Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Joint Project Südtirol- FWF (grant number 23623; Italy-Austria) and the Department of Innovation, Research and University of the Autonomous Province of Bolzano-South Tyrol (Italy) Background Mitochondria are dynamic organelles that sense and respond to environmental, nutritional, and pathological changes. These organelles can smartly adapt to different stressful conditions, by changing the way they network or the energy sources they use. Over the past years, numerous disorders have been associated with mitochondrial dysfunction, including cardiomyopathies. Arrhythmogenic Cardiomyopathy (ACM) is a cardiac disease characterized by the progressive replacement of the myocardium with fibro-fatty tissue. The current treatment primarily focuses on managing the symptoms rather than addressing the underlying issue of tissue substitution. Purpose: Therefore, in this study, we explored the mitochondrial and metabolic phenotype in ACM and tested the efficacy of a mitochondria-targeting library to rescue the fibro-fatty phenotype in ACM Methods: Human primary cardiac stromal cells (CStCs) were cultured in adipogenic medium (AM), a condition used to model the ACM fibro-fatty phenotype. Mitochondria-related analyses of ultrastructure, membrane potential, network connection, respiratory capacity, ROS production and redox system were performed at day (d) d0, d3, d7, d15 of AM exposure. A mitochondria-targeting library (MedChem Express) was tested on ACM-CStCs exposed to AM for 7 days. The extent of lipid accumulation was determined using the neutral lipid staining dye BODIPY 493/503, cell numbers were measured using Hoechst counterstaining, and images acquired using a high-content imaging system Results: At d0, the mitochondrial network was more fragmented in ACM compared to healthy CStCs (CTR). This result was in line with the observation of a decrease in mitochondrial respiratory capacity and an increase in ROS production. In addition, AM exposure led to faster and higher lipid accumulation in ACM CStCs, which was associated with higher mitochondrial oxygen consumption rates observed in ACM CStCs after 7 days of AM exposure. When ACM CStCs were simultaneously exposed to AM and 407 mitochondrial-directed compounds, about 25% of the compounds demonstrated the ability to reduce lipid accumulation Conclusion: ACM CStCs demonstrate an increased risk for mitochondrial network impairment. Moreover, prolonged exposure of ACM CStCs cells to AM negatively affects lipid deposition as well as mitochondrial and metabolic parameters. This highlights the potential for using specific mitochondria-targeting compounds to reduce lipid accumulation.

Kinetochores assemble on centromeres via histone H3 variant CENP-A and low levels of centromere transcripts (cenRNAs). The latter are ensured by the downregulation of RNA polymerase II (RNAPII) activity, and cenRNA turnover by the nuclear exosome. Using S. cerevisiae , we now add protein kinase Rio1 to this scheme. Yeast cenRNAs are produced either as short (median lengths of 231 nt) or long (4458 nt) transcripts, in a 1:1 ratio. Rio1 limits their production by reducing RNAPII accessibility and promotes cenRNA degradation by the 5’−3’exoribonuclease Rat1. Rio1 similarly curtails the concentrations of noncoding pericenRNAs. These exist as short transcripts (225 nt) at levels that are minimally two orders of magnitude higher than the cenRNAs. In yeast depleted of Rio1, cen- and pericenRNAs accumulate, CEN nucleosomes and kinetochores misform, causing chromosome instability. The latter phenotypes are also observed with human cells lacking orthologue RioK1, suggesting that CEN regulation by Rio1/RioK1 is evolutionary conserved.

