Role of iron in Nramp1-mediated inhibition of mycobacterial growth

Abstract

Innate resistance to mycobacterial growth is mediated by a gene, Nramp1. We have previously reported that Nramp1 mRNA from macrophages of Mycobacterium bovis BCG-resistant (Bcgr) mice is more stable than Nramp1 mRNA from macrophages of BCG-susceptible (Bcgs) mice. Based on these observations and on reports that show that the closely related Nramp2 gene is a metal ion transporter, we evaluated the effect of iron on the growth of Mycobacterium avium within macrophages as well as on the stability of Nramp1 mRNA. The addition of iron to macrophages from Bcgs mice resulted in a stimulation of mycobacterial growth. In contrast, iron increased the capacity of macrophages from Bcgr mice to control the growth of M. avium. When we treated recombinant gamma interferon (IFN-gamma)-activated macrophages with iron, we found that iron abrogated the growth inhibitory effect of IFN-gamma-activated macrophages from Bcgs mice but that it did not affect the capacity of macrophages from Bcgr mice to control microbial growth. A more detailed examination of the effect of iron on microbial growth showed that the addition of small quantities of iron to resident macrophages from Bcgr mice stimulated antimicrobial activity within a very narrow dose range. The effect of iron on the growth inhibitory activity of macrophages from Bcgr mice was abrogated by the addition of catalase or mannitol to the culture medium. These results are consistent with an Fe(II)-mediated stimulation of the Fenton/Haber-Weiss reaction and hydroxyl radical-mediated inhibition of mycobacterial growth.

FIG. 1

Effect of iron on the growth of M. avium in macrophages from Bcg^r and Bcg^s mice. Resident peritoneal macrophages were infected with M. avium and then treated with ferric ammonium sulfate. After 5 days of culture, the macrophages were lysed and the bacteria were labeled with [³H]uracil. The radioactivity incorporated by the bacteria is an indication of the number of metabolically active bacteria remaining in the culture. The data are expressed as the percentages of inhibition of growth compared to growth of cultures of macrophages not treated with iron. The amount of radioactivity (mean ± standard deviation) incorporated by M. avium from Bcg^r macrophages was 18,627 ± 2,899 cpm, while that incorporated by M. avium from macrophages from Bcg^s mice was 31,095 ± 2,602 cpm. There were no differences noted in the numbers of bacteria phagocytized by the macrophages at the beginning of the experiment. The effect of iron on the growth of the bacteria in macrophages from Bcg^s mice was significant as determined by analysis of variance (ANOVA). Similarly, the effect of iron on the inhibition of mycobacterial growth in macrophages from Bcg^r mice was also significant as determined by ANOVA. Thus, Fe stimulated the growth of the mycobacteria in macrophages from Bcg^s mice. In contrast, at low levels iron suppressed the growth of the mycobacteria in macrophages from Bcg^r mice.

FIG. 2

The addition of iron to cultures of macrophages infected with M. avium results in the inhibition of mycobacterial growth. For comparison with macrophages treated with rIFN-γ, resident peritoneal macrophages were treated with 100 U of rIFN-γ/ml overnight prior to infection with M. avium. Other cultures were infected with M. avium and treated with iron but not treated with rIFN-γ. After 5 days, the macrophages were lysed and the bacteria were labeled with [³H]uracil. The amount of radioactivity is an indication of the amount of metabolically active bacteria in the cultures. The data are expressed as the percentages of inhibition of bacterial growth compared to growth of cultures not treated with rIFN-γ or iron. The effect of rIFN-γ treatment was significant, as was the effect of iron, on mycobacterial growth as determined by analysis of variance.

FIG. 3

Iron differentially affects the capacity of IFN-γ-activated macrophages from Bcg^r or Bcg^s mice to inhibit the growth of mycobacteria. Macrophages were activated by treatment with 100 U of rIFN-γ/ml prior to infection with M. avium. The radioactivity incorporated by the bacteria was determined after 5 days in culture. The data are expressed as the percentages of inhibition of mycobacterial growth based on the growth of the mycobacteria in macrophages not treated with rIFN-γ. The mycobacteria isolated from macrophages of Bcg^s mice that had not been treated with rIFN-γ incorporated 42,025 ± 2,561 cpm (mean ± standard deviation), while those isolated from macrophages of Bcg^r mice incorporated 31,689 ± 2,882 cpm. The effect of iron on the growth of M. avium in macrophages from Bcg^s mice was significant. Iron did not affect the capacity of the rIFN-γ-treated macrophages from Bcg^r mice to inhibit the growth of the bacterium.

FIG. 4

Inhibitors of hydroxyl radical generation prevent the increased antimycobacterial activity induced by iron. Resident peritoneal macrophages from Bcg^r mice were infected with M. avium and cultured with 0.05 μM Fe in the presence or absence of the following: SOD, 1 mg/ml; MMLA, 5 mM; mannitol, 50 mM; catalase, 2 mg/ml; and DMSO, 150 mM. Several concentrations were used for each of the inhibitors; the effect of the optimal concentration is shown. The effects of mannitol, catalase, and DMSO were significant at a P of <0.05 or lower. The effect of SOD or MMLA was not significant.

FIG. 5

Manipulation of intracellular iron affects mRNA stability. Splenic macrophages from Bcg^r or Bcg^s mice were stimulated with 100 U of rIFN-γ/ml in the presence of 100 μM desferrioxamine (Desferox) or 50 μM ferric ammonium sulfate. After 20 h, the macrophage cultures were treated with 20 μg of actinomycin D (Act D) or medium for 2, 4, 8, 16, 32, or 48 h prior to extraction and isolation of total RNA. Control cultures (lane 1) were treated with rIFN-γ only. The corresponding decay curve of Nramp1 mRNA expression, representing the percentages of mRNA remaining following actinomycin D treatment, is derived from densitometric analysis of Northern blots. ■, Mutant (Bcg^s) plus desferrioxamine or Fe; □, mutant (Bcg^s) without desferrioxamine or Fe; ▴, wild-type (Bcg^r) plus desferrioxamine or Fe; ▵, wild-type (Bcg^r) without desferrioxamine or Fe.

FIG. 6

Iron transport by phagosomes isolated from the RAW264.7 macrophage cell lines, transfected with Nramp1^Gly169 (resistance) or Nramp1^Asp169 (susceptibility) alleles and infected with M. avium. Macrophage cell lines were labeled with ⁵⁵Fe-labeled iron citrate prior to infection with M. avium. Phagosomes were isolated following lysis of the cells and differential centrifugation on a sucrose gradient. The radioactivity incorporated into the phagosomes is adjusted for total protein, which includes bacterial protein, and the data are expressed as picomoles of Fe per milligram of protein. The number of moles of Fe was calculated based on the specific activity of the isotope and does not take into account endogenous Fe levels.