Progressive replacement of embryo-derived cardiac macrophages with age

Abstract

Cardiac macrophages (cMΦ) are critical for early postnatal heart regeneration and fibrotic repair in the adult heart, but their origins and cellular dynamics during postnatal development have not been well characterized. Tissue macrophages can be derived from embryonic progenitors or from monocytes during inflammation. We report that within the first weeks after birth, the embryo-derived population of resident CX3CR1(+) cMΦ diversifies into MHCII(+) and MHCII(-) cells. Genetic fate mapping demonstrated that cMΦ derived from CX3CR1(+) embryonic progenitors persisted into adulthood but the initially high contribution to resident cMΦ declined after birth. Consistent with this, the early significant proliferation rate of resident cMΦ decreased with age upon diversification into subpopulations. Bone marrow (BM) reconstitution experiments showed monocyte-dependent quantitative replacement of all cMΦ populations. Furthermore, parabiotic mice and BM chimeras of nonirradiated recipient mice revealed a slow but significant donor contribution to cMΦ. Together, our observations indicate that in the heart, embryo-derived cMΦ show declining self-renewal with age and are progressively substituted by monocyte-derived macrophages, even in the absence of inflammation.

Figure 1.

cMΦ develop into 4 subpopulations after birth. (A and B) Cytometry analysis of cMΦ from adult CX3CR1^GFP/+ mice. (A) FACS profiles of cMΦ populations, pregated on living single CD11b⁺ cells with low CD11c and Ly6C expression. (B) Mean of total and subpopulations of cMΦ in absolute numbers (left) or as percentage of total (right) as determined by bead-normalized flow cytometry. Error bars represent SEM (n = 4). (C and D) Cytometry analysis (C) and mean percentage (D) of cMΦ subpopulations from CX3CR1^GFP/+ mice of indicated age. Error bars represent SEM (n = 3–4). (E) MHCII⁻ and MHCII⁺ cMΦ from adult CX3CR1^cre:R26-yfp mice (black line) were analyzed for YFP expression and compared with Cre⁻ littermate controls (gray area; n = 3–4). Data in all panels are representative of at least two independent experiments.

Figure 2.

Decreased contribution of embryo-derived macrophages to cMΦ with age. (A) CX3CR1^creER:R26-yfp embryos were treated with TAM on E9 or E13 and analyzed on the day of delivery (P0) or 6 wk after delivery (P42). (B) Percentage of microglia-normalized YFP⁺ cMΦ and representative cytometry plots pregated on total cMΦ at P0 and P42 after E13 labeling. Bars show median (n = 9–13). ***, P ≤ 0.005, Mann-Whitney test. Data were pooled from two independent experiments. (C) Representative cytometry plot showing YFP⁺ within total cMΦ at P42 and percentage of microglia-normalized YFP⁺ cMΦ at P0 and P42 after E9 labeling. Bars show median (n = 7) *, P ≤ 0.05. Data were pooled from three independent experiments. (D) Representative cytometry plots showing YFP⁺ cells within MHCII⁻ and MHCII⁺ cMΦ at P0 and P42 after E13 labeling (n = 9–13). (E) Quantification of YFP⁺ cells within MHCII⁻ and MHCII⁺ cMΦ at P42 after E13 labeling. Bars show the median (n = 9–13). ***, P ≤ 0.005, Mann-Whitney test. Data were pooled from two independent experiments. (F) Adult TAM-treated CX3CR1^creER:R26-yfp mice were analyzed for YFP⁺ cMΦ and microglia at 1 and 4 wk after treatment. Percentage of microglia-normalized YFP⁺ cMΦ is shown as median with extreme samples as error bars (n = 3). Representative cytometry plots show YFP⁺ within total cMΦ. Unless otherwise indicated data in all panels are representative of two independent experiments.

Figure 3.

Decreased proliferation rate of embryo-derived cMΦ with age. (A) cMΦ subpopulations of adult CX3CR1^GFP/+ mice were analyzed for BrdU incorporation and expression of Ki67 by flow cytometry four hours after BrdU injection (i.p.). Bars show median (n = 12–13). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005; Wilcoxon test. (B and C) Total (B) or CX3CR1⁺MHCII⁻ (C) cMΦ of CX3CR1^GFP/+ mice of indicated age were analyzed for BrdU incorporation and Ki67 expression by flow cytometry 4 h after BrdU injection (i.p.). Bars show median (n = 4–15). ***, P ≤ 0.005, Mann-Whitney test. Data in all panels were pooled from 2–3 independent experiments.

Figure 4.

Monocytes contribute to four cMΦ subpopulations in adult mice. (A) WT mice were lethally irradiated and reconstituted with BM from Ubow:CX3CR1^GFP/+ mice. cMΦ were analyzed for contribution of dTomato⁺ cells at indicated time points after reconstitution and graft-derived cells were analyzed for CX3CR1 and MHCII expression. Data are presented as mean percentage ± SEM (n = 3–7) and derived from two independent experiments. (B) Mixed BM chimeras were generated by reconstituting lethally irradiated WT mice (CD45.1/CD45.2) with BM from CCR2^−/−:CX3CR1^GFP/+ (CD45.2) and WT CX3CR1^GFP/+ (CD45.1) mice. cMΦ, circulating CD11b⁺CD115⁺ monocytes (Ly6C⁺ and Ly6C⁻) and B220⁺ B cells were analyzed for WT (CD45.1) and CCR2^−/− (CD45.2) contribution. Host- and graft-derived cMΦ were analyzed for the ratio of MHCII⁺/MHCII⁻ cMΦ. Bars show median (n = 4). *, P ≤ 0.05, Mann-Whitney test. (C) Parabiosis was established between adult CCR2^−/− (CD45.2) and WT (CD45.1) mice. After 10 wk, CCR2^−/− mice were analyzed for contribution of CD45.1⁺ non-host cells to cMΦ (total, MHCII⁺, and MHCII⁻), circulating monocytes (Ly6C⁺ and Ly6C⁻), and B cells. For CD45.1⁻ host and CD45.1⁺ non-host cMΦ, the ratio of MHCII⁺/MHCII⁻ cMΦ was calculated and compared. Bars show median (n = 4). **, P ≤ 0.01, Mann-Whitney test. (D) BM chimeras were generated by transfer of LT-HSC isolated from panYFP mice into nonirradiated Rag2^−/−γ_c^−/−Kit^W/Wv mice. cMΦ (total, MHCII⁺, and MHCII⁻) and neutrophils (Gr1^hi and SSC^hi) were analyzed for contribution of grafted YFP⁺ cells 8 wk after transplantation. Bars show median (n = 6). **, P ≤ 0.01, Mann-Whitney test. Data in all panels are representative of two independent experiments.