Fig 2: PcrV reeducates tumor-associated macrophages (TAMs) into a tumoricidal M1 phenotype in vivo. Mice bearing LLC cell-derived tumors were treated with PBS or PcrV. (A–D) FACS analysis of the percentages of M1 (CD45+F4/80+CD11c+CD206-, CD45+F4/80+MHCII+, and CD45+F4/80+CD86+) and M2 (CD45+F4/80+CD11c-CD206+) TAMs in tumor tissues. (E, F) Immunofluorescence staining for visualizing F4/80+iNOS+ M1 and F4/80+Arg1+ M2 TAMs in tumor tissues. Data were expressed as means ± SD [(B, D), n = 6] and analyzed by unpaired Student’s t-test. *p < 0.05 and ***p < 0.001. Ctrl indicates control. LLC, Lewis lung cancer; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorting.

Fig 3: PcrV-primed macrophages suppress tumor growth by increasing the levels of apoptosis and iNOS expression in tumor tissues. (A) Schematic of the mouse models used. (B) Immunofluorescence staining of F4/80+ macrophages in the tumor tissues (harvested on day 16) of mice treated with PBS liposomes or clodronate liposomes (day 0 and 7). Tumor growth (C) and weight (D) were measured in LLC cell-derived tumor-bearing mice treated with PBS, PcrV, or bone marrow-derived macrophages (BMDMs) primed or not with PcrV. (E) Apoptosis was detected by TUNEL assay. (F) iNOS protein level and F4/80+iNOS+ M1 TAMs in tumor tissues were detected by immunofluorescence staining. Data were expressed as means ± SEM (C, n = 4) or means ± SD (D, n = 4), and were analyzed by two-way ANOVA (C) or unpaired Student’s t-test (D). **p < 0.01. s.c, subcutaneous injection; i.p., intraperitoneal injection; i.t., intratumoral injection; BM, BMDMs; PBS-lipo, PBS liposomes; Clo-lipo, clodronate liposomes; iNOS, inducible nitric oxide synthase; PBS, phosphate-buffered saline; LLC, Lewis lung cancer. n.s, no significance.

Fig 4: Cellular recruitment to the bronchoalveolar lavage fluid (BALF) of mice and effects of an iNOS inhibitor on the survival curve. (A) The number of total macrophages (CD45+ CD11b+ F4/80+); (B) total macrophage MFI (CD11b+); (C) MFI of activated macrophages (MHCII+) and (D) MFI of iNOS+ macrophages; (E) Representative histograms of CD11b+ (left), MHC-II (center) and iNOS (right) staining in macrophages (CD45+F4/80+CD11b+ cells) from lung homogenates of mono and co-infected mice; (F) Number of total neutrophils (CD11b+ LY6G+); (G) MFI of activated neutrophils (MHCII+) and (H) MFI of iNOS+ neutrophils; (I) Representative histograms of MHC-II (top) and iNOS (bottom) staining in neutrophils CD45+Ly6G+CD11b+ cells) from BALF of mono and coinfected mice. *p<0.05 compared with the NI group. #p<0.05 compared with the Cg group. (ANOVA/Newman-Keuls multiple comparisons test). NI: non-infected group; Pa3d: group infected with P. aeruginosa and euthanized 3 days after; Cg: group infected with C. gattii; Pa+Cg: group infected with P. aeruginosa 3 days before C. gattii infection. Cg and Pa+Cg groups were euthanized 10 days after Cg infection. MFI (Median Fluorescence Intensity). (J) Survival curve of mice treated and non-treated (NT) with Aminoguanidine Hydrochloride. *** p <0.001 compared with all the other groups. ** p <0.01 compared with the CgNT group (Log-rank test). Flow cytometry data are representative of three independent experiments.

Fig 5: S-IT MSCs efficiently promote polarization toward the M2 phenotype in vitro. (a) The percentage of CD206-positive, CD86-positive, Arg-1-positive and iNOS-positive cells in macrophages after coculture with DMEM, S-MSCs or S-IT MSCs by flow cytometry. (b) The corresponding bar chart. *P < 0.05, **P < 0.001, ***P < 0.0001.