Monocytes give rise to mucosal, but not splenic, conventional dendritic cells

Abstract

The mononuclear phagocyte (MP) system is a body-wide macrophage (MPhi) and dendritic cell (DC) network, which contributes to tissue homeostasis, inflammation, and immune defense. The in vivo origins of MPs remain poorly understood. Here, we use an adoptive precursor cell transfer strategy into MP-depleted mice to establish the in vivo differentiation sequence from a recently identified MPhi/DC-restricted bone marrow (BM) precursor (MDP) via BM and blood intermediates to peripheral MPhis and DCs. We show that MDPs are in vivo precursors of BM and blood monocytes. Interestingly, grafted Gr1high "inflammatory" blood monocytes shuttle back to the BM in the absence of inflammation, convert into Gr1low monocytes, and contribute further to MP generation. The grafted monocytes give rise to DCs in the intestinal lamina propria and lung, but not to conventional CD11chigh DCs in the spleen, which develop during homeostasis from MDPs without a monocytic intermediate.

Figure 1.

Characterization of CX₃CR1^gfp BM and IBC transfer of MDPs. (A) Flow cytometry analysis of CX₃CR1^gfp/+ BM. The cells in the dot plots to the right are gated according to CD115 and GFP expression. Note the presence of three main CD115⁺ GFP⁺ populations: CD11b⁻ CD117⁺ MDP, CD11b⁺ Gr1^high, and CD11b⁺ Gr1^low BM monocytes. (B) Flow cytometry analysis of an MDP graft isolated by high speed sorting according to CD117 and GFP expression. (C) Analysis of WT recipient BM (right, injected femur) at the indicated time points after IBC transfer of 2.5 × 10⁴ MDPs (purity: 85%, devoid of Gr1^high CD11b⁺ cells). Note the distinct GFP intensity pattern of differentiated graft-derived (CD45.1⁺) CD11b⁺ Gr1^high and Gr1^low BM monocytes and the decrease in the Gr1^high/Gr1^low ratio with time: 14 (day 3) to 2.7 (day 6). (D) Analysis of WT recipient BM (left, noninjected femur) at the indicated time points after IBC transfer. Gating as in C. (E) Analysis of recipient blood (day 6). The cells were gated according to CD115 surface marker expression. The data are representative of at least two independent experiments per time point.

Figure 2.

IBC transfer of BM monocytes. (A) Flow cytometry analysis of Gr1^high BM monocyte graft. (B) Analysis of WT recipient BM (right, injected femur) at the indicated time points after IBC transfer of 5 × 10⁵ Gr1^high BM monocytes (purity: 90%). Note the distinct GFP intensity pattern of differentiated graft-derived (CD45.1⁺) CD11b⁺ Gr1^high and Gr1^low cells and the decrease in the Gr1^high/Gr1^low ratio with time: 5 (day 1) to 1.8 (day 3). (C) Analysis of WT recipient BM (left, noninjected femur) at the indicated time points after IBC transfer. Gating as in C. The data are representative of at least two independent experiments per time point. (D) Analysis of recipient blood (day 3). Note the sizeable presence of graft-derived (CD45.1⁺) CD11b⁺ Gr1^low monocytes.

Figure 3.

Gr1^high blood and BM monocytes shuttle between blood and BM. (A) Flow cytometry analysis of a blood monocyte graft isolated from Rag^−/− CX₃CR1^gfp mice. The GFP⁻ cells are Ly6G⁺ CX₃CR1⁻ granulocytes (reference 18). (B) Analysis of recipient BM and blood 4 d after adoptive cell transfer of Gr1^high blood monocytes (10⁵ cells). Note the presence of graft-derived (GFP⁺) Gr1^low monocytes in the blood. Fractions of graft-derived GFP⁺ cells of total CD115⁺ cells in recipient BM (0.12%) and in recipient blood (0.76%). (C) Analysis of BM and blood of WT recipients of Gr1^high BM monocytes (5 × 10⁵ cells; purity: 96%) days 1 and 3 after i.v. transfer. Note the loss of Ly6C/G expression on grafted Gr1^high BM monocytes. Bar diagrams summarize data obtained from three mice per time point. Note the difference of GFP intensity of BM and blood monocytes on day 1 (mean fluorescence intensity: 274.75 ± 13.9 vs. 376.6 ± 20.5; P = 0.003). (D) Flow cytometry analysis of BM and peritoneal cavity (PC) lavage of WT recipients of MACS-purified CD115⁺ BM monocytes (10⁶ cells; purity: 88%) that were left untreated or had been inoculated with thioglycollate. Note the recruitment of grafted Gr1^high BM monocytes to the inflamed peritoneal cavity. The data are representative of at least two independent experiments per time point.

