The rapid deployment of fifth-generation (5G) wireless networks has raised societal concerns regarding potential biological effects, particularly on human skin, due to the use of higher carrier frequencies that penetrate tissue less deeply. Consequently, whether 5G-modulated radiofrequency (RF) electromagnetic fields (EMFs) at 3.5 GHz affect oxidative stress and DNA repair in skin cells remains an open question. Using genetically encoded Bioluminescence Resonance Energy Transfer (BRET)-based biosensors targeted to the cytoplasm and mitochondria, we assessed whether exposure of human fibroblasts to 5G RF-EMF at specific absorption rates (SAR) of 0.08 and 4 W/kg for 24 h could alter basal reactive oxygen species (ROS) levels or potentiate the effects of known ROS inducers, including H₂O₂, Kp372-1, and Antimycin A. We also evaluated whether pre-exposure to 5G RF-EMF could induce an adaptive response (AR), by modulating ROS production following a subsequent challenge with arsenic trioxide (As₂O₃). Additionally, we investigated the impact of combined RF-EMF and ultraviolet-B (UV-B) exposure on the formation and repair of cyclobutane pyrimidine dimer (CPD) lesions in HaCaT keratinocytes. Our results showed no significant effect of 5G RF-EMF exposure, either alone or in combination with chemical ROS inducers, on oxidative stress markers in either compartment. Likewise, RF-EMF exposure did not induce an adaptive response to oxidative challenge, nor did it alter the kinetics or the efficiency of CPD repair by the nucleotide excision repair (NER) pathway. These findings support the conclusion that the exposure to 5G RF-EMF at 3.5 GHz up to 4 W/kg does not induce oxidative stress or impair DNA repair efficiency in human skin cells, within the experimental conditions tested.

Xeroderma pigmentosum group C (XPC) is a versatile protein crucial for sensing DNA damage in the global genome nucleotide excision repair (GG-NER) pathway. This pathway is vital for mammalian cells, acting as their essential approach for repairing DNA lesions stemming from interactions with environmental factors, such as exposure to ultraviolet (UV) radiation from the sun. Loss-of-function mutations in the XPC gene confer a photosensitive phenotype in XP-C patients, resulting in the accumulation of unrepaired UV-induced DNA damage. This remarkable increase in DNA damage tends to elevate by 10,000-fold the risk of developing melanoma and non-melanoma skin cancers. To date, creating accurate and reproducible models to study human XP-C disease has been an important challenge. To tackle this, we used CRISPR-Cas9 technology in order to knockout the XPC gene in various human skin cells (keratinocytes, fibroblasts, and melanocytes). After validation of the knockout in these edited skin cells, we showed that they recapitulate the major phenotypes of XPC mutations: photosensitivity and the impairment of UV-induced DNA damage repair. Moreover, these knockout cells demonstrated a reduced proliferative capacity compared to their respective controls. Finally, to better mimic the disease environment, we built a 3D reconstructed skin using these XPC knockout skin cells. This model exhibited an abnormal behavior, showing an extensive remodeling of its extracellular matrix compared to normal skin. Analyzing the composition of the fibroblast secretome revealed a significant augmented shift in the inflammatory response following XPC knockout. Our innovative "disease on a dish" approach can provide valuable insights into the molecular mechanisms underlying XP-C disease, paving the way to design novel preventive and therapeutic strategies to alleviate the disease phenotype. Also, given the high risk of skin cancer onset in XP-C disease, our new approach can serve as a link to draw novel insights into this elusive field.

•CRISPR-Cas9 is a valuable tool for creating XPC-deficient primary melanocytes •XPC-deficient primary melanocytes (©-XPC-Mel) are hypersensitive to UV •©-XPC-Mel pave the way for studying the effects of NER on melanocyte biology •About 500 proteins are differentially expressed in ©-XPC-Mel compared to ©-Ctrl-Mel •The balance between pro- and anti-inflammatory signals is disturbed in ©-XPC-Mel

