The Interleukin (IL) 17R/IL-22R Signaling Axis Is Dispensable for Vulvovaginal Candidiasis Regardless of Estrogen Status

Abstract

Candida albicans, a ubiquitous commensal fungus that colonizes human mucosal tissues and skin, can become pathogenic, clinically manifesting most commonly as oropharyngeal candidiasis and vulvovaginal candidiasis (VVC). Studies in mice and humans convincingly show that T-helper 17 (Th17)/interleukin 17 (IL-17)–driven immunity is essential to control oral and dermal candidiasis. However, the role of the IL-17 pathway during VVC remains controversial, with conflicting reports from human data and mouse models. Like others, we observed induction of a strong IL-17–related gene signature in the vagina during estrogen-dependent murine VVC. As estrogen increases susceptibility to vaginal colonization and resulting immunopathology, we asked whether estrogen use in the standard VVC model masks a role for the Th17/IL-17 axis. We demonstrate that mice lacking IL-17RA, Act1, or interleukin 22 showed no evidence for altered VVC susceptibility or immunopathology, regardless of estrogen administration. Hence, these data support the emerging consensus that Th17/IL-17 axis signaling is dispensable for the immunopathogenesis of VVC.

Mice lacking IL-17RA, Act1, or IL-22 showed no evidence for altered susceptibility or immunopathology during vulvovaginal candidiasis, regardless of estrogen administration. These data help resolve controversy over whether Th17/IL-17 axis signaling is dispensable for the immunopathogenesis of Candida vaginitis.

Fungal pathogens cause millions of deaths worldwide and enormous morbidity, not to mention a significant financial medical toll [1]. Despite this, immunity to fungi is understudied, and to date there are no licensed vaccines to any pathogenic fungal organisms.

Candida albicans is a commensal fungus that colonizes human mucosal surfaces and skin, but under some circumstances becomes pathogenic. Oropharyngeal candidiasis (OPC) and vulvovaginal candidiasis (VVC) are the most common infectious manifestations of this microbe, while disseminated infection is the most serious. Immunity to C. albicans varies strikingly by anatomic site [2]. Immunocompromise of T cells, as in human immunodeficiency virus (HIV)/AIDS or various primary immunodeficiencies, results in OPC and sometimes dermal (mucocutaneous) candidiasis [3, 4]. In contrast, neutrophil deficiency, in conjunction with barrier breaches from indwelling catheters or abdominal surgery, drives susceptibility to disseminated infection [5]. Evidence from animal models and clinical studies strongly supports a crucial protective role of the interleukin 17 (IL-17)/T-helper 17 (Th17) axis against such infections [3, 6].

While most manifestations of candidiasis are due to immunocompromised states, VVC occurs in otherwise healthy women. In a study of human volunteers, robust neutrophil recruitment was strongly associated with onset of symptoms (eg, vaginal itching, burning, redness, and discharge) [7]. Moreover, neutrophils are strongly proinflammatory at the vaginal mucosa, yet seemingly do not contribute to fungal clearance [8]. Thus, unlike OPC, VVC is considered to be an immunopathology, where the host response actually drives disease symptoms. To date, the nature of protective responses against C. albicans at the vaginal mucosa remain enigmatic.

VVC manifests primarily in women of childbearing age, likely due to a central role of estrogen (estradiol [E2]) effects on the vaginal mucosa. This paradigm is supported by the fact that prepubescent girls and postmenopausal women (low E2 producers) rarely develop VVC, yet women prescribed high-dose estrogen oral contraceptives or those undergoing estrogenic hormone replacement therapy are highly susceptible [9]. The impact of estrogen is reflected in all common animal models of VVC (mouse, rat, pig), where E2 administration is required to maintain fungal colonization at the vaginal mucosa [10]. E2 has a multitude of effects on the vaginal epithelium. It modulates cytokine production and causes upper epithelial cells to cornify, keratinize, and ultimately slough. Despite its widespread use in model systems, the mechanisms that allow for enhanced C. albicans colonization of the estrogen-treated vaginal epithelium are not well understood.

