Immune mechanisms in vulvodynia: key roles for mast cells and fibroblasts

Vulvodynia is a debilitating condition characterized by painful sensitivity to touch and pressure in the vestibular tissue surrounding the vaginal opening. It is often a “diagnosis of exclusion” of idiopathic pain made in the absence of visible inflammation or injury. However, the association between increased vulvodynia risk and a history of yeast infections and skin allergies has led researchers to explore whether immune mechanisms of dysregulated inflammation might underlie the pathophysiology of this chronic pain condition. Here we synthesize epidemiological investigations, clinical biopsies and primary cell culture studies, and mechanistic insights from several pre-clinical models of vulvar pain. Taken together, these findings suggest that altered inflammatory responses of tissue fibroblasts, and other immune changes in the genital tissues, potentially driven by the accumulation of mast cells may be key to the development of chronic vulvar pain. The association of increased numbers and function of mast cells with a wide variety of chronic pain conditions lends credence to their involvement in vulvodynia pathology and underscores their potential as an immune biomarker for chronic pain. Alongside mast cells, neutrophils, macrophages, and numerous inflammatory cytokines and mediators are associated with chronic pain suggesting immune-targeted approaches including the therapeutic administration of endogenous anti-inflammatory compounds could provide much needed new ways to treat, manage, and control the growing global pandemic of chronic pain

Contact Hypersensitivity to Oxazolone Provokes Vulvar Mechanical Hyperalgesia in Mice

The interplay among pain, allergy and dysregulated inflammation promises to yield significant conceptual advances in immunology and chronic pain. Hapten-mediated contact hypersensitivity reactions are used to model skin allergies in rodents but have not been utilized to study associated changes in pain perception in the affected skin. Here we characterized changes in mechanical hyperalgesia in oxazolone-sensitized female mice challenged with single and repeated labiar skin exposure to oxazolone. Female mice were sensitized with topical oxazolone on their flanks and challenged 1-3 times on the labia. We then measured mechanical sensitivity of the vulvar region with an electronic pressure meter and evaluated expression of inflammatory genes, leukocyte influx and levels of innervation in the labiar tissue. Oxazolone-sensitized mice developed vulvar mechanical hyperalgesia after a single labiar oxazolone challenge. Hyperalgesia lasted up to 24 hours along with local influx of neutrophils, upregulation of inflammatory cytokine gene expression, and increased density of cutaneous labiar nerve fibers. Three daily oxazolone challenges produced vulvar mechanical hyperalgesic responses and increases in nerve density that were detectable up to 5 days post-challenge even after overt inflammation resolved. This persistent vulvar hyperalgesia is resonant with vulvodynia, an understudied chronic pain condition that is remarkably prevalent in 18-60 year-old women. An elevated risk for vulvodynia has been associated with a history of environmental allergies. Our pre-clinical model can be readily adapted to regimens of chronic exposures and long-term assessment of vulvar pain with and without concurrent inflammation to improve our understanding of mechanisms underlying subsets of vulvodynia and to develop new therapeutics for this condition

Mast cells: Versatile Gatekeepers of Pain

Mast cells are important first responders in protective pain responses that provoke withdrawal from intense, noxious environmental stimuli, in part because of their sentinel location in tissue-environment interfaces. In chronic pain disorders, the proximity of mast cells to nerves potentiates critical molecular cross-talk between these two cell types that results in their synergistic contribution to the initiation and propagation of long-term changes in pain responses via intricate signal networks of neurotransmitters, cytokines and adhesion molecules. Both in rodent models of inflammatory pain and chronic pain disorders, as well as in increasing evidence from the clinic, it is abundantly clear that understanding the mast cell-mediated mechanisms underlying protective and maladaptive pain cascades will lead to improved understanding of mast cell biology as well as the development of novel, targeted therapies for the treatment and management of debilitating pain conditions

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic Von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen, or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis

TNF-Alpha Neutralizing Antibody Blocks Thermal Sensitivity Induced By Compound 48/80-Provoked Mast Cell Degranulation

