Objective
Our goal was to gain a better understanding of the inflammatory pathways affected during localized vulvodynia, a poorly understood, common, and debilitating condition characterized by chronic pain of the vulvar vestibule.
Study Design
In a control matched study, primary human fibroblast strains were generated from biopsies collected from localized provoked vulvodynia (LPV) cases and from age- and race-matched controls. We then examined intracellular mechanisms by which these fibroblasts recognize pathogenic Candida albicans ; >70% of vulvodynia patients report the occurrence of prior chronic Candida infections, which is accompanied by localized inflammation and elevated production of proinflammatory/pain-associated interleukin (IL)-6 and prostaglandin E2 (PGE 2 ). We focused on examining the signaling pathways involved in recognition of yeast components that are present and abundant during chronic infection.
Results
Dectin-1, a surface receptor that binds C albicans cell wall glucan, was significantly elevated in vestibular vs external vulvar cells (from areas without pain) in both cases and controls, while its abundance was highest in LPV cases. Blocking Dectin-1 signaling significantly reduced pain-associated IL-6 and PGE 2 production during the response to C albicans . Furthermore, LPV patient vestibular cells produced inflammatory mediators in response to low numbers of C albicans cells, while external vulvar fibroblasts were nonresponsive. Inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (proinflammatory transcription factor) nearly abrogated IL-6 and PGE 2 production induced by C albicans , in keeping with observations that Dectin-1 signals through the nuclear factor kappa-light-chain-enhancer of activated B cells pathway.
Conclusion
These findings implicate that a fibroblast-mediated proinflammatory response to C albicans contributes to the induction of pain in LPV cases. Targeting this response may be an ideal strategy for the development of new vulvodynia therapies.
Vulvodynia is a poorly understood and largely understudied pain condition that is common, chronic, and debilitating ; as high as an estimated 28% of women in the United States are afflicted, many of whom are of childbearing age. Vulvodynia is diagnostically categorized into 2 categories: generalized or localized. These categories are further differentiated by whether vulvodynia pain can be provoked, unprovoked, or both. Women with localized provoked vulvodynia (LPV), the most common diagnostic category, experience acute and lasting pain in response to (light) touching of specific areas of the vulva. In contrast, regions of the external vulva (labia minora, labia majora, mons pubis, and perineum) are relatively pain-free to touch. Pain associated with LPV frequently results in an almost complete disruption of sexual activity, discomfort with tampon insertion, dysuria, and depression, while the current medical therapies do little to permanently alleviate the symptoms of disease. Ultimately, treatments cannot resolve or address the underlying cause(s) of disease, because they are still largely unknown, and only recently has this disease been linked to any preceding immunomodulatory stimulus. A current report demonstrated that >70% of LPV patients report the occurrence of prior, often chronic (>4/y) vaginal yeast infections. However, limited empirical evidence exists to show a causal link between vulvovaginal yeast infection and the onset of LPV. Very recently, it was demonstrated that repeated yeast infection in mice can lead to an increase in proinflammatory mediator production in the vulvar vestibule associated with heightened pain sensitivity, which suggests that prior/chronic infections in human beings are related to the occurrence of LPV. However, we acknowledge that other previously unidentified mechanisms may also exist, as not all patients with LPV have histories of chronic yeast infection.
