Fig. 4. LP macrophages contribute to increased inflammation of DSS-treated miR-494^−/− mice.

a Subpopulations of colonic LP macrophage (gated for CD11b⁺CD11C^-F4/80⁺ cells), M1 (gated for CD11b⁺CD11C^-F4/80⁺CD206⁻ cells) and M2 (gated for CD11b⁺CD11C⁻F4/80⁺CD206⁺ cells) subsets were analyzed from miR-494^−/− and WT (+/+) mice on day 7 after DSS treatment. b IF staining of colon tissues for macrophage marker F4/80 on day 7 after DSS treatment. Scale bar: 50 μm. c The protein levels of each indicated cytokines were determined by ELISA in plasma from miR-494^−/− mice and WT mice on day 7 after DSS treatment. d–h Mice were provided with free access to 3% DSS in drinking water for 7 days, and empty liposomes (EL) or clodronate liposomes (CL) was administrated by ip (200 μl/mice) at days −4, 0, 2, 4, and 6. Day 0 is the initial time point of DSS treatment. d Body weight changes and DAI of the indicated miR-494^−/− mice and WT mice after DSS treatment in macrophage deletion assay. e Colon length was measured on day 7 after DSS treatment. f Typical images of H&E staining of colon tissues, and histological score (0–12), scale bar: 200 μm. g IHC staining of colon tissues on day 7 after DSS treatment for proliferation marker Ki67 and the quantification of Ki67⁺cells. Scale bar: 100 μm, scale bar of close-up image: 50 μm. h IF staining of colon tissues on day 7 after DSS treatment for macrophage marker F4/80 (scale bar: 50 μm, scale bar of close-up image: 10 μm) and quantification of the number of F4/80⁺ cells. Data are presented as mean ± S.D. and analyzed by non-paired two-tailed Student’s t-test in the graphs in (a, c, d–h). ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05; NS, no significance.