Epitope unmasking in vulvovaginal candidiasis is associated with hyphal growth and neutrophilic infiltration

RESEARCH ARTICLE Epitope unmasking in vulvovaginal candidiasis is associated with hyphal growth and neutrophilic infiltration Eva Pericolini1,2¤, Stefano Perito1, Anna Castagnoli3, Elena Gabrielli1, Antonella Mencacci1, Elisabetta Blasi2¤, Anna Vecchiarelli1, Robert T. Wheeler1,4,5* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Medicine, University of Perugia, Perugia, Italy, 2 Department of Diagnostic, Clinical and Public Health Medicine, University of Modena and Reggio Emilia, Modena, Italy, 3 School of Specialization in Microbiology and Virology, University of Modena and Reggio Emilia, Modena, Italy, 4 Department of Molecular and Biomedical Sciences, University of Maine, Orono, Maine, United States of America, 5 Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, Maine, United States of America ¤ Current address: Department of Surgical, Medical, Dental and Morphological Sciences with interest in Transplant, Oncological and Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy * Abstract OPEN ACCESS Citation: Pericolini E, Perito S, Castagnoli A, Gabrielli E, Mencacci A, Blasi E, et al. (2018) Epitope unmasking in vulvovaginal candidiasis is associated with hyphal growth and neutrophilic infiltration. PLoS ONE 13(7): e0201436. https://doi. org/10.1371/journal.pone.0201436 Editor: Joy Sturtevant, Louisiana State University, UNITED STATES Received: March 16, 2018 Accepted: July 16, 2018 Published: July 31, 2018 Copyright: © 2018 Pericolini 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. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Funding for this study was provided by the Fulbright Commission of Italy (RTW), the Burroughs Wellcome Fund (RTW), and the Azienda Ospedaliera Universitaria-Policlinico di Modena (EB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Vaginal candidiasis is a common disorder in women of childbearing age, caused primarily by the dimorphic fungus Candida albicans. Since C. albicans is a normal commensal of the vaginal mucosa, a long-standing question is how the fungus switches from being a harmless commensal to a virulent pathogen. Work with human subjects and in mouse disease models suggests that host inflammatory processes drive the onset of symptomatic infection. Fungal cell wall molecules can induce inflammation through activation of epithelial and immune receptors that trigger pro-inflammatory cytokines and chemokines, but pathogenic fungi can evade recognition by masking these molecules. Knowledge about which cell wall epitopes are available for immune recognition during human infection could implicate specific ligands and receptors in the symptoms of vaginal candidiasis. To address this important gap, we directly probed the surface of fungi present in fresh vaginal samples obtained both from women with symptomatic Candida vaginitis and from women that are colonized but asymptomatic. We find that the pro-inflammatory cell wall polysaccharide β-glucan is largely masked from immune recognition, especially on yeast. It is only exposed on a small percentage of hyphal cells, where it tends to co-localize with enhanced levels of chitin. Enhanced β-glucan availability is only found in symptomatic patients with strong neutrophil infiltration, implicating neutrophils as a possible driver of these cell wall changes. This is especially interesting because neutrophils were recently shown to be necessary and sufficient to provoke enhanced β-glucan exposure in C. albicans, accompanied by elevated immune responses. Taken together, our data suggest that the architecture of C. albicans cell wall can be altered by environmental stress during vaginal candidiasis. PLOS ONE | https://doi.org/10.1371/journal.pone.0201436 July 31, 2018 1 / 14 Fungal epitope unmasking in human vaginitis Competing interests: The authors have declared that no competing interests exist. Introduction C. albicans causes both mucosal and disseminated disease, but vaginitis is the only disease it causes in healthy adults [1]. Acute vulvovaginal candidiasis (VVC) is estimated to affect two-thirds of all women during their lifetimes, while recurrent VVC (RVVC) is more severe but less common. Unfortunately, we still know little about why some women suffer from VVC or RVVC and others do not [2–5]. Both VVC and RVVC are usually caused by C. albicans, but can be caused by C. glabrata, C. krusei, and many other species [2, 4]. Therapy for VVC relies on a variety of azoles, which are of limited use due to concerns about the use of fluconazole in pregnancy [6–8]. Vaginitis symptoms from C. albicans infection are thought to stem from host immune responses rather than pathogen-mediated damage [9]. Support for this idea comes from a unique human challenge study that found that neutrophilic inflammation, rather than fungal burden, was the greatest predictor of symptomatic infection in otherwise healthy women [10]. Host inflammatory processes can be triggered by fungal cell wall molecules and proteins that interact with receptors of innate immune and epithelial cells [11–15]. Despite their well-recognized capacity to stimulate inflammation, we still understand little about the fungal cell wall ligands that are actually available for immune recognition during vaginal infection, especially during human infection. Pattern recognition of fungal cell wall components is limited by pathogens that regulate the availability of specific epitopes for interaction with immune receptors [16, 17]. C. albicans and other fungi can mask cell wall β-glucan and chitin from recognition by Dectin-1 and other PRRs, but it is unknown if this mechanism of evasion is used in human infection [16]. Some in vivo and in vitro models suggest that β-glucan is largely masked [18, 19], but other in vitro experiments have documented some β-glucan exposure during epithelial infection [20]. These differences among models may arise because each model presents a unique subset of signals that regulate exposure of cell wall epitopes, including environmental cues, stresses, and immune-triggered cell wall remodeling [17]. This is especially intriguing for VVC, where in vitro-validated cues for masking (lactate) and unmasking (low pH) are both expected to be present [21, 22]. It is difficult to exactly replicate the environmental conditions during human infection in vitro, especially because they are likely to be dynamic and to depend on infection progression in individual patients. Clinical studies of epitope exposure are technically and logistically challenging because epitope exposure is sensitive to fixation (...truncated)