Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis

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.

Figure 1. Reporter gene expression profile of macrophage populations in Cx3cr1^gfp/+, Cx3cr1^cre/+:R26-yfp and Tamoxifen-treated Cx3cr1^creER/+:R26-yfp mice

(A) Schematic of modified Cx3cr1 loci. The Cx3cr1^cre and Cx3cr1^creER mice were crossed to R26-yfp mice, in which irreversible induction of YFP expression is induced upon Cre recombinase expression and activation, respectively. (B & C) Flow cytometric analysis of CX₃CR1⁺ (B) and CX₃CR1 (C) mononuclear phagocyte populations of Cx3cr1^gfp/+, Cx3cr1^cre/+:R26-yfp and Cx3cr1^creER/+:R26-yfp mice. Cx3cr1^creER/+:R26-yfp mice were treated for 4 weeks with Tamoxifen prior to analysis. Results are representative of 6 mice per group.

Figure 2. Dual origins of macrophages

(A) (i) Flow cytometric and histological analysis of livers obtained from Cx3cr1^cre/+:R26-rfp:Cx3cr1^gfp/+ mice. In these mice GFP expression acts as a direct reporter for CX₃CR1 expression, while RFP expression is controlled by Cre recombinase. (ii) Analysis of fetal liver and adult liver and Kupffer cells of Cx3cr1^gfp/+ mice. (B) Flow cytometric analysis of the intestinal lamina propria of DTx-treated Cd11c-dtr mice that received 7 days earlier a Ly6C⁺ YFP monocyte graft isolated from tamoxifen-treated Cx3cr1^creER/+:R26-yfp mice. Results are representative of two experiments involving 3 mice per group. (C) Flow cytometric analysis of peritoneal lavages of Cx3cr1^creER/+:R26-rfp mice administered a single tamoxifen gavage (5 mg) one day following an intra-peritoneal thioglycollate injection. Rightmost graph shows phenotypic shift of monocyte-derived cells between three weeks and 8 weeks. Results are representative of two independent experiments involving 3 mice per group.

Figure 3. Reporter gene expression profile of mononuclear phagocyte precursors and circulating monocytes

(A & B) Flow cytometric analysis of different mononuclear phagocyte populations, precursors (A) and blood monocytes (B) obtained from Cx3cr1^gfp/+, Cx3cr1^cre/+:R26-yfp and Cx3cr1^creER/+:R26-yfp mice. Cx3cr1^creER/+:R26-yfp mice were treated for 4 weeks with Tamoxifen prior to analysis. Results are representative of 4–6 mice per group. (C) Bar graph summarizing frequencies of GFP or YFP⁺ cells in indicated mononuclear phagocyte populations of Cx3cr1^gfp/+, Cx3cr1^cre/+:R26-yfp and Cx3cr1^creER/+:R26-yfp mice. Mean ± SEM are performed with n=4–6 mice per group.

Figure 4. Monocyte subset dynamics

(A & B) Flow cytometric analysis of Ly6C⁺ and Ly6C⁻ blood monocyte subsets pulsed three times with 2 mg BrdU 3 hrs apart and monitored over 5 days. (B) Mice were treated with the anti-CCR2 antibody MC21 and BrdU incorporation by Ly6C⁻ blood monocyte was analysed. (C) Analysis of time course of BrdU incorporation of Ly6C⁺ and Ly6C⁻ monocytes in blood (red) and BM (blue) following a single 2 mg pulse of BrdU; BrdU incorporation by monocytes in mice treated with the MC21 antibody (green). Mean ± SEM are performed with n=3–4 mice per group.

Figure 5. Impaired BM exit of Ly6C⁺ blood monocyte affects the Ly6C⁻ cell compartment

(A) Flow cytometric analysis of mixed [Ccr2^−/−:Cx3cr1^gfp/+ (CD45.2) / Ccr2^+/+:Cx3cr1^gfp/+ (CD45.1) > wt] BM chimeras. Distribution of cells within the mononuclear compartment were analysed for percentage of CD45.2⁺ cells. Cell definitions for gating: MP (Lin CD135⁺ CD115 CD117⁺) MDP (Lin CD135⁺ CD115⁺ CD117⁺ CDP (Lin CD135⁺ CD115⁺ CD117) PreDC (Lin CD11c^int MHCIICD135⁺ CD172a CX₃CR1⁺) monocyte (CD11b⁺ CD115⁺). Results are representative of 6 analyzed mice. (B) Analysis of mixed [Ccr2^−/−:Cx3cr1^gfp/+ (cd45.2) / Ccr2^+/+:Cx3cr1^gfp/+ (CD45.1) > wt] BM chimeras. Representative result of two experiments involving 3–5 mice per group.

Figure 6. Prevalence of Ly6C⁺ blood monocytes determines the circulation half-life of Ly6C⁻ blood cells

(A) Analysis of Ly6C⁺ and Ly6C⁻ blood monocyte subsets of Ccr2^+/+ Cx3cr1^gfp/+ and Ccr2^−/− Cx3cr1^gfp/+ mice. Mean ± SEM are performed with n=4–6 mice per group. (B) Mean fluorescent intensities of CD115 expression on Ly6C⁻ monocytes analyzed in (A). Mean ± SEM are performed with n=4–6 mice each. (C) Flow cytometric analysis of blood of Cx3cr1^creER/+:R26-yfp mice treated by tamoxifen gavage to induce excision of the STOP cassette from R26-YFP loci. Ly6C⁻ blood monocytes were analysed over a 2 week period for reporter gene expression. Mice were left untreated or treated with the CCR2 antibody MC21. Representative result of two experiments involving 3 mice per group. (D) Mean fluorescent intensities of CD115 expression on Ly6C⁻ YFP⁺ monocytes analyzed in (D). Mean ± SEM are performed with n=3 mice per group. (E) Mean fluorescent intensities of CD115 expression on Ly6C⁻ monocytes of mice that received an injection of recombinant CSF-1. Mean ± SEM are performed with n=3 mice each. (F) Flow cytometric analysis of blood of tamoxifen treated Cx3cr1^creER/+:R26-yfp mice. Ly6C⁻ blood monocytes were analysed over a 2 week period for reporter gene expression. Mice were left untreated, treated with the anti-CCR2 antibody, or treated with a combination of MC21 and anti-CSF-1 R. Table summarizes half-lives of Ly6C⁻ blood monocytes in the time window from 4 to 10 days, as determined by exponential trendline fitting.