metallomic analysis of macrophages infected with histoplasma

1136 • JID 2010:202 (1 October) • Winters et al

M A J O R A R T I C L E

Metallomic Analysis of Macrophages Infectedwith Histoplasma capsulatum Revealsa Fundamental Role for Zinc in Host Defenses

Michael S. Winters,1 Qilin Chan,2 Joseph A. Caruso,2 and George S. Deepe, Jr1,3

1Division of Infectious Diseases, University of Cincinnati College of Medicine, 2Department of Chemistry, University of Cincinnati,and 3Veterans Affairs Hospital, Cincinnati, Ohio

The fungal pathogen Histoplasma capsulatum evades the innate and adaptive immune responses and thriveswithin resting macrophages. Cytokines that induce antimicrobial activity, such as granulocyte macrophagecolony-stimulating factor (GM-CSF), inhibit H. capsulatum growth in macrophages. Conversely, interleukin4 inhibits the killing of intracellular pathogens. Using inductively coupled plasma mass spectrometry, weexamined alterations in the metal homeostasis of murine H. capsulatum–infected macrophages that wereexposed to activating cytokines. Decreases in the levels of iron (Fe2+ and Fe3+) and zinc (Zn2+) were observedin infected, GM-CSF–treated macrophages compared with those in infected controls. Interleukin 4 reversedthe antifungal activity of GM-CSF–activated macrophages and was associated with increased intracellular Zn2+

levels. Chelation of Zn2+ inhibited yeast replication in both the absence of macrophages and the presence ofmacrophages. Treatment of cells with GM-CSF altered the host Zn2+ binding species profile. These resultsestablish that Zn2+ deprivation may be a host defense mechanism utilized by macrophages.

Histoplasma capsulatum infection is initiated by inha-

lation of fungal spores followed by conversion to the

pathogenic yeast phase. The infection is controlled in

most immunocompetent individuals but establishes a

persistent state. Histoplasmosis can be life-threatening

for individuals with immune defects arising from hu-

man immunodeficiency virus (HIV) infection or those

receiving drugs for malignancy, graft rejection, or au-

toimmune diseases. More recently, lower levels of tu-

mor necrosis factor a antagonists have been linked to

a higher incidence of histoplasmosis [1].

Alveolar macrophages are the presumed first line of

Received 16 December 2009; accepted 30 April 2010; electronically published23 August 2010.

Potential conflicts of interest: none reported.Financial support: Department of Veterans Affairs (Merit Review); National

Institute of Allergy and Infectious Diseases (grants AI-73337 and AI-83313).Reprints or correspondence: Michael S. Winters, Division of Infectious Diseases,

Department of Medicine, University of Cincinnati College of Medicine, Cincinnati,OH 45267-0560 ([email protected]).

The Journal of Infectious Diseases 2010; 202(7):1136–1145� 2010 by the Infectious Diseases Society of America. All rights reserved.0022-1899/2010/20207-0018$15.00DOI: 10.1086/656191

cellular defense that H. capsulatum encounters in the

host. Macrophages are the only professional phagocytic

cell population in which H. capsulatum replicates freely

[2], although growth is inhibited following cytokine

activation of macrophages. Interferon g (IFN-g) and

granulocyte macrophage colony-stimulating factor

(GM-CSF) restrict growth in mouse and human mac-

rophages, respectively [3, 4]. One mechanism by which

IFN-g inhibits H. capsulatum intracellular growth is by

restricting iron [3]. Much of these data have been ac-

crued by adding exogenous iron to cells or restricting

access to iron. A missing analysis has been a direct

measurement of iron or any metal present within phag-

ocytes or pathogens harbored by these cells.

Since metals are critical to the functional integrity of

cells, we sought to determine whether there was a cor-

relation between metal uptake of H. capsulatum–in-

fected macrophages activated by cytokines. We ob-

served that zinc (Zn2+) and total iron (Fe2+ and Fe3+)

levels were restricted inside GM-CSF–treated macro-

phages infected with H. capsulatum compared with

those inside resting macrophages. Zn2+ binding species

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Zinc and Histoplasma capsulatum • JID 2010:202 (1 October) • 1137

were differentially regulated within resting macrophages versus

GM-CSF–treated cells. Chelating Zn2+ reduced H. capsulatum

growth in medium and within resting macrophages. Moreover,

pretreating GM-CSF–activated macrophages with interleukin 4

(IL-4) reversed growth inhibition and partially replenished Zn2+

levels. These data support an important role for Zn2+ restriction

as a host defense strategy against H. capsulatum infection.

