Epithelial Cell-Derived S100 Calcium-Binding Proteins as Key Mediators in the Hallmark Acute Neutrophil Response duringCandidaVaginitis

Abstract

Vulvovaginal candidiasis (VVC), caused by Candida species, is a significant problem in women of childbearing age. Similar to clinical observations, a robust vaginal polymorphonuclear neutrophil (PMN) migration occurs in a subset of mice without affecting vaginal fungal burden. We hypothesize that the vaginal PMN infiltrate and accompanying inflammation are not protective but instead are responsible for the symptoms of infection. The purpose of this study was to identify the signal(s) associated with the PMN response in the established mouse model. Vaginal lavage fluid from inoculated mice were categorized base on PMN counts, evaluated for PMN chemotactic activity and analyzed by SDS-PAGE and mass spectrometry (MS) for unique protein identification. The lavage fluid from inoculated mice with high, but not low, PMN levels showed increased chemotactic activity. Likewise, SDS-PAGE of lavage fluid with high PMN levels showed distinct protein patterns. MS revealed that bands at 6 and 14 kDa matched the PMN chemotactic calcium-binding proteins (CBPs), S100A8 and S100A9, respectively. The presence of the CBPs in lavage fluid was confirmed by Western blots and enzyme-linked immunosorbent assay. Vaginal tissues and epithelial cells from inoculated mice with high PMN levels stained more intensely and exhibited increased mRNA transcripts for both proteins compared to those in mice with low PMN levels. Subsequent antibody neutralization showed significant abrogation of the chemotactic activity when the lavage fluid was treated with anti-S100A8, but not anti-S100A9, antibodies. These results reveal that the PMN chemotactic CBP S100A8 and S100A9 are produced by vaginal epithelial cells following interaction with Candida and that S100A8 is a strong candidate responsible for the robust PMN migration during experimental VVC.Vulvovaginal candidiasis (VVC), caused by Candida species, is an opportunistic fungal infection that affects ca. 75% of healthy women of childbearing age (34). Menarchal women are predisposed to VVC through several exogenous factors, most of which involve elevated hormone levels (e.g., pregnancy, use of high estrogen oral contraception or hormone replacement therapies, and the luteal phase of the menstrual cycle) (34). Frequent antibiotic usage and uncontrolled diabetes mellitus are also known to be linked to increased susceptibility to VVC. Recurrent vulvovaginal candidiasis (RVVC), defined as three or more episodes of VVC per year, affects a separate 5 to 10% of menarchal women. Most cases of RVVC are primary RVVC, where idiopathic infection occurs with no predisposing factors, whereas secondary RVVC (repeated occurrence of acute VVC) could occur as a result of being unable to avoid certain predisposing factors (34).Although VVC and RVVC have historically been attributed to a putative local immune deficiency, several studies using a mouse model of Candida vaginitis and many cross-sectional clinical studies evaluating women with primary RVVC have shown that protection is not mediated by local or systemic adap...

Performing Vaginal Lavage, Crystal Violet Staining, and Vaginal Cytological Evaluation for Mouse Estrous Cycle Staging Identification

“…Sampling should be performed no more than once daily using the non-invasive protocol described here as compared to repeated penetration of the vaginal canal, aspiration, and agitation. This can cause vaginal irritation resulting in an inflammatory response 17 resulting in leukocytes and other cell types to be present in smears that may confound cytological assessment. Moreover, even in colony-housed females, it is normal to see extended diestrus and estrus stages in different mice as well induction of anestrous 18 and this identification is useful in the interpretation of hormonal impact in reproductive, gender, and disease studies.…”

Characterization of IL-22 and antimicrobial peptide production in mice protected against pulmonary Cryptococcus neoformans infection

“…Alternatively, the antimicrobial peptides may act as messengers during fungal infections rather than as effector molecules. In studies of Candida albicans vaginitis, the S100 antimicrobial peptides have been examined, and data have shown that their role is more important in neutrophil recruitment than in direct microbicidal activity against Candida (Johnston et al, 2013;Yano et al, 2010Yano et al, , 2012a. However, additional studies by other laboratories have shown that IL-22 is critical to protection in a vaginal model of Candida albicans infection (De Luca et al, 2010.…”

Dectin-1 isoforms contribute to distinct Th1/Th17 cell activation in mucosal candidiasis

“…33 In combination with IL-17A, IL-22 has been found to be crucially involved in the control of Candida growth in the gastrointestinal tract in conditions of Th1 and Th17 deficiency. 23 Moreover, vaginal epithelial cells also produced S100A8 and S100A9 following interaction with Candida, 34 suggesting the possible involvement of IL-22 in vaginal candidiasis. Thus, IL-22 1 cells, employing ancient effector mechanisms of immunity, may represent a primitive mechanism of resistance against the fungus under conditions of limited inflammation.…”