Emergence of New Concepts in Skin Physiopathology through the Use of in vitro Human Skin Explants Models.

BACKGROUND: This review summarizes uses and new applications for dermatological research of in vitro culture models of human skin explants (HSEs). In the last decade, many innovations have appeared in the literature and an exponential number of studies have been recorded in various fields of application such as process culture engineering, stem cell extractions methodology, or cell-to-cell interaction studies under physiological and pathological conditions, wound-healing, and inflammation. Most studies also concerned pharmacology, cosmetology, and photobiology. However, these topics will not be considered in our review. SUMMARY: A better understanding of the mechanisms driving intercellular relationships, at work in the maintenance of 3D tissue architectures has led to the improvement of cell culture techniques. Many papers have focused on the physiological ways that govern in vitro tissue maintenance of HSEs. The analysis of the necessary mechanical stress, intercellular and cell-matrix interactions, allows the maintenance and prolonged use of HSEs in culture for up to 15 days, regardless of the great variability of study protocols from one laboratory to another and in accordance with the objectives set. Because of their close similarities to fresh skin, HSEs are increasingly used to study skin barrier repair and wound healing physiology. Easy to use in co-culture, this model allows a better understanding of the connections and interactions between the peripheral nervous system, the skin and the immune system. The development of the concept of an integrated neuro-immuno-cutaneous system at work in skin physiology and pathology highlighted by this article represents one of the new technical challenges in the field of in vitro culture of HSE. This review of the literature also reveals the importance of using such models in pathology. As sources of stem cells, HSEs are the basis for the development of new tissue engineering models such as organoids or optical clearing tissues technology. This study identifies the main advances and cross-cutting issues in the use of HSE.

New human in vitro co-culture model of keratinocytes and sensory neurons like cells releasing substance P with an evaluation of the expression of ZIKV entry receptors: A potent opportunity to test Zika virus entry and to study Zika virus' infection in neurons?

During the course of acute ZIKV infection, pruritus is a cardinal symptom widely documented in the literature. Its frequent association with dysesthesia and several dysautonomic manifestations, suggests a pathophysiological mechanism involving the peripheral nervous system. The aim of this study was to develop a functional human model to potentially able to be infected by ZIKV: by demonstrating the functionality on a new human model of co-culture of keratinocyte and sensory neuron derived from induced pluripotent stem cells using a classical method of capsaicin induction and SP release, and verify the presence of ZIKV entry receptor in these cells. Depending of cellular type, receptors of the TAMs family, TIMs (TIM1, TIM3 and TIM4) and DC-SIGN and RIG1 were present/detected. The cells incubations with capsaicin resulted in an increase of the substance P. Hence, this study demonstrated the possibility to obtain co-cultures of human keratinocytes and human sensory neurons that release substance P in the same way than previously published in animal models which can be used as a model of neurogenic skin inflammation. The demonstration of the expression of ZIKV entry receptors in these cells allows to considerate the potent possibility that ZIKV is able to infect cells.

Basic mechanisms of itch.

Pruritus (or itch) is an unpleasant sensation leading to a desire to scratch. In the epidermis, there are selective C or Aδ epidermal nerve endings that are pruriceptors. At their other ends, peripheral neurons form synapses with spinal neurons and interneurons. Many areas in the central nervous system are involved in itch processing. Although itch does not occur solely because of parasitic, allergic, or immunologic diseases, it is usually the consequence of neuroimmune interactions. Histamine is involved in a minority of itchy conditions, and many other mediators play a role: cytokines (eg, IL-4, IL-13, IL-31, IL-33, and thymic stromal lymphopoietin), neurotransmitters (eg, substance P, calcitonin gene-related peptide, vasoactive intestinal peptide, neuropeptide Y, NBNP, endothelin 1, and gastrin-releasing peptide), and neurotrophins (eg, nerve growth factor and brain-derived neurotrophic factor). Moreover, ion channels such as voltage-gated sodium channels, transient receptor potential vanilloid 1, transient receptor ankyrin, and transient receptor potential cation channel subfamily M (melastatin) member 8 play a crucial role. The main markers of nonhistaminergic pruriceptors are PAR-2 and MrgprX2. A notable phenomenon is the sensitization to pruritus, in which regardless of the initial cause of pruritus, there is an increased responsiveness of peripheral and central pruriceptive neurons to their normal or subthreshold afferent input in the context of chronic itch.

