β-Glucan induces reactive oxygen species production in human neutrophils to improve the killing of Candida albicans and Candida glabrata isolates from vulvovaginal candidiasis

Abstract

Vulvovaginal candidiasis (VVC) is among the most prevalent vaginal diseases. Candida albicans is still the most prevalent species associated with this pathology, however, the prevalence of other Candida species, such as C. glabrata, is increasing. The pathogenesis of these infections has been intensely studied, nevertheless, no consensus has been reached on the pathogenicity of VVC. In addition, inappropriate treatment or the presence of resistant strains can lead to RVVC (vulvovaginal candidiasis recurrent). Immunomodulation therapy studies have become increasingly promising, including with the β-glucans. Thus, in the present study, we evaluated microbicidal activity, phagocytosis, intracellular oxidant species production, oxygen consumption, myeloperoxidase (MPO) activity, and the release of tumor necrosis factor α (TNF-α), interleukin-8 (IL-8), IL-1β, and IL-1Ra in neutrophils previously treated or not with β-glucan. In all of the assays, human neutrophils were challenged with C. albicans and C. glabrata isolated from vulvovaginal candidiasis. β-glucan significantly increased oxidant species production, suggesting that β-glucan may be an efficient immunomodulator that triggers an increase in the microbicidal response of neutrophils for both of the species isolated from vulvovaginal candidiasis. The effects of β-glucan appeared to be mainly related to the activation of reactive oxygen species and modulation of cytokine release.

Figure 1. Microbicidal activity of β-glucan-treated neutrophils activated by different isolates of C. albicans and C. glabrata.

Neutrophils (2.0×10⁶ cells/ml) were previously treated or not with 3 mg/ml β-glucan and incubated with the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (RVVC, VVC, and ASS; 2.0×10⁷ CFU/ml) at 37°C for different times (0, 30, 60, 90, and 120 min). The quantity of viable yeast was estimated by plating the samples in Sabouraund Dextrose Agar (SDA) at 37°C for 24 h. The data are expressed as the mean ± SD of three separate experiments. * p≤0.05, significant difference compared with the control group (yeast alone); ^# p≤0.05, significant difference compared with untreated and activated neutrophils.

Figure 2. Phagocytosis activity of β-glucan-treated neutrophils activated by different isolates of C. albicans and C. glabrata. Neutrophils (2.0×10⁶ cells/ml) were previously treated or not with 3 mg/ml β-glucan and incubated for 1 h at 37°C with the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (RVVC, VVC, and ASS; 2.0×10⁷ CFU/ml) labeled with FITC.

Phagocytosis was determined by flow cytometry, and the results are expressed as the mean fluorescence (in arbitrary units [au]) ± SD of three independent experiments. ^# p≤0.05, significant difference compared with untreated and activated neutrophils.

Figure 3. Intracellular oxidant species production by β-glucan-treated neutrophils activated by different isolates of C. albicans and C. glabrata determined by flow cytometry.

Neutrophils (2.0×10⁶ cells/ml) were previously treated or not with 3 mg/ml β-glucan and incubated for 1 h with the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (RVVC, VVC, and ASS; 2.0×10⁷ CFU/ml), followed by 30 min incubation with DHR. The data are expressed as the mean ± SD of at least three independent experiments. * p≤0.05, significant difference compared with the control group (neutrophils alone); ^# p≤0.05, significant difference compared with untreated and activated neutrophils. (C and D) Representative dot plot display of FL1 (green fluorescence) vs. FL2 on a logarithmic scale. (C – (a) ATCC, (c) ASS, (e) VVC,(g) RVVC) C. albicans with untreated neutrophils. (C – (b) ATCC,(d) ASS,(f) VVC,(h) RVVC) C. albicans with neutrophils previously treated with 3 mg/ml β-glucan. (D – (a’) ATCC,(c’) ASS,(e’) VVC,(g’) RVVC) C. glabrata with untreated neutrophils. (D – (b’) ATCC,(d’) ASS,(f’) VVC,(h’) RVVC) C. glabrata with neutrophils previously treated with 3 mg/ml β-glucan.

Figure 4. HOCl production by β-glucan-treated neutrophils activated by different isolates of C. albicans and C. glabrata determined by spectrophotometry.

Neutrophils (2.0×10⁶ cells/ml) were previously treated or not with 3 mg/ml β-glucan and activated or not by the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (RVVC, VVC, and ASS; 2.0×10⁷ CFU/ml) and read at 655 nm. The data are expressed as the mean ± SD of three independent experiments. * p≤0.05, significant difference compared with the control group (neutrophils alone); ^# p≤0.05, significant difference compared with untreated and activated neutrophils.

Figure 5. Myeloperoxidase activity of β-glucan-treated neutrophils activated by different isolates of C. albicans and C. glabrata (integrated light emission).

The inset represents kinetic study of MPO activity of β-glucan-treated neutrophils after 20 minutes of incubation. Neutrophils (2.0×10⁶ cells/ml) were previously treated or not with 3 mg/ml β-glucan and incubated with the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (2×10⁷ CFU/ml) for 30 min. (a,a’) ATCC. (b,b’) ASS. (c,c’) VVC. (d,d’) RVVC. After incubation, chemiluminescence was monitored for 20 min at 37°C in a microplate luminometer using luminol as a chemical light amplifier. The data are expressed as the mean ± SD of three independent experiments. *p≤0.05, significant difference compared with the control group (neutrophils alone); ^# p≤0.05, significant difference compared with untreated and activated neutrophils.

Figure 6. Oxygen consumption by β-glucan-treated neutrophils activated by different isolates of C. albicans and C. glabrata.

Neutrophils (2.0×10⁶ cells/ml) were previously treated or not with 3 mg/ml β-glucan and activated or not by the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (RVVC, VVC, and ASS; 2.0×10⁷ CFU/ml). Oxygen consumption was monitored for 5–10 min and calculated from the polarographic recordings using an initial concentration of dissolved oxygen of 190 µM at 37°C. The data are expressed as the mean ± SD of three independent experiments. *p≤0.05, significant difference compared with the control group (neutrophils alone); ^# p≤0.05, significant difference compared with untreated and activated neutrophils.

Figure 7. Cytokine release by β-glucan-treated neutrophils activated by different isolates of C. albicans and C. glabrata.

Neutrophils (2.0×10⁶ cells/ml) were previously treated or not with 3 mg/ml β-glucan and activated or not by the reference strain and different isolates of (A) C. albicans and (B) C. glabrata (RVVC, VVC, and ASS; 2.0×10⁷ CFU/ml) and 1 µg/ml LPS and cultured for 18 h. (a, a’) IL-8. (b, b’) IL-1β. (c, c’) IL-1Ra. (d, d’) TNF-α. The data are expressed as the mean ± SD of three independent experiments. *p≤0.05, significant difference compared with the control group (neutrophils alone); ^# p≤0.05, significant difference compared with untreated and activated neutrophils.

Figure 8. (A) Thiol levels in different isolates of C. albicans (ASS, VVC, and RVVC) and (B) thiol levels in different isolates of C. glabrata (ASS, VVC, and RVVC) incubated with DTNB.

The data are expressed as the mean ± SD of three independent experiments. *p≤0.05, compared with ASS or control group (neutrophils).