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Citation: Collette JR, Zhou H, Lorenz MC (2014) Candida albicans Suppresses Nitric Oxide Generation from Macrophages via a Secreted Molecule. PLoS ONE 9(4): e96203. doi:10.1371/journal.pone.0096203
Editor: Neeraj Chauhan, New Jersey Medical School, Rutgers University, United States of America
Received: February 14, 2014; Accepted: April 4, 2014; Published: April 22, 2014
Copyright: © 2014 Collette et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by U.S. Public Health Service awards NIH R01AI075091 and NIH R21AI071134 to MCL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Editor: Neeraj Chauhan, New Jersey Medical School, Rutgers University, United States of America
Received: February 14, 2014; Accepted: April 4, 2014; Published: April 22, 2014
Copyright: © 2014 Collette et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by U.S. Public Health Service awards NIH R01AI075091 and NIH R21AI071134 to MCL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Candida albicans is a commensal fungus residing on the skin and in the oral cavity, gastrointestinal tract, and vagina of the human host. C. albicans cause a variety of superficial infections in individuals with certain risk factors, including oral thrush, vulvovaginal candidiasis, and various cutaneous infections. People with a significant immunodeficiency are more vulnerable to disseminated infection, which causes severe disease frequently resulting in death. The various defenses of the human innate immune system are primarily responsible for prevention of disseminated candidiasis [1].Phagocytic cells such as macrophages and neutrophils are important mediators of innate immunity, initiating a robust antimicrobial response upon recognition and internalization of potential pathogens. Activation of macrophages and neutrophils results in the synthesis of antimicrobial effectors, including reactive oxygen species (ROS) and reactive nitrogen species (RNS), the latter generated by the inducible nitric oxide synthase (iNOS, NOS2). SCID mice are more susceptible to mucosal Candida infections upon iNOS inhibition and macrophages from these mice had a reduced candidacidal activity upon treatment with an iNOS inhibitor in vitro [2]. However, other studies have shown that NO production may not be required to prevent Candida infections [3]–[5], and the exact role of NO in antifungal defense remains unresolved.
Upon NO exposure, C. albicans initiates a significant transcriptional response including the upregulation of the NO-detoxifying flavohemoglobin gene YHB1 [6], [7]. Two additional genes, YHB4 and YHB5, also encode flavohemoglobins suggesting that C. albicans considers NO a significant enough danger to create and maintain such a detoxifying system. Strains lacking YHB1, but not YHB4 or YHB5, were NO hypersensitive in vitro, but had only a modest virulence defect in the mouse model of disseminated hematogenous candidiasis [6], [7] suggesting that the additional flavohemoglobin genes might also be required for the establishment of an infection and/or that NO plays a limited role in antifungal defenses.
C. albicans also modulates other macrophage responses. Phagocytosed cells appear to follow a non-classical endocytic pathway [8], though the exact route and mechanism remain unclear. They also actively modulate the pH of the phagolysosome, potentially through the excretion of ammonia generated from amino acid catabolism, creating a neutral pH compartment conducive to hyphal morphogenesis [9], [10]. Secreted superoxide dismutases (SODs) of C. albicans detoxify extracellular ROS species: deletion of the SOD4 and SOD5 genes results in extensive ROS accumulation and reduced viability during co-culture with macrophages [11]. In addition, C. albicans can block NO production from phagocytic cells [12], [13], however the identification of the NO-inhibitory activity and its mechanism of action have not yet been determined.
In this report, we demonstrate that C. albicans produces an NO-inhibitory activity upon culturing with primary (bone marrow-derived) and tissue culture macrophages. This NO-inhibitory activity is secreted into co-culture supernatants and requires direct contact between fungal cells and macrophages. The effects on NO production are likely mediated by a failure to fully induce iNOS upon stimulation in the presence of this inhibitor, which preliminary characterization indicates is a small, aqueous, heat-stable compound. These findings represent another layer of modulation of phagocyte function by this important pathogen.
