An Extracellular Matrix–Based Mechanism of Rapid Neutrophil Extracellular Trap Formation in Response to Candida albicans

Angel S. Byrd, Xian M. O’Brien, Courtney M. Johnson, Liz M. Lavigne and Jonathan S. Reichner
J Immunol April 15, 2013, 190 (8) 4136-4148; DOI: https://doi.org/10.4049/jimmunol.1202671

Abstract

The armament of neutrophil-mediated host defense against pathogens includes the extrusion of a lattice of DNA and microbicidal enzymes known as neutrophil extracellular traps (NETs). The receptor/ligand interactions and intracellular signaling mechanisms responsible for elaborating NETs were determined for the response to Candida albicans. Because the host response of extravasated neutrophils to mycotic infections within tissues necessitates contact with extracellular matrix, this study also identified a novel and significant regulatory role for the ubiquitous matrix component fibronectin (Fn) in NET release. We report that recognition of purified fungal pathogen-associated molecular pattern β-glucan by human neutrophils causes rapid (≤30 min) homotypic aggregation and NET release by a mechanism that requires Fn. Alone, immobilized β-glucan induces reactive oxygen species (ROS) production but not NET release, whereas in the context of Fn, ROS production is suppressed and NETs are extruded. NET release to Fn with β-glucan is robust, accounting for 17.2 ± 3.4% of total DNA in the cell population. Release is dependent on β-glucan recognition by complement receptor 3 (CD11b/CD18), but not Dectin-1, or ROS. The process of NET release included filling of intracellular vesicles with nuclear material that was eventually extruded. We identify a role for ERK in homotypic aggregation and NET release. NET formation to C. albicans hyphae was also found to depend on β-glucan recognition by complement receptor 3, require Fn and ERK but not ROS, and result in hyphal destruction. We report a new regulatory mechanism of NETosis in which the extracellular matrix is a key component of the rapid antifungal response.

Introduction

Polymorphonuclear leukocytes (PMNs, neutrophils) are innate immune cells responsible for host defense against opportunistic fungal infections such as Candida albicans. Patients who have a reduction in the number of peripheral blood PMNs are at risk for acquiring invasive candidiasis, a nosocomial infection that is associated with 40–80% mortality and causes ∼10,000 deaths per year in the United States (1–6). Neutrophil antimicrobial defense mechanisms are well studied and include phagocytosis, production of reactive oxygen species (ROS) (7–9), production of antimicrobial peptides (10), degranulation, and extrusion of neutrophil extracellular traps (NETs) (11). NET production, or NETosis, is a cell death pathway that involves the destruction of the cytoplasmic membrane by a process that is different from apoptosis or necrosis, releasing NETs from within the cell (11–14). NETs are a combination of DNA fibers and granular enzymes (15) such as elastase and myeloperoxidase (5, 16, 17). These fibers immobilize extracellular organisms, including fungi (18), and are able to induce effective death of the microbe owing to the high central concentration of antimicrobial molecules (15). In the context of invasive fungal disease, NET release plays an important role in antifungal defense (16, 18). It has previously been shown that NET extrusion is dependent on the respiratory burst, which has been reported to correlate with time points beyond 1 h after PMA stimulation (11, 13, 15, 17, 19). Additionally, rapid extrusion of NETs in response to Staphylococcus aureus has been reported to occur by an oxidant-independent mechanism (20).

Pathogenic fungi such as C. albicans undergo a dimorphic switch between cellular and hyphal forms (18). Whereas blastoconidia can be cleared by phagocytosis, hyphal forms are too large to be internalized, obviating this signature mechanism of antimicrobial host defense (16). In this regard, NET release provides the host with an effective, extracellular antifungal defense. The key receptor/ligand recognition events required for neutrophilic elaboration of NETs in response to fungal pathogens is not known and is the subject of this study.

β-glucan, a class of long-chain polymers of glucose, is a major structural component of the fungal cell wall that allows neutrophils to recognize fungi without the need for opsonization and, as such, is a pathogen-associated molecular pattern (PAMP) (21–24). The β2 integrin complement receptor 3 (CR3, αmβ2, Mac-1, CD11b/CD18) is a receptor for fungal β-glucan recognition by human PMNs (21–27). Leukocyte adhesion deficiency patients that lack CR3 and other β2 integrins have recurrent localized and systemic candidiasis (28). Therefore, we hypothesized that CR3 may also play an important role in fungal-stimulated NET release.

Antimycotic immune responses occur within infected tissues, necessitating contact of extravasated PMNs with matrix components. Prior work from our laboratory identified a regulatory effect of extracellular matrix (ECM) that modifies the CR3-dependent response to fungal β-glucan (23, 29). In this regard, presentation of β-glucan to neutrophils migrating on fibronectin (Fn) induced a conversion from random to directed migration to otherwise suboptimal concentrations of fMLF or IL-8, extending the range of chemoattractant detection and driving cell navigation to a point source (26, 29). In a previous report that is of particular relevance to the present study, we showed that neutrophils undergo a robust respiratory burst to immobilized, purified fungal β-glucan, which is actively suppressed by ECM (23). This suppression was hypothesized to limit consequent tissue damage of migrating neutrophils until multifocal contact with hyphae is established. In this study, we tested the hypothesis that ECM plays a regulatory role in fungal NET formation under conditions that we showed previously prevent generation of a respiratory burst. We now demonstrate that CR3 recognition of fungal β-glucan results in homotypic cell aggregation and rapid NET release, but only when β-glucan is presented to PMNs together with matrix. We thereby report a requirement for ECM, but not ROS, in mediating the rapid NET formation to purified, immobilized β-glucan as well as to the β-glucan expressed in the cell wall of C. albicans hyphae.

Materials and Methods

Reagents

Abs used were as follows: anti–phospho-44/42 MAPK (ERK 1/2) (Thr202/Tyr204), anti-total 44/42 MAPK (ERK1/2), and anti–phospho-tyrosine (p-Tyr-100) (Cell Signaling Technology, Danvers, MA); activating anti-CD11b (VIM12 F(ab′)2) (Caltag Laboratories, Burlingame, CA); blocking anti-CD11b clone 44 abc, hybridoma from American Type Culture Collection; blocking Ab to human Dectin-1, GE2 (AbCam, Cambridge, MA). Dulbecco’s PBS, Lebovitz’s L15 medium (L-15), RPMI 1640, Sytox Green, 2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), and IL-8 were from Invitrogen (Carlsbad, CA). PicoGreen dsDNA quantitation reagent was from Molecular Probes (Eugene, OR). TNF-α was from R&D Systems (Minneapolis, MN). Medium 199 with Earle’s balanced salt solution, l-glutamine, and 25 mM HEPES was from Lonza (Walkersville, MD). Pharmaceutical-grade purified, endotoxin-free, soluble yeast β-glucan (ImPrime PGG), anti-glucan mAb (clone BFDiv), and whole glucan particles (WGPs; Wellmune) isolated from Saccharomyces cerevisiae were provided by Biothera (Eagan, MN). The β-glucan preparation contained <0.02% protein, <0.01% mannan, and <1% glucosamine. Human Fn was from BD Biosciences (Bedford, MA). All other reagents were from Sigma-Aldrich unless otherwise noted.

Neutrophil isolation

Blood was obtained from healthy human volunteers with approval of the Rhode Island Hospital Institutional Review Board. Blood was collected in EDTA-containing Vacutainer tubes (BD Biosciences, San Jose, CA) and used within 5 min of venipuncture. Histopaque-1077 was used for initial cell separation followed by sedimentation through 3% dextran (400–500 kDa molecular mass). Contaminating erythrocytes were removed by hypotonic lysis, yielding a >95% pure neutrophil preparation of >90% viability by trypan dye exclusion. Neutrophils were suspended in HBSS (without Ca2+/Mg2+) and placed on ice until use.

Neutrophil adhesion

Six-well tissue culture plates (Falcon Labware, Becton Dickinson) were coated overnight with purified, endotoxin-free human Fn at a concentration of 6 μg/ml in TBS (25mM Tris [pH 7.2], 150 mM NaCl) pH 9.0 and/or 1 mg/ml β-glucan and air-dried. Neutrophils were resuspended to a concentration of 3.5 × 106 cells/ml in L-15 medium supplemented with 2 mg/ml glucose and 2 ml was added to each well. Where indicated, cells were preincubated with 20 μg/ml blocking Ab or isotype control, U0126 (50 μM) or PD 98059 (30 μM) (Calbiochem/EMD Chemicals, San Diego, CA), or diphenyleneiodonium (DPI; 6.25 μM) on ice for 20 min. Cells were pretreated on ice with fMLF (10−9 M) for 20 min and/or 1 mM Mn2+ was added to cells immediately before plating.