METHODS

Mice. Six- to eight-week-old C57BL/6 mice were purchased

from Jackson Laboratories. Animals were housed in isolator

cages and maintained by the Department of Laboratory Animal

Medicine, University of Cincinnati, which is accredited by the

Association for Assessment and Accreditation of Laboratory

Animal Care. All animal experiments were performed in ac-

cordance with the Animal Welfare Act guidelines of the Na-

tional Institutes of Health. All protocols were approved by the

University of Cincinnati Institutional Animal Care and Use

Committee.

Reagents. Sodium dodecyl sulfate (SDS), high-performance

liquid chromatography (HPLC)–grade water, methanol, diethy-

lenetriaminepentaacetic acid (DTPA), ethylene glycol tetraacetic

acid (EGTA), N,N,N ′,N ′-tetrakis(2-pyridylmethyl)ethylenedi-

amine (TPEN), zinc sulfate (ZnSO4), iron sulfate (FeSO4), and

F-12 Ham nutrient mixture were purchased from Sigma Al-

drich. Recombinant mouse GM-CSF, IFN-g, and IL-4 were

acquired from PeproTech. Arginase (Arg1), chitinase 3-like 3

(Chi3l3), and resistin like alpha (Retnla) primers for reverse-

transcription polymerase chain reaction (RT-PCR) analysis

were purchased from Applied Biosystems, and TRIzol was pur-

chased from Invitrogen. A Diff-Quik stain kit was obtained

from IMEB.

All solutions for inductively coupled plasma mass spectrom-

etry (ICPMS) analysis were prepared in 18 MQ/cm double

deionized water (Sybron Barnstead), in which no metal was

detected. The mobile phase for size exclusion chromatography

(SEC) was made by dissolving tris(hydroxymethyl)aminometh-

ane (Tris) in double deionized water and adjusting the pH with

hydrochloric acid. The SEC standard (Bio-Rad Laboratories)

is a lyophilized mixture of molecular weight markers ranging

from 1300 to 670,000 Da.

H. capsulatum intracellular growth assays. Murine alve-

olar macrophages, bone-marrow-derived (BMD) macrophages,

and peritoneal macrophages were used. Alveolar macrophages

were prepared by means of consecutive lung lavages, rinsing 5

times with 1 mL of phosphate-buffered saline. Peritoneal mac-

rophages were acquired by lavaging peritoneal cavities with 10

mL of phosphate-buffered saline. BMD macrophages were gen-

erated by extracting bone marrow cells from the femurs of mice,

and cells were incubated in Roswell Park Memorial Institute

(RPMI) 1640 growth medium at 37�C in the presence of GM-

CSF at a concentration of 10 ng/mL and 5% carbon dioxide

for 5–6 d. In some experiments, cells were incubated with 10

ng/mL of IL-4 for another 24 h following 5 d of incubation

with GM-CSF. Peritoneal macrophages were incubated with 10

ng/mL of either GM-CSF or IFN-g for 24 h. All macrophages

were plated at cells per well in a 96-well plate.51 � 10

H. capsulatum strain G217B yeasts were grown as described

elsewhere [5]. Macrophages were infected with H. capsulatum

at a multiplicity of infection (MOI) of 5 yeasts per macrophage;

growth inside macrophages was quantified by plating. Infected

macrophages were lysed in sterile water and lysates plated onto

Mycosel agar 5 (Becton Dickinson) plates containing 5% sheep

blood and 5% glucose. Plates were incubated at 30�C for 1 wk.

The leucine assay was used for metal chelation and supple-

mentation experiments [4]. For metal supplementation exper-

iments, ZnSO4 or FeSO4 was added at a concentration of 100

mmol/L to RPMI 1640 medium, and for metal chelation ex-

periments, TPEN or DTPA was added prior to infection. The

toxicity of TPEN, DTPA, and ZnSO4 was measured using trypan

blue stain.

Sample preparation for ICPMS analysis of infected mac-

rophages and intracellular yeasts. Macrophages were plated

at cells per well in a 12-well plate and infected with H.61 � 10

capsulatum at a MOI of 5 yeasts per macrophage. After 24 h,

cells were washed with Hank buffered salt solution (HBSS).

Infected macrophages were treated (duration, ∼1 min) with

250 mL of 0.1% SDS in water per well. Metal concentrations

in 0.1% SDS and wash buffer (HBSS) were !10 ppb (10

) for all metals except sodium.ppb p 10 mg/L

For metal analysis of intracellular H. capsulatum, yeasts were

isolated from infected macrophages following the addition of

0.1% SDS and centrifugation. Each group of intracellular

yeasts isolated were set at cells per 10 mL by use of a81 � 10

hemacytometer.

