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. 2012 Jan 16;209(1):123-37.
doi: 10.1084/jem.20111009. Epub 2012 Jan 2.

Rapid monocyte kinetics in acute myocardial infarction are sustained by extramedullary monocytopoiesis

Florian Leuschner  1 , Philipp J Rauch, Takuya Ueno, Rostic Gorbatov, Brett Marinelli, Won Woo Lee, Partha Dutta, Ying Wei, Clinton Robbins, Yoshiko Iwamoto, Brena Sena, Aleksey Chudnovskiy, Peter Panizzi, Edmund Keliher, John M Higgins, Peter Libby, Michael A Moskowitz, Mikael J Pittet, Filip K Swirski, Ralph Weissleder, Matthias Nahrendorf
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Rapid monocyte kinetics in acute myocardial infarction are sustained by extramedullary monocytopoiesis

Florian Leuschner et al. J Exp Med. .

Abstract (V体育2025版)

Monocytes (Mo) and macrophages (MΦ) are emerging therapeutic targets in malignant, cardiovascular, and autoimmune disorders. Targeting of Mo/MΦ and their effector functions without compromising innate immunity's critical defense mechanisms first requires addressing gaps in knowledge about the life cycle of these cells. Here we studied the source, tissue kinetics, and clearance of Mo/MΦ in murine myocardial infarction, a model of acute inflammation after ischemic injury. We found that a) Mo tissue residence time was surprisingly short (20 h); b) Mo recruitment rates were consistently high even days after initiation of inflammation; c) the sustained need of newly made Mo was fostered by extramedullary monocytopoiesis in the spleen; d) splenic monocytopoiesis was regulated by IL-1β; and e) the balance of cell recruitment and local death shifted during resolution of inflammation. Depending on the experimental approach, we measured a 24 h Mo/MΦ exit rate from infarct tissue between 5 and 13% of the tissue cell population. Exited cells were most numerous in the blood, liver, and spleen. Abrogation of extramedullary monocytopoiesis proved deleterious for infarct healing and accelerated the evolution of heart failure. We also detected rapid Mo kinetics in mice with stroke VSports手机版. These findings expand our knowledge of Mo/MΦ flux in acute inflammation and provide the groundwork for novel anti-inflammatory strategies for treating heart failure. .