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This research was funded by the Department of Innovation, Research and University of the Autonomous Province of Bolzano-South Tyrol (Italy), and by the Joint Project Südtirol- FWF (Italy-Austria) for A.R., J.T., A.M., R.P.. Background Arrhythmogenic cardiomyopathy (ACM) is a genetic disease associated with sudden cardiac death and fibro-fatty replacement of myocardium. Recently, it has been demonstrated that oxidized lipid contributes to cardiac adipogenesis and that ACM hearts are characterized by an increase in oxidative stress. Purpose As mitochondria are an important source of reactive oxygen species (ROS) within mammalian cells, the present work aims to evaluate if increased oxidative stress observed in ACM hearts is associated with altered mitochondrial function. Methods The oxidative stress marker 4HNE was investigated together with the cardiomyocyte marker cardiac Troponin T on paraffin embedded human ventricular samples and analyzed by confocal microscopy. Human primary cardiac stromal cells (CStCs), obtained from either right ventricle biopsies of ACM patients or healthy cadaveric tissue donor (CTR), were used as cellular model as they are known for their contribution to adipogenesis in the ACM pathology. CStCs were cultured either in basal medium or adipogenic medium (ADIPO) in presence or absence of 500 nM of the ROS scavenger MitoTEMPO. After 7 days of adipogenic differentiation, intracellular lipid droplets accumulation and mitochondrial superoxide levels were measured in CStCs by confocal microscopy using BODIPY 493/503 (0.5 µM) and MitoSOX Red (5 µM) dyes, respectively. The oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) were also evaluated in CStCs by high resolution respirometry. Results 4HNE staining was increased in heart tissues from ACM patients compared to CTRs and particularly evident in non-cardiomyocyte cells. In agreement, a higher MitoSOX fluorescence intensity was found in ACM-CStCs compared to CTR-CStCs in ADIPO medium, confirming higher ROS presence in patient cells. The treatment with MitoTEMPO was efficient in reducing ROS abundance, strongly suggesting mitochondrial origin. Lipid accumulation in ACM-CStCs was also prevented by MitoTEMPO treatment. Preliminary evidence further indicates that mitochondrial respiratory capacity is increased in ACM-CStCs versus CTR-CStCs exposed to adipogenic medium for 7 days. However, transmission electron microscopy analysis and Western blot analysis of Mfn1/2, Opa1, Fis1 and Drp1 did not show an evident alteration neither in mitochondria ultrastructure nor in the expression of proteins regulating mitochondrial dynamics. Conclusions Our data support an alteration of mitochondrial activity in CStCs from ACM patients, apparently not linked to a modified network or morphology of mitochondria but associated with a higher ROS production.

Specialized pro-resolving lipid mediators (SPMs) as lipoxins (LX), resolvins (Rv), protectins (PD) and maresins (MaR) promote the resolution of inflammation. We and others previously reported reduced levels of LXA4 in bronchoalveolar lavages from cystic fibrosis (CF) patients. Here, we investigated the role of CF airway epithelium in SPMs biosynthesis, and we evaluated its sex specificity. Human nasal epithelial cells (hNEC) were obtained from women and men with or without CF. Lipids were quantified by mass spectrometry in the culture medium of hNEC grown at air-liquid interface and the expression level and localization of the main enzymes of SPMs biosynthesis were assessed. The 5-HETE, LXA4, LXB4, RvD2, RvD5, PD1 and RvE3 levels were significantly lower in samples derived from CF patients compared with non-CF subjects. Within CF samples, the 12-HETE, 15-HETE, RvD3, RvD4, 17-HODHE and PD1 were significantly lower in samples derived from females. While the mean expression levels of 15-LO, 5-LO and 12-LO do not significantly differ either between CF and non-CF or between female and male samples, the SPMs content correlates with the level of expression of several enzymes involved in SPMs metabolism. In addition, the 5-LO localization significantly differed from cytoplasmic in non-CF to nucleic (or nuclear envelope) in CF hNEC. Our studies provided evidence for lower abilities of airway epithelial cells derived from CF patients and more markedly, females to produce SPMs. These data are consistent with a contribution of CF airway epithelium in the abnormal resolution of inflammation and with worse pulmonary outcomes in women.

Ciguatera fish poisoning is caused by the consumption of fish contaminated with ciguatoxins (CTXs). The most distressing symptoms are cutaneous sensory disturbances, including cold dysesthesia and itch. CTXs are neurotoxins known to activate voltage-gated sodium channels, but no specific treatment exists. Peptidergic neurons have been critically involved in ciguatera fish poisoning sensory disturbances. Protease-activated receptor-2 (PAR2) is an itch- and pain-related G protein-coupled receptor whose activation leads to a calcium-dependent neuropeptide release. In this study, we studied the role of voltage-gated sodium channels, PAR2, and the PAR2 agonist cathepsin S in the cytosolic calcium increase and subsequent release of the neuropeptide substance P elicited by Pacific CTX-2 (P-CTX-2) in rat sensory neurons and human epidermal keratinocytes. In sensory neurons, the P-CTX-2-evoked calcium response was driven by voltage-gated sodium channels and PAR2-dependent mechanisms. In keratinocytes, P-CTX-2 also induced voltage-gated sodium channels and PAR2-dependent marked calcium response. In the cocultured cells, P-CTX-2 significantly increased cathepsin S activity, and cathepsin S and PAR2 antagonists almost abolished P-CTX-2-elicited substance P release. Keratinocytes synergistically favored the induced substance P release. Our results demonstrate that the sensory effects of CTXs involve the cathepsin S-PAR2 pathway and are potentiated by their direct action on nonexcitable keratinocytes through the same pathway.