Yeasts and infected macrophages were further diluted at 1:

1.25 to set at a concentration of 20% nitric acid. Before ICPMS

analysis of infected macrophages and isolated organisms, each

sample was subjected to microwave digestion using a closed-

vessel Discover-Explorer microwave digestion system (CEM).

The digestion process was performed at 150�C for 2 min by

maintaining a pressure of !250 psi.

For SEC and ICPMS analysis, samples were not subjected to

microwave digestion. Infected macrophages were immediately

centrifuged following treatment with 0.1% SDS, and H. cap-

sulatum was removed. The supernatant was stored at �70�C

before ICPMS analysis.

ICPMS analysis. ICPMS-based quantification has been

commonly used to determine accurate concentrations of mul-

tiple metals simultaneously in biological samples [6, 7]. ICPMS

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Table 1. Operating Conditions for High-Performance Liquid Chromatography and Inductively Coupled Plasma Mass Spectrometry

Parameter Value

Inductively coupled plasma mass spectrometryForward power, W 1500Plasma gas flow rate, L/min 15.0Carrier gas flow rate, L/min 1.01Isotope monitored for size exclusion chromatography Zinc 66Isotopes monitored for flow injection Sodium 23, magnesium 24, potassium 39, calcium 44, manganese

55, iron 57, nickel 60, copper 63, and zinc 66Collision gas (H2) flow rate, mL/min 3.2Quadrupole bias, V �16Octopole bias, V �18

Size exclusion chromatographyColumn Superdex 200 10/300 GLMobile phase 30 mmol/L Tris-HCl buffer (pH, 7.5)Flow rate, mL/min 0.7Injection volume, mL 100

Flow injectionColumn Not applicableMobile phase 0.1% nitric acidFlow rate, mL/min 0.8Injection volume, mL 50

metal analysis was performed on an Agilent 7500ce ICPMS

instrument (Agilent Technologies). A conventional Meinhard

nebulizer, a Peltier-cooled spray chamber (temperature, 2�C),

and a shield torch constitute the sample introduction system

under standard plasma conditions.

HPLC was performed using flow injection, and SEC was

performed with an Agilent 1100 liquid chromatograph (Agilent

Technologies) equipped with a binary HPLC pump, an auto-

sampler, a vacuum degasser system, a temperature column

compartment, and a diode array detector. The outlet of the

HPLC UV detector was connected to a sample inlet of the

ICPMS nebulizer by use of 0.25-mm-diameter polyether ether

ketone tubing of 30 cm in length. Both UV (wavelength, 280

nm) and ICPMS signals were collected online.

A Superdex 200 10/300 GL column (area, mm2;10 � 300

bead size, 13 mm; Amersham Pharmacia Biotech) was used for

SEC analysis. The column was calibrated with a UV detector

(wavelength, 280 nm) by use of a gel filtration standard mixture

(molecular weight of thyroglobulin, 670 kDa; molecular weight

of g-globulin, 158 kDa; molecular weight of ovalbumin, 44

kDa; molecular weight of myoglobin, 17 kDa; molecular weight

of vitamin B12, 1.3 kDa), which was purchased from Bio-Rad

Laboratories. Lysed macrophages (treated with 0.1% SDS as

described above) were injected onto the column. ICPMS flow

injection analysis and SEC conditions are shown in Table 1.

Intracellular H. capsulatum counting. The number of or-

ganisms within each cytokine-treated macrophage population

after the addition of the yeasts (MOI, 5 yeasts per macrophage)

was counted by staining cells with Diff-Quik and counting at

least 100 infected macrophages.

Flow cytometry. Two hundred thousand macrophages were

treated with IL-4 for 24 h and then exposed to H. capsulatum.

Cells were then stained with peridinin chlorophyll protein com-

plex (PerCP)–conjugated CD11b and allophycocyanin-conju-

gated CD71 (BD Biosciences). Cells were then stained with

allophycocyanin-conjugated CD71 antibodies. Flow cytometry

analysis was performed on macrophages infected with H. cap-

sulatum (MOI, 5 yeasts per macrophage) that expressed green

fluorescent protein [8] and PerCP-CD11b to determine the

percentage of infected cells. Staining for both groups was per-

formed at 4�C for 15 min in HBSS containing 1% bovine serum

albumin and 0.01% sodium azide. Cells were washed and re-

suspended in 1% paraformaldehyde to fix. Isotype controls

were performed in parallel. The fluorescence intensity was as-

sessed using a FACSCalibur device (BD Biosciences) and an-

alyzed using FCS Express software (version 3; De Novo).

