Éditeur(s) : HAL CCSD
Résumé : International audience
STUDY QUESTION: Do freezing and in vitro culture procedures enhance the expression of proteins involved in apoptotic or autophagic pathways in murine pre-pubertal testicular tissue? SUMMARY ANSWER: IVM strongly modified apoptosis- and autophagy-related relative protein levels in mice testicular tissue whereas the impact of cryopreservation procedures was minimal at the end of the culture. WHAT IS KNOWN ALREADY: In vitro spermatogenesis remains a challenging technical issue as it imposes to find a very close balance between survival and death of germ cell natural precursors (i.e. gonocytes and spermatogonia), which will eventually undergo a complete spermatogenesis close to in vivo conditions. The establishment of efficient culture conditions coupled with suitable cryopreservation procedures (e.g. controlled slow freezing [CSF] and solid surface vitrification [SSV]) of pre-pubertal testicular tissue is a crucial step in the fields of fertility preservation and restoration to improve the spermatic yield obtained in vitro. STUDY DESIGN, SIZE, DURATION: Here, we study cryopreservation procedures (i.e. CSF or SSV) and the impact of culture media compositions. A first set of 66 mouse pre-pubertal testes were directly cultured during 30, 36, 38 and 60 days (D) from 2.5 to 6.5-day-old CD-1 mice to evaluate the impact of time-aspect of culture and to endorse the reverse phase protein microarrays (RPPM) technique as an adapted experimental tool for the field of in vitro spermatogenesis. Ninety others fresh, slow-frozen and vitrified pre-pubertal testes were cultured during 30 days for the principal study to evaluate the impact of cryopreservation procedures before and after culture. Thirty-four testes dissected from 2.5, 6.5, 36.5, 40.5, 42.5 and 62.5 days postpartum (dpp) mice, corresponding to the time frames of spermatogenesis orchestrated in vitro, were used as in vivo controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: After in vitro culture, testicular tissue samples originated from 2.5 or 6.5-day-old CD-1 male mice were analyzed using RPPM. This targeted proteomic technique allowed us to assess the expression level of 29 apoptosis- and autophagy-related factors by normalizing blank-corrected signal values. In addition, morphological analyses (e.g. HES, PAS, TRA98 and CREM) and DNA fragmentation in intra-tubular cells (i.e. terminal deoxynucleotidyl transferase dUTP nick end labeling; TUNEL) were assessed for the distinct experimental conditions tested as well as for in vivo control mouse testes. MAIN RESULTS AND THE ROLE OF CHANCE: A validation of the RPPM procedure in the field of in vitro spermatogenesis was completed with assay and array robustness before a principal study concerning the evaluation of the impact of in vitro culture and cryopreservation procedures. The proportion of elongated spermatids and the total cell number per seminiferous tubule tended to be very different between the in vivo and in vitro conditions (P < 0.05), suggesting the presence of a beneficial regulation on the first spermatogenesis wave by intrinsic apoptosis (Caspase₉) and autophagy (Atg5) factors (P < 0.0003 and r2 = 0.74). Concerning the impact of culture media compositions, a basic medium (BM) composed of αMEM plus 10% KnockOut™ serum replacement and gentamicin supplemented with retinol (Rol) and vitamin E (Vit. E) was selected as the best culture medium for fresh 6.5 dpp tissue cultured during 30D with 27.7 ± 8.10% of seminiferous tubules containing elongated spermatids. Concerning the impact of cryopreservation procedures, SSV did not have any impact on the morphological parameters evaluated after culture in comparison to fresh tissue (FT) controls. The proportion of tubules with elongated spermatids on testicular explants cultured with BMRol+Vit. E was not different between SSV (6.6 ± 1.6%) and CSF (5.3 ± 1.9%); however, round spermatids were observed more frequently for SSV (19 ± 6.2%) than CSF (3.3 ± 1.9%, P = 0.0317). Even if the proportion of TUNEL-positive cells for BMRol+Vit. E was higher at D30 after SSV (4.12 ± 0.26%) than CSF (1.86 ± 0.12%, P = 0.0022) and FT (2.69 ± 0.33%, P = 0.0108), the DNA damages observed at the end of the culture (i.e. D30) were similar to respective 6.5 dpp controls. In addition, the relative protein level expression ratio of an apoptotic factor, the phosphorylated FADD on Fas, was reduced by 64-fold in vitrified testes cultured with BMRol+Vit. E. Furthermore, we found in this study that the StemPro®-34 SFM culture medium supplemented with growth factors (e.g. EGF, bFGF, GDNF and LIF) prevented the differentiation of spermatogonial stem cells in favor of a significant proliferation with a better architectural pattern than in vivo 6.5 dpp controls with an increase of seminiferous tubules area for FT (P = 0.0357) and CSF (P = 0.0317). LIMITATIONS REASONS FOR CAUTION: Despite our promising results, the evaluation of apoptotic- and autophagic-related proteins was studied for a limited amount of proteins and on global testicular tissue. WIDER IMPLICATIONS OF THE FINDINGS: The data presented herein will help to improve apoptotic and autophagic understanding during the first spermatogenic wave. Moreover, our findings illustrate for the first time that, using finely-tuned experimental conditions, a testicular in vitro culture combined with proteomic technologies may significantly facilitate the study of cryopreservation procedures and in vitro culture evaluations. This study may also contribute to improve work on testicular tissues from pre-pubertal and adolescent cancer survivors. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by a Ph.D. grant from the Rouen Normandie Université and a financial support from 'la Ligue nationale contre le cancer' (both awarded to L.D.), funding from Institute for Research and Innovation in Biomedicine (IRIB), Agence de la Biomédecine, and co-supported by European Union and Région Normandie. Europe gets involved in Normandie with European Regional Development Fund (ERDF). The authors declare that there is no conflict of interest.
Molecular Human Reproduction

hal-01647081
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01647081
DOI : 10.1093/molehr/gax054
PUBMED : 29040674

Éditeur(s) : Inter-research
Résumé : Vibriosis is a major disease problem in shrimp aquaculture. 'Syndrome 93' is a seasonal juvenile vibriosis caused by Vibrio penaeicida which affects Litopenaeus stylirostris in grow-out ponds in New Caledonia. This study assessed the toxic activities of extracellular products (ECPs) from V: penaeicida, V. alginolyticus and V, nigripulchritudo using in vivo injections in healthy juvenile L. stylirostris (= Penaeus stylirostris) and in vitro assays on shrimp primary cell cultures and the fish cell line epithelioma papulosum cyprini (EPC). Toxic effects of ECPs were demonstrated for all pathogenic Vibrio strains tested both in vivo and in vitro, but for shrimp only; no effect was observed on the fish cell line. ECP toxicity for New Caledonian V. penaeicida was found only after cultivation at low temperature (20 degrees C) and not at higher temperature (30 degrees C). This points to the fact that 'Syndrome 93' episodes are triggered by temperature drops. The assays used here demonstrate the usefulness of primary shrimp cell cultures to study virulence mechanisms of shrimp pathogenic bacteria.
Diseases Of Aquatic Organisms (0177-5103) (Inter-research), 2000-03 , Vol. 40 , N. 2 , P. 101-107

