No abstract

A history of allergies doubles the risk of vulvodynia—a chronic pain condition of unknown etiology often accompanied by increases in numbers of vulvar mast cells. We previously established the biological plausibility of this relationship in mouse models where repeated exposures to the allergens oxazolone or dinitrofluorobenzene on the labiar skin or inside the vaginal canal of ND4 Swiss Webster outbred mice led to persistent tactile sensitivity and local increases in mast cells. In these models, depletion of mast cells alleviated pain. While exposure to cleaning chemicals has been connected to elevated vulvodynia risk, no single agent has been linked to adverse outcomes. We sensitized female mice to methylisothiazolinone (MI)—a biocide preservative ubiquitous in cosmetics and cleaners—dissolved in saline on their flanks, and subsequently challenged them with MI or saline for ten consecutive days in the vaginal canal. MI-challenged mice developed persistent tactile sensitivity, increased vaginal mast cells and eosinophils, and had higher serum Immunoglobulin E. Therapeutic and preventive intra-vaginal administration of Δ9-tetrahydrocannabinol reduced mast cell accumulation and tactile sensitivity. MI is known to cause skin and airway irritation in humans, and here we provide the first pre-clinical evidence that repeated MI exposures can also provoke allergy-driven genital pain.

Occupational exposure to toxic chemicals increases the risk of developing localized provoked vulvodynia—a prevalent, yet poorly understood, chronic condition characterized by sensitivity to touch and pressure, and accumulation of mast cells in painful tissues. Here, we topically sensitized female ND4 Swiss mice to the common household and industrial preservative methylisothiazolinone (MI) and subsequently challenged them daily with MI or acetone and olive oil vehicle on the labiar skin. MI-challenged mice developed significant, persistent tactile sensitivity and long-lasting local accumulation of mast cells alongside early, transient increases in CD4 + and CD8 + T cells, eosinophils, neutrophils, and increases in pro-inflammatory cytokines. Therapeutic administration of imatinib, a c-Kit inhibitor known to inhibit mast cell survival, led to reduced mast cell accumulation and alleviated tactile genital pain. We provide the first pre-clinical evidence of dermal MI-induced mast-cell dependent pain and lay the groundwork for detailed understanding of these intersections between MI-driven immunomodulation and chronic pain.

Shortly after priming, the fate of activated CD4 T cells is segregated into BCL6+ follicular helper T (Tfh) and BCL6− effector (Teff) cells. However, it remains unknown how these subsets are sustained in the presence of chronic antigen stimulation. Using a combination of single cell- and population-based approaches, we show that in chronic viral infection, activated CD4 T cells differentiate into BCL6-dependent TCF-1+ progenitor cells with superior capacity to expand and give rise to both Teff and Tfh. They share properties with progenitor-exhausted CD8 T cells and are required for the continued generation of Teff cells as antigen persists. In response to tumors, an analogous CD4 T cell population develops in draining lymph nodes. Our study reveals the heterogeneity and plasticity of CD4 T cells upon encountering persistent antigen and highlights their population dynamics through a stable bipotent intermediate state.

Primary human vulvovaginal fibroblast cell lines are useful for studying biological mechanisms underlying genital pain, pelvic organ prolapse, and the spread of sexually transmitted infections. However, the vaginal biopsies necessary for establishing these cell lines are invasive and relatively difficult to obtain. Primary mouse fibroblast cell lines derived from pre-clinical animal models of these conditions can be used for better controlled experiments that can help us dissect disease mechanisms. To our knowledge, there are no published protocols for establishing primary murine vaginal fibroblast cell lines to date. Here, we describe a protocol for the establishment of murine vaginal fibroblast cell lines via enzymatic digestion of vaginal canal tissue. Cell lines generated using this method can be used for in vitro studies of these important structural cells in a variety of pre-clinical mouse models; such studies are required to identify and characterize relevant regulatory and therapeutic targets in a wide array of diseases of interest. As shown in our representative data, this protocol yields viable cell lines from ND4 Swiss outbred mice. These cells bear surface markers characteristic of fibroblasts and are capable of producing inflammatory cytokines in response to treatment with bacterial and yeast antigens in vitro .