High Intracellular Concentrations of Posaconazole Do Not Impact on Functional Capacities of Human Polymorphonuclear Neutrophils and Monocyte-Derived Macrophages In Vitro

Abstract

Posaconazole is a commonly used antifungal for the prophylaxis and treatment of invasive fungal infections. We previously demonstrated that the intracellular concentration of posaconazole in peripheral blood mononuclear cells (PBMCs) and polymorphonuclear neutrophils (PMNs) was greatly increased compared to the plasma concentration. As these professional phagocytes are crucial to combat fungal infections, we set out to investigate if and how, beneficial or deleterious, this high loading of intracellular posaconazole impacts the functional capacities of these cells. Here, we show that high intracellular concentrations of posaconazole do not significantly impact PMN and monocyte-derived macrophage function in vitro. In particular, killing capacity and cytoskeletal features of PMN, such as migration, are not affected, indicating that these cells serve as vehicles for posaconazole to the site of infection. Moreover, since posaconazole as such slowed the germination of Aspergillus fumigatus conidia, infected neutrophils released less reactive oxygen species (ROS). Based on these findings, we propose that the delivery of posaconazole by neutrophils to the site of Aspergillus species infection warrants control of the pathogen and preservation of tissue integrity at the same time.

INTRODUCTION

Posaconazole is a broad-spectrum triazole antifungal approved for prophylaxis and treatment of opportunistic invasive fungal infections (IFI) in immunocompromised patients. The majority of all IFI (probably more than 95%) are attributable to Aspergillus spp. and Candida spp. (1, 2). Aspergillus spp. are ubiquitous soil-dwelling fungi, often found in walls and dust. Due to their small size (2- to 4-μm diameter), Aspergillus conidia (spores) tend to remain airborne. It is estimated that all humans inhale at least several hundred Aspergillus fumigatus conidia per day (3). Upon inhalation, these conidia may germinate at body temperature in the terminal airways and alveoli, causing invasive pulmonary aspergillosis (3). Alveolar macrophages constitute the first line of defense against invading conidia. They immediately phagocytize conidia, thereby restricting their initial spread into the alveoli. The process of phagocytosis is accompanied by secretion of proinflammatory cytokines, leading to autocrine activation of the macrophage and attraction of other immune cells to the site of infection (4). It has been shown that recruitment of neutrophils is essential for efficient clearing of Aspergillus fumigatus conidia (5). Moreover, one of the most disposing risk factors for invasive aspergillosis is a depletion and/or functional impairment of neutrophils (6–8). Killing of Aspergillus fumigatus by neutrophils is mediated by the release of reactive oxygen species and neutrophil granular content (9–12).

Therapeutic substances are differentially distributed to various compartments of the human body, which impacts their pharmacokinetics and pharmacodynamics. With regard to the question of whether cells of the peripheral blood constitute a relevant compartment for the pharmacokinetics of triazoles, we previously measured the intracellular concentrations of posaconazole. We found that the intracellular concentration of posaconazole in peripheral blood mononuclear cells (PBMCs) and polymorphonuclear neutrophils (PMNs) was greatly increased compared to the plasma concentration (13). Given the importance of these cells in the immune response to fungal infections, we were curious whether the intracellular accumulation of posaconazole impacts their functional capacities. Considering potential positive and negative effects of high intracellular concentrations of posaconazole, one could anticipate that posaconazole, with its antifungal properties, has a positive effect on intracellular killing of Aspergillus fumigatus conidia. Conversely, high loading of lipophilic posaconazole could certainly interfere with, e.g., transmembrane processes. A recently published in vitro study revealed that posaconazole causes an anti-inflammatory effect by inhibiting the efflux of leukotriene B4 and the influx of Ca²⁺ of activated PMNs (14), indicating that posaconazole interferes with cell autonomous defense mechanisms, which are important for neutrophil antifungal properties. Hence, we set out to systematically investigate the impact of high intracellular loading of posaconazole on various functional capacities of neutrophils and monocyte-derived macrophages upon challenge with Aspergillus fumigatus conidia.

MATERIALS AND METHODS

Donors.

Whole blood from healthy donors was collected as part of the Cologne biobank protocol (University of Cologne Ethics Committee, no. 08-160) (15). Buffy coats were obtained from healthy blood donors at the Department of Transfusion Medicine of Cologne University Hospital (University of Cologne Ethics Committee, no. 14-144).

