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. Author manuscript; available in PMC: 2017 Aug 15.
Published in final edited form as: J Immunol. 2016 Jul 1;197(4):1252–1261. doi: 10.4049/jimmunol.1501855

The Membrane Phospholipid Binding Protein Annexin A2 Promotes Phagocytosis and Non-lytic Exocytosis of Cryptococcus neoformans and Impacts Survival in Fungal Infection

Sabriya Stukes 1, Carolina Coelho 2,3, Johanna Rivera 2, Anne E Jedlicka 3, Katherine A Hajjar 4, Arturo Casadevall 1,3
PMCID: PMC5160961  NIHMSID: NIHMS793625  PMID: 27371724

Abstract

Cryptococcus neoformans (Cn) is a fungal pathogen with a unique intracellular pathogenic strategy that includes non-lytic exocytosis, a phenomenon whereby fungal cells are expunged from macrophages without lysing the host cell. The exact mechanism and specific proteins involved in this process have yet to be completely defined. Using murine macrophages deficient in the membrane phospholipid binding protein, annexin A2 (ANXA2), we observed a significant decrease in both phagocytosis of yeast cells and in the frequency of non-lytic exocytosis. Cryptococcal cells isolated from annexin A2-deficient (Anxa2−/−) bone marrow-derived macrophages and lung parenchyma displayed significantly larger capsules then those isolated from wild-type macrophages and tissues. Concomitantly, we observed significant differences in the amount of reactive oxygen species produced between Anxa2−/− and Anxa2+/+ macrophages. Despite comparable fungal burden, Anxa2−/− mice died more rapidly than wild-type mice when infected with C. neoformans, and Anxa2−/− mice exhibited enhanced inflammatory responses, suggesting that the reduced survival reflected greater immune-mediated damage. Together, these findings suggest a role for ANXA2 in the control of cryptococcal infection, macrophage function and fungal morphology.

Introduction

Cryptococcus neoformans (Cn) is an environmental fungus with a complex intracellular pathogenic strategy that is intimately related to its capacity for virulence (1–3). Fungal infection results from the inhalation of aerosolized infectious particles (4). Unlike other intracellular pathogens, C. neoformans can survive and replicate inside an acidic phagosome that undergoes normal maturation (5). The outcome of the interaction between this fungal pathogen and macrophages appears to be a critical determinant of virulence (3, 6–9).

The mechanisms responsible for fungal intracellular survival involve a combination of factors that include: 1) residence in a large phagosome where lysosomal contents are diluted, 2) the presence of powerful antioxidant mechanisms including a large polysaccharide capsule, cell wall-associated melanin that can absorb lysosome-generated oxidants and enzymes, such as superoxide dismutase and laccase, which deactivate microbicidal compounds; and 3) the ability of the fungus to damage the phagosomal membrane allowing efflux of phagosomal contents into the cytoplasm (1, 8, 10–13). However, perhaps the most unusual aspect of the intracellular strategy of this fungal pathogen is its ability to escape the macrophage in a process known as non-lytic exocytosis (14, 15).

Non-lytic exocytosis has been described in vitro for different cell types such as J774 murine macrophage-like cells, primary mouse and human macrophages, Drosophila S2 cells, and amoebae (4, 16–19). Non-lytic exocytosis was shown to occur in vivo and could provide a mechanism by which yeast cells migrating from the lung in Trojan-horse macrophages are released into the circulation to infect the brain by transcytosis of the blood-brain barrier (20, 21). This interaction appears to be a pathogen-driven phenomenon since it requires cryptococcal cell viability and is selectively confined to fungal cells when macrophages ingest both live yeast cells and FITC-labeled beads (15). The phagosome appears to play a crucial role during this process, as phagosome labeling experiments suggest that during non-lytic exocytosis, the entire organelle is expunged, allowing for the release of fungal cells into the extracellular environment (22). Non-lytic exocytosis is under the control of both host and fungal factors, including host actin (22), phagosomal pH (20), fungal SEC14 (23), cytokine stimulation (24), host autophagy (25) and the presence of a capsule on fungal cells (15).

In this study, we investigated the role of the membrane binding protein annexin A2 (ANXA2) during in vitro fungal interactions with macrophages and in cryptococcal pathogenesis. Annexins are a family of calcium-binding proteins that function by bringing cellular membranes in close contact with each other to promote fusion (26). They function in a wide variety of biological processes, some of which include membrane trafficking, phagocytosis, the endocytic pathway, and, of interest to us, exocytosis of secretory vesicles (27–29). Specifically, we focused on ANXA2, as this protein mediates endosomal membrane-membrane fusion and plays a role in the docking mechanism needed for vesicles to adhere to cellular membranes. ANXA2 is also hypothesized to function in the membrane fusion events leading to the release of vesicles in chromaffin cells, exocytosis of lamellar bodies in alveolar epithelial cells, and regulated exocytosis of von Willebrand factor packaged in Weibel-Palade bodies in bovine endothelial cells (30–32). In addition, ANXA2 mediates secretion of vesical-bound macromolecular collagen VI from bronchial epithelial cells (33)

There is minimal information linking ANXA2 and C. neoformans, and what has been shown mostly focuses on the interaction of fungal cells and endothelial cells. Vu et al showed that ANXA2 and S100A10 genes were both up-regulated upon contact and ingestion of fungal cells by brain endothelial cells. This up-regulation translated into a decrease in the integrity of the cellular membrane, suggesting that fungal cells enhanced their transmigration into the brain by compromising the underlying cytoskeleton structure as these binding proteins are known to interact with actin (34, 35).