LPV pain to light touch, also known as “allodynia,” is indistinguishable from well-documented experimental and clinical examples of neural pain fiber (nociceptor) sensitization. Allodynia can be induced by intradermal or subcutaneous proinflammatory factors such as interleukin (IL)-6 and prostaglandin E2 (PGE 2 ). IL-6 and PGE 2 are elevated in chronic pain conditions, IL-6 and PGE 2 induction elicits allodynia, IL-6 and PGE 2 suppression reduces allodynia, and in preclinical animal models of pain, IL-6 and PGE 2 factor/receptor knockout mice display reduced allodynia. In keeping with these observations, human fibroblasts isolated from LPV patients at sites of allodynia-type pain respond to yeast and yeast-derived products through the heightened production of IL-6 and PGE 2 associated with allodynia-type pain. Following a live yeast or yeast product challenge, fibroblasts from the vulvar vestibule of LPV patients produce significantly more IL-6 and PGE 2 than fibroblasts derived from the external vulva of the same patient and more than fibroblasts derived from the vulvar vestibule and external vulva of pain-free controls. Furthermore, fibroblast-produced IL-6 and PGE 2 were shown to precisely predict mechanical pain thresholds in cases and controls. However, little is known about how human external vulvar and vestibular fibroblasts respond to yeast or yeast products. Even less is known about whether or how the mechanism differs in pain-associated vestibular cells vs external vulvar cells not directly associated with pain.
Vulvar fibroblast strains produce IL-6 and PGE 2 in response to challenge with yeast components, such as zymosan, a commercially available mixture of yeast cell wall mannoproteins and β-glucan commonly used as an immune stimulus to model responses to yeast infection. Zymosan is purified from Saccharomyces cerevisiae , which can be found at sites of infection, although it is significantly less pathogenic. However, the structure of the S cerevisiae cell wall is highly similar to that of prevalent vaginal yeast pathogens, such as Candida albicans and C glabrata . With our in vitro fibroblast model, we have shown that zymosan or live virulent yeast challenges produce comparable proinflammatory responses. These findings suggest that the recognition of yeast mannoprotein and β-glucan moieties may be critical to the vulvovaginal response to yeast, of which chronic/repeated infection has been associated with the occurrence of LPV.
Mannoprotein is abundant in the outermost layer of the Candida cell wall and is a known pathogen-associated molecular pattern (PAMP) that is recognized by cognate pattern recognition receptors of the human immune system. Although β-glucan can be found interior to mannoprotein, it is exposed at bud scars during cell division and is actively secreted into the extracellular milieu. In turn, these proteins (also found in zymosan) are presumably abundant during vulvovaginal yeast infection and may serve as key stimuli that contribute to the heightened immune response observed in LPV patients. During infection, C albicans debrides the epithelial layer through protease secretion and tissue invasion, exposing the underlying tissue (eg, fibroblasts) to yeast and yeast products. Repeated exposure to yeast or yeast products may result in sensitization, so that even normal (culture-negative) levels of yeast could signal a proinflammatory response.
We set out to investigate the underlying mechanisms that may influence pain sensitivity in the vulvar vestibule by evaluating the response to heavy, moderate, and low infectious doses of C albicans , while focusing on the signaling pathways that influence the immune response to yeast PAMPs (β-glucan and mannoprotein). We elected to concentrate on the Dectin-1 receptor, because it is regarded as the paradigm receptor for β-glucan detection, and it triggers a signaling cascade that can activate nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) (a protein complex that can regulate proinflammatory gene expression), which leads to production of IL-6. Dectin-1 is expressed on macrophages, monocytes, B cells, and dendritic cells. In addition, it is expressed on gingival fibroblasts and may serve some role in the response to oral candidiasis. In light of these observations, we evaluated Dectin-1 expression on human external vulvar (from the perineum) and vestibular fibroblasts and its role in the production of proinflammatory mediators in response to challenge with zymosan or live yeast. We also explored the role of NFκB signaling in proinflammatory mediator production. This work represents an important step in identifying an underlying intracellular mechanism for LPV; understanding the mechanisms that govern LPV may lead to therapeutic advances.
Materials and Methods
Patient/sample selection
LPV-afflicted cases (fulfilling Friedrich’s criteria ) and age-/race-matched pain-free controls were recruited from the Division of General Obstetrics and Gynecology clinical practice at the University of Rochester from December 2012 through February 2014. All subjects provided informed consent, and the research was approved by the University of Rochester Institutional Review Board (RSRB no. 42136). Expanded details on our selection criteria and sampling procedures have been previously published. In brief, cases and controls were age- and race-matched with a mean age of 33.5 years. All case and control subjects were Caucasian, non-Hispanic. Furthermore, all subjects denied the use of corticosteroids and nonsteroidal anti-inflammatory medications and had no chronic inflammatory illnesses other than LPV. Pain levels (at the vaginal vestibule) using the cotton swab test ranged from 7–9 out of a maximal score of 10 for LPV cases and were 0 for all pain-free controls. All study subjects had negative yeast cultures at the time of study entry. Tissue was sampled from sites as diagrammed previously. A total of 4 paired (vulvar vestibule and external vulva) case and 4 paired control strains (16 total) were used in this study.
