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Originally published online as doi:10.1189/jlb.0606405 on October 12, 2006

Published online before print October 12, 2006
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(Journal of Leukocyte Biology. 2007;81:195-204.)
© 2007 by Society for Leukocyte Biology

Hereditary hemochromatosis results in decreased iron acquisition and growth by Mycobacterium tuberculosis within human macrophages

Oyebode Olakanmi*,{dagger}, Larry S. Schlesinger{ddagger} and Bradley E. Britigan*,{dagger},1

* Department of Internal Medicine and Research Service, VA Medical Center-Cincinnati, Cincinnati, Ohio, USA;
{dagger} Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA; and
{ddagger} Departments of Internal Medicine and Molecular Virology, Immunology, and Medical Genetics; and the Center for Microbial Interface Biology, Ohio State University, Columbus, Ohio, USA

1Correspondence: University of Cincinnati College of Medicine, Department of Internal Medicine, 231 Albert Sabin Way, P.O. Box 670557, Cincinnati, OH 45267-0557, USA. E-mail: bradley.britigan{at}uc.ed

ABSTRACT

Iron (Fe) acquisition is essential for the growth of intracellular Mycobacterium tuberculosis (M.tb). How this occurs is poorly understood. Hereditary hemochromatosis is an inherited disease in which most cells become overloaded with Fe. However, hereditary hemochromatosis macrophages have lower than normal levels of intracellular Fe. This suggests M.tb growth should be slower in those cells if macrophage intracellular Fe is used by M.tb. Therefore, we compared trafficking and acquisition of transferrin (Tf)- and lactoferrin (Lf)-chelated Fe by M.tb within the phagosome of monocyte-derived macrophages (MDM) from healthy controls and subjects with hereditary hemochromatosis. M.tb in both sets of macrophages acquired more Fe from Lf than Tf. Fe acquisition by M.tb within hereditary hemochromatosis macrophages was decreased by 84% from Tf and 92% from Lf relative to that in healthy control macrophages. There was no difference in Fe acquired from Tf and Lf by the two macrophage phenotypes. Both acquired 3 times more Fe from Lf than Tf. M.tb infection and incubation with interferon gamma (IFN-{gamma}) reduced macrophage Fe acquisition by 20% and 50%, respectively. Both Tf and Lf colocalized with M.tb phagosomes to a similar extent, independent of macrophage phenotype. M.tb growth was 50% less in hereditary hemochromatosis macrophages. M.tb growing within macrophages from subjects with hereditary hemochromatosis acquire less Fe compared with healthy controls. This is associated with reduced growth of M.tb. These data support a role for macrophage intracellular Fe as a source for M.tb growth.

Key Words: lactoferrin • transferrin • inflammation • cell trafficking • interferon

INTRODUCTION

Iron (Fe) metabolism is important for the growth of most microbes [1 2 3 ]. As a strategy of host defense to limit Fe availability to microbial pathogens, most extracellular Fe in vivo is chelated to transferrin (Tf) and lactoferrin (Lf) [4 ]. Tf is the major Fe binding protein in serum, whereas Lf predominates at mucosal surfaces, although Tf is also present [4 ]. Whereas most bacteria have access to extracellular Fe chelates, pathogens that live primarily within intracellular environments face unique challenges because they need to acquire Fe from within their intracellular locale. Consequently, successful pathogens have evolved effective strategies to acquire host Fe.

Mycobacterium tuberculosis (M.tb) is a human pathogen that enters and multiplies within human macrophages in a unique phagosomal compartment that undergoes limited fusion with lysosomes [5 6 7 ]. M.tb requires Fe for its growth in broth culture media, as well as in macrophages [8 , 9 ]. Siderophore production is crucial to this process [8 , 9 ]. The M.tb-containing macrophage phagosome is an Fe-poor environment [10 ], which M.tb must overcome to sustain growth. Increased access to Fe enhances the growth rate of M.tb both in vitro and in vivo [8 , 9 , 11 , 12 ]

Extracellular Tf traffics to M.tb-containing phagosomes through plasma membrane transferrin receptor (TfR) cycling to early endosomes [13 , 14 ]. The M.tb phagosome interacts with early endosomes and is accessible to exogenously administered Tf [13 ]. It has been proposed that this provides M.tb with Fe [13 ]. However, we have demonstrated that intraphagosomal M.tb can acquire Fe from sources within the macrophage cytoplasm (endogenous), as well as from extracellular Tf and Lf (exogenous) [15 ]. These data raise the possibility that a portion of the Fe uptake from exogenous Tf and Lf by M.tb may involve initial Fe transfer from endocytosed Tf-Fe and Lf-Fe to the macrophage cytoplasm, with subsequent transfer to M.tb.

Studying Fe acquisition by M.tb located within macrophages that lack key components of Fe acquisition mechanisms could prove useful in delineating how Fe traffics to intracellular pathogens such as M.tb. The most common form of hereditary hemochromatosis results from mutations in a membrane protein, HFE [16 , 17 ], which, in turn, lead to reduced or absent expression of HFE (HFE–) on the cell surface. In normal cells, HFE binds to ß2-microglobulin on the cell surface and forms a complex with TfR [18 ]. This process alters the affinity of TfR for Fe-loaded Tf [19 20 21 ].