Preparation of conidial suspensions.

Aspergillus fumigatus conidia (ATCC 46645) were plated on Sabouraud agar plates (Becton Dickinson, Heidelberg, Germany) and cultivated for 3 days at 37°C. Conidia were then harvested, filtered through a 40-μm nylon cell strainer (BD Biosciences, San Jose, CA), and resuspended in phosphate-buffered saline (PBS) to a final concentration of 1.5 × 10⁹ conidia/ml.

Preparation of fungal lysates.

Aspergillus fumigatus conidia were inoculated at a final concentration of 2 × 10⁶ conidia/ml in yeast extract-peptone-dextrose medium (Becton Dickinson, Heidelberg, Germany) and shaken at 37°C with 200 rpm overnight. Mycelia were recovered by filtration and disrupted in a Mikro-Dismembrator S (Sartorius Stedim Biotech GmbH, Göttingen, Germany) at 200 rpm for 10 min using 0.5-mm glass beads.

Preparation of Aspergillus-activated serum (AAS).

Aspergillus fumigatus conidia (1 × 10⁸/ml) were incubated with serum for 30 min at 37°C. Afterwards, the reaction mixture was centrifuged to remove conidia and incubated at 56°C for 30 min in order to inactivate residual complement.

Labeling of Aspergillus conidia with C₁₂FDG.

Aspergillus fumigatus conidia (7.5 × 10⁸/ml) were incubated at 37°C for 1 h with intermittent sonication (10 s every 10 min) in 0.1 M bicarbonate buffer containing 2.0 mM 5-dodecanoylaminofluorescein di-β-d-galactopyranoside (C₁₂FDG; Molecular Probes, Invitrogen, Karlsruhe, Germany). After labeling, conidia were washed twice with bicarbonate buffer (1,500 × g, 10 min, 4°C), resuspended in assay buffer, and used directly.

Purification of PMNs.

Polymorphonuclear neutrophils (PMNs) were isolated from 20 ml of venous blood. Blood was anticoagulated with 2 ml acidified citrate and mixed with 18 ml dextran (7% dextran 70 in 0.9% NaCl), and erythrocytes were allowed to sediment for 60 min at room temperature. The leukocyte-rich supernatant was underlayered with an equal volume of LymphoPrep (Axis-Shield PoC AS, Oslo, Norway) and centrifuged for 20 min at 800 × g. The PMN fractions were recovered, and contaminating erythrocytes were lysed with hypotonic saline. Afterwards, the suspension was immediately adjusted to a volume of 40 ml with Hanks balanced salt solution (without Mg²⁺ and Ca²⁺, i.e., HBSS^−/−) containing 0.1% chicken egg albumin. The suspension then was centrifuged for 5 min at 300 × g and 4°C. PMNs were resuspended and washed again using HBSS^−/− plus 0.1% chicken egg albumin, and cell counts were determined using a hemocytometer.

Generation of human monocyte-derived macrophages (hMDMs).

Blood from buffy coats (50 ml) was mixed with phosphate-buffered saline (PBS; 50 ml). Four 25-ml aliquots were layered over 15 ml LymphoPrep solution. PBMCs were separated by gradient centrifugation (20 min, 800 × g, 21°C) and washed twice (300 × g, 10 min) with PBS. The pellet was resuspended in 30 ml RPMI 1640 plus 5% fetal bovine serum (FBS), and cell counts were determined using a hemocytometer. Assuming that 5% of total PBMCs are monocytes, 4 × 10⁶ PBMCs were seeded in each well of 24-well plates precoated with human serum in order to yield approximately 2 × 10⁵ monocytes/well. For 12-well plates, 1 × 10⁷ PBMCs, i.e., 5 × 10⁵ monocytes/well, were used. Monocytes were allowed to attach for 24 h. Floating lymphocytes then were removed upon washing with warm PBS, and adherent monocytes were replenished with RPMI plus 10% FBS. Cells were maintained (washed and replenished every 48 h) for 6 to 8 days until macrophages were well differentiated and confluent in each well.

Chemotaxis of PMNs.

