Figure 5. Fbxw7 deficiency downregulates CCL2 and CCL7 in resident macrophages.

(A) Colon tissue was cultured overnight, and cytokines in the supernatant were measured by ELISA (n = 5). (B) Heatmap showing expression changes of inflammation-related genes in BMDMs from LysM⁺ Fbxw7^fl/fl mice and Fbxw7^fl/fl littermates. The expression profile is accompanied by a colored bar indicating the standardized log₂ intensities. BMDMs were isolated from LysM⁺ Fbxw7^fl/fl mice and Fbxw7^fl/fl littermates and stimulated with LPS (100 ng/mL) for the transcriptomic assay. (C) CD11b⁺CX3CR1^hi resident macrophages and CD11b⁺CX3CR1^int proinflammatory MPhs were sorted from CLP of Fbxw7^fl/fl (WT) and LysM⁺ Fbxw7^fl/fl (KO) mice (n = 5) after 5 days of DSS or H₂O administration, and gene expression levels were quantified by gene-specific amplification and qRT-PCR. The ratio of Ccl2 and Ccl7 expression in CX3CR1^hi resident macrophages to Ccl2 and Ccl7 expression in CX3CR1^int MPhs is shown. (D) Colonic tissues of WT and KO mice treated with DSS were cultured overnight, CCL2 and CCL7 expression in the supernatant was measured by ELISA (n = 5). (E) In a Transwell assay, the number of monocytes in the lower chamber recruited by supernatant of CLP-derived CX3CR1^hi resident macrophages and CX3CR1^int proinflammatory MPhs was counted by flow cytometry (n = 5). (F) Concentration of CCL2 and CCL7 in the culture medium of CX3CR1^hi macrophages and CX3CR1^int MPhs (n = 5). (G) Number of monocytes in the lower chamber recruited by the supernatant of CX3CR1^hi resident macrophages from WT mice, with or without anti-CCL2 and anti-CCL7 Ab treatment (n = 5). (H) Gross morphology of colons from Fbxw7^fl/fl and LysM⁺ Fbxw7^fl/fl mice, with or without anti-CCL2 and anti-CCL7 Ab treatment and colon length measurements on day 9 (n = 5). (I) Body weight changes were assessed daily (n = 5). (J) The percentages of labeled WT and KO monocytes gated on CD45⁺Ly6C^hi living cells from DSS-challenged recipient mice (n = 5) were analyzed by flow cytometry. Graph shows a summary of the percentages of labeled WT monocytes recruited to the CLP of WT and KO recipient mice on day 5 or 9 after DSS treatment. Data are presented as the mean ± SD and are representative of 3 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired, 2-tailed Student’s t test.