In contrast to most other types of cells where hereditary hemochromatosis leads to Fe overload, reticuloendothelial macrophages and monocytes from individuals with hereditary hemochromatosis exhibit decreased intracellular Fe levels due to excessive Fe efflux relative to cells from healthy control subjects [22 23 24 25 ]. In previous work, we demonstrated that M.tb cultured in monocyte-derived macrophages (MDM) from individuals with hereditary hemochromatosis acquire significantly less Fe from Tf than MDM from normal subjects [26 ]. However, whether this is due to alterations in the function of the TFR or to decreased intracellular Fe stores in the MDM is unknown. Fe acquisition from Lf by M.tb residing within macrophages from individuals with hereditary hemochromatosis has not been examined. Modification of TfR function resulting from the absence of HFE would not be expected to alter Fe acquisition from exogenous Lf since Fe acquisition from Lf is not mediated through the TfR [27 28 29 30 31 ]. However, such modification would be seen if intracellular Fe stores in MDM are the principal source of Fe used by the organism. Thus, comparing Fe acquisition by M.tb located in macrophages from individuals with hereditary hemochromatosis or healthy controls should provide insight into Fe acquisition mechanisms of phagosomal M.tb.

Therefore, we chose to test the hypothesis that the nature of extracellular Fe chelates, as well as the phenotype of the macrophages, may influence intraphagosomal Fe acquisition by M.tb. This may then affect the long-term growth of the bacterium within the phagosome. Herein, we report studies in which we examined the growth of M.tb within MDMs from hemochromatosis subjects compared with healthy controls using Fe chelated to the two major Fe sources that the organism would likely be exposed to in vivo—Tf and Lf. We also used confocal microscopy to compare the trafficking of exogenously added Tf and Lf to the M.tb phagosome in both types of macrophages.

MATERIALS AND METHODS

Mycobacterium tuberculosis
All experiments were performed with the virulent Erdman M.tb strain (ATCC 35801). GFP-expressing H37Rv was a gift from Dr. Vojo Deretic, University of New Mexico. M.tb was cultivated for 10 days and then was harvested in RPMI containing 10 mM HEPES to form a predominantly single-cell suspension using a described previously method [32 ]. The bacterial suspension was kept on ice and used within 2 h of preparation.

Macrophage culture
Using approved Institutional Review Board protocols, we obtained peripheral blood mononuclear cells from healthy adult volunteers, as described previously [26 , 33 ], or from hereditary hemochromatosis patient associated with mutations in HFE undergoing phlebotomy at the McGowin Blood Center of the University of Iowa Hospitals and Clinics. All healthy subjects were purified protein derivative negative and had no known previous history of infection with M.tb. The mononuclear cells were cultured for 5 days in RPMI supplemented with 20% autologous serum using Teflon wells (Savillex, Minnetonka, MN). The resultant MDM fraction was isolated by adherence to 24-well tissue culture plates (Falcon, Lincoln Park, NJ) at 1 x 105 MDM/well for 2 h with 10% autologous serum and washed. The cells were then maintained for 7 days at 37°C in RPMI (Gibco, Grand Island, NY), supplemented with 20% autologous serum [26 , 33 , 34 ]. This methodology consistently yields a population of cells that is 95-98% macrophages, as defined by nonspecific esterase staining.

Fe uptake by intraphagosomal M.tb
MDM monolayers were washed 3 times in RPMI and incubated with a single suspension of M.tb at a bacteria:MDM ratio of 5 (multiplicity of infection, MOI=5) for 2 h in RPMI containing 10 mM HEPES and 1 mg/ml of human serum albumin (HSA). Monolayers were washed in RPMI and repleted with RPMI supplemented with 1% autologous serum. After 24 h, 10 µM [59Fe2], chelated to Tf or Lf [35 ] (each from Sigma Chemical, St. Louis, MO) was added to the monolayers and incubated for another 24 h. Both Tf and Lf were loaded with 59Fe to achieve saturation of >95% of the proteins’ two Fe binding sites, as described previously [36 ]. Intracellular M.tb were harvested, as described previously [26 ]. Briefly, MDM were washed with media containing ascorbate (5 mM) at pH 5.0 to remove any 59Fe associated with the MDM plasma membrane. Bacilli were recovered by lysing the MDM using 0.1% SDS in the presence of 1000 U/ml DNase (Invitrogen, Carlsbad, CA) and EDTA-free Protease Inhibitor Cocktail Tablet (Boehringer Mannheim/Roche, Indianapolis, IN). 59Fe present in duplicate (30 µl) aliquots of the cell lysate was determined by gamma counter. The released bacilli were recovered by centrifuging the supernatant at 10,000 g for 10 min at 4°C and washing the resultant bacterial pellet 3 times with 0.01% SDS in RPMI. The bacilli were finally resuspended, filtered using a 0.22 µm spin-x filter (Millipore), and 59Fe associated with the filter containing the trapped bacilli determined by gamma counter [26 ].

In the experiments to analyze Fe acquisition by IFN-{gamma}-treated MDM and intraphagosomal M.tb harvested from these cells, 12 day-old MDM monolayers were treated with 1000 U/ml of IFN-{gamma} (or medium only for control) for 5 days before the addition of M.tb and subsequent addition (24 h later) of the desired [59Fe] chelate or with the [59Fe] chelate alone (no M.tb). MDM and M.tb were separated from one another, as described above, and the amounts of 59Fe acquired by MDM and M.tb were determined by gamma counter.

