Identification of novel mechanisms involved in generating localized vulvodynia pain

Abstract

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 (PGE2). 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 PGE2 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 PGE2 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.

Keywords: Dectin-1; cytokine; fibroblast; inflammation; interleukin 6; prostaglandin; vulvodynia.

Figure 1. C. albicans and components of the yeast cell wall are potent inducers of the pro-inflammatory response in vestibular fibroblasts

Panels A (IL-6) and B (PGE₂) depict the amount of pro-inflammatory cytokine released in response to decreasing doses of live C. albicans. A single asterisk denotes a difference between the vehicle control and a particular dose of C. albicans (for vestibular cells only), while two asterisks denote a significant difference between vestibular and vulvar cells for a particular dose. Significance was determined via ANOVA with a post hoc Tukey test; values less than 0.05 were considered significant. Vestibular cells show a strong response to infection with live yeast and yeast cell wall components (zymosan), with a significant response to as little at 100 CFUs, while vulvar cells show no significant response to a dose up to 1000 times greater. Panel C depicts the transcription of the gene encoding IL-6 in response to treatment with zymosan for 72 h. A single asterisk denotes a difference between paired vestibular and vulvar cells obtained from a patient, while two asterisks denote differences between the normal control and the case for vestibular or vulvar strains. Paired t-tests were used to compared between case and control and vestibular and vulvar strains (n = 8, p < 0.05). After zymosan treatment, expression of IL-6 was significantly elevated in vestibular versus vulvar cells and in the case versus control, with the highest level of expression occurring in patient vestibular cells. Data are represented as the mean ± one standard deviation.

Figure 2. Dectin-1 mRNA is expressed by patient vestibular strains and its expression is increased with zymosan treatment

This figure depicts expression profiles for CLEC7A, the gene encoding Dectin-1. A single asterisk denotes a difference between the vehicle and zymosan treatment for vestibular cells, while two asterisks denote a difference between vestibular and vulvar cells. Paired t-tests comparing vestibular versus vulvar and vehicle versus zymosan were used to determine significance (n = 9, p < 0.05). Dectin-1 is expressed on human vestibular and vulvar fibroblasts; expression is induced with zymosan treatment and is elevated in vestibular versus vulvar strains. Data is represented as the mean ± one standard deviation.

Figure 3. Dectin-1 is expressed on the surface of fibroblast strains and is more abundant in vestibular cells and cases

Panel A depicts a typical result from flow cytometry analysis examining surface expression of Dectin-1. Although all cells are strongly positive for Dectin-1, there are no obvious differences between case and control or vestibular and vulvar strains. Panel B depicts a Western blot probing for Dectin-1 examining 4 paired case and 4 paired control strains. The bands alternate between vestibular (vest.) and vulvar (vulv.) strains as noted. All samples were run on a single Western blot with a single exposure to autoradiography film; bands have been cropped and positioned for display purposes. Equivalent loading was confirmed using total protein staining (not shown). Visual assessment suggests that Dectin-1 is more highly expressed in vestibular versus vulvar strains and in cases versus controls, which was confirmed via densitometry analysis, depicted in panel C. A single asterisk denotes a difference between vestibular and vulvar cells for cases, while two asterisks denote a difference between cases and controls for vestibular strains. Paired t-tests (case versus control, vestibular versus vulvar) were used to determine significance (n = 4, p < 0.05). Data is represented as the mean ± one standard deviation.

Figure 4. Dectin-1 contributes to pro-inflammatory mediator production

Panels A (IL-6) and B (PGE₂) depict the impact of laminarin treatment on pro-inflammatory mediator production in response to zymosan. A single asterisk denotes a significant reduction in pro-inflammatory mediator released between zymosan alone and zymosan with laminarin pre-treatment, while two asterisks denote a difference between vestibular and vulvar strains. Paired t-tests (zymosan versus zymosan + laminarin, vestibular versus vulvar) were used to determine significance (n = 8, p <0.05). Panel C demonstrates that siRNA against CLEC7A (encoding Dectin-1) is effective in reducing the amount of Dectin-1 protein in total protein lysates compared to treatment control siRNA. Panels D (IL-6) and C (PGE₂) depict the impact of siRNA treatment on pro-inflammatory mediator release; asterisk designations are the same as for panels A and B. Data is represented as the mean ± one standard deviation. Inhibiting the function of Dectin-1 results in a reduction in the amount of pro-inflammatory mediator released by fibroblasts treated with zymosan.

Figure 5. Zymosan treatment activates the NFκB pathway

This figure depicts a typical result for a representative LPV case (more than one strain was tested). Panel A shows the relative DNA binding activity of NFκB subunits (p65, p50, c-Rel, and p52) detected in the nucleus. RelB was also detected, but the binding activity did not exceed background. Zymosan treatment induces the activity of p65 and p50, which is also higher in vestibular versus vulvar strains, while c-Rel and p52 activity may decline somewhat with zymosan treatment. Panel B depicts Western blotting analysis for p65; zymosan treatment induces translocation of p65 to the nucleus, while translocation may be slightly accentuated in vestibular versus vulvar strains following zymosan treatment. The sensation of zymosan results in the activation of the NFκB pathway, likely through the canonical arm of the pathway, evidenced by specific translocation/activation of the p50 and p65 subunits.

Figure 6. Infection with live yeast activates the NFκB pathway

This figure depicts luciferase activity of an NFκB reporter normalized to constitutive renilla luciferase expression for patient strains infected with C. albicans and S. cerevisiae. A single asterisk denotes a significant difference between the vehicle control and C. albicans-infected cells, while two asterisks denote a significant difference between C. albicans- and S. cerevisiae-infected cells. Significance was determined via paired t-tests (vehicle versus C. albicans-infected, C. albicans-infected versus S. cerevisiae-infected; n = 8, p < 0.05). Data is represented as the mean ± one standard deviation. Although not denoted by a symbol, activity was also induced by S. cerevisiae compared to vehicle control (p < 0.05 for vestibular and for vulvar cells). Clearly, live yeast infection induces the activation of the NFκB pathway, while C. albicans infection is particularly potent, and the response is generally greater in pain-associated vestibular cells.

Figure 7. Activation of the NFκB pathway is essential for pro-inflammatory mediator production

Panels A (IL-6) and B (PGE₂) show that inhibition of the NFƙB pathway with the BAY-11-7082 inhibitor results in a dramatic and significant reduction in the amount of pro-inflammatory mediators released in response to treatment with zymosan. A single asterisk denotes a significant difference between zymosan alone and zymosan with BAY-11-7082. Significance was determined by paired t-test (zymosan alone versus zymosan + BAY-11-7082; n = 8, p < 0.05). Data is represented as the mean ± one standard deviation. Inhibition of the NFκB pathway reduces pro-inflammatory mediators to near background levels.