A stock solution of posaconazole (312 mg/liter; Schering-Plough Research Institute, Kenilworth, NJ) was prepared in methanol-water (50:50, vol/vol) and further diluted using HBSS^−/−. Cells were prepared as described above and incubated with posaconazole at different concentrations (0, 600, and 1,200 ng/ml). For chemotaxis assays, calcein acetoxymethyl ester (calcein-AM; Molecular Probes, Invitrogen, Karlsruhe, Germany)-loaded PMNs were utilized. PMNs were incubated with calcein-AM (5 μM) in HBSS^−/− at 37°C and 5% CO₂ for 20 min. After incubation, free calcein-AM was removed by dilution and a subsequent washing of the cells. PMN were resuspended to 2 × 10⁷ cells/ml in HBSS with Ca²⁺ and Mg²⁺ (HBSS^+/+) and 0.1% chicken egg albumin. Chemotaxis of calcein-AM-loaded PMN was assessed using a modified 96-well Boyden chamber with a polycarbonate polyvinylpyrrolidone (PVP)-free filter with 3-μm pores (ChemoTx 101-3; Neuro Probe, Gaithersburg, MD). Bottom plate wells were loaded with chemoattractants, i.e., zymosan-activated serum (ZAS; 0.1 mg/ml), AAS (1:10), N-formylmethionyl-leucyl-phenylalanine (fMLP; 1 × 10⁻⁷ M), and Aspergillus fumigatus conidia (1.5 × 10⁷/ml). Random migration of PMNs was determined using a negative control, i.e., HBSS^+/+ plus 0.1% chicken egg albumin. Corresponding filter membrane wells were loaded with 25 μl of the pretreated PMN suspensions. After a 90-min incubation period (37°C and 5% CO₂), free cells on the top of the filter were wiped off and the relative fluorescent intensity of cells that had migrated to the underside of the filter was quantified using a fluorescence plate reader (Cytofluor 4000; Applied Biosystems, Foster City, CA) using excitation at 485 nm and emission at 530 nm. Each chemotactic condition was analyzed in quadruplicate. The chemotactic index was calculated in relation to the negative control.

ROS production of PMNs.

Cells were prepared as described above, using RPMI 1640 (without phenol red) plus 10% FBS and adjusted to 2 × 10⁶ cells/ml. Reactive oxygen species (ROS) production was determined by chemiluminescence in the presence of luminol (4-amino-2,3-dihydro-1,4-phthalazinedione; 4 × 10⁻⁴ M). Briefly, white 96-well microplates were loaded with cell suspension (50 μl; 1 × 10⁵ cells/well) and different concentrations of posaconazole (0, 300, 600, and 1,200 ng/ml), luminol (4 × 10⁻⁴ M), and horseradish peroxidase (HRP). Afterwards, the generation of ROS was stimulated with Aspergillus fumigatus conidia (multiplicity of infection [MOI], 1:5), lyophilized Escherichia coli (positive control) (2.5 ng/ml), and medium (negative control). The luminescence was measured every 2 min for 6 h at 37°C using a microplate reader (Infinite M1000 Pro; Tecan, Switzerland).

Phagosomal processing of A. fumigatus conidia by PMNs.

Cells were prepared as described above and adjusted to 5.0 × 10⁷ cells/ml using HBSS^+/+ plus 20 mM HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid) containing different concentrations of posaconazole and incubated (37°C) with C₁₂FDG-labeled A. fumigatus conidia (MOI, 1:2). After defined intervals (0, 30, 60, and 120 min), a fixing solution (460 μl; 5% formaldehyde in PBS plus 2% fetal calf serum [FCS]) was added and the samples were analyzed by flow cytometry. The enzymatic cleavage of C₁₂FDG by ß-galactosidase within phagolysosomes produces a green fluorescent product and indicates phagosomal processing of the labeled conidia. Hence, phagosome maturation is considered to be directly proportional to the increase in mean fluorescence intensity (MFI).

Killing of A. fumigatus conidia by hPMNs.

