Accepted Manuscript

Iron acquisition by Mycobacterium tuberculosis residing within myeloid dendritic cells

Oyebode Olakanmi, Banurekha Kesavalu, Maher Y. Abdalla, Bradley E. Britigan

PII: S0882-4010(13)00121-6

DOI: 10.1016/j.micpath.2013.09.002

Reference: YMPAT 1441

To appear in: Microbial Pathogenesis

Received Date: 19 June 2013

Revised Date: 4 September 2013

Accepted Date: 6 September 2013

Please cite this article as: Olakanmi O, Kesavalu1 B, Abdalla MY, Britigan BE, Iron acquisition byMycobacterium tuberculosis residing within myeloid dendritic cells, Microbial Pathogenesis (2013), doi:10.1016/j.micpath.2013.09.002.

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IRON ACQUISITION BY MYCOBACTERIUM TUBERCULOSIS RESIDING

WITHIN MYELOID DENDRITIC CELLS

Oyebode Olakanmi1,2, Banurekha Kesavalu11, Maher Y. Abdalla3-5, and Bradley E.

Britigan3-5#

Medical and Research Services, VA Medical Center-Cincinnati, Cincinnati, OH 452201,

Department of Internal Medicine University of Cincinnati College of Medicine2, Cincinnati, OH

45267, Research Service, VA Medical Center-Omaha Nebraska Western Iowa Omaha, NE

681053 and Departments of Internal Medicine4 and Pathology and Microbiology5, University of

Nebraska Medical Center College of Medicine, Omaha, NE 681984

Running Title: Iron uptake by M.tb in Dendritic Cells

Address Correspondence to:

Bradley E. Britigan, M.D.

985520 Nebraska Medical Center

Omaha, NE 68198-5520

E-mail: bradley.britigan@unmc.edu

Tel: (402) 559-4204

FAX: 402-559-4148

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ABSTRACT

The pathophysiology of Mycobacterium tuberculosis (M.tb) infection is linked to the ability of

the organism to grow within macrophages. Lung myeloid dendritic cells are a newly recognized

reservoir of M.tb during infection. Iron (Fe) acquisition is critical for M.tb growth. In vivo,

extracellular Fe is chelated to transferrin (TF) and lactoferrin (LF). We previously reported that

M.tb replicating in human monocyte-dervied macrophages (MDM) can acquire Fe bound to TF,

LF, and citrate, as well as from the MDM cytoplasm. Acccess of M.tb to Fe may influence its

growth in macrophages and dendritic cells. In the present work we confirmed the ability of

different strains of M.tb to grow in human myeloid dendritic cells in vitro. Fe acquired by M.tb

replicating within dendritic cells from externally added Fe chelates varied with the Fe chelate

present in the external media: Fe-citrate> Fe-LF>Fe-TF. Fe acquisition rates from each chelate

did not vary over 7 days. M.tb within dendritic cells also acquired Fe from the dendritic cell

cytoplasm, with the efficiency of Fe acquisition greater from cytoplasmic Fe sources, regardless

of the initial Fe chelate from which that cytoplasmic Fe was derived. Growth and Fe acquisition

results with human MDM were similar to those with dendritic cells. M.tb grow and replicate

within myeloid dendritic cells in vitro. Fe metabolism of M.tb growing in either MDM or

dendritic cells in vitro is influenced by the nature of Fe available and the organism appears to

preferentially access cytoplasmic rather than extracellular Fe sources. Whether these in vitro data

extend to in vivo conditions should be examined in future studies.

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1. INTRODUCTION

Pulmonary tuberculosis (TB) is a worldwide health problem. TB pathophysiology is

intimately linked to the ability of the causative agent, Mycobacterium tuberculosis (M.tb), to

enter and multiply within human macrophages while situated in a unique phagosomal

compartment. Acquiring Fe is critical to the metabolism and growth of most microbes, including

M.tb. Limiting Fe availability is a strategy of host defense [1, 2]. In vivo, nearly all extracellular

Fe is chelated to the serum and mucosal proteins transferrin (TF) and lactoferrin (LF), markedly

decreasing microbial access to Fe [1, 2]. Fe also shifts from serum to macrophages in the bone

marrow, liver and spleen during acute and chronic infections [2]. This process, responsible for

“anemia of chronic disease”, is mediated by hepcidin, a defensin family cationic peptide,

released by hepatocytes in response to IL-6 and other proinflammatory factors [2]. Hepcidin

inhibits release of Fe from macrophages and other cells by inducing the internalization and

breakdown of the plasma membrane Fe exporting protein, ferroportin [3].

Essentially all successful pathogens have evolved strategies to acquire host Fe. Whereas

extracellular Fe is available to most bacteria, pathogens such as M.tb that live within cells must

acquire Fe from their intracellular locale. Fe is needed for in vitro M.tb growth in culture media

and in macrophages [4]. M.tb siderophore production is critical to this process [4]. Although

some evidence exists for direct trafficking of extracellular TF to the phagosome [5-7], little is

known about how M.tb acquires Fe while sequestered within the macrophage phagosome.

Functional studies with different Nramp homologues indicated that Nramp1 is present in late

endosomes and lysosomes and functions to transport Fe into the bacterium-containing

phagosome [8-10], whereas other studies provided evidence that Nramp1 transports metal

cations out of the phagolysosome in an ATP-dependent process [11]. Furthermore, elegant

studies by Gros and Grinstein [12] show that Nramp1 functions primarily as a H+ and Mn2+

transporter. Thus, the role of human Nramp1 in the pathogenesis of human TB in general and Fe

acquisition by intracellular M.tb in particular, remains unclear at this time

We have provided in vitro evidence that M.tb located within human macrophages can

acquire Fe from extracellular TF, LF and citrate, as well as the macrophage cytoplasm [13].

