Autophagy and Reactive Oxygen Species Are Involved in Neutrophil Extracellular Traps Release Induced by C. albicans Morphotypes

Abstract

Neutrophil extracellular traps (NETs) are a combination of DNA fibers and granular enzymes, such as elastase and myeloperoxidase. In this study, we demonstrate that Candida albicans hyphal (CAH) cells and yeast (CAY) cells induce differential amounts, kinetics and mechanisms of NET release. CAH cells induced larger quantities of NET compared to CAY cells and can stimulate rapid NET formation up to 4 h of incubation. CAY cells are, also, able to induce rapid NET formation, but this ability was lost at 4 h. Both reactive oxygen species (ROS) and autophagy are implicated in NET induced by CAH and CAY cells, but with a time-different participation of these two mechanisms. In particular, in the early phase (15 min) CAH cells stimulate NET via autophagy, but not via ROS, while CAY cells induce NET via both autophagy and ROS. At 4 h, only CAH cells stimulate NET formation using autophagy as well as ROS. Finally, we demonstrate that NET release, in response to CAH cells, involves NF-κB activation and is strongly implicated in hyphal destruction.

Keywords: C. albicans; NET; ROS; autophagy; neutrophils.

FIGURE 1

NETs formation induced by C. albicans. (A) Extracellular DNA released, in response to PMNs not stimulated (NS) or incubated with C. albicans hyphae (CAH) or live yeast (CALY), for 15 min or 4 h at 37°C and 5% of CO₂, was determined using SYTOXgreen probe, as described in Section “Materials and Methods.” Bars are the mean ± SEM of n = 7 experiments with similar results. ^∗P < 0.05 PMNs + CAH vs. PMNs NS; ^#P < 0.05 PMNs + CALY vs. PMN NS. (B) NET components (green: extracellular DNA; red: neutrophil elastase) were visualized by fluorescence microscopy after 4 h of PMNs incubation without stimuli (NS) or with C. albicans hyphae (CAH), live yeast (CALY), or heat inactivated yeast (CAIY). In the presence of DNAse-1 (100 U/ml), NET was completely degraded. Microphotography from a representative experiment (n = 3) are shown.

FIGURE 2

Inhibition of ROS production causes a reduction of NETs formation C. albicans induced. PMNs, pre-treated or not with N-acetyl L Cysteine (NAC, 1mM), were incubated without stimuli (NS) or with C. albicans hyphae (CAH), live yeast (CALY) or heat inactivated yeast (CAIY), and ROS production (A) and NETs formation (B) were determined as described in Section “Materials and Methods.” (A) The lines represent the kinetic of ROS release. The bars represent the total amounts of produced ROS obtained by analyzing the area under the curve (AUC). Lines are representatives of n = 5 experiments with similar results. Bars are the mean ± SEM of n = 5 experiments with similar results. ^∗P < 0.05 PMNs NAC pre-treated vs. PMNs not pre-treated. (B) NET formation in response to CAH, to CALY or to CAIY after 15 min and 4 h of incubation. Bars are the mean ± SEM of n = 7 experiments with similar results. ^∗P < 0.05 PMNs pre-treated with NAC + CAH or CALY vs. PMNs not pre-treated; ^#P < 0.05 PMNs + CAIY vs. PMNs NS.

FIGURE 3

Inhibition of autophagy causes a reduction of NETs release C. albicans mediated. PMNs, pre-treated or not with WT (1 nM), were incubated without stimuli (NS) or with C. albicans hyphae (CAH), live yeast (CALY) or heat inactivated yeast (CAIY) and Western Blotting experiments for LC3B-II expression (A) and NET release determination (B) were performed as described in Section “Materials and Methods.” (A) LC3B-II expression in response to CAH, to CALY or to CAIY after 15 min and 4 h of incubation. Actin was used as loading control. Optical density of reactive bands was measured and LC3B-II/LC3B-I ratio was evaluated. PMNs treated with DMSO alone were also run in parallel and the results were similar to those obtained in absence of WT. Blots and bars are representative of experiments (n = 3) with similar results. ^#P < 0.05 PMNs + CAH or CALY or CAIY vs. PMNs NS. (B) NETs formation in response to CAH, to CALY or to CAIY after 15 min and 4 h of incubation. PMNs treated with DMSO alone were also analyzed in parallel and the results were similar to those obtained in absence of WT. Bars are the mean ± SEM of n = 5 experiments with similar results.^∗P < 0.05 PMNs pre-treated with WT + CAH or CALY or CAIY vs. PMN not pre-treated.

