Figure 3. Tissue resident macrophages repopulate locally independently of CCR2⁺ progenitors.

(A) CD169^DTR/+ mice were injected i.p. with 10 µg/kg DT (n=5, filled bars) or control diluent (n=3, open bars) on day 1 and 4 and the absolute numbers of lung macrophages were enumerated 2 days and 9 days after the last injection. Representative data from three separate experiments are shown as mean +/− SEM. **: P < 0.01. (B, C) CD169^DTR/+×Ccr2^+/− (open circles, n=5/time point), and CD169^DTR/+×Ccr2^−/− mice (filled circles, n=6/time point) were injected with DT on day 0 and the number of BM macrophages (B) and lung macrophages (C) were analyzed at 2, 9, and 16 days after DT administration. CD169^+/+×Ccr2^+/− (open triangle) and CD169^+/+×Ccr2^−/− (filled triangle) mice treated with DT were used as non-depleted controls. The absolute cell numbers are represented as relative values with the mean absolute number of lung macrophages in non-depleted CD169^+/+×Ccr2^+/− mice set at 100%. Data were pooled from two independent experiments and shown as mean +/− SEM. (D-E) CD169^DTR/+ (CD45.2⁺, n=5) mice were infused with 7.5 × 10⁷ congenic CD45.1⁺ BM cells, followed by DT (filled circles) or PBS (open circles) administration on days 4 and 7 after BM cell transfer. The percentage of donor-derived CD45.1⁺ cells among (D) BM progenitor cells (white bars) and blood monocytes (Mono, gray bars) and (E) lung monocytes and tissue macrophages were analyzed sixteen days after the transfer. Representative data from three independent experiments are shown as mean +/− SEM. *: P < 0.05; **: P < 0.01 compared to Gr1^high monocytes. (F-H) S100a4Cre×R26^Tomato mice were infected intranasally with 100 pfu PR8 influenza virus (F and H filled circles) or instilled intranasally with 50µg Poly (I:C) (G and H gray circles) or control PBS (F-H, open circles). Kinetics of lung macrophages number at different times after PR8 infection (F) or Poly (I:C) injection (G) and numbers of tdTomato⁻ lung macrophages at six days and four weeks following PR8 infection or Poly (I:C) injection are shown (H). Data are represented as mean +/− SEM. See also Figure S3.