Microscopy

Images were captured using a Nikon TE-2000U inverted microscope (Nikon, Melville, NY) coupled to an iXonEM+ 897E back illuminated EMCCD camera (Andor, Belfast, U.K.) outfitted with a Bioptechs (Butler, PA) stage heater and ×10, ×20, and ×40 Nikon Plan Apochromat objectives. Differential interference contrast (DIC) images were captured using Elements program (Nikon). For fluorescence microscopy, a xenon lamp illuminated cells through a 33-mm ND4 filter and ×10, ×20, and ×40 Nikon Plan Apochromat objectives using a Nikon B2-A long pass emission filter set cube.

Confocal microscopy was performed at the Brown University Leduc Bioimaging Facility. Z-stacks of both DIC and fluorescence images were acquired with a z-step of 0.9 μm, using a Zeiss LSM 710 confocal laser scanning microscope equipped with a 34-channel Quasar detector running ZEN 2009 software (Carl Zeiss, Jena, Germany). The confocal module was mounted on a Zeiss Axio Observer Z1 inverted microscope outfitted with 37°C heated stage and ×20 Plan Apochromat objective (numerical aperture, 1.2). Sytox Green was visualized using 488-nm laser excitation at 25% and 500 gain. Fluorescence images were imported into AutoQuant v.9 software (MediaCybernetics, Bethesda, MD) as a z-stack series of images and processed using a blind deconvolution algorithm with 20 iterations. Composite images of DIC and deconvoluted fluorescence sum projections, as well as z-stack video reconstructions, were generated using ImageJ v.1.41o (National Institutes of Health, Bethesda, MD).

Western blot analysis

Adherent neutrophils were scraped and centrifuged at 700 × g for 5 min. Pellets were lysed using RIPA buffer (100 mM Tris-HCl, 150 mM NaCl, 1% deoxycholic acid, 1% Triton X-100, and 0.1% SDS with protease and phosphatase inhibitors) and subjected to 10–12% SDS-PAGE gradient gels and transferred to nitrocellulose membranes. Membranes were blocked in TBST (25 mM Tris [pH 7.2], 150 mM NaCl, 0.1% Tween 20) with 3% non-fat dry milk for 1 h at room temperature and incubated overnight at 4°C with anti-pERK1/2 (Thr202/Tyr204) (1:1000), anti-total ERK (1:1000), or anti-actin (1:5000) Ab in blocking buffer. Thereafter, membranes were washed and incubated with peroxidase-conjugated anti-mouse or anti-rabbit IgG (1:10,000) in blocking buffer. A chemiluminescence kit (Thermo Fisher Scientific, Philadelphia, PA) was used to detect HRP. Relative intensities of immunoreactive bands were quantified by scanning densitometry using ImageJ v.1.41o analysis software (National Institutes of Health).

Visualization of PMN NETs

Neutrophils were adhered as previously described to Fn with and without β-glucan–coated plates. After aggregation, 150 U/ml DNaseI (Promega, Madison, WI) was added when indicated. NETs were visualized on adherent PMNs by addition of 5 μM Sytox Green.

C. albicans (SC5314, American Type Culture Collection) was cultured overnight at 37°C with agitation in yeast extract/peptone/dextrose medium consisting of 1% yeast extract, 2% bactopeptone (both from Difco), and 2% dextrose. Coverslips/culture dishes (MatTek, Ashland, MA) were coated with 40 μg/ml Fn or poly-l-lysine in TBS (pH 9.0) overnight at room temperature. C. albicans was added onto precoated coverslips/culture dishes and incubated in Medium 199 supplemented with Earle’s balanced salt solution, l-glutamine, and 25 mM HEPES to induce differentiation into hyphae at 37°C for 4–5 h. Filamentous phenotype was confirmed by light microscopy. Coverslip was placed in a Delta T dish (Bioptechs, Butler, PA) and neutrophils added. When indicated, anti-glucan mAb (BFDiv) was added to hyphae prior to the addition of neutrophils. NETs were visualized by fluorescent microscopy using 5 μM Sytox Green.

Transmission electron microscopy

Samples for transmission electron microscopy (TEM) were fixed by gently layering 2.5% glutaraldehyde in 0.15 M sodium cacodylate buffer, rinsed with buffer, and postfixed with 1% osmium tetroxide. Slides were rinsed, dehydrated, and covered with resin and placed over Epox 812 filled slide-duplicating molds (Electron Microscopy Sciences, Hatfield, PA) overnight. Selected areas of interest were mounted on blocks for sectioning. Ultrathin sections (50–60 nm) were prepared using a Reichert Ultracut S microtome (Leica, Wetzlar, Germany), retrieved onto 300-mesh copper grids, and contrasted with uranyl acetate and lead citrate. Sections were examined using a Morgagni 268 transmission electron microscope (FEI, Hillsboro, OR) and images were collected with an AMT Advantage 542 CCD camera system (Advanced Microscopy Techniques, Danvers, MA).

NET quantification

Neutrophils were adhered as described above to Fn with and without β-glucan–coated plates. NETs were quantified after staining extracellular DNA with 2.5 μl PicoGreen stock reagent per milliliter, incubated for 6 min, and fluorescence was measured using an FL800 microplate fluorescence reader (BioTek) at 485 nm excitation/535 nm emission. Fluorescence measurements were determined from four to six independent experiments representing at least three donors. Blank reagent well readings were subtracted from each experimental well to obtain reported fluorescence, and results were confirmed by microscopy. Alternatively, after NET formation on Fn with β-glucan–coated plates, NETs were harvested by 30 min digestion at 37°C using 5 gel U/ml of micrococcal nuclease (New England Biolabs, Ipswich, MA), and the DNA from the resultant supernatants or the equivalent number of total cells was isolated using a DNeasy blood and tissue kit (Qiagen, Hilden, Germany). Isolated NET DNA content and total cellular DNA content from two to four replicate wells from five independent experiments were quantified by PicoGreen according to the manufacturer’s protocols using a LS50B luminescence spectrometer (PerkinElmer, Waltham, MA) at 480 nm excitation/520 nm emission. The percentage extracellular DNA was determined by dividing the amount of isolated NET DNA by the total DNA.

NET killing assay

Neutrophils were adhered as described above to either Fn with β-glucan–coated plates or Fn-coated plates that had been lightly seeded with C. albicans and that was allowed to differentiate overnight. Where indicated, cells were pretreated with 3% autologus serum or 6.25 μM DPI. After 30 min at 37°C, neutrophil-containing wells and control wells containing only Fn with β-glucan or hyphae were washed twice with RPMI 1640. For the hyphal system, 2 ml RPMI 1640 was added and hyphae were allowed to grow overnight at 37°C. For the immobilized system, 2 ml RPMI 1640 containing 70,000 C. albicans blastoconidia and 10 μM cytochalasin D (Sigma-Aldrich) were added to each well representing a multiplicity of infection of 1:100 and allowed to grow overnight at 37°C. Where indicated, overnight incubations were additionally carried out in the presence of 3% autologus serum, 6.25 μM DPI, 300 U/ml catalase (Sigma-Aldrich), 300 U/ml superoxide dismutase (Sigma-Aldrich), 1:1000 dilution micrococcal nuclease (Sigma-Aldrich), 150 U/ml DNaseI (Sigma-Aldrich), or equivalent PMN hypotonic lysate. Cells were scored microscopically for hyphal growth and viability was quantified by an MTT reduction assay (30). Briefly, after overnight growth, wells were washed with indicatorless RPMI 1640, incubated for 5 min at room temperature with 0.5% sodium deoxycholate (Sigma-Aldrich) to lyse remaining PMNs, and washed with indicatorless RPMI 1640. One milliliter 0.01 g/ml MTT was added to each well and allowed to incubate at 37°C for 2 h. Wells were washed twice with indicatorless RPMI 1640, the resultant formazan crystals were solubilized in 0.2 ml DMSO for 5 min at 37°C and diluted with 0.4 ml acidified isopropanol. Four to six 100 μl replicates per well were transferred to a 96-well plate and assayed at 540 nm using a microplate BioKinetics reader (BioTek Instruments) running DeltaSoft3 software. Data were obtained from 4 to 24 independent experiments representing at least three donors for each treatment condition.