Methods
Strains and media
C. albicans strains used in this study have been previously described and include the prototrophic wild-type strain SC5314, the non-filamentous cph1Δ efg1Δ double mutant HLC54, the arg1Δ mutant JRC12, and the arg1Δ + ARG1 complemented strain JRC29 [10], [14]–[16]. The arg4Δ (SN152) and arg5,6Δ (CNC44) strains were kindly provided by S. Noble and J. Pla [17], [18]. The S. cerevisiae strains EM93 and BY4741 have also been described [19]–[21]. Fungal strains were propagated in YPD (1% yeast extract, 2% peptone, and 2% glucose) under standard conditions [22]. RAW264.7 and J774A.1 macrophages (ATCC) were cultured at 37°C with 5% CO2 in RPMI culture media supplemented with 10% fetal bovine serum (FBS), 100U/mL penicillin and 100µg/mL streptomycin (Pen-Strep).Bone marrow-derived macrophages (BMDMs) were isolated from sacrificed ICR mice as described previously [23] and cultured at 37°C with 5% CO2 in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% FBS, Pen-Strep, and 10 ng/mL mouse granulocyte-macrophage colony-stimulating factor (GM-CSF). All animal work was performed in accordance with protocols approved by the Animal Welfare Committee of the University of Texas Health Science Center at Houston.
Fungal:macrophage co-cultures and NO determination
Nitric oxide (NO) was assayed in supernatants of macrophage-C. albicans cocultures in a 24-well plate format using the Greiss reagent to detect nitrite, the product of spontaneous degradation of NO. Macrophages were collected and counted using a hemocytometer and 3×105 cells were seeded into wells of a 24-well plate at least 2 hours prior to the initiation of co-culture with fungi. C. albicans or S. cerevisiae were grown overnight in YPD, diluted 1:100 in fresh YPD, and grown for up to 4 hours at 30°C to allow cells to reach exponential growth. Cells were pelleted, resuspended in phosphate-buffered saline (PBS), counted, and incubated with macrophages at the indicated ratios. One hour following addition of the fungi, co-cultures were treated with LPS (100 ng/mL) and IFNγ (100U/mL) and incubated for ~24 hours at 37°C/5% CO2. Nitrite was quantified using the Greiss Reagent following the manufacturer's protocol (Molecular Probes).To assess the role of C. albicans viability on NO production by macrophages, exponentially growing fungal cells were heat-killed via incubation at 65°C for 20 minutes prior to co-culturing with macrophages. The requirement for direct contact was assayed by measuring NO production from RAW264.7 macrophages cultured in wells of a 24-well plate with fungal cells present either in the same chamber or separated by a membrane in Transwell inserts (0.4µm polycarbonate membrane, Corning Inc.).
Collection and manipulation of co-culture supernatants
Culture media was collected from LPS/IFNγ-treated C. albicans:RAW264.7 macrophage co-cultures, centrifuged to pellet any cellular debris/dead cells, and supernatant sterilized by passage through a 0.2 µm nylon syringe filter. To assess the ability of this conditioned media to inhibit NO production, naïve macrophages were cultured in a 1:1 mixture of the sterilized supernatant and fresh media for at least 2 hours, then stimulated with LPS and IFNγ.Conditioned media from C. albicans:macrophage co-cultures were size-fractionated using Centricon centrifugal filtration devices with a 3 kDa molecular weight cutoff (MWCO) membrane, separating the supernatant into flow-through (less than 3 kDa) and retained (over 3 kDa) fractions. These samples were dried, resuspended in water, and added to naïve RAW264.7 macrophages stimulated with LPS/IFNγ. Separately, conditioned media was also mixed 1:1 with chloroform to generate aqueous and organic phases. Aqueous and organic layers were separated, dried, resuspended in water, and added to macrophages stimulated with LPS/IFNγ. In addition, macrophages were incubated with conditioned media that had been boiled for 20 minutes at 100°C. For all conditions, nitrite concentrations in the culture media were assayed after 24 hours.
Stability of nitrite supernatant over time was determined by assaying nitrite levels in sterilized co-culture conditioned media for 1–24 hours using the Greiss reagent. Separately, the NO donor DETA-Nonoate (Cayman Chemical) was added to fresh cell-free media and its concentration was assayed over 24 hours.
