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Eukaryotic Cell, July 2008, p. 1109-1117, Vol. 7, No. 7
1535-9778/08/$08.00+0     doi:10.1128/EC.00036-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

A Monoclonal Antibody to Histoplasma capsulatum Alters the Intracellular Fate of the Fungus in Murine Macrophages ^,

Li Shi,¹ Priscila C. Albuquerque,¹ Eszter Lazar-Molnar,² Xintao Wang,³ Laura Santambrogio,³ Attila Gácser,^1, and Joshua D. Nosanchuk^1,2*

Departments of Medicine (Division of Infectious Diseases),¹ Microbiology & Immunology,² Pathology, Albert Einstein College of Medicine, Bronx, New York³

Received 30 January 2008/ Accepted 15 April 2008

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Monoclonal antibodies (MAbs) to a cell surface histone on Histoplasma capsulatum modify murine infection and decrease the growth of H. capsulatum within macrophages. Without the MAbs, H. capsulatum survives within macrophages by modifying the intraphagosomal environment. In the present study, we aimed to analyze the affects of a MAb on macrophage phagosomes. Using transmission electron and fluorescence microscopy, we showed that phagosome activation and maturation are significantly greater when H. capsulatum yeast are opsonized with MAb. The MAb reduced the ability of the organism to regulate the phagosomal pH. Additionally, increased antigen processing and reduced negative costimulation occur in macrophages that phagocytose yeast cells opsonized with MAb, resulting in more-efficient T-cell activation. The MAb alters the intracellular fate of H. capsulatum by affecting the ability of the fungus to regulate the milieu of the phagosome.

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Histoplasma capsulatum var. capsulatum is a pathogenic dimorphic fungus with a worldwide distribution. H. capsulatum is the leading cause of fungal respiratory disease, infecting approximately 500,000 individuals in the United States annually (6, 10, 43). Infection is frequently asymptomatic or results in a mild pulmonary illness, but it may progress to life-threatening systemic disease, particularly in individuals with AIDS (24, 58). In 2002, there were 3,370 hospitalizations for histoplasmosis in the United States, with a crude mortality rate of 8% (15). In the setting of significant immunosuppression, such as with AIDS, the fatality rate in severe disease (e.g., shock and respiratory failure) with administration of appropriate antifungals is extremely high (47 to 70%) (56, 57). Hence, new therapies are urgently needed.

H. capsulatum is a pathogen that normally survives within phagosomes by regulating the intracellular milieu of macrophages (39, 47, 49). By maintaining a neutral pH in macrophages, H. capsulatum yeast avoid damage by host defenses, such as lysosomal hydrolases. A neutral pH has other salutatory affects for the fungus during infection of macrophages, such as inhibiting intracellular trafficking and antigen presentation, which are thought to depend on acidification of the phagosome (18). H. capsulatum is unique among the fungal pathogens in its ability to tightly regulate phagosomes of macrophages. For instance, the important facultative intracellular yeast-like fungus Cryptococcus neoformans resides in an acid phagosome (34). Evolution of mechanisms for the inhibition of phagosome acidification has occurred in diverse microbes. In Mycobacterium tuberculosis and Mycobacterium avium, acidification is inhibited in part by preventing vacuolar proton-ATPase accumulation at the phagosomal membrane (46, 51) and via alterations in cell signaling, such as by Ca²⁺ (37). Similarly, intraphagosomal acidification in H. capsulatum-infected murine macrophages is blocked by the inhibition of vacuolar ATPase insertion into the phagosomal membrane (49). For other pathogens, such as Legionella pneumophila and Toxoplasma gondii, inhibition of phagosome lysosome fusion prevents acidification (27, 48). H. capsulatum alters phagolysomal fusion to various degrees in different macrophage populations (19, 38, 40, 41, 49, 53), with the greatest inhibition occurring in human macrophages.

We have previously described monoclonal antibodies (MAbs) to histone 2B (H2B) on the cell surface of H. capsulatum yeast cells that modify the course of murine histoplasmosis (42). The MAbs reduce the fungal burden, decrease pulmonary inflammation, and prolong survival of lethally infected mice. Additionally, the MAbs increase phagocytosis and inhibit the growth of H. capsulatum in macrophages. The MAbs do not directly affect H. capsulatum growth or viability. In the present work, we describe downstream effects of a MAb to an H. capsulatum cell surface protein on the fate of the fungus within murine macrophages.

