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[ES] La dieta es la principal vía de exposición a mercurio (Hg) para la mayoría de la población. Las principales formas de Hg en los alimentos son el metilmercurio (MeHg) y el Hg inorgánico divalente [Hg(II)]. Aunque el intestino es la principal puerta de entrada del tóxico en la circulación sistémica, los estudios sobre su toxicidad a nivel intestinal son escasos. El objetivo de la presente tesis ha sido evaluar la toxicidad y los mecanismos de acción del Hg(II) y el MeHg a nivel intestinal. Adicionalmente, se ha ensayado la eficacia de cepas de bacterias ácido- lácticas (BAL) como estrategia de reducción de esta toxicidad. Para los estudios in vitro se ha desarrollado un modelo celular en el que se han combinado enterocitos, células mucosecretoras y macrófagos en un sistema bicameral. Las células se han expuesto a Hg(II) y MeHg (0.1-1 mg/L) durante 10 días. Los datos han evidenciado que ambas especies generan un aumento de la respuesta inflamatoria, incrementando la liberación de la citoquina IL-8 e IL-1β, y una respuesta pro-oxidante, con un aumento de las especies reactivas de oxígeno/nitrógeno (ROS/RNS) y una sobreexpresión de proteínas de estrés (HSP70, HSP90 y MT2A). Esta respuesta ha sido más notable en macrófagos, indicando que posiblemente sean las células inmunitarias las que gobiernen la respuesta al tóxico. La situación de estrés va acompañada de una alteración de la expresión de la proteína de las uniones estrechas ZO-1 y una modificación de la morfología de la monocapa. Además, la exposición genera un aumento de la secreción de mucus y una sobreexpresión de su principal mucina, principalmente a MeHg. Los mecanismos de esta hipersecreción podrían estar relacionados con la ruta IL4/IL13/STAT6. Todos estos efectos sobre la monocapa epitelial conllevan un aumento de la permeabilidad y una reducción de la capacidad de regeneración. En los ensayos in vivo se han expuesto ratones BALB/c a Hg(II) y MeHg (1- 10 mg/L) durante 4 meses a través del agua de bebida. Los datos obtenidos han confirmado lo observado in vitro. La exposición a ambas especies (especialmente a 5 y 10 mg/L) induce un proceso inflamatorio en el colon, con un aumento de las citoquinas TNF-α e IL1-β y la presencia de infiltrados de neutrófilos. Paralelamente, la exposición genera estrés oxidativo con un incremento de las ROS/RNS y los peróxidos lipídicos. Se ha confirmado in vivo la participación en esta respuesta de algunas rutas apuntadas in vitro, en concreto p38 MAPK y JNK. Además, también se evidencian alteraciones en la expresión de proteínas de las uniones estrechas y de la mucina MUC2 en el colon, acompañada de una hiperplasia de las células mucosecretoras, especialmente en los tratamientos con MeHg. En estos animales se observa, además, un aumento de la expresión de IL13 e IL4, lo que apunta a la participación de la ruta IL4/IL13/STAT6, tal y como indicaban los ensayos in vitro. Además, estos ensayos in vivo muestran un efecto de ambas especies sobre el metabolismo de la microbiota intestinal con una reducción de los contenidos luminales de los ácidos grasos de cadena corta (SCFA), aunque los cambios detectados en la composición de la microbiota son mínimos. Finalmente, los animales tratados presentan un aumento de la permeabilidad. Todos los datos obtenidos in vitro e in vivo ponen de manifiesto que una exposición continuada a Hg(II) y MeHg genera una disrupción de la barrera intestinal. Los ensayos in vitro realizados para determinar la eficacia de las cepas de BAL de origen murino LE1 y LE2 como estrategia de protección se han realizado coexponiendo el modelo celular a Hg(II) o MeHg (1 mg/L) junto con las cepas durante 7 días. Los datos obtenidos han mostrado un efecto protector de ambas cepas, con una reducción de la respuesta inflamatoria, el estrés y los efectos sobre las uniones estrechas y la producción de mucus. Asimismo, la presencia de ambas bacterias restaura parcialmente la permeabilidad y la capacidad de regeneración de las monocapas. Teniendo