Genetic priming of sensory neurons in mice that overexpress PAR2 enhances allergen responsiveness.

Pruritus is a common symptom of inflammatory skin conditions, including atopic dermatitis (AD). Although primary sensory neurons that transmit pruritic signals are well-cataloged, little is known about the neuronal alterations that occur as a result of skin disruption in AD. To address this question, we examined the molecular and behavioral consequences of challenging Grhl3PAR2/+ mice, which overexpress PAR2 in suprabasal keratinocytes, with serial topical application of the environmental allergen house dust mite (HDM). We monitored behavior and used RNA sequencing, qPCR, and in situ hybridization to evaluate gene expression in trigeminal ganglia (TG), before and after HDM. We found that neither Grhl3PAR2/+ nor wild-type (WT) mice exhibited spontaneous scratching, and pruritogen-induced acute scratching did not differ. In contrast, HDM exacerbated scratching in Grhl3PAR2/+ mice. Despite the absence of scratching in untreated Grhl3PAR2/+ mice, several TG genes in these mice were up-regulated compared to WT. HDM treatment of the Grhl3PAR2/+ mice enhanced up-regulation of this set of genes and induced additional genes, many within the subset of TG neurons that express TRPV1. The same set of genes was up-regulated in HDM-treated Grhl3PAR2/+ mice that did not scratch, but at lesser magnitude. Finally, we recorded comparable transcriptional changes in IL31Tg mice, demonstrating that a common genetic program is induced in two AD models. Taken together, we conclude that transcriptional changes that occur in primary sensory neurons in dermatitis-susceptible animals underlie a genetic priming that not only sensitizes the animal to chronic allergens but also contributes to pruritus in atopic skin disease.

Decreased p53 is associated with a decline in asymmetric stem cell self-renewal in aged human epidermis.

With age, the epidermis becomes hypoplastic and hypoproliferative. Hypoproliferation due to aging has been associated with decreased stem cell (SC) self-renewal in multiple murine tissues. The fate of SC self-renewal divisions can be asymmetric (one SC, one committed progenitor) or symmetric (two SCs). Increased asymmetric SC self-renewal has been observed in inflammatory-mediated hyperproliferation, while increased symmetric SC self-renewal has been observed in cancers. We analyzed SC self-renewal divisions in aging human epidermis to better understand the role of SCs in the hypoproliferation of aging. In human subjects, neonatal to 78 years, there was an age-dependent decrease in epidermal basal layer divisions. The balance of SC self-renewal shifted toward symmetric SC self-renewal, with a decline in asymmetric SC self-renewal. Asymmetric SC divisions maintain epidermal stratification, and this decrease may contribute to the hypoplasia of aging skin. P53 decreases in multiple tissues with age, and p53 has been shown to promote asymmetric SC self-renewal. Fewer aged than adult ALDH+CD44+ keratinocyte SCs exhibited p53 expression and activity and Nutlin-3 (a p53 activator) returned p53 activity as well as asymmetric SC self-renewal divisions to adult levels. Nutlin-3 increased Notch signaling (NICD, Hes1) and DAPT inhibition of Notch activation prevented Nutlin-3 (p53)-induced asymmetric SC self-renewal divisions in aged keratinocytes. These studies indicate a role for p53 in the decreased asymmetric SC divisions with age and suggest that in aged keratinocytes, Notch is required for p53-induced asymmetric SC divisions.

Single-Cell Profiling Reveals Divergent, Globally Patterned Immune Responses in Murine Skin Inflammation.

Inflammatory response heterogeneity has impeded high-resolution dissection of diverse immune cell populations during activation. We characterize mouse cutaneous immune cells by single-cell RNA sequencing, after inducing inflammation using imiquimod and oxazolone dermatitis models. We identify 13 CD45+ subpopulations, which broadly represent most functionally characterized immune cell types. Oxazolone pervasively upregulates Jak2/Stat3 expression across T cells and antigen-presenting cells (APCs). Oxazolone also induces Il4/Il13 expression in newly infiltrating basophils, and Il4ra and Ccl24, most prominently in APCs. In contrast, imiquimod broadly upregulates Il17/Il22 and Ccl4/Ccl5. A comparative analysis of single-cell inflammatory transcriptional responses reveals that APC response to oxazolone is tightly restricted by cell identity, whereas imiquimod enforces shared programs on multiple APC populations in parallel. These global molecular patterns not only contrast immune responses on a systems level but also suggest that the mechanisms of new sources of inflammation can eventually be deduced by comparison to known signatures.

