Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Exploitation of synthetic mRNA to drive immune effector cell recruitment and functional re-programming in vivo .

doi: 10.4049/jimmunol.1800924

Figure Lengend Snippet: We examined the biological impact of introducing BMDMs transfected with synthetic mRNA encoding CCL3 into the peritoneal cavity of mice. A: Experiment setup. In brief, we transfected synthetic mRNAs encoding either CCL2 or CCL3 into bone marrow-derived macrophage (BMDM) generated from CD45.1+ congenic mice. As control, we transfected BMDM with VR without mRNA. 8h after transfection, the cells were harvested, washed with PBS, and injected intraperitoneally into CD45.2+ congenic mice. 16h later, we collected the cells from the peritoneal cavity and analyzed them by flow cytometry. The use of the congenic CD45.1+/CD45.2+ mouse strains enabled us to discriminate between the donor BMDM transfected with the chemokine mRNAs and the resident tissue macrophages. B: Demonstration of the presence of CCL3 protein in the supernatant from BMDMS transfected with Ccl3 mRNA, as well as in the peritoneal fluid from mice inoculated with these BMDMs. C: Ccl3 synthetic mRNA transfected BMDM recruited a population of Ly6ChiCD11b+ monocytes (Gate 1), increased the number of Ly6CintCD11b+ monocytes (Gate 2), and decreased the number of F4/80hi large peritoneal macrophages. Experiments were repeated 3 times. Data showed representative flow plot from one experiment. D: The numbers of Ly6Chi and Ly6CintCD11b+ monocytes, F4/80hi large peritoneal macrophage and Ly6G+ neutrophil were quantified. E: As an additional control to demonstrate the recruitment phenotype was due to CCL3-encoding mRNA we compared the levels of cellular recruitment to VR alone, VR coated with Gfp mRNA and VR coated with Ccl3 mRNA. The recruitment of the cell population of significance, the Ly6Cint CD11b+ monocytes was clearly specific to the presence of the Ccl3 mRNA. Data represent mean ± SD. ns: p>0.05, **: p < 0.01, ***: p < 0.001, Unpaired t test with Wench correction.

Article Snippet: D : Representative flow plots of iNOS and RELM-α expression in these monocytes recruited by Viromer Red complexed mRNAs.

Techniques: Transfection, Derivative Assay, Generated, Injection, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Exploitation of synthetic mRNA to drive immune effector cell recruitment and functional re-programming in vivo .

doi: 10.4049/jimmunol.1800924

Figure Lengend Snippet: In order to demonstrate that the recruited cells can be polarized towards M1- and M2-like macrophage phenotypes we co-transfected BMDMs with cytokine-encoding mRNAs. A: Representative flow plots showing the expression of iNOS and RELM-α in recruited monocytes (Ly6C+CD11b+, upper panel) and in large peritoneal macrophages (F4/80hiCD11b+, lower panel) following their induction of Ccl3 mRNA in combination with Ifn-γ or Il-4 mRNA. B: The numbers of iNOS+ or RELM-α+ recruited monocytes and F4/80hi macrophages were quantified and shown to increase in the presence of appropriate cytokine-encoding mRNAs Experiments were repeated three times with comparable results. Data represent mean ± SD. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001, One-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: D : Representative flow plots of iNOS and RELM-α expression in these monocytes recruited by Viromer Red complexed mRNAs.

Techniques: Transfection, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Exploitation of synthetic mRNA to drive immune effector cell recruitment and functional re-programming in vivo .

doi: 10.4049/jimmunol.1800924

Figure Lengend Snippet: To generate functional data demonstrating the polarization of the recruited macrophages we quantified the intensity of their superoxide burst. A: The kinetics and magnitude of superoxide burst generated by BMDMs treated with or without recombinant IFN-γ and IL-4. Activation with IFN-γ enhanced the superoxide burst (Oxyburst beads), while alternative activation with IL-4 resulted in its decrease. Analysis was performed on a Perkin Elmer Envision fluorescent plate reader and the experiment was repeated three times. B: The kinetics and magnitude of superoxide burst generated by peritoneal cells from mice (n=3) injected with BMDMs transfected with mRNAs encoding CCL3 alone and in combination with either Ifn-γ and Il-4 mRNAs. The experiment was repeated twice. C: Peritoneal cells from mice (n=3) injected with different mRNA transfected BMDMs were analyzed by flow cytometry. Representative flow plots showing the gating of positive Oxyburst signal. Oxyburst beads without cells served as the negative control (upper panel). The lower panels were gated on recruited monocytes (Ly6C+CD11b+) from 4 groups incubated with Oxyburst beads and harvested at different times (10min, 30min and 60min). The experiment was repeated three times. D: Quantification of fluorescent index of mean Oxyburst bead fluorescent intensity. The mean Oxyburst fluorescent intensity of all the recruited monocytes that were positive for calibration fluor were calculated. The number was divided by the mean calibration fluorescent intensity to generate a fluorescent index. The experiment was repeated three times with comparable results. Data represent mean ± SD. *: p < 0.05, **: p < 0.01, ***: p < 0.001, One-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: D : Representative flow plots of iNOS and RELM-α expression in these monocytes recruited by Viromer Red complexed mRNAs.

Techniques: Functional Assay, Generated, Recombinant, Activation Assay, Injection, Transfection, Flow Cytometry, Negative Control, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Exploitation of synthetic mRNA to drive immune effector cell recruitment and functional re-programming in vivo .

doi: 10.4049/jimmunol.1800924

Figure Lengend Snippet: The potential of this approach for therapeutic applications is supported by the demonstration that the mRNA-coated VR particles can mediate comparable effects following direct inoculation. A: Representative flow plots of monocyte populations in the peritoneal cavity after injection of naked, Ccl3 mRNA or Ccl3 mRNA-coated Viromer Red particles. B: Representative flow plots of F4/80hi large peritoneal macrophage in the peritoneal cavity after injection of naked, Ccl3 mRNA or Ccl3 mRNA-coated Viromer Red particles. C: Quantification of the numbers of Ly6Chi and Ly6Cint monocytes, F4/80hi large peritoneal macrophage and Ly6G+ neutrophil. D: Representative flow plots of iNOS and RELM-α expression in these monocytes recruited by Viromer Red complexed mRNAs. E: Quantification of iNOS+ and RELM-α+ number of monocyte recruited by Viromer Red complexed mRNAs. Experiment was repeated two times. Data represent mean ± SD. ns: p>0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001, One-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: D : Representative flow plots of iNOS and RELM-α expression in these monocytes recruited by Viromer Red complexed mRNAs.

Techniques: Injection, Expressing