Macrophage lysates and Western blotting
2×106 RAW264.7 macrophages were seeded into wells of a 12-well plate for at least 2 hours prior to the initiation of LPS/IFNγ stimulation. Immediately prior to the addition of LPS/IFNγ, cells were washed with PBS and incubated with either fresh RPMI or undiluted co-culture supernatant. Following 8 hours of LPS/IFNγ stimulation, cells were washed with PBS and collected by scraping. Cell pellets were resuspended in RIPA buffer (25 mM TRIS-HCl pH 7.6, 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS) and incubated on ice for 5 minutes. Lysates were centrifuged at 14,000×g for 10 minutes at 4°C after which supernatants were transferred to new tubes. Protein concentrations of cell lysates were determined using the Bio-Rad Protein Assay Dye Reagent, and 20 µg of total cellular protein per sample was loaded onto an 8% SDS-polyacrylamide gel. Blots were probed with iNOS antiserum (Santa Cruz Biotechnologies) at a 1:500 dilution followed by HRP-conjugated anti-rabbit IgG antiserum at a 1:3000 dilution. As a control for equivalent protein loading, membranes were also probed with anti-tubulin antiserum (a gift from Dr. Eric Wagner, UT-Houston).iNOS activity assays
Intracellular NO production was measured via the fluorimetric Nitric Oxide Synthase Detection System (Sigma-Aldrich), which is based on the iNOS-dependent conversion of a cell-permeable diacetate derivative of 4,5-diamino-fluorescein (DAF-2 DA) to the membrane- impermeant fluorescent molecule triazolofluorescein (DAF-2T) via a 4,5-diamino-fluorescein (DAF2) intermediate. Fluorescence of DAF-2T was measured using a Synergy MX multimode plate reader (Bio-Tek, Winooski, VT) with an excitation wavelength of 490 nm and an emission wavelength of 520 nm.RAW264.7 macrophages were seeded into wells of a black-walled, clear bottom 96-well tissue culture plates and incubated for 2 hours to allow for adherence. Macrophages were washed with PBS and stimulated with LPS/IFNγ in either RPMI media or co-culture supernatant for ~24 hours before initiating the iNOS activity assay according to the manufacturer's protocol.
Results
C. albicans actively suppresses NO production from macrophages
Nitric oxide (NO) is a key component of the antimicrobial burst of phagocytic cells. To assess whether C. albicans affects NO production, RAW264.7 mouse macrophages were stimulated with lipopolysaccharide (LPS) and interferon-γ (IFNγ) to upregulate iNOS [24] and co-cultured with increasing amounts of C. albicans strain SC5314 for 24 hours. NO is rapidly oxidized in aqueous solutions to nitrite, which was detected in co-culture supernatants via the Greiss reagent. When increasing numbers of fungal cells are co-cultured with a fixed number of macrophages, a corresponding decrease in the amount of NO produced is observed (Figure 1A). This phenomenon is quite robust as a ratio of one C. albicans cell per 100 macrophages (0.01:1) resulted in a ~75% reduction in NO production, and an almost complete block was observed at fungal cell:macrophage ratios of 0.1:1. To determine if live fungal cells were necessary for this NO suppression, LPS/IFNγ-stimulated RAW264.7 macrophages were co-cultured with heat-killed fungal cells and assessed for NO production. As shown in Figure 1A, inviable C. albicans cells reduced NO production by only ~10–20% regardless of the fungal cell:macrophage ratio. This is in sharp contrast to the dose-dependent suppression obtained by live C. albicans cells and strongly supports the notion that NO inhibition by C. albicans is an active process requiring fungal viability.
Figure 1. Candida albicans actively inhibits nitric oxide (NO) synthesis by macrophages.
A. Exponentially growing C. albicans cells were mock-treated (Live) or heat-killed prior to co-culturing with LPS/IFNγ-stimulated RAW264.7 mouse macrophages at the indicated C. albicans:macrophage ratio. B. Exponentially growing C. albicans or S. cerevisiae cells were co-cultured with RAW264.7 macrophages at the indicated ratios as in panel A. C. Exponentially growing wildtype (SC5314) or the non-filamentous cph1Δ efg1Δ mutant (HLC54) C. albicans cells were co-cultured with J774A.1 mouse macrophages as in panel A. D. Exponentially growing C. albicans cells were co-cultured with mouse bone marrow-derived mouse macrophages as in panel A. For all panels, supernatants were collected following ~24 hours of co-culture, and nitrite levels were determined using the Greiss Reagent.
doi:10.1371/journal.pone.0096203.g001
Following phagocytosis by macrophages, C. albicans fungal cells will initiate hyphal growth to facilitate escape from acidic phagosomes and then ultimately escape from the phagocyte. To address whether or not the reduction in macrophage NO production by C. albicans is due to extensive macrophage lysis during the course of these experiments, we utilized the non-filamentous mutant C. albicans strain HLC54 [16] in co-cultures with a second murine macrophage-like cell line, J774A.1. Dose-dependent suppression of NO production was observed with these non-filamentous fungal cells (Figure 1C), however greater numbers of HLC54 cells were required to observe this suppression as compared to the hyphal-competent SC5314 strain. We conclude from this experiment that hyphal morphogenesis is not required for this phenomenon, but that it contributes to NO inhibition, potentially because of increased macrophage damage.
