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. 2001 Apr;69(4):2692-9.
doi: 10.1128/IAI.69.4.2692-2699.2001.

Antimycobacterial agent based on mRNA encoding human beta-defensin 2 enables primary macrophages to restrict growth of Mycobacterium tuberculosis

K O Kisich  1 , L Heifets, M Higgins, G Diamond
Affiliations

Antimycobacterial agent based on mRNA encoding human beta-defensin 2 enables primary macrophages to restrict growth of Mycobacterium tuberculosis

K O Kisich et al. Infect Immun. 2001 Apr.

Abstract

Human macrophages are hosts for Mycobacterium tuberculosis, the causative agent of tuberculosis, which killed approximately 1.87 million people in 1997. Human alveolar macrophages do not express alpha- or beta-defensins, broad-spectrum antimicrobial peptides which are expressed in macrophages from other species more resistant to infection with M. tuberculosis. It has been previously reported that M. tuberculosis is susceptible to killing by defensins, which may explain the difference in resistance. Defensin peptides have been suggested as a possible therapeutic strategy for a variety of infectious diseases, but development has been hampered by difficulties in their large-scale production. Here we report the cellular synthesis of human beta-defensin 2 via highly efficient mRNA transfection of human macrophages. This enabled mycobactericidal and mycobacteristatic activity by the macrophages. Although human macrophages are difficult to transfect with plasmid vectors, these studies illustrate that primary macrophages are permissive for mRNA transfection, which enabled expression of a potentially therapeutic protein.

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Figures

FIG. 1
FIG. 1
Fluorescence of human-monocyte-derived macrophages after mRNA transfection. (a) Phase-contrast image of MDM transfected with yeast tRNA as negative control for autofluorescence. Magnification, ×400. (b) Fluorescence image of panel a showing dim autofluorescence. (c) Fluorescence image of macrophages transfected with mRNA encoding eGFP/showing enhanced fluorescence for the majority of the macrophages. The images shown are representative of five experiments.
FIG. 2
FIG. 2
Intensity and frequency of eGFP expression following mRNA transfection. (a) Fluorescence intensity of human MDM following transfection with increasing amounts of eGFP mRNA-Oligofectin G complex. The fluorescence intensity is represented as relative light units (RLU), with the average background of the image subtracted. (b) Frequency of eGFP-positive cells as a function of increasing concentration of transfection complex. Each point represents the percentage of cells in three fields which are more than two standard deviations above the RNA control for that concentration.
FIG. 3
FIG. 3
Cell viability after transfection with mRNA lipid complexes. Cell viability was measured by incubation with 5 mg of MTT. After 24 h the absorbance of reduced MTT was measured at 585 nm for macrophages treated with Oligofectin G–HBD-2 mRNA complex (■) or Oligofectin G-eGFP mRNA complex (⧫). Cell viability was measured via reduction of MTT in at least three experiments, and representative results are presented here.
FIG. 4
FIG. 4
Association of HBD-2 with intracellular M. tuberculosis following transfection with mRNA encoding HBD-2. (a) Nomarski image (magnification, ×1,000) of cells transfected with mRNA encoding HBD-2 immunostained with preimmune serum and counterstained with auramine-O to show M. tuberculosis (arrows). (b) Fluorescence image of panel a showing locations of macrophages (dim green) and M. tuberculosis (bright green). (c) Cells transfected with control mRNA and stained with anti-HBD-2 monoclonal antibody, showing a lack of specific staining for HBD-2 corresponding to the M. tuberculosis shown in panel d. (d) Fluorescence image of panel c showing locations of M. tuberculosis. (e) Nomarski image of cells transfected with mRNA encoding HBD-2 immunostained with specific anti-HBD-2 serum and counterstained with auramine-O. The brown precipitate of the diaminobenzidine appears black under Nomarski optics and partially quenches the fluorescence from auramine-O seen in panel f. (f) Fluorescence image of panel c, showing the locations of M. tuberculosis.
FIG. 5
FIG. 5
Inhibition of mycobacterial growth in MDM monolayers. (a) Growth of M. tuberculosis following transfection with mRNA encoding HBD-2 or eGFP. The percent growth or decline of the mycobacteria was calculated by dividing the average number of colonies counted on four replicate plates on day 4 by the average number from the time zero lysates, with the result multiplied by 100, e.g., (day 4/day 0) × 100. The results are representative of three experiments. (b) Duration of growth inhibition following mRNA transfection. Macrophage monolayers were infected with M. tuberculosis Erdman at a 10:1 ratio for 1 h. Infected cells were then transfected with either eGFP mRNA or HBD-2 mRNA at the indicated concentrations. Cultures were lysed at the days indicated, and mycobacterial CFU were measured as in panel a. Error bars indicate the standard deviation of four samples. These results are representative of four experiments.
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References

    1. Aurup H, Siebert A, Benseler F, Williams D, Eckstein F. Translation of 2′-modified mRNA in vitro and in vivo. Nucleic Acids Res. 1994;22:4963–4968. - PMC - PubMed
    1. Clemens D L, Horwitz M A. The Mycobacterium tuberculosis phagosome interacts with early endosomes and is accessible to exogenously administered transferrin. J Exp Med. 1996;184:1349–1355. - PMC - PubMed
    1. Cole A M, Dewan P, Ganz T. Innate antimicrobial activity of nasal secretions. Infect Immun. 1999;67:3267–3275. - PMC - PubMed
    1. Couto M A, Liu L, Lehrer R I, Ganz T. Inhibition of intracellular Histoplasma capsulatum replication by murine macrophages that produce human defensin. Infect Immun. 1994;62:2375–2378. - PMC - PubMed
    1. Deretic V, Fratti R A. Mycobacterium tuberculosis phagosome. Mol Microbiol. 1999;31:1603–1609. - PubMed

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