Due to its functionality and previous research showing a role during regulated exocytosis, we hypothesized that this cellular membrane binding protein could be part of the machinery that enables non-lytic exocytosis of C. neoformans. Our results demonstrate that ANXA2 is involved in multiple cellular processes during the intracellular control of C. neoformans infection, including internalization of fungal cells and non-lytic exocytosis from macrophages.

Materials and Methods

Growth Conditions of Cryptococcus neoformans

A streak from a single colony of C. neoformans strain H99 (serotype A) was grown in Sabouraud dextrose broth (Difco) for 24 h at 30° C with constant agitation. This strain of C. neoformans was originally obtained from Dr. John Perfect (Durham, NC) and has been maintained in the Casadevall laboratory for several years. Unless otherwise specified, yeast cells were washed 3 times in sterile PBS, counted, and used at an effector: target ratio of 5:1 for all in vitro experiments unless otherwise noted.

C57BL/6J and Anxa2−/− Mouse Strains

Homozygous breeding pairs of annexin A2 knockout (Anxa2−/−) mice from a C57BL/6J background were obtained from the Hajjar laboratory (New York, NY) (36) and were maintained at the Animal Institute of Albert Einstein College of Medicine. Male and female C57BL/6J (8–10 weeks old) were obtained from NCI (Fredrick, MD). In all experiments, mice were used at 8–10 wk of age. Mice were housed in sterile microisolator cages in a barrier environment and were maintained in a specific pathogen-free barrier facility in microisolator cages, fed irradiated rodent food, provided with autoclaved bedding, and routinely monitored for serologic evidence of exposure to common murine pathogens.

Isolation of Bone Marrow Derived Macrophages (BMDM) and Peritoneal Macrophages

C57BL/6J and Anxa2−/− mice were used for all primary macrophage experiments. Femurs and tibias from both strains of mice were dissected and all muscle tissues were removed. Intact bones were disinfected by immersion in 70% ethanol for 3 min and rinsed in PBS. Both ends of each bone were cut off and the cells were flushed out using cold Dulbecco’s Modified Eagle’s Media (DMEM)(Corning Cellgro) using a 25 g needle and collected into tubes. The cell suspension was centrifuged for 5 min at 650 × g at room temperature and washed once with cold DMEM. Cell clumps were disrupted by passing the solution through a 70μm cell strainer. Red blood cells were removed by incubation of the cell suspension in ACK lysis solution (150 mM NH4CL, 10 mM KHCO3, 0.1 mM EDTA pH 7.4) for 10 min at room temperature and centrifuged to pellet cells for 5 min at 650 × g. Cells were plated on tissue culture treated petri dishes in growth media (DMEM supplemented with 20% L929 growth conditioning media, 10% Fetal Calf Serum (Atlanta Biologicals) 10% NCTC- 109 (Invitrogen), 1% HEPES (Corning Cellgro), Glutamax (Invitrogen), penicillin-streptomycin (Gibco), Non-essential amino acids (Corning Cellgro), and 0.1% β-mercaptoethanol (Invitrogen)). Fresh media was added after 3 d of growth, and media was again removed and replaced on day 6 of culture. BMDM were collected on day 7 and used for subsequent experiments.

To induce peritoneal macrophage recruitment, mice were injected with 3% sodium thioglycolate 3 days prior to isolation. Peritoneal macrophages were isolated by creating a small incision into the peritoneal cavity and aspirating with 10 ml cold PBS supplemented with EDTA (1 mM) and 1% penicillin/streptomycin (Gibco). Cell suspensions were kept cold and pelleted via centrifugation at 650 × g for 10 minutes. Macrophages were plated at a density of 106 cells/ml and allowed to adhere overnight in DMEM (Corning CellGro) supplemented with 10% fetal calf serum (Atlanta Biologicals) 10% NCTC- 109 (Invitrogen), 1% Penicillin-Streptomycin (Gibco), Non-essential amino acids (Corning Cellgro).