Mucocutaneous candidiasis due to T-cell or IL-17 deficiency is typically not associated with VVC, either in humans or in mice, but the involvement of IL-17 remains controversial. Studies in mice show associations with IL-17 expression and VVC [11–13]. An experimental C. albicans vaccine tested in women with recurrent VVC (RVVC) modestly increased time to recurrence and was associated with enhanced T-helper 1 (Th1) and Th17 responses and anti-Candida antibodies [14]. Conversely, cross-sectional clinical studies have largely demonstrated that HIV status is not positively correlated to VVC incidence, suggesting that adaptive immunity does not confer protection [15]. These data contrast with clinical data and animal models of OPC demonstrating that adaptive Th17-driven immunity is protective [16, 17]. Studies using the estrogen-dependent mouse model of VVC find no strong role for protection driven by interleukin 23 (IL-23) or IL-17 signaling [18].

Our goal here was to revisit the question of whether IL-17/Th17 signaling is protective during VVC and, in particular, to determine if the use of estrogen in the mouse model of VVC might mask a protective contribution of the IL-17/interleukin 22 (IL-22) axis. To that end, we compared results in mice lacking components of IL-17 signaling or other Th17-related cytokines in the presence and absence of estrogen in an established murine model of VVC and confirmed that there is no apparent requirement for this pathway in this setting.

METHODS

Microorganism Growth and Preparation

Candida albicans strains SC5314, DAY286, and DAY286-IL-17 were maintained as glycerol stocks stored at −80°C [19]. A single colony was grown overnight in Yeast Peptone Dextrose (YPD) at 30°C at 200 rpm. Cells were washed in nonpyrogenic phosphate-buffered saline (PBS), counted on a hemocytometer, and adjusted to 2.5 × 10⁸ cells/mL in PBS. In some cases, strains were grown overnight in 1X Yeast Nitrogen Base containing 0.5% glucose (YNB), centrifuged at 8000 rpm, and supernatants passed through a 0.22-µm syringe filter.

Mouse Model of VVC

Animal use protocols were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. All mice were on the C57BL/6 background. Wild-type (WT) mice were from the Jackson Laboratory, Taconic Farms, or generated from breeding colonies. Age-matched female mice aged 6–10 weeks were used for all procedures. Il17ra^-/- mice were a gift from Amgen and Il22^-/- mice were kindly provided by Genentech, and both strains were bred in-house. Mice were cohoused for at least 2 weeks prior to infection. Mice were subcutaneously administered 0.1 mg of estrogen (β-estradiol 17-valerate; Sigma) dissolved in 0.1 mL sesame oil vehicle or vehicle alone 72 hours prior to and weekly after intravaginal inoculation with 5 × 10⁶C. albicans blastospores as described elsewhere [20].

Quantitative Reverse-Transcription Polymerase Chain Reaction

RNA from whole vaginal tissue was homogenized in TRI Reagent and isolated by chloroform-ethanol precipitation. Complementary DNA was generated using random hexamers (RevertAid, Thermo) and 20 ng was amplified using Maxima SYBR green mix (Bio-Rad) on the Applied Biosystems 7500 platform. Proprietary primers from Integrated DNA Technologies spanning exon–exon junctions were used to measure messenger RNA expression.

Tissue Histology

Whole vaginal tissue was surgically excised and fixed in 4% buffered formalin for at least 24 hours. Samples were paraffin embedded, sectioned, mounted onto glass slides, and stained with hematoxylin and eosin. Slides were prepared by the Research Histology Core at the University of Tennessee Health Science Center or the University of Toledo.

