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International audience

Skin conventional dendritic cells (cDCs) exist as two distinct subsets, cDC1s and cDC2s, which maintain the balance of immunity to pathogens and tolerance to self and microbiota. Here, we examined the roles of dermal cDC1s and cDC2s during bacterial infection, notably Propionibacterium acnes (P. acnes). cDC1s, but not cDC2s, regulated the magnitude of the immune response to P. acnes in the murine dermis by controlling neutrophil recruitment to the inflamed site and survival and function therein. Single-cell mRNA sequencing revealed that this regulation relied on secretion of the cytokine vascular endothelial growth factor alpha (VEGF-alpha) by a minor subset of activated EpCAM(+)CD59(+) Ly-6D(+) cDC1s. Neutrophil recruitment by dermal cDC1s was also observed during S. aureus, bacillus Calmette-Guerin (BCG), or E. coli infection, as well as in a model of bacterial insult in human skin. Thus, skin cDC1s are essential regulators of the innate response in cutaneous immunity and have roles beyond classical antigen presentation.

International audience

Rosacea is a common, chronic inflammatory skin disease that can present with a variety of signs and symptoms. The potentially simultaneous occurrence of different signs and symptoms is due to different underlying inflammatory pathways, emphasizing the need for complementary treatment approaches. Topical ivermectin cream (10 mg/g) and systemic, oral anti-inflammatory doxycycline (40 mg modified-release) are both approved for the treatment of papulopustular rosacea (PPR). Whether or not a combined therapeutic approach may be more beneficial than monotherapy for patients with PPR remains to be tested. Here, we summarize underlying inflammatory pathways implicated in rosacea and clarify the impact of these two agents on selective pathways during inflammation, due to specific characteristics of their individual mechanisms of action (MoA). Based on the complementary MoA of doxycycline modified-release and ivermectin, a scientific rationale for a combined therapy targeting inflammatory lesions in rosacea is given. We propose that topical ivermectin cream is a promising new candidate as first-line treatment to target the inflammatory lesions of rosacea, which can be used in combination with systemic doxycycline modified-release to provide an optimal treatment approach considering all inflammatory pathways involved in PPR. Funding Galderma.

International audience; Testing hepatotoxicity is a crucial step in the development and toxicological assessment of drugs and chemicals. Bio-activation can lead to the formation of metabolites which may present toxicity for the organism. Classical cytotoxic tests are not always appropriate and are often insufficient, particularly when non metabolically-competent cells are used as the model system, leading to false-positive or false-negative results. We tested over 24 h the effects of eight reference compounds on two different cell models: primary cultures of rat hepatocytes and FAO hepatoma cells that lack metabolic properties. We performed inter-assay validation between three classical cytotoxicity assays and real-time cell impedance data. We then complemented these experiments with high-content screening (HC5) to determine the cell function disorders responsible for the observed effects. Among the different assays used, the neutral red test seemed to be well suited to our two cell models, coupled with real-time cellular impedance which proved useful in the detection of bio-activation. Indeed, impedance monitoring showed a high sensitivity with interesting curve profiles yet seemed unsuitable for evaluation of viability on primary culture. Finally, HCS in the evaluation of hepatotoxicity is likely to become an essential tool for use in parallel to a classical cytotoxic assay in the assessment of drugs and environmental chemicals.

International audience; Pesticides as well as many other environmental pollutants are considered as risk factors for the initiation and the progression of cancer. In order to evaluate the in vitro effects of chemicals present in the diet, we began by combining viability, real-time cellular impedance and high throughput screening data to identify a concentration "zone of interest" for the six xenobiotics selected: endosulfan, dioxin, carbaryl, carbendazim, p ' p ' DDE and hydroquinone. We identified a single concentration of each pollutant allowing a modulation of the impedance in the absence of vital changes (nuclear integrity, mitochondrial membrane potential, cell death). Based on the number of observed modulations known to be involved in hepatic homeostasis dysfunction that may lead to cancer progression such as cell cycle and apoptosis regulators, EMT biomarkers and signal transduction pathways, we then ranked the pollutants in terms of their toxicity. Endosulfan, was able to strongly modulate all the studied cellular processes in HepG2 cells, followed by dioxin, then carbendazim. While p,p,p ' DDE, carbaryl and hydroquinone seemed to affect fewer functions, their effects nevertheless warrant close scrutiny. Our in vitro data indicate that these xenobiotics may contribute to the evolution and worsening of hepatocarcinoma, whether via the induction of the EMT process and/or via the deregulation of liver key processes such as cell cycle and resistance to apoptosis.