Oxidative burst assay

Respiratory burst activity was determined by measuring the colorimetric change caused by superoxide anion reduction of ferricytochrome c. Cells were plated at a concentration of 3 × 105 cells/well, in replicates of 3–6 wells, at a volume of 100 μl/well onto 96-well tissue culture plates. When indicated, PMNs were assayed in the presence of fMLF, DPI, and/or WGPs as an additional stimulant added at time 0 of the assay. WGPs were sonicated before use to produce single particle suspensions. Stimulus concentrations of 1.4 × 107 WGPs/well were from published reports and optimized for maximal generation of superoxide through preliminary work. PMA (20 nM) was used as a positive control for superoxide production, and wells blocked with endotoxin-free BSA and untreated cells served as negative controls. For Ab-blocking experiments, cells were preincubated with 10−9 M fMLF for 10 min at room temperature. Cells were then incubated with 20 μg/ml indicated mAb or isotype control for 20 min on ice before being plated. Finally, 100 μl/well 100 μM ferricytochrome c was added to each well and absorbance was measured every 10 min for 90 min at dual wavelengths of 550 and 630 nm at 37°C using a microplate BioKinetics reader (BioTek Instruments) running DeltaSoft3 software. Superoxide production was calculated for 60 min with the following formula: (ΔAbsorbance550–630nm) × 15.166 = nmol/well; 15.166 is a predetermined absorbance constant and the final units are nmol superoxide anion/3 × 105 cells/h. To account for variability in donor response to WGPs, superoxide production for each donor is expressed as percentage of donor superoxide production to WGPs.

To measure the generation of ROS by PMNs against live yeast, PMNs were suspended in HBSS (5 × 106/ml) containing 8 μM CM-H2DCFDA and equilibrated at room temperature in the dark for 30 min. CM-H2DCFDA is a cell-permeable indicator for the respiratory burst that becomes fluorescent upon oxidation. Cells were washed once in an excess volume of HBSS without cations and suspended to a concentration of 3.5 × 106/ml in L-15/2 mg/ml glucose. Seven million cells were added to C. albicans hyphae that were differentiated on 6-well tissue culture plates coated with Fn to promote adhesion to the surface and minimize disturbance through multiple assay steps as earlier described and visualized microscopically as explained above.

FACS analysis

Samples of 1 × 106 isolated PMNs were blocked in ice-cold PBS containing 1% normal goat serum and Fc block (Accurate Chemical, Westbury, NY) for 30 min on ice. Cells were stained with 20 μg/ml purified mAb for 1 h on ice in a total volume of 100 μl. Cells were then washed twice and incubated with 30 μg/ml PE-labeled goat F(ab′)2 anti-mouse IgG (Sigma-Aldrich) for 30 min on ice. Cells were washed twice and resuspended in 1% paraformaldehyde in PBS. Analysis was performed on a FACScan (Becton Dickinson) using Becton Dickinson Lysis II software and FlowJo software (Tree Star, Ashland, OR) and gated on neutrophils.

Statistical analysis

Data were pooled from a minimum of three independent experiments representing at least three different donors, as indicated. ANOVA analysis with Newman–Keuls post hoc analysis or a paired-sample Student t test as appropriate were performed using Matlab (Mathworks, Natick, MA) or Excel (Microsoft, Redmond, WA) running the statistiXL data package (statistiXL, Nedlands, WA, Australia). The null hypothesis was rejected if p < 0.01.

Results

Neutrophil adhesion to the fungal PAMP β-glucan results in rapid homotypic aggregation by a matrix-dependent mechanism

To model immune recognition of β-glucan in fungal hyphae, which are too large for internalization, soluble β-glucan was immobilized onto a tissue culture surface. This reductionist system allowed for the interrogation of neutrophil binding and responsiveness in the absence of phagocytosis. Because neutrophil recognition of fungal hyphae during the response to mycotic tissue necessarily involves contact with ECM, the cellular effect of binding to β-glucan was studied in the presence and absence of the ubiquitous tissue matrix component Fn. When human neutrophils were primed with 1 nM fMLF in the presence of Mn2+ (0.5–1 mM), rapid homotypic cell aggregation was observed in response to Fn with β-glucan, but not Fn or β-glucan alone (Fig. 1A). Neutrophil aggregation was β-glucan specific and required corecognition of Fn, as primed cells that were adhered to plastic, BSA, fibrinogen with and without β-glucan, or Fn with and without either dextran or mannan did not aggregate (data not shown). Time course studies identified a 30 min time point in which 90–95% of the cells exposed to Fn with β-glucan were involved in aggregates (Supplemental Fig. 1, Video 1). Again, it is noteworthy that cell aggregation does not occur to either Fn or β-glucan alone at 30 min (Fig. 1A, Supplemental Fig. 1), which was the time point used for the balance of the studies described. Additionally, we determined that there is a requirement for both fMLF and Mn2+ to promote PMN aggregation responding to Fn with β-glucan (data not shown). Because neutrophils reach a primed state upon diapedesis, fMLF was used as a canonical priming agent; however, rapid aggregate formation on Fn with β-glucan (but not to either ligand alone) was also observed when cells were primed with TNF-α (30 ng/ml) or IL-8 (7 ng/ml) (data not shown). Therefore, findings are not a unique consequence of fMLF stimulation, but are a generalized response of the primed neutrophil to β-glucan in the context of Fn. Collectively, this aggregation to fungal β-glucan requires conditions consistent with a tissue-based extravasated neutrophil including ECM, priming, and Mn2+.

FIGURE 1.
FIGURE 1.

Primed neutrophils in the context of Fn with β-glucan, but not Fn or β-glucan alone, form aggregates, and Ab inhibition and activation of β2 integrin CR3 modulates cell aggregation. (A–C) Micrographs show PMNs that were adhered to Fn (6 μg/ml), β-glucan (1 mg/ml), or Fn with β-glucan precoated wells. (A) PMNs were pretreated with 10−9 M fMLF for 20 min on ice and Mn2+ immediately before adhering cells. Cells formed aggregates in the context of Fn with β-glucan, but not Fn or β-glucan alone. Additionally, aggregation required priming with both fMLF and Mn2+ (data not shown). All experiments were incubated for 30 min at 37°C. Data represent at least 10 separate experiments done using neutrophils from different individual donors. (B) Aggregation is inhibited with CR3, but not Dectin-1–blocking mAb. PMNs were pretreated as described in (A) before adhering cells to Fn with and without β-glucan–coated wells for 30 min at 37°C. When PMNs were pretreated with 20 μg/ml anti-CR3–blocking mAb (clone 44abc), cell aggregation was prevented; anti-Dectin–blocking mAb (GE2) and IgG1 (which was used as an isotype control) have no effect on neutrophil aggregation. (C) fMLF- and Mn2+-primed cells were pretreated with an anti-CR3–activating mAb [VIM12 F(ab′)2] that mimics β-glucan. Treated cells aggregate on Fn-coated wells and exhibit an exaggerated cell aggregation phenotype on wells coated with Fn with β-glucan. These data represent at least four independent experiments using neutrophils from different individual donors. Original magnification ×20. Scale bars, 100 μm.

Neutrophil aggregation is dependent on CR3

To determine the neutrophil receptor responsible for β-glucan–induced aggregation, cells were pretreated with a CR3-blocking mAb before exposure to Fn with β-glucan. As shown in Fig. 1B, cell aggregation was inhibited by a CR3 function-blocking mAb (clone 44 abc). A Dectin-1 function-blocking mAb GE2 had no effect on neutrophil aggregation on Fn with β-glucan when used under conditions where Ab binding was demonstrable by FACS and effected a partial inhibition of the respiratory burst to WGPs (as reported by others; see Ref. 31) (Fig. 1B, Supplemental Fig. 2).

β-glucan binding to CR3 has been mapped to the lectin-like site of CD11b that is recognized by the anti-CD11b–activating mAb, VIM12 (32). In the presence of Fn alone, VIM12 activation mimicked β-glucan binding by causing cell aggregation (Fig. 1C). Interestingly, in the presence of Fn with β-glucan, VIM12 mAb activation showed a synergistic effect, forming cell aggregates that were highly enlarged relative to either agonist alone (Fig. 1C), suggesting that VIM12 is binding to a subset of CR3 integrins that are not fully occupied by β-glucan. These data together support a CR3-dependent mechanism of neutrophil homotypic aggregation in response to Fn with β-glucan.

