Role of ferroportin in macrophage-mediated immunity

Abstract

Perturbations in iron metabolism have been shown to dramatically impact host response to infection. The most common inherited iron overload disorder results from defects in the HFE gene product, a major histocompatibility complex class I-like protein that interacts with transferrin receptors. HFE-associated hemochromatosis is characterized by abnormally high levels of the iron efflux protein ferroportin. In this study, J774 murine macrophages overexpressing ferroportin were used to investigate the influence of iron metabolism on the release of nitric oxide (NO) in response to infection. Overexpression of ferroportin significantly impaired intracellular Mycobacterium tuberculosis growth during early stages of infection. When challenged with lipopolysaccharide (LPS) or M. tuberculosis infection, control macrophages increased NO synthesis, but macrophages overexpressing ferroportin had significantly impaired NO production in response to LPS or M. tuberculosis. Increased NO synthesis in control cells was accompanied by increased iNOS mRNA and protein, while upregulation of iNOS protein was markedly reduced when J744 cells overexpressing ferroportin were challenged with LPS or M. tuberculosis, thus limiting the bactericidal activity of these macrophages. The proinflammatory cytokine gamma interferon reversed the inhibitory effect of ferroportin overexpression on NO production. These results suggest a novel role for ferroportin in attenuating macrophage-mediated immune responses.

FIG. 1.

Ferroportin overexpression limits early intracellular M. tuberculosis growth. (A) Ferroportin protein expression in J774-GFP.RV or J774-FPN.RV2 cells was determined using immunofluorescent detection with a polyclonal anti-ferroportin (FPN) antibody. Bar, 10 μm. (B) Intracellular iron retention was determined through ferritin detection via Western blot following treatment with 50 μM Fe-NTA. Ferritin levels were quantified and normalized to actin using a Li-Cor infrared imaging system. The means ± the SEM from a single experiment (n = 3) are shown. *, P ≤ 0.05 (Student t test). (C) J774-GFP.RV (▪) and J774-FPN.RV2 (□) cells were infected with M. tuberculosis at an MOI of 1. The number of intracellular bacteria surviving in each cell line at the indicated time points was determined using CFU. The mean ± the SEM from a single experiment (n = 3) are shown. Statistical analysis using two-way ANOVA determined that the curves were significantly different (P = 0.0008).

FIG. 2.

Ferroportin overexpression inhibits NO production and iNOS protein expression after M. tuberculosis infection. J774-GFP.RV (▪) or J774-FPN.RV2 (□) cells were infected with M. tuberculosis at an MOI of 5. (A) Cell medium was recovered to measure NO levels by using the Griess reaction. (B) iNOS protein expression was also determined 24 h postinfection using immunofluorescent detection with a monoclonal anti-iNOS antibody. Infected cells were visualized using GFP-M. tuberculosis. Bar, 10 μm. For statistical analyses, the means ± the SEM from a single experiment (n = 3) are shown. **, P ≤ 0.01 (Student t test). For all experiments, similar results were obtained on separate occasions.

FIG. 3.

Ferroportin overexpression inhibits NO production and iNOS protein expression after LPS treatment. (A) J774-GFP.RV (▪) or J774-FPN.RV2 (□) cells were treated with 1 μg of LPS/ml for 24 h. Medium was recovered, and NO levels were determined by using the Griess reaction. (B) iNOS protein expression was determined at 0, 4, and 24 h posttreatment with 1 μg of LPS/ml by Western blotting with a monoclonal anti-iNOS antibody. Membranes were stripped and reprobed with an anti-actin antibody as a loading control (C) iNOS protein expression was also determined 24 h posttreatment using immunofluorescent detection with a monoclonal anti-iNOS antibody. Cells were visualized using GFP. For statistical analyses, the means ± the SEM from a single experiment (n = 3) are shown. ***, P ≤ 0.001 (Student t test). For all experiments, similar results were obtained on separate occasions.

FIG. 4.

iNOS mRNA is induced in ferroportin overexpressing cells. (A) J774-GFP.RV or J774-FPN.RV2 cells were infected with M. tuberculosis or treated with LPS for 4 h. NF-κB activation was determined using nuclear localization with a polyclonal anti-NF-κB antibody. (B) Cells were infected with M. tuberculosis at an MOI of 5 or treated with 1 μg of LPS/ml. Cells were lysed in TRIzol reagent at 0, 4, and 24 h postinfection or treatment. Relative iNOS expression was determined by using qRT-PCR analysis. Gene expression is represented as the 2^−ΔCT between iNOS and a reference gene (36B4). For statistical analysis, the means ± the SEM from replicate experiments (n = 5) are shown. Statistical significance was determined by using the Student t test.

FIG. 5.

AG enhances M. tuberculosis growth in J774-GFP.RV control cells. (A) J774-GFP.RV cells were treated with LPS for in the presence or absence of 200 μg of AG/ml for 24 h. iNOS activity was determined by using the Griess reaction. (B) J774-GFP.RV (closed symbols) or J774-FPN.RV2 (open symbols) cells were infected with M. tuberculosis at an MOI of 1 in the presence of 200 μg of AG/ml. The number of intracellular bacteria surviving in each cell line at the indicated time points was determined using CFU. Statistical analysis using two-way ANOVA determined that the curves from the treated and non-treated control cells were significantly different (P = 0.0011). AG did not impact the growth of the J774.FPN.RV2 cells (P = 0.4483).

FIG. 6.

IFN-γ treatment stimulates production of NO and iNOS expression in J774-FPN.RV2 cells. (A) J774-GFP.RV (closed symbols) or J774-FPN.RV2 (open symbols) cells were infected with M. tuberculosis at an MOI of 1 in the presence of 5 ng of IFN-γ/ml. The number of intracellular bacteria surviving in each cell line at the indicated time points was determined by using CFU count. Statistical analysis using two-way ANOVA determined that the curves were significantly different (P < 0.0001). (B) J774-GFP.RV or J774-FPN.RV2 cells were treated with 5 ng of IFN-γ/ml. Cells were lysed in TRIzol reagent at 4 or 24 h as indicated. iNOS expression was determined by using qRT-PCR analysis as described previously. (C) iNOS protein expression was determined by Western blotting with a monoclonal anti-iNOS antibody. Membranes were stripped and reprobed with an anti-actin antibody as a loading control. (D) Medium was recovered following IFN-γ treatment to determine the NO levels. The means ± the SEM from a single experiment (n = 3) are shown. Statistical significance was determined by using the Student t test. ***, P ≤ 0.001.