Phagocytosis Assay

BMDM were plated in triplicate on glass bottomed 96-well Matricalplate at a density of 5 × 104 cells/ml and allowed to adhere overnight in 20% L929 feeding media containing 50 U/ml recombinant murine IFN-γ and 10 ng/ml LPS. A cold mixture of fungal cells and 10 μg/ml of mAb 18B7 (IgG1) was prepared and placed on ice. This mAb binds to capsular polysaccharide and is opsonic (37). BMDM were placed on ice and cold fungal cells were added at an effector to target ratio of 2:1. Cells were allowed to sit on ice for 15 min to allow settling of cryptococcal cells. Warm media was then added and phagocytosis was allowed to occur at two time intervals, 0.5 and 2 h, at 37°C in 10% CO2. At each time interval, extracellular fungal cells were removed and macrophages were washed 3 times with PBS, fixed with cold methanol for 30 min at −20°C, and washed 2 times with sterile PBS before adding appropriate dyes as detailed before (38). Briefly, wheat germ agglutinin (WGA) conjugated to Alexa 633 (Invitrogen) was added at 10 μg/ml and incubated overnight at 4°C. Uvitex 2B (Polysciences, Inc., Warrington, PA) was then added at 0.1 μg/ml and incubated for 1 min at room temperature. Subsequently, propidium iodide (Sigma-Aldrich, St. Louis, MO) was added at 5 μg/ml for a total volume of 400 μl per well and analyzed in the propidium iodide solution.

Non-lytic Exocytosis Assay

Non-lytic exocytosis assays were carried out as previously described (16). Briefly, primary bone marrow derived macrophages were plated on mat-Tek glass petri dishes and allowed to adhere overnight in growth media supplemented with 10 μg of LPS and 50 u/ml of IFN-γ. The next day, cells were washed, and live cryptococcal cells opsonized with 10 μg/ml of mAb 18b7 were added to each dish. After a co-infection period of two hours, cells were washed thoroughly with PBS and fresh activating media without mAb 18B7 was added to the dish. A time lapse movie was set up to acquire images every 4 min for 24 h using a Zeiss Axiovert 200 M inverted microscope in an enclosed chamber with conditions at 5% CO2 at 37°C.

Survival Studies

C57BL/6J and Anxa2−/− mice aged 8–10 weeks old were infected intratracheally (IT) as previously described (39) with 105 or 106 C. neoformans. Mice were monitored daily for signs of stress and deterioration of health throughout the experiment.

Capsule Growth Measurement in vitro and in vivo

For in vitro experiments, C57BL/6J and Anxa2−/− bone marrow derived macrophages were plated in 100 mm petri dishes, infected with opsonized C. neoformans as described for non-lytic exocytosis assays and incubated at 37°C for 24 hr. Extracellular fungal cells were washed away, macrophages lysed with water, and cryptococcal cells collected by centrifugation. Capsule size was visualized using India ink and analyzed with an Olympus AX70 microscope at a magnification of 40X. Individual cells were measured using ImageJ software. For each cell, the diameter of the cell body was subtracted from the diameter of the entire cell to determine the capsule radial dimension. For in vivo measurements, mice were infected as described above, sacrificed at 7, 14, 21 and 28 days, and their right lung lobes removed. Lung tissue was homogenized in 1 ml of sterile PBS, and capsule dimensions measured as described above. Left lung lobes were fixed in formaldehyde and sections stained with hematoxylin and eosin for histological studies..

Immunofluorescence Microscopy

BMDM were plated as described for non-lytic exocytosis assays. Macrophage monolayers were infected with C. neoformans at an effector:target ratio of 2:1. After a2 h incubation the extracellular fungal cells were washed away. At specific time intervals thereafter, samples were permeabilized and fixed with methanol at −20°C for 20 minutes. Uvitex 2B was used to highlight internalized fungal cells, and an LC3 rabbit polyclonal antibody (Santa Cruz) was used to determine localization. Cells were visualized using a Zeiss Axio Observer CLEM at 63X objective.

Reactive Oxygen Species (ROS) Measurement

Peritoneal macrophages were infected with C. neoformans and incubated for various time intervals. Extracellular fungal cells were washed away, and the macrophage monolayer stained with media supplemented with CellRox Deep Red Reagent (Life Technologies) for 30 minutes at 37°C. The medium was removed, and macrophages washed three times with PBS. Fluorescence was measured using a Becton Dickinson LSRII (BD Biosciences).