Fibroblast strains
Primary fibroblast strains were established from fresh biopsy tissues, which were minced and immobilized on culture dishes and then cultured in RPMI 1640 medium supplemented with 10 mmol/L HEPES, 50 μg/mL gentamicin, 1 mmol/L sodium pyruvate, 2 mmol/L L-glutamine, antibiotic/antimycotic solution (Gibco/Invitrogen, part of Thermo Fisher Scientific, Waltham, MA), 10% fetal bovine serum (FBS), and 50 μmol/L DMSO (Thermo Fisher Scientific) until fibroblasts proliferated onto the culture surface, which was followed by subsequent passages in minimum essential medium (MEM) with 10% FBS, GlutaMAX, gentamicin, and antibiotic/antimycotic solution (all reagents from Thermo Fisher Scientific) as previously described. Early passage (4-10) external vulvar and vestibular fibroblast strains were seeded at 5 × 10 4 cells/well. After achievement of confluence, fibroblasts were serum-reduced for 48 hours in fresh media containing 0.05% FBS. Fibroblast cellular identity was confirmed by microscopic inspection and with fibroblast-specific markers (eg, vimentin, collagen). At the same time, the cells were confirmed to be negative for epithelial cell markers (eg, cytokeratin), smooth muscle and myofibroblast markers (eg, α-smooth muscle actin), endothelial cell markers (eg, CD34), and bone marrow–derived cell markers (eg, CD45).
Dose response to live infection with C albicans
Cultures of fibroblast strains were seeded to 24-well tissue culture plates at roughly half confluence and were allowed to grow until confluent (∼3-4 days) at 37°C and 5% carbon dioxide in MEM supplemented with 10% FBS, GlutaMAX, gentamicin, and antibiotic/antimycotic solution (Thermo Fisher Scientific). Once confluent, cells were transitioned to serum-reduced media (supplemented with 0.05% FBS) and incubated for 48 hours. The evening prior to infection, C albicans SC5314 (a virulent wild type strain) yeast cells were inoculated into a 10-mL culture of yeast peptone dextrose (YPD) broth (Thermo Fisher Scientific) from a YPD plate culture <2 weeks old. Yeast cultures were incubated overnight at 37°C and 220 rpm. After ∼18 hours’ growth, the culture was diluted to OD 600 = 1.0 in fresh YPD broth. Inoculums were prepared by diluting these yeast cultures to ∼1 × 10 4 colony-forming unit (CFU)/mL and then serially diluting (10-fold dilutions) to 1 × 10 1 CFU/mL in antibiotic/antimycotic-free MEM supplemented with 0.05% FBS and GlutaMAX. Confluent fibroblast wells were then infected with 1 mL of each inoculum (1 × 10 4 , 1 × 10 3 , 1 × 10 2 , and 1 × 10 1 blastoconidia) and incubated for 24 hours at 37°C and 5% carbon dioxide. At the same time, additional wells were treated with 100 μg/mL zymosan (Sigma-Aldrich, St Louis, MO), which was diluted in MEM from a 250X stock dissolved in 100% EtOH. A corresponding vehicle control was also prepared. Standard sandwich enzyme-linked immunosorbent assays (ELISAs) were performed to measure production of IL-6 (BD Biosciences, San Jose, CA) and competitive EIA assays were performed to measure PGE 2 production (Cayman Chemical Company, Ann Arbor, MI). Experiments were performed a minimum of 2 times in quadruplicate.