Droits : info:eu-repo/semantics/openAccess
http://archimer.ifremer.fr/doc/00000/10543/7600.pdf
DOI:10.3354/dao040101
http://archimer.ifremer.fr/doc/00000/10543/

Éditeur(s) : HAL CCSD
Résumé : During the course of their life cycle organisms are exposed to natural environment variations capable of inducing physiological, morphological and behaviour changes, thus a phenotypic plasticity. Phenotypic plasticity is the ability of a genotype to generate a new phenotype following exogeneous or endogeneous stress. Here, we investigated the phenotypic plasticity of the non-calcifying symbiotic cnidarian Anemonia viridis at multiple levels of structural complexity, in vivo and in vitro. In vivo, we determined the mechanisms behind the phenotypic plasticity under expected future climate change (i.e. ocean acidification and ocean warming). Our results show physiological changes in the inorganic carbon use of the sea anemone A. viridis exposed to high pCO2 during a long-term stress in natura or a short-term stress in controlled conditions. We then observed an equivalent decrease in carbonic anhydrase activity, a key enzyme of cnidarian carbon concentrating mechanisms. Also, we demonstrated that an increase in seawater temperature modified the response observed during a high pCO2 scenario. In vitro, we established a viable primary cell culture from regenerating tentacles of A. viridis. We determined the gastrodermal tissue origin of the cultivated cells and validated the use of this new tool to the in vitro study of stress response at the cellular level. The set-up of this powerful in vitro tool will open a multitude of perspectives for the study of cellular responses to exogeneous stress (as global change perturbations) and to endogeneous stress (as the symbiosis constraints experienced by symbiotic cnidarians)
Durant leur cycle de vie, les organismes sont exposés à des variations environnementales capables d'induire des changements physiologiques, morphologiques et comportementaux, résultant d’une plasticité phénotypique. La plasticité phénotypique est la capacité d'un génotype à générer un nouveau phénotype suite à un stress. Ici, nous avons étudié la plasticité phénotypique d’un Cnidaire symbiotique et non-calcifiant, l’anémone de mer Anemonia viridis, à de multiples niveaux de complexité structurale, in vivo et in vitro. In vivo, nous avons identifié les mécanismes sous-jacents de la plasticité phénotypique potentiellement induits par les futurs changements climatiques (acidification et réchauffement des océans). Nos résultats montrent des modifications dans l'utilisation du carbone inorganique par A. viridis exposée à une forte pCO2 lors d’un stress chronique in natura ou lors d’un stress court en conditions contrôlées. Nous avons ainsi observé une diminution des activités anhydrase carbonique, une enzyme clé des mécanismes de concentration du carbone chez les Cnidaires. Nous avons aussi démontré que l'augmentation concomittante de la température modifie la réponse observée lors d'une élévation seule de la pCO2. In vitro, nous avons établi une culture de cellules primaires viables issue de tentacules d’A. viridis en régénération. Nous avons déterminé l'origine gastrodermale des cellules cultivées et validé l'utilisation de ce nouvel outil pour l'étude de la réponse au stress au niveau cellulaire. Ce nouvel outil ouvre une multitude de perspectives pour l'étude des réponses cellulaires aux stress exogènes (changement climatique) et endogènes (contraintes dues à la symbiose)
https://tel.archives-ouvertes.fr/tel-01674220

Droits : info:eu-repo/semantics/OpenAccess
NNT : 2016AZUR4136
tel-01674220
https://tel.archives-ouvertes.fr/tel-01674220
https://tel.archives-ouvertes.fr/tel-01674220/document
https://tel.archives-ouvertes.fr/tel-01674220/file/2016AZUR4136.pdf

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Ethnopharmacological relevancePsidium acutangulum Mart. ex DC is a small tree used by the Wayana Amerindians from the Upper-Maroni in French Guiana for the treatment of malaria.Aim of the studyIn a previous study, we highlighted the in vitro antiplasmodial, antioxidant and anti-inflammatory potential of the traditional decoction of P. acutangulum aerial parts. Our goal was then to investigate on the origin of the biological activity of the traditional remedy, and eventually characterize active constituents.Materials and methodsLiquid-liquid extractions were performed on the decoction, and the antiplasmodial activity evaluated against chloroquine-resistant FcB1 ([3H]-hypoxanthine bioassay) and 7G8 (pLDH bioassay) P. falciparum strains, and on a chloroquine sensitive NF54 ([3H]-hypoxanthine bioassay) P. falciparum strain. The ethyl acetate fraction (D) was active and underwent bioguided fractionation. All the isolated compounds were tested on P. falciparum FcB1 strain. In vitro anti-inflammatory activity (IL-1β, IL-6, IL-8, TNFα) of the ethyl acetate fraction and of an anti-Plasmodium active compound, was concurrently assessed on LPS-stimulated human PBMC and NO secretion inhibition was measured on LPS stimulated RAW murine macrophages. Cytotoxicity of the fractions and pure compounds was measured on VERO cells, L6 mammalian cells, PBMCs, and RAW cells.ResultsFractionation of the ethyl acetate soluble fraction (IC50 ranging from 3.4 to <1 µg/mL depending on the parasite strain) led to the isolation of six pure compounds: catechin and five glycosylated quercetin derivatives. These compounds have never been isolated from this plant species. Two of these compounds (wayanin and guaijaverin) were found to be moderately active against P. falciparum FcB1 in vitro (IC50 5.5 and 6.9 µM respectively). We proposed the name wayanin during public meetings organized in June 2015 in the Upper-Maroni villages, in homage to the medicinal knowledge of the Wayana population. At 50 µg/mL, the ethyl acetate fraction (D) significantly inhibited IL-1β secretion (−46%) and NO production (−21%), as previously observed for the decoction. The effects of D and guiajaverin (4) on the secretion of other cytokines or NO production were not significant.ConclusionsThe confirmed antiplasmodial activity of the ethyl acetate soluble fraction of the decoction and of the isolated compounds support the previous results obtained on the P. acutangulum decoction. The antiplasmodial activity might be due to a mixture of moderately active non-toxic flavonoids. The anti-inflammatory activities were less marked for ethyl acetate fraction (D) than for the decoction.
ISSN: 0378-8741

hal-01313006
http://hal.upmc.fr/hal-01313006
http://hal.upmc.fr/hal-01313006/document
http://hal.upmc.fr/hal-01313006/file/Houel_Wayanin_and.pdf
DOI : 10.1016/j.jep.2016.04.053