Cole disease (CD) is a rare genodermatosis featuring punctate palmoplantar keratosis and patchy hypopigmentation. This disorder results from pathogenic variants in ENPP1 which encodes ectonucleotide pyrophosphatase/ phosphodiesterase 1a. ENPP1 is responsible for the biosynthesis of an inhibitor of ectopic calcification. CD-causing variants affect a specific domain of the protein which may regulate response to insulin. We have previously shown that ENPP1 is expressed in keratinocytes. Downregulation of ENPP1 in organotypic skin cultures and MNT1 cells was associated with hyperproliferation and decreased melanin synthesis, respectively. We then aimed to investigate the pathomechanism by which CD-causing variants result in epidermal proliferation and decrease in melanin synthesis. Loss-of-function of ENPP1 in murine articular cartilage results in hedgehog pathway activation and overexpression of sonic hedgehog signaling in the skin causes epidermal basal proliferation and decrease in melanin synthesis as seen with ENPP1 downregulation, suggesting a mechanism underlying CD pathogenesis. A significant increase in luciferase activity was measured in HeLa cells transfected with a construct expressing the CD-causing c.530G>A ENPP1 variant along with a sonic hedgehog luciferase reporter as compared to cells transfected with wild-type ENPP1 cDNA. Similarly, downregulation of ENPP1 with specific siRNAs in the same system was also associated with a significant increase in luciferase activity as compared with cells transfected with a control siRNA, suggesting a loss-of-function effect. In summary, the present data substantiate the notion that ENPP1 regulates epidermal differentiation and melanogenesis by modulation of the hedgehog pathway activity.

Segmental vitiligo (SV) is a unilateral subtype of vitiligo which is clinically characterized by a cutaneous depigmentation and histologically by a melanocyte loss from the epidermis and hair follicle reservoirs. To date, its pathogenesis remains a mystery. In many cases, this skin depigmentation shares several clinical features and dysfunctions with herpes zoster (HZ). So, for the first time, we examined whether any nucleus and cell fusion associated with a positive immunolabelling of varicella-zoster virus (VZV) and VZV mature virions could be found in SV skin samples as in herpes zoster (HZ). A total of 40 SV samples were used for histological and immunochemical studies. Control samples were obtained from three HZ, and 10 generalized vitiligo lesions. For ultrastructural study, three recent SV and one HZ as controls were recruited. Here, we report that nuclear fusion in epidermal cells were statistically associated with recent SV (p < .001), whereas syncytia formation was associated with long-lasting SV (p = .001). A positive detection of VZV antigen was statistically associated in the epidermis with recent SV and in the dermis with long-lasting SV (p = .001). Finally, the discovery of mature virions in 3/3 recent SV samples provides additional arguments for our viral hypothesis.

Vitiligo is a T cell-mediated inflammatory skin disorder characterized by the loss of epidermal melanocytes. However, the contribution of melanocytes to the physiopathology of the disease in response to the T-cell microenvironment remains unclear. Here, using NanoString technology and multiplex ELISA, we show that active vitiligo perilesional skin is characterized by prominent type 1 and 2 associated immune responses. The vitiligo skin T-cell secretome downregulated melanocyte function and adhesion while increasing melanocyte mitochondrial metabolism and expression of inflammatory cytokines and chemokines by epidermal cells. The Jak1/2 inhibitor ruxolitinib strongly inhibited such effects on epidermal cells. Our data highlight that vitiligo is more complex than previously thought, with prominent combined activities of both T helper type 1- and T helper type 2-related cytokines inducing inflammatory responses of epidermal cells. Melanocytes do not appear only to be a target of T cells in vitiligo but could actively contribute to perpetuate inflammation. Jak inhibitors could prevent the impact of T cells on epidermal cells and pigmentation, highlighting their potential clinical benefit in vitiligo.

