Fig 2: E2-P4 C7 concentrations favor CC17 GBS crossing of the intestinal barrier through Peyer’s patches.SPF 3-week-old mice were administered E2-P4 cocktails for hormonal impregnation as described in Figure 1. (a, b, and f to h) Mice were gavaged by 2.1010 CFU of CC17 GBS. (a, b) Bacteria were enumerated 2 hr after infection in the intestine and Peyer’s patches either as tissue-associated bacteria (a) or as intratissular bacteria following tissue treatment with gentamicin (b). Bacterial counts are expressed in CFU per gram of tissue and were obtained following tissue homogenization and plating on Granada medium (n = 10 mice per group). (c to e) Representative confocal images (out of 12) of E2-P4 C7 mice small intestine ligated loops infected by 2.1010 CFU of CC17 GBS for 1 hr and showing intratissular bacteria (n = 3 mice). Small intestine sections showing bacteria associated with M cells (c) and CD11c positive cells (d). (e) View of a tridimensional representation of CC17 GBS associated with a CD11c positive cell. M cells were labeled with anti-UEA-I antibody, dendritic cells (DC) and macrophages with anti-CD11c antibody and nuclei with DAPI. White triangles indicate GBS cocci. (f, g) Intestinal mRNA levels of genes involved in leukocytes recruitment (f) and DC activation and migration (g) 2 hr after mouse oral gavage. Results are normalized to Actin and expressed as mean fold change relative to control condition ± SEM (n = 10 mice per group). (h) Flow cytometry analysis of CD11c and MHC-II positive cells in MLN of non-infected (NI, n = 3 mice per group) and CC17-infected mice (n = 7 mice per group) 2 hr after oral gavage of E2-P4 C0 and E2-P4 C7 mice. (a, b, and f to h) Lines are represented at median value. Multiple-group comparisons were performed by non-parametric two-way ANOVA (a, b) and Kruskal-Wallis test (f to h). *p<0.05; **p<0.01, ***p<0.001; ns: not significant.

Fig 3: Effect of CHA on glioblastoma progression in G422 xenograft model.ICR mice were injected subcutaneously in the flank with G422 glioma cells in and were treated with CHA (20 mg/kg or 40 mg/kg per day) for 14 days or cisplatinum (CPT) (6 mg/kg per week). The tumor weight (A) and body weight (B) in individual mice were detected. The infiltration of M1 macrophages (C) in the tumor tissues were evaluated using flow cytometry. The percentage of CD11c, MHC-II IA/IE and F4/80 positive macrophages was presented in the histogram (D). The infiltration of M2 macrophages (E) in the tumor tissues were evaluated using flow cytometry. The percentage of CD206 and F4/80 positive macrophages was presented in the histogram (F). Furthermore, M1 macrophages infiltrating in tumor sections were visualized by immunofluorescence staining of F4/80 and CD11c (G). M2 macrophages infiltrating in tumor sections were visualized by immunofluorescence staining of F4/80 and CD206 (H). The F4/80+CD11c+ area (I) and the F4/80+CD206+ area (J) (% of F4/80+ tumor area) were qualified as area value of overlapping fields. Five random fields from sections of each mouse tumor xenograft were examined. The data are presented as the mean ± SD. *p-value < 0.05, **p-value < 0.01 vs. control.

Fig 4: The effects of liposomal honokiol (Lip-HNK) on the progression of glioblastomas in a G422 xenograft model. G422 glioma cells were administered subcutaneously into the flanks of ICR mice, which were then treated with Lip-HNK (12.5 or 25 mg/kg per day) or temozolomide (TMZ) (50 mg/kg). The weights of the tumors (A) and tumor volume changes (B) were measured. Moreover, visualization of the infiltrating M1 macrophages in tumor sections was achieved using immunofluorescence staining of CD11c and F4/80 (C). Visualization of the tumor sections infiltrated by M2 macrophages was achieved using immunofluorescence staining of F4/80 and CD206 (D). The F4/80+CD11c+ area (E) and the F4/80+CD206+ area (F) (% of F4/80+ tumor area) were quantified as the area of intersecting fields. Five randomly selected fields from mouse xenograft tumor sections were examined. *, P<0.05; **, P<0.01; ***, P<0.001.

Fig 5: DCs are a significant source of CXCR3 ligands during chronic infection(A) Representative flow cytometry plots of chemokine expression in REX3 spleens before infection, 3 weeks after infection, and 6 weeks after infection.(B and C) Summary data of the percentages (B) and absolute numbers (C) of chemokine-producing splenocytes during infection.(D and E) Splenocytes from REX3 mice were stained with the indicated surface markers. Cells were then gated on chemokine expression, and the phenotype of different chemokine-expressing subsets was measured. Shown are (D) representative plots of CD11c × CD11b staining and (E) summary data. The mean and standard deviation of the percent of CD11c+ cells in each population is displayed above each chart.(F) Subsets of DCs were identified (Figure S6), and their chemokine expression was analyzed in uninfected mice and 6 weeks after infection.Data in (A)–(E) are pooled from three separate experiments. Data in (F) are combined from two separate experiments (n = 3 mice for uninfected, n = 8 mice for 6 weeks post infection).