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Predictive toxicology aims at developing methodologies to relate the results obtained from in vitro experiments to in vivo exposure. In the case of polycyclic aromatic hydrocarbons (PAHs), a substantial amount of knowledge on effects and modes of action has been recently obtained from in vitro studies of gene expression. In the current study, we built a physiologically based toxicokinetic (PBTK) model to relate in vivo and in vitro gene expression in case of exposure to benzo(a)pyrene (BaP), a referent PAH. This model was calibrated with two toxicokinetic datasets obtained on rats exposed either through intratracheal instillation or through intravenous administration and on an in vitro degradation study. A good agreement was obtained between the model's predictions and the concentrations measured in target organs, such as liver and lungs. Our model was able to relate correctly the gene expression for two genes targeted by PAHs, measured in vitro on primary human macrophages and in vivo in rat macrophages after exposure to BaP. Combining in vitro studies and PBTK modeling is promising for PAH risk assessment, especially for mixtures which are more efficiently studied in vitro than in vivo.
Toxicology Letters

hal-00873317
https://hal.archives-ouvertes.fr/hal-00873317
https://hal.archives-ouvertes.fr/hal-00873317/document
DOI : 10.1016/j.toxlet.2010.11.017
PUBMED : 21134426

Éditeur(s) : HAL CCSD
Résumé : Benzophenone derivatives (BPs) are used as UV filters in many different products. These lipophilic compounds were recently described as ubiquitous contaminant of different compartiments of aquatic systems, including fish. In this study, we evaluated estrogenic activity of ten BPs in a model fish species, the zebrafish (Danio rerio), by combining newly developed in vitro and in vivo models. In vitro, three specific zebrafish reporter gene models expressing zebrafish estrogen receptors (ZfERs) in zebrafish hepatic cell line (ZFL) were used.Estrogenic activity of BPs was quantify in vivo by using a newly developed transgenic zebrafish line that expresses green fluorescent protein (GFP) under the control of the cyp19a1b promoter. The cyp19a1b gene codes for the brain aromatase B that is under the control of estrogens. Vitellogenin (Vtg), a marker of estrogenic compound, was used to assess the potential estrogenic effect of certain BPs in male fish. Five of 10 BPs induced an estrogenic activity on the three models: BP2, THB, BP1, 4BP, while BP and 234 BP were non active. We noted diffrence in BPs ranking depending on the zfER subtypes suggesting ?/? receptor selectivity by certain BPs. By quantifying GFP expression in live zebrafish, we confirm the estrogenic activity of some BP, i.e., BP1, THB, 4BP and 44'BP, as shown by their ability to induce GFP in a concentration-dependent manner. However, the ranking of BPs as a function of their estrogenic activity assay is different between models. For instance, 4BP was the most estrogenic compound in vivo but not in vitro. Differences were also noticed regarding the estrogenic activity of BP2. In vitro, BP2 has strong activity but had no effect on GFP in cyp19a1b-GFP embryo. However, BP2 induced strongly Vtg in male. The lack of estrogenic activity of BP2 in embryo may be related to difference of biodisponibility and/or metabolisation of this compound between embryo and adult fish. Our approach allows to characterize the estrogenic potency of emerging pollutants, using innovative in vitro and in vivo assays within a unique model fish species. The differences observed between assays deserve further studies to characterize the behavior of compounds in the different biological models in term of biodisponibility and metabolism.
1. European Conference on the Replacement, Reduction and Refinement of Animal Experiments in Ecotoxicology
Dübendorf, Switzerland

ineris-00971019
https://hal-ineris.archives-ouvertes.fr/ineris-00971019
INERIS : EN-2012-243

Éditeur(s) : HAL CCSD BioMed Central
Résumé : International audience
BACKGROUND: Estrogen receptors (ER) α and β are found in both women and men in many tissues, where they have different functions, including having roles in cell proliferation and differentiation of the reproductive tract. In addition to estradiol (E2), a natural hormone, numerous compounds are able to bind ERs and modulate their activities. Among these compounds, phytoestrogens such as isoflavones, which are found in plants, are promising therapeutics for several pathologies. Glyceollins are second metabolites of isoflavones that are mainly produced in soybean in response to an elicitor. They have potentially therapeutic actions in breast cancer by reducing the proliferation of cancer cells. However, the molecular mechanisms driving these effects remain elusive. METHODS: First, to determine the proliferative or anti-proliferative effects of glyceollins, in vivo and in vitro approaches were used. The length of epithelial duct in mammary gland as well as uterotrophy after treatment by E2 and glyceollins and their effect on proliferation of different breast cell line were assessed. Secondly, the ability of glyceollin to activate ER was assessed by luciferase assay. Finally, to unravel molecular mechanisms involved by glyceollins, transcriptomic analysis was performed on MCF-7 breast cancer cells. RESULTS: In this study, we show that synthetic versions of glyceollin I and II exert anti-proliferative effects in vivo in mouse mammary glands and in vitro in different ER-positive and ER-negative breast cell lines. Using transcriptomic analysis, we produce for the first time an integrated view of gene regulation in response to glyceollins and reveal that these phytochemicals act through at least two major pathways. One pathway involving FOXM1 and ERα is directly linked to proliferation. The other involves the HIF family and reveals that stress is a potential factor in the anti-proliferative effects of glyceollins due to its role in increasing the expression of REDD1, an mTORC1 inhibitor. CONCLUSION: Overall, our study clearly shows that glyceollins exert anti-proliferative effects by reducing the expression of genes encoding cell cycle and mitosis-associated factors and biomarkers overexpressed in cancers and by increasing the expression of growth arrest-related genes. These results reinforce the therapeutic potential of glyceollins for breast cancer.
ISSN: 1478-811X

hal-01560288
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01560288
DOI : 10.1186/s12964-017-0182-1
PUBMED : 28666461