ERK MAPK is regulated depending on differential ligation of neutrophils

To characterize the intracellular signaling mechanisms by which primed neutrophils formed aggregates in response to differential ligation by Fn versus Fn with β-glucan, a global tyrosine phosphoproteomic analysis was undertaken (J.S. Reichner, unpublished observations). Western blotting validated the proteomic finding of elevated ERK phosphorylation in cells exposed to Fn with β-glucan as compared with Fn alone (Fig. 2A, 2B). To assess a functional role for ERK in neutrophil aggregation, the upstream kinase responsible for ERK phosphorylation, MEK, was inhibited. Neutrophils pretreated with inhibitors of MEK (U0126 or PD 98059) failed to form aggregates (Fig. 2C), supporting a role for ERK phosphorylation in the aggregation of primed neutrophils exposed to Fn with β-glucan. Supplemental Fig. 3 demonstrates complete pharmacologic inhibition of pERK under the experimental conditions employed.

FIGURE 2.
FIGURE 2.

Phosphorylated ERK levels are increased in Fn with β-glucan–treated PMNs. (A) PMNs were pretreated as previously described in the legend for Fig. 1A before they were adhered to Fn with and without β-glucan–precoated wells and incubated at 37°C for 5, 10, 20, and 30 min. Cells were harvested at the appropriate time point, lysed, separated on 10% SDS-PAGE gels, and transferred to a nitrocellulose membrane. Membranes were immunoblotted with anti-pERK, anti-total ERK, and anti-actin mAb. Open bar indicates noncontiguous samples that were run on the same gel. (B) A representative densitometric analysis was determined by scanning densitometry using ImageJ analysis software and expressed as phosphorylated ERK over actin. (C) Neutrophils were incubated on ice for 20 min with MEK inhibitors U0126 (50 μM) and PD 98059 (30 μM) and subsequently pretreated as previously described in the legend for Fig. 1A before they were adhered to Fn with and without β-glucan–precoated wells and incubated at 37°C for 30 min. Inhibition of ERK phosphorylation prevented PMN aggregation when adhered to Fn with β-glucan. Original magnification ×20. Scale bar, 100 μm. Blots were derived from the same protein samples. These results represent at least four independent experiments using neutrophils from different individual donors.

Immobilized Fn with β-glucan induces PMN NET formation

Given that human neutrophils release NETs as part of the response to fungal pathogens (16, 18), we tested the hypothesis that homotypic cell aggregation to the fungal PAMP β-glucan is caused wholly or in part by NET release following β-glucan recognition. Supporting this hypothesis, when neutrophil aggregates that were formed in response to Fn with β-glucan were subsequently treated with DNaseI, aggregates were significantly disrupted, suggesting that extracellular DNA may be entrapping PMNs into aggregates (Fig. 3A). A significant increase in extracellular NET extrusion from neutrophils exposed to Fn with β-glucan compared with Fn alone was observed by confocal microcopy using Sytox Green, a molecule that is fluorescent when bound to dsDNA (Fig. 3B, Supplemental Video 1) (20, 33). Z-stack reconstruction of this can be seen in Supplemental Videos 2 and 3. NET production in response to Fn with and without β-glucan was quantified under our assay conditions using the highly sensitive PicoGreen dsDNA staining revealing a 17-fold increase in extracellular DNA on Fn with β-glucan as compared with Fn alone (Fig. 3B, bar graph). Using an alternative method to quantitate NET release, extracellular DNA from cells on Fn with β-glucan was harvested by micrococcal nuclease digestion, purified, and stained with PicoGreen (as described in Materials and Methods). DNA concentration was determined by fluorometery and the DNA released on Fn with β-glucan was determined to represent 17.2 ± 3.4% (n = 5 independent experiments) of the total cellular DNA. Fig. 4 (arrowhead) shows intracellular NET formation and release from within neutrophils adhered to Fn with β-glucan by TEM. In concert with other reports, in our study neutrophils showed characteristic rounded and condensed multilobular nuclei, blebbing with extensive dilation between the inner and outer nuclear membranes, and budding vesicles, which contain strands of DNA with attached nucleosomes. The nuclei are circular with homogeneous condensed chromatin simultaneous with vesicles continuing to bud from the nuclear envelope. The intact vesicles extrude into the extracellular space, rupture, and release chromatin. Along the plasma membrane are dense cytoplasmic granules, which also release their contents in the extracellular space, contributing to the granule content of NETs. NETs can be seen in the cytoplasm and extracellularly (Fig. 4Ai, increased magnification) as well as in cytoplasmic vesicles (20) (Fig. 4Aii, increased magnification).

FIGURE 3.
FIGURE 3.

Rapid PMN NET formation of PMNs adhered to immobilized Fn with β-glucan. PMNs were pretreated as previously described in the legend for Fig. 1A before they were adhered to Fn with and without β-glucan–precoated wells and incubated at 37°C for 30 min. (A) After aggregates formed, DNaseI was added directly to the wells, which significantly disrupted PMN aggregates. Original magnification ×20. Scale bar, 100 μm. (B) Sytox Green staining shows NET formation in the context of Fn with β-glucan, corresponding to PMN aggregates. Aggregates and NET formation are not seen in cells responding to Fn alone. Original magnification ×20 (using confocal microscopy as described; see Materials and Methods). Scale bar, 100 μm. Bar graph shows 17-fold increase in extracellular DNA on Fn with β-glucan as compared with Fn alone as quantified by plate fluorometer under our assay conditions using PicoGreen dsDNA staining. Error bars represent SEM. These results represent six independent experiments using neutrophils from at least three individual donors. *p < 0.01, paired sample Student t test.

FIGURE 4.
FIGURE 4.

NETs formed by PMNs adhered to immobilized Fn with β-glucan decrease yeast viability. (A) Neutrophils were prepared as previously described in the legend for Fig. 1A, and following glutaraldehyde fixation, samples were processed and examined with TEM. Visualization of changes in the nuclear envelope and NET formation (arrowhead) are shown. Original magnification ×7100. Scale bar, 2 μm. The nuclear envelope undergoes a division of the inner nuclear membrane (INM) and the outer nuclear membrane (ONM). (i) DNA material of NETs shown extracellularly. Original magnification ×44,000. Scale bar, 100 nm. (ii) Enlarged view of cytoplasmic vesicles containing NETs using a voltage of 80 kV. Original magnification ×36,000. Scale bar, 500 nm. Sections were stained with uranyl acetate and lead citrate (see Materials and Methods). These results represent at least four independent experiments using neutrophils from different individual donors. (B) Yeast viability by reduction of MTT. PMNs were pretreated as described in the legend Fig. 1A before they were adhered to Fn with β-glucan–precoated wells and incubated at 37°C for 30 min. Wells were washed and RPMI 1640, C. albicans blastoconidia, and 10 μM cytochalasin were added to wells with and without DNaseI and incubated at 37°C overnight. For comparison, blastoconidia were incubated with the hypotonic lysate of an equivalent number of PMNs. Wells were scored microscopically for yeast growth (left), and viability was quantified by MTT reduction (bar graph). Error bars represent SEM. Results represent 6–24 independent experiments from at least three donors. Scale bar, 100 μm. *p < 0.01 versus PMN lysate and **p < 0.01, PMN NETs with and without DNaseI, ANOVA full factorial, post hoc Newman–Keuls test.

Immobilized Fn with β-glucan–induced PMN NET formation has fungicidal activity against C. albicans

The importance of NETosis in killing C. albicans has been shown by others (16, 18). We sought to extrapolate the mechanisms driving this response. We have reported previously that the respiratory burst produced by neutrophils in response to immobilized β-glucan is suppressed to undetectable levels by Fn (23), conditions that were shown in this study to be permissive for homotypic cell aggregation and NET formation. We assayed NET killing to confirm that for NETs released by PMNs exposed to immobilized Fn with β-glucan, when ROS production was inhibited, the NET composition maintained its expected fungicidal activity. As shown in Fig. 4B, there is NET-dependent killing of C. albicans. Yeast grown in the presence of PMN NETs elaborated on Fn with β-glucan resulted in a significant decrease in viability (62.8 ± 11.2%) as measured by MTT reduction when compared with yeast grown in the presence of the hypotonic lysate of an equal number of PMNs. Additionally, when NET integrity is disrupted by the addition of DNaseI, there is a significant increase (100.8 ± 13.2%) in yeast viability, supporting a NET-specific mechanism of killing. In this system, neutrophils were additionally treated with cytochalasin to minimize the phagocytic component of killing.