Bioinformatics

Previously published RNA-seq datasets were used to compare differential gene expression during murine OPC and VVC [21, 22]. Sequencing reads were aligned to the University of California Santa Cruz (UCSC) mouse reference genome (mm10, GRCm38.75) using TopHat2. Alignment files from TopHat2 were subsequently used to generate read counts for each gene in each experimental group, and a statistical analysis of differential gene expression was performed between infected groups and uninfected control groups using the DESeq package from Bioconductor. A gene was considered differentially expressed if the P value for differential expression was <.05. No cutoff was imposed for fold-change between experimental groups. Venn diagrams displaying the comparison of differentially expressed genes in each direction between datasets were generated using Venny (https://bioinfogp.cnb.csic.es/tools/venny/). Heatmaps displaying the expression of the IL-17 pathway target genes were constructed using MeV_4_8 (version 10.2) software.

Quantitation of Fungal Burden, Neutrophil Recruitment, and IL-17

Vaginal lavage fluid was serially diluted 10-fold and plated onto YPD agar containing 50 μg/mL chloramphenicol, followed by colony enumeration. Lavage fluid was smeared onto glass slides and stained by the Papanicolaou technique to assess Polymorphonuclear (PMN) leukocyte recruitment (defined as small blue cells with multilobed nuclei). PMNs were counted in 5 nonadjacent fields using a ×40 objective by observers blinded to the sample identity. IL-17A was measured in clarified, lavage fluid or filtered serially diluted YNB medium by enzyme-linked immunosorbent assay (ELISA) (eBioscience, R&D Systems).

Statistical Analyses

Quantitative real-time polymerase chain reaction was analyzed by analysis of variance (ANOVA) and Student t test. Fungal burdens are presented with geometric means and geometric standard deviation. Significance was determined by ANOVA and Dunn multiple comparison tests or Mann–Whitney U test analysis. Bioinformatics analysis is described above.

RESULTS

An IL-17 Gene Signature Is Induced in VVC That Parallels Oral Candidiasis

In an effort to understand immune events occurring in murine VVC, we challenged mice intravaginally for 3 days with PBS vehicle (sham) or C. albicans strain SC5314. As expected, administration of E2 led to consistent colonization with C. albicans (Figure 1A) and vaginal neutrophil recruitment as indicated by quantitative and histological analyses (Figure 1B). RNA-seq analysis from total vaginal tissue of C. albicans–infected and naive mice revealed that 593 genes were up-regulated and 948 were down-regulated during infection [21]. Among the up-regulated genes were cytokines classically associated with the type 17 pathway, including Il17a, Il17f, and Il22a (Figure 1C). IL-17 induction at the transcript and protein level in infected mice was also confirmed (Figure 1D).

Figure 1.

Interleukin 17 (IL-17) is induced during murine vulvovaginal candidiasis (VVC). Wild-type C57BL/6 mice (n = 5) were challenged intravaginally with Candida albicans or phosphate-buffered saline (sham). At day 3 postinoculation, vaginal lavage fluid was assessed for fungal burden (geometric mean ± standard deviation; A) and neutrophil recruitment (mean ± standard error of the mean; B). Data were compared using 2-tailed Student t test. ***P < .001. Whole vaginal tissue was stained with hematoxylin and eosin. Scale bar indicates 300 µm. Data are representative of 2 independent experiments. C, Total RNA was extracted from whole vaginal tissue (n = 3 per group), prepared for RNA-seq, and differentially expressed IL-17–related cytokines were reported as fold-change of C. albicans infected over naive. D, IL-17A messenger RNA was assessed in vaginal tissue (light gray) by quantitative polymerase chain reaction (n = 5) and IL-17A protein (dark gray) in vaginal lavage fluid by enzyme-linked immunosorbent assay (n = 5 per group). Data were compared using 2-tailed Student t test. ***P < .001. Data are representative of 2 independent experiments. Abbreviations: CFU, colony-forming units; Ct, cycle threshold; IL, interleukin; n.d., not detected; PMN, polymorphonuclear leukocyte; VVC, vulvovaginal candidiasis; WT, wild-type.