Quantitative RT-qPCR

Anax2+/+ and Anxa2−/− mice infected with C. neoformans were sacrificed at days 14 and 21 post infection. Lung and brain were removed and immediately placed in RNAlater (Ambion, Austin, Tx). RNA from 10 mg of tissue was extracted with Rneasy Mini Plus kit (Qiagen, Hilden, Germany). RNA concentration and integrity was checked by High Sensitivity Screen Tape on Agilent TapeStation 2200 and Nanodrop®. RNA Integrity Numberfor brain was above 7.5 for all samples, while for lung samples it ranged from 2 to 8. For cDNA preparation, 250 μg of RNA in a total volume of 20 μL was added to cDNA EcoDry Premix with Random Hexamers (Clontech, Mountain View, CA). Primers (Supplemental Table 1) were designed to span an exon-exon junction and qPCR was performed with Sybr Green Master Mix (ABI, Warrington, UK) in a StepOne instrument (ABI, Warrington, UK) by performing a cycle of 95°C for 10 min, followed by 40 cycles of 15s at 95°C, and 1 min at annealing temperature. Analysis was performed in StepOne Software v2.3 with automatic threshold and Cq determination. Cq values were exported to Prism, and the sample target gene level was calculated by normalizing expression level to the arithmetic mean of the levels of Actin and TataBox Binding Protein as reference genes. Fold change was calculated using d14 WT1 as reference. Statistical significance was calculated from expression levels using an unpaired two-way ANOVA with Sidak post-correction on Graph Pad Prism for MacOS X v6.0c.

Cytokine Studies

Anxa2+/+ and Anxa2−/− mice (n = 6 per group) were infected with C. neoformans as described previously, and sacrificed at 14 and 21 days post infection. The lungs were homogenized in 2 ml of PBS in the presence of protease inhibitors (Complete Mini; Roche Applied Science). Homogenates were centrifuged at 2000 × g for 10 min to remove cell debris, and the supernatant frozen at −80°C until tested. Supernatants were assayed using mouse cytokine protein array C3 (Ray Biotech, Norcross, GA), per the manufacturer’s instructions. Briefly, membranes were blocked with 1× blocking buffer, washed three times, and then incubated with samples. Membranes were washed again and incubated for 1 h with biotin-conjugated cytokines, which were detected by incubation with HRP-conjugated streptavidin. Unbound reagents were removed by washing and the membranes developed. All incubations were done at 37 °C for 1 h.

Histology

Lung tissue sections from infected Anxa2+/+ and Anxa2−/− mice were stained for arginase. Sections were deparaffinized in xylene followed by graded alcohols. Antigen retrieval was performed by incubating sections in 10 mM sodium citrate buffer (pH 6.0), and heated to 96 °C for 20 min. Endogenous peroxidase activity was quenched using 3% hydrogen peroxide in PBS for 30 min at room temperature. Sections were blocked with 5% BSA in PBS for 1 h. The primary antibody to arginase (Abcam Inc, Cambridge, MA) was used at 1:50 for 1 h at room temperature. Sections were stained by routine immunohisthochemistry methods using goat anti-mouse HRP conjugate (Southern tech) to localize the antibody bound to antigen with diaminobenzidine as the final chromogen. All immunostained sections were lightly counterstained with hematoxylin.

Statistical analysis

Statistical analyses were performed using Graph Pad Prism V6. Tests included Student’s two tailed t-test and unpaired two-way ANOVA with Sidak post-correction.

Results

Anxa2−/− macrophages are less efficient at phagocytosis

To determine the cellular processes affected by the absence of ANXA2, we examined one of the first steps of an intracellular infection, namely internalization of fungal cells. Using BMDM macrophages in conjunction with a previously described staining protocol optimized for light scanning cytometry (38), we could readily distinguish macrophages that had internalized yeast cells from those without fungi (Figure 1A). We observed a small but significant decrease in phagocytosis by Anxa2−/− macrophages at both time intervals compared to Anxa2+/+ macrophages (Figure 1B). At 30 minutes, 30% of Anxa2−/− macrophages remained uninfected, while only 20% of wild-type macrophages contained no fungal cells (Figure 1C). At 2 h, the difference decreased with 20% of the Anxa2−/− macrophages containing no fungal cells (Figure 1D). Therefore, Anxa2−/− macrophages could phagocytose IgG-opsonized C. neoformans cells but Anxa2−/− macrophages had phagocytic defects compared to wild-type macrophages during early stages of internalization.

Figure 1. Efficiency of phagocytosis by Anxa2+/+ and Anxa2−/− murine macrophages.

Figure 1

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(A) Macrophages were stained with propidium iodide (red, nuclear stain). Fungal cells were stained with Uvitex2B (cyan, chitin-binding for the fungal cell well) and analyzed as in (4, 16, 17, 31). Uninfected macrophages (white arrow) could be readily distinguished from macrophages that had internalized fungal cells (black arrow). (B) Percent phagocytosis was determined by calculating the number of macrophages that had internalized Cn divided by the total number of macrophages counted. (C, D) Number of Cn cells ingested by macrophages at (C) 30 min and (D) 2 hours. Each bar represents 25 fields of cells counted in 5 separate wells with an average of 1,000 cells counted per well. Each xperiment was performed 3 times. The data are shown as mean +/- SEM with the statistical significance indicated by **** (p < 0.0001) or ** (p< 0.001) using Student’s two tailed t-test.

