Fig. 5: LiMacs are present in murine milk and contribute to neutrophil recruitment in mastitis.

a, Representative immunofluorescence image of a lactating mammary gland (day 15 pp). DAPI (blue), SMA (green) and Iba1 (yellow). Arrowhead shows an Iba1⁺ cell inside an alveoli. Scale bar, 70 µm. b,c, Representative flow cytometry plots and gating strategy (b) and violin plots showing frequencies of eosinophils, neutrophils, Ly6C^hi monocytes and liMacs within CD45⁺ cells (c) in lactating mammary glands (days 7–11 and 12–15 pp) from wild-type mice; n = 3 for days 7–11 pp, and n = 6 for days 12–15 pp, data pooled from two independent experiments. Two-tailed Mann–Whitney test was performed. d, Survival curve and weight of WT pups nursed by WT dams treated with CSF-1 antibodies or isotype control at E18.5 and every other day pp; n = 15, pooled data from three independent experiments. e, Survival curve and weight of pups (Cd11c^CreCsf1r^fl/+ or Csf1r^fl/+) nursed by CD11c^CreCsf1r^fl/fl or Csf1r^fl/fl mice; n = 30, pooled data from four experiments. f, H&E staining of mammary gland sections from unchallenged (control) WT mice or WT mice injected with LPS into the mammary gland (18 h post LPS challenge). Scale bar, 100 µm; n = 2–3 mice per group. g, UMAP plots and bar graph showing the frequencies of eosinophils, F4/80^hi macrophages (Mac), F4/80^lo macrophages, liMacs, Ly6C^hi monocytes (Mo), Ly6C^lo monocytes, neutrophils and CD11b^intMHCII⁺ cells (pregated on CD45⁺CD3^–CD19^–NK1.1^– and CD11b⁺ and/or CD11c⁺ and/or F4/80⁺ cells) in lactating mammary glands (days 11–14 pp) of WT mice, injected with LPS into the fourth mammary glands 18 h earlier or left untreated (control). The corresponding heatmap shows the median marker expression level. Data were transformed and quantile normalized, n = 2–3 per group. h, Representative flow cytometry plots and violin plots showing total cell counts of liMacs (top) (pregated on CD45⁺CD19^–NK1.1^–CD3^–SiglecF^–Ly6G^–) and neutrophils (bottom) (pregated on CD45⁺LiMacs^–) in lactating mammary glands (days 10–14 pp) in WT mice left untreated or pretreated with CSF-1 antibodies on days 7–11 pp, and injected with LPS into the fourth mammary glands 18 h earlier or left uninjected. Data (n = 5–6 per group) were pooled from two independent experiments. One-way ANOVA test was applied, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.