Furthermore, M.tb Fe acquisition from extracellular TF, LF and citrate may involve Fe

movement to the macrophage cytoplasm, followed by Fe transfer to M.tb [13-15]. The nature of

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the extracellular Fe chelate (e.g. TF vs. LF) may influence both the ability of M.tb to acquire that

Fe and the route that the Fe takes to get to the bacteria [15]. In addition, we showed that some

conditions that lower macrophage intracellular Fe, such as hereditary hemochromatosis (due to

genetic mutations altering the protein HFE), but not all (e.g. IFN-γ), decrease Fe trafficking to

phagosomal M.tb [7, 14].

Although the macrophage has historically been viewed as the principal site of M.tb growth,

M.tb also have been shown to be capable of growing in dendritic cells [16, 17] in vivo, recent

data suggest that lung myeloid dendritic cells may also be a major M.tb reservoir during lung

infection [18]. M.tb growing within dendritic cells would still need access to Fe. Yet, the cell

biology of dendritic cells is distinct from macrophages [19]. Paradigms of M.tb biology in human

macrophages may not apply to dendritic cells. For example, although still controversial, evidence

has been published that is consistent with the ability of M.tb to escape the dendritic cell

phagosome and enter the cytoplasm [20].

Therefore, we hypothesized that the nature of Fe sources and/or the mechanism of Fe

acquisition employed by M.tb growing within dendritic cells might be different from that for

M.tb residing within macrophages. For example, it seemed likely that M.tb growing within a

dendritic cell may be able to more readily acquire Fe residing in the dendritic cell cytoplasm

compared to extracellular Fe. There are essentially no prior studies of Fe acquisition by M.tb in a

cell other than a macrophage. Accordingly, the above hypothesis was examined in vitro and the

results are the subject of this report.

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2. MATERIALS AND METHODS

2.1. Mycobacterium tuberculosis strains and cultivation

Mycobacterium tuberculosis strains H37Ra (ATCC 25177), H37Rv (ATCC 27294) and

Erdman (ATCC 35801) were cultivated on 7H11 agar plates for 10 d. They were then harvested

into 7H9 medium containing 10mM HEPES, to form predominantly single-cell suspensions, as

previously reported [21], and used shortly thereafter.

2.2. Human Myeloid Dendritic Cells and Monocyte-Derived Macrophages

In order to generate human myeloid dendritic cells, heparinized blood was obtained from

healthy adult volunteers, who were without a history of infection with M.tb. Peripheral blood

mononuclear cells were isolated as previously described [7, 14, 22]. The mononuclear cells were

adhered onto a Petri dish for 4 h after which the monolayer was washed 3 times to remove the

non-adherent lymphocytes. The monocytes were collected with 0.5 mM EDTA in PBS without

Ca2+ or Mg2+. Cells were washed in RPMI 3 times and then cultured at 1x106 cells/mL for 6 d in

Teflon wells (Savillex, Minnetonka, MN) containing RPMI supplemented with 20% autologous

serum, GM-CSF (300 U/mL) and IL-4 (200U/mL,), both from PeproTech, Rocky Hill, NJ.

Additional GM-CSF and IL-4 were added on days 2 and 4. On day 6, the dendritic cells were

collected and washed twice in RPMI (500 x g for 10 min), counted and re-suspended at 1x106

cells/mL in RPMI supplemented with 1% serum.

In order to generate monocyte-derived macrophages (MDM), monocytes collected as above

were cultured in Teflon wells containing RPMI supplemented with 20% autologous serum for 5

d. Cells were harvested, washed, and counted. The 5 d old MDM were adhered on to six-well

plates for 2 h, repleted with 20% serum in RPMI overnight, and then washed.

Where desired, cells, dendritic cells or MDM, were infected with M.tb bacilli at an MOI of 2

as per our standard protocol [7, 14, 22].

2.3. Surface Expression of Proteins on Cellular Membrane

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To ascertain the differentiation of monocytes into dendritic cells, the 6-day old cells cultured

in the presence of GM-CSF and IL-4, as described above, were tested for expression of

membrane proteins specific for dendritic cells by FACS using a FACSDiva Version 6.1.2

(Becton, Dickenson Company, San Jose, CA). The cells were stained for various membrane

markers including mouse anti-human HLA ABC, CD11C, CD 86, CD 80 and CD 83 (all

antibodies were obtained from AbD serotec, Raleigh, NC). Background was assessed for

autofluorescence by staining cells with IgG1 isotype (AbD serotec) as negative control, which

was subtracted from specific positive event populations. A total of 20,000 events with 10,000

specific events for each marker were collected. Data are reported as mean +/- SD (n=4) of

positive events after correction for isotype.

2.4. Acquisition of Iron by M. tb, dendritic cells, and macrophages

Myeloid dendritic cells were incubated with M.tb at MOI of 2 for 2 h at 37oC in RPMI

containing 10 mM HEPES. 1 mg/mL human serum albumin (HAS), and washed to remove non-

phagocytosed M.tb. The cells were then incubated in RPMI supplemented with 1% autologous

serum for 24 h after which 10 µM 59Fe chelated to TF, LF, or citrate was added. The cells were

incubated for a specified time and processed for the measurement of total cellular 59Fe and

bacterial-associated 59Fe as previously described [7, 14, 22]. Briefly, cells were washed twice

with warm RPMI containing 5 mM ascorbate that our previous studies showed removed

membrane-associated, but non-internalized, Fe. Cells were then lysed with 0.1% SDS in the

presence of EDTA-free Protease Inhibitor Cocktail Tablet (Boehringer Mannheim/Roche,

