Figure 4.

The activation of the host immune response was significantly weakened for the flagellum-deficient bacteria. (A) The weight of spleens of immunocompetent mice bearing melanoma allografts 6 days after the intraperitoneal treatment of wild-type VNP20009 (labelled as VNP in the figure), ΔflhD and ΔfliE strains. Data are mean ± SD, n = 3. Representative images of resected spleens were shown below the columns. The percentage of CD69⁺ cells in total CD4⁺ T cells (B) and total CD8⁺ T cells (C) were measured by flow-cytometry 6 days after the intraperitoneal treatment of wild-type VNP20009, ΔflhD and ΔfliE strains. Data are mean ± SD, n = 3. The percentage of CD4⁺ T cells (D), CD8⁺ T cells (E) and F4/80⁺ macrophages (F) infiltrating in tumors were measured by flow-cytometry 6 days after the intraperitoneally treatment of wild-type VNP20009, ΔflhD and ΔfliE strains. Data are mean ± SD, n = 3. (G) The intratumoral concentrations of various inflammatory cytokines (C_a) were measured by Bio-Plex Multiplex Suspension Array 6 days after the intraperitoneal treatment of wild-type VNP20009, ΔflhD and ΔfliE strains. The fold change of the cytokine concentration (CC_a) relative to the mean of all mice (C_mean) was calculated by the formula: CC_a=(CC_a–C_mean)/C_mean × 100%. The values were converted into colors and plotted. Green indicates reductions, and red indicates increase. (H) The RNA levels of Il4, Il5, Il13, Il17α, Il21, Il22 and Ifng in the tumor-infiltrating T cells were compared by RT-PCR 6 days after the intraperitoneal treatment of wild-type VNP20009, ΔflhD and ΔfliE strains. (I) The activation status of ERK/JNK/MAPK and NF-κB signaling pathways was evaluated by Western blots. ∗∗∗P < 0.001; n.s. stands for nonsignificant. PBS: phosphate-buffered saline; i.p. stands for intraperitoneally.