IL-17 is critical for immunity in OPC, and an IL-17–dependent gene signature is strongly induced in the oral mucosa following infection [22–26]. In comparing genes induced in VVC and OPC, it was evident that that there were significant overlapping transcripts either up- or down-regulated in these model systems (Figure 2A). Among the shared genes were multiple IL-17 family cytokines (Il17a, Il17c, Il17f), as well as genes characteristic of a classic IL-17–dependent gene signature [27, 28], including cytokines (Il1b, Il6, Il22, Il23A), chemokines (Cxcl2, Cxcl3, Cxcl6, Ccl2, Ccr1), and antimicrobial peptides (S100a8, S100a9, Defb3) (Figure 2B). Not surprisingly, there were also substantial numbers of immunity-related genes whose expression was induced in OPC but not in VVC, including Csf2, Ccl20, Csf3, Lcn2, Cxcl10, Il1a, Csf3r, Ccl7, Sele, Cxcr2, and Ccl22 (Figure 2A).

Figure 2.

The interleukin 17 (IL-17) gene signature induced in vulvovaginal candidiasis (VVC) exhibits similarities with oropharyngeal candidiasis (OPC). Genome-wide and IL-17–focused comparisons of the host response during VVC and OPC were undertaken. A, Venn diagrams comparing the sets of genes that are up-regulated and down-regulated during infection compared to uninfected controls. B, Differential gene expression of IL-17 target genes during OPC or VVC (n = 3 mice per group). Values represent log (base 2)–transformed ratios of expression in infected mice compared to uninfected controls. White indicates infection-induced expression; gray indicates infection-repressed expression; black indicates no change.

Mice With IL-17 Signaling Deficiencies Do Not Show Elevated Fungal Burdens or Altered Immunopathology in VVC

The mouse model of VVC uses estrogen (E2) to synchronize the estrus cycles of recipient mice and render the vaginal mucosa receptive to C. albicans infection. Prior studies indicated that WT, Il23p19^-/-, Il17ra^-/-, and Il22^-/- mice administered E2 did not show differences in fungal burden or neutrophil recruitment following intravaginal C. albicans challenge [18]. However, given the strong influence of IL-17 on other manifestations of C. albicans infection (oral, dermal, and systemic), we questioned whether the potent impact of E2 might mask any contribution of IL-17 signaling in VVC. To address this issue, we administered either vehicle (sesame oil) or E2 to WT and Il17ra^-/- mice, followed by intravaginal inoculation of C. albicans. Vaginal lavage fluid was collected at days 3 and 7 postinoculation and the fungal burden was assessed. As expected, E2 treatment was associated with increased fungal loads, seen as early as day 3 and more strongly by day 7 postinoculation (Figure 3A). Consistent with prior reports, there was no difference in fungal colonization between WT and Il17ra^-/- mice treated with E2. Similarly, there was no change in C. albicans colonization in WT or Il17ra^-/- mice that were not given estrogen, though fungal burdens were lower and more variable than E2-treated animals (Figure 3A). Given the variability in fungal loads, we compared the numbers of mice with any detectable fungal burden over time (Figure 3B). As shown, vehicle-treated WT and Il17ra^-/- mice cleared C. albicans at the same rates, and E2-treated mice all maintained fungal loads during the course of the experiment. These data suggest that IL-17 signaling is not required for fungal clearance at the vaginal mucosa.

Figure 3.

Interleukin 17R (IL-17R) signaling is not required for fungal clearance in vulvovaginal candidiasis (VVC). A, The indicated mice (wild-type [WT], n = 28; Il17ra^-/-, n = 24; WT + estradiol [E2], n = 23; Il17ra^-/- + E2, n = 10) were intravaginally challenged with Candida albicans and fungal loads were assessed days 3 and 7 postinoculation by microbiological plating and colony enumeration. Data are presented as geometric mean ± standard deviation. Data are pooled from 4 independent experiments and analyzed by analysis of variance (ANOVA) and Dunn multiple comparisons test. ***P < .001. B, Percentage of mice with detectable fungal burdens at days 3 and 7 is depicted, derived from data in A. ***P < .001, ****P < .0001 by log-rank Mantel–Cox test. C, Indicated mice were analyzed as described in A. (WT, n = 13; Act1^-/-, n = 11; WT + E2, n = 14; Act1^-/-, n = 6). Data are pooled from 2 independent experiments and analyzed by ANOVA and Dunn multiple comparisons test. Abbreviations: CFU, colony-forming units; KO, knockout; n.s., not significant; veh, vehicle; WT, wild-type.