Growth of intraphagosomal M.tb with an exogenous source of Fe
M.tb was added to 12-day-old MDM monolayers, as previously reported [34 ]. Briefly, the MDM monolayers were washed 3 times in RPMI and incubated with single-cell suspensions of M.tb at a MOI = 1 for 2 h in RPMI containing 10 mM HEPES and 1 mg/ml of HSA. Monolayers were washed in RPMI and repleted with RPMI supplemented with 1% autologous serum. M.tb does not replicate extracellularly in this culture medium (data not shown). After 24 h, 10 µM [56Fe2] in the form of Tf (holoTf) or Lf (holoLf), each from Sigma Chemical, St. Louis, MO, was added to the monolayers and incubated for specific time period. M.tb not associated with the MDM were removed by repetitive washing of the monolayer. Intracellular M.tb were recovered by lysing the MDM using 0.1% SDS in the presence of 1000 U/ml DNase (Invitrogen, Carlsbad, CA) [26 ]. Aliquots of the lysates were withdrawn for colony forming unit (CFU) determination.

Growth of intraphagosomal M.tb with an endogenous source of Fe
MDM monolayers were incubated with the desired [56Fe2] chelate for 24 h and washed. After an additional 24 h period (chase), M.tb bacilli were added to the monolayers at an MOI of 1 for 2 h, after which bacilli not associated with the MDM were removed by repetitive washing of the monolayer. Intracellular M.tb bacilli were harvested from MDM at various time periods, and their CFUs were determined.

Determination of colony forming units
One-hundred microliters of diluted MDM lysates, prepared as above, were plated on 7H11 agar in 6-well plates in duplicate. The plates were incubated under 100% humidity, 5% CO2, and 37°C for three weeks, and colonies were counted. To address the possibility of failure to detect slower growing M.tb, in certain cases, the plates were returned to the incubator for an additional 2 wk. No difference in the number of colonies detected was ever observed.

Confocal microscopy
To assess for the potential colocalization of M.tb-containing phagosome with Tf and Lf, we used confocal microscopy. Macrophages were adhered onto Biocoat cell environment culture slides (Falcon, Franklin Lakes, NJ) at 1x105 MDM/well in 20% autologous serum for 7 days. Cells were incubated with GFP-M.tb (H37Rv) at a MOI = 5 as above and washed. After overnight incubation, 10 µM Fe2Tf or Fe2Lf was added for 2, 8, or 24 h. The cells were fixed in situ with 1% paraformaldehyde for 10 min and then after removal of fixative, for an additional 10 min with 2% paraformaldehyde. The fixed monolayers were permeabilized with 100% methanol for 5 min and washed 3 times with Dulbecco PBS (PD). Cells were blocked overnight at 4°C with PD containing 10% heat-inactivated FCS (hiFCS) and 5 mg/ml BSA. Blocking buffer was removed, and the monolayers were washed 3 times in blocking buffer and incubated with mouse anti-human Tf or Lf (RDI Research Diagnostics, Flanders, NJ) overnight at 4°C on a rocking platform. After washing 6 times in blocking buffer, the monolayers were incubated with Texas red-conjugated donkey anti-mouse antibody (Jackson ImmunoResearch Labs, West Grove, PA) at room temperature for 1 h in the dark. The monolayers were then washed 6 times in blocking buffer and once with ddH2O. The slides were separated from the wells, immersed gently and briefly into a beaker of water, and let dry in the dark and covered with aluminum wrap. Vectashield was added to each sample on the slide, which was then overlayed with a coverslip and sealed with nail polish. The slides were viewed within three days on a Zeiss 510 confocal microscope (40x). Microscopic fields were selected at random and M.tb-containing MDM phagosomes were scored (positive or negative) for the presence of Tf or Lf.

Statistical analysis
Results obtained under different experimental conditions were compared by Student’s paired t test when independent variables were being assessed or by ANOVA when trends were being determined. For both types of analyses, results were considered significant at P ≤ 0.05. Because absolute results vary from MDM donor to donor, each experiment was analyzed relative to its own control group(s).

RESULTS

M.tb residing within macrophages from hemochromatosis patients acquired less Fe compared with healthy control cells
Our previous studies have shown that M.tb residing within MDM from subjects with hemochromatosis acquire less Fe from Tf than bacilli located in MDM from healthy control subjects [26 ]. This could be related to alterations in TfR function or to the decrease in the labile pool of Fe in the macrophage cytoplasm that has been shown to occur in these cells [22 23 24 25 ]. To address these two possibilities, we examined whether there is a difference in Fe acquisition from Lf, by intraphagosmal M.tb residing within MDM derived from individuals with hereditary hemochromatosis relative to those from healthy control subjects. Fe acquisition from LF would not be expected to be directly affected by a change in TfR function since Fe acquisition from LF is not mediated by the TfR [27 28 29 30 31 ]. Because Lf is a major form of chelated Fe in the human lung, this question also has significance for understanding the pathogenesis of M.tb infection in the human lung.

MDM from normal and hemochromatosis subjects were infected with M.tb. After 24 h of infection, the MDM monolayers were incubated with 59Fe chelated to Tf or Lf for an additional 24 h, and then MDM monolayers were lysed. Intracellular M.tb were harvested and M.tb-associated 59Fe was determined.

M.tb recovered from both hemochromatosis and normal MDMs exhibited greater Fe acquisition from Lf relative to Tf (Fig. 1 ). Fe uptake by intracellular M.tb isolated from hemochromatosis cells was significantly lower (P<0.05) than uptake by bacilli residing within healthy control cells, demonstrating a 84.4 ± 4.5% reduction from Tf (Fig. 1A) and 92.1 ± 4.4% decrease from Lf (Fig. 1 , P<0.05).