Indianapolis, IN) and 250 U/mL DNase (Invitrogen, Carlsbad, CA). Aliquots of the lysate were

withdrawn to determine the total cellular 59Fe. In addition, M.tb CFUs were determined by serial

dilution onto 7H11 agar plates. The rest of the lysate was centrifuged (10,000 x g, 10 min, 4o C)

and the supernatant was discarded. The pellet was washed three times in RPMI containing 5 mM

ascorbate and .01% SDS. The pellet was finally re-suspended in the same medium and added to

spin-x (Millipore, Billerica, MA), and centrifuged. The filter was cut into a 5 mL gamma tube

and counted for the presence of 59Fe. Results for M.tb-associated 59Fe were expressed as 59Fe/CFU and for eukaryotic cells 59Fe/106 cells. This washing procedure has been previously

found to eliminate Fe bound to the cell surfaces[14]. For 59Fe acquisition from an endogenous

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source within the cells, the cells were incubated with the desired [59Fe2] chelate for 24 h and

washed. M.tb bacilli were added at an MOI of 2 for 2 h, following which bacilli not associated

with the cells were removed by washing. No additional 59Fe chelate was present during the time

following M.tb infection of the cells. The remainder of the assay was then performed as above.

2.5. Analysis of Data

Statistical analysis was done using GraphPad Prism version 4 for Windows (GraphPad Software

San Diego, CA). For analysis limited to two groups, Student’s t-test was employed. To determine

differences between three or more means, we used one-way analysis of variance (ANOVA) with

Bonferroni post-hoc tests. Error bars represent mean ± SEM. All statistical analyses are

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).

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3. RESULTS

3.1. Growth of M.tb in dendritic cells and MDM shows equivalent rates regardless

the nature of the extracellular Fe chelate present.

It has been reported that M.tb has the ability to grow and replicate within myeloid dendritic

cells [18, 20]. Fe availability is critical to the ability of M.tb to grow and replicate in

macrophages [4] and cellular Fe content has been reported to influence the function of

macrophages [23, 24]. We previously demonstrated that the nature of exogenous Fe presented to

the macrophage impacts the ability of M.tb to access that Fe and influences the growth of M.tb

within these phagocytes [13-15]. Furthermore, we found that infection of macrophages by M.tb

alters the magnitude of Fe acquired from extracellular sources by these cells [14, 15].

We hypothesized that similar variation in Fe utilization would occur for M.tb located within

myeloid dendritic cells and that infection with M.tb would alter myeloid dendritic cell Fe

metabolism. In order to test these hypotheses, myeloid dendritic cells were generated by

incubating human peripheral blood monocytes in the presence of IL-4 and GM-CSF for 6 days.

As expected, this treatment resulted in the generation of cells with surface marker expression of

myeloid dendritic cells: CD80+ (5.4 +/- 1.4%); CD83+ (0.0 +/- 2.1%); CD86+ (10.8 +/- 7.9%);

CD11c+ (75.3 +/- 6.2%); and HLAABC+ (82.7 +/- 6.6%) [25, 26]. Furthermore, consistent with

earlier literature [18, 20], M.tb grew well in these cells and at rates equivalent to those observed

in human MDM, regardless of the nature of the supplemental Fe source provided (Figure 1).

3.2. Iron Acquisition by Myeloid Dendritic Cells Varies with the Nature of the Iron

Chelate

In advance of examining the nature of Fe acquisition by M.tb growing in dendritic cells, we

first evaluated the magnitude of Fe acquired by dendritic cells themselves from three physiologic

sources of extracellular Fe. Dendritic cells were incubated with 59Fe bound to TF, LF, or citrate

and the amount of cell-associated 59Fe determined at various time points. Dendritic cell 59Fe

acquisition was found to be greatest when the 59Fe was initially bound to citrate, with Fe

acquisition from LF and TF similar (Figure 2).

We previously reported that infection of MDM with M.tb results in an approximately 50%

decrease in the magnitude of Fe acquired by these cells from TF, LF, or citrate [14, 15]. In

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contrast to results with MDM, infection of dendritic cells with M.tb had no effect on the

magnitude of Fe acquired by these cells from TF, LF, or citrate (Figures 2A, 2B and 2C).

Overall hierarchy of Fe acquisition by dendritic cells from the different Fe chelates was similar

to that of MDM, although on a per cell basis the amount of 59Fe acquired by the dendritic cells

from each chelate ranged from 10-50% lower than that seen with MDM (Figure 2D).

3.3. Iron Acquisition by M. tuberculosis Within Myeloid Dendritic Cells Varies with

the Nature of the External Iron Chelate

We previously found that M.tb varies in its ability to acquire 59Fe from extracellular sources

when growing within human MDM [13-15]. The extent to which this observation applies to

M.tb growing in myeloid dendritic cells was explored. The magnitude of 59Fe acquisition

normalized per number of CFU at 24 h was similar from citrate and LF, which was in turn

greater than that from TF (Figure 3). The Erdman strain appeared to be better able to acquire Fe

from TF than H37Ra. Results with the H37Rv strain were similar to Erdman (data not shown).

We found that the pattern of Fe acquisition per 24 h period was similar at each time point

examined from 1 to 5 days post-infection (Figures 4A-C) in terms of the magnitude of Fe

acquired each chelate: citrate>LF>TF. The amount of Fe acquired from LF and TF remained

stable or decreased slightly at later time intervals (Figure 4A and 4B). This was somewhat

surprising in that, as noted above, the amount of Fe acquired from TF and LF by the dendritic

cells themselves increased with each additional 24 h period post-infection (Figure 2). This

suggests that factors in addition to dendritic cell absolute Fe content influence the magnitude of

Fe acquisition by M.tb. Results of Fe acquisition by M.tb within dendritic cells and human

MDM were quite similar in magnitude and hierarchy of preferred chelates at each time interval

(Figures 4A-C).