IL-17RA signaling is mediated by the proximal multifunctional adaptor Act1, which binds to IL-17RA through mutual SEFIR domain interactions [24]. Mice and humans with mutations in Act1 are highly susceptible to mucocutaneous candidiasis, similar to IL-17RA deficiency [29, 30]. Additionally, Act1 is implicated in signaling through BAFF and CD40 [31], suggesting that IL-17R–independent activities may also exist. To date, only 1 female patient with Act1 deficiency has been identified who did not report evidence of VVC (A. Puel, personal communication) [29]. Accordingly, we asked whether Act1 deficiency in mice caused altered susceptibility to VVC. As shown, Act1^-/- mice had the same fungal burdens as WT mice, regardless of E2 administration, with levels similar to Il17ra^-/- mice (Figure 3C).

Lack of an apparent robust protective role for IL-17 signaling at the vaginal mucosa was further investigated by challenging mice with a strain of C. albicans expressing secreted mouse IL-17A (DAY286-IL-17A) [19]. As previously reported, DAY286-IL-17 secreted substantial IL-17A into culture medium as measured by ELISA (Figure 4A), whereas no signal was detectable using the isogenic control strain DAY286. Nonetheless, infection with DAY286-IL-17 resulted in fungal burdens and neutrophil levels similar to those obtained by challenge with DAY286 (Figure 4B), verifying that IL-17 does not contribute to VVC pathology.

Figure 4.

An interleukin 17A (IL-17A)–expressing strain of Candida albicans does not alter colonization or neutrophil recruitment in vulvovaginal candidiasis. Wild-type C57BL/6 mice were intravaginally challenged with a C. albicans strain engineered to secrete murine IL-17A (DAY286-IL-17) or an isogenic control (DAY286). A, IL-17A was measured by enzyme-linked immunosorbent assay in culture supernatants to confirm secretion (mean ± standard error of the mean [SEM]). Experiments were repeated in triplicate. Mice (n = 5 per group) were assessed for vaginal fungal burden (geometric mean ± standard deviation; B) and neutrophil recruitment (mean ± SEM; C) at days 3 and 7 postinfection. Data was compared using a 2-tailed Student t test. Data are representative of 2 independent experiments. Abbreviations: CFU, colony-forming units; E2, estradiol; IL, interleukin; n.d., not detected; n.s., not significant; PMN, polymorphonuclear leukocyte.

The emerging view of VVC pathology is that much of the symptomatology is mediated by an overexuberant neutrophil dominated immune response [32]. IL-17 is a strong activator of neutrophil responses in many mucosal immune settings, including OPC [24, 33, 34]. Accordingly, we asked whether estrogen masked vaginal neutrophil recruitment in Il17ra^-/- mice during VVC. Control E2-treated mice showed elevated PMN counts, as typically observed in this model (Figure 5) [20, 35]. Although variable, there was no statistical difference in PMN counts between WT and Il17ra^-/- mice (Figure 5). Thus, a loss of IL-17 signaling in the absence of estrogen does not render mice more susceptible either to fungal colonization or altered PMN counts.

Figure 5.

Loss of interleukin 17R (IL-17R) signaling does not impact neutrophil recruitment to the vaginal mucosa. The indicated mice (wild-type [WT] + estradiol, n = 13; WT + vehicle, n = 18; IL-17RA knockout + vehicle, n = 16) were intravaginally challenged with Candida albicans and neutrophils were assessed from Papanicolaou-stained vaginal smears by an investigator blinded to sample identity. Data were averaged from 5 nonadjacent fields of view (geometric mean ± standard deviation). Representative qualitative images are shown for reference. Data were analyzed by analysis of variance and Tukey post hoc analysis. **P < .01. Data are pooled from 2 independent experiments. Abbreviations: E2, estradiol; IL, interleukin; KO, knockout; PMN, polymorphonuclear leukocyte; Veh, vehicle; WT, wild-type.