Do Merkel complexes initiate mechanical itch?

Itch is a common sensation which is amenable to disabling patients' life under pathological and chronic conditions. Shared assertion easily limits itch to chemical itch, without considering mechanical itch and alloknesis, its pathological counterpart. However, in recent years, our understanding of the mechanical itch pathway, particularly in the central nervous system, has been enhanced. In addition, Merkel complexes, conventionally considered as tactile end organs only responsible for light touch perception due to Piezo2 expressed by both Merkel cells and SA1 Aß-fibres - low threshold mechanical receptors (LTMRs) -, have recently been identified as modulators of mechanical itch. However, the tactile end organs responsible for initiating mechanical itch remain unexplored. The consensus is that some LTMRs, either SA1 Aß- or A∂- and C-, are cutaneous initiators of mechanical itch, even though they are not self-sufficient to finely detect and encode light mechanical stimuli into sensory perceptions, which depend on the entire hosting tactile end organ. Consequently, to enlighten our understanding of mechanical itch initiation, this article discusses the opportunity to consider Merkel complexes as potential tactile end organs responsible for initiating mechanical itch, under both healthy and pathological conditions. Their unsuspected modulatory abilities indeed show that they are tuned to detect and encode light mechanical stimuli leading to mechanical itch, especially as they host not only SA1 Aß-LTMRs but also A∂- and C-fibres.

Complication rates of proximal hypospadias: meta-analyses of four surgical repairs.

INTRODUCTION: Proximal hypospadias surgery is impacted by a high complication rate. The goal of this work was to assess the overall composite complication rate, fistula rate and stenosis rate following proximal hypospadias surgery realized according to onlay urethroplasty, Duckett, Koyanagi and Bracka techniques. METHODS: The databases MEDLINE, EMBASE, SCOPUS, Cochrane Library, the Cochrane Database of Systematic Reviews and the Cochrane Central Register of Controlled Trials (CENTRAL) and Sciencedirect were searched. Studies had to report data about the mean age of population, the average duration of patient follow-up and the number of procedures required for surgical treatment of primary and proximal hypospadias. Two independent including one urologist reviewers screened all the articles and selected the articles to be included. RESULTS: Overall composite complication rates were 32%, 34%, 49%, and 43%, for Onlay urethroplasty, Duckett's tubularized flaps urethroplasty, Koyanagi repair and Bracka 2 stages repair, respectively. Fistula rates were 13%, 18%, 21% and 23% respectively. The heterogeneity of complication rates reported in the different studies was not moderated by age, country, or patient's continent origin. DISCUSSION: The classifications of complications used in articles were disparate and make comparisons between techniques difficult. The report of post-surgical complications in the literature is often poorly coded and follow-up times were often too short. CONCLUSION: This meta-analysis attempts to determine to the extent possible, given the serious weaknesses in the hypospadias literature, plausible estimates of complication rates after skin flap urethroplasty. The patched onlay skin flap, the Duckett's tubularized skin flap technique, the Koyanagi's technique, and the Bracka's two-stage urethroplasty procedure lead to very high complication rates. Reported complication rates are comparable across techniques.

Chronic itch: emerging treatments following new research concepts.

Until recently, itch pathophysiology was poorly understood and treatments were poorly effective in relieving itch. Current progress in our knowledge of the itch processing, the numerous mediators and receptors involved has led to a large variety of possible therapeutic pathways. Currently, inhibitors of IL-31, IL-4/13, NK1 receptors, opioids and cannabinoids, JAK, PDE4 or TRP are the main compounds involved in clinical trials. However, many new targets, such as Mas-related GPCRs and unexpected new pathways need to be also explored.