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.

Introduction: Ciguatera fish poisoning (CFP), the most common seafood poisoning worldwide, is caused by the consumption of seafood contaminated with ciguatoxins (CTXs). Pruritus is one of the most distressing symptoms, associated with other cutaneous sensory disorders, including paresthesia and cold dysesthesia. No specific treatment exists. CTXs are known to primarily activate voltage-gated sodium channels, but the downstream molecular events that lead to sensory disturbances remain poorly defined. Peptidergic sensory neurons were recently identified as major players in CFP sensory disturbances. Methods: In this study, we examined the role of molecular actors in 2 effects induced by Pacific CTX-2 (P-CTX-2): the increase in cytosolic calcium levels in rat primary sensory neurons; and the release of the neuropeptide substance P (SP) in sensory neurons co-cultured with keratinocytes. Results: Our results (i) rule out the involvement of the Na+/Ca2+ exchanger (NCX) and the transient receptor potential channels transient receptor potential ankyrin 1 and and transient receptor potential vanilloid 1; (ii) show that N-type voltage-gated calcium (Cav) channels contribute to the initiation of the calcium signal elicited by P-CTX-2 in rat sensory neurons, while N-type and L-type Cav channels play equal parts in the SP release in the co-culture; and (iii) identify store-operated calcium entry supported by Orai calcium release-activated calcium modulator 1 (ORAI1) as a critical effector of the late phase of the calcium signal and the subsequent SP release elicited by P-CTX-2. Discussion: Our in vitro findings indicate that Cav and ORAI1 channels may be promising pharmacological targets for specifically relieving the sensory effects of CTXs.