Quantitative real-time polymerase chain reaction
Expression profiles of IL-6 and CLEC7A messenger RNA (mRNA) sequences were evaluated at 30 minutes, and 6, 24, and 72 hours following treatment with 100 μg/mL zymosan or vehicle control in fibroblast strains obtained from LPV cases. Cells were propagated and treated in 24-well plates. At each time point, cells were lysed and total mRNA was extracted using the Qiagen RNeasy kit following the manufacturers’ instructions (Qiagen Corp, Carlsbad, CA). A NanoDrop ND-1000 (NanoDrop/Thermo Fisher Scientific) was used to quantify the mRNAs, which were used as templates for cDNA synthesis using the iScript cDNA synthesis kit (BioRad, Hercules, CA); 300 ng total RNA template was used in each reaction. Negative reverse transcriptase controls (where no enzyme was added to the reaction) were also prepared to confirm the absence of DNA contamination. cDNA samples were diluted 1:5 in RNase-free molecular grade water (Qiagen Corp) and used as templates for quantitative real-time polymerase chain reaction reactions (5 μL/reaction). A standard curve was constructed for each primer set by preparing 5-fold serial dilutions of a reference set of cDNAs prepared from RNAs purified from cells treated with zymosan. Reactions were prepared in a total of 12 μL using SsoAdvanced Universal SYBR Green Supermix (BioRad). Previously published primer sequences for human CLEC7A were used to quantify Dectin-1 expression, while primers for human IL-6 (sense: 5’-GTACATCCTCGACGGCATC and anti-sense: 5’-ACCTC AACTCCAAAAGACCAG) were designed using IDT oligo design tools (OligoAnalyzer, http://www.idtdna.com ). All quantitative real-time polymerase chain reaction values were normalized to the 18S rRNA signal amplified using previously published primer sequences. Each experiment was performed a minimum of 2 times in quadruplicate.
Dectin-1 protein expression on human external vulvar and vestibular fibroblasts
Fibroblast strains (external vulvar and vestibular strains from both cases and controls) were grown to confluency in 6-well culture dishes (Thermo Fisher Scientific), which were then released from the culture surface using trysin-EDTA solution and trypsin inhibitor (Gibco/Invitrogen, part of Thermo Fisher Scientific) and then washed with PBS before blocking nonspecific antibody binding with 5% human Fc receptor blocker (Miltenyi Biotech Inc, San Diego, CA) in PBS containing 1% BSA and 0.1% sodium azide. Cells were then either unstained or incubated with phycoerythrin-conjugated anti-human Dectin-1 antibody (GeneTex Inc, Irvine, CA). Unstained and positively stained cells were analyzed on a FACS Canto II flow cytometer running FACSDiva software (BD Biosciences, San Jose, CA) using a 488-nm excitation laser and a 585-/42-nm band pass detector and subsequently analyzed using FlowJo software (TreeStar Data Analysis Software, Ashland, OR). A typical result from several flow experiments is depicted ( Figure 3 , A).
Additional 6-well cultures of fibroblast strains were prepared as described earlier, then washed with PBS, and lysed in 0.1 mol/L Tris with 2% sodium dodecyl sulfide and protease inhibitor cocktail at a 1:10 dilution (Sigma-Aldrich). Protein concentrations were determined using a BioRad DC protein assay, and 10 μg of each protein lysate was run on a 4-20% pre-cast Criterion Tris-HCl gel (BioRad) with Spectra multicolor broad range protein ladder (Thermo Fisher Scientific) and electro-transferred to a 0.45-μm EMD Millipore Immobilon PVDF membrane (Thermo Fisher Scientific). Membranes were stained with Ponceau S (Sigma-Aldrich) for 10 minutes to visualize total protein on the membrane. Membranes were destained in 5% acetic acid, then washed in western wash buffer (PBS with 0.1% Tween-20) several times before blocking with 2% bovine serum albumin for 30 minutes (reagents from Sigma-Aldrich). After blocking, membranes were incubated with a mouse monoclonal antibody specific for the Dectin-1 receptor (GeneTex Inc) for 1 hour at room temperature. Membranes were washed and then incubated with a goat antimouse antibody (Jackson ImmunoResearch Laboratories Inc, West Grove, PA) for 30 minutes. Dectin-1 receptor expression was visualized using enhanced chemiluminescent HRP substrate (Thermo Fisher Scientific) and exposure to x-ray film, followed by densitometric analysis using Quantity One 1-D Analysis software, Version 4.6.9 (BioRad).