Quantitative RT-PCR. A total of macrophages55 � 10

were infected at a MOI of 5 yeasts per macrophage. After 24

h, the total RNA from macrophages was isolated using TRIzol.

Oligo(deoxythymidylic acid)-primed complementary DNA was

prepared using the reverse transcriptase system (Promega).

Quantitative RT-PCR analysis for was performed using Taq-

Man Master Mix and the following primers: hydroxyphos-

phoribosyl transferase (Hprt), Arg1, Retnla (Fizz1), Chi3l3

(Ym1), and calprotectin (S100). Samples were analyzed on an

ABI Prism 7500 instrument (Applied Biosystems). In each ex-

periment, Hprt was used as an internal control. The conditions

for amplification were 50�C for 2 min, then 95�C for 10 min,

followed by 40 cycles of 95�C for 15 s and 60�C for 1 min.

Nitric oxide detection. Nitric oxide levels in infected mac-

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Figure 1. Activation of macrophages by granulocyte macrophage colony-stimulating factor (GM-CSF) to exert antifungal activity and inhibition of activationby interleukin 4 (IL-4). Shown are colony counts of Histoplasma capsulatum in culture alone, with resting alveolar macrophages, with GM-CSF–treated alveolarmacrophages, with resting peritoneal macrophages, with GM-CSF–treated peritoneal macrophages, and with GM-CSF–treated bone-marrow-derived macrophagespretreated with IL-4 for 24 h. Data represent the mean (� standard error of the mean) from 3 experiments. * (Student t test) compared with restingP ! .05peritoneal macrophages. AMf, alveolar macrophages; BMf, bone-marrow-derived macrophages; Mf, macrophages; PMf, peritoneal macrophages.

Figure 2. Expression of interleukin 4 (IL-4)–regulated genes in bone-marrow-derived (BMD) macrophages. Shown are the results of quantitative reverse-transcription polymerase chain reaction of arginase, resistin like alpha (FIZZ1), and chitinase 3-like 3 (YM1) expression in BMD macrophages pretreatedwith 10 ng of IL-4 for 24 h, infected with Histoplasma capsulatum (without IL-4), or pretreated with IL-4 and infected. Expression levels were normalizedto those of uninfected BMD macrophages without IL-4. Data represent the mean (� standard error of the mean) from 3 experiments. Hc, H. capsulatum.

rophages (MOI, 5 yeasts per macrophage) were measured using

the Cayman Nitrate/Nitrite colorimetric assay kit. Experiments

were performed in triplicate with BMD macrophages55 � 10

per well in a 96-well plate.

Statistical analysis. The Student t test was used to compare

two groups, whereas analysis of variance was used to compare

multiple groups. Differences for which were consideredP ! .05

to be statistically significant.

RESULTS

Intracellular yeast growth in resting versus GM-CSF–activated

macrophages. Elsewhere we reported that GM-CSF enhanced

host defenses to H. capsulatum [9]. Hence, we sought to de-

termine whether it would act on macrophages. The exposure

of several populations of macrophages to GM-CSF inhibited

the intracellular growth of H. capsulatum (Figure 1). IL-4 blunts

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Table 2. Metal Levels in Macrophages and Histoplasma capsulatum Yeasts

Cell type, metal

Mean metal level, ppb (RSD, %)

Resting PMsGM-CSF–

treated PMsGM-CSF–treated

BMDMs

GM-CSF–treatedBMDMswith IL-4 Culture alone

Uninfected macrophages,cells per 50 mL51.5 � 10

Magnesium 2240 (2) 790 (2) 1210 (3) 1270 (2) NACalcium 3700 (3) 1750 (5) 2220 (5) 2044 (2) NAIron 1360 (2) 664 (5)a 932 (3) 1002 (5) NAZinc 190 (6) 89 (6) 179 (5) 153 (2) NACopper 57 (5) 23 (6) 22 (3) 20 (1) NAManganese 8 (9) !1 (3) !1 (4) !1 (2) NA

H. capsulatum–infected macrophages,cells per 50 mL51.5 � 10

NA

Magnesium 778 (2) 954 (2) 1300 (1) 1310 (3) NACalcium 1780 (8) 1350 (4) 2110 (1) 2032 (2) NAIron 680 (6) 275 (7)a 566 (3) 597 (7) NAZinc 131 (8) 28 (6)a 63 (2)a 162 (10)b NACopper 24 (11) 22 (2) 26 (3) 24 (8) NAManganese !1 (9) !1 (12) !1 (2) !1 (1) NA