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Reagents, cell lines, and H. capsulatum. The murine MAb 9C7 of immunoglobulin M (IgM) isotype, specific for H2B of H. capsulatum, was used in all assays (42). Isotype-matched controls were MAb to TEPC183 (ICN Pharmaceuticals, Aurora, OH) and MAb 5c11, specific for lipoarabinomannan of M. tuberculosis (22). The fluorescent probes 5-(and 6)-carboxyfluorescein succinimidyl (NHS-CF) and 5-(and 6)-carboxytetramethylrhodamine succinimidyl (NHS-Rho) were obtained from Molecular Probes (Eugene, OR). Triton X-100, fluorescein isothiocyanate (FITC)-dextran (molecular weight, 70,000), and paraformaldehyde were from Sigma (St. Louis, MO). SuperBlock blocking buffer in phosphate-buffered saline (PBS) was from Pierce (Rockford, IL). Anti-major histocompatibility complex (MHC) class II β-chain (clone KL-295) was obtained from ATCC (Rockville, MD); rhodamine red-X-conjugated AffiniPure F(AB')₂ goat anti-rat IgG(H+L) was from Jackson ImmunoResearch Laboratories (West Grove, PA); cathepsin S (clone M19) was from Santa Cruz Biotechnology (California); and CD107a LAMP-1 (clone 1D4B), CD74 invariant chain (clone In-1), and CD 71 transferrin receptor (clone C2) were from Becton Dickinson (San Diego, CA).

H. capsulatum strain G217B was obtained from the ATCC and was cultured at 37°C for 3 days in Ham's F-12 medium prior to use as described previously (1). For all experiments, H. capsulatum yeast were washed at least three times with PBS, unless otherwise specified, between incubations. H. capsulatum yeast cell viability was not affected by labeling with the various antibodies and fluorescent dyes (data not shown).

The macrophage-like cell line J774.16 (derived from a reticulum cell sarcoma) and RAW 264.7 cells (BALB/c mouse macrophage transformed with Abelson leukemia virus) were obtained from the ATCC. The cell lines were selected since they have been extensively used to study the pathogenesis of intracellular organisms, including H. capsulatum. The J774.16 and RAW cells were grown in phenol-red-free Dulbecco's modified Eagle medium (DMEM)/F-12 (Mediatech, Herndon, VA) with 5% fetal calf serum (Gemini Bio-Products, Woodland, CA) at 37°C in 5% CO₂. The cell counts for the experiments were determined using a hemacytometer.

Peritoneal macrophages were selected for use as primary cells due to the relative ease of their isolation and the fact that they are major effector cells during disseminated infection, such as occurs in our murine infection model (42). Alveolar macrophages were also examined, since they are the initial phagocytes engaging H. capsulatum during pulmonary infection. For primary peritoneal macrophage isolation, the abdominal cavities of euthanized mice were lavaged five times with sterile PBS using a Pasteur pipette. For isolation of alveolar macrophage, the tracheas were cannulated with a 20-gauge Angiocath catheter (Becton Dickinson, Sandy, UT) and the lungs were lavaged 10 times with sterile Hanks balanced salt solution without phenol red (Life Technologies, Grand Island, NY) with 1 mM EGTA (Sigma). The lavage fluids were pooled, cells were collected by centrifugation, and erythrocytes were lysed by incubating the cell preparation in 0.17 M NH₄Cl for 10 min in ice. A 10-fold excess of RPMI 1640 solution was then added to make the solution isotonic, and the cells were collected by centrifugation and suspended in DMEM (Life Technologies), 10% NCTC-109 (Life Technologies), and 1% nonessential amino acids (Mediatech). Cell viability was >95%, as measured by trypan blue vital dye exclusion.

Fluorescence microscopy. Fluorescence microscopy was initially used to study lysosomal fusion and phagosome maturation. We first analyzed lysosomal fusion with phagosomes using FITC-dextran. Phagolysosomal fusion was inferred by the accumulation of FITC-dextran within H. capsulatum-containing phagosomes as previously described (19, 21). J774.16 cells, RAW cells, or primary peritoneal macrophages (1.6 x 10⁵) were adhered to culture slides in fresh non-phenol-red medium for 4 h at 37°C at 5% CO₂. The cells were then incubated with 0.5 mg/ml FITC-dextran (molecular weight, 70,000) overnight. H. capsulatum cells were collected and washed, and then 1.6 x 10⁷ yeast cells were incubated with 40 µg/ml NHS-Rho at 4°C for 1 h. The cells were washed and incubated with 100 µg/ml 9C7, 5C11, or medium only for 1 h at 37°C. The cells were washed and then were added to the cultures of macrophages (1.6 x 10⁶/well for a yeast:macrophage ratio of 5:1). After a 1-h incubation, uningested yeasts were removed by washing and the cocultures were incubated for an additional 2 h at 37°C at 5% CO₂. The samples were fixed in 3.75% paraformaldehyde for 20 min at room temperature, coverslips were mounted, and the slides were viewed with an Olympus AX70 microscope with fluorescent filters attached. Phagolysosomal fusion was considered to have occurred when the red fluorescence-labeled yeast were surrounded by a green fluorescent rim.