en cuenta que en este estudio las BAL están inactivadas térmicamente, esta protección se asocia principalmente a su capacidad de quelación del Hg, aunque no hay que descartar que algún componente estructural de la pared bacteriana pueda ejercer otro tipo de efecto beneficioso. Los ensayos in vivo para confirmar el efecto protector de las BAL se han realizado en ratones BALB/c expuestos durante 2 meses a MeHg (5 mg/L) a través del agua de bebida. Las bacterias, en este caso viables, se han administrado por sonda gástrica diariamente. Los datos obtenidos muestran que, aunque las dos cepas reducen por igual los contenidos colónicos de MeHg, la reducción de los efectos tóxicos es mucho más notable con la cepa LE1. Se observan reducciones de los contenidos de mediadores de inflamación y estrés en el colon, hay una recuperación de la expresión de proteínas constituyentes de las uniones estrechas y del mucus. Se reestablecen los niveles luminales de los metabolitos de la microbiota y se restaura parcialmente la permeabilidad intestinal. Los resultados muestran que, además del proceso de quelación, la cepa LE1 activa la ruta de señalización Nrf2/Keap1/ARE y la producción de la citoquina anti-inflamatoria IL10. Los datos obtenidos in vitro e in vivo, apuntan a que la cepa LE1 podría ser una buena alternativa para reducir la toxicidad intestinal del Hg; reducción que puede tener también repercusiones beneficiosas a nivel sistémico. / [CA] La dieta és la principal via d'exposició a mercuri (Hg) per a la majoria de la població. Les principals formes de Hg en els aliments són el metilmercuri (MeHg) i el Hg inorgànic divalent [Hg(II)]. Encara que l'intestí és la principal porta d'entrada del tòxic a la circulació sistèmica, els estudis sobre la seua toxicitat a nivell intestinal són escassos. L'objectiu de la present tesi ha sigut avaluar la toxicitat i els mecanismes d'acció del Hg(II) i el MeHg a nivell intestinal. Addicionalment, s'ha assajat l'eficàcia de soques de bacteris àcid-làctics (BAL) com a estratègia de reducció d'aquesta toxicitat. Per als estudis in vitro s'ha desenvolupat un model cel·lular en el qual s'han combinat enteròcits, cèl·lules mucosecretores i macròfags en un sistema bicameral. Les cèl·lules s'han exposat des de l'inici a Hg(II) i MeHg (0.1-1 mg/L) durant 10 dies. Les dades han evidenciat que totes dues espècies generen un augment de la resposta inflamatòria, incrementant l'alliberament de la citocina IL- 8 i IL-1β, i una resposta pro-oxidant, amb un augment de les espècies reactives d'oxigen/nitrogen (ROS/RNS) i una sobreexpressió de proteïnes d’estrès (HSP70, HSP90 i MT2A). Aquesta resposta ha sigut més notable en macròfags, indicant que possiblement són les cèl·lules immunitàries les que governen la resposta al tòxic. La situació d’estrès va acompanyada d'una alteració de l'expressió de la proteïna de les unions estretes ZO-1 i una modificació de la morfologia de la monocapa. A més, l'exposició, principalment a MeHg, genera un augment de la secreció de mucus i una sobreexpressió de la seua principal mucina. Els mecanismes d'aquesta hipersecreció podrien estar relacionats amb la ruta IL4/IL13/STAT6. Tots aquests efectes sobre la monocapa epitelial comporten un augment de la permeabilitat i una reducció de la capacitat de regeneració. En els assajos in vivo s'han exposat ratolins BALB/c a Hg(II) i MeHg (1-10 mg/L) durant 4 mesos a través de l'aigua de beguda. Les dades obtingudes han confirmat l'observat in vitro. L'exposició a totes dues espècies (especialment a 5 i 10 mg/L) indueix un procés inflamatori en el còlon, amb un augment de les citocines TNF-α i IL1-β i la presència d'infiltrats de neutròfils. Paral·lelament, l'exposició genera estrès oxidatiu amb un increment de les ROS/RNS i els peròxids lipídics. S'ha confirmat in vivo la participació en aquesta resposta d'algunes rutes apuntades in vitro, en concret p38 MAPK i JNK. A més, també s'evidencien alteracions en l'expressió de proteïnes de les unions i de la mucina MUC2 en el còlon, acompanyada d'una hiperplàsia de les