Proderm technology: a water- based lipid delivery system for dermatitis that penetrates viable epidermis and has antibacterial effects.

BACKGROUND: A defective skin barrier and bacterial colonization are two important factors in maintenance and progression of atopic dermatitis and chronic allergic/irritant hand dermatitis. A water-based lipid delivery system containing physiologic lipids was previously shown to be a useful adjunct in the treatment of hand dermatitis. We tested the ability of this formulation to penetrate into the viable epidermis and in addition assessed its antibacterial properties. METHODS: Epidermal penetration of the product was assessed by fluorescence microscopy. Recovery of Escherichia coli and Staphylococcus aureus MRSA from skin treated with Neosalus® foam was quantified. RESULTS: Components of Neosalus® penetrated the stratum corneum and were distributed throughout the viable epidermis. Neosalus® significantly decreased recovery of both Staphylococcus aureus and Escherichia coli from the skin surface. CONCLUSIONS: The ability of components of Neosalus® to be taken up into the viable epidermis and potentially made available for incorporation into the barrier lipids, combined with antibacterial properties, indicate that this formulation may be valuable not only in chronic hand dermatitis, but also in various other forms of dermatitis. TRIAL REGISTRATION: Current Controlled Trials ISRCTN18191379 , 28/12/2018, retrospectively registered.

Influence of pH on Skin Stem Cells and Their Differentiation.

Intracellular pH influences proliferation and differentiation in a range of stem-like and progenitor cells, including embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, and cancer stem cells. Sodium hydrogen exchanger (NHE1), a glycoprotein that plays a major role in regulating intracellular pH, has a major role in the proliferation and cell differentiation in multiple cell types. We review observations collected on the influence of pH on multiple stem-like cell populations. Altering pH, either intracellular or extracellular, can influence stem cell maintenance, self-renewal, differentiation, and pluripotency. Study of the influence of NHE1 and intracellular pH on epidermal stem cell behavior could lead to the discovery of new targets to use in order to manipulate stem cell divisions. This is highly relevant for skin conditions such as psoriasis, wound healing, and melanoma where stem cell proliferation and migration are key factors.

Brief Report: Interleukin-17A-Dependent Asymmetric Stem Cell Divisions Are Increased in Human Psoriasis: A Mechanism Underlying Benign Hyperproliferation.

The balance between asymmetric and symmetric stem cell (SC) divisions is key to tissue homeostasis, and dysregulation of this balance has been shown in cancers. We hypothesized that the balance between asymmetric cell divisions (ACDs) and symmetric cell divisions (SCDs) would be dysregulated in the benign hyperproliferation of psoriasis. We found that, while SCDs were increased in squamous cell carcinoma (SCC) (human and murine), ACDs were increased in the benign hyperproliferation of psoriasis (human and murine). Furthermore, while sonic hedgehog (linked to human cancer) and pifithrinα (p53 inhibitor) promoted SCDs, interleukin (IL)-1α and amphiregulin (associated with benign epidermal hyperproliferation) promoted ACDs. While there was dysregulation of the ACD:SCD ratio, no change in SC frequency was detected in epidermis from psoriasis patients, or in human keratinocytes treated with IL-1α or amphiregulin. We investigated the mechanism whereby immune alterations of psoriasis result in ACDs. IL17 inhibitors are effective new therapies for psoriasis. We found that IL17A increased ACDs in human keratinocytes. Additionally, studies in the imiquimod-induced psoriasis-like mouse model revealed that ACDs in psoriasis are IL17A-dependent. In summary, our studies suggest an association between benign hyperproliferation and increased ACDs. This work begins to elucidate the mechanisms by which immune alteration can induce keratinocyte hyperproliferation. Altogether, this work affirms that a finely tuned balance of ACDs and SCDs is important and that manipulating this balance may constitute an effective treatment strategy for hyperproliferative diseases. Stem Cells 2017;35:2001-2007.