A second fluorescence microscopy technique for detecting lysosomal fusion and phagosome maturation studied the accumulation of the membrane glycoprotein LAMP-1 at phagosomes containing H. capsulatum yeast and was performed according to a modification of methods described previously (4, 34). LAMP-1 is a type I transmembrane glycoprotein that is localized in lysosomes and endosomes (14, 44). Briefly, J774.16 cells or primary macrophages (1.6 x 10⁵/well) were incubated in eight-chamber tissue culture-treated glass slides (Becton Dickinson) overnight at 37°C at 5% CO₂. H. capsulatum yeast were grown and washed as described above, and 1.6 x 10⁷ H. capsulatum cells were incubated with 30 µg/ml NHS-CF for 1 h at 4°C. After washing, 3.2 x 10⁶ labeled H. capsulatum cells were incubated with 100 µg/ml MAb 9C7, IgM control, or medium for 1 h at 37°C. The medium was removed from the macrophage cultures, and 1.6 x 10⁶ labeled H. capsulatum cells were added to per well. After incubation at 37°C for 2 h, uningested yeasts were removed by washing and the macrophages were fixed with 3.75% paraformaldehyde for 20 min at room temperature. Samples were washed, permeabilized in –20°C methanol for 10 s, and incubated in blocking buffer (SuperBlock blocking buffer containing 2% goat serum) for 1 h at 37°C. The cells were incubated with antimouse CD107a specific to LAMP-1 diluted 1:100 in blocking solution for 1 h at 37°C. The samples were washed three times with blocking buffer and then incubated with rhodamine red-X-conjugated AffiniPure F(AB')₂ goat anti-rat IgG(H+L) for 1 h at 37°C. The slides were washed, coverslips were affixed, and the samples viewed with an Olympus AX70 microscope (Melville, NY) with fluorescent filters attached.

Subcellular fractionation and Western blot analysis. To evaluate phagosomal maturation and processing of H. capsulatum antigens, subcellular fractionations of J774.16 and RAW cells were analyzed. One hundred million macrophage cells were lysed in homogenization buffer (250 mM sucrose, 1 mM EDTA [pH 7.4]). Early and late endosomes and lysosomes were prepared over consecutive Percoll gradients (27% and 10%). Each 1-ml fraction was tested for β-hexosaminidase to locate lysosomes and late endosomes. The late endocytic marker LAMP-1 and the early endosome/plasma membrane marker transferrin receptor (TrfR) were used to assess the purity of the endosomal preparations. Pulled fractions 6 and 7 from the 27% Percoll gradient (lysosomes) and 3 and 4 from the 10% Percoll gradient (late endosomes) were lysed in NP-40 lysis buffer (150 mM NaCl, 50 mM Tris HCl, 5 mM EDTA, 1% NP-40). Proteins were resolved through 12% sodium dodecyl sulfate polyacrylamide-gel electrophoresis, transferred to a nitrocellulose membrane, and probed with anti-MHC class II β-chain, CD107a Lamp-1, cathepsin S, CD74 invariant chain, and CD 71 transferrin receptor. Additionally, membranes were probed with H. capsulatum-specific MAbs to heat shock protein 60 (Hsp60) and H2B (42). The amounts of the Hsp60 and H2B proteins in the various fractions were semiquantitated using ImageJ (http://rsb.info.nih.gov/ij/).

Transmission electron microscopy. Electron microscopy was used to evaluate the morphology of phagosomes in the presence or absence of a disease-modifying MAb. J774.16 cells were grown for 24 h on tissue culture plates (Becton Dickinson Laboratories, Franklin Lakes, NJ) at 37°C in 5% CO₂ overnight. H. capsulatum yeast cells were collected by centrifugation and washed three times in PBS, and 5 x 10⁷ cells were incubated with 100 µg/ml MAb 9c7 or MAb 5C11 in 100 µl PBS for 1 h at 37°C. A second control consisted of yeast cells not exposed to MAb. The yeast and J774.16 cells were separately collected, washed, and combined in DMEM/F-12 medium in a 1.5-ml microcentrifuge tube at a 1:1 effector-to-target ratio with 10⁷ cells of each cell type/ml for 2 at 37°C. The medium was aspirated, and the cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate at room temperature overnight. The samples were processed for electron microscopy as previously described (11). The samples were viewed with a JEOL (Tokyo, Japan) 100CX transmission electron microscope.