Figure 4.

Whole body imaging of Gr1^high BM monocyte recipients. (A) Color-coded near-infrared fluorescent image overlaid on a photographic image of a CD1 nude mouse 16 h after tail vein injection of DiR-labeled BM monocytes (3 × 10⁶ cells; purity: 94%; right). Control (acquired on IVIS 100) is shown on the left. Note the presence of label in femura. (B) Color-coded near-infrared fluorescent image overlaid on a photographic image of isolated lung, spleen, and femura of CD1 nude recipients of DiR-labeled BM monocytes (right) and noninjected control (left; 3 × 10⁶ cells; purity: 94%). (C) Monochrome fluorescent microscopy image of a cranium of a CD1 nude mouse that received DiR-labeled BM monocytes (3 × 10⁶ cells; purity: 94%). Note the presence of labeled cells in bone cavities. (D) Dual-channel fluorescent microscopic image of a cranium of a C57BL/6 WT recipient mouse that had received an i.v. injection of CFSE-labeled BM monocytes (3 × 10⁶ cells; purity: 96%). Arrows indicate two graft-derived cells that extravasated into the BM parenchyma. Bar, 100 μm. The data are representative of two independent experiments.

Figure 5.

MDPs, but not monocytes, act as precursors of splenic DCs. (A) Flow cytometry analysis of CX₃CR1^gfp donor spleen and spleens of DTx-treated, MP-depleted mice with and without engraftment of CX₃CR1^gfp MDP (8 × 10⁴ cKit⁺ CD115⁺ Gr1⁻ CD11b⁻ cells; purity: 80%) and Gr1^high BM monocytes (8 × 10⁵ CD115⁺ Gr1⁺ CD11b⁺ cells; purity: 88%). Note the presence of graft-derived (CD45.1⁺) DCs (CD11c^high) in MDP recipients that, as in donor mice CD11b⁻ DCs, are split into CX₃CR1/GFP⁺ and CX₃CR1/GFP⁻ cells. CD11c^high/CD11c^int ratios in an MDP recipient: 2.7 in Gr1^high monocyte recipient 0.05 (as determined by the indicated gates). (B) Flow cytometry analysis of the spleen of an MP-depleted mouse with and without intra-splenic injection of CX₃CR1^gfp BM MDPs (2.5 × 10⁴ cells; purity: 96%). Note the presence of graft-derived (CD45.1⁺) DCs (CD11c^high) in MDP recipients. The data are representative of two independent experiments.

Figure 6.

Monocytes are precursors of MPs in the intestine and lung. (A) Flow cytometry analysis of small intestinal lp cells isolated from a CX₃CR1^gfp donor mouse, as well as of WT, MP-depleted, and MP-deficient recipients of CX₃CR1^gfp Gr1^high BM monocytes and MDPs (7 d after i.v. transfer of 8 × 10⁵ CD115⁺ CD11b⁺ Gr1⁺ monocytes [purity: 88%] or 8 × 10⁴ CD117⁺ Gr1⁻ CD11b⁻ MDPs [purity: 80%]). Note the presence of graft-derived (CD45.1⁺) CD11c⁺ CX₃CR1/GFP⁺ lpDCs and CD11c⁺ CX₃CR1/GFP⁻ lpMΦs in donors and MP-depleted and MP-deficient recipients of precursor grafts. The data are representative of three independent experiments. (B) Fluorescence microscopy analysis of small intestinal villi of mice constitutively lacking lpDCs and lpMΦs with or without adoptive transfer of CX₃CR1^gfp BM monocytes (10⁶ CD115⁺ Gr1^high CD11b⁺ cells; purity: 85%). Note the presence of graft-derived CX₃CR1/GFP⁺ lpDCs in the live tissue of a BM monocyte recipient. Bar, 100 μm. The data are representative of three independent experiments. (C) Flow cytometry analysis of parenchymal lung cells isolated from CX₃CR1^gfp donor mouse, an MP-depleted mouse, and MP-depleted recipients of CX₃CR1^gfp Gr1^high BM monocytes (7 d after i.v. transfer of 7.5 × 10⁵ CD115⁺ Gr1⁺ CD11b⁺ cells; purity: 85%). Note the presence of graft-derived (CD45.1⁺) CD11c⁺ CD11b⁺ CX₃CR1/GFP⁺ lung DCs in the graft recipient. The data are representative of two independent experiments.