CR3 blockade significantly attenuates neutrophil NET formation to immobilized Fn with β-glucan

As shown in Fig. 1B, cell aggregation in response to Fn with β-glucan was abrogated when pretreated with a CR3-blocking mAb. Fig. 5 shows that NET formation in response to Fn with β-glucan was also significantly reduced when cells were pretreated with a CR3-blocking mAb but not an isotype control, as measured by a fluorescent plate reader using PicoGreen dsDNA stain (Fig. 5). Taken together, our data suggests that CR3 mediates aggregation and NET formation to fungal β-glucan presented in the context of Fn.

FIGURE 5.
FIGURE 5.

CR3 blockade attenuates neutrophil aggregation and rapid NET formation of PMNs adhered to immobilized Fn with β-glucan. PMNs were pretreated as previously described in the legend for Fig. 1A. PMNs additionally pretreated with 20 μg/ml anti-CR3–blocking mAb (clone 44abc) did not aggregate. Sytox Green staining after the samples were incubated at 37°C for 30 min demonstrated attenuation of NET formation; IgG1, which was used as an isotype control, has no effect on neutrophil aggregation or NET formation. Scale bar, 100 μm. Bar graph shows significant inhibition of NET production on Fn with β-glucan when cells are pretreated with 20 μg/ml anti-CR3–blocking mAb (clone 44abc), as quantified by a plate fluorometer using PicoGreen dsDNA staining. Error bars represent SEM. These results represent at least three independent experiments using neutrophils from different individual donors. *p < 0.01 untreated versus anti-CR3–blocking mAb and nd, no significant difference versus untreated, ANOVA full factorial, post hoc Newman–Keuls.

C. albicans in the presence of Fn induces PMN NET formation through recognition of hyphal β-glucan by neutrophil CR3

Prior work in our laboratory showed that when C. albicans infection was established by direct inoculation of rat kidney, a robust neutrophilic response included pronounced cell aggregates surrounding the hyphal filaments that are visually similar to the aggregates we observed on Fn with β-glucan–coated surfaces (22). It has also been demonstrated that NETs enhance bacteria trapping and interact with C. albicans in vivo (18, 33, 34). As mentioned above, the response of neutrophils to Candida-infected tissues includes contact with matrix; therefore, Candida hyphae were grown on Fn to track NET formation to the intact pathogen. As shown in Fig. 6 and Supplemental Video 4, neutrophils aggregate and form NETs rapidly (within 30–50 min) in response to C. albicans hyphae grown on Fn. Additional representative fields of this observation can be seen in Supplemental Fig. 4, and complementary z-stack images can be viewed in Supplemental Video 5.

FIGURE 6.
FIGURE 6.

C. albicans induced rapid PMN NETs that are inhibited by anti-CR3–blocking mAb. C. albicans hyphae were grown on culture dishes coated with 40 μg/ml Fn. Neutrophils were pretreated as previously described in Fig. 1A and blocked with anti-CR3 mAb (clone 44abc) for 20 min on ice before adding them to the dishes containing yeast hyphae and incubated at 37°C for 30–50 min. Neutrophils conform to the hyphae, and confocal microscopy shows NET formation by Sytox Green staining except when inhibited by anti-CR3 mAb, indicating a functional role for CR3. Original magnification ×20 (using confocal microscopy as described; see Materials and Methods). Scale bar, 100 μm. These results represent at least five independent experiments using neutrophils from different individual donors.

The receptor/ligand interactions leading to NET release in response to fungal hyphae have not yet been determined. To assess the role of CR3 recognition of β-glucan in mediating NET formation, neutrophils were pretreated with an anti-CR3–blocking mAb before adding them to C. albicans hyphae. As shown in Fig. 6 and Supplemental Video 6, CR3 receptor blocking significantly reduced the formation of diffuse NETs in response to C. albicans hyphae that was consistent with our finding showing CR3 to be essential for aggregate and NET formation to immobilized Fn with β-glucan (Figs. 1B, 5). By observation, blocking CR3 seemingly reduces PMN binding to the hyphae, but it does not obliterate binding as observed when β-glucan in the hyphae cell wall is blocked. More interestingly, although there is still cell binding to the hyphae when CR3 is blocked, it is insufficient to promote NET extrusion. This suggests that there are receptors other than CR3 that mediate cell adhesion to intact hyphal filaments, but do not necessarily lead to NET release.

Consistent with yeast viability data obtained using the immobilized system (Fig. 4B), PMNs added directly to C. albicans hyphae under NET-producing conditions similarly restrict hyphal viability (Fig. 7A). Supporting a NET-specific mechanism of killing, there is a significant increase in hyphal viability (102.6 ± 17.3%) when DNaseI is added to disrupt PMN NETs using the hyphal system. Taken together, these data confirm the fungicidal capacity of NETs elicited under our experimental conditions.

FIGURE 7.
FIGURE 7.

Rapid PMN NET formation in response to C. albicans decreases hyphal viability, is matrix-dependent, and is inhibited by a β-glucan–specific mAb. (A) Hyphal viability by reduction of MTT. Neutrophils were pretreated as previously described in Fig. 1A and added to lightly seeded C. albicans hyphae grown on Fn-coated wells and incubated at 37°C for 30 min. Wells were washed and RPMI 1640 with and without DNaseI was added and incubated at 37°C overnight. Wells were scored microscopically for yeast growth (left) and viability was quantified by MTT reduction (bar graph). Error bars represent SEM. These data represent eight independent experiments with at least three donors. *p < 0.01 versus hyphae with PMNs, paired sample Student t test. (B and C) C. albicans hyphae were grown on coverslips coated with 40 μg/ml Fn. Neutrophils were pretreated as previously described in Fig. 1A, added to the hyphae, and visualized by Sytox Green staining. (B) PMN NET formation is induced when hyphae and neutrophils are adhered to Fn, but not poly-l-lysine. (C) Neutrophil NET formation is prevented when fungal β-glucan is preblocked by the β-glucan–specific mAb BFDiv before PMNs are added to hyphae and incubated at 37°C for 30 min. Original magnification ×20. Scale bar, 100 μm. These data represent four independent experiments using neutrophils from different individual donors.

To further determine the regulatory role of ECM, C. albicans hyphae were grown on poly-l-lysine as a nonspecific adhesive control and were found not to support rapid NET formation (Fig. 7B).

To test the hypothesis that β-glucan within fungal filaments is mediating NET formation, C. albicans hyphae were incubated with β-glucan–specific mAb (BFDiv) to block exposed β-glucan. As shown in Fig. 7C, NET formation is abrogated with BFDiv treatment, indicating that β-glucan is mediating the PMN NET response to fungal hyphae.

Cell aggregation and NET formation to immobilized Fn with β-glucan and C. albicans are independent of the respiratory burst

A number of initial reports posited that NET formation is functionally coupled to the release of ROS (11, 13, 15, 17, 19). Prior work from our laboratory showed that neutrophils exposed to immobilized β-glucan alone induced a dose-dependent respiratory burst (22), whereas in this study we demonstrated that β-glucan alone is insufficient for aggregation and hence NET formation (Supplemental Fig. 1). We also reported that addition of Fn to immobilized β-glucan completely suppressed respiratory burst (23) under conditions that we now show result in rapid and robust aggregation and NET formation, suggesting that the response is dependent on Fn but independent of ROS. This independence of ROS is in agreement with the findings of Pilsczek et al. (20). Moreover, in experiments to exclude any possible vestigial respiratory burst activity, we found that addition of the NADPH oxidase inhibitor DPI did not impede aggregation and NET formation on immobilized Fn with β-glucan (Fig. 8A, 8B). The complete inhibition of the respiratory burst at the concentrations of DPI used in this study was confirmed both by cytochrome c colorimetric assay as shown in Fig. 8C and by using the fluorescent oxidative stress indicator CM-H2DCFDA (data not shown). Consistent with observations on immobilized Fn with β-glucan, NET formation in response to fungal hyphae was also found to be independent of the PMN respiratory burst and unperturbed by use of DPI (Fig. 9A). Treatment with DPI, superoxide dismutase, or catalase did not prevent PMN killing by NET formation. When 6.25 μM DPI, 300 U/ml superoxide dismutase, or 300 U/ml catalase were added to the yeast viability assay described in Fig. 4B, there was no significant change in percentage viability after NET exposure as measured by reduction of MTT relative to treatment controls (data not shown; see Materials and Methods).

FIGURE 8.
FIGURE 8.