3.4. Iron Acquisition From Extracellular Versus Intracellular Iron Sources by M.

tuberculosis Within Myeloid Dendritic Cells

The above results indicated that M.tb located within dendritic cells is able to gain access to

Fe initially bound to extracellular proteins or other chelates. However, the route by which that

Fe reaches the bacteria is not clear. Does Fe traffic directly to the bacterium or first move to a

cytosolic pool of the cell to which the organism has access? To examine these possibilities, we

measured 59Fe acquired from dendritic cells that had been exposed to 59Fe bound to TF, LF, or

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citrate for 24 h prior to infection with M.tb. The cells were washed free of extracellular 59Fe prior

to infection with M.tb. Hence the only 59Fe to which the organism could potentially have access

was that internalized by the cell prior to infection. Under these experimental conditions, termed

the endogenous paradigm, 59Fe acquisition by M.tb was detected. The magnitude of 59Fe uptake

by M.tb from the dendritic cell intracellular sites (endogenous paradigm) was considerably less

than occurred when the 59Fe was present extracellularly during the time of infection, termed the

exogenous paradigm (Figure 5A). Interestingly, the relative magnitude of 59Fe uptake when the 59Fe was provided initially bound to TF, LF, or citrate was the same for both the endogenous and

endogenous paradigms – citrate > LF > TF (Figure 5A). The ability of M.tb to acquire Fe to a

lesser extent with the endogenous and exogenous paradigm, as measured by Fe/CFU, paralleled

the amount of 59Fe present in the dendritic cells under the same conditions (Figure 5B).

However, if the iron uptake by M.tb is normalized per the mount of 59Fe available to it as

reflected by total dendritic cell 59Fe (calculated as the ratio of M.tb 59Fe: dendritic cell 59Fe), the

data show that M.tb is better able to acquire Fe from endogenous sources regardless of the

extracellular Fe chelate provided (Figure 5C).

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4. DISCUSSION

Acquiring Fe is critical to the metabolism and growth of most microbes, including M.tb.

Limiting Fe availability is a strategy of host defense [1, 2]. In vivo, nearly all extracellular Fe is

chelated to the serum and mucosal proteins TF and LF, markedly decreasing Fe availability for

use by invading microbes [1, 2]. In addition, a small portion of extracellular Fe is in the form of

low molecular chelates, bound to small anions such as citrate [27]. 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. Intracellular Fe is generally split into two broad categories of storage. Once

internalized, Fe enters a cytoplasmic labile iron pool (LIP) that is comprised of a variety of low

molecular weight Fe chelates, such as Fe-citrate [28]. Once in the LIP, Fe is used for cellular

needs or transferred to ferritin, the principal storage form of intracellular Fe [29].

Macrophages are reservoirs for host Fe storage. With Fe overload or infection, extracellular

Fe moves into macrophages, from where it can be mobilized back into the circulation as needed

[29]. Over the last several decades the mechanism(s) that result in Fe acquisition from TF have

been increasingly understood, particularly that linked to the presence of TF receptors on the cell

surface [30]. Although, it has been clearly shown that cells can acquire Fe from LF and citrate,

the mechanism(s) responsible remains poorly defined, but it does not appear to involve TF

receptors [31, 32]. In contrast to macrophages, little is known about the Fe metabolism of

dendritic cells, except that they express some of the key components of general cellular Fe

metabolism, including TF receptors, heme oxygenase-1, ferroportin, DMT-1 and HFE [33, 34].

In the present work, we found that human myeloid dendritic cell exhibited the ability to

acquire Fe in vitro from TF, LF, or citrate. The magnitude of Fe acquisition varied with the

chelate: TF<LF<citrate. When compared to current and prior studies in human MDM [7, 14,

15], the magnitude of Fe acquisition by myeloid dendritic cells was slightly lower under the

same conditions compared to that seen with M.tb-infected MDM. In prior work, we reported that

Fe acquisition by MDM from TF and LF decreases approximately 50% when the cells are

infected by M.tb [14, 15]. In contrast to our experience with MDM, Fe acquisition from all

chelates by dendritic cells was not significantly affected by M.tb infection.

M.tb growing within macrophages requires access to Fe to grow and replicate [35, 36] and

we have previously shown that M.tb varies in its ability to acquire 59Fe from extracellular sources

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when growing within human MDM [7, 14, 15]. Evidence has been presented that lung that M.tb

can also grow in dendritic cells [16, 17] and that myeloid dendritic cells may be a major site of

M.tb growth during lung infection [18]. It should be noted that previous reports have shown that

unlike macrophages, IL-10 converted DC induces growth inhibition of intracellular

mycobacterial pathogen [16], and these monocyte-derived DCs do not allow intracellular growth

of M.tb (H37Rv), although the bacteria are not killed [17].

M.tb would need to have access to Fe to grow in dendritic cells and that the cell biology of

dendritic cells and macrophages are different [19]. Published data suggest that, in contrast to

their behavior in macrophages, M.tb is able to move effectively from the phagosome to the

cytoplasm when growing in dendritic cells. However these data remain controversial. Other data

indicate that M.tb that resides within phagosomes and blocks or delays lysosomal fusion by

interfering signal transduction pathways [37, 38]. Recently it was shown that phagolysosomal

rupture followed by necrotic cell death of the infected macrophages might help M.tb to escape

innate host defenses and favor their spread to new cells [39, 40].