Phagosomal pH. The pHs of H. capsulatum-containing phagosomes were determined as described previously (49) with minor modifications. Briefly, H. capsulatum yeast cells were collected and washed three times in PBS, and 5 x 10⁷ cells were incubated in 100 µl PBS with 200 µg/ml NHS-CF and 50 µg/ml NHS-Rho for 1 h at 4°C. NHS-CF is pH sensitive, whereas NHS-Rho is not. According to the manufacturer (Invitrogen), the pK_a for NHS-CF is 6.5, providing for quantitative measurements between 5 and 8. The cells were washed, and 5 x 10⁶ labeled cells were incubated with 100 µg/ml MAb 9c7, control IgM, or macrophage medium alone for 1 h at 37°C. To generate confluent monolayers, 5 x 10⁴ macrophages per well were incubated on 96-well black cell culture plates (Corning Incorporated, Corning, NY) overnight. The medium was removed, and 5 x 10⁵ H. capsulatum cells were added per well. After 2 h, the cells were washed with PBS. To generate a standard curve, wells were treated with 100 µl 0.1% Triton X-100 solution with a defined pH (pH 4.0 to 8.0). The fluorescence intensity was measured at an excitation wavelength of 495 nm and emission wavelength of 520 nm for NHS-CF and excitation of 540 nm and emission of 580 nm for NHS-Rho with a FlUOstar Optima spectrofluorometer (BMG, Durham, NC).

Measurement of nitric oxide and superoxide dismutase release by macrophages. J774.16 and RAW cells were plated at 10⁵ cells per well in a 96-well polystyrene tissue-culture plate, flat bottom (Becton Dickinson), and grown in DMEM with 10% heat-inactivated fetal calf serum, 10% NCTC-109 medium, and 1% nonessential amino acids overnight at 37°C in a 5% CO₂-95% air atmosphere. Yeast cells were preincubated with MAb 9C7, nonspecific IgM, or PBS for 1 h at 37°C prior to addition to the macrophage monolayer. After 1 h of incubation, aliquots from the supernatant were collected at different intervals. To investigate nitric oxide formation, we measured nitrite (NO₂), one of two primary, stable, and nonvolatile breakdown products of NO, using a commercial Griess reagent kit (Promega) (13). Similarly, superoxide dismutase activity was determined using a commercial kit from Cayman Chemical (Ann Arbor, MI) that uses tetrazolium salt to quantify superoxide radicals generated by xanthine oxidase and hypoxanthine (17).

T-cell assays. H. capsulatum yeast cells were incubated with PBS, 150 µg/ml MAb 9C7, or an equivalent concentration of isotype control MAb for 1 h at 37°C prior to infection of C57BL/6 mice with 1.25 x 10⁷ yeast cells/mouse (42). At day 2 postinfection, macrophages were obtained by peritoneal lavage and enriched to more than 80% purity by adherence to plastic dishes. T cells from spleens of naive C57BL/6 mice were purified using nylon wool fiber columns (Polysciences, Warrington, PA). T cells were plated in 96-well plates at a density of 10⁵ cells/well and activated for 2 h with 0.5 µg/ml plate-bound anti-CD3 (BD Biosciences). Activation was also attempted with histoplasmin (a protein filtrate from H. capsulatum [61]), but limited activation occurred (data not shown). The purified macrophages were added to the T cells at a ratio of 1:4. Supernatants were collected after 24 h to measure interleukin 2 (IL-2) and 48 h to measure gamma interferon (IFN-) by enzyme-linked immunosorbent assay. After 48 h, cultures were pulsed with 1 µCi/well [³H]thymidine (Amersham Pharmacia Biotech, Piscataway, NJ) for another 14 h. Cells were harvested, and cell proliferation was determined by [³H]thymidine incorporation.

PD-L1 expression. The expression of the negative costimulatory ligand PD-L1 was measured on macrophages as described previously (33). Briefly, macrophages were isolated through alveolar lavage 2 days postinfection from control and infected mice, respectively. For fluorescence-activated cell sorter staining, cells were first incubated with 2.4G2 cell culture supernatant to block Fc receptors and then stained with fluorescently labeled antibodies. Phycoerythrin-conjugated anti-PD-L1 and allophycocyanin-conjugated CD11b (control antibody) were purchased from eBiosciences (San Diego, CA). Samples were processed on a Becton Dickinson FACScan (upgraded dual-laser) flow cytometer and analyzed using FlowJo software.

Statistical analysis. Data were analyzed using Student's t test or the Kruskal-Wallis test (GraphPad [San Diego, CA] Prism v. 3).

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Phagosomal maturation and lysosomal fusion. Fusion of lysosomes with phagosomes was indicated by the detection of FITC-dextran around H. capsulatum yeast within macrophages. Similar results were seen with J774.16 cells (Fig. 1), RAW cells (data not shown), and primary macrophages (Fig. 2). The majority of phagosomes containing H. capsulatum opsonized with MAb 9C7 demonstrated colocalization of FITC-dextran with NHS-Rho-labeled yeast (Fig. 1B and 2B). In J774.16 macrophages, the NHS-Rho-labeled H. capsulatum yeast cells with colocalization of FITC-dextran and the total labeled yeast cells per condition were counted to determine the percentage of phagosomes fused with lysosomes. The median percentage of phagolysosomal fusion was 58% in the presence of MAb 9C7, compared to 19% with medium alone and 14% with irrelevant control MAb (Fig. 1C).