S | Innate Immunity and Inflammation 332 Plasma exosomal miR-375-3p regulates ferroptosis in keratinocytes by targeting GPX4 in SJS/TEN C Zhang, G Wang and M Fu Dermatology, Xijing Hospital, Fourth Military Medical University, Shaanxi, Xi’an, China, Xian, China Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening, cutaneous adverse drug reactions that are accompanied by keratinocyte cell death. Ferroptosis is a recently recognized form of regulated cell death driven by lipid-based reactive oxygen species (ROS) accumulation. However, the molecular mechanisms of ferroptosis regulation are still largely unknown in SJS/TEN. Exosomes are nanometre-sized vesicles with a lipid bilayer membrane and present in body fluids, containing functional proteins, mRNAs and miRNAs. Nevertheless, the potential roles of plasma exosomes and the underlying mechanisms in SJS/TEN have yet to be explored. In our study, we uncovers that exosomes isolated from plasma of SJS/TEN patients showed the expected size between 30 and 150nm. Then, exosomes were shown to be positive for exosomal markers CD9, CD63 and TSG101. Deep sequencing analysis and qRT-PCR of plasma exosomal derived miRNAs demonstrated that miR-375-3p level was the markedly upregulated in SJS/TEN patients and was positively correlated with clinical features. Then, plasma derived exosomes from patients were internalized by human primary keratinocytes and promoted malondialdehyde (MDA) and lipid ROS accumulation and ferroptotic cell death. Additionally, ectopic expression of miR-375-3p suppressed glutathione peroxidase 4 (GPX4), an enzyme converts lipid hydroperoxides to lipid alcohols, resulting in increased ferroptotic cell death. Importantly, knockdown of miR375-3p inhibited ferroptosis, which completely prevented SJS/TEN-like responses in a mouse model of SJS/TEN. Our results indicate that GPX4 downregulated by the overexpressed miR375-3p mediates keratinocyte ferroptosis in SJS/TEN patients. Circulating exosomal miR-3753p might be used as a potential disease marker for the diagnosis of SJS/TEN. S272 Journal of Investigative Dermatology (2019), Volume 139 333 Effect of thermal spring water on human dendritic cell inflammatory responses 2 1 1 1 Y Eliasse , M Galliano , H Hernandez-Pigeon , S Bessou-Touya , N Castex-Rizzi, D Redoules and E Espinosa 1 Pierre Fabre Dermocosmetic, Toulouse, France, 2 INSERM U1043, CPTP CHU Purpan, Toulouse, France and 3 Laboratoires Avène, Lavaur, France Hydrotherapy appears as a valuable therapeutic tool in the management of patients suffering from chronic skin inflammatory diseases. Nevertheless, the underlying immune mechanisms of these beneficial effects remain poorly understood. To better understand the biological effects of thermal spring water on the immune system, we investigated the effects of Avène thermal spring water (ASW) on dendritic cells as key cells participating in the control of the immune response. DCs were generated from human monocytes and matured with LPS in ASW-based culture medium or in dexamethasone supplemented culture medium as an antiinflammatory treatment. The phenotypes and abilities of these DCs to produce cytokines and induce allogeneic T cell response was next assessed. We showed that ASW modulated the differentiation of monocytes into DCs and impacted the DC maturation upon LPS priming. We observed a reduction of the CD83, CD86, CD1a and HLA-DR molecule expression and a decrease of IL-12 and IL-23 production whereas IL-10 production was increased. LPS-primed DCs generated in presence of ASW exhibited a reduced capacity to induce naive CD4 T cell proliferation and IFN-g and IL-17 production. Our study showed that ASW is endowed with an immunomodulatory potential. ASW limits the DC stimulatory capacity of Th1 and Th17 cell responses by impairing their maturation, IL-12 and IL-23 production and accessory cell function. 334 Involvement of cathepsin S and protease-activated receptor-2 in ciguatoxin-induced substance P release: new promising targets to treat ciguatera pruritus O Pierre, K L’Herondelle, S Fouyet, R Leschiera, C Le Gall Ianotto, R Philippe, P Buscaglia, O Mignen, L Misery and R Le Garrec 1 LIEN, EA4685, University of Western Brittany, Brest, France, 2 INSERM U1227, University of Western Brittany, Brest, France and 3 Department of Dermatology, University Hospital of Brest, Brest, France Ciguatera Fish Poisoning (CFP) is a widespread tropical intoxication consecutive to ciguatoxin (CTX) ingestion, which is characterized by persistent neuro-cutaneous disturbances, including an intense pruritus (itch). Currently, there is no specific treatment. The primary target of CTXs is the voltage dependent sodium channel (Nav), which is largely expressed in sensory nerves and, to a lesser extent, in keratinocytes. By activating Nav, CTXs induce neuronal hyper-excitability but the downstream molecular mechanisms leading to sensory disorders are poorly understood. Recent advances in dermatology