Absence of ANXA2 in bone marrow derived macrophages reduces non-lytic exocytosis

We then analyzed whether the absence of ANXA2 had an effect on the frequency of non-lytic exocytosis. As previously described (16), time lapse movies of multiple infected macrophages were analyzed to determine the outcome of the C. neoformans-macrophage interaction. For each movie, all the cells in a field were categorized into three groups: (a) no change, indicating that the cryptococcal cells remained in the macrophage and there was no release of the fungal burden (data not shown), (b) cell lysis, in which the macrophages burst releasing the cryptococcal cells into the extracellular environment effectively killing the host cell, or (c) non-lytic exocytosis, whereby cryptococcal cells were released from the confines of the macrophage leaving both host and pathogen alive. Anxa2−/− macrophages were eight times more likely to lyse during exocytosis of cryptococcal cells than wild type macrophages, and had on average of 20% fewer non-lytic exocytosis events (p <0.008) (Figure 2). This phenomenon was specific to macrophages containing live fungal cells, because neither uninfected Anxa2−/− macrophages nor macrophages infected with beads or heat-killed C. neoformans underwent lysis (data not shown).

Figure 2. Lytic exocytosis is increased while non-lytic exocytosis is decreased in Anxa2−/− macrophages.

Figure 2

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Primary macrophages were infected with fungal cells for two hours, after which extracellular fungal cells were washed away and video acquisition was initiated with images taken at 4-minute intervals for a total of 24 hours.. Events were placed into two categories: Cellular Lysis, and Non-Lytic Exocytosis. Approximately 100–300 events were counted from five experiments with the statistical significance indicated by * (p<0.04 or ** (p<0.008) using Student’s two tailed t-test.

Our previous work categorized the process of non-lytic exocytosis into three subcategories based on both the number of fungal cells released, and the manner in which they were extruded (16). In Type I release, complete emptying of fungal cells from the macrophage occurred, whereas, in Type II release, partial emptying of fungal cells from the macrophage was noted. In Type III release, transfer of fungal cells between individual macrophages was observed. Anxa2−/− macrophages displayed all three types of non-lytic exocytosis at rates and frequencies that were similar to those observed in Anxa2+/+ macrophages, indicating that, although ANXA2 had an effect on the overall process, it was not specific for any single type of non-lytic exocytosis (data not shown).

Cryptococcus neoformans capsule is enlarged in Anxa 2−/− deficient macrophages

In our in vitro microscopy experiments, we observed that after 24 h yeast cells inside bone marrow derived Anxa2−/− macrophages had larger capsules than yeast cells found within Anxa2+/+ macrophages (Figure 3A). To explore this phenomenon, bone marrow derived macrophages from wild type and Anxa2−/− mice were infected in vitro for 24 h and then lysed to recover intracellular organisms. Fungal cells recovered from Anxa2−/− macrophages had significantly larger capsules than those recovered from Anxa2+/+ macrophages (p < 0.0001) (Figure 3B). When individual capsule sizes were categorized into groups according to their individual capsule size, those associated with wild type macrophages were predominantly in the range of 0.5–1.5 μM, while those associated with Anxa2−/− macrophages were predominantly 1–3 μM in size (Figure 3C). To ascertain whether this phenomenon also occurred in vivo, we infected mice intratracheally, and analyzed lung fungal burden 7 and 14 days after infection. Lungs were excised and homogenized to isolate and measure capsule size. We observed a significant increase (p < 0.0002) in capsule thickness at days 7 and 14 (Figure 4A), which translated into an increase in capsule volume (data not shown). The largest difference between the two murine environments occurred at day 7 (Figure 4B). C. neoformans capsule sizes in Anxa2−/− mice were comparable between days 7 and 14 suggesting that the capsule size enlargement occurred earlier in Anxa2−/− than in wild type mice.

Figure 3. C. neoformans cells co-incubated in vitro with Anxa2−/− macrophages for 24 hours show enlarged capsule sizes.

Figure 3

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(A) Microscopic analysis of Cn capsule size revealed smaller capsule size in yeast cells associated with Anxa2+/+ macrophages compared to yeast cells associated with Anxa2−/− macrophages. (B) Cn capsule size is smaller in yeast cells isolated from Anxa2+/+ macrophages. Infected macrophages were lysed, and capsule size calculated by from the diameter of the whole cell minus the diameter of the inner cell body (n=200). Statistical significance is represented by **** (p < 0.0001), and calculated using Student’s two tailed t-test. (C) Distribution of capsule sizes for each genotype.

Figure 4. Fungal capsule is enlarged in Anxa2−/− lung tissue on days 7 and 14.

Figure 4

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Anxa2+/+ and Anxa2−/− mice were infected intratracheally and sacrificed at day 7 and 14. (A) Fungal cells with varying capsule sizes can be seen at day 14 for Anxa2+/+ macrophages, whereas Anxa2−/− macrophages show cells that have a larger, more uniform size. (B) Capsule thickness was quantified for both time points, and a significant difference between the two mouse strains was observed. *** (p < 0.0002; using Student’s two tailed t-test).

