Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Jan 15;202(2):608-617.
doi: 10.4049/jimmunol.1800924. Epub 2018 Dec 12.

Exploitation of Synthetic mRNA To Drive Immune Effector Cell Recruitment and Functional Reprogramming In Vivo

Yitian Xu  1   2 , Lu Huang  2 , Jonathan L Kirschman  3 , Daryll A Vanover  3 , Pooja M Tiwari  3 , Philip J Santangelo  3 , Xiling Shen  1   4   5 , David G Russell  6
Affiliations

Exploitation of Synthetic mRNA To Drive Immune Effector Cell Recruitment and Functional Reprogramming In Vivo

Yitian Xu et al. J Immunol. .

Abstract

Therapeutic strategies based on in vitro-transcribed mRNA (IVT) are attractive because they avoid the permanent signature of genomic integration that is associated with DNA-based therapy and result in the transient production of proteins of interest. To date, IVT has mainly been used in vaccination protocols to generate immune responses to foreign Ags. In this "proof-of-principle" study, we explore a strategy of combinatorial IVT to recruit and reprogram immune effector cells to acquire divergent biological functions in mice in vivo. First, we demonstrate that synthetic mRNA encoding CCL3 is able to recruit murine monocytes in a nonprogrammed state, exhibiting neither bactericidal nor tissue-repairing properties. However, upon addition of either Ifn-γ mRNA or Il-4 mRNA, we successfully polarized these cells to adopt either M1 or M2 macrophage activation phenotypes. This cellular reprogramming was demonstrated through increased expression of known surface markers and through the differential modulation of NADPH oxidase activity, or the superoxide burst. Our study demonstrates how IVT strategies can be combined to recruit and reprogram immune effector cells that have the capacity to fulfill complex biological tasks in vivo.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.. mRNA with N1-methyl pseudouridine substitution induces high protein expression levels with limited target cell activation.
We explored different chemical modifications of synthetic mRNA in Hela cells to assess both efficiency of expression and minimization of target cell activation. A: Expression of GFP and Luciferase by mRNAs with different chemical modifications (5meC, Y, 5moC, m1Y, 5moU and combinations of these modifications) in Hela cells. B: Percentage of G3BP+ Hela cells (contain stress granules) following transfection with mRNAs with different modifications. C: Expression of IFN-β and IL-6 in Hela cells when transfected with Gfp mRNAs with different modifications as demonstration of immune activation.
Figure 2.
Figure 2.. J774 cells transfected with synthetic mRNA encoding GFP, CCL2 or CCL3, respectively.
We examined the expression of synthetic mRNAs encoding GFP, CCL2, and CCL3 in the murine macrophage-like cell line J774. A: Cells were transfected with 1μg of Gfp mRNA and fixed 24 hours post-transfection. Cells were stained for GFP (green) and nuclei were stained with DAPI (blue). Scale bar represents 12μm. B: CCL2 and CCL3 were detected by ELISA in in tissue culture supernatants following transfection of cells with either 1μg of Ccl2 or Ccl3 mRNA (blue) or following incubation with 10μg/mL LPS for 24 h. Scale bars represent standard deviation. Data represents mean of two independent experiments. Statistical significance was measured by two-way ANOVA. *: p<0.05. **: p<0.01. ***: p<0.001. ****: p<0.0001.
Figure 3.
Figure 3.. Ccl3 synthetic mRNA transfected BMDM modify the cellular populations in the peritoneal cavity.
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.
Figure 4.
Figure 4.. Peripheral blood-derived monocytes are recruited to the peritoneal cavity.
We demonstrate that the cells recruited into the peritoneal cavity in response to CCL3 had their origin in the blood. A: Representative flow plots showing mice in which peripheral blood monocytes were isolated from CD45.1+ mice and transferred intravenously (1X106 monocytes transferred per mouse) to CD45.2+ mice. These CD45.2+ mice were injected intraperitoneally with BMDMs transfected with or without Ccl3 mRNA and the presence of recruited CD45.1+ monocytes in the peritoneal cavity was assessed. Experiments were repeated 2 times. B: The number of donor CD45.1+ monocytes, Ly6Chi, Ly6Cint and Ly6Clow monocytes were quantified. Data represent mean ± SD. ns: p>0.05, *: p < 0.05, ***: p < 0.001, Unpaired t test with Wench correction.
Figure 5.
Figure 5.. Co-transfection of Ifn-γ or Il-4 synthetic mRNA, in combination with Ccl3 mRNA, reprograms both resident and recruited cell phenotypes.
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.
Figure 6.
Figure 6.. Quantification of NADPH Oxidase activity in the reprogrammed phagocytes correlates with the M1 or M2 activation status.
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.
Figure 7.
Figure 7.. mRNA complexed with Viromer Red particles can recruit similar populations of monocytes and differentiate them towards different activation statues.
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.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Kuhn AN, Beibetaert T, Simon P, Vallazza B, Buck J, Davies BP, Tureci O, and Sahin U. 2012. mRNA as a versatile tool for exogenous protein expression. Curr Gene Ther 12: 347–361. - PubMed
    1. Sahin U, Kariko K, and Tureci O. 2014. mRNA-based therapeutics--developing a new class of drugs. Nat Rev Drug Discov 13: 759–780. - PubMed
    1. Kormann MS, Hasenpusch G, Aneja MK, Nica G, Flemmer AW, Herber-Jonat S, Huppmann M, Mays LE, Illenyi M, Schams A, Griese M, Bittmann I, Handgretinger R, Hartl D, Rosenecker J, and Rudolph C. 2011. Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nat Biotechnol 29: 154–157. - PubMed
    1. Anderson BR, Muramatsu H, Jha BK, Silverman RH, Weissman D, and Kariko K. 2011. Nucleoside modifications in RNA limit activation of 2’−5’-oligoadenylate synthetase and increase resistance to cleavage by RNase L. Nucleic Acids Res 39: 9329–9338. - PMC - PubMed
    1. Andries O, Mc Cafferty S, De Smedt SC, Weiss R, Sanders NN, and Kitada T. 2015. N(1)-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice. J Control Release 217: 337–344. - PubMed

Publication types

MeSH terms