reveal protease-activated receptor-2 (PAR-2) involvement in itch pathophysiology. Interestingly, this receptor is expressed in sensory neurons and keratinocytes. To better understand the pathophysiology of CFP pruritus, the purpose of the present study was to identify cellular and molecular actors involved in the substance P (SP) release elicited by P-CTX-2 from co-cultured sensory neurons and keratinocytes. First, P-CTX-2 is able to induce calcium signal in both sensory neurons and keratinocytes. We show that antagonism of PAR-2 significantly inhibited the P-CTX-2-evoked calcium signal in both cells. Then, our results demonstrate the synergistic role of keratinocytes in the SP release elicited by P-CTX-2 in the coculture. The P-CTX-2-induced SP was almost completely abolished by PAR-2 or cathepsin S (Cat S) antagonists, and Cat S activity was significantly increased after P-CTX-2 treatment. Finally, P-CTX-2 is able to internalise PAR-2 in keratinocytes. Taken together, this work reveals that keratinocytes, PAR-2 and Cat S are novel actors in the P-CTX-2-induced release of SP, suggesting those are promising pharmacological targets for specifically treating CFP neuro-cutaneous disorders. 335 Anti-inflammatory effects of potassium iodide in sodium dodecyl sulphate-induced inflammatory murine skin through interleukin-10 regulation S Hayashi, S Ishikawa, M Koike, T Kaminaga, Y Hamasaki and I Ken Dermatology, Dokkyo medical university, Mibu, Japan Background: Applications of potassium iodide (KI) to inflammatory skin diseases have not been prevalent, most likely because the mechanism of its therapeutic effects has been unknown. Currently, information on effects of KI on skin diseases is available only in case reports, and few studies report on its mechanism. Recently, we reported 25 cases of palmoplantar pustulosis (PPP) treated with KI and its success and effectiveness. PPP is a common, chronic and very refractory inflammatory skin disorder. However, none of the many different remedies that exist are generally accepted as being reliably effective, and almost all PPP cases are resistant to treatment. This old drug has been replaced by powerful and expensive new drugs, such as biological or immunosuppressive agents, in current dermatology practice. KI is inexpensive with few side effects and, although it has long been used as a drug, it has not been used frequently in clinical practice. Elucidation of the pharmacologic mechanism for the therapeutic effects of KI would lead to its wide use in clinical practice with high evidence, which also would be very cost-effective. Methods: We applied 5% sodium dodecyl sulphate (SDS) and 3% KI to the shaved backs of BALB/C mice. In SDS-induced inflammatory murine skin, inflammatory leukocyte invasion is observed, such as macrophage and the neutrophilic permeation other than a lymphocyte. In addition, primary inflammatory cytokine such as TNF-a or IL-1 are increasing in this model mouse. We speculated that the effect of KI, a drug whose effectiveness is known for several diseases, can be easily verified using a simple dermatitis model. In addition, small interfering RNA (siRNA), which was designed to knockdown interleukin IL-10, was injected to same mice. Expressions of the genes encoding inflammatory cytokines and inflammatory cell counts were measured by realtime reverse transcription-polymerase chain reaction fluorescence-activated cell sorting analysis, respectively. 336 Mutualistic skin bacteria protect against dermatitis via the induction of steroid biosynthesis pathways Y Ito, T Sasaki, E Kawakami, W Suda, K Atarashi, M Amagai and K Honda 1 Keio Univ., RIKEN-IMS, Yokohama, Japan, 2 Keio Univ., Tokyo, Japan, 3 RIKEN-MIH, Yokohama, Japan and 4 RIKEN-IMS, Yokohama, Japan The skin microbiota plays a role in homeostasis and inflammatory diseases such as atopic dermatitis. While the pathogenic potential of several skin microbiota members has been wellcharacterized, an emerging body of evidence suggests certain commensals possess properties that benefit the host. To elucidate novel host-commensal relationships that endow protection against inflammation, we established a preventable, microbiota-driven dermatitis mice model using gnotobiotic techniques and transgenic mice. Specifically, we used Tmem79 knockout mice which, under specific pathogen-free conditions, develop spontaneous dermatitis accompanied by an increase in both Staphylococcus aureus colonization density and number of IL-17-expressing T cells in the skin. In contrast, we found that dermatitis does not develop when the mice are kept under germ-free conditions. We examined the influence of mouseorigin skin microbiota on the development of dermatitis by monocolonizing germ-free mice with various bacterial isolates. Gnotobiotic mice monocolonized with S. aureus or Corynebacterium mastitidis exhibited dermatitis, whereas those monocolonized with other Staphylococci strains did not. We also found that Sta