Éditeur(s) : Université de La Rochelle, Ecole doctorale Gay Lussac
Résumé : The thesis work is part of the very current issue on mass mortality outbreaks affecting Pacific oyster, C. gigas, spat and juveniles and questions about the involvement of pesticides in this phenomenon. The first part of this thesis was devoted to study the effects of pesticides on hemocyte parameters in the Pacific oyster (in vitro and in vivo). The immunomodulator effect of selected pesticides (alone or in mixture) has been explored principally through flow cytometry. The second part concerned the study of the effects of a pesticides mixture on OsHV-1 itself. No direct effects have been reported on the viral particles in presence of the pollutants. Moreover, pesticide effects on Pacific oysters were also explored through experimental pathology assays after treatment of animals with a polluant mixture. Results showed that pesticide treated oysters appeared more susceptible to the viral infection in experimental conditions. The third part concerned the study of autophagy in the Pacific oyster, C. gigas. The publication of the complete genome in 2012 has opened up new possibilities for the study of innate immunity in this species. The study of autophagy for the first time in oysters consisted in a first step in the in silico search for genes involved in this pathway and the corresponding proteins by Western blotting. Then, the role of this important process in innate immunity has been explored through reproduction infections tests with or without modulators of autophagy. Results showed that autophagy appeared as involved in defence mechanisms against viral and bacterial infection in Pacific oysters.
Le travail de thèse s’inscrit dans la problématique très actuelle des mortalités massives de naissain et de juvéniles d’huîtres creuses, C. gigas, et des questionnements autour de l’implication des pesticides dans ce phénomène. La première partie de la thèse a été consacrée à l’étude des effets de pesticides sur les capacités hémocytaires de l’huître creuse (in vitro et in vivo). L’effet immunomodulateur des xénobiotiques sélectionnés (seul ou en mélange) a été exploré principalement au travers de la cytométrie en flux. La deuxième partie de la thèse a concerné l’étude des effets d’un mélange de pesticides sélectionnés sur le virus OsHV-1 et l’infection qu’il induit chez l’huître creuse. Les effets des pesticides sur le virus lui-même ont été évalués. Les expériences réalisées non pas permis de mettre en évidence dans les conditions testées, de dégradation des particules virales en présence des polluants. D’autre part, leurs effets sur les huîtres elles-mêmes ont été explorés après traitement des animaux avec un mélange de pesticides lors d’essais de pathologie expérimentale. Il a ainsi pu être montré que des huîtres préalablement comtaminées par un mélange de pesticides à des concentrations retrouvées dans l’environnement semblaient être plus sensibles à l’infection virale. La troisième partie de la thèse a concerné l’étude de l’autophagie chez l’huître creuse, C. gigas. La publication du génome complet de cette espèce en 2012 a ouvert de nouvelles possibilités pour étudier l'immunité innée. L’étude de l’autophagie réalisée pour la première fois chez l’huître creuse a consisté lors d’une première étape en la recherche in silico de gènes impliqués dans cette voie et des protéines correspondantes par western blotting. Puis, le rôle de ce processus important dans l’immunité innée a été exploré au travers d’essais de reproduction d’infections en présence ou non de modulateurs de l’autophagie. Les résultats obtenus semblent montrer que l’autophagie soit un processus important pouvant être impliqué dans les capacités de défense de l’huître creuse vis-à-vis d’infections virales et bactériennes.

Droits : 2014 Université de La Rochelle
http://archimer.ifremer.fr/doc/00226/33740/32149.pdf
http://archimer.ifremer.fr/doc/00226/33740/

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Sexually transmitted viruses infect the genital and colorectal mucosa of the partner exposed to contaminated genital secretions through a wide range of mechanisms, dictated in part by the organization of the mucosa. Because understanding the modes of entry into the organism of viruses transmitted through sexual intercourse is a necessary prerequisite to the design of treatments to block those infections, in vitro modeling of the transmission is essential. The aim of this review is to present the models and methodologies available for the in vitro study of the interactions between viruses and mucosal tissue and for the preclinical evaluation of antiviral compounds, and to point out their advantages and limitations according to the question being studied.
ISSN: 0042-6822

Droits : http://creativecommons.org/licenses/by/
hal-01663650
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01663650
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01663650/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01663650/file/Frouard%20-%20In%20vitro%20models%20for%20deciphering.pdf
DOI : 10.1016/j.virol.2017.11.023

Éditeur(s) : HAL CCSD
Résumé : International audience
Primary cultures of hepatocytes are powerful models in studying the sequence of events that are necessary for cell progression from a G0-like state to S phase. The models mimic the physiological process of hepatic regeneration after liver injury or partial hepatectomy. Many reports suggest that the mitogen-activated protein kinase (MAPK) ERK1/2 can support hepatocyte proliferation in vitro and in vivo and the MEK/ERK cascade acts as an essential element in hepatocyte responses induced by the EGF. Moreover, its disregulation has been associated with the promotion of tumor cell growth of a variety of tumors, including hepatocellular carcinoma. Whereas the strict specificity of action of ERK1 and ERK2 is still debated, the MAPKs may have specific biological functions under certain contexts and according to the differentiation status of the cells, notably hepatocytes. In this paper, we will focus on MEK1/2-ERK1/2 activations and roles in normal rodent hepatocytes in vitro and in vivo after partial hepatectomy and in human hepatocarcinoma cells. The possible specificity of ERK1 and ERK2 in normal and transformed hepatocyte will be discussed in regard to other differentiated and undifferentiated cellular models.
International Journal of Hepatology

Droits : info:eu-repo/semantics/OpenAccess
hal-00875664
https://hal.archives-ouvertes.fr/hal-00875664
https://hal.archives-ouvertes.fr/hal-00875664/document
https://hal.archives-ouvertes.fr/hal-00875664/file/TheMAPKMEK12-accepted.pdf
DOI : 10.1155/2012/328372

Éditeur(s) : HAL CCSD
Résumé : International audience
The present study was conducted to assess the effects of Cd exposure on estrogen signaling in the zebrafish brain, as well as the potential protective role of Zn against Cd-induced toxicity. For this purpose, the effects on transcriptional activation of the estrogen receptors (ERs), aromatase B (Aro-B) protein expression and molecular expression of related genes were examined in vivo using wild-type and transgenic zebrafish embryos. For in vitro studies, an ER-negative glial cell line (U251MG) transfected with different zebrafish ER subtypes (ERα, ERβ1 and ERβ2) was also used. Embryos were exposed either to estradiol (E2), Cd, E2+Cd or E2+Cd+Zn for 72 h and cells were exposed to the same treatments for 30 h. Our results show that E2 treatment promoted the transcriptional activation of ERs and increased Aro-B expression, at both the protein and mRNA levels. Although exposure to Cd, does not affect the studied parameters when administered alone, it significantly abolished the E2-stimulated transcriptional response of the reporter gene for the three ER subtypes in U251-MG cells, and clearly inhibited the E2 induction of Aro-B in radial glial cells of zebrafish embryos. These inhibitory effects were accompanied by a significant downregulation of the expression of esr1, esr2a, esr2b and cyp19a1b genes compared to the E2-treated group used as a positive control. Zn administration during simultaneous exposure to E2 and Cd strongly stimulated zebrafish ERs transactivation and increased Aro-B protein expression, whereas mRNA levels of the three ERs as well as the cyp19a1b remained unchanged in comparison with Cd-treated embryos. In conclusion, our results clearly demonstrate that Cd acts as a potent anti-estrogen in vivo and in vitro, and that Cd-induced E2 antagonism can be reversed, at the protein level, by Zn supplement.
Journal of Applied Toxicology

Droits : info:eu-repo/semantics/OpenAccess
hal-01274217
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01274217
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01274217/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01274217/file/Inhibitory%20effect%20of%20Cadmium%20on%20estrogen_accepted.pdf
DOI : 10.1002/jat.3285