Dectin-1 receptor blockade
Two independent methods were employed to block the function of the Dectin-1 receptor in fibroblast strains derived from women diagnosed with LPV. The impact of blockade was evaluated by quantifying the amount of proinflammatory mediators released (IL-6 and PGE 2 ) in response to challenge with 100 μg/mL zymosan in cells with functional receptor vs those receiving treatment to impair function. IL-6 and PGE 2 were assayed because they are abundantly produced by fibroblast strains from LPV cases and have been generally associated with the evolution of pain during inflammation. For both methods, cells were grown in 24-well plates in MEM with 10% FBS until confluent, then transitioned to low serum media (0.05% FBS) for 48 hours. To physically block receptor function, cells were first incubated for 1 hour at 37°C with 1 mg/mL laminarin, a commercially purified soluble β-glucan that binds to Dectin-1, yet fails to signal an inflammatory response (Sigma-Aldrich); cells in control wells with laminarin alone failed to produce IL-6 or PGE 2 as anticipated. Cells were then challenged with vehicle control or 100 μg/mL zymosan by direct addition to the culture media from concentrated stock; after addition, cells were incubated at 37°C for another 24 hours, at which point culture supernatants were collected and assayed for IL-6 and PGE 2 , as described earlier. A second molecular approach was used to block the expression of the gene encoding Dectin-1 (CLEC7A). A small interfering RNA (siRNA) against human CLEC7A and Silencer negative control no. 1 siRNA was purchased from Ambion (a division of Thermo Fisher Scientific). The lipofectamine 2000 reagent (Ambion) was used to transfect cells with control and anti-Dectin-1 siRNAs according to the manufacturer’s instructions; cells were first washed with PBS, then transfected with a total of 500 ng siRNA/well in MEM with 0.05% FBS. Following the transfection procedure, cells were incubated for 48 hours at 37°C. At this time, cells were then challenged with 100 μg/mL zymosan or vehicle control in fresh media for another 24 hours, at which point supernatants were collected for detection of IL-6 and PGE 2 . The adherent cells were then washed in PBS, and protein was collected for Western blotting against Dectin-1. Western blots confirmed that Dectin-1 protein levels were not affected by the control siRNA, while the levels were dramatically reduced by anti-CLEC7A siRNA. Each experiment was performed a minimum of 2 times in quadruplicate.
NFκB subunit translocation assays
LPV patient strains were propagated and treated with zymosan or vehicle for a total of 3 hours, then the nuclear and cytoplasmic protein fractions were purified separately using a nuclear extract kit per the manufacturer’s instructions (Active Motif, Carlsbad, CA). Nuclear extracts were then applied to an NFκB family TransAM assay (Active Motif) to measure NFκB subunit levels in the nucleus. Nuclear and cytoplasmic protein fractions were also analyzed by Western blotting for p65 subunit expression by probing with an anti-p65 monoclonal rabbit IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Rabbit monoclonal ant-histone H3 in the nucleus, and monoclonal rabbit anti-β-tubulin in the cytoplasm were used as loading controls and to assess the efficiency of nuclear and cytoplasmic separation (antibodies from Cell Signaling Technology, Danvers, MA). Visualization of protein expression was performed as described previously using appropriate secondary antibodies and was performed for >1 set of patient strains.
