Echinocandin Treatment of Candida albicans Biofilms Enhances Neutrophil Extracellular Trap Formation

The nosocomial pathogen Candida albicans forms biofilms on medical devices that persist in the face of antifungals and host defenses. Echinocandins, the most effective antibiofilm drugs, have recently been shown to augment the activity of neutrophils against biofilms through an unknown mechanism.

ABSTRACT

The nosocomial pathogen Candida albicans forms biofilms on medical devices that persist in the face of antifungals and host defenses. Echinocandins, the most effective antibiofilm drugs, have recently been shown to augment the activity of neutrophils against biofilms through an unknown mechanism. Here, we show that treatment of C. albicans biofilms with subinhibitory concentrations of echinocandins promotes the formation of neutrophil extracellular traps (NETs), structures of DNA, histones, and antimicrobial proteins with antifungal activity.

TEXT

Candida albicans forms resilient biofilms on vascular catheters, urinary catheters, and dentures (1, 2). In these protective communities, C. albicans resists immune attack and withstands high concentrations of antifungals (3–8). Biofilm formation frequently complicates the treatment of candidiasis, and retained infected devices can lead to prolonged infections and poor outcomes (9, 10). Given the profound multidrug resistance observed for C. albicans biofilms, novel approaches to treatment are of great interest.

One antibiofilm tactic is to devise strategies that augment the host immune response to these infections. Neutrophils, which are critical for control of numerous fungal infections, exhibit very little activity against C. albicans biofilms (6–8). However, Katragkou et al. (11) found that anidulafungin, an antifungal of the echinocandin drug class, can enhance the activity of neutrophils against C. albicans biofilms. In the current report, we analyze neutrophil-biofilm interactions to identify the mechanism of the synergy observed between echinocandins and neutrophils against biofilms.

We first identified subinhibitory concentrations of echinocandins against mature C. albicans biofilms to use in biofilm-neutrophil studies. We utilized an XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay to examine the impact of caspofungin (Apexbio Technology LLC), anidulafungin (Apexbio Technology LLC), and micafungin (Astellas) on 24-h C. albicans SC5314 biofilms (12, 13). Antifungals diluted in RPMI 1640 (without phenol red) were applied to biofilms for 24 h. The results of XTT metabolic assays revealed a dose-dependent inhibition of mature C. albicans biofilms after exposure to each echinocandin (Fig. 1A to C). The subinhibitory concentrations selected for further study were not statistically different from those of untreated controls and were 4-fold lower than the lowest inhibitory concentrations. These included 0.05, 0.025, and 0.0015 μg/ml for micafungin, caspofungin, and anidulafungin, respectively.

FIG 1.

Identification of subinhibitory concentrations of echinocandins. (A to C) The impact of 24 h of echinocandin treatment on 24-h C. albicans biofilms was assessed by XTT assay (n = 3). Replicate with SD is shown. *, P < 0.05 by Student's t test compared to untreated control. (D) By scanning electron microscopy, treatment of C. albicans biofilms with micafungin (0.05 μg/ml), caspofungin (0.025 μg/ml), and anidulafungin (0.0015 μg/ml) did not impact biofilm architecture. Bars represent 10 and 1 μm for the 2,000× and 10,000× images, respectively.

Because echinocandin treatment can influence fungal morphology, we examined the cellular architecture of biofilms that had been exposed to these subinhibitory concentrations by scanning electron microscopy using a coverslip model of biofilm formation (6). Briefly, C. albicans biofilms were grown in RPMI-MOPS (morpholinepropanesulfonic acid) on coverslips for 24 h at 37°C before a 24-h treatment with media containing subinhibitory concentrations of echinocandins, followed by processing for imaging (6). On scanning electron microscopy, biofilms treated with these concentrations were similar in appearance, consisting primarily of hyphae (Fig. 1D). Abnormalities in cellular morphology were not observed.