CD133 is a marker for long-term repopulating murine epidermal stem cells.

Maintenance, repair, and renewal of the epidermis are thought to depend on a pool of dedicated epidermal stem cells (EpiSCs). Like for many somatic tissues, isolation of a nearly pure population of stem cells is a primary goal in cutaneous biology. We used a quantitative transplantation assay, using injection of keratinocytes into subcutis combined with limiting dilution analysis, to assess the long-term repopulating ability of putative murine EpiSC populations. Putative EpiSC populations were isolated by FACS sorting. The CD133(+) population and the subpopulation of CD133(+) cells that exhibits high mitochondrial membrane potential (DΨm(hi)) were enriched for long-term repopulating EpiSCs versus unfractionated cells (3.9- and 5.2-fold, respectively). Evidence for self-renewal capacity was obtained by serial transplantation of long-term epidermal repopulating units derived from CD133(+) and CD133(+)ΔΨm(hi) keratinocytes. CD133(+) keratinocytes were multipotent and produced significantly more hair follicles than CD133(-) cells. CD133(+) cells were a subset of the previously described integrin α6(+)CD34(+) bulge cell population, and 28.9±8.6% were label-retaining cells. Thus, murine keratinocytes within the CD133(+) and CD133(+)ΔΨm(hi) populations contain EpiSCs that regenerate the epidermis for the long term, are self-renewing, multipotent, and label-retaining cells.

Transit-amplifying cell frequency and cell cycle kinetics are altered in aged epidermis.

Aged epidermis is less proliferative than young, as exemplified by slower wound healing. However, it is not known whether quantitative and/or qualitative alterations in the stem and/or transit-amplifying (TA) compartments are responsible for the decreased proliferation. Earlier studies found a normal or decreased frequency of putative epidermal stem cells (EpiSCs) with aging. We show, using long-term repopulation in vivo and colony formation in vitro, that, although no significant difference was detected in EpiSC frequency with aging, TA cell frequency is increased. Moreover, aged TA cells persist longer, whereas their younger counterparts have already differentiated. Underlying the alteration in TA cell kinetics in the aged is an increase in the proportion of cycling keratinocytes, as well as an increase in cell cycle duration. In summary, although no significant difference in EpiSC frequency was found, TA cell frequency was increased (as measured by in vivo repopulation, growth fraction, and colony formation). Furthermore, the proliferative capacity (cellular output) of individual aged EpiSCs and TA cells was decreased compared to that of young cells. Although longer cell cycle duration contributes to the decreased proliferative output from aged progenitors, the greater number of TA cells may be a compensatory mechanism tending to offset this deficit.

Decreased ceramide transport protein (CERT) function alters sphingomyelin production following UVB irradiation.

Increased cellular ceramide accounts in part for UVB irradiation-induced apoptosis in cultured human keratinocytes with concurrent increased glucosylceramide but not sphingomyelin generation in these cells. Given that conversion of ceramide to non-apoptotic metabolites such as sphingomyelin and glucosylceramide protects cells from ceramide-induced apoptosis, we hypothesized that failed up-regulation of sphingomyelin generation contributes to ceramide accumulation following UVB irradiation. Because both sphingomyelin synthase and glucosylceramide synthase activities were significantly decreased in UVB-irradiated keratinocytes, we investigated whether alteration(s) in the function of ceramide transport protein (or CERT) required for sphingomyelin synthesis occur(s) in UVB-irradiated cells. Fluorescently labeled N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (C(5)-DMB-ceramide) relocation to the Golgi was diminished after irradiation, consistent with decreased CERT function, whereas the CERT inhibitor N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (1R,3R isomer) (HPA-12) produced an equivalent effect. UVB irradiation also induced the rapid formation of a stable CERT homotrimer complex in keratinocytes as determined by Western immunoblot and mass spectrometry analyses, a finding replicated in HeLa, HEK293T, and HaCaT cells and in murine epidermis. Ceramide binding activity was decreased in recombinant CERT proteins containing the UVB-induced homotrimer. The middle region domain of the CERT protein was required for the homotrimer formation, whereas neither the pleckstrin homology (Golgi-binding) nor the START (ceramide-binding) domains were involved. Finally like UVB-treated keratinocytes, HPA-12 blockade of CERT function increased keratinocyte apoptosis, decreased sphingomyelin synthesis, and led to accumulation of ceramide. Thus, UVB-induced CERT homotrimer formation accounts, at least in part, for apoptosis and failed up-regulation of sphingomyelin synthesis following UVB irradiation, revealing that inactive CERT can attenuate a key metabolic protective mechanism against ceramide-induced apoptosis in keratinocytes.