Loss of melanocytes is the pathological hallmark of vitiligo, a chronic inflammatory skin depigmenting disorder induced by exaggerated immune response, including autoreactive CD8 T cells producing high levels of type 1 cytokines. However, the interplay between this inflammatory response and melanocyte disappearance remains to be fully characterized. Here, we demonstrate that vitiligo skin contains a significant proportion of suprabasal melanocytes, associated with disruption of E-cadherin expression, a major protein involved in melanocyte adhesion. This phenomenon is also observed in lesional psoriatic skin. Importantly, apoptotic melanocytes were mainly observed once cells were detached from the basal layer of the epidermis, suggesting that additional mechanism(s) could be involved in melanocyte loss. The type 1 cytokines IFN-γ and TNF-α induce melanocyte detachment through E-cadherin disruption and the release of its soluble form, partly due to MMP-9. The levels of MMP-9 are increased in the skin and sera of patients with vitiligo, and MMP-9 is produced by keratinocytes in response to IFN-γ and TNF-α. Inhibition of MMP-9 or the JAK/STAT signaling pathway prevents melanocyte detachment in vitro and in vivo. Therefore, stabilization of melanocytes in the basal layer of the epidermis by preventing E-cadherin disruption appears promising for the prevention of depigmentation occurring in vitiligo and during chronic skin inflammation.

Le vitiligo est une dermatose inflammatoire chronique caractérisée par la perte des mélanocytes et associée à une réponse exagérée du système immunitaire, avec production locale de cytokines inflammatoires, en particulier l’IFNγ et le TNFα, produites notamment par les lymphocytes T résidents mémoires (LTRM) cutanés. Cependant l’impact des facteurs solubles produits par les LT cutanés infiltrant la peau périlésionnelle sur la réponse épidermique reste à ce jour mal évalué. Nous montrons que les LT cutanés périlésionnels de patient vitiligo produisent non seulement des cytokines de type Th1 mais également de type Th2. Le sécrétome de ces LT induit une forte expression de gènes associés à l’inflammation par le mélanocyte, une dérégulation de l’activité métabolique et l’apparition d’un stress oxydatif, démontrant un rôle majeur du mélanocyte lui-même dans cette pathologie. Enfin, le ruxolitinib, un inhibiteur de JAK1/JAK2, permet d’inhiber les effets des sécrétomes sur la réponse mélanocytaire. Ainsi, nos résultats mettent en évidence le rôle du mélanocyte, de la voie JAK/STAT et la complexité des cytokines produites au cours du vitiligo, permettant d’envisager une voie thérapeutique grâce aux inhibiteurs de JAK dans cette pathologie encore orpheline de traitement.

Cole disease is a unique genodermatosis featuring combined abnormal pigmentation and keratinization. It is characterized by congenital or early-onset punctate keratoderma associated with irregularly shaped hypopigmented macules. Cole disease results from heterozygous mutations in ENPP1 encoding a protein that has been shown to play a critical role in bone mineralization and insulin resistance. Here, we aimed at investigating ENPP1 involvement in epidermal differentiation and pigmentation, which are abnormal in Cole disease. Using qPCR and Western blot analysis, we observed that ENPP1 is expressed in keratinocytes (KCs) in a calcium-dependent manner. In organotypic skin cultures, downregulation of ENPP1 was associated with hyperproliferation and decreased differentiation. In line with these data, immunostaining of ENPP1-silenced skin equivalents revealed a significant increase in KRT5 and a significant reduction in KRT10 and loricrin expression. Increase in KRT5 was also observed in hypopigmented macules of a patient carrying the c.530G>A mutation and in skin equivalents reconstructed with melanocytes from this patient. Furthermore, Ki67-positive cells were significantly more numerous in ENPP1-downregulated models. Downregulation of ENPP1 in MNT1 cells (a melanocytic cell line) resulted in decreased melanin synthesis as measured by immunohistochemistry and a melanin synthesis assay. Overexpression of c.530G>A mutation in primary melanocytes decreased the expression of TRP-1 and tyrosinase at the protein level. Primary melanocytes overexpressing the c.530G>A mutation induced hyperproliferation in skin equivalents. In summary, we found that ENPP1 not only regulates ectopic calcification, it also promotes keratinocyte differentiation and melanogenesis, which may in part contribute to the pathogenesis of Cole disease.