Although recognizing its controversial nature, given some evidence that M.tb is able to

move effectively from the phagosome to the cytoplasm when growing in dendritic cells, we

hypothesized that the Fe sources and/or the mechanism of Fe acquisition employed by M.tb

growing within dendritic cells versus macrophages might be different. Fe acquisition by M.tb

growing within dendritic cells was slightly greater from citrate than LF, which was in turn much

greater than that from TF. Results were similar for all the M.tb strains examined, except that the

virulent Erdman strain of M.tb exhibited greater Fe acquisition from TF at 24 h than the avirulent

H37Ra strain. Whether or not this observation contributes to virulence is unclear at this point and

will require further investigation. The overall results with dendritic cells are slightly different

from those observed for M.tb growing in MDM. For M.tb growing in MDM, M.tb Fe acquisition

from TF was closer to that observed with LF, but the overall pattern (TF<LF<citrate) was

similar. It has been revealed by comparative genomic analysis of M.tb strain H37Ra versus

H37Rv that there are at least 35 PE/PPE/PE-PGRS family members that differ between H37Ra

and H37Rv [41]. Moreover, it was reported that IdeR, a regulator of genes responding to Fe and

siderophore synthesis are connected to the PE/PPE family genes in M.tb [42]. Therefore, it is

possible that differences in one or more genes between H37Ra and H37Rv might contribute to

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the lesser ability of H37Ra to efficiently use TF-bound Fe as an Fe source during its intracellular

replication in the dendritic cells.

We also studied the ability of M.tb to acquire Fe over 24 h in daily increments post

infection. Somewhat surprisingly, the magnitudes of Fe acquisition from each of the three

chelates studied were similar at each time point. Similar stability of Fe uptake rates were seen

with M.tb in MDM at the various time intervals, with the exception that Fe acquisition from

citrate was higher in the 120 h infection period than at earlier time points.

When the ability of M.tb growing within dendritic cells to acquire Fe from the dendritic cell

cytoplasm was compared to Fe acquired from chelates added extracellularly, considerably less

M.tb Fe uptake occurred from the dendritic cell cytoplasm. This appears to relate to the lesser

amount of dendritic cell-associated 59Fe that resulted from the endogenous paradigm.

Interestingly, the relative magnitude of Fe uptake from dendritic cell cytoplasm when only the

cytoplasmic Fe pool was labeled by 59Fe that was provided initially bound to TF, LF, or citrate

remained identical to that using the exogenous paradigm: citrate > LF > TF. When M.tb Fe

uptake was normalized to the amount of Fe available to the bacteria, as defined by total dendritic

cell 59Fe, the efficiency of Fe acquisition by M.tb was significantly greater from the endogenous

Fe source than from the external one. This was true regardless of whether Fe was initially

provided externally as Fe-TF, Fe-LF, or Fe citrate. These data suggest that access to cytoplasmic

Fe may be more important to intracellular M.tb than access to extracellular Fe. Extrapolation of

these data would also suggest that Fe acquired from the extracellular environment may involve

initial movement of Fe to the cytoplasm of the dendritic cell where it is then acquired by M.tb,

rather than direct movement of Fe to the M.tb containing phagosome. Although the work

reported used the attenuated M.tb strain (H37Ra), limited studies with virulent strains H37Rv

and Erdman have yielded similar results.

In summary, our work confirms the ability of M.tb to grow in human myeloid dendritic

cells, with rates observed to be similar to that in MDM. This work is the first to demonstrate and

quantify Fe acquisition by M.tb replicating within a cell other than a classic macrophage and

shows a correlation between the ability of the organism to acquire Fe and its rate of growth in

that cell type. These data suggest that the nature of Fe available within the local environment

may influence the growth of M.tb within both macrophages and myeloid dendritic cells. It also

appears that Fe present in the dendritic cell cytoplasm is preferably accessed by the bacteria

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relative to that present in the extracellular space. Several conditions, most notably cigarette

smoking, is known to increase the amount of low molecular weight Fe chelates in the airway and

increasing the Fe content of reticuloendothelial cells [43-45]. In addition, M.tb is also able to

utilize Fe derived from hemoglobin to meet its metabolic needs [46], which could be important

given that macrophages degrade senescent erythrocytes [47]. Future studies addressing the

potential impact of various Fe chelates within the lung, as well as the trafficking of that Fe to the

myeloid cell cytoplasm, on M.tb infection in vivo could improve our understanding of factors

that influence the pathogenesis of infection with this important human pathogen.

Acknowledgements

This work is based upon work supported in part by the Department of Veterans Affairs through

a Merit Review grant to BEB. The contents do not necessarily represent the view of the

Department of Veterans Affairs or U.S. Government.

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References

[1] Finkelstein RA, Sciortino CV, McIntosh MA. Role of iron in microbe-host interactions. Rev Infect Dis 1983; 5:5759-77.

[2] Nemeth E, Ganz T. Regulation of Iron Metabolism by Hepcidin. Annu Rev Nutr

2006; 26(1):323-42. [3] Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, Ganz T,

Kaplan J. Hepcidin Regulates Cellular Iron Efflux by Binding to Ferroportin and Inducing Its Internalization. Science 2004; 306(5704):2090-93.

[4] Ratledge C. Iron, mycobacteria and tuberculosis. Tuberculosis (Edinburgh,

Scotland) 2004; 84(1):110-30. [5] Clemens DL, Horwitz MA. The Mycobacterium tuberculosis phagosome interacts

with early endosomes and is accessible to exogenously administered transferrin. J Exp Med 1996; 184(4):1349-55.

[6] Wagner D, Maser J, Lai B, Cai Z, Barry CE, Höner zu Bentrup K, Russell DG,

Bermudez LE. Elemental Analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-Containing Phagosomes Indicates Pathogen-Induced Microenvironments within the host cell's endosomal system. J Immunol 2005; 174(3):1491-500.

[7] Olakanmi O, Schlesinger LS, Britigan BE. Hereditary hemochromatosis results in

decreased iron acquisition and growth by Mycobacterium tuberculosis within human macrophages. J Leukocyte Biol 2007; 81(1):195-204.

