassical monocytes make up 5% and 8%, respectively. 99% of classical monocytes leave the circulation, and 1% go onto become intermediate monocytes. 100% of intermediate monocytes mature in the circulation to become nonclassical monocytes under steady state.
Figure 1. In vivo labeling and a methodological approach of modeling human monocyte subset kinetics at steady state. (a) Polychromatic flow cytometry gating strategy for blood monocyte subsets. Peripheral blood mononuclear phagocyte cells were identified as Lin− (CD3, CD19, CD20, CD56, CD66b) HLA-DR+ cells. This population comprises classical monocytes (CD14+ CD16−: black gated population), intermediate monocytes (CD14+ CD16+: gray gated population), and nonclassical monocytes (CD14lo CD16+: red gated population) representative of >10 subjects. Representative cytospin images from 10 healthy volunteers stained with hematoxylin and eosin (bottom). Bar, 25 µm. (b) Flow cytometry viSNE analysis of monocyte subsets illustrating membrane expression for CCR2, CD62L, CD36, CD64, CD11b, CD11c, HLA-DR, SLAN, and CX3CR1, representative of eight healthy volunteers. (c) Schematic of protocol for labeling newly divided cells. Healthy volunteers received 20 g deuterium-labeled glucose over 3 h. Monocytes subsets were then sorted from whole blood over a 30-d period, DNA was extracted to quantify the deuterium enrichment in each monocyte subset by gas chromatography mass spectrometry. (d) Percentage of deuterium label in peripheral blood classical (black), intermediate (gray), and nonclassical (red) monocytes following oral admission of deuterated glucose in four healthy volunteers; values shown are mean ± SEM. (e) Model of circulating monocyte kinetics. Cartoon depicts the sequential model for the fate of circulating monocyte subsets. Monocytes mature in the bone marrow, where their precursors proliferate at a rate p. Classical monocytes leave the bone marrow at rate r1, after a delay of Δ1 days between the last proliferation and release into the circulation. In the blood, classical monocytes either mature into intermediate monocytes at rate α2 r2, where α = proportion of the subset, or they disappear from the blood (either by death or by moving to other organs) at rate (1–α2)r2. The total disappearance rate is thus r2. Likewise, a proportion α3 of the intermediate monocyte subset develop into nonclassical monocytes, the remainder disappearing from blood. A parameter Δ3 has been included to allow for a potential delay in the differentiation of intermediate monocyte to nonclassical monocytes. (f) Polychromatic flow cytometry comparing BM with circulating monocyte subsets. Human BM was initially gated as Lin−HLA-DR+. Human BM obtained as either an aspirate or femoral head excavated biopsy was examined by flow cytometry to identify resident monocyte subsets. Only classical monocytes could be detected in the biopsy specimen. These data are representative of three donors for each procedure. (g) Summary of the steady-state kinetics for monocyte subsets. Figures in black bold text denote lifespans in each compartment figures in italics denote the relative probability of each cell undergoing the respective fate (death/disappearance versus phenotype transition). Progenitor cells in the bone marrow proliferate at rate of 0.42/d (blue), where the postmitotic cells remain within the bone marrow for 1.6 d before being released into the circulation as classical monocytes. Classical monocytes contribute 87% to the total monocyte pool, whereas intermediate and nonclassical monocytes make up 5% and 8%, respectively. 99% of classical monocytes leave the circulation, and 1% go onto become intermediate monocytes. 100% of intermediate monocytes mature in the circulation to become nonclassical monocytes under steady state.CC BY · PubMed Central
Figure 2. Sequential reappearance of monocytes subsets after endotoxin challenge. (a) Schematic protocol for administrating deuterium-labeled glucose 20 h before i.v. endotoxin 2 ng/kg in healthy volunteers. Classical monocytes were then sorted from whole blood, DNA extracted, and deuterium enrichment quantified by gas chromatography mass spectrometry over the ensuing 8 d. (b) Flow cytometry analysis of human monocyte subsets at 0, 2, 4, 8, 24, 48, and 72 h and 7 d after i.v. administration of endotoxin, representative of 10 individuals. (c) Time course of absolute monocyte numbers at selected time points following endotoxin challenge for classical, intermediate, and nonclassical monocytes (mean ± SEM × 109/L of three individual subjects; note the different scale for each subset). (d) Comparison of deuterium-labeled classical monocyte egression from the BM under normal physiological conditions (triangles, dashed line, four subjects) and after endotoxin challenge (circles, solid line, three subjects). Values represent mean ± SEM.
Figure 2. Sequential reappearance of monocytes subsets after endotoxin challenge. (a) Schematic protocol for administrating deuterium-labeled glucose 20 h before i.v. endotoxin 2 ng/kg in healthy volunteers. Classical monocytes were then sorted from whole blood, DNA extracted, and deuterium enrichment quantified by gas chromatography mass spectrometry over the ensuing 8 d. (b) Flow cytometry analysis of human monocyte subsets at 0, 2, 4, 8, 24, 48, and 72 h and 7 d after i.v. administration of endotoxin, representative of 10 individuals. (c) Time course of absolute monocyte numbers at selected time points following endotoxin challenge for classical, intermediate, and nonclassical monocytes (mean ± SEM × 109/L of three individual subjects; note the different scale for each subset). (d) Comparison of deuterium-labeled classical monocyte egression from the BM under normal physiological conditions (triangles, dashed line, four subjects) and after endotoxin challenge (circles, solid line, three subjects). Values represent mean ± SEM.CC BY · PubMed Central
Figure 3. Development of intermediate and nonclassical human monocytes from classical monocytes. (a) Classical human monocyte LIN− HLA-DR+ CD14+ CD16− cells were sorted from healthy blood by FACS. (b) 1.5 × 106 sorted classical monocytes were grafted i.v. into the humanized MISTRG mouse. Grafted cells could be readily identified by expression of the human isoform of CD45 compared with recipient leukocytes expressing mouse CD45. (c) Flow cytometry analysis identified human CD45+ circulating monocytes from MISTRG recipients following adoptive transfer of human CD14+CD16− classical monocytes at 10 min and 24, 72, and 96 h after infusion. Results are representative of three analyzed mice per time point.
Figure 3. Development of intermediate and nonclassical human monocytes from classical monocytes. (a) Classical human monocyte LIN− HLA-DR+ CD14+ CD16− cells were sorted from healthy blood by FACS. (b) 1.5 × 106 sorted classical monocytes were grafted i.v. into the humanized MISTRG mouse. Grafted cells could be readily identified by expression of the human isoform of CD45 compared with recipient leukocytes expressing mouse CD45. (c) Flow cytometry analysis identified human CD45+ circulating monocytes from MISTRG recipients following adoptive transfer of human CD14+CD16− classical monocytes at 10 min and 24, 72, and 96 h after infusion. Results are representative of three analyzed mice per time point.CC BY · PubMed Central

