Tetrahydrocannabinol Reduces Hapten-Driven Mast Cell Accumulation and Persistent Tactile Sensitivity in Mouse Model of Allergen-Provoked Localized Vulvodynia

Abstract

Vulvodynia is a remarkably prevalent chronic pain condition of unknown etiology. An increase in numbers of vulvar mast cells often accompanies a clinical diagnosis of vulvodynia and a history of allergies amplifies the risk of developing this condition. We previously showed that repeated exposures to oxazolone dissolved in ethanol on the labiar skin of mice led to persistent genital sensitivity to pressure and a sustained increase in labiar mast cells. Here we sensitized female mice to the hapten dinitrofluorobenzene (DNFB) dissolved in saline on their flanks, and subsequently challenged them with the same hapten or saline vehicle alone for ten consecutive days either on labiar skin or in the vaginal canal. We evaluated tactile ano-genital sensitivity, and tissue inflammation at serial timepoints. DNFB-challenged mice developed significant, persistent tactile sensitivity. Allergic sites showed mast cell accumulation, infiltration of resident memory CD8⁺CD103⁺ T cells, early, localized increases in eosinophils and neutrophils, and sustained elevation of serum Immunoglobulin E (IgE). Therapeutic intra-vaginal administration of Δ⁹-tetrahydrocannabinol (THC) reduced mast cell accumulation and tactile sensitivity. Mast cell-targeted therapeutic strategies may therefore provide new ways to manage and treat vulvar pain potentially instigated by repeated allergenic exposures.

Keywords: chronic pain; dinitrofluorobenzene; hypersensitivity; mast cells; vulvodynia; Δ9-tetrahydrocannabinol.

Figure 1

Repeated topical dinitrofluorobenzene (DNFB) exposure on the labiar skin in previously sensitized ND4 mice results in increased abundance of local mast cells, higher levels of IL-6 and IFN-γ transcripts, elevated serum IgE, greater myeloperoxidase and eosinophil peroxidase activity, and increased numbers of infiltrating T cells. (A) Representative sections at 200x magnification showing FITC (fluorescein-isothiocyanate)-Avidin-stained mast cell granules in labiar tissues of DNFB vs. saline-challenged mice 1 day and 21 days after 10 challenges; (B) Relative intensity of Avidin-stained mast cell granules in the labiar skin of DNFB vs. saline-challenged mice 1 day and 21 days after 10 labiar DNFB challenges. n= 3; (C) Total IgE levels in serum measured 1 day, 21 days, and 42 days after 10 challenges with DNFB or saline in previously sensitized mice and in age-matched untreated mice (NT). ** and * represent p-values of p < 0.01 and p < 0.05 respectively. n = 3–6; (D) Eosinophil peroxidase and (E) myeloperoxidase levels in the labiar tissue, normalized to tissue weight, 1 and 21 days after 10 DNFB or saline challenges. *** represents p-values of p < 0.001. n = 3–6; (F) CsD3⁺, CD4⁺, CD4⁺CD25⁺, CD4⁺CD44⁺, CD4⁺CD25⁺CD44⁺, CD8⁺, and CD8⁺CD103⁺ cell numbers in flank tissues derived from previously sensitized mice challenged with either DNFB or saline vehicle on the flank. ** and * represent p-values of p < 0.01 and p < 0.05 respectively. n = 6; nd = not detected; (G) Relative abundances of IL-6 and IFN-γ mRNAs in the labiar tissue of DNFB- vs. saline-challenged, previously sensitized mice 1 day and 21 days after 10 DNFB challenges, each normalized to β2-microglobulin (housekeeping) mRNA levels. n = 3.

Figure 2

Ten daily DNFB challenges on the labiar skin and in the vaginal canal results in increased sensitivity that persists 21 days post tenth challenge. (A) Percent decrease from baseline measurements 1, 21, and 42 days after 10 labiar challenges, n = 18–27 and (B) 1, 14, 21, and 28 days post vaginal canal challenge. Only post-treatment withdrawal responses that show a 33%, represented by blue dotted line, or greater decrease from baseline withdrawal thresholds are considered hyperalgesic. *** and * represent a p-value of p < 0.001 and p < 0.05 respectively n = 9–18.

Figure 3

Repeated DNFB exposure in the vaginal canal provokes increased local mast cell density, upregulation of IL-6 and IFN-γ expression, increased IgE concentration in the serum, and increased numbers of B cells, T cells, and mast cells in the iliac lymph nodes. (A) Representative immunofluorescent images at 200X magnification of FITC-Avidin stained mast cells in vaginal canal tissue of DNFB and saline challenged mice 1 day and 21 days post 10th challenge; (B) Relative fluorescent intensity of DNFB challenged mice 1 day and 21 days post 10 challenges compared to saline challenged mice. n = 3; (C) Relative transcript abundance of IL-6 and IFN-γ mRNAs in the vaginal canal tissue normalized to β2-microglobulin (housekeeping) mRNA levels. n = 5–9; Local (D) eosinophil peroxidase and (E) myeloperoxidase levels in the vaginal canal, normalized to tissue weight, 1 day after 10 DNFB or saline challenges. * represents a p-value of p < 0.05. n = 3; (F) Total IgE levels in the serum measured 1 day after 10 challenges with DNFB or saline in previously sensitized mice and in age-matched untreated mice (NT). ** represents a p-value of p < 0.01. n = 4–6; Cell counts of (G) mast cells, and (H) B cells, CD4⁺ T cells, and CD8⁺ T cells in the iliac lymph nodes 1 day, 3 days, and 7 days post 10th challenge. ** and * represent a p-value of p < 0.01 and p < 0.05 respectively. n = 6.

Figure 4

Repeated local Δ⁹-tetrahydrocannabinol (THC) application after ten DNFB challenges in the vaginal canal results in decreased sensitivity and local mast cell densities compared to controls. (A) Sensitivity measured 1 day after 10 DNFB challenges and either 6 THC treatments or no treatment (NT). Only post-treatment withdrawal responses that show a 33%, represented by the dotted line, or greater decrease from baseline withdrawal thresholds are considered hyperalgesic. ** represents a p-value of p < 0.01. n = 17–18; (B) Representative immunofluorescence images at 200x magnification showing fluorescent FITC-Avidin-stained mast cell granules in vaginal canals of THC treated and not treated mice; (C) Relative intensity of Avidin-stained mast cell granules in vaginal canals of THC treated mice calculated as fold change over non-THC treated mice. n = 3.

Figure 5

Repeated DNFB exposures on the skin lead to accumulation of regulatory T cells, resident memory T cells and mast cells. Mast cell increases are correlated with increased tactile sensitivity; therapeutic intra-vaginal THC application reduces mast cell density and tactile pain. Solid arrows indicate the hypothesized mechanisms proposed in this model. Dotted lines indicate previously published experimental observations of the contributions of regulatory T cells, IFN-γ and circulating IgE to mast cell recruitment, survival and activity in the tissue. T-bars indicate suppression of mast cell accumulation and tactile sensitivity following THC application. Artwork by Amy Pelz.