Poly- and Oligosaccharide Ulva sp. Fractions from Enzyme-Assisted Extraction Modulate the Metabolism of Extracellular Matrix in Human Skin Fibroblasts: Potential in Anti-Aging Dermo-Cosmetic Applications.

Ulva sp. is known to be a source of bioactive compounds such as ulvans, but their biological activity on human dermal fibroblast extracellular matrix (ECM) is poorly reported. In this work, the regulation of ECM has been investigated for the first time at both proteomic and transcriptomic levels in normal human skin dermal fibroblasts, after 48 h of incubation with poly- and oligosaccharide fractions from Ulva sp. obtained after enzyme-assisted extraction and depolymerization. Cell proliferation enhancement (up to +68%) without exhibiting any cytotoxic effect on fibroblasts was demonstrated at 50 and 1000 µg/mL by both fractions. At the proteomic level, polysaccharide fractions at 1000 µg/mL enhanced the most the synthesis of glycosaminoglycans (GAGs, up to +57%), total collagen, especially types I (up to +217%) and III, as well as the synthesis and activity of MMP-1 (Matrix Metalloproteinase-1, up to +309%). In contrast, oligosaccharide fractions had no effect on GAGs synthesis but exhibited similarities for collagens and MMP-1 regulation. At the transcriptomic level, the decrease of COL1A1 and COL1A2 expression, and increase of COL3A1 and MMP-1 expression, confirmed the modulation of ECM metabolism by both fractions. Our research emphasizes that poly- and oligosaccharide Ulva sp. fractions exhibit interesting biological activities and supports their potential use in the area of skin renewal for anti-aging dermo-cosmetic applications.

The p.E152K-STIM1 mutation deregulates Ca2+ signaling contributing to chronic pancreatitis.

Since deregulation of intracellular Ca2+ can lead to intracellular trypsin activation, and stromal interaction molecule-1 (STIM1) protein is the main regulator of Ca2+ homeostasis in pancreatic acinar cells, we explored the Ca2+ signaling in 37 STIM1 variants found in three pancreatitis patient cohorts. Extensive functional analysis of one particular variant, p.E152K, identified in three patients, provided a plausible link between dysregulated Ca2+ signaling within pancreatic acinar cells and chronic pancreatitis susceptibility. Specifically, p.E152K, located within the STIM1 EF-hand and sterile α-motif domain, increased the release of Ca2+ from the endoplasmic reticulum in patient-derived fibroblasts and transfected HEK293T cells. This event was mediated by altered STIM1-sarco/endoplasmic reticulum calcium transport ATPase (SERCA) conformational change and enhanced SERCA pump activity leading to increased store-operated Ca2+ entry (SOCE). In pancreatic AR42J cells expressing the p.E152K variant, Ca2+ signaling perturbations correlated with defects in trypsin activation and secretion, and increased cytotoxicity after cholecystokinin stimulation.This article has an associated First Person interview with the first author of the paper.

Keratinocytes Communicate with Sensory Neurons via Synaptic-like Contacts.

OBJECTIVE: Pain, temperature, and itch are conventionally thought to be exclusively transduced by the intraepidermal nerve endings. Although recent studies have shown that epidermal keratinocytes also participate in sensory transduction, the mechanism underlying keratinocyte communication with intraepidermal nerve endings remains poorly understood. We sought to demonstrate the synaptic character of the contacts between keratinocytes and sensory neurons and their involvement in sensory communication between keratinocytes and sensory neurons. METHODS: Contacts were explored by morphological, molecular, and functional approaches in cocultures of epidermal keratinocytes and sensory neurons. To interrogate whether structures observed in vitro were also present in the human epidermis, in situ correlative light electron microscopy was performed on human skin biopsies. RESULTS: Epidermal keratinocytes dialogue with sensory neurons through en passant synaptic-like contacts. These contacts have the ultrastructural features and molecular hallmarks of chemical synaptic-like contacts: narrow intercellular cleft, keratinocyte synaptic vesicles expressing synaptophysin and synaptotagmin 1, and sensory information transmitted from keratinocytes to sensory neurons through SNARE-mediated (syntaxin1) vesicle release. INTERPRETATION: By providing selective communication between keratinocytes and sensory neurons, synaptic-like contacts are the hubs of a 2-site receptor. The permanent epidermal turnover, implying a specific en passant structure and high plasticity, may have delayed their identification, thereby contributing to the long-held concept of nerve endings passing freely between keratinocytes. The discovery of keratinocyte-sensory neuron synaptic-like contacts may call for a reassessment of basic assumptions in cutaneous sensory perception and sheds new light on the pathophysiology of pain and itch as well as the physiology of touch. ANN NEUROL 2020;88:1205-1219.