IL-22 Deficiency Does Not Alter VVC Fungal Burden or Immunopathology

IL-17 is the eponymous cytokine of the Th17 lineage, but IL-22 is also produced by type 17 cells and mediates many overlapping processes in inflammation. Like IL-17, IL-22 acts on nonhematopoietic target cells of epithelial origin [36]. Similar to IL-17, we observed no differences in vaginal colonization of C. albicans in Il22^-/- mice compared to WT animals (Figure 6A). Fungal clearance rates were also indistinguishable between these cohorts (Figure 6B). Collectively, these data suggest that neither IL-17 nor IL-22 signaling is required for fungal clearance at the vaginal mucosa.

Figure 6.

Interleukin 22 (IL-22) is not required for immunity to vulvovaginal candidiasis. A, The indicated mice (wild-type [WT], n = 12; Il22^-/-, n = 11; WT + estradiol [E2], n = 12; Il22^-/- + E2, n = 9) were intravaginally challenged with Candida albicans and fungal loads were assessed at days 3 and 7 postinoculation by microbiological plating and colony enumeration. Data are presented as geometric mean ± standard deviation and analyzed by analysis of variance and Dunn multiple comparisons test. B, Percentage of mice with detectable fungal burdens at days 3 and 7 is depicted, derived from data in A. *P < .05 by log-rank Mantel–Cox test. Data are pooled from 2 independent experiments. Abbreviations: CFU, colony-forming units; E2, estradiol; IL, interleukin; KO, knockout; n.s., not significant; veh, vehicle; WT, wild-type.

DISCUSSION

Despite its prevalence, VVC remains a major unmet clinical need. While not lethal, VVC causes significant quality-of-life issues for affected women. Estimates state that 75% of all women will experience at least 1 episode of VVC. Unlike other forms of mucosal and systemic candidiasis, where breach of protective intrinsic, innate, or adaptive immunity precipitates pathogenesis, the underlying factors that drive acute or relapsing susceptibility during VVC are still poorly understood. Gaining mechanistic insight into human VVC is obfuscated by variable symptomatic presentation, disease onset kinetics, host genetics, fluctuation in hormones, disease mimicry, lifestyle, and a sometimes mistaken assumption that coincidental colonization of C. albicans is a cause of vaginal inflammation.

The mouse model of VVC has been an indispensable tool for understanding the sequence of events that occur in this disease, notwithstanding some acknowledged shortcomings to the system (eg, elevated vaginal pH compared to humans, nonasymptomatic carrier). Work by the Fidel laboratory demonstrated that Th1-type responses were up-regulated during experimental vaginitis, findings that were recapitulated during challenge of human volunteers using C. albicans cell wall antigens [37]. Despite strong induction of this Th1 profile in humans, systemic depletion of CD4⁺ or CD8⁺ T cells in mice did not impact vaginal fungal burdens during murine VVC [38]. Similarly in humans, HIV-positive individuals with low T-cell counts do not experience a higher incidence of VVC [15].