BACKGROUND:Neuro-inflammatory circuits in the tissue regulate the complex pathophysiology of pain. Protective nociceptive pain serves as an early warning system against noxious environmental stimuli. Tissue-resident mast cells orchestrate the increased thermal sensitivity following injection of basic secretagogue compound 48/80 in the hind paw tissues of ND4 mice. Here we investigated the effects of pre-treatment with TNF-α neutralizing antibody on compound 48/80-provoked thermal hyperalgesia.METHODS:We treated ND4 Swiss male mice with intravenous anti-TNF-α antibody or vehicle 30 minutes prior to bilateral, intra-plantar compound 48/80 administration and measured changes in the timing of hind paw withdrawal observed subsequent to mice being placed on a 51oC hotplate. We also assessed changes in tissue swelling, TNF-α gene expression and protein abundance, mast cell degranulation, and neutrophil influx in the hind paw tissue.FINDINGS:We found that TNF-α neutralization significantly blocked thermal hyperalgesia, and reduced early tissue swelling. TNF-α neutralization had no significant effect on mast cell degranulation or neutrophil influx into the tissue, however. Moreover, no changes in TNF-α protein or mRNA levels were detected within 3 hours of administration of compound 48/80.INTERPRETATION: The neutralizing antibodies likely target pre-formed TNF-α including that stored in the granules of tissue-resident mast cells. Pre-formed TNF-α, released upon degranulation, has immediate effects on nociceptive signaling prior to the induction of neutrophil influx. These early effects on nociceptors are abrogated by TNF-α blockade, resulting in compromised nociceptive withdrawal responses to acute, harmful environmental stimuli

Recurrent Yeast Infections and Vulvodynia: Can We Believe Associations Based on Self-Reported Data?

Objective: We determined whether self-reported new or recurrent yeast infections were a risk factor for and/or consequence of vulvodynia and then determined the extent to which various levels of misclassification of self-reported yeast infections influenced these results. Materials and Methods: In this case–control study we retrospectively assessed self-reported new and recurrent yeast infections prior and subsequent to first vulvar pain onset among 216 clinically confirmed cases and during a similar time period for 224 general population controls. Results: A history of \u3e10 yeast infections before vulvodynia onset was strongly but imprecisely associated with currently diagnosed vulvodynia after adjustment for age, age at first intercourse, and history of urinary tract infections [adjusted odds ratio = 5.5, 95% confidence interval (CI) 1.7–17.8]. Likewise, a history of vulvodynia was associated with a twofold risk of subsequent new or recurrent onset of yeast infections after adjustment for age, age at first intercourse, and history of yeast infections before vulvodynia onset (comparable time period among controls, 95% CI 1.5–2.9). Bias analyses showed that our observed associations were an underestimation of the true association when nondifferential misclassification of self-reported yeast infections and certain differential misclassification scenarios were present. However, if women with vulvodynia more frequently misreported having them when they truly did not, our observed associations were an overestimate of the truth. Conclusions: There appears to be a positive relationship between yeast infections preceding and following the diagnosis of vulvodynia, but this relationship varies from strong to nonexistent depending on the relative accuracy of the recalled diagnosis of yeast infections among cases and controls. To better understand the bidirectional associations between yeast infections and vulvodynia, future validation studies are needed to determine the extent to which misclassification of self-reported yeast infections differs between women with and without vulvodynia

The C–C chemokine receptor 7: An immune molecule that modulates central nervous system function in homeostasis and disease

The interaction between the central nervous system (CNS) and the peripheral immune system is key for brain function in homeostasis and disease. Recent studies have revealed that the C–C chemokine receptor 7 (CCR7) is expressed in both CNS resident cells and peripheral immune cells, and plays an important role in regulating behavior in homeostasis and neuroinflammation in disease. This review integrates studies examining the role of CCR7 in CNS resident and peripheral immune cells in homeostasis and disease, as well as the pathways of peripheral immune cell migration in and out of the brain via CCR7. A special emphasis is placed on the CCR7-dependent migration of peripheral immune cells into the recently discovered meningeal lymphatic vessels surrounding the brain and nasal lymphatics, its migration into cervical lymph nodes, and the implications that this migration might have for CNS function