Molecular Mapping of Hydrogen Sulfide Targets in Normal Human Keratinocytes.

Although sulfur-rich thermal waters have ancestrally been used in the context of dermatological conditions, a global mapping of the molecular effects exerted by H2S on human keratinocytes is still lacking. To fill this knowledge gap, we subjected cultured human keratinocytes to distinct amounts of the non-gaseous hydrogen sulfur donor NaHS. We first checked that H2S accumulated in the cytoplasm of keratinocytes under our experimental conditions andused a combination of proteomics, genomics and biochemical approaches to unravel functionally relevant H2S targets in human keratinocytes. We found that the identified targets fall into two main categories: (i) the oxidative stress response molecules superoxide dismutase 2 (SOD2), NAD(P)H quinone dehydrogenase 1 (NQO1) and culin 3 (CUL3) and (ii) the chemokines interleukin-8 (IL-8) and CXCL2. Interestingly, NaHS also stimulated the caspase-1 inflammasome pathway, leading to increased secretion of the pro-inflammatory molecule interleukin-18 (IL-18). Interestingly, the secretion of interleukin-1 beta (IL-1ß) was only modestly impacted by NaHS exposure despite a significant accumulation of IL-1ß pro-form. Finally, we observed that NaHS significantly hampered the growth of human keratinocyte progenitors and stem cells cultured under clonogenic conditions or as epidermal cell sheets. We conclude that H2S exerts specific molecular effects on normal human keratinocytes.

In Vitro Differentiation of Human Skin-Derived Cells into Functional Sensory Neurons-Like.

Skin-derived precursor cells (SKPs) are neural crest stem cells that persist in certain adult tissues, particularly in the skin. They can generate a large type of cell in vitro, including neurons. SKPs were induced to differentiate into sensory neurons (SNs) by molecules that were previously shown to be important for the generation of SNs: purmorphamine, CHIR99021, BMP4, GDNF, BDNF, and NGF. We showed that the differentiation of SKPs induced the upregulation of neurogenins. At the end of the differentiation protocol, transcriptional analysis was performed on BRN3A and a marker of pain-sensing nerve cell PRDM12 genes: 1000 times higher for PRDM12 and 2500 times higher for BRN3A in differentiated cells than they were in undifferentiated SKPs. Using immunostaining, we showed that 65% and 80% of cells expressed peripheral neuron markers BRN3A and PERIPHERIN, respectively. Furthermore, differentiated cells expressed TRPV1, PAR2, TRPA1, substance P, CGRP, HR1. Using calcium imaging, we observed that a proportion of cells responded to histamine, SLIGKV (a specific agonist of PAR2), polygodial (a specific agonist of TRPA1), and capsaicin (a specific agonist of TRPV1). In conclusion, SKPs are able to differentiate directly into functional SNs. These differentiated cells will be very useful for further in vitro studies.

Intra-epidermal nerve endings progress within keratinocyte cytoplasmic tunnels in normal human skin.

Intra-epidermal nerve endings, responsible for cutaneous perception of temperature, pain and itch, are conventionally described as passing freely between keratinocytes, from the basal to the granular layers of the epidermis. However, the recent discovery of keratinocyte contribution to cutaneous nociception implies that their anatomical relationships are much more intimate than what has been described so far. By studying human skin biopsies in confocal laser scanning microscopy, we show that intra-epidermal nerve endings are not only closely apposed to keratinocytes, but can also be enwrapped by keratinocyte cytoplasms over their entire circumference and thus progress within keratinocyte tunnels. As keratinocytes must activate intra-epidermal nerve endings to transduce nociceptive information, these findings may help understanding the interactions between the keratinocytes and nervous system. The discovery of these nerve portions progressing in keratinocyte tunnels is a strong argument to consider that contacts between epidermal keratinocytes and intra-epidermal nerve endings are not incidental and argue for the existence of specific and rapid paracrine communication from keratinocytes to sensory neurons.