Éditeur(s) : HAL CCSD
Résumé : Inorganic arsenic is an environmental human carcinogen, but is also studied these days because of its potential effectiveness in curing chronic inflammatory disease. Indeed, this metalloid possesses immunosuppressive properties which can dysregulate physiological mechanisms involved in immune defense, or reduce inflammation associated with those inflammatory diseases. Inorganic arsenic is known mainly to alter functions of T cells and macrophages. However, it is unknown whether arsenic targets dendritic cells (DCs). Yet, this antigen presenting cells plays a major role in the immunosurveillance, and is involved in the physiopathology of chronic inflammatory diseases. So, the aim of my thesis work was to study the effects of inorganic arsenic on in vitro differenciation and maturation of dendritic cells from human monocytes. Our results mainly shows that concentrations corresponding to those measured in environmentally exposed people, inhibits DCs secretion of proinflammatory cytokines, which plays a major role in activation and polarization of T cells. Particularly, arsenic strongly impairs expression and secretion of interleukine 12 (IL-12) by an underlying molecular mechanism involving Nrf2. Finally, this work shows that inorganic arsenic has immunosuppressive properties on the physiology in vitro of human dendritic cells. Immunotoxicity may then contribute to the metalloid toxicity in environmentally exposed people. This element could be taken into account when determining arsenic effects in curing some chronic inflammatory diseases.
L’arsenic inorganique est un contaminant environnemental, cancérogène pour l’homme, mais également un métalloïde étudié, aujourd’hui, dans le traitement de maladies inflammatoires chroniques. Il possède des propriétés immunosuppressives pouvant déréguler les mécanismes physiologiques de défense ou bloquer l’exacerbation de réponses inflammatoires chroniques. L’arsenic inorganique altère principalement les fonctions des lymphocytes T et des macrophages. En revanche, l’impact du métalloïde sur la physiologie des cellules dendritiques (DCs) est peu connu. Pourtant, ces cellules présentatrices d’antigène jouent un rôle fondamental dans les processus d’immunosurveillance et sont très impliquées dans la physiopathologie des maladies inflammatoires chroniques. Dans ce contexte, les objectifs de mon travail de thèse étaient d’étudier les effets de l’arsenic inorganique sur la différenciation et la maturation in vitro de DCs générées à partir de monocytes humains. Nos résultats démontrent principalement que des concentrations de métalloïde, compatibles avec les taux plasmatiques d’arsenic mesurés chez les individus exposés, répriment la capacité des DCs à sécréter différentes cytokines pro-inflammatoires jouant un rôle essentiel dans l’activation et la polarisation des lymphocytes T. En particulier, l’arsenic inhibe l’expression et la sécrétion de l’interleukine-12 par un mécanisme moléculaire impliquant le facteur de transcription Nrf2. Au total, ces travaux de thèse démontrent que l’arsenic inorganique possède des propriétés immunosuppressives sur la physiologie in vitro des DCs humaines. Cette immunotoxicité pourrait contribuer à la toxicité du métalloïde chez l’homme exposé par voie environnementale, et être prise en compte pour déterminer les effets de l’arsenic dans le traitement de certaines maladies inflammatoires chroniques
https://tel.archives-ouvertes.fr/tel-01135887

NNT : 2013REN1B013
tel-01135887
https://tel.archives-ouvertes.fr/tel-01135887
https://tel.archives-ouvertes.fr/tel-01135887/document
https://tel.archives-ouvertes.fr/tel-01135887/file/pdf2star-1392896105-Th--se-M--linda-Macoch2.pdf

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
High-mobility group box-1 (HMGB1) is remarkably mobile in living cells, which reflects its ability to interact only transiently with both DNA and protein. This property is likely essential for HMGB1 nuclear activities. Nonetheless the weak interaction of HMGB1 with DNA and/or protein partners has also been a major limitation for investigating HMGB1 subnuclear localisation and for the identification of HMGB1 containing complexes by conventional biochemical approaches. In the present study, FRAP experiments demonstrated that DsRed-mediated oligomerization strongly reduces HMGB1 mobility due to an increased affinity for cellular chromatin. Moreover, oligomerized DsRed HMGB1 exhibited a higher affinity for supercoiled DNA in vitro compared to its monomeric counterpart. These results indicate that DsRed-meditated oligomerization is prone to stabilize labile interactions involving HMGB1 both in vivo and in vitro.
ISSN: 0300-9084

hal-01546170
https://hal.archives-ouvertes.fr/hal-01546170
DOI : 10.1016/j.biochi.2012.11.001

Éditeur(s) : HAL CCSD Public Library of Science
Résumé : International audience
Few studies have been undertaken to assess the possible effects of bisphenol A (BPA) on the reproductive hormone balance in animals or humans with often contradictory results. We investigated possible direct endocrine disruption by BPA of the fetal testes of 2 rat strains (14.5-17.5 days post-coitum) and humans (8-12 gestational weeks) and under different culture conditions. BPA concentrations of 10-8M and 10-5M for 72h reduced testosterone production by the Sprague-Dawley fetal rat testes, while only 10-5M suppressed it in the Wistar strain. The suppressive effects at 10-5M were seen as early as 24h and 48h in both strains. BPA at 10-7-10-5M for 72h suppressed the levels of fetal rat Leydig cell insulin-like factor 3 (INSL3). BPA exposure at 10-8M, 10-7M, and 10-5M for 72h inhibited testosterone production in fetal human testes. For the lowest doses, the effects observed occurred only when no gonadotrophin was added to the culture media and were associated with a poorly preserved testicular morphology. We concluded that (i) BPA can display anti-androgenic effects both in rat and human fetal testes; (ii) it is essential to ascertain that the divergent effects of endocrine disruptors between species in vitro do not result from the culture conditions used, and/or the rodent strain selected; (iii) the optimization of each in vitro assay for a given species should be a major objective rather than the search of an hypothetical trans-species consensual model-system, as the organization of the testis is intrinsically different between mammalian species; (iv) due to the uncertainty existing on the internal exposure of the human fetal testis to BPA, and the insufficient number of epidemiological studies on the endocrine disruptive effects of BPA, caution should be taken in the extrapolation of our present results to the human reproductive health after fetal exposure to BPA.
ISSN: 1932-6203

hal-01121759
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01121759
PUBMED : 25706302
DOI : 10.1371/journal.pone.0117226