[8] North RJ, Medina E. How important is Nramp1 in tuberculosis? Trends Microbiol

1998; 6(11):441-43. [9] Kuhn DE, Baker BD, Lafuse WP, Zwilling BS. Differential iron transport into

phagosomes isolated from the RAW264.7 macrophage cell lines transfected with Nramp1Gly169 or Nramp1Asp169. J Leukocyte Biol 1999; 66(1):113-9.

[10] Kuhn DE, Lafuse WP, Zwilling BS. Iron transport into Mycobacterium avium -

containing phagosomes from an Nramp1 Gly169 -transfected RAW264.7 macrophage cell line. J Leukocyte Biol 2001; 69(1):43-49.

[11] Peracino B, Wagner C, Balest A, Balbo A, Pergolizzi B, Noegel AA, Steinert M,

Bozzaro S. Function and Mechanism of Action of Dictyostelium Nramp1 (Slc11a1) in Bacterial Infection. Traffic 2006; 7(1):22-38.

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[12] Jabado N, Jankowski A, Dougaparsad S, Picard V, Grinstein S, Gros P. Natural Resistance to Intracellular Infections: Natural Resistance–Associated Macrophage Protein 1 (Nramp1) Functions as a Ph-Dependent Manganese Transporter at the Phagosomal Membrane. The Journal of Experimental Medicine 2000; 192(9):1237-48.

[13] Olakanmi O, Britigan BE, Schlesinger LS. Gallium Disrupts Iron Metabolism of

Mycobacteria Residing within Human Macrophages. Infect Immun 2000; 68(10):5619-27.

[14] Olakanmi O, Schlesinger LS, Ahmed A, Britigan BE. Intraphagosomal

Mycobacterium tuberculosis acquires iron from both extracellular iron and intracellular iron pools: impact of interferon- g and hemochromatosis. J Biol Chem 2002; 277:49727-34.

[15] Olakanmi O, Schlesinger LS, Ahmed A, Britigan BE. The Nature of Extracellular

Iron Influences Iron Acquisition by Mycobacterium tuberculosis Residing within Human Macrophages. Infect Immun 2004; 72(4):2022-28.

[16] Förtsch D, Röllinghoff M, Stenger S. IL-10 Converts Human Dendritic Cells into

Macrophage-Like Cells with Increased Antibacterial Activity Against Virulent Mycobacterium tuberculosis. J Immunol 2000; 165(2):978-87.

[17] Tailleux L, Neyrolles O, Honoré-Bouakline Sp, Perret E, Sanchez Fo, Abastado J-

P, Lagrange PH, Gluckman JC, Rosenzwajg M, Herrmann J-L. Constrained Intracellular Survival of Mycobacterium tuberculosis in Human Dendritic Cells. The Journal of Immunology 2003; 170(4):1939-48.

[18] Wolf AJ, Linas B, Trevejo-Nuأ±ez GJ, Kincaid E, Tamura T, Takatsu K, Ernst JD.

Mycobacterium tuberculosis Infects Dendritic Cells with High Frequency and Impairs Their Function In Vivo. J Immunol 2007; 179(4):2509-19.

[19] Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. Nat Rev Immunol

2005; 5(12):953-64. [20] van der Wel N, Hava D, Houben D, Fluitsma D, van Zon M, Pierson J, Brenner M,

Peters PJ. M. tuberculosis and M. leprae Translocate from the Phagolysosome to the Cytosol in Myeloid Cells. Cell 2007; 129(7):1287-98.

[21] Schlesinger LS, Bellinger-Kawahara CG, Payne NR, Horwitz MA. Phagocytosis of

Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3. J Immunol 1990; 144(7):2771-80.

[22] Gaynor CD, McCormack FX, Voelker DR, McGowan SE, Schlesinger LS.

Pulmonary surfactant protein A mediates enhanced phagocytosis of

MANUSCRIP

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17

Mycobacterium tuberculosis by a direct interaction with human macrophages. J Immunol 1995; 155:5343-51.

[23] Ward RJ, Wilmet S, Legssyer R, Crichton RR. The influence of iron homoeostasis

on macrophage function. Biochem Soc Trans 2002; 30(4):762-65. [24] Weiss G, Wachter H, Fuchs D. Linkage of cell-mediated immunity to iron

metabolism. Trends Immunol 1995; 16:495-500. [25] Freudenthal PS, Steinman RM. The distinct surface of human blood dendritic cells,

as observed after an improved isolation method. Proc Natl Acad Sci 1990; 87(19):7698-702.

[26] Dauer M, Obermaier B, Herten J, Haerle C, Pohl K, Rothenfusser S, Schnurr M,

Endres S, Eigler A. Mature Dendritic Cells Derived from Human Monocytes Within 48 Hours: A Novel Strategy for Dendritic Cell Differentiation from Blood Precursors. J Immunol 2003; 170(8):4069-76.

[27] De Jong G, Van Dijk JP, Van Eijk HG. The biology of transferrin. Clin Chim Acta

1990; 190:1-46. [28] Hentze MW, Muckenthaler MU, Andrews NC. Balancing Acts: Molecular Control

of Mammalian Iron Metabolism. Cell 2004; 117(3):285-97. [29] Hentze MW, Muckenthaler MU, Galy B, Camaschella C. Two to Tango: Regulation

of Mammalian Iron Metabolism. Cell 2010; 142(1):24-38. [30] Birgens HS. The interaction of lactoferrin with human monocytes. Dan Med Bull

1991; 38:244-52. [31] Olakanmi O, Rasmussen GT, Lewis TS, Stokes JB, Kemp JD, Britigan BE.

Multivalent metal-induced iron acquisition from transferrin and lactoferrin by myeloid cells. J Immunol 2002; 169:2076-84.