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FIG. 1. Immunofluorescence analysis of phagolysosomal fusion in J774.16 macrophages by FITC-dextran colocalization with NHS-Rho-labeled H. capsulatum yeast cells. (A) H. capsulatum yeast (red) previously incubated with irrelevant control MAb within a J774.16 cell with FITC-dextran (green) within the J774.16 cell but not the phagosome. Bar, 5 µm. (B) Yeast cells opsonized with MAb 9C7 with colocalization of the FITC-dextran within the J774.16 phagosomes. (C) Percentage of phagolysosomal fusion in J774.16 cells as a measure of NHS-Rho-labeled H. capsulatum yeast cells with FITC-dextran colocalization within phagosomes of J774.16 cells. The data are the means ± the standard deviations for 100 H. capsulatum yeast cells for three wells for each condition tested. *, P < 0.001. The experiments were performed four times with similar results.

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FIG. 2. Immunofluorescence analysis of phagolysosomal fusion in primary peritoneal macrophages by FITC-dextran colocalization with NHS-Rho-labeled H. capsulatum yeast cells. (A) H. capsulatum yeast (red) previously incubated with irrelevant control MAb within primary peritoneal macrophages with FITC-dextran (green) within the cytoplasm of the macrophages but not the phagosome. (B) Yeast cells opsonized with MAb 9C7 with colocalization of the FITC-dextran within the primary macrophage's phagosomes. Bar, 5 µm. The experiments were performed twice with similar results.

To further characterize the level of phagosomal maturation, we examined the association of the lysosome-associated membrane glycoprotein LAMP-1 with phagosomes containing H. capsulatum yeast cells. This was accomplished by the labeling of LAMP-1 with rhodamine and H. capsulatum yeast with NHS-CF (Fig. 3 and 4). Figures 3C and 4C show that the fluorescent staining of LAMP-1 in macrophages infected with H. capsulatum yeast opsonized with MAb 9C7 brightly colocalized with the labeled yeast cells. This pattern was seen in 64% of the J774.16 phagosomes containing H. capsulatum labeled with MAb 9C7 (Fig. 3D). LAMP-1 could also be appreciated diffusely in macrophages containing yeast opsonized with MAb 9C7. Significantly less LAMP-1 staining occurred in J774.16 cells without ingested yeast or in yeast incubated with irrelevant antibody (Fig. 3B). However, LAMP-1 did colocalize with yeast in approximately 15% of the MAb control H. capsulatum-infected J774.16 phagosomes (Fig. 3B, inset, and 3D). Although LAMP-1 colocalization was uncommon in primary macrophages that phagocytosed H. capsulatum incubated in medium alone or with control MAb, there was significant LAMP-1 staining in the cytoplasm (Fig. 4A and B). LAMP-1 colocalization similarly occurred in alveolar macrophages that phagocytosed yeast opsonized with MAb 9C7 (85% of the time) but rarely with control yeast (<10%) (see Fig. S1 in the supplemental material).

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FIG. 3. Fluorescence analysis of phagolysosomal fusion by localization of LAMP-1 (green) to J774.16 phagosomes containing H. capsulatum yeast cells labeled with NHS-CF (red). (A) Stacked light and fluorescence showing intracellular H. capsulatum yeast within the phagocytic cell. Bar, 5 µm. (B) Fluorescence images of the yeast previously incubated with irrelevant control MAb within phagosomes of J774.16 cells. Insets in panels A and B show the minority of J774.16 cells were in LAMP-1 localized to phagosomes containing H. capsulatum cells. (C) Yeast cells opsonized with MAb 9C7 with intense LAMP-1 staining at the phagosomes. The LAMP-1 studies were performed three times with J774.16 cells with similar results. (D) Percentage of colocalization of LAMP-1 with H. capsulatum in phagosomes of J774.16 cells. The data are the means ± the standard deviations for 100 H. capsulatum yeast cells for three wells for each condition tested. *, P < 0.001. The experiments were performed three times with similar results.

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FIG. 4. Fluorescence analysis of phagolysosomal fusion by localization of LAMP-1 (green) to primary peritoneal macrophage phagosomes containing H. capsulatum yeast cells labeled with NHS-CF (red). Fluorescence images of H. capsulatum yeast previously incubated with irrelevant control MAb (A) or cells exposed to medium alone (B) within phagosomes of primary macrophages are shown. (C) Yeast cells opsonized with MAb 9C7, with intense LAMP-1 staining at the phagosomes. Bar, 5 µm. The experiments were performed twice with similar results.