Éditeur(s) : Elsevier Science Bv
Résumé : In New Caledonia, shrimp hatcheries are confronted with mass mortality in the larval stages, a phenomenon poorly understood as no specific causative agent has been identified. This has resulted in an excessive use of prophylactic antibiotics, although their adverse effects in aquaculture are notorious. The present work was thus aimed at selecting potential probiotic strains for penaeid hatcheries. From a pool of more than 400 marine bacterial isolates sampled from the local marine environment seven strains exhibited in vitro antagonistic activity towards Vibrio harveyi. These isolates were characterized both phenotypically and genotypically using a biochemical approach and 16S rDNA sequencing. Six out of these seven strains were found to belong to the genus Pseudoalteromonas, the last one belonging to the Vibrionaceae family and related to the Harveyi clade. Selected probiotic candidates were individually tested for antagonistic activities in vitro using a green fluorescent protein (GFP)-labelled transconjugant of V. harveyi and for inherent pathogenicity towards cultured shrimp larvae at two different developmental stages. This approach enabled rapid processing and selection of candidates to be tested in our experimental hatchery. Four different experiments were conducted to test candidate strains either alone or in combination. Repeated trials showed that postlarval survival was significantly improved by adding the strain NC201 individually to the rearing water compared to unchallenged controls. The analysis of immune-related gene expressions showed that the Litsty PEN3 transcript abundance of larvae was significantly increased after being reared in probiotic-containing water; however no significant difference in lysozyme gene expression was recorded in this study. Together, these results open new insights into the use of these strains as potential substitutes to antibiotherapy in shrimp larval rearing in New Caledonia.
Aquaculture (0044-8486) (Elsevier Science Bv), 2014-08 , Vol. 432 , P. 475-482

Droits : 2014 Elsevier B.V. All rights reserved.
http://archimer.ifremer.fr/doc/00190/30129/28591.pdf
DOI:10.1016/j.aquaculture.2014.04.031
http://archimer.ifremer.fr/doc/00190/30129/

Éditeur(s) : HAL CCSD
Résumé : Croton urucurana Baillon (Euphorbiaceae), plant species popularly known in Brazil as “sangra-d’água”, is commonly found in Mato Grosso do Sul State (midwestern Brazil), where its latex is popularly used to treat several types of cancer. Despite the ethnopharmacological significance of the species, no phytochemical or cytotoxic studies of its latex have been published. In this context, the aim of the present study was to assess the cytotoxic activity of crude extracts, fractions and isolated compound from the latex of C. urucurana and to investigate the effects of crourorb A1 on the viability, apoptosis, cell cycle and cell migration of human hepatocarcinoma cell lines (Huh-7). Also, we have evaluated the viability of Huh-7 cells treated with crourorb A1 in 3D collagen gels. The cytotoxicity was detected using the sulforhodamine B (SRB) assay against six human cancer cells lines and one nontumor NIH/3T3 cell line (murine fibroblast). Studies on the mechanism of cell death of crourorb A1 were conducted in different test systems in vitro. The bioactive ethyl acetate phase obtained from the latex of C. urucurana afforded a novel cyclic peptide, [1−9-NαC]-crourorb A1, that proved active against NCIADR/ RES (ovary, multidrug-resistance phenotype) cells with the same potency as doxorubicin (positive control) and inactive up to the highest concentration tested against nontumor NIH/3T3 cells (GI50 = > 250 μg/mL). Crourorb A1 treatment decreases the cell viability of Huh-7 at dose and time dependent manner associated with induction of apoptosis by caspase 3/7 activation and increasing expression of pro-apoptotic proteins: Bak, Bid, Bax, Puma, Bim, and Bad and clived-caspase3 protein. The JNK/MAP pathway is involved in this induced cell death. Crourorb A1 effect is also associated with G2/M phase cell cycle arrest. In parallel, we observed that crourorb A1 treatment resulted in an increase of cyclin-dependent kinase (CDK1), cyclin B1 and cyclin D1 expressions. A significant reduction of Huh-7 cells migration by crourorb A1 can be assessed and we also show that cells grown on 3D collagen gels exhibited increased resistance to crourorb A1 when compared to 2D cell culture.
Croton urucurana Baillon (Euphorbiaceae), est une plante très connue et utilisée en médecine traditionnelle au Brésil, la «sangra-d’água» retrouvée dans l'État du Mato Grosso do Sul (région Centre-Ouest du Brésil). Son latex est abondamment utilisé en médecine traditionnelle dans le traitement de plusieurs types de cancer. En dépit de son importance ethnopharmacologique, on ne trouve aucune étude sur sa composition chimique ou sur sa potentielle activité anti-cancéreuse. L’objectif de ce travail a été d'évaluer l'activité cytotoxique des extraits totaux, fractions d’extraits et de molécules purifiées, isolées du latex de C. urucurana. Nous avons étudié plus particulièrement les effets du crourorb A1 sur la viabilité, l'apoptose, le cycle cellulaire et la migration de cellules issues de carcinome hépatocellulaire humaine (Huh-7). Nous avons également évalué l’effet du crourorb A1sur la viabilité des cellules Huh-7 cultivées dans les gels de collagène en 3D. L' activité cytotoxique ont été réalisées par le test à la sulforhodamine B sur six lignées cellulaire cancéreuses et sur une lignée cellulaire non cancéreuse de fibroblastes murins, les cellules NIH/3T3. L'étude phytochimique de la fraction ‘acétate d'éthyle’ du latex de C. urucurana bioguidé a permis d’obtenir un nouveau cyclopeptide que nous avons nommé [1-9-NαC]-crourorb A1. Le crourorb présente une cytotoxicité puissante sur la lignée cellulaire cancéreuse NCI-ADR/RES (ovaire, le phénotype de multirésistance) à un niveau équivalent à celle de la doxorubicine (témoin positif). Ce composé est de plus pratiquement inactif (GI50 = >250 μg/mL) sur la lignée non cancéreuse NIH/3T3. L'évaluation de la cytotoxicité in vitro du crourorb A1 (test wst1) sur les cellules Huh-7 a démontré que la viabilité des cellules est inhibée de manière dose et temps-dépendant. Nous avons mesuré une forte activation des caspases 3 et 7 et une nette augmentation de l’expression des protéines pro-apoptotiques (Bak, Bid, Bax, Puma, Bim et Bad) ainsi que de la caspase 3 clivée par le crourorb A1. Nous avons également déterminé que la voie de signalisation JNK-MAPkinase semble nécessaire à l’induction de la mort cellulaire des cellules Huh-7 traitées au crourorb A1. Le crourob A1 induit un arrêt du cycle cellulaire en phase G2/M. En parallèle, les expressions de la cycline-dépendante kinase CDK1, des cyclines B1 et D1 sont augmentées après traitement au crourorb A1. De plus, le crourorb A1 a un effet inhibiteur significatif de la migration des cellules Huh-7, en présence ou absence de mitomycine C. Nous avons également confirmé, l’effet inhibiteur du crourob A1 sur la viabilité des cellules Huh7 cultivées en 3D dans les gels de collagène 1 et démontré que les cellules cultivées en 3D semblent plus résistantes au crourorb A1 par rapport aux cultures en monocouche 2D.
Croton urucurana Baillon (Euphorbiaceae) é uma espécie arbórea conhecida popularmente no Brasil como “sangra d´água”, comumente encontrada no estado de Mato Grosso do Sul, onde seu látex tem sido utilizado na medicina popular no tratamento de vários tipos de câncer. Apesar de sua importância etnofarmacológica, não há relatos de estudos relacionados à sua composição química ou atividades anticâncer na literatura. Nesse contexto, o objetivo do presente estudo foi avaliar a atividade citotóxica de extratos, fases, frações e da substância isolada do látex de C. ururucurana e investigar os mecanismos celulares e moleculares in vitro responsáveis pela citotoxicidade da substância isolada considerando seu potencial apoptótico e influência sobre a progressão do ciclo celular em uma linhagem de hepatocarcinoma celular humano (Huh-7), bem como, os efeitos desta substância sobre a migração e viabilidade de células Huh-7 cultivadas em geis de colágeno em 3D. O ensaio de citotoxicidade in vitro foi realizado com o corante sulforrodamina B (SRB) em seis linhagens de células neoplásicas humanas e uma linhagem de células não neoplásicas NIH/3T3 (fibloblasto murinho). Os estudos de mecanismos de ação com crourorb A1 foram conduzidos em diferentes sistemas-teste in vitro. O estudo fitoquímico biomonitorado da fase bioativa acetato de etila do látex de C. urucurana resultou no isolamento de um novo ciclopeptídeo denominado [19-NαC]-crourorb A1, que apresentou potente atividade citotóxica frente à linhagem de células neoplásicas NCI-ADR/RES (ovário com fenótipo de resistência a múltiplos fármacos), com a mesma potência que a doxorrubicina (controle positivo), e foi inativa até a maior concentração testada (GI50 = >250 μg/mL) frente à linhagem de células não neoplásicas 3T3. A exposição de células Huh-7 ao crourorb A1 resultou em uma diminuição da viabilidade celular de maneira dose e tempo-dependentes. Nós observamos um aumento da atividade de caspases 3 e 7 e um aumento da expressão de proteínas próapoptóticas (Bak, Bid, Bax, Puma, Bim e Bad) e caspase 3 clivada após tratamento com crourorb A1. Além disso, observamos que a via de sinalização celular MAP/JNK foi necessária para induzir a morte celular em células Huh-7 tratadas com crourorb A1. Os efeitos de crourorb A1 foram também associados com o acúmulo de células na fase G2/M do ciclo celular. Em paralelo, as expressões da cinase dependente da ciclina (CDK1), ciclina B1 e ciclina D1 foram aumentadas após tratamento com crourorb A1. Foi demonstrado também o envolvimento de crourorb A1 na redução da migração de células Huh-7 cultivadas na presença e ausência de mitomicina C. A avaliação da viabilidade de células Huh-7 cultivadas em geis de colágeno em 3D apontou uma maior resistência destas células ao crourorb A1 quando comparadas com a cultura de células em 2D.
https://tel.archives-ouvertes.fr/tel-01478286