For the killing of A. fumigatus conidia by human PMNs (hPMNs), cells were prepared as described above, adjusted to 1.0 × 10⁷ cells/ml using HBSS^+/+ plus HEPES (20 mM) containing different concentrations of posaconazole, and incubated (37°C) with serum-opsonized A. fumigatus conidia (MOI, 1:4). After 3 h, the neutrophils were lysed by adding cold Tween 20 (460 μl; 0.1% Tween 20 in sterile water; 4°C) and stored at −80°C. The amount of viable conidia/CFU was determined by serial dilution plating. In brief, samples were thawed, serially diluted (1:10 with 0.1% Tween 20), and plated on Sabouraud agar plates containing gentamicin and chloramphenicol (Becton Dickinson, Heidelberg, Germany). The plates were incubated for 20 h at 37°C, and CFU were counted afterwards. A negative control of the inoculum without neutrophils was kept under the same conditions as those applied in the infection experiment. The killing index was then calculated in relation to the CFU of that negative control.

Metabolic activity of A. fumigatus conidia challenged by hPMNs.

Cells were prepared as described above, adjusted to 2.0 × 10⁶ cells/ml using HBSS^+/+ plus 20 mM HEPES containing different concentrations of posaconazole, and incubated (37°C, 3 h) with serum-opsonized A. fumigatus conidia (MOI, 1:5). Neutrophils then were centrifuged (1,500 × g, 4°C, 5 min), lysed by adding a cold Tween 20 solution (0.1% Tween 20 in sterile water, 4°C), and stored at −80°C. The metabolic activity of conidia was assessed colorimetrically based on the reduction of the tetrazolium salt 2,3-bis(2-methoxy-4-nitro-5-[(sulfenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) in the presence of menadione as published elsewhere, with some modifications (16). Briefly, the samples were thawed, supplemented with RPMI (without phenol red), transferred to 96-well microplates (100 μl/well), and incubated at 37°C. After 16 h, XTT (167 mg/ml) and menadione (167 μM) were added and the microplates were further incubated for 3 h at 37°C before the optical density at 450 nm (OD₄₅₀) was measured using a microplate reader (Infinite M1000 Pro; Tecan, Switzerland).

IL-8 production of hPMNs stimulated by A. fumigatus conidia.

Neutrophils were isolated from whole blood using the MACSxpress neutrophil isolation kit (Miltenyi Biotec, Germany) according to the manufacturer's instructions. Cells were counted using a hemocytometer, centrifuged (350 × g, 10 min), and adjusted to 4.0 × 10⁶ cells/ml using RPMI 1640 (without phenol red) containing different concentrations of posaconazole. Equal volumes of cell suspension (250 μl) and A. fumigatus conidia (250 μl; 2.0 × 10⁷ conidia/ml; MOI, 1:5) were admixed and incubated at 37°C. After 6 h, the infected neutrophils were centrifuged (350 × g, 4°C, 5 min), and then the supernatant was centrifuged again (14,000 × g, 4°C, 5 min), aliquoted, and stored at −80°C until use. The supernatants were thawed and analyzed using the human CXCL8/interleukin-8 (IL-8) DuoSet kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

ROS production by MDMs.

6-Carboxy-2′,7′-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (DCF; Molecular Probes, Invitrogen, Karlsruhe, Germany) was used as an indicator for ROS production within human MDMs. Briefly, MDMs were grown in RPMI plus 10% FBS for 6 to 8 days in 24-well plates (as described above), washed (PBS), and incubated with 250 μl 16 μM DCF for 15 min at 37°C. Plates were washed twice (RPMI plus 10% FBS) and treated with posaconazole at different concentrations (0, 200, 600, and 1,200 ng/ml). ROS production was stimulated with Aspergillus fumigatus conidia (2.0 × 10⁶ conidia/well; MOI, 1:10), phorbol myristate acetate (4 μM PMA; P8139; Sigma-Aldrich, Munich, Germany) (positive control), and medium (negative control). After 3 h of incubation at 37°C, MDMs were carefully scraped off and analyzed by flow cytometry. Macrophages were identified and gated based on their appearance within the cellular forward and sideward scatter (FSC/SSC). The purity of cells within this gate was confirmed by CD45/CD14 staining. At least 98% of the cells within the respective gate were CD45⁺/CD14⁺ cells, i.e., evidently human MDMs. Only cells within the MDM gate were analyzed. ROS production was assessed as relative change of the MFI, calculated in relation to the negative control.

Phagosomal processing of A. fumigatus conidia by hMDMs.