Figure 1
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  		Figure 1. Fe acquisition from transferrin and lactoferrin by M.tb bacilli residing in the MDM from healthy control and hemochromatosis patients. Twelve-day-old MDM monolayers were incubated with M.tb (+M.tb) at a multiplicity of infection of 5 (MOI=5) for 2 h and washed. Ten micromolar [59Fe2]Tf or [59Fe2]Lf was added. The monolayers were incubated for 24 h and then lysed. The lysate was centrifuged at 10,000 g for 10 min at 4°C. The supernatant was removed, and the pellet was washed 3 times in RPMI containing 5 mM ascorbate, pH 5. The pellet was finally suspended in 500 µl of the washing medium, pipetted into spin-x filter tube and centrifuged. The filter, containing trapped bacilli, was cut into a gamma counter tube. Bacteria-associated 59Fe was assessed on a gamma counter. Results from M.tb harvested from healthy control MDM (solid bar) and hemochromatosis subjects (cross-hatched) were then analyzed for each Fe chelate and are expressed as mean ± SEM, n = 4. Acquisition was expressed as picomoles of 59Fe in intracellular M.tb per million MDM. A significant decrease in M.tb Fe acquisition from Tf and Lf was seen when results obtained with MDM from controls and HFE– individuals were compared: P < 0.02 for both Tf and Lf.

 
Macrophages from both hemochromatosis patients and healthy controls acquire more Fe from Lf than from Tf
In vitro studies of the Fe metabolism of macrophages from individuals with hereditary hemochromatosis have focused on Fe from Tf [23 , 24 , 37 ]. To what extent mutations in HFE could impact macrophage Fe acquisition from Lf has not been extensively examined. Potential differences in the amount of Fe acquired from Tf and Lf by HFE– vs. control MDM could explain in part the above impact of the HFE– state on M.tb Fe acquisition. Therefore, we compared Fe acquisition from Tf and Lf by MDM from hemochromatosis patients and healthy control individuals in the presence and absence of M.tb infection. MDM from hemochromatosis patients and healthy controls were washed and incubated for 24 h with 59Fe2 (10 µM) chelated to either Tf or Lf. The cells were lysed 24 h later, and the total cell-associated Fe was determined. Consistent with our previous findings, net Fe uptake from Tf by hemochromatosis cells was not significantly different from that of healthy control MDM (Fig. 2 ) (P>0.05 for each comparative test condition). For both HFE– and healthy control MDM, the magnitude of Fe acquired was greater from Lf than Tf (Fig. 2) . Fe acquired from Lf by hemochromatosis macrophages was similar to that from healthy control cells (P>0.05). These observations held regardless of whether macrophages had or had not been infected with M.tb (Fig. 2) .


Figure 2
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  		Figure 2. Fe acquisition from transferrin and lactoferrin by MDM from healthy control and hemochromatosis patients. Twelve-day-old MDM monolayers from healthy controls (solid bars) or hemochromatosis patients (cross-hatched) were incubated with M.tb (+M.tb) at a multiplicity of infection of 5 (MOI=5) for 2 h and washed or treated in the same manner except that they were not infected with M.tb (untreated). 10 µM [59Fe2]Tf or [59Fe2]Lf was added to all groups of MDM, following which, they were incubated for 24 h and then lysed. To determine total lysate Fe, 1% of each lysate was withdrawn into gamma counter tubes in duplicate. Cell-associated 59Fe was assessed by gamma counter. Acquisition was expressed as mean ± SEM, nanomoles of 59Fe per million MDM (n=4). No significant differences (P>0.05) were observed in comparison to results between MDM from healthy controls and HFE– individuals for either Tf or Lf.

 
Thus, the decreased ability of M.tb residing within MDM from subjects with hemochromatosis to acquire Fe from either Tf or Lf cannot be explained on the basis of a decrease in Fe acquisition from these compounds by the macrophage

Fe-loaded Tf and Lf traffic to the M.tb phagosome in macrophages
Fe acquisition from Lf in hemochromatosis macrophages was decreased, in spite of the fact that it would not be expected to be impacted by alterations in TfR-dependent Fe acquisition. This suggested that the decreased ability of M.tb to acquire Fe from both Lf and Tf in macrophages from subjects with hemochromatosis reflects the effect of the HFE– mutation on a common downstream component of macrophage Fe metabolism that is altered by the absence of HFE.

Previous studies have demonstrated trafficking of Tf to the M.tb phagosome in macrophages [13 , 14 ], purportedly as a result of movement of Tf/TfR complex-containing early endosomes to M.tb containing phagosomes. Since HFE forms a complex with the TfR [38 ], it seemed possible that the decrease in Fe acquisition by M.tb in HFE– MDM could be related to a change in trafficking of Tf to the M.tb phagosome. Also, it was not known whether Lf is also trafficked to the M.tb phagosome, and if so, whether this process could also be impacted by the absence of HFE. To further understand the mechanism of Fe delivery to intraphagosomal M.tb in normal and HFE– MDM, we studied the trafficking of Tf and Lf using confocal microscopy. Cells were incubated with GFP-M.tb (H37Rv) for 2 h, washed and incubated overnight. Fe chelated to Tf or Lf was added for defined time periods. MDM were then fixed, permeabilized, and stained for Tf or Lf. MDM from both hemochromatosis patients and healthy controls contain varying number of bacteria with no difference in morphology (Fig. 3A 3B ). Immunostaining for Tf and Lf revealed colocalization with M.tb phagosomes, and the amount of colocalization was similar regardless of whether macrophages were from hemochromatosis or healthy control subjects [22.7±6.3% and 17.0±0.4% of M.tb demonstrated colocalization with Tf and 16.0±8.8% and 23.3±2.4% with Lf, for healthy control (Fig. 3A) and HFE– (Fig. 3B) , respectively, (P>0.05 for all comparisons)]. Thus, the HFE phenotype does not appear to alter the trafficking of Tf or Lf to the M.tb-containing phagosome of human MDM.