The experiments in Figs. 1A–B used RAW264.7 cells while those in Fig. 1C used J774A.1 cells. These cell line are the most common used for in vitro studies with C. albicans, but both have alterations that may affect fungal recognition. RAW264.7 cells, for instance, lack the ASC component of the inflammasome while J774A.1 cells do not express the mannose receptor [25]. The ability of wild-type C. albicans to inhibit NO production is essentially the same in both cell lines (Fig. 1A–C). We also investigated whether NO inhibition by C. albicans is a phenomenon observed in primary macrophages. Mouse bone marrow-derived macrophages (BMDMs) were stimulated with LPS/IFNγ and co-cultured with increasing amounts of C. albicans. Thus, C. albicans suppresses NO production from two different cell lines as well as primary cells (Fig. 1), indicating that this is a general phenomenon. For simplicity, cultured macrophages were utilized for the remainder of the experiments.
Macrophages activated by LPS/IFNγ stimulation induce robust iNOS expression, but in vivo C. albicans recognition by phagocytes is mediated by a variety of receptors that recognize fungal associated molecular patterns, including Dectin-1, Dectin-2, the Mannose Receptor, and several Toll-Like Receptors [1]. To verify that the failure to induce NO production is not due to a lack of recognition of the fungal cells or interference with LPS/IFNγ signaling, we stimulated RAW264.7 macrophages either with IFNγ/LPS or with live cells of S. cerevisiae strain BY4741, in the presence or absence of the non-filamentous C. albicans strain HLC54 (used to minimize macrophage damage from hyphal growth). As shown in Fig. 2, S. cerevisiae cells induced robust NO production, comparable to LPS/IFNγ, indicating that these cells are activated in response to fungal antigens. In contrast, little NO was detected when macrophages were incubated with S. cerevisiae in the presence of C. albicans. Nearly identical results were obtained using J774A.1 cells. Thus, C. albicans inhibits NO synthesis induced by multiple signaling pathways.
Figure 2. NO inhibition by C. albicans in the absence of LPS/IFNγ stimulation.
Unstimulated RAW264.7 macrophages were co-cultured with either live S. cerevisiae strain BY4741, live C. albicans strain HLC54, or both (at a 1:1 ratio). Following ~24 hours of co-culture, supernatants were collected and nitrite levels assayed via the Greiss Reagent. Nitrite levels are expressed as a percentage of that generated by RAW264.7 macrophages stimulated with LPS/IFNγ for ~24 hours in the absence of fungi. The ‘*’ indicates a p-value of <0.05 relative to IFNγ/LPS or S. cerevisiae alone.
doi:10.1371/journal.pone.0096203.g002
Direct contact between C. albicans and macrophages required for full NO suppression
Macrophages generate antimicrobials such as NO following recognition and phagocytosis of various pathogenic microbes. The requirement for direct contact between C. albicans and macrophages in order to block NO production was assessed by culturing each cell type on opposite sides of a Transwell barrier containing pores too small for the diffusion of whole cells but suitable for the diffusion of small molecules. Nitric oxide levels in co-cultures of LPS/IFNγ-stimulated macrophages and C. albicans on the same side of the Transwell were dramatically lower than in control macrophage cultures (Figure 3A, Regular Well). However, when macrophages and fungal cells were cultured on opposite sides of the Transwell barrier NO synthesis was unchanged (Figure 3A, Transwell), indicating that direct contact was necessary for the NO inhibitory activity of C. albicans.
Figure 3. NO inhibition by C. albicans requires direct C. albicans-macrophage contact and is mediated by a secreted activity.
A. C. albicans and LPS/IFNγ-stimulated RAW264.7 macrophages were co-cultured on either the same side (Regular Well) or the opposite side (Transwell) of polycarbonate membrane Transwell inserts containing 0.4µm pores. Supernatants were collected following ~24 hours of co-culture, and nitrite levels were determined using the Greiss Reagent. B. LPS/IFNγ-stimulated RAW264.7 macrophages were cultured with either live C. albicans (Fungi) at the indicated ratio or with culture media (co-culture supernatant) previously collected from C. albicans: RAW264.7 macrophage co-cultures. Supernatants were collected following ~24 hours of culture, and nitrite levels were determined using the Greiss Reagent.