In contrast to the clear protective role for the IL-17 axis in OPC, its importance during VVC remains controversial [4]. Mice genetically deficient for IL-17RA, IL-23p19, or IL-22 showed similar colonization levels and immunopathology as WT mice when challenged intravaginally with C. albicans [18], a finding that we reproduced here. However, Pietrella et al reported that mice treated with halofuginone, which (among other functions) inhibits Th17 cell development, showed higher and more persistent vaginal fungal burden [11]. Another study using Il17a^-/- and Il17f^-/- mice demonstrated elevated Candida burden and neutrophil recruitment at the vaginal mucosa [11]. One report indicated that Il-22^-/- mice showed increases in vaginal neutrophil recruitment in VVC [39], but others saw no impact of IL-22 [18]. Notably, all of these prior studies were conducted with estrogen-treated mice to induce disease. In testing whether use of E2 might have masked a more subtle impact of these cytokines, we demonstrate that the IL-17/IL-22 axis is dispensable for antifungal defense at the murine vaginal interface, irrespective of estrogen administration. It is worth noting that vehicle-treated mice presumably maintained endogenous estrogen levels, but these were clearly inadequate to inhibit fungal clearance regardless of IL-17/IL-22 sufficiency. These data are in line with accumulating data in humans that loss of IL-17, either from congenital or acquired immunodeficiency affecting IL-17 or Th17 cells, does not predispose women to VVC [3, 32].

Potential variation in fungal strains, genetic drift in mouse backgrounds by vendor, and composition of vaginal microbiota may partially explain observed differences among these studies. For example, the vaginal colonizer Streptococcus agalactiae can reportedly impair C. albicans–mediated activation of the local Th17 response via inhibition of hyphal growth and may moderately increase vaginal fungal burden [13]. Although we did not explicitly test for S. agalactiae, our cohousing approach should have reduced this potential confounding factor.

A growing body of literature reveals complex interactions between estrogen, neutrophil recruitment, and the IL-17 axis. Naive T cells isolated from mice treated with estrogen exhibited reduced capacity to polarize into Th17 subsets due to down-regulation of retinoid acid receptor–related orphan receptor C (encoding RORγt), a transcription factor required for Th17 cell development and IL-17A and IL-17F expression [40, 41]. Mice, like humans, progress through a cyclical hormonal cycle mediated by alternating levels of estrogen and progesterone. In rodents, this cycle does not culminate in menstruation, but causes recruitment of neutrophils to the vaginal lumen to debride the endometrium. This progesterone-dependent neutrophil migration is achieved by a CXCL1/CXCL2 gradient in the vaginal stroma that is sensed by CXCR2 on the phagocyte surface [42]. Estrogen signaling through ER⍺ disrupts the CXCL1 signal, preventing transepithelial migration of neutrophils into the vaginal lumen. Therefore, C. albicans must overcome this CXCL1 defect to reestablish the gradient, perhaps by inducing its expression or another CXCR2 agonist (eg, CXCL3). Regardless, this begs the question as to why the host has evolved to potentially compromise mucosal immunity at the vaginal interface in response to circulating sex hormones. Recognition of nonself antigens by the female immune system (eg, sperm proteins) induces Th17 responses that might otherwise be detrimental for reproduction [43]. Thus, estrogen may establish a tolerogenic vaginal microenvironment during phases of peak mating receptiveness. Findings in this study verify that estrogen inhibits fungal clearance, but independent of IL-17/IL-22 signaling.

Estrogen establishes fungal colonization in several ways. Concomitant with epithelial thickening and cornification during estrogen administration, vaginal glycogen content also increases. Candida albicans assimilates glycogen when grown on media using this as a sole carbon source, although not all Candida species appear to retain this capacity [44]. Therefore, the estrogen-modulated epithelial surface may represent a nutrient-rich environment that favors high fungal turnover. Heparan sulfate (HS) present at the vaginal mucosa may also promote VVC susceptibility. Neutrophils treated with vaginal lavage from estrogen-resistant CD-1 (low HS) mice exhibit robust antifungal activity. However, when treated with lavage fluid from highly estrogen-sensitive C57BL/6 (high HS) mice, PMN killing was largely inhibited [45]. Although vaginal heparan sulfate levels were not explicitly quantified in this study, the phenotype was partially estrogen dependent, as vaginal fluid obtained from C57BL6 mice administered E2 demonstrated less killing capacity than sesame oil–treated controls. Therefore, vaginal HS may impair PMN function, leading to extended colonization.