Depigmentation observed in vitiligo results from the loss of epidermal melanocytes, involving the combination of several factors, including exaggerated inflammatory and immune response with overproduction of the type-1 cytokines interferon (IFN)-g and tumor necrosis factor (TNF)-a. Nonetheless, the precise mechanism leading to such disappearance remains controversial. We used ex vivo, in vitro and in vivo approaches to decipher the interplay between type-1 cytokines and melanocyte loss. We show that basal melanocytes from vitiligo patients skin are not apoptotic but suffer from a disruption of E-cadherin expression, the main protein involved in melanocyte adhesion to keratinocytes, explaining the presence suprabasal melanocytes. Importantly, we found that type-1 cytokines IFNg and TNFa reproduced this phenotype both in vitro in a 3D model of human pigmented epidermis, and vivo in mice. Such effect was mediated through the promotion of epidermal production of matrix metalloprotease MMP9, resulting in disruption of E-cadherin expression and subsequent destabilization of melanocytes from the basal layer of the epidermis. In addition, inhibition of MMP9 or of JAK signaling prevented such detachment of melanocytes. Therefore, preventing E-cadherin disruption appears as an attractive therapeutic approach to stabilize melanocytes in the basal layer of the epidermis and prevent depigmentation.

Trypanosoma brucei gambiense, an extracellular eukaryotic flagellate parasite, is the main etiological agent of human African trypanosomiasis (HAT) or sleeping sickness. Dendritic cells (DCs) play a pivotal role at the interface between innate and adaptive immune response and are implicated during HAT. In this study, we investigated the effects of T gambiense and its excreted/secreted factors (ESF) on the phenotype of human monocyte‐derived DCs (Mo‐DCs). Mo‐DCs were cultured with trypanosomes, lipopolysaccharide (LPS), ESF derived from T gambiense bloodstream strain Biyamina (MHOM/SD/82), or both ESF and LPS. Importantly, ESF reduced the expression of the maturation markers HLA‐DR and CD83, as well as the secretion of IL‐12, TNF‐alpha and IL‐10, in LPS‐stimulated Mo‐DCs. During mixed‐leucocyte reactions, LPS‐ plus ESF‐exposed DCs induced a non‐significant decrease in the IFN‐gamma/IL‐10 ratio of CD4 + T‐cell cytokines. Based on the results presented here, we raise the hypothesis that T gambiense has developed an immune escape strategy through the secretion of paracrine mediators in order to limit maturation and activation of human DCs. The identification of the factor(s) in the T gambiense ESF and of the DCs signalling pathway(s) involved may be important in the development of new therapeutic targets.

Melanocyte loss is the pathological hallmark of vitiligo, the archetype of a chronic depigmenting inflammatory skin disorder. Yet, whether such disappearance results from melanocyte death and/or detachment is still a matter of debate. We previously showed that vitiligo skin is imprinted with resident memory T cells producing elevated levels of IFNγ and TNFα, while displaying moderate cytotoxicity. Therefore, we investigated the interplay between the inflammatory response characterizing vitiligo disease and melanocyte loss. We found that the combined activity of IFNγ and TNFα induced melanocyte detachment rather than their death in reconstructed pigmented human epidermis through defect of E-cadherin expression, the major protein involved in melanocyte attachment to keratinocytes, and the release of soluble E-cadherin. Such phenomenon was undeniably observed in vitiligo patients skin and was further confirmed in vivo following dermal injection of both IFNγ and TNFα. Additional experiments demonstrated that these two cytokines induced the production of active matrix metalloproteinase 9 (MMP9) by keratinocytes, leading to the cleavage of E-cadherin and instability of melanocytes. MMP9 levels were found increased in vitiligo skin and patients sera and positively correlated with the body surface area involved. Lastly, we showed that MMP9 inhibition downregulated melanocyte detachment in vitro and in vivo. These results emphasize a new mechanism to explain depigmentation associated with inflammation and highlight MMP9 as a new therapeutic target in vitiligo, a disease that still suffers from a lack of effective treatment.

Vitiligo is a chronic autoimmune depigmenting skin disorder that results from a loss of melanocytes. Multiple combinatorial factors have been involved in disease development, with a prominent role of the immune system, in particular T cells. After repigmentation, vitiligo frequently recurs in the same area, suggesting that vitiligo could involve the presence of resident memory T cells (TRM). We sought to perform a thorough characterization of the phenotype and function of skin memory T cells in vitiligo. We show that stable and active vitiligo perilesional skin is enriched with a population of CD8 TRM expressing both CD69 and CD103 compared with psoriasis and control unaffected skin. CD8 TRM expressing CD103 are mainly localized in the epidermis. Expression of CXCR3 is observed on most CD8 TRM in vitiligo, including the population of melanocyte-specific CD8 T cells. CD8 TRM displayed increased production of IFN-γ and tumor necrosis factor-α with moderate cytotoxic activity. Our study highlights the presence of functional CD8 TRM in both stable and active vitiligo, reinforcing the concept of vitiligo as an immune memory skin disease. The CD8 TRM that remain in stable disease could play a role during disease flares, emphasizing the interest in targeting this cell subset in vitiligo.