FIGURE 4

Inhibition of NF-κB activation causes a reduction of NETs release C. albicans induced. PMNs, were incubated without stimuli (NS) or with C. albicans hyphae (CAH), live yeast (CALY) or heat inactivated yeast (CAIY) and (A) Western Blotting analysis for pNF-κB and (B) NET release determination were performed as described in Section “Materials and Methods.” (A) NF-κB activation in response to CAH, to CALY or to CAIY after 15 min and 4 h of incubation. Optical density of reactive bands was measured and normalized by the NF-κB density in the same line. pNF-κB was quantified relative to PMNs NS. Actin was used as loading control. Blots are representative of experiments (n = 3) with similar results. Bars are the mean ± SEM of experiments (n = 3) with similar results. ^∗P < 0.05 PMNs + CAH vs. PMN NS; ^#P < 0.05 PMNs + CALY or CAIY vs. PMN NS. (B) NET release determination in PMNs stimulated as above described, but pre-treated with NF-κB inhibitor (2.5 μM). The bars are the mean ± SEM of n = 5 experiments with similar results. ^∗P < 0.05 PMNs pre-treated with NF-κB inhibitor + CAH or CAIY vs. PMNs stimulated with CAH or CAIY.

FIGURE 5

Flow-cytometry analysis of pNF-κB. PMNs, pre-treated or not with NF-κB inhibitor (2.5 μM), were incubated without stimuli (NS) or with C. albicans hyphae (CAH), live yeast (CALY) or heat inactivated yeast (CAIY) for 15 min and 4 h. Then cytofluorimetric analysis (events = 5000) was performed as described in Section “Materials and Methods.” Filled histograms represent fluorescence in absence of NF-κB inhibitor pre-treatment, black lines represent fluorescence in presence of NF-κB inhibitor pre-treatment. A PE-conjugated isotype-matched mAb was used as a negative control. Neutrophils untreated or treated with PE-conjugated isotype-matched mAb showed similar results. Histograms are representative of n = 3 experiments with similar results.

FIGURE 6

Role of ROS production, autophagy and NF-κB in NETs released C. albicans induced. PMNs, pre-treated with NAC (A), or WT (B), or NF-κB inhibitor (2.5 μM) (C), were incubated without stimuli (NS) or with C. albicans hyphae (CAH), live yeast (CALY), or heat inactivated yeast (CAIY) for 15 min and 4 h. NETs formation was determined and percentage of NETs release reduction was calculated. The bars are the mean ± SEM of n = 5 experiments with similar results. ^∗P < 0.05 PMNs + CAH or CALY or CAIY vs. PMNs without stimuli.

FIGURE 7

NETs involvement on PMNs C. albicans killing. PMNs killing activity vs. C. albicans hyphal and yeast forms. The PMNs, pre-treated, or not (total killing), with DNase-1 (100 U/ml; extracellular killing without NET-mediated killing), or with Cytochalasin D (cytD; 10 μg/ml; external killing), were incubated, for 2 h, with CA1398 hyphal (A) or yeast (B) forms as described in Section “Materials and Methods.” Bars are the mean ± SEM of experiments (n = 3) with similar results. ^∗P < 0.05 CA1398 hyphal form killing by PMNs pre-treated with DNAse-I vs. PMNs untreated (TOT). (C) Percentage of killing NET mediated was calculated as described in Section “Materials and Methods.” Bars are the mean ± SEM of experiments (n = 3) with similar results.^∗P < 0.05 CA1398 hyphal form vs. CA1398 yeast.

FIGURE 8

Schematic representation of NET’s mechanism formation in response to C. albicans. C. albicans hyphae induces rapid NET via autophagy (LC3B-II) and C. albicans yeast both via autophagy than reactive oxygen species (ROS). Later, after 4 h of contact with PMNs, only hyphal form induces NET formation by using autophagy as well as reactive oxygen species.