Electron micrographs show significant differences in the phagosomes containing H. capsulatum yeast cells incubated with or without the disease-modifying MAb (Fig. 5). All the vacuoles had well-defined membranes, and several contained more than one organism. As shown in Fig. 5B, fusion of lysosomes and endosomes with phagosomes containing H. capsulatum yeast opsonized with MAb 9C7 occurred more frequently than with controls. However, limited engagement and fusion of lysosomes were seen in the controls (Fig. 5A and C), though the degree of fusion varied among phagosomes within each condition.

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FIG. 5. Transmission electron micrographs of J774.16 cells with H. capsulatum yeast cells. The H. capsulatum yeast cells were preincubated with PBS (A), MAb 9C7 (B), or isotype-matched MAb 5C11 (C) prior to infection. The arrows indicate fusion of a lysosome. The inset in panel B is a magnification of the marked region of a phagosome depicting high levels of lysosomal fusion. Bar, 2 µm. The experiment was performed twice, and similar results were obtained.

In support of the microscopic data, the subcellular fractionation studies show that MAb 9C7 increases the quantity of H. capsulatum proteins within late endosomes and lysosomes (Fig. 6), which is consistent with increased antigen processing. Endosomes and lysosomes were successfully isolated from macrophages following phagocytosis of H. capsulatum in the presence or absence of opsonizing MAb (Fig. 6A and B). The efficiency of H. capsulatum delivery to endosomal compartments was determined by quantification of the pathogen-specific proteins in each isolated organelle. Similar amounts of the H. capsulatum-specific proteins H2B and HSP60 were found in early endosomes, whereas increased amounts of both proteins were found in macrophages infected with H. capsulatum yeast opsonized with MAb 9C7 (Fig. 6C). For example, twice as much H. capsulatum H2B was present in lysosomes from the macrophages exposed to H. capsulatum with MAb 9C7 than in lysosomes from cells exposed to the fungus incubated with isotype-matched control MAb. Similar results were obtained with J774.16 and RAW cells.

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FIG. 6. Subcellular fractionation from RAW cells. (A) Representative hexosaminidase activity measured in each fraction (1 ml) of a 10/27% two-step Percoll gradient to separate lysosomes and late endosomes. (B) Western blot analysis of pulled fractions 1 and 2 of the 27% Percoll gradient (lysosomes), fraction 2 of the 10% Percoll gradient (late endosomes) total cell, and total lysate (total) as a control. (C) Western blot analysis of early endosomes, late endosomes, and lysosomes from macrophages with or without infection in the presence or absence of MAb 9C7. The bands were given numerical values based on semiquantitative determinations using ImageJ (http://rsb.info.nih.gov/ij/). The experiments were performed twice, with consistent results. An additional experiment was performed using J774.16 cells, and the results were similar.

pH of H. capsulatum-containing phagosomes. The acidification of phagosomes of J774.16 and RAW cells containing H. capsulatum yeast labeled with pH-sensitive and -insensitive probes was determined using a spectrofluorometer. Standard curves were generated with the fluorophores (see Fig. S2 in the supplemental material). The pH of phagosomes in macrophages infected with the fungus for 2 h were analyzed. No detectable shedding of the dyes from the organism during the incubations with antibody or after infection of the macrophages was seen using fluorescence microscopy. This is important, since shedding could affect the pH determinations. The phagosomal pH of J774.16 cells infected with H. capsulatum in the absence of antibody or with irrelevant antibody was 6.0 or 5.9, respectively. In contrast, a pH of 5.3 was measured for phagosomes containing H. capsulatum opsonized with MAb 9C7. For RAW cells, the pH of phagosomes containing H. capsulatum exposed to medium alone, irrelevant antibody, or MAb 9C7 was 7.3, 7.4, or 6.5, respectively.

Nitric oxide and superoxide. The release of nitric oxide by J774.16 or RAW macrophages cocultured with H. capsulatum yeast opsonized with or without MAb 9C7 was similar (see Fig. S3 in the supplemental material). Similarly, superoxide production was not affected by the addition of our MAb in comparison to results for controls (see Fig. S4 in the supplemental material).