Calumenin is a secretory pathway protein regulating different endoplasmic reticulum (ER) proteins such as the sarco-endoplasmic reticulum calcium ATPase (SERCA) pumps. Combined with its diverse cellular distribution, its calcium-binding ability, and its interaction with proteins involved in calcium signaling, it is easy to speculate on future description of important roles of calumenin in calcium homeostasis in many cell types, as it was initially observed in muscle cells. In this chapter, we describe basic techniques to modulate calumenin expression and detect its impact on ER calcium content using classic transfection and Western blot techniques, as well as ER calcium measurement using microplate reader.

Ciguatera Fish Poisoning (CFP) is a widespread tropical intoxication consecutive to ciguatoxin (CTX) ingestion, which is characterized by persistent neuro-cutaneous disturbances, including an intense pruritus (itch). Currently, there is no specific treatment. The primary target of CTXs is the voltage dependent sodium channel (Nav), which is largely expressed in sensory nerves and, to a lesser extent, in keratinocytes. By activating Nav, CTXs induce neuronal hyper-excitability but the downstream molecular mechanisms leading to sensory disorders are poorly understood. Recent advances in dermatology reveal protease-activated receptor-2 (PAR-2) involvement in itch pathophysiology. Interestingly, this receptor is expressed in sensory neurons and keratinocytes. To better understand the pathophysiology of CFP pruritus, the purpose of the present study was to identify cellular and molecular actors involved in the substance P (SP) release elicited by P-CTX-2 from co-cultured sensory neurons and keratinocytes. First, P-CTX-2 is able to induce calcium signal in both sensory neurons and keratinocytes. We show that antagonism of PAR-2 significantly inhibited the P-CTX-2-evoked calcium signal in both cells. Then, our results demonstrate the synergistic role of keratinocytes in the SP release elicited by P-CTX-2 in the coculture. The P-CTX-2-induced SP was almost completely abolished by PAR-2 or cathepsin S (Cat S) antagonists, and Cat S activity was significantly increased after P-CTX-2 treatment. Finally, P-CTX-2 is able to internalise PAR-2 in keratinocytes. Taken together, this work reveals that keratinocytes, PAR-2 and Cat S are novel actors in the P-CTX-2-induced release of SP, suggesting those are promising pharmacological targets for specifically treating CFP neuro-cutaneous disorders.

In cystic fibrosis (CF), impaired airway surface hydration (ASL) and mucociliary clearance that promote chronic bacterial colonization, persistent inflammation, and progressive structural damage to the airway wall architecture are typically explained by ion transport abnormalities related to the mutation of the gene coding for the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. However, the progressive and unrelenting inflammation of the CF airway begins early in life, becomes persistent, and is excessive relative to the bacterial burden. Intrinsic abnormalities of the inflammatory response in cystic fibrosis have been suggested but the mechanisms involved remain poorly understood. This review aims to give an overview of the recent advances in the understanding of the defective resolution of inflammation in CF including the abnormal production of specialized pro-resolving lipid mediators (lipoxin and resolvin) and their impact on the pathogenesis of the CF airway disease.

PAR2 activation in basal keratinocytes stimulates inflammation via the Ca2+-dependent production of mediators such as IL-1β, 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-1β, 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.

Cystic Fibrosis (CF) is the most frequent fatal genetic disease in Caucasian populations. Mutations in the chloride channel CF Transmembrane Conductance Regulator (CFTR) gene are responsible for functional defects of the protein and multiple associated dysregulations. The most common mutation in patients with CF, F508del-CFTR, causes defective CFTR protein folding. Thus minimal levels of the receptor are expressed at the cell surface as the mutated CFTR is retained in the endoplasmic reticulum (ER) where it correlates with defective calcium (Ca2+) homeostasis. In this study, we discovered that the Ca2+ binding protein Calumenin (CALU) is a key regulator in the maintenance of ER-Ca2+ calcium homeostasis in both wild type and F508del-CFTR expressing cells. Calumenin modulates SERCA pump activity without drastically affecting ER-Ca2+ concentration. In addition, reducing Calumenin expression in CF cells results in a partial restoration of CFTR activity, highlighting a potential function of Calumenin in CFTR maturation. These findings demonstrate a pivotal role for Calumenin in CF cells, providing insights into how modulation of Calumenin expression or activity may be used as a potential therapeutic tool to correct defects in F508del-CFTR.

