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

Uncovering a MYC-driven Tumor-suppressive Program in Proliferating Lymphocytes

Rapid cell proliferation is a hallmark feature of adaptive immune cells lymphocytes. It is essential for the establishment of diverse antigen receptor repertoires and amplification of antigen-specific immune responses. While such proliferation is beneficial for host protection from infections and cancers, it inevitably elevates the risk of oncogenic transformation. In developing and germinal center B lymphocytes, the risk is further increased by endogenous, genomic insults due to antigen receptor rearrangements and somatic mutations, with which expression of the proto-oncogene c-MYC is closely associated. Nonetheless, frequencies of cancers originated from B lymphocytes are relatively low, suggesting that they are protected from transformation through a putative tumor-suppressive program coupled with their cell proliferation. In this work, we found that the proliferative driver c-MYC not only facilitates rapid cell proliferation but also unexpectedly engages a counteracting tumor-suppressive program through its downstream protein TFAP4. Missense mutations of TFAP4, including loss of function variants, were detected in human lymphoid malignancies, particularly in 12% of Burkitt lymphomas driven by overexpression of c-MYC. Furthermore, B-ALL patients with c-MYC overexpression and reduced expression of TFAP4 had worse survival compared to patients with high TFAP4 expressing B-ALL. In mice, haploinsufficiency for AP4 drastically accelerated tumor development in MYC-driven B cell lymphoid tumor models in a cell-intrinsic manner. Mechanistically, TFAP4 restricted the expression of protooncogene Erg, which, together with c-MYC, is required for B cell development. The risk of transformation was thus limited by blocking inappropriate co-expression of the two oncogenic proteins. TFAP4-mediated restriction of Erg also ensured appropriate coupling of c-MYC-mediated proliferation and the loss of self-renewing capacity in developing B cells. Thus, c-MYC suppresses the stemness of B cell progenitors by inducing TFAP4 while driving proliferation, and this transcription factor cascade functions as a tumor suppressor module that safeguards against the transformation of developing B cells. Thus, c-MYC concurrently engages proliferative and tumor-suppressive programs in B lymphocytes

Repeated hapten exposure induces persistent tactile sensitivity in mice modeling localized provoked vulvodynia.

BACKGROUND:Vulvodynia is a remarkably prevalent chronic pain condition of unknown etiology. Epidemiologic studies associate the risk of vulvodynia with a history of atopic disease. We used an established model of hapten-driven contact hypersensitivity to investigate the underlying mechanisms of allergy-provoked prolonged sensitivity to pressure. METHODS:We sensitized female ND4 Swiss mice to the hapten oxazolone on their flanks, and subsequently challenged them four days later with oxazolone or vehicle for ten consecutive days on the labia. We evaluated labiar sensitivity to touch, local mast cell accumulation, and hyperinnervation after ten challenges. RESULTS:Oxazolone-challenged mice developed significant tactile sensitivity that persisted for over three weeks after labiar allergen exposures ceased. Allergic sites were characterized by mast cell accumulation, sensory hyper-innervation and infiltration of regulatory CD4+CD25+FoxP3+ T cells as well as localized early increases in transcripts encoding Nerve Growth Factor and nerve-mast cell synapse marker Cell Adhesion Molecule 1. Local depletion of mast cells by intra-labiar administration of secretagogue compound 48/80 led to a reduction in both nerve density and tactile sensitivity. CONCLUSIONS:Mast cells regulate allergy-provoked persistent sensitivity to touch. Mast cell-targeted therapeutic strategies may provide novel means to manage and limit chronic pain conditions associated with atopic disease

A Newton Algorithm for the Nearest Correlation Matrix

Firstly, we describe and investigate the algorithm of Qi and Sun which solves the problem of finding the nearest correlation matrix to a symmetric matrix. This algorithm claims a quadratic convergence. We discuss improving this algorithm's efficiency and reliability and detect a problem when we are aiming at a nearest correlation matrix with a high accuracy, using small error tolerences. As a consequence, we suggest a modified version, based on the algorithm of Qi and Sun, which is also a quadratically convergent algorithm, has improved efficiency and is modified so that the algorithm can return the nearest correlation matrix to high accuracy showing a robust and reliable behaviour. Secondly, we investigate the general alternating projections method and also Higham's alternating projections method for the nearest correlation matrix. We discuss variations of the latter and include a further projection which allows more constraints to be added to the problem. We introduce a new algorithm and compare its convergence behaviour with Higham's alternating projections method