Neutrophil aggregation and rapid PMN NET formation in the context of immobilized Fn with β-glucan are independent of the PMN respiratory burst. (A and B) PMNs were prepared as previously described in Fig. 1A. Cells were pretreated with DPI (6.25 μM) on ice for 20 min before they were adhered to Fn with and without β-glucan–precoated wells and incubated at 37°C for 30–50 min. (A) Inhibition of the respiratory burst with DPI does not prevent PMN aggregation. Original magnification ×10. Scale bar, 100 μM. (B) Inhibition of the respiratory burst with DPI does not attenuate NET formation in the context of Fn with β-glucan. Sytox Green was added to the sample after aggregate formation to assess NET formation. Original magnification ×20. Scale bar, 100 μm. (C) DPI inhibits the PMN respiratory burst to PMA. Error bars represent SD. These results represent at least four independent experiments using neutrophils from different individual donors.

FIGURE 9.
FIGURE 9.

Rapid PMN NET formation in the context of C. albicans is independent of the PMN respiratory burst. (A–C) Neutrophils and yeast hyphae were prepared as previously described in Fig. 6. (A) Cells were pretreated with DPI (6.25 μM) on ice for 20 min before adding them to coverslips containing yeast hyphae and incubated at 37°C for 30–50 min. Inhibition of the respiratory burst with DPI does not attenuate NET formation in the context of C. albicans hyphae. Original magnification ×10. Scale bar, 100 μm. (B) Neutrophils were incubated the MEK inhibitor U0126 for 20 min on ice before adding them to hyphae to prevent ERK phosphorylation. This treatment resulted in a significant decrease in NET formation in response to C. albicans hyphae. Sytox Green was added after the samples were incubated at 37°C for 30–50 min. Original magnification ×20. Scale bar, 100 μm. (C) PMNs were loaded with the respiratory burst indicator CM-H2DCFDA, treated with U0126, and then added to hyphae. Inhibition of ERK phosphorylation with U0126 does not inhibit the respiratory burst (top) but it does inhibit the formation of NETs (bottom) as visualized by Sytox Green staining. Original magnification ×10. Scale bar, 100 μm. Data represent four independent experiments with neutrophils from different individual donors.

With respect to the neutrophilic response to intact C. albicans hyphae, inhibition of ERK phosphorylation significantly reduced NET formation (Fig. 9B, 9C, lower panels) without affecting the ability of neutrophils to undergo the respiratory burst (Fig. 9C, upper panel).

Taken together, these data show that neutrophil rapid NET formation in response to fungal hyphae is mediated by CR3 recognition of fungal β-glucan and corecognition of Fn. NET formation facilitates neutrophil aggregation, is independent of the PMN respiratory burst, and is modulated in part through ERK phosphorylation.

Discussion

Neutrophils play a central role in host defense against fungal pathogens; indeed, neutropenia or deficient cell function is a predisposing factor for mycotic infection (1). Unlike phagocytic clearance of unicellular microbes, neutrophils are challenged with the physical reality that fungal hyphae are too large to ingest and must be combated by alternative mechanisms (16). β-glucans are the major structural component of the fungal cell wall and are important PAMPs for innate immune sensing of fungi (21–24). Immobilization of purified β-glucan onto tissue culture surfaces enables mechanistic studies of β-glucan recognition by human neutrophils to be conducted in the absence of phagocytosis, thereby modeling the response to hyphae. In this study we report that neutrophils undergo rapid (≤30 min) homotypic aggregation and extrusion of NETs in response to β-glucan when immobilized together with the ECM protein Fn, but not to either ligand alone (Figs. 1A, 3B, Supplemental Fig. 1 and Videos 1–3). The physiological relevance of the regulated response to β-glucan by Fn is considerable because recognition of fungi by extravasated neutrophils occurs within afflicted tissues and necessitates matrix contact (23). Moreover, the rapid release of NETs requires neutrophil priming, again consistent with the state of the extravasated cell. Because the response to fungi as shown in this study depends on CR3, the requirement for priming may also allow the translocation of the large intracellular store of this receptor to the cell surface to heighten the response. In a similar vein, the effect of Mn2+ is consistent with an integrin-mediated response as it maintains CR3 in a permissive state. Moreover, neither immobilized Fn or Mn2+ is found in the circulation but is demonstrable in damaged or infected tissues, and neither one alone effects the cellular response to fungal β-glucan. Together, the combinatorial requirements of matrix, cell priming, and CR3 activation for a rapid NET response to fungal components are thoroughly consistent with a host defense mechanism selective for a host response within infected tissues and not in the peripheral bloodstream.

Work shown in this study, as well as that reported previously, identifies consistent regulation of neutrophil host defense functions when β-glucan is presented in the context of ECM as compared with cells exposed to either ligand alone. We showed that cells migrating on β-glucan together with Fn resulted in a conversion from random to directed motility (29) and a suppressed respiratory burst as compared with β-glucan alone (23). We now show promotion of homotypic aggregation and NET formation to ligand combinations suggesting a broad regulatory role for ECM in mediating host response to a fungal PAMP. Finding that NET formation in response to C. albicans hyphae or to immobilized β-glucan did not occur with poly-l-lysine in place of Fn (Fig. 7B) suggests that adhesion without matrix activation is an insufficient signal for rapid NET release to intact hyphae. The need for the ECM protein Fn in PMN aggregation and rapid NET formation is an unprecedented finding that renders insight into understanding this host defense mechanism against problematic fungal infections within tissues. It remains to be determined whether matrix proves to be a generalized regulator of the rapid NET response to other microbes.

Urban et al. (16) found NETs released from PMA-activated neutrophils capture and kill C. albicans in the absence of exogenous ECM at time points beyond 1 h. Although their experiments did not include the addition of purified Fn, serum was present and thus a role for Fn in temporal regulation of NET formation to PMA cannot be dismissed. Moreover, they used a nonphysiological stimulant as opposed to C. albicans used in our present study. Because of these data and the preponderance of literature already showing killing, our contribution is to understand the mechanisms mediating this response. We have shown that NETs formed in response to immobilized Fn with β-glucan, as well as in response to intact C. albicans, have fungicidal activity (Figs. 4B, 7A). The role of the respiratory burst is nonessential, as inhibition with DPI, superoxide dismutase, and catalase has no effect on the ability of PMNs to kill C. albicans hyphae (data not shown). Additionally, there does not appear to be an essential role for complement, as NET production and hyphal killing experiements in the presence autologus serum showed robust NET production and percentages of hyphal viability that did not statistically differ from those in the absence of serum (data not shown). Future studies will recapitulate these data to corroborate the findings we have reported in this study and to further understand the intricacies of NET formation and killing.

The NET response to β-glucan and Fn was found to depend on the integrin CR3 (CD11b/CD18), as Ab blockade of the integrin prevented aggregation and NET release (Figs. 1B, 5, 6). By using our reductionist system, we demonstrate that recognition of β-glucan by CR3, and not by Dectin-1 (Fig. 1B, 1C), in the context of Fn is necessary and sufficient for homotypic aggregation and hence NET production. This is consistent with the finding that purified recombinant Dectin-1 only recognizes β-glucan–containing or unicellular yeast particles and yeast but not hyphae (35). Furthermore, blockade of β-glucan within C. albicans hyphae in the presence of Fn substantially reduced the NET response to the intact pathogen, demonstrating that β-glucan is a key PAMP in the NET response (Fig. 7B). Blockade of neutrophil CR3 prevented NET formation to both immobilized Fn with β-glucan and fungal hyphae (Figs. 5 and 6, respectively). This is in support of van Bruggen et al. (28), who showed that CR3, not Dectin-1, is the primary receptor on human neutrophils for the phagocytosis and response to β-glucan–containing particles. Therefore, our present study identified both the fungal ligand as well as the neutrophil counterreceptor that leads to NETosis.

Intracellular mechanisms leading to NET formation are not well understood. Neutrophil NET formation has been shown to occur after several hours of PMA treatment by a ROS-dependent mechanism (11, 15, 17, 19) even though PMA stimulates the respiratory burst instantaneously (36, 37). An alternate rapid (5–60 min) pathway of NET formation to S. aureus that is ROS-independent has also been described (20) and resembles the kinetics of NET release we report in this study. We also find rapid, ROS-independent neutrophil aggregation and NET formation when cells adhere to immobilized Fn with β-glucan in ≤30 min and in ≤60 min in response to Fn with hyphae. Furthermore, we observe similar changes in the morphology of NET-producing neutrophils as described in that report (20), such as blebbing with extensive dilation between the inner and outer nuclear membranes containing strands of DNA with attached nucleosomes (Fig. 4A).