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C. albicans secretes an NO inhibitory activity
While direct contact between C. albicans and macrophages is required for suppression of NO production, this may be due to a cell autonomous secreted molecule produced only upon contact/phagocytosis or through a cell-dependent mechanism. To address this question, we assayed cell-free co-culture conditioned media for suppressive activity. To do this, culture media was collected from C. albicans-macrophage co-cultures of LPS/IFNγ-stimulated macrophages and C. albicans, filter-sterilized, and mixed 1:1 with fresh RPMI + serum to ensure sufficient nutrients were present to support macrophage growth. In this media, naïve macrophages stimulated with LPS/IFNγ in the absence of fungal cells produced significantly less NO than controls (Figure 3B). The decrease in NO production observed in macrophages cultured with sterilized co-culture supernatant was roughly half than seen with live cells, which is consistent with the 1:1 dilution of the conditioned media. Supernatant taken from monocultures of C. albicans or macrophages alone in RPMI + serum did not inhibit NO production from stimulated macrophages (data not shown). Thus, the inhibitory activity is produced only after direct cell contact but is then secreted into the supernatant where it can act autonomously.It remained possible that the secreted activity was not truly blocking NO production by iNOS, but rather was masking the actual amount of nitrite present in the culture media by converting it into a form not detectable by the Greiss reaction. If so, the amount of detectable nitrite in conditioned media should decay over time through the action of the inhibitor. To address this possibility, we collected and sterilized conditioned media from a co-culture and incubated it at 37°C for up to 24 hours. Nitrite concentrations in this media were stable over time (Figure 4A), indicating that the C. albicans-derived activity did not affect nitrite once it was generated.
Figure 4. The secreted NO inhibitory activity of C. albicans does not sequester NO upon addition of an exogenous NO donor.
A. Conditioned media from a 24-culture was filter sterilized and incubated for 24 hours at 37°C. At the indicated times, aliquots were removed and assayed for nitrite concentration. B. 100 µM DETA-NONOate was added to fresh media or filter sterilized conditioned media from a 24 hour co-culture (CCS) and incubated at 37°C. Nitrite concentrations were assayed at the indicated times using the Greiss Reagent.
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NO inhibition is mediated by a small, aqueous, heat-stable compound
Identification of the secreted compound(s) responsible for this NO suppression by C. albicans will be critical for understanding the mechanism of action. The composition of the co-culture supernatant is quite extensive, containing a broad spectrum of molecular species including ions, co-factors, vitamins, carbohydrates, and proteins. Experiments were conducted to discriminate among these numerous possibilities. To obtain information about the molecular weight of the NO inhibitory activity, filter-sterilized conditioned media from C. albicans: macrophage co-cultures were size fractionated using centrifugal filtration devices with a 3 kDa MWCO, separating the supernatant into flow-through and retained fractions. Fractions were incubated with LPS/IFNγ-stimulated RAW264.7 macrophages and nitrite concentrations in the culture media were assayed following 24 hours. Nitrite levels in macrophages treated with the flow-through fraction were comparable to the levels observed in macrophages treated with co-culture supernatant (CCS) (Figure 5A), indicating that this NO inhibitory activity passed through the filter and was less than ~3 kDa in size. A small amount of activity was observed upon addition of the retained fraction (Figure 5A), however the presence of this weaker activity can be attributed to the retention of a small volume of the residual liquid in the upper chamber of the filter following centrifugation.
Figure 5. C. albicans NO inhibition is mediated by a small, aqueous, and heat-stable activity.
A. Conditioned media from C. albicans:macrophage co-cultures were size fractionated using Centricon centrifugal filtration devices containing a 3 kDa MWCO, separating the supernatant into flow-through and retained fractions. These samples were dried, resuspended in water, and added to naïve RAW264.7 macrophages stimulated with LPS/IFNγ. Stimulated macrophages, macrophages co-cultured with live C. albicans, and the complete co-culture supernatant (CCS) are used as controls. B. Conditioned media was prepared as in panel A and mixed 1:1 with chloroform. The organic and aqueous layers were separated, dried, and resuspended in water, then added to stimulated cultures of naïve macrophages. Separately, stimulated macrophages were incubated in conditioned media that had been boiled for 20 minutes at 100°C. Stimulated macrophages and complete co-culture supernatant (CCS) are used as controls. For both panels A and B, nitrite concentrations in the culture media were determined after 24 hours. Data statistically significant from the control stimulated macrophages (p<0.05) is indicated by ‘*’. In panel A, the ‘†’ indicates data that is not significantly different from the positive (C. albicans) or negative (stimulated) controls, likely because of the dilution of the supernatant into fresh media. In panel B, the ‘†’ indicates data intermediate between the stimulated control and the unfractionated co-culture supernatant.
doi:10.1371/journal.pone.0096203.g005
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