H. capsulatum yeasts,c cells per 50 mL72.5 � 10Potassium 25,500 (1) 14,000 (1) 13,400 (1) 28,200 (2) 13,400 (1)Magnesium 17,000 (1) 3400 (9) 2400 (8) 4100 (12) 1800 (9)Calcium 38,300 (1) 11,800 (7)a 5600 (12)a 12,500 (10)b 4400 (15)Iron 6400 (4) 4300 (3) 1700 (5)a 2160 (7) 4650 (2)Zinc 1900 (1) 1200 (2)a 500 (1)a 1100 (1)b 600 (1)Copper 406 (2) 219 (2) 73 (6) 50 (25) 46 (3)Manganese 42 (16) 53 (15) 21 (26) 55 (2) 36 (15)Nickel 175 (9) 147 (15) 59 (23) 102 (7) 56 (19)

NOTE. Data are the mean of 3 experimental replicates represented in parts per billion ( ). BMDMs, bone-marrow-derived macrophages; GM-1 ppb p 1 mg/LCSF, granulocyte macrophage colony-stimulating factor; IL-4, interleukin 4; NA, not applicable; PMs, peritoneal macrophages; RSD, relative standard deviation.

a (analysis of variance) compared with infected resting PMs.P ! .05b (analysis of variance) compared with infected GM-CSF–treated BMDMs.P ! .05c Metal levels in H. capsulatum yeast cells after isolation from infected macrophages or grown in culture alone.

immunity to H. capsulatum and is associated with the emer-

gence of alternatively activated macrophages [10]. We asked

whether pretreatment of macrophages with IL-4 would alter

intracellular growth in activated macrophages [11]. We found

that exposure of macrophages to IL-4 for 24 h enhanced yeast

growth in BMD macrophages (Figure 1).

We examined whether IL-4 alternatively activated infected

macrophages. We assessed the expression levels of Arg1, Retnla,

and Chi3l3 [12]. Each gene manifested enhanced expression

following exposure of BMD macrophage cells to IL-4 and in-

fection with H. capsulatum (Figure 2). The abundance of an-

other marker of IL-4 activation, transferrin receptor (TfR), was

analyzed by flow cytometry. IL-4 treatment of macrophages 24

h before H. capsulatum infection did not increase total TfR

expression levels as determined by the mean fluorescence in-

tensity (MFI). The MFI of macrophages exposed to medium

(926.6) did not differ from that of cells exposed to 10 ng/mL

IL-4 (952.3). Moreover, no differences in the percentage of cells

expressing TfR were observed between the two groups (data

not shown). IL-4 did enhance TfR expression on uninfected

cells in a dose-dependent manner. The MFI of cells exposed

to medium only (2055.2) was less than that of cells exposed

to IL-4 at a concentration of 0.1 ng/mL (3763.4), 1 ng/mL

(4378.7), or 10 ng/mL (4333.9). Likewise, there was an increase

in the percentage of cells expressing TfR; 79.5% of cells in-

cubated in medium only were positive for TfR, whereas those

values were 81.5%, 86.1%, and 88.1% for IL-4 concentrations

of 0.1, 1, and 10 ng/mL, respectively.

We did not detect a statistically significant difference in the

level of nitric oxide production between IL-4–treated macro-

phages ( mmol/L nitrite) and untreated macrophages5.3 � 0.2

( mmol/L nitrite).5.7 � 0.4

GM-CSF regulates macrophage metal levels. We queried

whether GM-CSF would alter metal levels in cells. We undertook

an unbiased approach using ICPMS to assess levels of magnesium

(Mg2+), calcium (Ca2+), total Fe, Zn2+, copper (Cu2+), and man-

ganese (Mn2+) during infection of macrophages.

We determined metal levels in uninfected and infected peri-

toneal macrophages and in those pretreated with GM-CSF [4].