La ciguatéra est une intoxication des régions tropicales provoquée par l’ingestion de poissons contaminés par des ciguatoxines (CTXs). Les signes cliniques caractéristiques de cette maladie sont des troubles sensitifs cutanés, tels qu’une allodynie au froid et un prurit intense qui peuvent persister des semaines, voire des mois. C’est la raison pour laquelle la ciguatéra est communément appelée « la gratte ». Les CTXs se fixent aux canaux Na+ des neurones. Il a été montré que les fibres sensorielles activées par les CTXs lors de l’allodynie au froid sont de type Aδ et C, expriment le CGRP et que l’activation implique le canal cationique TRPA1. Le prurit est une sensation désagréable qui conduit au besoin de se gratter. Bien que sa physiopathologie complexe reste incomplètement connue, nous savons que certains éléments y participent, tels que le TRPA1, les TRPV (Transient Receptor Potential Ankyrin/Vanilloid) et les PARs (Protease Activated Receptors). De plus, les fibres nerveuses du prurit possèdent comme neuropeptides de référence la SP et le CGRP. Les anti-histaminiques ne fonctionnent que dans 30 % des cas de prurit et il est nécessaire de trouver de nouvelles voies d’activation. L’étude du prurit ciguatérique, non histaminergique, apparaît comme une approche intéressante pour mieux comprendre la physiopathologie du prurit. Nous avons établi une co-culture de kératinocytes humains et de neurones sensoriels de rat afin de mieux appréhender les signes sensitifs périphériques de la ciguatéra. Des dosages de SP et CGRP dans les surnageants ont été réalisés pour explorer l’effet d’une CTX sur la sécrétion de neuropeptides. Sur ce même modèle, différents inhibiteurs ont été utilisés pour identifier des acteurs potentiels dans cet effet neurosécretoire, comme le TRPV1, le TRPA1 et le PAR2. Comme l’exocytose de neuropeptides est consécutive à une augmentation de calcium intracellulaire, nous avons réalisé des expériences d’imagerie calcique. Afin de tester si les kératinocytes pouvaient être une cible directe des CTXs, nous avons étudié leur réponse calcique à une CTX, avec ou non pré-incubation d’inhibiteurs spécifiques. Nous avons montré que les neuropeptides de référence du prurit, SP et CGRP, sont sécrétés sous l’influence des CTXs. Nous avons aussi montré que la neurosécrétion induite par les CTXs est liée à l’activation de récepteurs spécifiques encore inconnus en tant que cibles des CTXs. Les expériences d’imagerie calcique ont révélé que les kératinocytes sont une nouvelle cible cellulaire des CTXs, en plus des terminaisons nerveuses. Cette étude met pour la première fois en évidence une libération de neuropeptides et une activation directe des kératinocytes par une CTX. Elle permet d’envisager de nouvelles cibles thérapeutiques dans la ciguatéra mais aussi plus largement dans un certain nombre de prurits non histaminergiques. Travail réalisé avec le soutien de la SFD.

Cystic Fibrosis (CF) disease is caused by mutations in the CFTR gene (CF transmembrane conductance regulator). F508 deletion is the most represented mutation, and F508del-CFTR is absent of plasma membrane and accumulates into the endoplasmic reticulum (ER) compartment. Using specific Ca2+ genetics cameleon probes, we showed in the human bronchial CF epithelial cell line CFBE that ER Ca2+ concentration was strongly increased compared to non-CF (16HBE) cells, and normalized by the F508del-CFTR corrector agent, VX-809. We also showed that ER F508del-CFTR retention increases SERCA (Sarcoplasmic/Reticulum Ca2+ ATPase) pump activity whereas PMCA (Plasma Membrane Ca2+ ATPase) activities were reduced in these CF cells compared to corrected CF cells (VX-809) and non-CF cells. We are showing for the first time CFTR/SERCA and CFTR/PMCA interactions that are modulated in CF cells and could explain part of Ca2+ homeostasis deregulation due to mislocalization of F508del-CFTR. Using ER or mitochondria genetics Ca2+ probes, we are showing that ER Ca2+ content, mitochondrial Ca2+ uptake, SERCA and PMCA pump, activities are strongly affected by the localization of F508del-CFTR protein.

We previously we attempted to make a three-dimensional human skin model consisting of three different cells, dendritic cells, keratinocytes and fibroblasts (KDF-Skin) to evaluate immunoreactions in human skin; however, this model had various problems; for example (1) the incubation period for the construction of this model is long (about three weeks); (2) to construct the collagen gel, high amounts of fibroblasts are needed; and (3) the horny layer of keratinocytes in this skin model is thinner than that of keratinocytes in real human skin.In order to overcome these problems, a new three-dimensional human skin model utilizing a handy scaffold of collagen vitrigel membrane (VG-KDF-Skin) was constructed. As a result, after 14 days incubation, the epidermis layer of normal human keratinocytes was thicker than the keratinocyte layer of KDF-Skin. The incubation period for VG-KDF-Skin construction was 7 days shorter than that of KDF-Skin, and the number of fibroblasts needed to seed VG-KDF-Skin was four times fewer than that of KDF-Skin. After the application of sensitizers such as DNCB, VG-KDF-Skin induced the expression of CD86 and cytokine release.These results suggest that the new three-dimensional human skin model consisting of dendritic cells, keratinocytes, fibroblasts and collagen vitrigel membrane was more useful for alternative animal testing than the KDF-Skin model.