We then evaluated the lung histology of Anxa2−/− and Anxa2+/+ mice infected with C. neoformans. Lung histology at 7, 14, 21 and 28 d showed a striking difference in the capsule size for cells recovered from the two strains of mice. The lungs of Anxa2−/− mice harbored large collections of extracellular organisms with large capsules starting at day 14 and continuing until day 28. In contrast, yeast cells in lungs of Anxa2+/+ mice showed enlarged capsules only at later time points in the infection (Figure 5). At day 14, Anxa2+/+ mice exhibited granulomatous infiltrates composed of macrophages with some polymorphonuclear cells (PMN), primarily eosinophils. By day 21, the response was more organized and more lymphocytes were present. At day 7, Anxa2−/− mice exhibit minimal inflammation with large collections of C. neoformans cells within alveolar spaces. By day 14, there was an increase in inflammatory cell infiltrate composed of macrophages and PMNs. Perivascular cuffing was present, but not as organized compared to Anxa2+/+ mice and did not improve with time. Examination of the lungs of Anxa2+/+ and Anxa2−/− mice at day 28 revealed large collections of yeast cells within the alveolar space with significant damage to the lung. This phenotype did not translate into differences in fungal burden, as no statistical difference was observed when CFU’s were quantified from the lung, brain and liver of infected mice at all-time s intervals (Supplemental Figure 1).

Figure 5. Anxa2−/− mice show increased inflammation, and contain fungal cells with large capsule sizes following intratracheal infection with C. neoformans.

Figure 5

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Anxa2+/+ mice have a granulomatous inflammatory response to yeast cells with infiltration of macrophages and eosinophils. By day 21, the response is more organized and there are more lymphocytes present. In Anxa2−/− mice, some macrophages, eosinophils are present along with perivascular cuffing are present at day 7, but granuloma formation is not as organized as in Anxa2+/+ mice. By days 21 and 28, the lungs are filled with yeast cells, and lung architecture is significantly damaged, in both genotypes. Original magnification = 40×.

Reactive oxygen species (ROS) production and LC3 recruitment to phagosomes

Given that free radicals can shave C. neoformans capsules (40) and that ANXA2 also functions as a cellular redox regulatory protein (41), we evaluated the production of oxidative bursts in Anxa2−/− and Anxa2+/+ peritoneal macrophages. ROS production by these macrophages did not differ dramatically throughout the infection in either genotype, showing only a small statistical difference for uninfected cells (Figure 7). However, when quantifying the total amount of ROS produced, we observed that Anxa2−/− macrophages had reduced production compared to Anxa2+/+ macrophages. Overall, these results show that the yield of reactive oxygen species from Anxa2−/− peritoneal macrophages is lower than wild type macrophages.

Figure 7. Reactive oxygen species production in infected peritoneal macrophages.

Figure 7

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Reactive oxygen species (ROS) produced by thyoglicolate-induced peritoneal macrophages were measured for both uninfected and infected macrophages using CellRox Deep Red Reagent. (A) Macrophages of the two genotypes displayed similar amounts of ROS at baseline. (B) Peritioneal macrophages were infected with opsonized C. neoformans, which led to an an increase in ROS. Values represent combined results from two independent experiments. * p <0.05, ** < 0.01 using a two-tailed Student t test.

In addition, we examined infected phagosomes of bone marrow derived macrophages at different time intervals using two different markers, the phagosomal marker LAMP-1, and the autophagy marker LC3-II, as well as the acidification dye LysoTracker. We observed no difference between LAMP-1 localization or the degree of phagosomal acidification between the two genotypes (data not shown). However, we did observe that fewer phagosomes accumulated LC3 at later times in the infection in Anxa2−/−macrophages (Figure 6A). Based upon LC3 fluorescence around individual internalized fungal cells, we observed a significant reduction in LC3 recruitment for yeast cells within Anxa2−/− macrophages (Figure 6B).

Figure 6. LC3 localization in Cn phagosomes is decreased in Anxa2−/− macrophages.

Figure 6

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(A) Primary murine macrophages were infected with C. neoformans stained with Uvitex2B (shown here in blue). After 2 hours of phagocytosis, extracellular yeast cells were washed away, and macrophage monolayers stained for LC3 localization (shown in green) at the indicated time intervals. (B) Fluorescence of internalized fungal cells with LC3 localization was quantified using Image J software. The experiment was repeated three times and statistical significance assessed for each time point using Student’s two-tailed t test (* p = 0.0103, ** p=0.0032, **** p < 0.0001).

Mice deficient in ANXA2 show decreased survival when infected with C. neoformans

It is known that the capsule of C. neoformans exerts immunomodulatory effects on host cells. Given the histological differences observed between Anxa2+/+ and Anxa2−/− mice, we evaluated survival after fungal infection in the two genotypes. When infected with 106 Cn/mouse, Anxa2−/− mice exhibited a non-significant trend towards susceptibility to infection (p < 0.17) (Figure 8A). When we repeated this experiment at lower inocula, (105/mouse), Anxa2−/− mice showed increased susceptibility to cryptococcal infection compared to wild type controls (p <0.05) (Figure 8B). Given the various processes affected by the absence of ANXA2, our results suggest a working model that implicates a multi-functional role for ANXA2 during murine infection with C. neoformans (Figure 9).