cèl·lules mucosecretores, especialment en els tractaments amb MeHg. En aquests animals hi ha, a més, un augment de l'expressió d'IL13 i IL4, la qual cosa apunta a la participació de la ruta IL4/IL13/STAT6, tal com indicaven els assajos in vitro. A més, aquests assajos in vivo mostren un efecte de les espècies de Hg sobre el metabolisme de la microbiota intestinal, amb una reducció dels continguts luminals dels àcids grassos de cadena curta (SCFA), encara que els canvis en la composició de microbiota intestinal son minims. Finalment, els animals tractats presenten un augment de la permeabilitat. Totes les dades obtingudes in vitro i in vivo posen de manifest que una exposició continuada a Hg(II) i MeHg genera una disrupció de la barrera intestinal. Els assajos in vitro realitzats per a determinar l'eficàcia de les soques de BAL d'origen murí LE1 i LE2 com a estratègia de protecció s'han realitzat coexposant el model cel·lular combinat a Hg(II) o MeHg (1 mg/L) juntament amb les soques durant 7 dies. Les dades obtingudes han mostrat un efecte protector de totes dues soques, amb una reducció de la resposta inflamatòria, l'estrès i els efectes sobre les unions estretes i la producció de mucus. Així mateix, la presència dels bacteris restaura parcialment la permeabilitat i la capacitat de regeneració de les monocapes. Tenint en compte que en aquest estudi les BAL estan inactivades tèrmicament, aquesta protecció s'associa principalment a la seua capacitat de quelació del Hg, encara que no cal descartar que algun component estructural de la paret bacteriana puga exercir un altre tipus d'efecte beneficiós. Els assajos in vivo per a confirmar l'efecte protector de les BAL s'han realitzat en ratolins BALB/c exposats durant 2 mesos a MeHg (5 mg/L) a través de l'aigua de beguda. Els bacteris, en aquest cas viables, s'han administrat per sonda gàstrica diàriament. Les dades obtingudes en aquest assaig mostren que, encara que els dues soques redueixen per igual els continguts colònics de MeHg, la reducció dels efectes tòxics és molt més notable amb la soca LE1. S'observen reduccions dels continguts dels mediadors d'inflamació i estrès en el còlon, hi ha una recuperació de l'expressió de proteïnes constituents de les unions estretes i el mucus. Es restableixen els nivells luminals dels metabòlits de la microbiota i es restaura parcialment la permeabilitat intestinal. Els resultats mostren que a més del procés de quelació, la soca LE1 activa la ruta de senyalització Nrf2/Keap1/ARE i la producció de la citocina anti-inflamatòria IL10. Les dades obtingudes in vitro i in vivo apunten al fet que la soca LE1 podria ser una bona alternativa per a reduir la toxicitat intestinal del Hg; reducció que pot tindre també repercussions beneficioses a nivell sistèmic. / [EN] A cell model for in vitro studies in which enterocytes, mucosecretory cells and macrophages have been combined in a bicameral system has been developed. Cells have been exposed to Hg for 10 days. The data have shown that both species induce an inflammatory response and a pro-oxidant response, with an increase in reactive oxygen/nitrogen species and an overexpression of stress proteins. The situation of stress is accompanied by alterations in the expression of the tight junction protein ZO-1 and changes in the morphology of the intestinal monolayer. In addition, exposure, especially to MeHg, generates an increase in mucus secretion and an overexpression of its main mucin. The mechanisms of this hypersecretion could be related to the IL4/IL13/STAT6 pathway. All these effects on the epithelial monolayer lead to and increased permeability and a reduced regeneration capacity. In vivo assays have been performed in BALB/c mice exposed to Hg (1-10 mg/L) for 4 months through drinking water. The data obtained have confirmed the previous results observed in vitro. Exposure to both species induces an inflammatory process in the colon and generates oxidative stress with an increase in ROS/RNS and lipid peroxides. The participation in this response of some signaling