T-cell assays. Figure 7 shows that MAb 9C7 affects immunoactivation of T cells during murine histoplasmosis. Naive T cells purified from spleens of C57BL/6 mice were activated, and macrophages from uninfected control mice and from mice infected with H. capsulatum or infected with H. capsulatum opsonized with MAb or isotype control MAb were added to them. Interestingly, the presence of naive macrophages greatly increased T-cell proliferation in response to anti-CD3 activation. In contrast, the same number of macrophages from H. capsulatum-infected mice without MAb pretreatment were less able to induce T-cell proliferation. However, macrophages from mice infected with H. capsulatum opsonized with MAb significantly increased the secretion of IL-2 and IFN- from the anti-CD3 activated naive T cells compared to results for infected controls (Fig. 7A and B). IL-2 release is indicative of increased early cytokine responses and IFN- late responses. The presence of MAb 9C7 increased IL-2 levels by 44% and IFN- levels by 36%. Furthermore, MAb 9C7 significantly increased T-cell proliferation (Fig. 7C). Counts per minute were 24% greater under conditions with macrophages from MAb 9C7-opsonized H. capsulatum than with infected controls (P = 0.007).

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FIG. 7. In vitro activation of naive T cells cocultured with macrophages from mice. Noninfected controls (PBS) or mice infected for 48 h with H. capsulatum (Hc), H. capsulatum opsonized with MAb 9C7 (Hc and MAb 9C7), or H. capsulatum incubated with irrelevant control MAb (Hc and control MAb) were studied. IL-2 (A) or IFN- (B) was measured as an indicator of early or late cytokine response, respectively. (C) In vitro proliferation of T cells cocultured with macrophages from H. capsulatum-infected mice was determined by measuring the incorporation of [3H]thymidine. Data points represent mean values from individual mice, and horizontal bars represent the overall average from four mice. Kruskal-Wallis test: *, P = 0.009; **, P = 0.01; ***, P = 0.007. Results were similar in three different experiments.

PD-L1 expression. Figure 8 demonstrates that alveolar macrophages from mice infected with H. capsulatum opsonized with MAb 9C7 have reduced expression of PD-L1 in comparison to results for mice infected with nonopsonized yeast cells. However, PD-L1 expression in the macrophages from mice infected with opsonized yeast was elevated compared to the baseline constitutive expression of the ligand on control macrophages from uninfected mice.

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FIG. 8. PD-L1 upregulation on macrophages is lower after infection with H. capsulatum yeast preincubated with MAb 9C7. Macrophages isolated through alveolar lavage were stained for PD-L1 expression. PD-L1 constitutively expressed on uninfected macrophages (green line) is highly upregulated after infection (blue line). Upregulation is reduced in the presence of the MAb 9C7 (orange line). The specificity of the staining was verified using the isotype control antibody coupled to the same fluorophore (red line).

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In contrast to immunocompetent individuals, where dissemination with H. capsulatum is uncommon, 95% of individuals with AIDS present with disseminated histoplasmosis (56, 58). Patients with AIDS are more likely to require hospitalization and have higher fatality rates (25). Even with highly active antiretroviral therapy and the availability of prophylactic triazoles, there have not been significant changes in the rates of morbidity or mortality in patients with human immunodeficiency virus and histoplasmosis (25). Hence, new therapeutics are critically important for combating this disease, and MAbs to H. capsulatum antigens could provide a substantial benefit to individuals with severe histoplasmosis. Understanding the effects of MAbs that modify the progression of histoplasmosis is vital to the development of these antibodies as therapeutics and also for advancing our knowledge of the pathogenesis of disease.

Although professional phagocytes routinely internalize inert particles into membrane-bound vacuoles and hydrolyze the particles in mature phagosomes, H. capsulatum yeast cells manage to avoid degradation within phagosomes and thrive within the nutrient-rich environment of the host cell. We have shown that administration of MAbs to H. capsulatum H2B reduces intracellular growth and survival of the fungus (42). The disease-modifying MAbs are of the IgM isotype, and phagocytosis of H. capsulatum yeast cells opsonized with these antibodies occurs via CD18 of CR3 (42), which is the same receptor utilized in the ingestion of the fungus in the absence of antibody (36). Hence, the initial phagosome may be similar in the two instances.

For H. capsulatum, a major focus on intracellular survival has been on avoidance of lysomsal hydrolases, and the fungus accomplishes this by maintaining a neutral intraphagosomal pH (18) where most lysosomal hydrolases are presumably inactive (19). Additionally, H. capsulatum reduces exposure to these hydrolases by inhibiting phagolysosomal fusion to various degrees in macrophages, particularly in human macrophages (40, 41). In the present study, we have shown that phagolysosomes containing H. capsulatum opsonized with MAb are more acidic than yeast cells incubated with PBS or control MAb. In particular, the pH of phagosomes containing yeast opsonized with MAb 9C7 in J774.16 macrophage-like cells was 5.3 after a 2-h incubation, at which pH lysosomal hydrolases should be active.