Recent studies indicate that VVC immunopathology is largely driven by the pore-forming peptide toxin candidalysin [20]. This fungal virulence factor is produced strictly by hyphae and activates cellular damage and innate immune responses at the vaginal mucosa. Candidalysin activates NLRP3 inflammasome signaling, driving neutrophil recruitment and immunopathogenesis [46]. Even though candidalysin can signal synergistically with IL-17 to induce innate inflammation [25], this may be insufficient to promote VVC pathology. Several translational studies using systems genomics approaches have identified human susceptibility alleles that correlate inflammasome activation with elevated incidence of RVVC. Subjects with RVVC were found to express significantly more IL-1B and less IL-1RA, positive and negative regulators of NLRP3 activity, compared to healthy controls [47]. Similar findings were made in another study of women who had vaginal swabs negative for C. albicans, those with positive swabs but who were asymptomatically colonized, or those with active infection [48]. Expression of the inflammasome effectors NLRP3, CASP1, and IL-1B in vaginal epithelial cells were elevated in the symptomatic group compared to asymptomatic or uncolonized controls. Higher expression of fungal inflammasome inducers (eg, candidalysin and secreted aspartyl proteinases) were also found in the symptomatic group. A polymorphism in the SIGLEC15 gene was also observed at a higher frequency in RVVC subjects. This gene encodes a lectin that can bind sialic acid residues on the fungal surface, resulting in hyperactivation of NLRP3 and IL1B following C. albicans challenge [49]. Silencing of SIGLEC15 in mice led to increased fungal burden and vaginal neutrophil recruitment. Thus, it is plausible that reduced function of this gene in the RVVC population would explain failure to adequately control Candida colonization and attenuate vaginal inflammation.

In summary, although a robust IL-17 gene signature is induced in the murine vagina during VVC, clearance phenotypes in the presence or absence of estrogen occur independent of the IL-17/IL-22 axis. Given the conservation of IL-17 gene signatures at both the oral and vaginal mucosa, the data herein reinforce the anatomic specificity of anti-Candida responses. Moreover, these data demonstrate that induction of an immune mediator does not necessarily imply a causative immunological role and highlight how physiological responses may intertwine with pathogenicity. Thus, caution must be taken to avoid universally applying findings across all C. albicans infection models and to devise highly targeted therapeutic strategies.

Notes

Acknowledgments. The authors thank K. Fairhurst (University of Pittsburgh) for technical assistance, and H. Conti (University of Toledo), J. Naglik (King’s College London), and Anne Puel (Institut national de la santé et de la recherche médicale, France) for helpful discussions. U. Siebenlist (National Institutes of Health [NIH]) kindly provided Act1^-/- mice. Il17ra^-/- mice were a kind gift from Amgen and Il22^-/- mice were generously provided by Genentech.

Author contributions. B. M. P., S. L. G., and B. M. C. designed the experiments. B. M. C., H. M. E. W., K. S. B., B. M. P., F. E. Y. A., E. C., and A. V. performed the experiments. V. B. and B. M. P. performed bioinformatic analyses. All authors helped analyze and interpret data. S. L. G. and B. M. P. wrote the manuscript with input from B. M. C., H. M. E. W., K. S. B., F. E. Y. A., E. C., A. V., A. H. H., S. B., A. P., and V. M. B.

Disclaimer. The content within is solely our responsibility and does not necessarily represent the official views of the NIH or other funding agencies.

Financial support. This work was supported by the National Institute of Dental and Craniofacial Research, NIH (grant numbers DE022550 and DE023815 to S. L. G. and DE026189 to A. H. H.); the National Institute of Allergy and Infectious Diseases, NIH (grant numbers AI134796 and AI141829 to B. M. P.); and the Crohn’s and Colitis Foundation (Career Development Award to S. B.).

Potential conflicts of interest. The IL-17–expressing Candida albicans strain used here is the subject of US patent 10160974 (“Engineered cytokine- and chemokine-expressing Candida albicans strains and methods of use”). All authors: No reported conflicts of interest.

All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.