Fig 1: IRF5/IKKß mRNA nanoparticles double survival of mice with ovarian cancer. a Time lines and dosing regimens. b Sequential bioluminescence imaging of tumor growth. c Kaplan–Meier survival curves. Statistical analysis was performed using the log-rank test. N = 10 biologically independent animals. d Flow cytometric quantitation of in vivo transfection rates in different immune cell subpopulations 48 h after a single i.p. dose of untargeted versus Di-mannose-coated NPs carrying GFP mRNA: macrophages (CD45+, CD11b+, MHCII+, CD11c-, Ly6C-/low, Ly6G-), monocytes (CD45+, CD11b+, MHCII+, CD11c-, Ly6C+, Ly6G-), dendritic cells (CD45+, CD11c+, CD11b-, MHCII+), neutrophils (CD45+, CD11b+, MHCII+, CD11c-, Ly6G+), CD4+ T cells (CD45+, TCR-ß chain+, CD4+, CD8-), CD8+ T cells (CD45+, TCR-ß chain+, CD4-, CD8+), and natural killer cells (CD45+, TCR-ß chain-, CD49b+) were measured. e Flow cytometric analysis of macrophage phenotypes in the peritoneum of mice with disseminated ID8 ovarian cancer. Animals were either treated with 4 doses of IRF5/IKKß NPs or PBS. f Box plots summarizing relative percent (left panel) and absolute numbers (right panel) of Ly6C-, F4/80+, and CD206+ (M2-like) macrophages. Corresponding numbers for Ly6C-, F4/80+, and CD206- (M1-like) macrophages are shown in (g). h Representative hematoxylin and eosin (H&E)-stained sections of ovarian tumor-infiltrated mesenteries isolated from PBS controls (left panel) or IRF5/IKKß NP-treated animals (right panel; scale bar 100 µm). Tenfold magnifications of representative malignant lesions are shown on the right (scale bar 50 µm). i Luminex assay measuring cytokines produced by isolated peritoneal macrophages from each treatment group. In parallel experiments, FACS-sorted CD11b+, F4/80+ peritoneal macrophages were directly analyzed by NanoString gene expression analysis. j Results are depicted as a Volcano plot. k Heat map of signature gene expression in macrophages isolated from mice treated with IRF5-NPs versus control PBS. All boxes in boxplots in this figure represent the mean values and the line in the box represents median. The bars across the boxes show the minimum and maximum values. Whiskers represent 95% confidence intervals. N = 5 biologically independent samples
Fig 2: Characterization of the neutrophils and MDSCs of EGFP TIIs ex vivo by flow cytometry.The markers of neutrophils are CD11b+Ly6G+Ly6Clow, and markers of MDSCs are CD11b+Gr1+.DOI: http://dx.doi.org/10.7554/eLife.14756.071
Fig 3: Long-Term Anti-tumor Effects of Trained Granulopoiesis(A) WT mice were treated with ß-glucan or PBS, and after 28 days were subcutaneously inoculated with B16-F10 melanoma cells. Tumor volume was monitored for another 14 days after tumor inoculation (n = 5 mice in the PBS group; n = 6 mice in the ß-glucan group).(B–D) As indicated in the experimental scheme (B), WT CD45.1+ mice were injected with ß-glucan or PBS, and after 7 days BM cells were isolated and were transplanted into CD45.2+ mice. Six weeks after transplantation, recipient mice were inoculated with tumors. In (C), (left) tumor volume and (right) the weight of B16-F10 melanoma tumors at the end of the experiment are shown (n = 6 mice per group). Shown in (D), 14 days after the tumor injection in recipient mice, TANs (CD45+CD11c-CD11b+Ly6c-Ly6g+) were sorted and relative mRNA expression of the “trained TAN1-like signature” was performed. Relative mRNA expression was normalized against 18S rRNA and was set as 1 in TANs from recipients that were transplanted with cells from PBS-treated donor mice (n = 4 mice per group).Data are presented as mean ± SEM; n.s.. non-significant; *p < 0.05, **p < 0.01, ***p < 0.001.See also Figure S3.
Fig 4: Effect of ethanol exposure on neutrophil and S. pneumoniae localization and bacterial phagocytosis after infection. (A) IHC staining was performed onformalin-fixed lung sections with antibodies against Ly6G for neutrophils (brown) and S. pneumoniae (pink) and counterstained with hematoxylin. Representative images are at 100x magnification for the top panel (scale bar = 200µm) and 400x magnification for the bottom panel (scale bar = 50 µm). (B) Percent phagocytosis of S. pneumoniae as measured by the ratio of cells with internalized bacteria to total nucleated cells. Black arrows denote cells with internalized S. pneumoniae. Data are presented as mean ± SEM. n = 3-4 mice per group per experiment and are representative of 2 individual experiments. *p < 0.05 by unpaired t test.
Fig 5: DDR1 induces CXCL5 production in pancreatic cancer cells.(A) Chemokine array analysis in cell lysate and supernatant of MDA-PATC 148 cells with knockdown DDR1. (B and C) MDA-PATC 148 cells with knockdown or reexpressed DDR1 were treated with collagen I for 3 hours. (B) CXCL5 mRNA level by using real-time PCR. (C) CXCL5 protein level by using ELISA. (D) CXCL5 expression in overexpressed DDR1 in 5 pancreatic cancer cell lines. Upper: DDR1 levels were checked by western; middle: CXCL5 mRNA level were detected by real-time PCR; lower: CXCL5 protein levels were analyzed by ELISA. (E–G) Mice were orthotopically injected with MDA-PATC 148 (control, DDR1-deficient or DDR1-reexpression clones) cells for 9 weeks. (E) IHC staining with anti-DDR1 (upper panel) and anti-CXCL5 (bottom panel) antibodies in pancreas. (F) ELISA showed CXCL5 level in plasma harvest from mice. (G) FACS by using anti-CD11b and anti-Ly6G antibodies to determine the presence of CD11b+Ly6G+ neutrophils infiltration in pancreas. (B–D) Data are mean ± SD. n = 3–4, 3 independent experiments; (B and C) 1-way ANOVA with Sidak post hoc testing; (D) Unpaired 2-tailed Student’s t test. *P < 0.05; **P < 0.01. (F and G) n = 5–10 mice, data performed in triplicate; 1-way ANOVA with Sidak post hoc testing. **P < 0.01; ***P < 0.001. Data show signal after membrane exposed to x ray film for 2 minutes.
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