To gain insight into the synergism observed between neutrophils and echinocandins against C. albicans biofilms, we cocultured neutrophils with untreated biofilms and those treated with subinhibitory concentrations of echinocandins. Human peripheral blood was obtained from donors with written informed consent through a protocol approved by the University of Wisconsin Internal Review Board, and neutrophils were purified using the MACSxpress neutrophil isolation and MACSxpress erythrocyte depletion kits (Miltenyi Biotec, Inc., Auburn, CA). Neutrophils (5 × 10⁵) in RPMI 1640 supplemented with 2% heat-inactivated fetal bovine serum (FBS) and glutamine (0.3 mg/ml) were added to coverslip biofilms for 4 h and processed for imaging (6). On scanning electron microscopy, neutrophils applied to untreated biofilms for 4 h appeared rounded, as previously described (6). In contrast, neutrophils exposed to echinocandin-treated biofilms produced weblike lattices consistent with the formation of NETs (Fig. 2A) (14).

FIG 2.

Echinocandin treatment of C. albicans biofilms triggers neutrophils to form NETs. C. albicans biofilms were incubated in the presence of subinhibitory concentrations of echinocandins for 24 h and then cocultured with human neutrophils for 4 h. (A) By scanning electron microscopy, NETs were triggered in response to echinocandin-treated biofilms. Bars represent 10 and 1 μm for the 2,000× and 10,000× images, respectively. (B) NET release was estimated by Sytox green detection of free DNA. NET formation in response to a potent inducer of NETs, PMA, is shown for comparison. Experiments were performed in triplicate on six occasions (SEM shown). *, P < 0.05 by paired t test.

To quantify the production of NETs, we utilized Sytox green staining of free DNA (Fig. 2B) (6). Neutrophils were added to echinocandin-treated and untreated biofilms in a 96-well plate to a final concentration of 2 × 10⁵ cells/well. After 4 h of incubation, Sytox green was added at a final concentration of 1 μM, and fluorescence (excitation 500 nm/emission 528 nm) was measured. Background fluorescence for each condition was subtracted from total fluorescence values. In response to untreated biofilms, neutrophils produced very little free DNA, suggesting minimal NET formation, as previously described for biofilm-neutrophil interactions (6). However, treatment of biofilms with subinhibitory concentrations of echinocandins resulted in elevated free DNA, approximately 6- to 8-fold above the levels measured for untreated biofilms and reaching up to a third of the levels observed in response to 100 nM phorbol 12-myristate 13-acetate (PMA), a potent stimulus for NET formation. These findings are consistent with the release of NETs, an observation confirmed by scanning electron microscopy (Fig. 2A).

Neutrophils have been shown to form NETs in response to nonbiofilm C. albicans, and the importance of this process for control of candidiasis and other invasive fungal infections has been demonstrated (15–17). However, biofilm formation by C. albicans inhibits this process, which contributes to the resistance of biofilms to neutrophil attack (18, 19). Interestingly, biofilms exhibit susceptibility to NETs if induced through other stimuli, suggesting that they would be an effective mechanism of killing if initiated on biofilm. Here, we show that neutrophils release NETs in response to echinocandin-treated biofilms. This altered response likely contributes to the synergy observed for neutrophils uniquely with this drug class (11).

During planktonic growth, treatment of C. albicans infection with subinhibitory concentrations of echinocandins disrupts cell wall assembly and leads to a greater exposure of β-glucan, a proinflammatory polysaccharide that is normally masked (20). This unmasking of glucan occurs preferentially in hyphal cells and is unique to treatment with the echinocandin drug class (21). Because β-glucan has been shown to induce neutrophils to form NETs, unmasked glucan following echinocandin treatment of biofilms may serve as a trigger for NET release (22). Considering that echinocandin treatment of Candida parapsilosis biofilms also augments the activity of neutrophils, a similar mechanism of neutrophil activation may be triggered in response to this pathogen (23). Understanding how antifungal therapy modulates the innate immune response may open new avenues to augment the host response to invasive fungal infections.