The phagosomal processing of A. fumigatus conidia by posaconazole-loaded MDMs was studied by flow cytometry. MDMs were incubated (37°C) with C₁₂FDG-labeled A. fumigatus conidia (1.25 × 10⁷ conidia/ml; MOI, 1:10) at different concentrations of posaconazole (0, 200, 600, and 1,200 ng/ml). After defined intervals (0, 30, 60, and 120 min), the MDMs were washed twice (in PBS at 37°C), resuspended in cold PBS (4°C), and immediately analyzed by flow cytometry. The increase of green (FL1) fluorescence caused by the enzymatic cleavage of C₁₂FDG by ß-galactosidase within phagolysosomes is considered to be directly proportional to phagosomal processing of the labeled conidia.

Killing of A. fumigatus conidia by hMDMs.

MDMs were grown in RPMI plus 10% FBS for 6 to 8 days in 12-well plates (as described above), washed with PBS, and incubated (37°C) with 500 μl RPMI plus 10% FBS containing serum-opsonized A. fumigatus conidia (5 × 10⁶ conidia/ml; MOI, 1:5). After 1 h, unbound conidia were washed away with PBS (37°C). Macrophages of four wells were lysed and processed (as described below), and the macrophages within the remaining wells were incubated for an additional 6 h in 500 μl RPMI plus 10% FBS containing different concentrations of posaconazole (0, 200, 600, and 1,200 ng/ml). Afterwards, the MDMs were washed twice with PBS (37°C), lysed by adding cold Tween 20 (500 μl; 0.1% Tween 20 in sterile water; 4°C), and stored at −80°C until use. The amount of viable conidia/CFU was determined by serial dilution plating. In brief, samples were thawed, serially diluted (1:10 with 0.1% Tween 20), and plated on Sabouraud agar plates containing gentamicin and chloramphenicol (Becton Dickinson, Heidelberg, Germany). CFU were counted after 20 h of incubation at 37°C. The killing index, i.e., the percentage of Aspergillus fumigatus conidia killed after 6 h in relation to the amount of phagocytized conidia (after 1 h), was calculated.

Statistical analysis.

Statistical analysis was performed using GraphPad Prism 5.02 (GraphPad Software Inc., CA, USA). Values were compared using analysis of variance (ANOVA); intergroup differences were confirmed by a Bonferroni post hoc test with multiple-test corrections. P values lower than 0.05 were considered statistically significant. Figures were computed with GraphPad Prism 5.02 (GraphPad Software Inc., CA, USA).

RESULTS

PMNs do not migrate toward Aspergillus fumigatus conidia.

The migration capacity of unloaded neutrophils toward Aspergillus fumigatus conidia, as well as Aspergillus fumigatus lysate, was studied first. However, conidia in concentrations up to 1.5 × 10⁸ did not induce PMN migration. In fact, the level of migration toward conidia was equal to that of random migration (which is the negative control) (data not shown), as previously described by Waldorf et al. (17).

Intracellular loading of posaconazole does not impact neutrophil migration capacities.

Migration to the site of infection is crucial for PMNs in order to combat fungal pathogens. Thus, we investigated whether intracellular loading of posaconazole has an influence on neutrophil chemotaxis toward physiological chemoattractants. To this end we used serum which had been activated by Aspergillus fumigatus (AAS) in our assay. We applied N-formylmethionyl-leucyl-phenylalanine (fMLP) as an additional positive control. Whenever cells remained that were not needed for the actual assay, intracellular concentrations were determined as previously described (18); the ratio between the cellular and the applied concentration was well within the expected range (4.95 ± 1.90 versus 7.66 ± 6.50) (13). The results of the chemotaxis assay are shown in Fig. 1; there was no significant effect of posaconazole on the migration capacity of neutrophils toward AAS or fMLP.

FIG 1.

Chemotactic activity of posaconazole-loaded neutrophils. The migration capacity of posaconazole-loaded neutrophils (0, 0.6, and 1.2 μg/ml) toward zymosan-activated serum (ZAS; 0.1 mg/ml), Aspergillus fumigatus-activated serum (AAS; 1:10), N-formylmethionyl-leucyl-phenylalanine (fMLP; 1 × 10⁻⁷ M), and Aspergillus fumigatus conidia (1.5 × 10⁷/ml) was measured using a modified 96-well Boyden chamber. The chemotactic index (means ± standard errors of the means [SEM]) was calculated in relation to the negative control, indicated by the dashed line (n = 6).

Phagosomal maturation is not affected by intracellular concentrations of posaconazole.