In a previous report that is of particular relevance to the present study we showed that neutrophils undergo a robust respiratory burst to immobilized, purified fungal β-glucan, which is actively suppressed by ECM. This suppression was hypothesized to limit consequent tissue damage of migrating neutrophils until multifocal contact with hyphae is established (23). In the present study, we tested the hypothesis that ECM plays a regulatory role in fungal NET formation under conditions that we showed previously prevent generation of a respiratory burst. Although contrary to other reports (11, 13, 15, 17, 19), we provide several lines of evidence for oxidant-independent NET formation. First, we have reported previously that the respiratory burst produced by neutrophils in response to immobilized β-glucan is suppressed to undetectable levels by Fn (23), conditions that were shown in the present study to be permissive for homotypic cell aggregation and NET formation. The use of DPI to block any vestigial ROS did not affect neutrophil aggregation and NET formation (Fig. 8). Because DPI did not limit cell migration, aggregation, or NET release, its use with neutrophils to assess the role of ROS in NET production was not pharmacologically contraindicated. Taken together with the biological inhibition of ROS by Fn with β-glucan, our conclusions are not solely reliant on chemical inhibition of NADPH oxidase. Second, NETs were formed by neutrophils responding to C. albicans hyphae in the presence of DPI under conditions where production of ROS was inhibited (Fig. 9A). Third, ERK-inhibited cells responding to C. albicans hyphae were competent for respiratory burst, but NET formation was attenuated (Fig. 9C). Interestingly, these data are in support of Dikshit and colleagues (19), who demonstrated that inhibition of ERK phosphorylation does not significantly reduce PMA-induced ROS production but prevents the release of NETs. Fourth, cells plated on immobilized β-glucan alone undergo the respiratory burst, but not clustering, and hence do not release NETs. These lines of evidence show that the oxidative burst and rapid NET formation are uncoupled with respect to the response to fungal β-glucan. These data support the independence of ROS in mediating the fungicidal activity of NETs as previously discussed. We cannot rule out that at time points beyond 1 h, NET formation may include oxidant-dependent mechanisms. It is noteworthy that a recent report showed that the requirement for oxidant production for NET formation is not absolute and depends on the stimulus. Keeping this in mind when contrasting our work to that of Dikshit and colleagues (19), who attest a ROS-dependent activation of ERK and MAPK in NET formation, time and stimulus are crucial differences, which might explain the apparent discrepancy in our reported results. Ligand/receptor complexes may ultimately determine the intracellular signaling that leads to NET release either in the presence or absence of ROS.

To begin to understand intracellular signaling mechanisms affected by cells responding to β-glucan supplemented with Fn, as compared with Fn alone, we undertook a quantitative global tyrosine phosphorylation approach (J.S. Reichner, unpublished observations). Tyrosine kinases, particularly of the Src and Syk families, are activated upon ligation of β1 and β2 integrins and transduce signals that have functional effects on leukocytes (38–41), and hence phosphorylation of tyrosine is germane to our studies. From our analysis, we characterized phosphorylated ERK (Y204) as a potential regulator of PMN homotypic aggregates and NET formation. To begin to dissect the intracellular signaling mechanisms affected by cells responding to β-glucan supplemented with Fn, as compared with Fn alone, we took advantage of pharmacological inhibitors. For this study and the phosphoproteomic analysis, a 30 min time point was chosen because at this time we observed maximal cellular response to β-glucan–supplemented Fn compared with Fn alone. This is considered an early step to mapping the signaling profile of human neutrophils upon binding to a PAMP, characterizing a single time point that will ultimately render an extensive pathway analysis.

In conclusion, we show promotion of homotypic aggregation and NET formation to ligand combinations suggesting a regulatory role for CR3 in mediating the host response to a fungal PAMP. In this study, we have found that this phenomenon depends on ERK MAPK but is independent of the respiratory burst allowing NET production in the absence of ROS, which in turn may minimize collateral tissue damage. We have successfully shown a correlation between PMN defense mechanisms within a reductionist model and responses to a more physiologically relevant stimulus, C. albicans hyphae. Further work will recapitulate an in vivo representation, clarifying how diverse mechanisms converge and optimizing the host defense against pathological fungal infections.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. Nicole Morin, Dr. Meredith Crane, Annalisa Wilde, and Maggie Chung for technical assistance, Carol Ayala (Core Research Laboratories at Rhode Island Hospital) for TEM, the Brown University Leduc Bioimaging Facility, and Drs. Jorge Albina and Crane for critically reading the manuscript.

Footnotes

  • This work was supported by National Institutes of Health Grant GM066194 (to J.S.R.); A.S.B. is supported by a United Negro College Fund/Merck graduate science research dissertation fellowship.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    CM-H2DCFDA
    2′,7′-dichlorodihydrofluorescein diacetate
    CR3
    complement receptor 3
    DIC
    differential interference contrast
    DPI
    diphenyleneiodonium
    ECM
    extracellular matrix
    Fn
    fibronectin
    NET
    neutrophil extracellular trap
    PAMP
    pathogen-associated molecular pattern
    PMN
    polymorphonuclear leukocyte
    ROS
    reactive oxygen species
    TEM
    transmission electron microscopy
    WGP
    whole glucan particle.

  • Received September 24, 2012.
  • Accepted February 11, 2013.