Activation of NFκB with live yeast challenge
To assess the effect of live yeast infection on the activation of the NFκB pathway, LPV patient fibroblast strains were transfected with an NFκB-firefly luciferase reporter and pRL-SV40, a commercially available control renilla luciferase reporter (Promega, Madison, WI). Nucleofection of these constructs was performed as previously described. Cells were allowed to reach confluence in 24-well plates and were then serum-starved for 48 hours prior to transfection. After transfection, cells were allowed to recover for 24 hours before transitioning to fresh media (MEM + 0.05% FBS) containing 1 × 10 4 C albicans SC5314 cells/mL, 1 × 10 4 S cerevisiae S288C cells/mL, or a matched volume of YPD (vehicle control). Both yeast inoculums were prepared from overnight cultures of each strain diluted to an OD 600 of 1 in fresh YPD; the overnight culture of C albicans was grown at 37°C, while S cerevisiae was grown at 30°C. After 24 hours of infection, reporter activity was determined using the Dual-Glo Luciferase Assay System (Promega). Luminescence was quantified on a VarioSkan Flash Multimode Reader (Thermo Fisher Scientific). Firefly luciferase activity was then normalized to constitutive renilla activity to adjust for any differences in the final numbers of cells in each well. Assays were performed a minimum of 2 times in quadruplicate.
Impact of NFƙB inhibition on proinflammatory mediator release
Patient fibroblast strains were cultured in 24-well plates as described and then simultaneously treated with either zymosan alone or with 100 μg/mL zymosan and 5 μg/mL BAY-11-7082 (NFκB inhibitor; Cayman Chemical) in MEM + 0.05% FBS. Cells were incubated for 24 hours at 37°C prior to supernatant collection. The amount of IL-6 and PGE 2 in the supernatant was determined with ELISAs/EIAs as detailed earlier. Assays were performed a minimum of 2 times in quadruplicate.
Statistical analysis
GraphPad Prism 4 (GraphPad Software Inc, La Jolla, CA) was used to conduct the statistical analysis. For most cases, paired t tests were used to compare between vestibular and external vulvar cells, cases and controls, or treated and vehicle control samples. An analysis of variance with a post hoc Tukey test was used to determine differences in the responses to specific C albicans doses ( Figure 1 ).
Results
Patient vestibular fibroblasts respond strongly to low infectious doses of live C albicans and to isolated yeast components present in zymosan
At the time of diagnosis, LPV cases often show no signs of active yeast infection, yet the cells associated with pain produce proinflammatory/pro-pain mediators. Therefore, we examined the response of LPV patient fibroblasts to decreasing doses of live yeast to assess whether they may respond to clinically undetectable amounts of yeast in the vulvar vestibule.
In keeping with our hypothesis, we found that vestibular cells from LPV cases respond strongly, through the production of both IL-6 and PGE 2 , to as few as 100 C albicans CFUs, which translates to a multiplicity of infection (number of yeast cells per each mammalian cell) of ∼0.001 ( Figure 1 , A and B), which is lower than the numbers of yeast present during active infection in vivo. There is a statistically significant difference between the vehicle control and a dose of 100 CFUs for both IL-6 and PGE 2 in vestibular strains ( P < .05), while the difference between the vehicle control and a dose of 10 CFUs approaches significance, yet is not statistically different from the vehicle control ( P < .1). External vulvar cells (not typically associated with pain) show a less potent response to even higher doses of C albicans (eg, 10 4 CFUs), consistent with previous observations. There are no statistically significant differences between vehicle control and any dose of yeast for both IL-6 and PGE 2 in external vulvar cells ( P > .05). Furthermore, with the exception of the vehicle control, the amount of PGE 2 and IL-6 released by vestibular cells is significantly higher than in external vulvar cells ( P < .05). These data support the notion that pain-associated vestibular cells may be able to sense and respond to a very small population of yeast cells normally present in the vagina not actively associated with infection and potentially below the threshold of detection by standard yeast screening techniques, such as DNA probe or culture.
