Fig. 3: Neonatal intestinal macrophages promote endothelial cell sprouting/proliferation in vitro via IGF-1.

a 4 × 10⁴ intestinal endothelial cells (from mT/mG mice—red) and 2 × 10⁴ CD11b⁺ intestinal myeloid cells (from CX3CR1-GFP mice—green) were cultured in duplicated wells separately or together in Matrigel and images were taken at day 1–4 of culture. scale bar: 100 μm. b When CD31 and CD11b were cultured together, endothelial cell sprout length per area was assessed over time (mean ± SEM). For each time point, 7–10 sprouts per area were measured. Sprouts were unmeasurable in individually cultured cells. c Small intestinal tissue of a 24 h-old CX3CR1-GFP reporter neonatal dam-fed pup was stained for CD31 (red) and GFP (CX3CR1 -green). Scale bar = 100 μm. d–h 5 × 10⁴ intestinal CX3CR1⁺ macrophages and 10 × 10⁴ endothelial cells cultured separately or together in 24-well plates were collected at 48 h of culture and stained with anti-Tie-2, CX3CR1, Ki-67, IGF-1 antibodies or isotype controls for flow cytometric analysis. d Contour plots showing the percentage of proliferating (Ki-67⁺) endothelial cells. e Percentage of proliferating (Ki-67⁺) endothelial cells (Tie-2⁺ CX3CR1⁻ and macrophages (Tie-2⁻CX3CR1⁺). f Relative number of macrophages and endothelial cells in co-culture to their respective number when grown separately. g Histogram showing IGF-1 expression of neonatal intestinal endothelial cells and macrophages when cultured separately or together compared to isotype antibody control. h Mean fluorescence intensity of IGF-1 of the two cell populations when cultured separately or together. Data indicate two separate experiments combined (mean ± SEM for e, f, h), n = 6/group (e, f, h). See supplemental Fig. 3 for gating strategy for d and g. P values were calculated using multiple t tests (e, f, h). *p < 0.05, ***p < 0.001, ****p < 0.0001. Source data are provided as a Source Data file.