Perinuclear Anti-Neutrophil Cytoplasmic Antibodies (pANCA) Impair Neutrophil Candidacidal Activity and Are Increased in the Cellular Fraction of Vaginal Samples from Women with Vulvovaginal Candidiasis

Abstract

Vulvovaginal candidiasis (VVC) is primarily caused by Candida albicans and affects 75% of childbearing age women. Although C. albicans can colonize asymptomatically, disease is associated with an increased Candida burden, a loss of epithelial tolerance and a breakdown in vaginal microbiota homeostasis. VVC symptoms have been ascribed to a powerful inflammatory response associated with the infiltration of non-protective neutrophils (PMN). Here, we compared the immunological characteristics of vaginal fluids and cellular protein extracts obtained from 28 VVC women and from 23 healthy women colonized by Candida spp. We measured the levels of antibodies against fungal antigens and human autoantigens (anti-Saccharomyces cerevisiae antibodies (ASCA), C. albicans germ tube antibodies (CAGTAs) and perinuclear anti-neutrophil cytoplasmic antibodies (pANCA)), in addition to other immunological markers. Our results show that the pANCA levels detected in the cellular protein extracts from the vaginal fluids of symptomatic women were significantly higher than those obtained from healthy colonized women. Consistent with a potential physiologically relevant role for this pANCA, we found that specific anti-myeloperoxidase antibodies could completely neutralize the ex vivo killing capacity of polymorphonuclear cells. Collectively, this preliminary study suggests for the first time that pANCA are found in the pathogenic vaginal environment and can promptly impair neutrophil function against Candida, potentially preventing a protective response.

Keywords: ASCA; CAGTA; Candida; VVC; pANCA.

Figure 1

PMN number, CD11b and MPO gene expression and S100A8 determination. (A) Mean ± SEM of PMN number/field from vulvovaginal candidiasis (VVC) or colonized women. (B,C) Mean ± SEM of the relative expression of CD11b (B) and MPO (C) genes from cDNA obtained from cellular pellets of VVC and colonized women (data were obtained from the analysis of samples indicated in Table S1). (D) S100A8 was assessed in vaginal fluids (VF) of VVC and colonized women. The samples from women colonized by non-albicans Candida (NAC) species are colored in magenta. Level of significance is indicated by **** p < 0.0001 and by * p = 0.0127.

Figure 2

C. albicans germ tube antibody (CAGTA) IgA determination. (A): Pie charts of CAGTA IgA in positive (magenta) and negative (black) samples from VF of VVC women and C. albicans- or NAC-colonized women. (B): Representative images from samples with no CAGTA IgA (colonized women: CAGTA IgA negative, SP-8605) and with CAGTA IgA (VVC women: CAGTA IgA positive, SP-4900). The Candida cells shown are used to capture specific CAGTA and are part of the analysis kit. Magnification = 40X; scale bar = 50 μm.

Figure 3

pANCA levels in VF and PE. (A,B) Mean ± SEM of pANCA IgG (pg/mL) in vaginal fluids (VF) and in protein extracts (pg/μg) (PE). (C) pANCA IgG index in VF + PE expressed as arbitrary units (A.U.) from VVC and colonized women. The samples from women colonized by NAC species are colored in magenta. Levels of significance are indicated by ** p < 0.01 and * p = 0.0496.

Figure 4

Candidacidal activity. (A) Mean ± SEM of the percentage killing activity of human neutrophils (PMN) against C. albicans SC5314 in the presence or absence of pANCA. Significance is indicated with ** p < 0.01. (B) Oxidative burst of neutrophils in the presence or absence of pANCA was kinetically analyzed (for 80 cycles, where 1 cycle was 5 min) after in vitro exposure to C. albicans (SC5314). B-inset highlights the oxidative burst of PMN against C. albicans SC5314 (already shown in Panel B) without pANCA treatment. Results are the means of two independent experiments with duplicate samples.