Total metal concentrations were highest in resting uninfected

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Figure 3. Inhibition of Histoplasma capsulatum growth by zinc chelation. A, Counts per minute (CPM) from tritiated leucine assays of H. capsulatumin medium containing diethylenetriaminepentaacetic acid (DTPA) (filled circles), in the presence of DTPA plus iron sulfate (FeSO4) (triangles), or in thepresence of N,N,N ′,N ′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) plus zinc sulfate (ZnSO4) (open circles). B, CPM from tritiated leucine assays ofH. capsulatum with peritoneal macrophages in the presence of DTPA (filled circles), DTPA plus FeSO4 (triangles), or DTPA plus ZnSO4 (open circles).C, CPM from tritiated leucine assays of H. capsulatum in medium containing TPEN (filled circles), in the presence of TPEN plus FeSO4 (triangles), orTPEN plus ZnSO4 (open circles). D, CPM from tritiated leucine assays of H. capsulatum in medium containing ethylene glycol tetraacetic acid (EGTA)(filled circles) or medium containing EGTA plus peritoneal macrophages (open circles). Data represent the mean (� standard error of the mean [SEM])of triplicates from 1 representative experiment of 3. PMf, peritoneal macrophages.

peritoneal macrophages compared with those in all other pop-

ulations (Table 2). We observed decreases in the overall con-

centrations of metals upon infection, but this restriction was

enhanced for total Fe and Zn2+ by GM-CSF activation (Table

2). GM-CSF did induce a decrease in Zn2+ levels in uninfected

peritoneal macrophages, but this was not statistically significant

(Table 2).

Macrophages from various organs may not regulate metals

similarly; hence, Zn2+ concentrations in BMD macrophages and

alveolar macrophages were measured. Similar to infected peri-

toneal macrophages exposed to GM-CSF, infected BMD mac-

rophages grown in the presence of GM-CSF yielded lower Zn2+

and total Fe levels compared with those of infected resting

peritoneal macrophages (Table 2). A Zn2+ concentration of 246

ppb (relative standard deviation [RSD], 3%; ) was mea-n p 3

sured in resting alveolar macrophages, whereas in GM-CSF–

treated alveolar macrophages the Zn2+ concentration was 124

ppb (RSD, 6%; ). The level of total Fe was 1200 ppbn p 3

(RSD, 1.9%; ) in resting alveolar macrophages, whereasn p 3

that in GM-CSF–treated alveolar macrophages was 540 ppb

(RSD, 4%; ).n p 3

To ensure that the altered metal levels observed in this study

were not a result of different numbers of yeasts initially ingested

by the macrophages, we counted the number of yeasts within

each cytokine-treated macrophage population. We observed

that 1 h after infection, each macrophage population contained

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Figure 4. Regulation of host zinc binding species by macrophage activation and Histoplasma capsulatum infection. Shown are the results of sizeexclusion chromatography (SEC) followed by inductively coupled plasma mass spectrometry (ICPMS) analysis of zinc species in resting peritonealmacrophages (A), resting peritoneal macrophages infected with H. capsulatum (B ), peritoneal macrophages treated with granulocyte macrophagecolony-stimulating factor (GM-CSF) (C ), and infected GM-CSF–treated peritoneal macrophages (D ). Yeasts were removed before SEC and ICPMSanalysis. Molecular weight standards are listed above panels A and B. CPS, counts per second; Hc, H. capsulatum; PMf, peritoneal macrophages.

a mean (� standard error of the mean) of yeasts.12 � 3

Moreover, using H. capsulatum that expressed green fluorescent

protein, we found that ∼90% of macrophages were infected

after 24 h.

We questioned whether our measurements of the overall

cellular metal concentrations were acceptable. Reports estimate

eukaryotic cell free cytosolic zinc levels to be in the ∼10�12 mol/

L range, whereas total cellular zinc levels are ∼10�4 mol/L; we

measured a concentration of ∼10�6 mol/L in ∼105 cells diluted

in 50 mL of buffer [13–15].

Metal levels within intracellular yeast cells. The analysis

of the metal levels in total macrophage populations may not

reveal the levels within ingested yeasts. Therefore, we analyzed

concentrations of potassium (K+), Mg2+, Ca2+, total Fe, Zn2+,

Cu2+, Mn2+, and nickel (Ni2+) within H. capsulatum yeasts iso-

lated from macrophages. Lower amounts of Zn2+ and Ca2+

( ) were detected in yeasts isolated from BMD macro-P ! .05

phages and GM-CSF–exposed peritoneal macrophages com-

pared with those in resting peritoneal macrophages or H. cap-

sulatum grown in culture alone (Table 2).

To validate our method, we assessed whether IFN-g pre-

treatment of peritoneal macrophages decreased total Fe levels.

A mean total Fe concentration of 2440 ppb (RSD, 7%; n p

) was detected in H. capsulatum cells isolated from IFN-g–3

pretreated peritoneal macrophages, compared with a mean total

Fe concentration of 6240 ppb (RSD, 4%; ) detected inn p 3

resting peritoneal macrophages. Thus, these data support the

utility of this method for assaying intracellular metal levels.