pathways targeted in vitro has been confirmed in vivo. In addition, alterations in the expression of tight junction proteins and the mucin MUC2 in the colon are also evidenced, accompanied by a hyperplasia of the mucosecretory cells, especially in MeHg treatments. In these animals, there is also an increase in the expression of IL-13 and IL-4 cytokines, which points to the participation of the IL4/IL13/STAT6 pathway. In addition, these in vivo assays show an effect of both species on the metabolism of the intestinal microbiota, with a reduction in the luminal contents of short-chain fatty acids, although changes in the intestinal microbiota composition are minimal. Finally, the treated animals showed an increase in permeability. The in vitro studies carried out to determine the efficacy of the BAL strains of murine origin LE1 and LE2 as a strategy of protection have been performed by co-exposing the combined cell model to Hg (1 mg/L) together with the strains for 7 days. The data obtained have shown a protective effect of both strains, with a reduction in the inflammatory response, oxidative stress and the effects on tight junctions and mucus production. In the same way, the presence of both bacteria partially restores the permeability and the regenerative capacity of the monolayers. Bearing in mind that in this study the LABs are heat-inactivated, this protection is mainly associated with their ability to bind Hg, although it cannot be ruled out that some structural components of the bacterial wall may exert another type of beneficial effect. In vivo assays carry out to confirm the protective effect of LAB have been performed in BALB/c mice exposed for 2 months to MeHg (5 mg/L) through drinking water. The strains of LAB have been administered daily by gavage. Data show that, the reduction of the toxic effects is much more pronounced when LE1 strain is administrated. Reductions in the contents of inflammatory and stress mediators in the colon are observed, together with a recovery in the expression of proteins of the tight junctions and the mucus layer. The luminal levels of microbiota metabolites are restored, and intestinal permeability is partially reestablished. The results show that in addition to the chelation process, LE1 strain activates the Nrf2/Keap1/ARE signaling pathway and the production of the anti-inflammatory cytokine IL10. The data obtained in vitro and in vivo suggest that LE1 strain could be a good strategy to reduce Hg intestinal toxicity; reduction that can also have beneficial repercussions at a systemic level. / Rodríguez Viso, P. (2023). Estrategias para mitigar la toxicidad asociada a la exposición crónica al mercurio a través de la dieta [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/192865

A tuberculose continua sendo um grave problema de saúde pública mundial. O principal agente etiológico da doença é o Mycobacterium tuberculosis, infectando entre outras células, os macrófagos. A infecção pode manter-se latente por vários anos sem causar sintomas clínicos aparentes. Para esta pesquisa, foi proposto o estudo de indução de marcadores de resposta inflamatória em células THP-1 diferenciadas em macrófagos utilizando isolados micobacterianos sensíveis, uni ou multidrogas resistentes transfectados com o plasmídeo pFPCAGFP carreando o gene da proteína verde fluorescente, como marcador de infecção celular. Objetivou também avaliar a relação entre isolados clínicos de M.tuberculosis sensíveis ou com resistência a uma ou multidrogas e sua relação à virulência. Para isso, culturas da linhagem monocítica humana (THP-1) foram mantidas para posterior infecção por isolados clínicos, genotipicamente identificados pertencentes à nossa micobacterioteca. A expressão do RNAm foi quantificada e avaliada por RTPCR e posteriormente, PCR em tempo real foi realizado. Após infecção das células THP-1 diferenciadas em macrófagos, observou-se que houve um aumento na expressão das citocinas pró-inflamatórias (IL-1β, IL-6 e TNF-α) e TLRs (TLR2 e TLR4) em relação ao controle da infecção. As