We evaluated the effect of posaconazole on the phagosomal-lysosomal fusion with C₁₂FDG-coated A. fumigatus conidia. C₁₂FDG is a self-quenched nonfluorescent substrate that is hydrolyzed upon exposure to ß-galactosidase found in phagolysosomes, producing highly fluorescent fluorescein. Therefore, C₁₂FDG-coated conidia become fluorescent only as they are processed within phagolysosomes (19). The flow-cytometric profiles of human neutrophils 2 h after internalization of C₁₂FDG-coated A. fumigatus are shown in Fig. 2A (one representative of three individual experiments). The mean fluorescence intensity (MFI) increased with phagosomal maturation following uptake of conidia; this effect was partially reversed by bafilomycin A1, which inhibits the phagosomal-lysosomal fusion.

FIG 2.

(A) Flow-cytometric analysis of phagolysosomal fusion. Human neutrophils were incubated (2 h, 37°C) with 5-dodecanoylaminofluorescein di-β-d-galactopyranoside (C₁₂FDG)-coated Aspergillus fumigatus conidia (MOI, 1:2) in the presence of 0, 0.2, 0.6, and 1.2 μg/ml posaconazole, as depicted by the blue, green, orange, and red lines, respectively. The brown line shows neutrophils with C₁₂FDG-coated Aspergillus fumigatus conidia (MOI, 1:2) in the presence of bafilomycin A1 (1 μM). Neutrophils were acquired by flow cytometry to measure the release of green (FL1) fluorescence, which is directly proportional to the cleavage of C₁₂FDG and phagosome-lysosome formation. The gray curve represents untreated human neutrophils (2 h, 37°C). The gray area represents the negative control, i.e., neutrophils with C₁₂FDG-coated Aspergillus fumigatus conidia, which were acquired immediately (time point zero). (B) Flow-cytometric analysis of phagolysosomal fusion. Human monocyte-derived macrophages (hMDM) were incubated with C₁₂FDG-coated Aspergillus fumigatus conidia (MOI, 1:10; 2 h, 37°C) in the presence of 0, 0.2, 0.6, and 1.2 μg/ml posaconazole, as depicted by the blue, green, orange, and red lines, respectively. The brown line shows hMDMs with C₁₂FDG-coated Aspergillus fumigatus conidia (MOI, 1:10) in the presence of bafilomycin A1 (1 μM). HMDMs were acquired by flow cytometry to measure the release of green (FL1) fluorescence, which is directly proportional to the cleavage of C₁₂FDG and phagosome-lysosome formation. The gray curve represents untreated hMDMs (2 h, 37°C). The gray area represents the negative control, i.e., hMDMs with C₁₂FDG-coated Aspergillus fumigatus conidia, which were acquired immediately (time point zero).

Posaconazole inhibits germination of conidia, thereby impacting release of ROS from PMNs.

Release of ROS from PMNs is crucial for extracellular killing of pathogens as well as for the initiation of several intracellular signaling processes. However, ROS also drives inflammation in the microenvironment at the site of infection, which may be harmful to the host. Figure 3 shows the relative change in ROS production by human neutrophils upon stimulation with conidia of different Aspergillus fumigatus strains after 3 h of incubation. While posaconazole did not affect the production of ROS upon stimulation with a highly azole-resistant strain, M-491 (MIC of >8 mg/ml), there was a significant reduction of ROS produced by human neutrophils upon stimulation with conidia of a posaconazole-susceptible strain (ATCC 46645) (Fig. 4). Upon stimulation with another azole-resistant strain, the M-850 strain (MIC of ≥2 mg/ml), only the highest posaconazole concentration (1.2 mg/ml) showed a significant reduction in ROS production. Both azole-resistant strains (M-491 and M-850) have been described elsewhere (20). The production of ROS upon stimulation with lyophilized E. coli (2.5 ng/ml) was not affected by posaconazole (see Fig. S1 in the supplemental material). The amount of IL-8 released by human neutrophils was not affected by posaconazole, as shown in Fig. S2 in the supplemental material. In addition, the decline in ROS production does not seem to be attributable to a decrease in A. fumigatus conidia due to the antifungal efficacy of posaconazole (and hence elimination of the stimulus). In fact, posaconazole did not affect killing of A. fumigatus conidia by human neutrophils, as shown in Fig. 5A. However, it is known from previous studies that posaconazole inhibits the germination of Aspergillus fumigatus conidia (21), and ROS release from neutrophils is significantly lower upon stimulation with resting conidia than with swollen conidia or hyphae (12, 22). In order to confirm inhibition of germination of A. fumigatus conidia by posaconazole in our experimental setting, we assessed the metabolic activity of A. fumigatus conidia in the presence of posaconazole and/or neutrophils.