[32] Tamaki T, Konoeda Y, Tanaka M, Takahashi Y, Uchida Y, Kaizu T, Kawamura A.

Heme oxygenase-1 expression in rat kupffer cells by immunomodulating agents: Role in the pathogenesis of hepatic ischemia/reperfusion injury. Transplant Proc 2000; 32(7 PT 1):1665-66.

[33] Brinkmann M, Teuffel R, Laham N, Ehrlich R, Decker P, Lemonnier FA, Pascolo S.

Expression of iron transport proteins divalent metal transporter-1, Ferroportin-1, HFE and transferrin receptor-1 in human monocyte-derived dendritic cells. Cell Biochem Funct 2007; 25(3):287-96.

[34] Chauveau C, Rémy S, Royer PJ, Hill M, Tanguy-Royer S, Hubert F-X, Tesson L,

Brion R, Beriou G, Gregoire M, Josien R, Cuturi MC, Anegon I. Heme oxygenase-1

MANUSCRIP

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ACCEPTED MANUSCRIPT

18

expression inhibits dendritic cell maturation and proinflammatory function but conserves IL-10 expression. Blood 2005; 106(5):1694-702.

[35] De Voss JJ, Rutter K, Schroeder BG, Barry CE, III. Iron acquisition and

metabolism by mycobacteria. J Bacteriol 1999; 181(15):4443-51. [36] De Voss JJ, Rutter K, Schroeder BG, Su H, Zhu YQ, Barry CE, III. The salicylate-

derived mycobactin siderophores of Mycobacterium tuberculosis are essential for growth in macrophages. Proc Natl Acad Sci 2000; 97(3):1252-57.

[37] Kumar D, Rao KVS. Regulation between survival, persistence, and elimination of

intracellular mycobacteria: a nested equilibrium of delicate balances. Microbes and Infection 2011; 13(2):121-33.

[38] Ehrt S, Schnappinger D. Mycobacterial survival strategies in the phagosome:

defence against host stresses. Cellular Microbiology 2009; 11(8):1170-78. [39] de Chastellier C. The many niches and strategies used by pathogenic mycobacteria

for survival within host macrophages. Immunobiology 2009; 214(7):526-42. [40] Simeone R, Bobard A, Lippmann J, Bitter W, Majlessi L, Brosch R, Enninga J.

Phagosomal Rupture by <italic>Mycobacterium tuberculosis</italic> Results in Toxicity and Host Cell Death. PLoS Pathog 2012; 8(2):e1002507.

[41] Zheng H, Lu L, Wang B, Pu S, Zhang X, Zhu G, Shi W, Zhang L, Wang H, Wang S,

Zhao G, Zhang Y. Genetic Basis of Virulence Attenuation Revealed by Comparative Genomic Analysis of Mycobacterium tuberculosis Strain H37Ra versus H37Rv. PLoS One 2008; 3(6):e2375.

[42] Rodriguez GM, Voskuil MI, Gold B, Schoolnik GK, Smith I. ideR , an essential gene

in Mycobacterium tuberculosis : Role of IdeR in iron-dependent gene expression, iron metabolism, and oxidative stress response. Infect Immun 2002; 70(7):3371-81.

[43] McGowan SE, Murray JJ, Parrish MG. Iron binding, internalization, and fate in

human alveolar macrophages. J Lab Clin Med 1986; 108:587-95. [44] McGowan SE, Henley SA. Iron and ferritin content and distribution in human

alveolar macrophages. J Lab Clin Med 1988; 111:611-17. [45] Thompson AB, Bohling T, Heires A, Linder J, Rennard SI. Lower respiratory tract

iron burden is increased in association with cigarette smoking. J Lab Clin Med 1991; 117:493-99.

[46] Tullius MV, Harmston CA, Owens CP, Chim N, Morse RP, McMath LM, Iniguez

A, Kimmey JM, Sawaya MR, Whitelegge JP, Horwitz MA, Goulding CW.

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Discovery and characterization of a unique mycobacterial heme acquisition system. Proc Natl Acad Sci 2011; 108(12):5051-56.

[47] Knutson M, Wessling-Resnick M. Iron metabolism in the reticuloendothelial system.

Crit Rev Biochem Mol Biol 2003; 38(1):61-88.

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Figure legends

Figure 1: Growth of M.tb in dendritic cells and MDM varies with nature of the

extracellular Fe chelate present: Human myeloid dendritic cells or MDM were incubated with

M.tb (strain H37Ra) for 2 h at a MOI of 2. After 2 h, uningested bacteria were removed by

washing and the infected cells were placed in culture in medium supplemented with 10 µM Fe

chelated to TF, LF, or citrate. At defined time points (24-120 h) after infection the cells were

lysed and M.tb CFU determined by serial dilution. Shown are results in dendritic cells and

MDMs as a function of the nature of the Fe chelate present: Fe-TF (Panel A); Fe-LF (Panel B);

and Fe-citrate (Panel C). As shown in panel D that compares the 96h results from panels A-C for

dendritic cells and MDM, M.tb growth in both dendritic cells and MDM was similar, with slight

variations in M.tb growth depending upon the nature of the extracellular Fe chelate present. The

only significant difference between dendritic cells and MDM was that M.tb growth was greater

in greater with Fe-LF with dendritic cells than MDM (p<0.01), (n=3-5 independent

experiments).