Figure 8. Survival of Anxa2−/− mice is decreased when infected with C. neoformans (Cn).

Figure 8

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Mice (n=10) were infected intratracheally with varying doses of C. neoformans. (A) Survival when mice were infected with 106 Cn (B) Survival when mice were infected with 105 Cn. (p < 0. 05). The experiment was terminated when the last Anxa2−/− mouse died.

Figure 9. A multi-functional role of annexin A2 during a fungal infection: a model.

Figure 9

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The depletion of this membrane phospholipid-binding protein affects several aspects of host cell function when controlling a fungal infection.. The absence of annexin A2 causes an initial decrease in the number of fungal cells internalized. Once ingested, fungal capsule size is increased within these phagosomes, as the time of infection progresses. Knockout macrophages also exhibit differences in their phagosomal environment, as there is a reduction in both LC3 recruitment and ROS production. Overall, there is a decrease in non-lytic exocytosis and an increase in cellular lysis due to the host cell’s inability to contain the fungal cells.

To investigate the contribution of macrophage ANXA2 to decreased host survival, we performed lung and brain qPCR and arginase immunostaining (Supplemental Fig. 1), as well as cytokine arrays (Supplemental Figure 2). We found that neither total lung gene expression nor CFU counts changed significantly between Anxa2+/+ and Anxa2−/− animals. However, we did observe that Anxa2−/− mice demonstrated a widespread increase in inflammatory cytokines such as IL12p40/p70 and XCL-1, without a discernible shift of Th1 vs Th2 polarization when compared to Anxa2+/+ animals. We found that the Arg1 expression in the lungs of infected Anxa2−/− mice was delayed in comparison with Anxa2+/+ animals. Therefore, the presence or absence of ANXA2 does not alter mouse control of yeast burden, but instead affects cytokine signaling. Its absence results in inflammatory dysregulation relative to what is observed in the wild type mice.

Discussion

In this study, we show that several specific aspects of the host response to fungal infection with Cryptococcus neoformans are deficient in macrophages lacking ANXA2, a phospholipid binding protein that promotes intracellular membrane fusion. A role for ANXA2 during phagocytosis was previously suggested based on the observation that this protein localized to the phagocytic cup during ingestion of photoreceptor outer segments in retinal pigment cells (42). In our system, the absence of this protein caused a subtle defect in the number of fungal cells that macrophages are able to phagocytose in the early stages of infection. However, because overall phagocytosis by Anxa2−/− macrophages was comparable to wild-type macrophages, Anxa2−/− macrophages may compensate for this subtle phagocytic defect by other mechanisms, possibly including the recruitment of other annexins.

Since deficiency of ANXA2 did not abrogate the release of fungal cells into the extracellular environment, other annexins may have provided redundancy for the missing protein, as macrophages contain multiple annexins with similar structures and functions. In contrast to wild-type macrophages, infected Anxa2−/− macrophages were more likely to undergo lytic exocytosis. Annexins are also involved in the cellular membrane repair pathway, specifically during Listeria monocytogenes cell-cell infection of primary murine macrophages, as ANXA2 is recruited to bacterial vacuoles filled with phosphatidylserine to promote membrane repair (43, 44). Based on this insight, it is possible that the absence of ANXA2 could inhibit the macrophage’s ability to repair the physical disruption that may occur between membranes during exocytosis leading to lysis of host cells.

The molecular mechanisms that drive non-lytic exocytosis are unknown, but they could be similar to those known to underlie some elements of regulated exocytosis, including vesicle docking, membrane fusion and subsequent release of secretory vesicles. Even though annexins are not fusogenic proteins, they interact with SNAPs (soluble NSF attachment proteins), membrane-binding proteins that merge membranes together to facilitate fusion. The phagosome formation and maturation process is regulated by SNAP-23, and the interaction between SNAP-23 and ANXA2 is crucial for exocytosis of lung surfactant from alveolar epithelial cells (45, 46), as well as secretion of large, multimeric, membrane-enclosed proteins such as collagen VI and von Willebrand factor (33, 47). Hence, it is conceivable that ANXA2 forms a complex with SNAP-23 located on fungal phagosomes, bringing them in close contact with the cellular membrane. In our system, ANXA2 appears critical for controlling of C. neoformans infection, since the number of macrophages that lysed was increased greatly over the period studied. Since we also observed a decrease in non-lytic exocytosis, this could suggest that the mechanism of non-lytic exocytosis is protective for the macrophage in the sense that the host cell is more likely to survive the interaction with C. neoformans. In addition, interference with autophagy is associated with reduced non-lytic exocytosis and the reduction in LC3 recruitment observed in Anxa2−/− macrophages could be an additional mechanism contributing to this effect (25).