Figure 3
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  		Figure 3. Colocalization of M.tb with transferrin and lactoferrin in macrophages. Cells were incubated with GFP-M.tb (H37Rv) for 2 h, washed, and incubated overnight. Fe chelated to Tf or Lf was added for defined time periods. Control (untreated) received medium only. MDM were then fixed, permeabilized, and stained for Tf or Lf. Monolayer was immunostained with Texas red and viewed using a confocal microscope. Fifty M.tb phagosomes were examined for determination of colocalization. Shown are results from one experiment representative of three. (A) Result using healthy control MDM. (B) Result with MDM from hemochromatosis patients.

 
Interferon-{gamma} and infection with M.tb decrease Fe acquisition by macrophages from healthy individuals and patients with hemochromatosis
IFN-{gamma} plays an important role in host defense against M.tb in mouse macrophages [39 ]. At least, some of the antimicrobial effects of IFN-{gamma} have been attributed to alterations of macrophage-labile Fe pool stores and a decrease in surface expression of TfR [40 41 42 ]. We previously found that infection of MDM with M.tb decreased MDM Fe uptake by 15-40% depending on the chelate used [15 ]. Whether IFN-{gamma} or infection with M.tb has a similar effect on Fe acquisition and storage by MDM from hemochromatosis subjects has not been investigated.

Accordingly, we incubated MDM from hemochromatosis subjects and healthy controls with 1,000 U/mL IFN-{gamma} over 5 days, or infected them with M.tb for 24 h, or subjected them to both conditions. Fe acquisition from Tf and Lf by M.tb was then determined. Consistent with previous studies, Fe uptake from Tf and Lf by M.tb-infected MDM was significantly lower when healthy control cells were pretreated with IFN-{gamma} compared with untreated cells (P<0.01, Fig. 4A 4B ). A significant decrease (P≤0.05) in Fe acquisition was also seen with IFN-{gamma} treatment of MDM from subjects with hemochromatosis (Fig. 4A 4B) . These effects were somewhat less pronounced in HFE– cells relative to healthy control cells. The combination of M.tb infection and IFN-{gamma} resulted in Fe acquisition that was not significantly different from IFN-{gamma} alone for both healthy and HFE– cells (Fig. 4A 4B) .


Figure 4
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  		Figure 4. Fe acquisition from transferrin (A) and lactoferrin (B) by MDM from healthy control and hemochromatosis patients. Twelve-day-old MDM monolayers from healthy controls (solid bars) or hemochromatosis patients (cross-hatched) were incubated with 1000 U/ml of IFN-{gamma} or medium (untreated) for 5 days. Cells were then incubated with M.tb (+M.tb and +M.tb+IFN-{gamma}) at a multiplicity of infection of 5 (MOI=5) for 2 h and washed. Ten micromolar [59Fe2]Tf or [59Fe2]Lf was added to all groups, incubated for 24 h and lysed. To determine total lysate Fe, 1% of each lysate was withdrawn into gamma counter tubes in duplicate. Cell-associated 59Fe was assessed by gamma counter. Acquisition was expressed as mean ± SEM nanomoles of 59Fe per million MDM (n=4). The data for the non-IFN-{gamma} treated cells are reproduced from Figure 2 . (A) Fe acquisition from Tf *, P < 0.003; comparison between untreated and treated healthy control. **, P < 0.03; comparison between untreated and treated hemochromatosis. #, P < 0.05; comparison between healthy control and hemochromatosis. (B) Fe acquisition from Lf, a, P < 0.01–0.04; comparison between untreated and treated healthy control. b, P < 0.05; comparison between untreated and treated hemochromatosis. c, P < 0.01; comparison between healthy control and hemochromatosis.

 
Treatment of MDM from healthy controls with IFN-{gamma} decreased Fe uptake by M.tb from Tf to 29.5 ± 4.8% (P<0.02) and from Lf to 42.0 ± 6.1% (P< 0.04) of control (untreated) MDM (Fig. 5A 5B ). However, IFN-{gamma} treatment of MDM did not alter Fe acquisition by M.tb from Tf or Lf for HFE– MDM (P > 0.05, Fig. 5A 5B ). Thus, the addition of IFN-{gamma} treatment is unable to further limit Fe acquisition by M.tb beyond that which occurs as a consequence of the HFE– phenotype. This suggests that the absence of HFE and IFN-{gamma} may be decreasing M.tb Fe acquisition by modifying the same component of the Fe acquisition pathway. The labile Fe pool of the macrophage is felt to be altered by both processes, suggesting it to be a likely candidate.