Lifting the veil on the keratinocyte contribution to cutaneous nociception.

Cutaneous nociception is essential to prevent individuals from sustaining injuries. According to the conventional point of view, the responses to noxious stimuli are thought to be exclusively initiated by sensory neurons, whose activity would be at most modulated by keratinocytes. However recent studies have demonstrated that epidermal keratinocytes can also act as primary nociceptive transducers as a supplement to sensory neurons. To enlighten our understanding of cutaneous nociception, this review highlights recent and relevant findings on the cellular and molecular elements that underlie the contribution of epidermal keratinocytes as nociceptive modulators and noxious sensors, both under healthy and pathological conditions.

Cutaneous nociception: Role of keratinocytes.

Recent years have brought an enhanced understanding of keratinocyte contribution to cutaneous nociception. While intra-epidermal nerve endings were classically considered as the exclusive transducers of cutaneous noxious stimuli, it has now been demonstrated that epidermal keratinocytes can initiate nociceptive responses, like Merkel cells do for the innocuous mechanotransduction. In the light of recent in vivo findings, this article outlines this paradigm shift that points to a not yet considered population of sensory epidermal cells.

Intraepidermal nerve fibres are not the exclusive tranducers of nociception.

The goal of this article is to provide an insight of the most recent data on cutaneous nociception. Indeed recent studies have demonstrated that keratinocytes closely participate in sensory transduction, and therefore, intraepidermal free nerve endings are not exclusive transducers of pain. Therefore, the results of neurophysiological studies should be interpreted in the light of this information.

New insights into the roles of myofibroblasts and innervation during skin healing and innovative therapies to improve scar innervation.

During the resolution phase of normal skin wound healing, there is a considerable loss of various cell types, including myofibroblasts by apoptosis. Inappropriate delay of apoptosis, and thus increased survival of myofibroblasts, may be a factor leading to pathologies and excessive scarring. Considerable data now clearly suggest that innervation plays a major role in wound healing, including the modulation of fibroblast cellular activity. An abnormal level of neuromediators is implicated not only in the development of chronic wounds but also in excessive scar formation. Understanding interactions between neuromediators and myofibroblasts, allowing normal reinnervation and having adequate levels of neuromediators during the healing process are clearly important to avoid the appearance of pathological healing or fibrosis/scarring. The aim of this review was first to discuss the mechanisms leading to normal or excessive scarring and then to present the roles of innervation during wound healing. Finally, the latest therapeutic strategies to help wound repair and reinnervation after skin damage will be introduced. Advantages and limitations in the use of neuropeptides, growth factors and biomaterials will be discussed as well as the most recent studies on electrostimulation and the potential of targeting resident skin mesenchymal stem cells.

Major Role for TRPV1 and InsP3R in PAR2-Elicited Inflammatory Mediator Production in Differentiated Human Keratinocytes.

PAR2 activation in basal keratinocytes stimulates inflammation via the Ca2+-dependent production of mediators such as IL-1ß, TNF-α, and TSLP. In this study, we investigated PAR2 calcium signaling and the consequent production of inflammatory mediators in differentiated human primary keratinocytes (DhPKs). Stimulation with the PAR2-activating peptide SLIGKV promoted Ca2+ store depletion in both undifferentiated human primary keratinocytes and DhPKs. SLIGKV-evoked Ca2+ store depletion did not trigger the store-operated Ca2+ entry (i.e., SOCE) through ORAI1 in DhPKs compared with undifferentiated human primary keratinocytes. The inhibition of phospholipase C and the concomitant inhibition of TRPV1 and inositol triphosphate receptor in DhPKs abrogated the SLIGKV-evoked Ca2+ store depletion; NF-κB activity; and the production of inflammatory mediators such as IL-1ß, TNF-α, and TSLP. Taken together, these results indicate a key role for both InsP3R and TRPV1 in Ca2+ internal stores in the PAR2-evoked Ca2+ release and consequent skin inflammation in DhPKs. These findings may provide clues to understanding the pathological role of DhPKs in skin disorders in which PAR2 is known to be involved, such as atopic dermatitis, Netherton syndrome, and psoriasis.