To further investigate the response to specific yeast components, we measured IL-6 mRNA levels before and after treatment with zymosan, which contains the yeast cell wall PAMPs, β-glucan, and mannoprotein. We found that IL-6 mRNA levels were strongly induced in as little as 6 hours following zymosan treatment and that IL-6 was significantly more highly expressed in vestibular vs external vulvar cells and more highly expressed in cases vs controls ( P < .05); the expression of IL-6 was >300-fold more highly expressed in vestibular cells from cases compared to those from controls ( P < .05) ( Figure 1 , C). These results suggest that human vestibular fibroblasts obtained from LPV patients are exquisitely sensitive to components of the yeast cell wall, namely β-glucan and mannoprotein.
Human vestibular and external vulvar fibroblasts express the yeast β-glucan receptor Dectin-1
Our first step in examining the mechanism by which host fibroblasts recognize yeast components was to test for the expression of Dectin-1, a paradigm receptor for yeast β-glucan, which is a chief component of zymosan and is also present in the cell wall and biofilm matrix of C albicans . We used published primer sequences for the CLEC7A gene encoding Dectin-1 to examine expression in vestibular and vulvar strains obtained from a case demonstrated to respond strongly to challenge with yeast/yeast products. We determined that: (1) CLEC7A mRNA is expressed in both vestibular and external vulvar cells, (2) zymosan increases CLEC7A expression in vestibular cells after 72 hours of treatment ( P < .05), and (3) CLEC7A is more highly expressed in vestibular vs external vulvar cells following treatment with zymosan, while the expression is slightly higher, but not significantly different between vestibular and external vulvar cells prior to treatment ( P > .05) ( Figure 2 ).
We then went on to detect Dectin-1 protein on the surface of vestibular and external vulvar cells from both patient and control strains using flow cytometry analysis with a fluorescently labeled antibody against Dectin-1. Using this approach, we identified Dectin-1 on the surface of vestibular and external vulvar strains from both case and controls, demonstrating that Dectin-1 is present in these strains in a location that may allow it to readily interact with yeast β-glucan moieties present in vivo. However, we did not identify any significant differences in Dectin-1 surface expression between the case and control or between the vestibular and external vulvar strains ( Figure 3 , A). Therefore, we elected to expand our survey to multiple case and control strains using Western blotting to assess Dectin-1 expression in total protein lysates.
Western blotting, loading equivalent amounts of protein and using a second distinct antibody to Dectin-1, showed that a wider survey of fibroblasts strains from both patients and controls express readily detectable levels of Dectin-1 ( Figure 3 , B). Furthermore, densitometry analysis of the Dectin-1 bands revealed that Dectin-1 was slightly more abundant in patient lysates from vestibular cells than from external vulvar cells ( P < .05) and slightly more abundant in cases vs controls ( P < .05) ( Figure 3 , C). These data demonstrate that while Dectin-1 is present on and in human vestibular and external vulvar strains (a new finding), its abundance may also be slightly elevated in fibroblast strains obtained from patients at sites where LPV pain is localized.
Functional Dectin-1 receptor plays a role in proinflammatory cytokine production
After detecting the Dectin-1 receptor on human fibroblast strains, we sought to investigate its function in proinflammatory mediator production by monitoring the release of IL-6 and PGE 2 after impairing the ability of the receptor to recognize zymosan or after inhibiting its transcription/expression. In the first approach, we used laminarin to saturate the binding sites on Dectin-1 prior to challenge with zymosan. Laminarin is a soluble β-glucan moiety that readily binds Dectin-1, while the strength of its interaction with Dectin-1 is not sufficient to elicit an inflammatory response. We found that laminarin was highly effective in reducing both IL-6 and PGE 2 release in vestibular and external vulvar fibroblasts; treated values were significantly lower than vehicle control ( P < .05). Furthermore, there was a significant difference in the amount of IL-6 and PGE 2 produced by vestibular vs external vulvar cells for both laminarin-treated and vehicle-treated cells ( P < .05). Even after treatment, vestibular cells continued to produce significantly more proinflammatory mediators than their external vulvar counterparts ( Figure 4 , A and B).