Macrophage-derived nitric oxide (8, 30) and superoxide species (7, 8, 59, 60) can inhibit H. capsulatum. However, in the absence of antibody, H. capsulatum does not affect nitric oxide or superoxide release from murine macrophages, although increased production of reactive species occurs if macrophages are exposed to gamma interferon (30-32). Our data show that free-radical generation is also not significantly altered in murine macrophages by the presence of MAb to H. capsulatum H2B.

The electron microscopy and immunofluorescence experiments show that the phagosomes containing H. capsulatum yeast cells opsonized with the MAb are significantly more active than controls. Prior research has shown that the degree of phagosome-lysosome fusion in H. capsulatum-containing phagosomes varies significantly depending on time, inoculum, strain, and cell type (16, 19, 39-41). In the studies with LAMP-1 and FITC-dextran, we found that opsonization with MAb increases J774.16 lysosomal fusion with phagosomes by 35% compared to results with controls. Antibody can also increase phagolysosomal fusion events in the intracellular pathogen M. tuberculosis, although this did not translate into increased antibacterial activity of the macrophages since this organism is extremely resistant to lysosomal products (5).

Importantly, the colocalization of LAMP-1 with the H. capsulatum cells opsonized with MAb suggests that more-effective antigen presentation may be occurring. The subcellular fractionation results confirm that increased processing of H. capsulatum-specific products occurs in the presence of MAb 9C7-opsonized yeast cells. Compartments containing LAMP-1 can efficiently process protein antigens into peptides that can be recognized by class II MHC molecules (45, 54). In turn, this could stimulate a more-effective cellular response to the invading fungus, since antigen interaction with class II MHC molecules is important in optimal control of histoplasmosis (9). Effective resistance H. capsulatum requires the activation of T cells (3, 35).

T cells are largely responsible for the induction of cytokines in tissues infected with H. capsulatum (3, 23). Here we demonstrate that opsonization of H. capsulatum increases phagocytosis by macrophages that can efficiently process fungal antigens that induce T-cell proliferation and increased Th1-type cytokines. For instance, the T-cell proliferation assays showed a significant increase in IFN- from macrophages cultured with opsonized yeast. This cytokine is required for protection against lethal histoplasmosis (2, 62). Collaboration between the humoral and cellular immune systems aids host control of disease in additional bacterial, parasitic. and fungal intracellular pathogens, including Burkholderia pseudomallei (26), Toxoplasma gondii (52), Candida albicans (12, 28), and Cryptococcus neoformans (55). It is notable that the T-cell proliferative response was suppressed in macrophages infected with nonopsonized H. capsulatum. We recently reported that macrophages infected with H. capsulatum have increased expression of the costimulatory ligand PD-L1 (33). PD-L1 interacts with PD-1 on T cells in a negative fashion that suppresses T-cell activation (29). The opsonization of H. capsulatum with MAb 9C7 interferes with negative costimulation, albeit incompletely. Hence, our data show that MAb 9C7 increases antigen processing of macrophages (Fig. 6) and reduces negative costimulation (Fig. 8), resulting in improved T-cell function (Fig. 6).

Our studies have begun to elucidate the complex impact of a MAb to the pathogenic fungus H. capsulatum. In our murine macrophage system, the MAb alters the intracellular fate of the fungus by enhancing phagosomal maturation and increasing lysosome-phagosome fusion, which appears to diminish the capacity of H. capsulatum to modify the phagosomal milieu. These effects translate into a reduction in the survival of ingested H. capsulatum yeast cells in macrophages and improved survival of lethally infected mice (42). Phagosomes are structurally complex, being comprised of several hundred proteins (20, 50) that interact with the organelle's contents to effect and define dynamic host responses. Furthermore, there are important differences between responses in human and murine macrophages to H. capsulatum (38-40), and further study of these processes in additional systems for a broader understanding of the effects of the MAbs is needed. Hence, there are numerous avenues open for future studies to pursue that will further define the effects of MAbs on the intracellular fate of H. capsulatum and other microbes.

 
J.D.N. is supported in part by NIH AI056070-01A2, a Wyeth Vaccine Young Investigator Research Award from the Infectious Disease Society of America, and the Einstein MMC Center for AIDS Research (NIH NIAID AI51519). P.C.A. is supported by an Interhemispheric Research Training Grant in Infectious Diseases from the Fogarty International Center (NIH D43-TW007129). E.L.-M. was supported by a postdoctoral fellowship from the Cancer Research Institute.

The authors do not have any conflicts of interest.

 
* Corresponding author. Mailing address: Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: (718) 430-2366. Fax: (718) 430-8968. E-mail: nosanchu{at}aecom.yu.edu

Published ahead of print on 16 May 2008.

Supplemental material for this article may be found at http://ec.asm.org/.

Present address: Department of Microbiology, University of Szeged, Szeged, Hungary.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Eukaryotic Cell, July 2008, p. 1109-1117, Vol. 7, No. 7
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