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  1. van Furth, 1972, [Mononuclear phagocytic system: new classification of macrophages, monocytes and of their cell line], Bull. World Health Organ., 47, 651

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Metadata

Authors
Patel, A. A., Zhang, Y., Fullerton, J. N., Boelen, L., Rongvaux, A., Maini, A. A., Bigley, V., Flavell, R. A., Gilroy, D. W., Asquith, B., Macallan, D., & Yona, S.
Affiliations
  • University of London (GB)
  • St George's, University of London (GB)
  • Imperial College London (GB)
  • Yale University (US)
  • Newcastle University (GB)
  • Howard Hughes Medical Institute (US)
Corresponding author
Yona, Simon
ORCID
Venue
Journal of Experimental Medicine
Publisher
Rockefeller University Press
Published
2017-06-12
Volume
214
Issue
7
Pages
1913-1923
ISSN
  • 0022-1007 (ISSN-L)
  • 1540-9538
DOI
10.1084/jem.20170355
PMC
PMC5502436
OpenAlex
W2626120438
Cited by
946
References
64
Citations by year
2026: 67 · 2025: 111 · 2024: 126 · 2023: 104 · 2022: 120 · 2021: 114 · 2020: 126 · 2019: 94 · 2018: 76 · 2017: 8
FWCI
37.62
Type
journal article
Language
en
Open access
Licence
Locations
  • The Journal of Experimental Medicine (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • St George's Online Research Archive (St George's University of London) (acceptedVersion)
  • UCL Discovery (University College London) (submittedVersion)
  • Spiral (Imperial College London) (submittedVersion, cc-by)
  • Newcastle University ePrints (Newcastle Univesity) (cc-by)
Funders
  • Engineering and Physical Sciences Research Council
  • Wellcome Trust (093053/Z/10/Z, 103865)
  • Medical Research Council (G1001052, J007439)
  • Bloodwise (15012)
  • European Union Seventh Framework Program (FP7/2007–2013, 317040)
  • Leukemia and Lymphoma Research (15012)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Immune cells in cancer
  • Life Sciences › Immunology and Microbiology › Immunology › Immune Response and Inflammation
  • Life Sciences › Immunology and Microbiology › Immunology › Immune responses and vaccinations
Keywords
monocyte subsets, classical monocytes, intermediate monocytes, non-classical monocytes, systemic inflammation, endotoxemia, monocytopenia, monocyte differentiation, fate mapping, humanized mice, deuterium labeling
MeSH terms
Animals, Bone Marrow Cells, Cell Differentiation, Cell Survival, Cells, Cultured, Deuterium, Flow Cytometry, Homeostasis, Humans, Inflammation, Isotope Labeling, Monocytes, Time Factors, Endotoxemia, Mice

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