NNT : 2016REN1B010
tel-01478286
https://tel.archives-ouvertes.fr/tel-01478286
https://tel.archives-ouvertes.fr/tel-01478286/document
https://tel.archives-ouvertes.fr/tel-01478286/file/DE-MATOS-CANDIDO-BACANI-Priscila.pdf

Éditeur(s) : HAL CCSD Springer Verlag
Résumé : International audience
Interleukin-27 (IL-27) belongs to the IL-6/IL-12 family of cytokines, associated with different inflammatory diseases and orchestrates its biological activity via common heterodimeric receptor composed of WSX-1 (IL-27Rα) and gp130. The present study was aimed to investigate the regulation of CXCL9, CXCL10, and CXCL11 chemokines in hepatic cells (human LX-2 cell line derived from normal human stellate cells (HSC), primary human hepatocytes, HSC, and HepG2 cells) and concanavalin A (ConA)-induced liver inflammation. We demonstrated that IL-27, but not IL-6, induced/up-regulated CXCR3 ligand genes (CXCL9, CXCL10, and CXCL11; out of 26 selected genes) in a STAT1-dependent manner in hepatic cells in vitro both at transcript and protein levels. In ConA-induced T cell-mediated hepatic model, we showed that soluble IL-27/IFNγ was elevated following ConA hepatitis in association with increased CXCL9, CXCL10, and CXCL11 expression in the liver. The exogenous IL-27 administration induced CXCR3 ligands in mouse liver at 4 h with any significant effect on recruitment of CXCR3(+) immune cells in the liver. The neutralization of IL-27 during ConA hepatitis differentially modulated (transcript vs protein expression) CXCR3 ligands and IFNγ during ConA-induced hepatitis with down-regulated expression of CXCL9 and CXCL10 at transcript level. The IFNγ, complementary regulated the expression of CXCR3 ligands as their up-regulation during ConA hepatitis, was abolished in IFNγ KO mice. In summary, IL-27 up-regulated the CXCL9, CXCL10, and CXCL11 chemokine expression in hepatic cells. IL-27 regulated CXCR3 ligand expression in IFNγ-dependent manner during acute hepatitis suggesting a complementary role of IL-27 and IFNγ to moderate liver inflammation via regulation of CXCR3 ligands. KEY MESSAGE: IL-27 up-regulated CXCR3 ligand expression in human hepatic cells in vitro. IL-27 up-regulated CXCR3 ligand expression and secretion in ConA hepatitis in vivo. CXCR3 ligand expression was down-regulated by blocking IL-27 or IFNγ deficiency. IL-27 modulated liver injury by regulation of CXCR3 ligands in IFNγ-dependent manner.
ISSN: 0946-2716

hal-01214006
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01214006
DOI : 10.1007/s00109-015-1319-6
PUBMED : 26199110

Éditeur(s) : Université de Montpellier II
Résumé : Penaeid family shrimp constitute the first aquaculture product in the world in terms of commercial value. They are produced in third world countries of the sub-equatorial belt. Among the causes limiting their production, one is the presence of the WSSV (White Spot Syndrome Virus), a pathogenic agent that produces massive mortality. Our aim was to investigate the first stages of the viral infection, in order to be used as target of prophylactic actions. A fish cell line (SSN-1) was used as model to tentatively develop in vitro studies. Only defective particles were produced confirming the high specificity to crustacea of the infection with crustacean virus. Electron microscopy showed structural similarities between the WSSV and B, B2 and Baculo-B viruses of crabs. This suggests B2 may belong also to the family Nimaviridae, genus Whispovirus. This comparison with B2 virus gives the possibility to understand the role played by the tail-like extension of these viruses in the infectious process by attachment to the plasmic membrane at the beginning of the infection in its specific host.
Les crevettes de grande taille de la famille des Penaeidae constituent le premier produit aquacole en valeur commerciale à l'échelle planétaire. Elles sont produites à 99% dans les pays en voie de développement de la ceinture sub-équatoriale. Parmi les causes limitant leur production, le White Spot Syndrome Virus est l'agent pathogène ayant provoqué le plus des pertes dans le monde. Notre étude a été orientée vers la reconnaissance des premiers stades de l'infection, susceptibles d'être la cible d'une action prophylactique. Nous avons testé l'utilisation d'une lignée cellulaire de poisson SSN-1 afin d'étudier les possibilités d'un développement in vitro. Seules des particules défectives ont été produites confirmant la haute spécificité des infections à virus de crustacés. Les recherches en microscopie électronique ont montré une similarité structurale du WSSV avec les virus (B, B2 et Baculo-B) des crabes. Ceci suggère que ces agents (B2 et WSSV) seraient tout deux de la famille des Nimaviridae et du genre Whispovirus. La comparaison avec le virus B2 permet de comprendre le rôle clef joué par la partie caudale de ces virus dans l'infection par son attachement à la membrane plasmique lors de l'infection chez son hôte spécifique.

