Fig 1: The adoptive transfer of normal alveolar macrophages or CCL24 neutralization rescues anti-tumor immune impairment in Abt mice. The mice were given antibiotics for five weeks and then challenged with B16/F10 cells (1 × 105 cells/mouse, i.v.). Alveolar macrophages transfer and CCL24 neutralization were performed in the Abt mice. The mice were treated with antibiotics for the entire experimental period. (a) The lungs were analyzed on day 17 after the B16/F10 melanoma challenge. The graphs showed the total number of tumor colonies present in the lung lobes, and the numbers of tumor foci in the lungs were calculated. (b) The total number of MNCs in the lungs on day 17 after the B16/F10 challenge. (c) The frequency of ?dT cells among lymphocytes in the lungs, and the absolute number were shown. The isolated MNCs were analyzed by FACS and the lymphocytes were gated by FSC and SSC. (d) The frequency of IL-17A+ ?dT cells and IFN-?+ ?dT cells among total ?dT cells in the lungs, and their absolute numbers were shown. There were six mice in each group. The data are shown as the mean ± SEM. *p < 0.05 compared with the control group.
Fig 2: Decreased numbers but M2 polarization of alveolar macrophages in Abt mice challenged with B16/F10 melanoma. The mice were given antibiotics for five weeks and then challenged with B16/F10 cells (1 × 105 cells/mouse, i.v.). On day 17 after the B16/F10 challenge, the lung MNCs were isolated. (a) Frequency and number of alveolar macrophages (F4/80hi CD11chi) in the lung MNCs from mice challenged with B16/F10 cells (n = 5). (b) Purified alveolar macrophages (F4/80hi CD11chi) were analyzed by GeneChip. The pie chart showed the distribution of DEGs in Abt group compared with the control. Unknown and duplicated genes were filtered for the analysis (2 samples/group, 15 mice/sample). (c) Purified alveolar macrophages (F4/80hi CD11chi) (3 × 105 cells/well) were stimulated for 48 h in the DMEM containing 10% FBS with the stimulation of 1 µg/ml LPS, 100 µg/ml PolyI:C, 1 µg/ml flagellin or 20 ng/ml PMA, and then culture supernatants were analyzed for the cytokine expression by ELISA (Arg1 and CCL24) and CBA (IL-13, IL-6, IL-10 and IL-1ß) (n = 3). The data are shown as the mean ± SEM. *p < 0.05 compared with the control group.
Fig 3: Recovered commensal bacteria restore the functions of alveolar macrophages in Abt mice. The mice in Abt group were given antibiotics for five weeks. In the recovered group, the mice were given antibiotics for five weeks and then co-housed with the wild type mice for four weeks. Purified alveolar macrophages (F4/80hi CD11chi) were analyzed. (a) M1 and M2 specific gene expression in alveolar macrophages from Abt mice was compared with that in control mice by GeneChip analysis. (b and c) Bacterial loads in the upper respiratory tract and stool from the recovered mice compared with those in the control mice (n = 3/group) were measured by BAP culture. (d and e) Relative abundances (d) and a clustering map (e) for the bacteria in the upper respiratory tract were determined by 16S rRNA analysis (3 samples/group, 10 mice/sample). (f and g) Relative abundances (f) and a clustering map (g) for the bacteria in the stool were determined by 16S rRNA analysis (3 mice/group). (h) Purified alveolar macrophages (F4/80hi CD11chi) were stimulated for 48 h in the indicated culture medium (1 µg/ml LPS, 100 µg/ml PolyI:C, 1 µg/ml flagellinor 20 ng/ml PMA) and then analyzed for the protein expression by ELISA (Arg1 and CCL24) and CBA (IL-13, IL-6, IL-10 and IL-1ß) (n = 3). The data are shown as the mean ± SEM. *p < 0.05, **p < 0.01 compared with the control group.
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