Figure 5
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  		Figure 5. Fe acquisition from transferrin (A) and lactoferrin (B) by M.tb bacilli residing in the MDM from healthy control and hemochromatosis patients. Twelve-day-old MDM monolayers were incubated with 1000 U/ml of IFN-{gamma} or medium (untreated) for 5 days. Cells were then incubated with M.tb (+M.tb and +M.tb+IFN-{gamma}) at a multiplicity of infection of 5 (MOI=5) for 2 h and washed. Ten micromoles [59Fe2]Tf or [59Fe2]Lf was added to all groups, incubated for 24 h and lysed. The lysate was centrifuged at 10,000 g for 10 min at 4°C. The supernatant was removed and the pellet was washed 3 times in RPMI containing 5 mM ascorbate, pH 5. The pellet was finally suspended in 500 µl of the washing medium, transferred into spin-x filter tube, and centrifuged. The filter, containing trapped bacilli, was cut into a gamma counter tube. Bacteria-associated 59Fe was assessed on a gamma counter. Results from M.tb harvested from healthy control MDM (solid bar) and hemochromatosis subjects (cross-hatched) were then analyzed. Acquisition was expressed as means ± SEM picomoles of 59Fe in intracellular M.tb per million MDM (n=4). The data for the non-IFN-{gamma} treated cells are reproduced from Figure 1 . *, P < 0.04; comparison between INF-{gamma}-treated and untreated healthy control. @, P < 0.02; comparison between M.tb-infected control cells and HFE– cells. {alpha}, P < 0.004; comparison between INF-{gamma}-treated control and HFE– cells (Tf). ß, P < 0.03; comparison between INF-{gamma}-treated control cells and HFE– cells (Lf).

 
M.tb growth is decreased in macrophages from hemochromatosis individuals compared with healthy control macrophages
Although Fe is required for M.tb growth, it was not clear whether the decrease in Fe acquisition by M.tb located in HFE– MDM was sufficient to alter bacterial growth in the macrophage. In spite of decreasing access of M.tb to Fe, previous studies found that IFN-{gamma} treatment of human MDM does not inhibit the in vitro growth of M.tb [34 , 43 ]. Therefore, we compared growth of intracellular M.tb over time in MDM from hemochromatosis and healthy control subjects. The impact of the addition of exogenous Fe-Tf or Fe-Lf on this process was also examined.

The number of M.tb phagocytosed by HFE– and healthy control cells was equivalent based upon initial CFUs (Fig. 6 ), as well as by enumeration of MDM-containing GFP-expressing M.tb at 2 h using the methods used in Fig. 3 (data not shown). There was a steady increase in the number of intracellular M.tb over time, irrespective of the chelate or the phenotype of the macrophage. However, the growth was slower in hemochromatosis macrophages (~50%) than in healthy control macrophages. The slower growth rate was significant at all time points greater or equal to day 2. Somewhat surprisingly, although we found a greater amount of Fe acquired by M.tb from Lf than Tf (Fig. 1) , the rate of M.tb growth was quite similar when the media were supplemented with exogenous FeTf or FeLf (Fig. 6) .


Figure 6
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  		Figure 6. Time-dependent growth of intracellular M.tb isolated from MDM from healthy control and hemochromatosis patients. For the exogenous conditions of Fe treatment (A), Twelve-day-old MDM monolayers from healthy controls (solid bars) or hemochromatosis patients (cross-hatched) were incubated with M.tb (MOI=1) for 2 h and washed. Then, 10 µM Fe chelated by Tf or Lf were added and incubated with the monolayer for the desired time. Control cells had medium only. For the endogenous condition (B), cells were first treated with 10 µM Fe for 24 h and washed prior to incubation with M.tb. In both the exogenous and endogenous paradigms, duplicate wells were withdrawn from each group per day over 5 days and lysed and plated in three serial dilutions onto 7H11 agar plates, each dilution in duplicate. The plates were incubated under 5% CO2 at 37°C for 3 wk before counting the colonies. Growth was plotted as CFUs over time for untreated cells (i), Fe2Tf-treated cells (ii), and Fe2Lf-treated cells (iii). Results are the mean ± SEM of 3–8 experiments. a, P < 0.02; comparison between healthy control and hemochromatosis at specified time point. b, P < 0.05; comparison between healthy control and hemochromatosis at specified time point.

 
DISCUSSION

We previously demonstrated that M.tb are able to acquire Fe from both extracellular LF and TF, as well as from the macrophage cytoplasm that had been previously loaded with Fe from these two chelates [26 , 44 ]. The latter observation suggested that M.tb is able to access Fe from the macrophage cytoplasm. We have also reported that acquisition of Fe initially bound to extracellular Tf by intraphagosomal M.tb is dramatically decreased when the bacteria reside in macrophages from individuals homozygous for mutations in HFE relative to healthy controls [26 ]. This could have been due to alterations in TfR-binding kinetics associated with mutations in HFE or to the previously described decrease in macrophage labile pool Fe that occurs in hemochromatosis [22 23 24 25 ]. Therefore, to provide additional insight into the role of the macrophage-labile Fe pool as a source for M.tb Fe needs, we compared Fe acquisition from Tf and Lf in control macrophages and those from individuals with hereditary hemochromatosis. Alterations in the TfR would not be expected to impact Fe acquisition from Lf, which does not use this pathway. It was expected that these findings would also be of relevance to M.tb pathogenesis, as M.tb-infected macrophages would likely be exposed to Fe bound to Lf in vivo, as Lf is a major Fe-binding protein in the human airway [4 , 45 ].