IFN-g also decreased Zn2+ levels. Cytokine-treated cells con-

tained a mean Zn2+ level of 600 ppb (RSD, 6%; ), andn p 3

resting cells contained a mean Zn2+ level of 1900 ppb (RSD,

6%; ).n p 3

Increases in zinc levels in IL-4–treated macrophages and

intracellular yeast cells. We asked whether IL-4 enhancement

of fungal growth would be associated with a change in intra-

cellular metal abundance levels. Zn2+ levels increased by 12

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Figure 5. No increase of calprotectin expression in peritoneal macrophages by granulocyte macrophage colony-stimulating factor (GM-CSF). Shownare the results of quantitative reverse-transcription polymerase chain reaction of calprotectin expression of Histoplasma capsulatum–infected restingperitoneal macrophages, uninfected peritoneal macrophages pretreated with 10 ng of GM-CSF for 24 h, or infected GM-CSF–treated peritonealmacrophages. Expression levels were normalized to those of uninfected peritoneal macrophages without GM-CSF treatment. Data represent the mean(� standard error of the mean) from 3 experiments. Hc, H. capsulatum.

times inside infected BMD macrophages treated with IL-4,

whereas total Fe concentrations did not increase (Table 2). In

addition, higher Zn2+ and Ca2+ levels were detected in H. cap-

sulatum cells isolated from IL-4–pretreated BMD macrophages

compared with those detected in untreated cells (Table 2).

Zinc influences yeast growth. A decrease of Zn2+ levels in

activated macrophages, coupled with an increase following IL-

4 treatment, led us to hypothesize that restriction of Zn2+ is a

host defense mechanism against H. capsulatum infection. We

sought to determine the effects of the Zn2+ chelators DTPA and

TPEN on H. capsulatum growth.

DTPA inhibited H. capsulatum growth in a dose-dependent

manner in the presence and absence of peritoneal macrophages

(Figures 3A and 3B). DTPA was not toxic at the concentrations

used in this study. TPEN inhibited H. capsulatum growth in a

dose-dependent manner in the presence and absence of peri-

toneal macrophages (Figure 3C). TPEN is toxic to macrophages

at low concentrations; therefore, we could not adequately in-

terpret the influence of TPEN on intracellular yeast growth.

Because TPEN and DTPA bind Fe2+ [16], we determined

whether Fe2+ chelation by DTPA and TPEN influenced our

results. In the presence of DTPA, FeSO4 and ZnSO4 partially

rescued H. capsulatum growth in culture alone or inside resting

peritoneal macrophages. In the presence of TPEN, the addition

of FeSO4 partially enhanced H. capsulatum growth, whereas

ZnSO4 completely restored it.

We examined whether Ca2+ had an effect on yeast growth

inside macrophages because Ca2+ levels were decreased in H.

capsulatum cells isolated from GM-CSF–treated macrophages

and increased following IL-4 treatment. Chelation of Ca2+ with

EGTA did not statistically significantly influence yeast growth

in medium or inside peritoneal macrophages (Figure 3D).

GM-CSF regulation of macrophage zinc species. Zn2+ is a

cofactor and is required for numerous host processes [17]. We

hypothesized that the changes in total Zn2+ levels would need

to be tightly regulated. Thus, we should detect a change in the

total Zn2+ binding species of GM-CSF–treated peritoneal mac-

rophages and BMD macrophages compared with that in resting

peritoneal macrophages. Figure 4 shows SEC chromatograms

followed by ICPMS analysis representing the total Zn2+ binding

species of infected and uninfected resting peritoneal macro-

phages versus GM-CSF–treated peritoneal macrophages. A

higher amount of Zn2+ binding species was consistently detected

in GM-CSF–treated peritoneal macrophages compared with

that detected in resting peritoneal macrophages, especially Zn2+

species eluting at 27 min.

Effect of GM-CSF is independent of modulation of

calprotectin. Calprotectin has been shown to bind Zn2+ and

to possess antifungal activity [18]. We asked whether GM-CSF

would alter the expression of calprotectin. Exposure of cells to

GM-CSF did not statistically significantly increase the levels of

calprotectin expression ( ; Student t test) (Figure 5).P 1 .05

DISCUSSION

In this study, GM-CSF–activated macrophages restricted the

intracellular growth of H. capsulatum and decreased the con-

centrations of Zn2+ and total Fe. Moreover, IL-4 reversed the

growth-inhibitory properties of activated macrophages and in-

creased levels of Zn2+ in macrophages and yeast cells. Among

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the metals analyzed, Zn2+ was the most heavily influenced by

GM-CSF and IL-4 treatment. These findings prompted us to

examine the role of Zn2+ in the growth of H. capsulatum. Zn2+

chelation restricted growth both in medium alone and within

macrophages. Furthermore, GM-CSF activation was accom-

panied by changes in the abundance and number of Zn2+ bind-

ing species. The data provide a link between Zn2+ abundance

and the intracellular survival of H. capsulatum.