análises estatísticas permitiram a correlação na expressão destas citocinas e TLRs, e através desses resultados, foi possível constatar que TLR2 regula a expressão das citocinas pró-inflamatórias (IL-1β, IL-6 e TNF-α) e que os Isolados clínicos com maior resistência, sinalizam melhor a expressão das citocinas próinflamatórias e TLRs, ao passo que isolados clínicos sensíveis, possuem uma fraca sinalização. / Tuberculosis is the major causative of death and morbidity and still is a serious problem to the health in the world. Mycobacterium tuberculosis is the major causative agent of tuberculosis disease, infecting cells like macrophages. The infection can keep dormant for several years without any clinical symptoms. For this research, the study was proposed for induction of markers of inflammatory response in cells differentiated THP-1 in macrophages by clinical isolates from sensitive or multidrug resistant mycobacteria caring pFPCAGFP plasmid that contains green fluorescent protein gene, a marker of infection cell. Also objective assesses the relationship between clinical isolates of sensitive or with resistance to one or four drugs M.tuberculosis and its relationship to virulence. For this, cultures of monocitic lineage human (THP-1) were kept for subsequent infection by clinical isolates, genotypically identified belonging to our mycobacterioteca. The expression of mRNA was quantified and evaluated by RT-PCR and subsequently, real-time PCR was performed. After infection of the THP-1 differentiated cells in macrophages, it was observed that there was an increase expression of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and TLRs (TLR2 and TLR4) compared to infection control. Statistical analyses allowed expression correlation of these cytokines and TLRs, and through those results, it was possible to see that TLR2 regulates pro-inflammatory cytokines (IL-1β , IL-6 and TNF-α) expression and those clinical isolates with four resistance have better signal of pro-inflammatory cytokines and TLRs expression, while sensitive clinical isolates, has a weak signal.

L’inflammation joue un rôle clé dans de nombreuses pathologies, telles que les maladies inflammatoires chroniques, les désordres métaboliques et le cancer. L’un de ses médiateurs le plus puissant est l’interleukine-1β (IL-1β), qui est une cytokine pro-inflammatoire participant à tous les stades de l’inflammation et de l’immunité. Son activation est régulée par un complexe multi-protéique nommé inflammasome, dont la caspase-1 active en découlant est responsable du clivage et de la maturation de l’IL-1β. Huit types d’inflammasomes activant et clivant la pro-caspase-1 ont été identifiés et contiennent tous la protéine ASC (apoptosis-associated speck-like protein containing a CARD). Les inflammasomes partagent un signal intracellulaire commun et le mécanisme menant à leur assemblage et leur activation n’est pas totalement élucidé. L’utilisation de la lignée cellulaire humaine monocytaire, THP-1, différenciée en macrophages grâce à un ester de phorbol, le TPA, a permis la mise en place d’un modèle d’étude de modulateurs de l’inflammasome en conditions stériles. Ce modèle a permis l’étude des mécanismes impliqués suite à des signaux issus de l’inflammation chronique, tels que l’ATP et les espèces réactives de l’oxygène (ROS). Ce travail montre qu’il existe une synergie entre ATP et ROS, qui agissent grâce à une boucle d’activation impliquant probablement plusieurs inflammasomes, dont NLRP3. Des donneurs de NO connus (trinitrine et isosorbide dinitrate) ou nouveau (dérivé de purine) ont montré une activité anti-inflammatoire. D’autres composés ont été identifiés comme de potentiels inhibiteurs d’inflammasome (extraits de dattes et dérivé de purine portant un acide lipoïque) / Inflammation has a pivotal role in several disorders, such as chronic inflammatory diseases, metabolic disorders and cancer. Interleukin-1β (IL-1β) is one of the most powerful mediators in this mechanism. IL-1β is a pro-inflammatory cytokine which is implicated in