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Figure 1.
Figure 1.
Monocyte infarct residence time after ischemia. Cell tissue kinetics were studied using flow cytometric analysis (FCM) of heart transplants and BrdU pulse experiments. (A) Schematic set-up of the experiment (n = 16; experiment was performed four times). (B) Dot plots of FCM analysis showing the contribution of donor- and recipient-derived Mo in the infarcted heart at 6, 12, and 24 h after transplantation of the heart. (C) Number of Mo retrieved from digested infarcts on day 3 to 6 after MI by FCM. (D) Fitting of donor Mo (y-axis) over time (x-axis). Red circles indicate individual data, the gray line represents the fit described by the equation. (E) Flow cytometric DAPI staining of Mo in the infarct on day 3 after coronary ligation. (F) FCM analysis of the infarct 24 h after BrdU pulse. Dot plots are gated on Mo. (G) Fold-change in BrdU+ Mo and lineage+ cells in the heart after MI. Mean ± SEM (n = 3–10 per group, experiment was repeated twice). *, P < 0.05.
Figure 2.
Figure 2.
Kinetics of Mo subsets after MI. (A) FCM of BrdU pulse experiment in naive mice on day 3 after MI (B) and on day 5 after MI (C). Dot plots show Mo identified as CD11bhigh (CD90/B220/CD49b/NK1.1/Ly-6G)low (F4/80/CD11c)low, including separation into subsets using Ly-6C. Mean ± SEM (n = 4–6 per group, experiment was repeated twice).
Figure 3.
Figure 3.
Mo clearance. Cell exit and local death were studied as major contributors to cell clearance from the site of inflammation. (A) Bar graph shows number of exited CD45.2+ Mo in organs based on FCM; pie chart illustrates percentage of exited myeloid cells normalized to the number in the infarct (n = 5). (B) Left bar graph shows excited cell number in organs calculated using alternative scintillation data; pie chart illustrates the exit rate. The right bar graph reflects decay-corrected photon counts of the graft before and 24 h after heart transplantation. Experiment was done in duplicate, data are mean ± SEM. (C) Immunofluorescence of infarct tissue (DAPI, blue; TUNEL, green; CD11b, red). Pie charts indicate frequency of TUNEL signal in CD11b+ cells (arrows; n = 5 per group). (D) Analysis of cell death by DAPI FCM staining in the heart (n = 4 per group). (E) In vitro time lapse microscopy of representative splenic Mo to determine timing of cell death signals. Experiment was done in duplicates.
Figure 4.
Figure 4.
The spleen is a major source of Mo during acute inflammation. (A) Gate in dot plots of infarcted hearts shows myeloid cells on day 6 after MI (SPX indicates splenectomy 24 h before analysis; Lin indicates staining for lineage markers). Bar graphs enumerate total number of Mo in the heart, bone marrow, and blood (n = 6–9 per group, from three independent experiments). (B) Histograms gated on Mo in the heart. Bar graph shows the total number of BrdU+ Mo. Mean ± SEM (n = 4–6 per group, experiment performed twice). *, P < 0.05.
Figure 5.
Figure 5.
MI induces extramedullary monocytopoiesis in the spleen. (A) CFU assay of splenocytes from naive mice and from mice 6 d after MI (n = 3 per group, experiment performed twice). Top images show representative scans of the culture plate, bottom images show magnifications of colonies. (right) Bar graph enumerates colonies in cultures. (B) Representative dot plots from spleen and enumeration of splenic MDPs. lin* indicates lineage for myeloid progenitor staining as described in the Materials and methods section (n = 6–9 per group from three independent experiments). (C) Cell cycle analysis for splenic MDPs in mice after MI (n = 3 per group from one experiment). (D) Adoptive transfer of GMPs on day 3 after MI. CD45.2+ cells were transferred into infarcted CD45.1+ mice, which were analyzed 3 d later. Dot plots show adoptively transferred precursors in the splenic pool and the infarcted myocardium (n = 6 from one experiment). (E) Ly-6Chigh and Ly-6Clow Mo in the spleen after MI. Mean ± SEM (n = 4–10 per group from 4 independent experiments). *, P < 0.05.
Figure 6.
Figure 6.
Current systemic MPS flux model in subacute MI in the mouse. The model was populated with numbers and rates obtained in mice with coronary ligation and is a first attempt at a more systematic visualization of system-wide cell kinetics in acute inflammation.
Figure 7.
Figure 7.
Serial in vivo imaging reveals key function of splenic Mo/MΦ in infarct healing. Study groups: MI and no SPX (top row), MI and SPX on d0 (middle), and MI and SPX on d3 after MI (bottom). Three-channel FMT-CT was done on day 5 after MI. Bar graphs on the bottom show fluorescence signal in the infarct in respective channels. MR images from day 1 (inset) and day 21 (full size) are shown on the right, with bar graphs on the bottom. Arrows indicate gadolinium-DTPA enhanced infarct. Mean ± SEM (n = 8–12 per group from two experiments). *, P < 0.05.
Figure 8.
Figure 8.
Histological biomarkers of infarct healing after splenectomy. Experimental groups: MI and no SPX (top row), MI and SPX on d0 (middle), and MI and SPX on d3 after MI (bottom). IHC staining for myeloid cells (CD11b), MΦ (MAC-3), neo-vascularization (CD31), and collagen deposition (Masson trichrome, picrosirius red [PSR], and collagen I). ROI, region of interest. Mean ± SEM (n = 5 per group from one experiment). *, P < 0.05.
Figure 9.
Figure 9.
Rapid Mo kinetics and supply of splenic Mo in stroke. (A) Dot plots from brain tissue gated on CD11b+ and lineage− cells 24 h after BrdU pulse. Mean ± SEM (n = 4–5 per group; P < 0.05 stroke vs. no stroke). (B) Pie chart illustrates turnover of Ly-6Chigh Mo in the brain on day 2 after stroke. (C) Number of Mo in the spleen after stroke. Dot plots are gated on CD11b+ lin− cells and compare naive control mice to mice 2 d after stroke. Bar graph shows total Mo in the spleen. Mean ± SEM (*, P < 0.05; n = 4–5 per group from one experiment). (D) Increased MDP on day 10 after stroke in the spleen. Mean ± SEM (*, P < 0.05, n = 4–5 per group from one experiment).
Figure 10.
Figure 10.
IL-1β is a key cytokine for splenic monocytopoiesis. (A) rtPCR of IL-1β mRNA expression in the spleen throughout the first week after MI (n = 6–7 per group; *, P < 0.05). IL-1β protein by ELISA in the spleen on day 6 after MI (n = 4 per group; *, P < 0.05). (B) Cultured spleen cells from WT and IL-1R−/− mice colony forming capacity on day 6 after MI. Experiment was done in duplicates. (C) Enumeration after FCM analysis on day 6 after MI of splenic MDPs (left), BrdU+ Mo in the spleen (middle), and in the heart (right) of IL-1R−/− mice compared with WT. Mean ± SEM (n = 5 per group; *, P < 0.05). (D) Setup of the experiment: Adoptive transfer of CD45.2+ GMPs into infarcted CD45.1+ mice 1 d after MI. Analysis was performed 5 d later, comparing the capacity of transferred cells to generate Mo in the presence (WT) or absence (IL-1−/−) of the IL-1R. (E) Dot plots from spleens are gated on Mo, identified as CD11b+, lineage−, and CD11c−. Mean percentage ± SEM (n = 4 recipients per group). (F) Enumeration of CD45.2+ Mo in the spleens of CD45.1+ recipients 6 d after MI and 5 d after adoptive transfer. Mean ± SEM (n = 4 per group; *, P < 0.05).
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