Zn2+ is a cofactor for 1300 proteins and is required for the

survival of most microorganisms [17]. Salmonella enterica and

Aspergillus fumigatus utilize a Zn2+ uptake system that is re-

quired for Zn2+ homeostasis and full virulence [19, 20]. Several

pathogenic fungi including Candida albicans and A. fumigatus

display growth impairment to Zn2+ deprivation [21]. In our

study, the use of Zn2+ chelators DTPA and TPEN revealed that

Zn2+ is required for the growth of H. capsulatum in medium

and can influence intracellular growth. Although our data sug-

gest that this chelator also binds Fe2+, a clear advantage exists

for the host to restrict both Fe2+ and Zn2+ during infection with

H. capsulatum.

Activation of murine macrophages with IFN-g stimulates the

production of nitric oxide, which is believed to promote deg-

radation of several intracellular pathogens including H. cap-

sulatum [22–24]. Indirect and direct evidence also exists for

IFN-g–mediated host restriction of total Fe availability [3, 25–

27]. We detected lower levels of total Fe and Zn2+ in IFN-g–

treated macrophages and GM-CSF–treated macrophages. GM-

CSF is a pleiotropic cytokine that induces myelopoiesis, acts as

proinflammatory agent, and arms phagocytes to express anti-

microbial activity [28]. Much of the literature indicates that

GM-CSF enhances reactive oxygen intermediates as one mech-

anism of host defense. Our data reveal that another antimi-

crobial defense mechanism is the limitation of metals [29, 30].

We discovered that Zn2+ levels were lower in GM-CSF–treated

macrophages infected with H. capsulatum than those in resting

infected macrophages.

Host intracellular Zn2+ concentrations are controlled by Zn2+

importers, exporters, and metal-binding proteins such as me-

tallothionens [31]. Zn2+ is an essential element required for

mammalian cell function, but high concentrations of Zn2+ can

be toxic; therefore, regulation of this metal must be tightly

controlled [32]. Thus, fluctuations in intracellular Zn2+ levels

must be initiated and/or responded to by the host. Accordingly,

we detected changes in Zn2+ species abundance to accompany

GM-CSF–induced Zn2+ restriction and IL-4–induced Zn2+ re-

plenishment. Thus, the changes in the number of Zn2+ species

add further support to the Zn2+ level alterations detected. These

data also suggest the participation of a host zinc regulatory

mechanism influenced by different cytokines during H. cap-

sulatum infection.

IL-4 alternatively activates macrophages and thereby inhibits

the killing of intracellular and some extracellular organisms in

vitro [11, 33, 34]. Overproduction of IL-4 under conditions of

altered immunity accelerates H. capsulatum infection, but the

mechanism remains unclear [10, 35, 36]. One mechanism

through which IL-4 may contribute to organism survival during

infection is by reducing nitric oxide production [37], but in

this study IL-4 reversal of GM-CSF activity was independent

of nitric oxide production. Another potential mechanism of

organism growth enhancement by IL-4 is the alteration of metal

homeostasis. IL-4 increases Ca2+ movement and accumulation

in macrophages [38, 39]. Likewise, we detected increases in

Ca2+ levels in H. capsulatum cells isolated from IL-4–exposed

macrophages. However, Ca2+ chelation had only a minimal ef-

fect on intracellular yeast growth, thus suggesting that this metal

is not important in the effect of IL-4. Treatment of macrophages

with IL-4 has been suggested to make iron more available to

Mycobacterium tuberculosis in macrophages [40]. In this study,

increases in macrophage total Fe levels were not detected. In

contrast, IL-4 treatment partially restored intracellular yeast

growth and was associated with increased intracellular Zn2+

levels. Thus, zinc rather than iron replenishment following IL-

4 treatment of macrophages may contribute to a more per-

missive environment.

In summary, we have shown that cytokine-activated mac-

rophages manifest a modulation of metal levels. The data sup-

port a crucial role for Zn2+ limitation as a key element of host

defenses against H. capsulatum infection.

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metallomic analysis of macrophages infected with histoplasma

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