Cole disease is a rare autosomal dominant disorder characterized by hypopigmented macules and hyperkeratosis. However, patient may also have hyperpigmented macuIes. In hypopigmented macules, a normal number of pigmented melanocytes but decreased melanin content in keratinocytes, suggesting an impairment of melanosome transfer, have been reported. Five mutations in somatomedin-B-like domains of EctoNucleotide Pyrophospahatase /Phosphodiesterase 1 (ENPP1) has been identified in five families with Cole disease. To study the role of ENPP1 in pigmentation and skin differentiation, we, first, reconstructed skin with cells from one patient with Cole disease and surprisingly Cole melanocytes were able to induced thickening of epidermal reconstructs. Since this disease is rare, we transduced melanocytes and keratinocytes with lentivectors coding wild-type (WT) ENPP1 or coding the first three mutations (M) identified in ENPP1. In melanocytes, at the protein level, expression of TRP-1 and tyrosinase but not of MITF seemed inversely correlated to the level of expression of mutated ENPP1. Reconstructions with melanocytes transduced with mutated ENPP1 were not thicker than those reconstructed with melanocytes transduced with WT melanocytes. But reconstructions with cells co-transduced with WT and mutated ENPP1 forms seemed thicker than those with WT alone. We also used pharmacological inhibitor of ENPP1 on monolayer cell culture. Secretome from melanocytes treated with ENPP1 inhibitors modulated expression of keratin 5 in keratinocytes whereas direct inhibition of ENPP1 in keratinocytes seemed less effective. Thus mutations of ENPP1 seemed directly implicated in establishing and sustaining hypo or hyperpigmentation in Cole Disease. Furthermore melanocytes seemed implicated in establishment of hyperkeratosis in Cole Disease.

Plasmacytoid dendritic cells (pDCs) are a subset of dendritic cells specialized in the production of type I interferon (IFN-α/β) and involved in various cutaneous inflammatory and autoimmune disorders, such as cutaneous lupus erythematosus (CLE) and vitiligo. Heat shock proteins (HSPs) are molecular chaperones essential for maintaining cellular functions, but they can act as a danger signal during inflammation.To decipher the role of HSP70 in the production of IFN-α by pDCs in CLE and vitiligo.Expression of HSP70 and CD123+ pDCs was analysed by immunohistochemistry or immunofluorescence in CLE and vitiligo skin samples. Flow cytometry was performed to analyse expression of HSP70 receptors, activation markers on pDCs and DNA uptake by pDCs in the presence of HSP70. The impact of HSP70 on DNA-induced IFN-α secretion by pDCs was evaluated by enzyme-linked immunosorbent assay (ELISA). The effect of IFN-α on chemokine (C-X-C motif) ligand 9 (CXCL9)/10 gene and protein expression by keratinocytes was determined by real-time polymerase chain reaction and ELISA.Infiltration of pDCs in CLE and progressive vitiligo was primarily located in the epidermis, close to keratinocytes expressing HSP70. In vitro experiments revealed that the pDCs expressing HSP70 receptor Lox-1 (lectin-like oxidized low-density lipoprotein-receptor-1) were able to aggregate HSP70. Exogenous HSP70 induced activation of pDCs and increased the uptake of exogenous DNA. Furthermore, HSP70 potentiated DNA-induced IFN-α production by pDCs. Finally, IFN-α induced expression of CXCL9 and CXCL10 by keratinocytes.These data demonstrate that interaction between HSP70 and pDCs in CLE and vitiligo is a prerequisite for the enhancement of IFN-α production, and could be an interesting target.