PKCzeta protects against UV-C-induced apoptosis by inhibiting acid sphingomyelinase-dependent ceramide production.

In a recent study, we described that UV-C irradiation resulted in redox-dependent activation and relocalization of A-SMase (acid sphingomyelinase) to the external surface of raft membrane microdomains, hydrolysis of SM (sphingomyelin) associated with the plasma membrane outer leaflet, ceramide generation and apoptosis. In the present study, we have investigated the influence of PKCzeta (protein kinase Czeta), an atypical form of PKC on this pathway. This study shows that PKCzeta overexpression resulted in the abrogation of UV-C-induced A-SMase translocation and activation into the raft microdomains, lack of ceramide generation and apoptosis inhibition. Moreover, PKCzeta overexpression resulted in a decrease in UV-C-induced ROS (reactive oxygen species) production, which correlated with increased gene expression level of various antioxidant enzymes, including TRx (thioredoxin), TR (thioredoxin reductase) 1, TR2 and peroxiredoxin 1/TPx2 (thioredoxin peroxidase 2). Importantly, enforced TPx2 gene expression inhibited UV-C-induced A-SMase translocation. Finally, PKCzeta inhibition led to a significant reduction in TPx2 protein expression. Altogether, these results suggest that PKCzeta interferes with the UV-activated sphingolipid signalling pathway by regulating the TRx system. These findings may have important consequences for UV-induced carcinogenesis and resistance to phototherapy.

UVA induces granzyme B in human keratinocytes through MIF: implication in extracellular matrix remodeling.

In a previous study, we have described that UVB induces granzyme B (GrB) in human keratinocyte cells, and that confers potent cellular cytotoxicity against various cellular models, including immune cells (Hernandez-Pigeon, H., Jean, C., Charruyer, A., Haure, M. J., Titeux, M., Tonasso, L., Quillet-Mary, A., Baudouin, C., Charveron, M., and Laurent, G. (2006) J. Biol. Chem. 281, 13525-13532). Herein, we have found that, in contrast to UVB, UVA failed to enhance keratinocyte cellular cytotoxicity but was still able to trigger GrB production. We show that GrB is accumulated through a p38 MAPK-dependent transcriptional mechanism stimulated by redox-dependent migration inhibitory factor release. Moreover, GrB purified from UVA-treated cellular extracts was found to degrade fibronectin in vitro. Treatment with antisense oligonucleotide directed against GrB resulted in the inhibition of UVA-induced cell detachment and cell death and facilitated cell migration through fibronectin and vitronectin matrix upon UVA exposure. Altogether, these results suggest another function for GrB in the context of the UV response. Indeed, combined with our previous study, it appears that, whereas this enzyme mediates keratinocyte cellular cytotoxicity following UVB irradiation, GrB supports the capacity of keratinocyte to degrade extracellular matrix components following UVA irradiation. UV-mediated GrB production may thus have important consequences in photoaging and photocarcinogenesis.

Human keratinocytes acquire cellular cytotoxicity under UV-B irradiation. Implication of granzyme B and perforin.