Consistent with previous findings by ourselves and others [26 , 44 , 46 ], MDM from both healthy controls and subjects with hemochromatosis exhibited similar abilities to acquire Fe over 24 h, with both types of MDM acquiring 2- to 3-fold more Fe from Lf than Tf. The difference in MDM Fe acquisition from Tf and Lf is not surprising since the mechanism(s) whereby macrophages and other eukaryotic cells acquire Fe from Tf and Lf are known to be different. Fe uptake from Tf occurs via TfR-mediated endocytosis [47 ]. Fe uptake from Lf does not involve TfR, although the exact mechanism and identity of LF receptor(s) remains unclear [27 28 29 30 31 ].

Whereas we observed only minor differences in the ability of MDM from subjects with hemochromatosis or healthy controls to acquire Fe from Tf and Lf, dramatic differences were observed in the amount of Fe acquired by M.tb residing in the phagosomes of macrophages from these two populations. Eighty-four and ninety-two percent less Fe was acquired by M.tb from Tf and Lf, respectively, when residing in phagosomes of macrophages from individuals with hemochromatosis relative to those from healthy controls.

The defect in hemochromatosis is mediated by a protein that binds the TfR, which should not impact Fe acquisition from Lf. Yet, the decrease in Fe acquisition by M.tb in HFE– macrophages occurred with Fe bound to either chelate. This suggests that the mechanism does not involve interaction of Tf or Lf with the cell surface, and/or internalization of the two Fe-binding proteins. Instead, it likely involves a common process that impacts intracellular Fe availability and trafficking within the macrophage cytoplasm after it has been removed from either of the two proteins.

Tf has been shown to move to M.tb-containing macrophage phagosomes [13 , 14 ]. Using confocal microscopy, we found no difference in the number of M.tb-containing phagosomes that contained Tf or Lf. This is the first study of which we are aware to document transfer of extracellular Lf to M.tb phagosomes. The previous work of Sturgill-Koszycki and colleagues [14 ] reported that ~50% of M.tb phagosomes exhibited no immunogold particles directed against Tf, 28% contained 1, and 20% had 2 or more immunogold particles. The number of Tf-positive phagosomes detected by our immunofluorescence methodology is thus nearly identical to the heaviest immunostained group in the earlier study. We conclude that the difference in Fe acquisition by intraphagosomal M.tb within MDM of healthy controls vs. subjects with hemochromatosis is not explained by variation in the magnitude of Tf or Lf trafficking to the M.tb phagosome.

We have previously showed that M.tb can acquire Fe from the macrophage cytoplasm, most likely its labile Fe pool [15 , 26 ]. Significant portions of Fe acquired from both Tf and Lf are believed to enter the macrophage labile Fe pool; although it is not known to what extent macrophages handle Fe acquired from these two sources differently. In addition to its impact on Tf binding to TfR, hereditary hemochromatosis leads to a decrease in the magnitude of the labile Fe pool of mononuclear phagocytes [22 23 24 ]. We hypothesize that the failure of M.tb to acquire Fe as efficiently, while residing within macrophages of individuals with hemochromatosis compared with healthy controls, is due to differences in the amount, form, and/or accessibility of Fe present in the labile Fe pool of the two macrophage phenotypes. In this scenario, the effect of hemochromatosis on the labile pool would be independent of whether the Fe was acquired by the macrophage from Tf or Lf.

The impact of the HFE– phenotype on M.tb Fe acquisition is nevertheless more complex than simple alterations in macrophage Fe content. IFN-{gamma} decreased the net Fe acquired from both Tf and Lf by control MDM to levels below those of untreated MDM from individuals with hemochromatosis (Fig. 4) . Yet, the ability of intracellular M.tb to acquire Fe from exogenous Tf and Lf remained significantly less when they were residing in MDM from subjects with hemochromatosis relative to control MDM treated with IFN-{gamma} (Fig. 5) . Furthermore, there was no additive impact on M.tb Fe uptake from either Fe chelate in hemochromatosis subject macrophages as a consequence of IFN-{gamma} exposure (Fig. 5) .

Several previous studies have reported that M.tb infection inhibits some macrophage responses to IFN-{gamma} [48 49 50 ]. However, we did not find evidence that the ability of IFN-{gamma} to decrease MDM Fe was blunted by M.tb infection. This difference may be due to the fact that in the earlier studies, macrophages were treated with IFN-{gamma} after they were infected with M.tb, which is the opposite of our experimental protocol, in which the macrophages were exposed to IFN-{gamma} for 5 days before infection with M.tb.

If the macrophage-labile Fe pool is a primary source of Fe for intraphagosomal M.tb, how does it acquire that Fe? M.tb produces a membrane-bound siderophore, mycobactin, and a soluble one, exochelin [51 52 53 ]. Siderophore production is required for optimal growth of M.tb within macrophages [9 ], and exochelin can remove Fe from both Tf and Lf [54 ]. If M.tb siderophores are involved in acquiring Fe from the macrophage-labile pool, then exochelin would need to be able to gain access to that Fe. Other secreted products of intraphagosomal M.tb have been reported to be able to exit the phagosome [55 ]. However, this remains controversial [56 ], and there are no data of which we are aware demonstrating that exochelin crosses the phagosome membrane and enters the macrophage cytoplasm.

