The monocyte to macrophage transition in the murine sterile wound - PubMed (original) (raw)

The monocyte to macrophage transition in the murine sterile wound

Meredith J Crane et al. PLoS One. 2014.

Abstract

The origin of wound repair macrophages is incompletely defined and was examined here in sterile wounds using the subcutaneous polyvinyl alcohol sponge implantation model in mice. Phenotypic analysis identified F4/80(+)Ly6C(hi)CD64(+)MerTK(-) monocytes and F4/80(+)Ly6C(low)CD64(+)MerTK(+) macrophages in the wound. Circulating monocytes were the precursors of inflammatory Ly6C(hi) wound monocytes. Ly6C(low)MerTK(+) macrophages appeared later, expressed CD206, CD11c, and MHC class II, produced cytokines consistent with repair function, and lacked a gene expression profile compatible with mesenchymal transition or fibroblastic transdifferentiation. Data also demonstrated that Ly6C(hi) wound cells were precursors of Ly6C(low) macrophages, although monocytes did not undergo rapid maturation but rather persisted in the wound as Ly6C(hi)MerTK(-) cells. MerTK-deficient mice were examined to determine whether MerTK-dependent signals from apoptotic cells regulated the maturation of wound macrophages. MerTK-deficient mice had day 14 cell compositions that resembled more immature wounds, with a smaller proportion of F4/80(+) cells and higher frequencies of Ly6G(+) neutrophils and Ly6C(hi) monocytes. The cytokine profile and number of apoptotic cells in day 14 wounds of MerTK-deficient mice was unaffected despite the alterations in cell composition. Overall, these studies identified a differentiation pathway in response to sterile inflammation in which monocytes recruited from the circulation acquire proinflammatory function, persist in the wound, and mature into repair macrophages.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Phenotype of wound monocyte/macrophage subsets.

C57BL/6J mice were wounded for 1, 3, 7 or 14 days. Wound cells were isolated and examined by flow cytometry. FSC and SSC were used to gate viable cells. (A) Wound monocytes/macrophages were defined as F4/80+SSClow/int. F4/80intSSChi eosinophils were excluded from analysis. Gated F4/80+ wound monocytes/macrophages were additionally examined for expression of Ly6C. (B) Expression of CD64 and MerTK was examined on gated Ly6Chi and Ly6Clow monocyte/macrophage subsets. (C) Blood cells were isolated from naïve C57BL/6J mice and examined by flow cytometry. An identical gating strategy to that described for wound cells was employed to examine MerTK and CD64 expression on blood F4/80+Ly6Chi and Ly6Clow monocytes. Numbers accompanying gates are the mean ± SD, n = 3 mice per group, and are representative of 2–3 independent experiments. (D) Mertk gene expression was determined in day 14 Ly6Chi and Ly6Clow wound monocyte/macrophage subsets by qPCR. Mertk expression was normalized to Hprt and data are presented as ΔCt Expression. Data shown are the mean ± SD, n = 3 mice per group.

Figure 2

Figure 2. Surface marker expression on day 1 and day 14 wound macrophage subsets.

C57BL/6J mice were wounded for 1 or 14 days, and the surface expression of MHC class II, CD11c and CD206 on wound monocyte/macrophage subsets was determined by flow cytometry. Wound monocyte/macrophage subsets were defined as F4/80+Ly6Chi (Ly6Chi) and F4/80+Ly6Clow (Ly6Clow). Mean fluorescence intensities (MFI; geometric mean) were calculated and are presented as the mean ± SD, n = 4 mice per group. Data are representative of at least 3 independent experiments.

Figure 3

Figure 3. Migration of blood monocyte subsets to the wound.

Ly6Chi and Ly6Clow blood monocytes were selectively labeled with fluorescent microparticles (MP) following treatment with or without liposome-encapsulated clodronate (clo-lip) as described in Materials & Methods. C57BL/6J mice were then wounded for 1 or 7 days and accumulation of labeled monocytes/macrophages in the wound was assessed by flow cytometry. (A) Schematic of clo-lip and MP administration for the selective labeling of Ly6Chi monocytes. Mice were wounded 1 day after MP delivery. (B) F4/80+ blood and wound cells were examined by flow cytometry following clodronate treatment and MP labeling. In blood and the day 1 and day 7 wound, Ly6Chi monocytes were predominantly labeled with MPs. (C) Schematic of MP treatment for the selective labeling of circulating Ly6Clow monocytes prior to wounding for 1 or 7 days. (D) F4/80+ blood and wound cells were examined by flow cytometry following selective labeling of Ly6Clow monocytes. MP-labeled Ly6Clow monocytes were identified in the circulation but not in the day 1 or day 7 wound. The proportion of cells in quadrants is indicated by numbers on plots. N = 2 mice per group.

Figure 4

Figure 4. Chemokine receptor expression on circulating and wound monocytes/macrophages.

(A) Blood cells were isolated from naïve, day 1 and day 7-wounded C57BL/6J mice. Gated F4/80+SSClow blood monocytes were examined for Ly6C expression in naïve and day 1-wounded mice. Mean frequencies (±SD) are indicated above each gate. The mean number ± SD of F4/80+Ly6Chi and Ly6Clow blood monocytes is also shown. (B) Expression of CX3CR1 was compared on circulating and wound F4/80+ monocyte/macrophage subsets at 1 and 14 days after wounding by flow cytometry using CX3CR1-GFP transgenic mice. (C) C57BL/6J or CX3CR1-GFP transgenic mice were wounded for 1 or 14 days. Gated F4/80+Ly6Chi and Ly6Clow wound monocytes/macrophages were examined by flow cytometry for CCR2 and CX3CR1 expression. Average MFI ± SD (geometric mean) is indicated in each plot. N = 3–5 mice per group. Data are representative of at least 3 independent experiments.