Merkel Cells Sense Cooling with TRPM8 Channels.

In the skin, Merkel cells connect with keratinocytes and Aß nerve fibers to form a touch receptor that functions as a slow adapting mechanoreceptor (slow adapting type 1). In human and mouse Merkel cells, we observed an increased concentration of intracellular Ca2+ ions in response to cold temperature and transient receptor potential melastatine 8 (TRPM8) ion channel agonists. A reduction in the response to cooling and TRPM8 agonists occurred after the addition of TRPM8 antagonists, as well as in TRPM8 knockout mice. Cold temperature and TRPM8 agonists also induced a current that was inhibited by a TRPM8 antagonist. Our results indicate that Merkel cells sense cooling through TRPM8 channels. We hypothesized that cooling modulates the slow adapting type 1 receptor response. Cooling mouse skin to 22°C reduced the slow adapting type 1 receptor discharge frequency. Interestingly, we observed no such reduction in TRPM8 knockout mice. Similarly, in human skin, a temperature of 22°C applied to the slow adapting type 1 receptive field reduced the spiking discharge. Altogether, our results indicate that Merkel cells are polymodal sensory cells that respond to mild cold stimuli through the activation of TRPM8 channels. Thermal activation of Merkel cells, and possibly other TRPM8-expressing non-neuronal cells, such as keratinocytes, potentially adapts the discharge of slow adapting type 1 receptors during cooling.

What about physical contacts between epidermal keratinocytes and sensory neurons?

Recent studies have demonstrated that keratinocytes closely participate in sensory transduction, and therefore, intra-epidermal free nerve endings are not exclusive transducers of pain. This discovery implies the existence of close afferent communication from keratinocytes to sensory neurons. Although reciprocal interactions between keratinocytes and intra-epidermal free nerve endings via soluble mediators are well established, little attention has been paid to physical contacts between keratinocytes and intra-epidermal free nerve endings. This review proposes to consider the ultrastructural and functional knowledge of these contacts, in both human skin biopsies and keratinocyte-sensory neuron cocultures to speculate on the possible existence of synaptic contacts.

A new tool to test active ingredient using lactic acid in vitro, a help to understand cellular mechanism involved in stinging test: An example using a bacterial polysaccharide (Fucogel® ).

The stinging test is an in vivo protocol that evaluates sensitive skin using lactic acid (LA). A soothing sensation of cosmetics or ingredients can be also appreciated through a decrease in stinging score. To predict the soothing sensation of a product before in vivo testing, we developed a model based on an LA test and substance P (SP) release using a co-culture of human keratinocytes and NGF-differentiated PC12 cells. A bacterial fucose-rich polysaccharide present in Fucogel® was evaluated as the soothing molecule in the in vivo stinging test and our in vitro model. Excluding toxic concentrations, the release of SP was significant from 0.2% of lactic acid for the PC12 cells and from 0.1% of lactic acid for the keratinocytes. When the pH was adjusted to approximately 7.4, LA did not provoke SP release. At these concentrations of LA, 0.1% of polysaccharide showed a significant decrease in SP release from the two cellular types and in co-cultures without modifying the pH of the medium. In vivo, a stinging test using the polysaccharide showed a 30% decrease in prickling intensity vs the placebo in 19 women between the ages of 21 and 69. Our in vitro model is ethically interesting and is adapted for cosmetic ingredients screening because it does not use animal experimentation and limits human volunteers. Moreover, Fucogel® reduced prickling sensation as revealed by the in vivo stinging test and inhibits the neurogenic inflammation as showed by our new in vitro stinging test based on SP release.