Ultraviolet (UV) radiation from the sun is widely considered as a major cause of human skin photoaging and skin cancer. Granzyme B (GrB) and perforin (PFN) are two proteins contained in granules and implicated in one of the mechanisms by which cytotoxic lymphocytes and natural killer cells exert their cytotoxicity against virus-infected, alloreactive, or transformed cells. The distribution of GrB and PFN in the skin has received little attention. However, Berthou and co-workers (Berthou, C., Michel, L., Soulie, A., Jean-Louis, F., Flageul, B., Dubertret, L., Sigaux, F., Zhang, Y., and Sasportes, M. (1997) J. Immunol. 159, 5293-5300) described that, whereas freshly isolated epidermal cells did not express GrB or PFN, keratinocyte growth to confluence was associated with GrB and PFN mRNA and protein synthesis. In this work, we have investigated the possible role of UV-B on GrB and PFN expression in keratinocytes. We found that UV-B induces GrB and PFN expression in these cells through redox-, epidermal growth factor receptor-, and mitogen-activated protein kinase-dependent signaling. Furthermore, under UV irradiation, keratinocytes acquire a significant cytotoxicity, which is GrB and PFN dependent, toward a variety of cellular targets including transformed T-lymphocytes, melanocytes, and keratinocytes. This phenomenon may have important functional consequences in the regulation of skin inflammatory response and in the emergence of cancer skin.

UV-C light induces raft-associated acid sphingomyelinase and JNK activation and translocation independently on a nuclear signal.

The initiation of UV light-induced signaling in mammalian cells is largely considered to be subsequent to DNA damage. Several studies have also described ceramide (CER), a lipid second messenger, as a major contributor in mediating UV light-induced c-Jun N-terminal kinase (JNK) activation and cell death. It is demonstrated here that UV-C light irradiation of U937 cells results in the activation and translocation of a Zn2+-independent acid sphingomyelinase, leading to CER accumulation in raft microdomains. These CER-enriched rafts aggregate and play a functional role in JNK activation. The observation that UV-C light also induced CER generation and the externalization of acid sphingomyelinase and JNK in human platelets conclusively rules out the involvement of a nuclear signal generated by DNA damage in the initiation of a UV light response, which is generated at the plasma membrane.

Kit signaling inhibits the sphingomyelin-ceramide pathway through PLC gamma 1: implication in stem cell factor radioprotective effect.

Previous studies demonstrated that Kit activation confers radioprotection. However, the mechanism by which Kit signaling interferes with cellular response to ionizing radiation (IR) has not been firmly established. Based on the role of the sphingomyelin (SM) cycle apoptotic pathway in IR-induced apoptosis, we hypothesized that one of the Kit signaling components might inhibit IR-induced ceramide production or ceramide-induced apoptosis. Results show that, in both Ba/F3 and 32D murine cell lines transfected with wild-type c-kit, stem cell factor (SCF) stimulation resulted in a significant reduction of IR-induced apoptosis and cytotoxicity, whereas DNA repair remained unaffected. Moreover, SCF stimulation inhibited IR-induced neutral sphingomyelinase (N-SMase) stimulation and ceramide production. The SCF inhibitory effect on SM cycle was not influenced by wortmannin, a phosphoinositide-3 kinase (PI3K) inhibitor. The SCF protective effect was maintained in 32D-KitYF719 cells in which the PI3K/Akt signaling pathway is abolished due to mutation in Kit docking site for PI3K. In contrast, phospholipase C gamma (PLC gamma) inhibition by U73122 totally restored IR-induced N-SMase stimulation, ceramide production, and apoptosis in Kit-activated cells. Moreover, SCF did not protect 32D-KitYF728 cells (lacking a functional docking site for PLC gamma 1), from IR-induced SM cycle. Finally, SCF-induced radioprotection of human CD34(+) bone marrow cells was also inhibited by U73122. Altogether, these results suggest that SCF radioprotection is due to PLC gamma 1-dependent negative regulation of IR-induced N-SMase stimulation. Beyond the scope of Kit-expressing cells, it suggests that PLC gamma 1 status could greatly influence the post-DNA damage cellular response to IR, and perhaps, to other genotoxic agents.