FIG 3.

ROS production by neutrophils. Neutrophils were stimulated with conidia from different Aspergillus fumigatus strains (MOI, 1:5) in the presence of posaconazole (0, 0.2, 0.6, and 1.2 μg/ml) and incubated for 6 h at 37°C. ROS generation was detected by monitoring luminol chemiluminescence and expressed as a change in relative light units (RLU) compared to the negative control, i.e., neutrophils without (w/o) posaconazole (means ± SEM; n = 6).

FIG 4.

ROS generation by human monocyte-derived macrophages (hMDM). The generation of ROS was stimulated by phorbol myristate acetate (PMA) and Aspergillus fumigatus conidia (MOI, 1:10) in the presence of posaconazole (0, 0.2, 0.6, and 1.2 μg/ml) at 37°C for 3 h (means ± SEM; n = 3).

FIG 5.

(A) Percentage of Aspergillus fumigatus conidia killed by neutrophils determined by CFU counting after serial dilution plating. Human neutrophils were incubated (3 h, 37°C) with serum-opsonized Aspergillus fumigatus conidia (MOI, 1:4) in the presence of posaconazole (0, 0.2, 0.6, and 1.2 μg/ml) (means ± SEM; n = 5). (B) Percentage of Aspergillus fumigatus conidia killed by monocyte-derived macrophages (MDM) determined by CFU counting after serial dilution plating. Human MDMs were infected (1 h, 37°C) with serum-opsonized, Aspergillus fumigatus conidia (MOI, 1:10), washed, and incubated (6 h, 37°C) in the presence of posaconazole (0, 0.2, 0.6, and 1.2 μg/ml) (means ± SEM; n = 10).

As shown in Fig. 6, posaconazole at 0.2, 0.6, and 1.2 μg/ml led to reduced metabolic activity of A. fumigatus conidia by 12.7% ± 2.9%, 13.9% ± 1.9%, and 11.1% ± 2.3%, respectively. However, we could not show a dose-dependent effect on metabolic activity. In fact, since all studied concentrations of posaconazole were well above the MIC of the strain (MIC of 0.063 mg/liter), we did not expect to see any difference between those concentrations.

FIG 6.

Metabolic activity of Aspergillus fumigatus conidia. Human neutrophils were incubated (3 h, 37°C) with Aspergillus fumigatus conidia (MOI, 1:5) in the presence of posaconazole (0, 0.2, 0.6, and 1.2 μg/ml). Afterwards, the metabolic activity was assessed colorimetrically. Groups were compared using ANOVA, followed by a Bonferroni post hoc test. ***, P < 0.001. The dashed line indicates the negative control, and the gray bar represents the positive control, i.e., Aspergillus fumigatus conidia incubated without neutrophils (means ± SEM; n = 4). a.u., arbitrary units; n.s., not significant.

We also studied the functional capacities of human monocyte-derived macrophages in the presence of posaconazole.

Release of ROS from hMDMs is not affected by posaconazole.

Since A. fumigatus conidia are rapidly phagocytized by macrophages, we measured intracellular ROS production of macrophages using DCF (Fig. 4) instead of luminol, which is used to quantify intra- and extracellular ROS. Although there seemed to be a slight tendency toward smaller amounts of ROS with increasing concentrations of posaconazole, we did not see any significant effect of posaconazole on the ROS production by macrophages. Therefore, we studied the phagosomal processing of C₁₂FDG-coated conidia after 2 h of incubation. As shown in Fig. 2B, posaconazole did not affect the phagosomal processing.

Killing is equally effective in the presence or absence of posaconazole.

After 6 h of incubation, 90.3% ± 8.0% of the phagocytized conidia were killed by macrophages. As shown in Fig. 5B, posaconazole did not affect the killing of A. fumigatus conidia by human macrophages; however, a two-way analysis of variance (ANOVA) suggested that the killing was donor dependent (P < 0.0001) or at least depended on the quality of the buffy coat and hence the resulting MDMs. A mere 4.2% of the variance might have been caused by the treatment, i.e., posaconazole (P = 0.011).