Figure 2: Iron acquisition by M.tb-infected and uninfected dendritic cells and MDM varies

with nature of the extracellular Fe chelate present: Dendritic cells and MDM were incubated

with 10 µM 59Fe chelated to either TF, LF, or citrate for defined time periods following which

the cells were harvested and cell-associated 59Fe determined. Shown are results (n=4-10) at

different time points for control dendritic cells and M.tb H37Ra-infected dendritic cells as a

function of the presence of different exogenous Fe chelates: Fe-TF (Panel A); Fe-LF (Panel B);

and Fe-citrate (Panel C). Comparison of Fe acquisition by uninfected versus H37Ra infected

dendritic cells revealed no significant differences for any of the three Fe chelates examined

(panel A-C). For both uninfected and infected cells, at 120 h a significant difference in Fe

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acquisition by dendritic cells was seen for LF vs. citrate and TF vs. citrate (p < 0.04), but the

difference for TF vs. LF was not significant (p > 0.05). Panel D compares results with M.tb-

infected dendritic cells and MDM at 96h. For M.tb infected MDM, at 96 h a significant

difference in Fe acquisition was seen for LF vs. citrate (p<0.01)(**) and TF vs. citrate (p <

0.001)(**), but the difference for TF vs. LF was not significant (p > 0.05). For M.tb infected

dendritic cells, at 96 h a significant difference in Fe acquisition was seen for LF vs. citrate and

TF vs. citrate (p < 0.05)(*), but the difference for TF vs. LF was not significant (p > 0.05). (n=4-

7 independent experiments).

Figure 3: Fe acquisition by M.tb in dendritic cells varies with nature of the extracellular Fe

chelate present: Human myeloid dendritic cells were incubated with M.tb Erdman or H37Ra for

2 h at a MOI of 2. After 2 h, uningested bacteria were removed by washing and the infected

cells were placed in culture medium supplemented with 10 µM 59Fe chelated to TF, LF, or

citrate. At defined time points after infection the cells were lysed and M.tb-associated 59Fe and

CFU were determined. 59Fe per CFU was then calculated. Shown are results (n=3-4) as a

function of the external Fe chelate at 24 h for each of the two M.tb strains. Results with Erdman

show no significant difference in Fe acquisition from LF vs. TF or citrate vs. LF (p > 0.05) but a

significant difference with TF vs. citrate (p < 0.02). In the case of H37Ra at 24 h a significant

difference was observed for LF vs. TF (p < 0.03) and citrate vs. TF (p < 0.02), but no difference

was seen with LF vs. citrate (p > 0.05), (n=3-4 independent experiments)..

Figure 4: Fe acquisition rates by M.tb in dendritic cells and MDM are stable over time

post-infection: Human myeloid dendritic cells or MDM were incubated with H37Ra for 2 h at a

MOI of 2. After 2 h, uningested bacteria were removed by washing and the infected cells were

placed in culture medium supplemented with 10 µM 59Fe chelated to TF, LF, or citrate. At

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defined time points after infection the cells were lysed and M.tb-associated 59Fe and CFU were

determined. 59Fe per CFU was then calculated. Shown are results (n=3-4) as a function of the

external Fe chelate provided at each 24 h time point: Fe-TF (Panel A); Fe-LF (Panel B); and Fe-

citrate (Panel C). In DC, Fe acquisition by H37Ra was significantly greater for LF vs. TF (p <

0.02, panel A vs. panel B) and citrate vs. TF (p < 0.04, panel A vs. panel C), but not for LF vs.

citrate (p > 0.05). In the case of H37 Ra in MDM, Fe acquisition was significantly different for

LF vs. TF (p < 0.01, panel A vs. panel B), citrate vs. LF (p < 0.02, panel B vs. panel C) and

citrate vs. TF (p < 0.02, panel A vs. panel C). Fe acquisition for each 24 h time period was not

significantly different within each chelate/cell pair, (n=3-4 independent experiments).

Figure 5: Iron acquisition by M.tb in dendritic cells is more efficient from endogenous vs.

exogenous Fe sources: Human myeloid dendritic cells were incubated with M.tb H37Ra for 2 h

at a MOI of 2. After 2h uningested bacteria were removed by washing and the infected cells

were placed in culture in medium supplemented with 10 µM 59Fe chelated to TF, LF, or citrate.

At defined time points after infection the cells were lysed and M.tb-associated 59Fe and CFU

were determined. 59Fe per CFU was then calculated. This is referred to as the exogenous

paradigm as it measures Fe acquisition from an external Fe source. Alternatively, the cells were

incubated with 10 µM 59Fe chelated to TF, LF, or citrate for 24 h and then extensively washed to

remove all extracellular 59Fe. M.tb bacilli were then added at an MOI of 2 for 2 h, following

which bacilli not associated with the cells were removed by washing. The remainder of the assay

was then performed as above except that no exogenous 59Fe was provided during the subsequent

period of incubation. This is referred to as the endogenous paradigm as M.tb 59Fe acquisition

must come from 59Fe sources within the dendritic cell. Shown is M.tb (panel A, n=4-7

independent experiments) and dendritic cell (panel B, n=4-6 independent experiments)

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associated 59Fe as a function of the 59Fe chelate employed for the exogenous and endogenous

paradigms. M.tb 59Fe was normalized to the number of CFU. For all chelates Fe acquisition by

M.tb (panel A) was significantly greater (p < 0.04) in the exogenous versus endogenous

paradigm. The same was true for Fe acquisition from TF and LF with dendritic cell (panel B), p

< 0.04), but was not significant (p >0.05) for citrate. Panel C shows Fe uptake by M.tb as a

function of the amount of Fe available as defined by dendritic cell Fe (Panel B data divided by

Panel A data). M.tb Fe acquisition normalized for dendritic cell 59Fe was significantly higher

from endogenous sources (Fe-LF, or citrate) compared to exogenous sources (P<0.05). *

indicates significant differences between exogenous and endogenous paradigm as indicated

above.

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In vitro growth of M. tb is similar in human myeloid dendritic cells and macrophages • Fe acquired by intracellular M.tb from extracellular sources varies with the chelate

• M.tb in dendritic cells preferentially access cytoplasmic over extracellular Fe

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