Figure 5

Figure 5. Ly6Chi monocytes mature in situ into Ly6Clow wound macrophages.

Sponges were implanted into CD45.1 congenic mice. At 1 day post-wounding, sponges were transferred from donor CD45.1 congenic mice to recipient B6 (CD45.2) mice as described in Materials & Methods. Wound cells were isolated from recipient mice at 1, 3 or 7 days post-transfer and monocyte/macrophage subsets were analyzed by flow cytometry. CD45.1+ cells were identified on gated F4/80+SSClow/int cells and examined for F4/80+Ly6Chi and Ly6Clow subsets. Numbers accompanying gates are the mean frequency ± SD, n = 3 mice per group. Data are representative of at least 2 independent experiments.

Figure 6

Figure 6. Changes in monocyte phenotype upon entry to the wound.

Gated F4/80+Ly6Chi blood monocytes from naïve and day 1-wounded C57BL/6J mice were compared to wound monocytes from day 1-wounded mice by flow cytometry analysis. (A) Proportion of TNF-α+ cells and representative FACS plots, (B) proportion of CD14+ cells and representative histograms, (C) FSC median fluorescence intensity (MFI) and (D) SSC MFI with representative histograms. Data shown are the mean ± SD, n = 3 mice per group for (A) and (B); n = 2–3 mice per group for (C) and (D). Data are representative of 2–3 independent experiments.

Figure 7

Figure 7. Cytokine production by wound monocyte/macrophage subsets.

F4/80+Ly6Chi monocytes/macrophages were sorted from day 1 wound cells. F4/80+Ly6Chi and Ly6Clow monocytes/macrophages were sorted from day 14 wound cells. Sorted populations were cultured as described in Materials & Methods and supernatants were tested by ELISA for (A) IL-1β, (B) TNF-α, (C) TGF-β and (D) VEGF production. Data shown are the mean ± SD; n = 3 mice per experiment; data represent 2–3 independent experiments. # = below the level of detection.

Figure 8

Figure 8. Comparative gene expression analysis of day 14 Ly6Chi and Ly6Clow macrophages.

F4/80+Ly6Chi and F4/80+Ly6Clow macrophages were FACS-sorted from the day 14 wound. Expression of matrix metalloproteinase 9 (Mmp9), vimentin (Vim), tissue inhibitor of metalloproteinase 1 (Timp1), collagen 1 (Col1a1), collagen 3 (Col3a1) and α-smooth muscle actin (Acta2) was examined by qPCR. Target gene expression was normalized to Hprt. ΔΔCt Expression is presented as fold difference in relative gene expression of Ly6Clow relative to Ly6Chi cells as described in Materials & Methods. Equal gene expression between subsets is indicated by a grey dashed line placed at a fold change of 1. Data are means ± SD, n = 3 mice per group.

Figure 9

Figure 9. Cellular responses to sterile injury in MerTK−/− mice.

Control or MerTK−/− mice received sponges for 14 days. (A) Flow cytometry analysis was used to determine the proportion of F4/80+ macrophages and Ly6G+ neutrophils in the day 14 wounds of control and MerTK−/− mice. (B) The F4/80+ macrophage population was assessed for the frequency of Ly6Chi and Ly6Clow subsets. (C) The expression of activated caspase-3/7 was determined by FACS in control (blue histogram) and MerTK−/− (red histogram) mice at 14 days after wounding. The negative control is shown as a grey histogram. As an indication of neutrophil phagocytosis by wound monocytes/macrophages (Mo/Mφ), the frequency (D) and number (E) of Ly6Chi and Ly6Clow cells expressing intracellular Ly6G was determined by FACS. Representative dot plots showing intracellular Ly6G expression in Ly6Chi and Ly6Clow wound monocytes/macrophages are shown in (D). Plots were gated on F4/80+ cells. Proportion of Ly6ChiLy6G+ and Ly6ClowLy6G+ cells (±SD) is indicated in quadrants. Data shown in (D) are representative of 6 mice. Data shown in A–D are the mean ± SD, n = 6 mice per group. * p<0.05.

References

    1. Geissmann F, Jung S, Littman DR (2003) Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 19: 71–82. - PubMed
    1. Gordon S, Taylor PR (2005) Monocyte and macrophage heterogeneity. Nat Rev Immunol 5: 953–964 10.1038/nri1733 - DOI - PubMed
    1. Auffray C, Sieweke MH, Geissmann F (2009) Blood monocytes: development, heterogeneity, and relationship with dendritic cells. Ann Rev Immunol 27: 669–692 10.1146/annurev.immunol.021908.132557 - DOI - PubMed
    1. Geissmann F, Gordon S, Hume DA, Mowat AM, Randolph GJ (2010) Unravelling mononuclear phagocyte heterogeneity. Nat Rev Immunol 10: 453–460 10.1038/nri2784 - DOI - PMC - PubMed
    1. Arnold L, Henry A, Poron F, Baba-Amer Y, van Rooijen N, et al. (2007) Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis. J Exp Med 204: 1057–1069 10.1084/jem.20070075 - DOI - PMC - PubMed

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