Droits : info:eu-repo/semantics/openAccess
http://archimer.ifremer.fr/doc/2008/these-6650.pdf
http://archimer.ifremer.fr/doc/00000/6650/

Éditeur(s) : HAL CCSD
Résumé : Macrophages (MΦ), well-known to play a key role in immune response, also respond to environmental toxic chemicals such as diesel exhaust particles (DEP), an air pollutant recently classified as carcinogenic to humans. MΦ are heterogeneous and plastic cells which activate according to their microenvironment into either an M1 subtype (so called classically activated or pro-inflammatory) under IFNγ and LPS stimulation or an M2 subtype (so called alternatively activated or anti-inflammatory) under IL-4 and/or IL-13 stimulation. However, potential effects of DEPs on M1/M2 MΦ polarization remain poorly documented. First, we characterized the expression marker of in vitro M-CSF-differentiated MΦ from human monocytes and activated into the M1 or M2 subtypes. Our main results show that M-CSF-generated MΦ considered as anti-inflammatory are actually able to switch to an M1 phenotype after IFNγ/LPS stimulation. Furthermore, the markers identified in this study were used to assess the impact of organic extracts of DEP (DEPe) on MΦ polarization and more generally on their physiology. DEPe alter some M1 and M2 markers expressed by polarized MΦ, without causing the overall inhibition of the M1 and M2 polarization process or the switch to a different phenotype. This phenotype alteration is associated with a decrease in the LPS-dependent inflammatory response in M1 MΦ and the chemotactic capacities in M2 MΦ. DEPe decrease the secretion of some cytokines and chemokines such as IL-6, IL-12p40 and CCL18 via AhR and/or Nrf2 activation. At the same time, we show that M1 and M2 MΦ in response to DEPe are able to secrete a sufficient level of a pro-fibrotic growth factor, the platelet derived growth factor B (PDGF-B) via AhR activation, leading to stimulation of lung fibroblast proliferation. Finally, these works show that DEPe have immunotoxic properties with regards to the physiology of human in vitro polarized MΦ. This immunotoxicity may then contribute to the deleterious effects of these urban environmental contaminants on human health.
Les macrophages (MΦ), des cellules clefs de la réponse immunitaire peuvent répondre à des contaminants environnementaux comme les particules diesel (DEP), des polluants atmosphériques récemment classés cancérigènes pour l'Homme. Les MΦ sont des cellules hétérogènes et plastiques qui s'activent en fonction de leur microenvironnement soit en MΦ M1 (dits classiquement activés ou pro-inflammatoires) sous l'effet de l'INFγ et du LPS soit en MΦ M2 (dits alternativement activés ou réparateurs) sous l'effet de l'IL-4 et/ou de l'IL-13. Les effets des DEP sur la polarisation M1/M2 des MΦ restent peu documentés. Nous avons dans un premier temps caractérisé l'expression des marqueurs des MΦ différenciés in vitro en présence de M-CSF à partir de monocytes humains et polarisés en sous-type M1 ou M2. Nos principaux résultats montrent que les MΦ différenciés au M-CSF considérés comme des MΦ anti-inflammatoires, sont en réalité capables de s'activer vers un phénotype M1 après une stimulation au LPS/IFNγ. De plus, les marqueurs mis en évidence au cours de ce travail ont permis d'évaluer l'impact d'extraits organiques de DEP (DEPe) sur la polarisation des MΦ et plus généralement sur leur physiologie. Les DEPe altèrent l'expression de certains marqueurs M1 et M2 des MΦ, sans toutefois provoquer d'inhibition globale des processus de polarisation M1 et M2 ou de transition d'un phénotype vers un autre. Cette altération du phénotype est associée à une diminution de la réponse inflammatoire LPS-dépendante dans les MΦ M1 et des capacités chimiotactiques des MΦ M2. Les DEPe diminuent la sécrétion de certaines cytokines et chimiokines comme l'IL-6, l'IL-12p40 et le CCL18 via l'activation d'AhR et/ou de Nrf2. Parallèlement, nous montrons que les MΦ M1 et M2 exposés aux DEPe sécrètent le platelet deried growth factor B (PDGF-B), un facteur de croissance profibrosant, via l'activation d'AhR en quantité suffisante pour stimuler la prolifération de fibroblastes pulmonaires. Au total, ces travaux démontrent que les DEP possèdent des propriétés immunotoxiques vis-à-vis de la physiologie des macrophages humains polarisés in vitro. Cette immunotoxicité pourrait participer aux effets délétères de ces contaminants environnementaux urbains sur la santé humaine.
https://tel.archives-ouvertes.fr/tel-01188679

NNT : 2015REN1S011
tel-01188679
https://tel.archives-ouvertes.fr/tel-01188679
https://tel.archives-ouvertes.fr/tel-01188679/document
https://tel.archives-ouvertes.fr/tel-01188679/file/JAGUIN_Marie.pdf

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
From 15 to 17 June 2011, a dedicated workshop was held on the subject of in vitro models for mammalian spermatogenesis and their applications in toxicological hazard and risk assessment. The workshop was sponsored by the Dutch ASAT initiative (Assuring Safety without Animal Testing), which aims at promoting innovative approaches toward toxicological hazard and risk assessment on the basis of human and in vitro data, and replacement of animal studies. Participants addressed the state of the art regarding human and animal evidence for compound mediated testicular toxicity, reviewed existing alternative assay models, and brainstormed about future approaches, specifically considering tissue engineering. The workshop recognized the specific complexity of testicular function exemplified by dedicated cell types with distinct functionalities, as well as different cell compartments in terms of microenvironment and extracellular matrix components. This complexity hampers quick results in the realm of alternative models. Nevertheless, progress has been achieved in recent years, and innovative approaches in tissue engineering may open new avenues for mimicking testicular function in vitro. Although feasible, significant investment is deemed essential to be able to bring new ideas into practice in the laboratory. For the advancement of in vitro testicular toxicity testing, one of the most sensitive end points in regulatory reproductive toxicity testing, such an investment is highly desirable.
ISSN: 0890-6238

hal-00876979
https://hal.archives-ouvertes.fr/hal-00876979
DOI : 10.1016/j.reprotox.2013.04.004
PUBMED : 23612449