References

  1. ↵
    1. Aratani Y.,
    2. H. Koyama,
    3. S. Nyui,
    4. K. Suzuki,
    5. F. Kura,
    6. N. Maeda
    . 1999. Severe impairment in early host defense against Candida albicans in mice deficient in myeloperoxidase. Infect. Immun. 67: 1828–1836.
    OpenUrlAbstract/FREE Full Text
    1. Bassetti M.,
    2. E. Righi,
    3. A. Costa,
    4. R. Fasce,
    5. M. P. Molinari,
    6. R. Rosso,
    7. F. B. Pallavicini,
    8. C. Viscoli
    . 2006. Epidemiological trends in nosocomial candidemia in intensive care. BMC Infect. Dis. 6: 21–26.
    OpenUrlCrossRefPubMed
    1. Cheng M. F.,
    2. Y. L. Yang,
    3. T. J. Yao,
    4. C. Y. Lin,
    5. J. S. Liu,
    6. R. B. Tang,
    7. K. W. Yu,
    8. Y. H. Fan,
    9. K. S. Hsieh,
    10. M. Ho,
    11. H. J. Lo
    . 2005. Risk factors for fatal candidemia caused by Candida albicans and non-albicans Candida species. BMC Infect. Dis. 5: 22–26.
    OpenUrlCrossRefPubMed
    1. Fraser V. J.,
    2. M. Jones,
    3. J. Dunkel,
    4. S. Storfer,
    5. G. Medoff,
    6. W. C. Dunagan
    . 1992. Candidemia in a tertiary care hospital: epidemiology, risk factors, and predictors of mortality. Clin. Infect. Dis. 15: 414–421.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Metzler K. D.,
    2. T. A. Fuchs,
    3. W. M. Nauseef,
    4. D. Reumaux,
    5. J. Roesler,
    6. I. Schulze,
    7. V. Wahn,
    8. V. Papayannopoulos,
    9. A. Zychlinsky
    . 2011. Myeloperoxidase is required for neutrophil extracellular trap formation: implications for innate immunity. Blood 117: 953–959.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Aratani Y.,
    2. F. Kura,
    3. H. Watanabe,
    4. H. Akagawa,
    5. Y. Takano,
    6. K. Suzuki,
    7. M. C. Dinauer,
    8. N. Maeda,
    9. H. Koyama
    . 2002. Relative contributions of myeloperoxidase and NADPH-oxidase to the early host defense against pulmonary infections with Candida albicans and Aspergillus fumigatus. Med. Mycol. 40: 557–563.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Chen Y.,
    2. W. G. Junger
    . 2012. Measurement of oxidative burst in neutrophils. Methods Mol. Biol. 844: 115–124.
    OpenUrlCrossRefPubMed
    1. Nathan C.
    2006. Neutrophils and immunity: challenges and opportunities. Nat. Rev. Immunol. 6: 173–182.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Nathan C.,
    2. S. Srimal,
    3. C. Farber,
    4. E. Sanchez,
    5. L. Kabbash,
    6. A. Asch,
    7. J. Gailit,
    8. S. D. Wright
    . 1989. Cytokine-induced respiratory burst of human neutrophils: dependence on extracellular matrix proteins and CD11/CD18 integrins. J. Cell Biol. 109: 1341–1349.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Lehrer R. I.,
    2. W. Lu
    . 2012. α-Defensins in human innate immunity. Immunol. Rev. 245: 84–112.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Brinkmann V.,
    2. A. Zychlinsky
    . 2007. Beneficial suicide: why neutrophils die to make NETs. Nat. Rev. Microbiol. 5: 577–582.
    OpenUrlCrossRefPubMed
    1. Amulic B.,
    2. G. Hayes
    . 2011. Neutrophil extracellular traps. Curr. Biol. 21: R297–R298.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Lee W. L.,
    2. S. Grinstein
    . 2004. Immunology: the tangled webs that neutrophils weave. Science 303: 1477–1478.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Phillipson M.,
    2. P. Kubes
    . 2011. The neutrophil in vascular inflammation. Nat. Med. 17: 1381–1390.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Fuchs T. A.,
    2. U. Abed,
    3. C. Goosmann,
    4. R. Hurwitz,
    5. I. Schulze,
    6. V. Wahn,
    7. Y. Weinrauch,
    8. V. Brinkmann,
    9. A. Zychlinsky
    . 2007. Novel cell death program leads to neutrophil extracellular traps. J. Cell Biol. 176: 231–241.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Urban C. F.,
    2. U. Reichard,
    3. V. Brinkmann,
    4. A. Zychlinsky
    . 2006. Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms. Cell. Microbiol. 8: 668–676.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Papayannopoulos V.,
    2. K. D. Metzler,
    3. A. Hakkim,
    4. A. Zychlinsky
    . 2010. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps. J. Cell Biol. 191: 677–691.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Urban C. F.,
    2. D. Ermert,
    3. M. Schmid,
    4. U. Abu-Abed,
    5. C. Goosmann,
    6. W. Nacken,
    7. V. Brinkmann,
    8. P. R. Jungblut,
    9. A. Zychlinsky
    . 2009. Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans. PLoS Pathog. 5: e1000639.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Keshari R. S.,
    2. A. Verma,
    3. M. K. Barthwal,
    4. M. Dikshit
    . 2013. Reactive oxygen species-induced activation of ERK and p38 MAPK mediates PMA-induced NETs release from human neutrophils. J. Cell. Biochem. 114: 532–540.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Pilsczek F. H.,
    2. D. Salina,
    3. K. K. Poon,
    4. C. Fahey,
    5. B. G. Yipp,
    6. C. D. Sibley,
    7. S. M. Robbins,
    8. F. H. Green,
    9. M. G. Surette,
    10. M. Sugai,
    11. et al
    . 2010. A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus. J. Immunol. 185: 7413–7425.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Xiang D.,
    2. V. R. Sharma,
    3. C. E. Freter,
    4. J. Yan
    . 2012. Anti-tumor monoclonal antibodies in conjunction with β-glucans: a novel anti-cancer immunotherapy. Curr. Med. Chem. 19: 4298–4305.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Lavigne L. M.,
    2. J. E. Albina,
    3. J. S. Reichner
    . 2006. β-glucan is a fungal determinant for adhesion-dependent human neutrophil functions. J. Immunol. 177: 8667–8675.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Lavigne L. M.,
    2. X. M. O’Brien,
    3. M. Kim,
    4. J. W. Janowski,
    5. J. E. Albina,
    6. J. S. Reichner
    . 2007. Integrin engagement mediates the human polymorphonuclear leukocyte response to a fungal pathogen-associated molecular pattern. J. Immunol. 178: 7276–7282.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Goodridge H. S.,
    2. A. J. Wolf,
    3. D. M. Underhill
    . 2009. β-glucan recognition by the innate immune system. Immunol. Rev. 230: 38–50.
    OpenUrlCrossRefPubMed
    1. O’Brien X. M.,
    2. K. E. Heflin,
    3. L. M. Lavigne,
    4. K. Yu,
    5. M. Kim,
    6. A. R. Salomon,
    7. J. S. Reichner
    . 2012. Lectin site ligation of CR3 induces conformational changes and signaling. J. Biol. Chem. 287: 3337–3348.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Tsikitis V. L.,
    2. J. E. Albina,
    3. J. S. Reichner
    . 2004. β-glucan affects leukocyte navigation in a complex chemotactic gradient. Surgery 136: 384–389.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Leal S. M., Jr..,
    2. C. Vareechon,
    3. S. Cowden,
    4. B. A. Cobb,
    5. J. P. Latgé,
    6. M. Momany,
    7. E. Pearlman
    . 2012. Fungal antioxidant pathways promote survival against neutrophils during infection. J. Clin. Invest. 122: 2482–2498.
    OpenUrlCrossRefPubMed
  28. ↵
    1. van Bruggen R.,
    2. A. Drewniak,
    3. M. Jansen,
    4. M. van Houdt,
    5. D. Roos,
    6. H. Chapel,
    7. A. J. Verhoeven,
    8. T. W. Kuijpers
    . 2009. Complement receptor 3, not Dectin-1, is the major receptor on human neutrophils for β-glucan-bearing particles. Mol. Immunol. 47: 575–581.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Harler M. B.,
    2. E. Wakshull,
    3. E. J. Filardo,
    4. J. E. Albina,
    5. J. S. Reichner
    . 1999. Promotion of neutrophil chemotaxis through differential regulation of β1 and β2 integrins. J. Immunol. 162: 6792–6799.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Levitz S. M.,
    2. R. D. Diamond
    . 1985. A rapid colorimetric assay of fungal viability with the tetrazolium salt MTT. J. Infect. Dis. 152: 938–945.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Kennedy A. D.,
    2. J. A. Willment,
    3. D. W. Dorward,
    4. D. L. Williams,
    5. G. D. Brown,
    6. F. R. DeLeo
    . 2007. Dectin-1 promotes fungicidal activity of human neutrophils. Eur. J. Immunol. 37: 467–478.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Whitlock B. B.,
    2. S. Gardai,
    3. V. Fadok,
    4. D. Bratton,
    5. P. M. Henson
    . 2000. Differential roles for αMβ2 integrin clustering or activation in the control of apoptosis via regulation of Akt and ERK survival mechanisms. J. Cell Biol. 151: 1305–1320.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Clark S. R.,
    2. A. C. Ma,
    3. S. A. Tavener,
    4. B. McDonald,
    5. Z. Goodarzi,
    6. M. M. Kelly,
    7. K. D. Patel,
    8. S. Chakrabarti,
    9. E. McAvoy,
    10. G. D. Sinclair,
    11. et al
    . 2007. Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood. Nat. Med. 13: 463–469.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Ma A. C.,
    2. P. Kubes
    . 2008. Platelets, neutrophils, and neutrophil extracellular traps (NETs) in sepsis. J. Thromb. Haemost. 6: 415–420.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Gantner B. N.,
    2. R. M. Simmons,
    3. D. M. Underhill
    . 2005. Dectin-1 mediates macrophage recognition of Candida albicans yeast but not filaments. EMBO J. 24: 1277–1286.
    OpenUrlAbstract
  36. ↵
    1. Steinberg B. E.,
    2. S. Grinstein
    . 2007. Unconventional roles of the NADPH oxidase: signaling, ion homeostasis, and cell death. Sci. STKE 2007: pe11.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    1. Parker H.,
    2. M. Dragunow,
    3. M. B. Hampton,
    4. A. J. Kettle,
    5. C. C. Winterbourn
    . 2012. Requirements for NADPH oxidase and myeloperoxidase in neutrophil extracellular trap formation differ depending on the stimulus. J. Leukoc. Biol. 92: 841–849.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Mócsai A.,
    2. M. Zhou,
    3. F. Meng,
    4. V. L. Tybulewicz,
    5. C. A. Lowell
    . 2002. Syk is required for integrin signaling in neutrophils. Immunity 16: 547–558.
    OpenUrlCrossRefPubMed
    1. Miranti C. K.,
    2. J. S. Brugge
    . 2002. Sensing the environment: a historical perspective on integrin signal transduction. Nat. Cell Biol. 4: E83–E90.
    OpenUrlCrossRefPubMed
    1. Berton G.,
    2. C. A. Lowell
    . 1999. Integrin signalling in neutrophils and macrophages. Cell. Signal. 11: 621–635.
    OpenUrlCrossRefPubMed
  41. ↵
    1. Gakidis M. A.,
    2. X. Cullere,
    3. T. Olson,
    4. J. L. Wilsbacher,
    5. B. Zhang,
    6. S. L. Moores,
    7. K. Ley,
    8. W. Swat,
    9. T. Mayadas,
    10. J. S. Brugge
    . 2004. Vav GEFs are required for β2 integrin-dependent functions of neutrophils. J. Cell Biol. 166: 273–282.
    OpenUrlAbstract/FREE Full Text
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