An unexpected finding was the observation that co-incubation of fungal cells with Anxa2−/− macrophages was associated with a significant increase in capsule size both in vitro and in vivo. This finding is consistent with, and complementary tothe report that when ANXA2’s subcellular ligand, S100A10, is down-regulated in murine brain endothelial cells, there is both a decrease in phagocytosis and significant increase in the capsule size of cryptococcal cells (48). The mechanism for the enlarged capsule phenotype is unknown but it is conceivable that it is related to lower ROS in Anxa2−/− macrophages and deficient recruitment of LC3 to the phagosome when compared to wild-type macrophages. Phagocytosis of fungal cells is accompanied by an oxidative burst and free radicals can shave outer parts of the cryptococcal capsule and reduce its apparent size (40). ANXA2 has also been reported to play a role in modulating cellular oxidative damage (49). In this scenario, the larger capsules in ANXA2- and S100A10-deficient cells could reflect the fact that there is reduced damage to fungal cells from weaker oxidative fluxes in Anxa2−/− phagolysosomes, thus allowing them to express larger capsules. An increase in capsule size could also offer protection by making fungal cells less susceptible to killing, by buffering ROS employed by macrophages to kill intracellular fungal cells (50). Furthermore these data combined with the recent discovery that ANXA2-deficient dendritic cells show a reduction in autophagic flux (51) would suggest that Anxa2−/− macrophages are presumably at an increased risk for cellular damage that, over time, could translate into impaired phagolysosomal function. Variations in both the cytosolic and phagosomal environments could allow for the dramatic increase in fungal capsular size and therefore facilitate its damage to the host cell. Presumably, if yeast cells with large capsules are released into the extracellular environment, they would also be less likely to be taken up by other phagocytes as the larger capsules reduce the efficiency of phagocytosis (52). Therefore, it is possible that ANXA2 and S100A10 are involved in a pathway that influences C. neoformans capsule growth. Our initial hypothesis was based on the assumption that ANXA2 might function to bring the phagosomal membrane in close proximity to the cellular membrane to initiate the key steps of non-lytic exocytosis. While that hypothesis could still hold true, our results hint at another non-exclusive possibility for a reduction in non-lytic exocytosis: depletion of the protein causes an enlargement of the polysaccharide capsule which hinders the release of the fungal cells and could cause cellular stress such that the macrophage lyses. The amount of capsule needed for non-lytic exocytosis to occur could be thought of as a “Goldilocks volume,” whereby too much capsule hinders non-lytic exocytosis, as does too little because acapsular fungal cells residing within macrophages are not readily exocytosed.

Our in vivo data provides further support for the notion that ANXA2 plays an important role in controlling a fungal infection as Anxa2−/− mice showed reduced survival after infection. We did not observe a noticeable difference in the types of inflammatory cells recruited to fungal granulomas, but did see changes in the structural organization of the granulomas. It is possible that having enlarged capsules in lung tissues could affect granuloma formation, and translate into reduced mouse survival since cryptococcal polysaccharide causes numerous deleterious effects on host immunity (53). Anxa2−/− mice infected with C. neoformans displayed an overall increase in cytokine levels in the tissue and a delayed activation of arginase, but had comparable fungal tissue burden in both the lung and the brain. We hypothesize that the increased susceptibility of Anxa2−/− mice reflects an overwhelming or dysregulated inflammatory response, which translates into increased tissue damage and reduced survival, as posited by the damage-response framework (54). It is most likely that ANXA2 plays a role in the macrophage/C. neoformans interaction, possibly at the macrophage lysosome level and the brain endothelial transmigration level. However, it may concomitantly affect the biology of other immune cells. In this regard, and consistent with our observations, ANXA2 has been shown to be involved in the regulation of cytokine responses (55, 56). Overall, our data indicate that ANXA2 and associated proteins are important molecules used in defense against fungal infections, and that further experiments need to be performed.

In summary, our results implicate a role for the membrane fusion protein, ANXA2, in non-lytic exocytosis and other processes that occur in the macrophage during fungal infection. ANXA2 contributes to phagocytic efficiency, and its absence could reduce the macrophage’s ability to control a fungal infection. We show that the presence and absence of ANXA2 is associated with differences in fungal capsule size within the confines of the macrophage, a phenomenon that could impact non-lytic exocytosis. Given that annexins are expressed in a wide variety of cells and that non-lytic exocytosis has been observed in different cell types and for various pathogens, it is likely that this protein has protean roles in host-fungal interactions in different settings.

Supplementary Material

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Acknowledgments

Funding Information

This research was funded in part by National Institutes of Health awards 5R01A1033774, 5R37AI033142, and 5T32A107506. This work was also supported in part by CTSA grants 1 ULI TR001073-01, 1 TLI 1 TR001072-01, and 1 KL2 TR001071 from the National Center for Advancing Translational Sciences (NCATS).

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