Fig. 3

Monocytes gradually differentiate into Ly6C^-MHCII^hiCX3CR1^hi macrophages in the normal small intestine. a Endogenous monocyte and monocyte-derived cells in the small intestinal lamina propria (siLP) were identified as Ly6G⁻SiglecF⁻CD11b⁺CD11c^lo/intCD64⁺ leukocytes (MNP^CD64+ cells). MNP^CD64+ cells were further divided into four subpopulation based on expression of Ly6C, MHCII, and CX3CR1/GFP; namely Ly6C⁺MHCII⁻, Ly6C⁺MHCII⁺, Ly6C⁻MHCII^hiCX3CR1^int, and Ly6C⁻MHCII^hiCX3CR1^hi. b Representative histograms demonstrating expression of CX3CR1, CD64, CD11c, and CD206 on subpopulations shown in a and neutrophils (CD11b⁺Ly6G⁺) for comparison. c Schematic illustration of the experimental setup for d–g. Purified monocytes were adoptively transferred to recipient mice and donor cells were recovered from siLP at day 1, 2, and 5 after transfer. d Donor MNP^CD64+ cells recovered from siLP of the recipients were identified by congenic markers followed by gating as described in left panel of a. e Representative FACS plots demonstrating donor MNP^CD64+ cells that gradually lost expression of Ly6C and gained expression of MHCII from day 1 to day 5 after transfer. f Frequency of Ly6C- and MHCII-defined subsets among donor MNP^CD64+ cells in the intestine at the indicated time points. Data were pooled from at least 4 independent experiments with a total of 9–10 mice each. g Expression of CX3CR1/GFP, CD64, and CD11c on indicated subsets of donor MNP^CD64+ cells at day 1, 2, and 5 post-transfer compared to endogenous neutrophils. Results in g are representative of 4 independent experiments each. h Heat map showing hierarchal clustering of gene expression profiles of adoptively transferred MNP^CD64+ cells. Donor monocyte-derived cells were recovered from siLP of recipient mice at day 1, 2, or 5 after transfer and transcriptomic analysis was performed by nanostring nCounter analysis. Differentially expressed genes were determined by Qlucore Omics explorer with a statistical significance p-value of 0.05. Data were pooled from 2–3 independent experiments with a total of 4–5 mice for each time point