Human plasmacytoid dendritic cells (pDCs) are capable of producing large amounts of type I interferon (IFN-α/β) under appropriate activation. pDCs are involved in a wide range of cutaneous disorders such as cutaneous lupus erythematosus (CLE) and psoriasis. Recently we have demonstrated that this cell population is also present in peri-lesional skin of progressive vitiligo and could play an important role during initiation of depigmentation. Heat-shock proteins (HSP) are molecular chaperones essential for maintaining cellular functions. However during inflammation, HSP can be released extracellularly upon cellular injury or necrosis. Several works support the role of exogenous HSPs as a danger-signal to the host, providing a link between the innate and adaptive immune system. Therefore, we sought to characterize the interplay between HSP70, pDCs and IFNα production in the context of skin inflammatory disorders. We confirmed an increased production of HSP70 in the epidermis of CLE and progressive vitiligo compared to stable vitiligo, psoriasis and healthy controls. Importantly, pDCs were primarily localized in the epidermis in CLE and progressive vitiligo. These cells interacted with keratinocytes expressing HSP70. In vitro experiments revealed that pDCs expressing HSP70 receptor LOX-1 were able to aggregate HSP70. pDCs cultured with exogenous HSP70 underwent maturation and activation as assessed by measuring the expression of costimulatory molecules CD80, CD86 and HLA-DR. Furthermore, exogenous HSP70 increased the uptake of exogenous DNA by pDCs. Lastly, HSP70 potentiated the production of IFNα induced by DNA in pDCs. All together these data demonstrate the interaction between HSP70 and pDCs in CLE and vitiligo is a prerequisite for the enhancement of IFNα production, and could be an interesting target for future therapy.

Introduction Le vitiligo est une dermatose dépigmentante multifactorielle, caractérisée par la disparition progressive des mélanocytes. Cette pathologie reste à ce jour orpheline de traitement efficace. Le rôle de l’auto-immunité dans la perte des mélanocytes a été bien décrit dans la littérature, impliquant à la fois des lymphocytes T (LT) CD4+ et CD8+. Toutefois, le phénotype et la fonction de ces cellules restent à ce jour controversés en raison de l’absence d’une analyse approfondie sur les modèles humains et animaux reproduisant clairement la complexité de la maladie. Ainsi, l’objectif de notre travail est d’étudier le rôle des lymphocytes T effecteurs mémoires (LTEM) dans la dépigmentation au cours du vitiligo. Matériel et méthodes L’analyse multiparamétrique du profil phénotypique et fonctionnel des populations lymphocytaires TEM CD4+ et CD8+ circulantes et cutanées de patients atteints de vitiligo et de sujets sains a été réalisée par cytométrie en flux. Les effets des cytokines produites par ces populations lymphocytaires ont ensuite été évalués in vitro sur des cultures primaires de mélanocytes et des épidermes reconstruits pigmentés par PCR quantitative et par Elisa. Résultats Nos résultats montrent une diminution de la fréquence des LTEM circulants CD4+ et CD8+ exprimant le CXCR3 chez les patients atteints du vitiligo par rapport aux sujets sains, suggérant un recrutement de ces populations du compartiment sanguin vers la peau, ce que nous avons confirmé au niveau cutané. Par ailleurs, un pourcentage important de ces lymphocytes TEM présente un phénotype résident mémoire, exprimant les marqueurs CD103 et CD69. Au niveau cutané, ces LTEM produisent des niveaux élevés d’interféron (IFN)-g et de « tumor necrosis factor » (TNF)-a, deux cytokines inflammatoires connues pour leur implication dans plusieurs pathologies auto-immunes. L’étude des effets de ces deux cytokines in vitro a révélé un effet synergique dans l’inhibition de la fonction et l’adhésion du mélanocyte, reproduisant les observations réalisées chez les patients. Par ailleurs, l’IFNg et le TNFa induisent la production de chimiokines et cytokines pro-inflammatoires à la fois par les kératinocytes et les mélanocytes, permettant ainsi le maintien de la réponse inflammatoire à l’origine de la dépigmentation. Conclusion Nos résultats apportent une meilleure compréhension des mécanismes physiopathologiques impliqués dans la pathogénie du vitiligo et permettent ainsi d’établir un lien direct entre immunité, facteurs solubles inflammatoires et perte des mélanocytes afin de définir de nouvelles cibles thérapeutiques prometteuses.