Repeated hapten exposure induces persistent tactile sensitivity in mice modeling localized provoked vulvodynia - Fig 1

<p><b>Timeline of oxazolone sensitization, challenge, and post-challenge outcome measures</b> (A) To measure oxazolone-driven vulvar tactile sensitivity, mice were topically sensitized with 2% Ox on the shaved flank (day 1) and subsequently challenged on the shaved labiar skin (days 5–14) with 1% Ox or EtOH vehicle for a total of 10 challenges. Tactile sensitivity was assessed in the ano-genital ridge area 1, 21, and 42 days after challenge cessation. Labiar skin was harvested at these time points from a separate cohort of mice for assessing molecular and cellular changes in the tissue. (B) To characterize T cell infiltration in Ox-challenged skin, mice were topically sensitized on their shaved back with 2% Ox (day 1) and challenged on both shaved flanks with 1% Ox or EtOH (days 5–14). Flank skin was harvested from both sides 1 day after challenge cessation for flow cytometric analysis of T cell infiltration. (C) To assess the effects of local mast cell depletion on Ox-induced tactile sensitivity and hyperinnervation, mice were topically sensitized with 2% Ox on the shaved flank (day 1), challenged on the shaved labia with 1% Ox or EtOH (days 5–14) and treated with intralabiar injection of saline or c48/80 (days 5–8 after Ox challenge cessation). Tactile sensitivity, mast cell levels and innervation were assessed 9, 21, and 35 days after the final Ox challenge.</p

Injection of c48/80 after challenges depletes mast cells and reduces CGRP⁺ nerve density and sensitivity.

<p>Density of Avidin⁺ mast cells (A) and CGRP⁺ cutaneous nerves (D) on day 9 after 4 treatments with c48/80 or saline (administered on days 5–8 after cessation of 10 Ox challenges) in 10 μm labiar cryo-sections, displayed as mean ± SEM (n = 2-3/treatment group). Representative images for mast cells (B-C) and nerves (E-F); 20x magnification; scale bar represents 50 μm. Means are compared to Ox/EtOH (** = p<0.01, *** = p<0.001); significance determined using one-way ANOVA and Tukey Kramer <i>post hoc</i> analysis. (G) Tactile sensitivity in mice treated with either saline or c48/80 (n = 6–9 mice per treatment group; two independent experiments). Means are compared to Ox/Ox/Saline (* = p<0.05, ** = p<0.01, *** = p<0.001); significance determined using an unpaired Student’s T test at each time point.</p

Injection of c48/80 after challenges depletes mast cells and reduces CGRP⁺ nerve density and sensitivity.

<p>Density of Avidin⁺ mast cells (A) and CGRP⁺ cutaneous nerves (D) on day 9 after 4 treatments with c48/80 or saline (administered on days 5–8 after cessation of 10 Ox challenges) in 10 μm labiar cryo-sections, displayed as mean ± SEM (n = 2-3/treatment group). Representative images for mast cells (B-C) and nerves (E-F); 20x magnification; scale bar represents 50 μm. Means are compared to Ox/EtOH (** = p<0.01, *** = p<0.001); significance determined using one-way ANOVA and Tukey Kramer <i>post hoc</i> analysis. (G) Tactile sensitivity in mice treated with either saline or c48/80 (n = 6–9 mice per treatment group; two independent experiments). Means are compared to Ox/Ox/Saline (* = p<0.05, ** = p<0.01, *** = p<0.001); significance determined using an unpaired Student’s T test at each time point.</p

Ten oxazolone challenges provoke tactile sensitivity that persists for 21 days after cessation of challenges.

<p>Sensitized mice that received ten daily Ox challenges on the labiar skin had increased tactile sensitivity in their ano-genital ridge area compared to controls. Percent decrease in labiar withdrawal threshold for each treatment group is displayed as mean ± SEM. Significance at each time point was determined using one-way ANOVA and Tukey Kramer <i>post hoc</i> analysis based on comparisons to previously sensitized mice challenged with EtOH (Ox/EtOH; * = p<0.05, *** = p<0.001) and untreated controls (NT; ## = p<0.01, ### = p<0.001). n = 9–12 mice per treatment group; data represent two independent experiments. Raw withdrawal thresholds at baseline and post Ox-challenge cessation for each animal are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169672#pone.0169672.s001" target="_blank">S1 Fig</a> and summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169672#pone.0169672.s004" target="_blank">S1 Table</a>.</p