Endosulfan is an organochlorine insecticide described as a potential carcinogen in humans. This insecticide was recently reported to alter the mitogen-activated protein (MAP) kinase signaling pathways and is suspected to affect cell growth and differentiation in human keratinocytes. This study was designed to assess the mitogenic, apoptogenic, and genotoxic effects of endosulfan on the HaCaT cell line. We first found that 25 microM endosulfan led to persistent extracellular signal-regulated kinase (ERK)1/2 phosphorylation with an accumulation of the phosphorylated form in the nucleus, probably caused by MAP kinase phosphatase (MKP) inhibition. As previously described under sustained ERK1/2 activation, cell growth was decreased: delayed confluency and 35% decrease of BrdU incorporation was demonstrated in endosulfan-treated keratinocytes. In addition, endosulfan has been shown to generate transient reactive oxygen species (ROS), and blocking this oxidative stress by N-acetyl cysteine (NAC) strongly prevented both persistent nuclear ERK1/2 phosphorylation and cell growth decrease. Additional experiments demonstrated that unchanged endosulfan rather than its metabolites has mutagenic effects (Ames positive without S9) and increased DNA strand breaks (Comet assay) in HaCaT cells, via a ROS-dependent mechanism. Therefore, to assess the putative pro-apoptotic response of damaged cells, caspases 3/7 activity and poly(ADP-ribose)-polymerase (PARP) cleavage were measured. The results clearly indicated that endosulfan inhibited both spontaneous and staurosporine-induced apoptosis. Taken together, these findings strongly support that endosulfan induces ROS generation leading to sustained ERK1/2 phosphorylation and decrease in cell growth. Moreover, endosulfan was found to inhibit apoptosis and this could contribute to mutant cell survival and therefore have possible carcinogenic effects.

Many persistent pesticides have been implicated in reproductive and developmental adverse effects, in man and wildlife. It has been hypothesized that these so-called xeno-hormones could upset the endocrine system function by binding to human estrogen receptor alpha and beta (ERalpha, beta) and thus be responsible for the higher incidence of breast and cervical cancer, infertility and endometriosis. In this report, forty-nine pesticides were tested for ERalpha and beta activation or inhibition in stable reporter cell lines, HELN ERalpha and ERbeta. Stable transfection of the ERalpha and ERbeta constructs together with an estrogen reporter luciferase vector into the HeLa cell line resulted in two estradiol-sensitive cell lines. In our model, fifteen of the tested pesticides were found to agonize the ERalpha-mediated transcription in a dose-dependent manner and DDT, trans-nonachlor, chlordane, fenvalerate and toxaphene were also capable to activate ERbeta. Antagonistic activities toward hERalpha and hERbeta were shown in three (carbaryl, pentachlorophenol and 2,4,5-trichlorophenoxyacetic acid) and seven (chlordecone, methoxychlor, carbaryl, endosulfan, endrin, dieldrin, aldrin) pesticides, respectively. Remarkably chlordecone and methoxychlor which were the most effective antagonist compounds for hERbeta, were agonists for hERalpha. Although the ERalpha activation potential of the pesticides was lower than that of estradiol, the overall body scale response might be amplified by the ability of pesticides to act via several mechanisms and by frequent and prolonged exposure to different pesticides, even at low concentrations.

Organochlorine pesticides (OCs) are reported as potential carcinogens in humans. The aim of this study was to investigate the effects of four OCs (dieldrin, endosulfan, heptachlor, and lindane) on mitogen-activated protein kinase (MAPK) cascades and more specifically to identify the mechanism underlying OC-induced ERK1/2 activation. Organochlorine pesticides increased phosphorylated Raf, MEK1/2, ERK1/2, and c-Jun in human HaCaT cells, but they had no effect on p38 MAPK activation. Moreover, blockade of Raf, MEK1/2, or PKC activation with geldanamycin, U0126, or calphostin C inhibited ERK1/2 phosphorylation, demonstrating a PKC-Raf-MEK1/2 pathway. We also showed that these insecticides induced the production of reactive oxygen species (ROS). Pre-treatment with the antioxidant molecule N-acetyl cysteine sharply decreased the level of phospho-ERK1/2 and had no effect on Raf and MEK1/2 activation, suggesting a Raf-independent mechanism. This study indicates that OCs strongly activate the ERK1/2 pathway, and it identifies a critical role of ROS in OC-induced ERK activation, probably by stabilizing its phosphorylation.

Five organochlorine pesticides, namely, chlordane, dieldrin, aldrin, endrin, and endosulfan, activate human retinoic acid receptor (RAR)-mediated gene transcription via a retinoic acid response element (RARE). Transactivation studies were performed with stable RARalpha, beta, or gamma reporter cell lines in which the RAR DNA-binding domain (DBD) was replaced by that of estrogen receptor alpha (ERalpha)? Five of the organochlorine pesticides tested activated RARbeta and RARgamma but not RARalpha; their half-maximal luciferase activity (EC(50)) was determined. Furthermore, that activity was RAR-specific and organochlorine pesticides did not activate the retinoid X receptor (RXR) pathway. However, competitive binding experiments with [(3)H]-CD367, a pan-RAR agonist, showed that only chlordane could bind RARbeta and RARgamma, albeit with low affinity. In addition, organochlorine pesticides strongly induce cytochrome P450RAI1 (P450RAI1), a key factor of retinoic acid level regulation in many tissues and whose expression and activity are strongly induced by retinoic acid. This study shows that organochlorine pesticides can activate two RAR homologues, with low-binding affinity. Although the agonistic potential of organochlorine pesticides is lower than that of (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl] benzoic acid (TTNPB), they are able to induce RAR-mediated gene transcription as P450RAI1 and may disrupt the retinoid signaling pathway. Because these chemicals are extremely persistent and tend to accumulate in biological tissues, these results support the hypothesis that the increase in teratogenicity observed in some developing countries could be due to prolonged exposure to organochlorine pesticides ubiquitously present in the environment.