Alternatively, Fe could be transported into the phagosome by macrophage cation transporters located on the phagosomal membrane. DMT-1 (NRAMP2, DCT-1) is a divalent metal transporter present at the surface of phagosomes that plays an important role in Fe acquisition from Tf. However, DMT-1 pumps Fe out of endosomes [57 58 59 60 ]. Polymorphisms of a related metal transporter, NRAMP1, are linked to resistance to M.tb infection in human populations [61 , 62 ]. Zwilling and colleagues reported that Fe can be pumped into M. avium-containing phagosomes, a process that they linked to Nramp1 [63 , 64 ]. However, prevailing data indicate that NRAMP1 is primarily a Mn2+ and H+, not an Fe, transporter and that it too pumps metals out of the phagosome [65 ]. Further studies are needed to define the mechanism(s) whereby M.tb gains access to macrophage cytoplasmic Fe.

Access to Fe is important for growth and replication of human pathogens, including M.tb [1 2 3 , 8 , 9 ]. In addition to decreased access to Fe, M.tb residing within macrophages from individuals with hemochromatosis exhibited a 50% decrease in the rate of M.tb growth relative to that seen in macrophages from healthy controls. The fact that this was observed with exogenous supplementation of the media with Fe chelated to either Tf or Lf suggests that the effect is not mediated by HFE’s effect on the interaction of Tf with TfR, but rather impacts Fe acquisition from multiple sources of Fe, e.g., the macrophage-labile pool. It is worth noting that expression of hepcidin, a protein secreted by the liver that plays an important role in regulating intestinal Fe transport and which has direct antibacterial activity, appears to be inappropriately low in hereditary hemochromatosis [66 , 67 ]. The impact of low expression of hepcidin on susceptibility to infection with M.tb has not yet been addressed. Recent data with Salmonella suggest that modulations of macrophage ferroportin expression, which is regulated by hepcidin, impact Fe availability and growth of that pathogen within the macrophage [68 ].

Similarly, it is possible that other HFE-dependent alterations in the macrophage phagosome or macrophage cell function that are independent of Fe transport could be responsible for the slower rates of growth of M.tb in HFE– MDM. For example, TNF has been postulated to play a role in macrophage antimycobacterial activity, and macrophage TNF production has been shown to be impacted by Fe availability. However, the effect of TNF on M.tb growth is unclear—inhibiting it in some systems [69 ] and promoting it in others [70 ]. Interestingly, HFE– monocytes reportedly produced decreased amounts of TNF in response to LPS than control macrophages [71 ]. Whether this would extend to other stimuli such as M.tb is unknown. Whatever the specific mechanisms(s) responsible, our data support the contention that the HFE– phenotype may result in enhanced resistance to infection with intracellular pathogens of macrophages.

The above conclusion is counter to a recent study [12 ] that identified a role for Fe in the mechanism of the previously known increase in susceptibility of ß2-microglobulin knockout mice to infection with M.tb [72 ]. ß2-microglobulin is the binding site for HFE on cell membranes and the absence of ß2-microglobulin leads to a state of increased tissue Fe [73 ]. Schaible and colleagues [12 ] found that the increased susceptibility of ß2-microglobulin knockout mice to infection with M.tb is reversed by treating the animals with deferoxamine or intranasal apoLf. This treatment was associated with increased nitric oxide production [12 ]. Exogenous apoLf also slowed the rate of in vitro M.tb growth in macrophages from ß2-microglobulin-deficient mice. The authors attributed their results to decreased availability of Fe for microbial growth, and they proposed that hemochromatosis may be associated with increased susceptibility to infection with M.tb and other intracellular pathogens [12 ].

It is important to note that the increase in the tissue and serum Fe levels of ß2-microglobulin knockout mouse requires them to be placed on high-Fe diets [73 ]. In addition to Fe overload of parenchymal cells, it also results in increased Fe content of Kuppfer cells [73 ], a feature not seen until very late-stage hemochromatosis. Schaible and colleagues [12 ] only studied ß2-microglobulin knockout mice that had received a high-Fe diet. Thus, the model may be more applicable to late-stage hemochromatosis in which the high tissue levels of Fe may negate the inherent beneficial effects of the limited intracellular Fe levels in macrophages from individuals with hemochromatosis that would be unopposed early in life. HFE– mouse strains have been created, and it will be important to determine whether these animals are as susceptible to M.tb infection in the presence or absence of Fe overload. Even then, murine macrophages generate much more nitric oxide than human macrophages, and therefore murine studies may be difficult to extrapolate to human disease.

In summary, we find that M.tb residing within the phagosomes of macrophages from individuals with hereditary hemochromatosis exhibit a profound defect in their ability to acquire Fe from exogenous Tf and Lf relative to M.tb-infected macrophages from healthy control subjects. There is also a corresponding decrease in M.tb growth. Our studies provide further evidence in support of the possibility that the macrophage-labile Fe pool is a major, and perhaps predominant source, of Fe used by intraphagosomal M.tb. Further studies to define the routes whereby M.tb gains access to host Fe and the impact of the HFE– phenotype on the process are needed. Such work could prove beneficial in the development of strategies to disrupt M.tb Fe metabolism as a means of therapy against this important human pathogen.

ACKNOWLEDGEMENTS

This work was supported in part by VA Merit Review Grants (B.E.B. and L.S.S.) and NIH Grants [AI24954 (B.E.B.), AI33004 (L.S.S.), and AI43870 (L.S.S.)].

Received June 20, 2006; revised July 28, 2006; accepted August 14, 2006.

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