DISCUSSION

In this study, we assessed key functional capacities of human neutrophils and human monocyte-derived macrophages in the presence of posaconazole at various concentrations in vitro. We hypothesized that high intracellular loading of posaconazole either supports the professional phagocytes in their mission of pathogen killing or has a toxic effect and thereby hampers neutrophil and MDM function. Despite high intracellular concentrations of posaconazole, we did not see any significant effect, neither beneficial nor deleterious, on the functional capacities of these cells, except for ROS release. The total amount of ROS released by human neutrophils was decreased in the presence of posaconazole. We attributed this effect to reduced metabolic activity, i.e., germination of the A. fumigatus conidia in response to the drug than to an immunomodulatory effect of posaconazole on the neutrophil. This conclusion is supported by the fact that, when stimulated with conidia of azole-resistant Aspergillus fumigatus strains or E. coli, neutrophils showed equal ROS release in the presence and absence of posaconazole. However, the observation of decreased ROS release in the presence of posaconazole indicates that patients who experience control of fungal germination by sufficient drug levels suffer less collateral tissue damage at the site of infection, as it is associated with excessive ROS release. Based on the available literature, overall the data on ROS release by neutrophils in the presence of azoles are inconsistent. The effect of fluconazole on the phagocytic response of polymorphonuclear leukocytes was studied in a rat model of acute bacterial sepsis. In these experiments a significant and dose-dependent inhibition of the ROS-mediated proinflammatory cascade was observed (23). In contrast, Velert et al. measured the generation of free radicals by human PMNs in vitro upon PMA stimulation with and without fluconazole (at 0.1, 1, 5, and 50 mg/ml) and itraconazole (at 0.05, 0.5, and 5 mg/ml); neither compound had a significant effect on ROS release (24).

Neutrophils are attracted to the site of Aspergillus infection, e.g., the alveoli, by the release of distinct cytokines. This scenario happens early in the infection process and is crucial for efficient clearing of Aspergillus fumigatus conidia (5). We assessed directed and random migration of human neutrophils in a modified 96-well Boyden chamber system. PMNs exhibited only random migratory activity toward Aspergillus fumigatus conidia as such. Applying fMLP, Aspergillus fumigatus-activated serum (AAS), as well as zymosan-activated serum (ZAS) as chemoattractants, we observed equal directed migration of neutrophils independent of the presence of posaconazole. In contrast, Vuddhakul et al. described a significant suppression of neutrophil chemotaxis for human neutrophils in vitro in the presence of itraconazole, which is closely related to posaconazole (25). However, these examinations were carried out in protein-free media, and it was shown by Perfect et al. that the ratio of intracellular to extracellular concentrations (C/E) of itraconazole in alveolar macrophages (AC) depends on the composition of the surrounding medium. The C/E ratio of itraconazole within protein-free medium was significantly higher than that in media which contained serum, with C/E ratios of 87.9, 18, and 3 in 0%, 5%, and 100% serum, respectively (26). Hence, the intracellular concentration of itraconazole used in the experiments by Vuddhakul et al. (25) were much higher than those under physiological conditions. This might have contributed to the observation that itraconazole suppressed neutrophil functions to a greater extent than fluconazole.

Our in vitro study used protein-supplemented medium, and neutrophil migration was not affected by high concentrations of posaconazole. Given the fact that under physiological conditions protein concentrations are even higher, we propose that neutrophils are the perfect vehicles to deliver the drug to the site of infection. The inevitable death of neutrophils would subsequently lead to the release of posaconazole, thus increasing drug concentrations directly at the infection site. This then would contribute to slowing the germination of Aspergillus spp., which leads to diminished release of ROS from further recruited neutrophils. If this hypothesis holds true, the delivery of posaconazole by neutrophils to the site of Aspergillus infection would warrant control of the pathogen and preservation of tissue integrity at the same time. Since infections with Aspergillus spp. pose a life-threatening event to patients with neutropenia, e.g., after chemotherapy, neutrophil transfusions that are preloaded with posaconazole might provide a new perspective on the treatment of fungal infections.

Funding Statement

The funder had no role in study design, data collection and interpretation, or the decision to submit the work for publication.