every step of inflammation and immunity. IL-1β is regulated by a multi-protein complex named inflammasome, in which active caspase-1 is responsible for IL-1β processing and maturation. Eight inflammasomes processing and activating pro-caspase-1 has been identified and all contain the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD). Inflammasomes share a common intracellular signal and the mechanism for their structural assembly and activation is not totally clear. The human monocytic cell line, THP-1, has been used and cells have been differentiated in macrophages by the phorbol ester, TPA. Based on this, a cellular pattern has been established for studying inflammasome modulators under sterile conditions. This pattern allowed the study of implicated mechanisms in chronic inflammatory signals, such as ATP and reactive oxygen species (ROS). This study shows that ATP and ROS synergize for activating inflammasomes and NLRP3 and act in an autocrine loop. Stimulation with others natural or synthetic compounds has been realized for characterization of their inflammatory activity. Therapeutic NO donors (trinitrin and isosorbide dinitrate) and a new NO donor (purine nitrate ester) have been characterized for their anti-inflammatory activity. Others compounds from a new category of inflammasome inhibitors have been identified (date fruit extracts and lipoic acid purine derivative)

A tuberculose continua sendo um grave problema de saúde pública mundial. O principal agente etiológico da doença é o Mycobacterium tuberculosis, infectando entre outras células, os macrófagos. A infecção pode manter-se latente por vários anos sem causar sintomas clínicos aparentes. Para esta pesquisa, foi proposto o estudo de indução de marcadores de resposta inflamatória em células THP-1 diferenciadas em macrófagos utilizando isolados micobacterianos sensíveis, uni ou multidrogas resistentes transfectados com o plasmídeo pFPCAGFP carreando o gene da proteína verde fluorescente, como marcador de infecção celular. Objetivou também avaliar a relação entre isolados clínicos de M.tuberculosis sensíveis ou com resistência a uma ou multidrogas e sua relação à virulência. Para isso, culturas da linhagem monocítica humana (THP-1) foram mantidas para posterior infecção por isolados clínicos, genotipicamente identificados pertencentes à nossa micobacterioteca. A expressão do RNAm foi quantificada e avaliada por RTPCR e posteriormente, PCR em tempo real foi realizado. Após infecção das células THP-1 diferenciadas em macrófagos, observou-se que houve um aumento na expressão das citocinas pró-inflamatórias (IL-1β, IL-6 e TNF-α) e TLRs (TLR2 e TLR4) em relação ao controle da infecção. As análises estatísticas permitiram a correlação na expressão destas citocinas e TLRs, e através desses resultados, foi possível constatar que TLR2 regula a expressão das citocinas pró-inflamatórias (IL-1β, IL-6 e TNF-α) e que os Isolados clínicos com maior resistência, sinalizam melhor a expressão das citocinas próinflamatórias e TLRs, ao passo que isolados clínicos sensíveis, possuem uma fraca sinalização. / Tuberculosis is the major causative of death and morbidity and still is a serious problem to the health in the world. Mycobacterium tuberculosis is the major causative agent of tuberculosis disease, infecting cells like macrophages. The infection can keep dormant for several years without any clinical symptoms. For this research, the study was proposed for induction of markers of inflammatory response in cells differentiated THP-1 in macrophages by clinical isolates from sensitive or multidrug resistant mycobacteria caring pFPCAGFP plasmid that contains green fluorescent protein gene, a marker of infection cell. Also objective assesses the relationship between clinical isolates of sensitive or with resistance to one or four drugs M.tuberculosis and its relationship to virulence. For this, cultures of monocitic lineage human (THP-1) were kept for subsequent infection by clinical isolates, genotypically identified belonging to our mycobacterioteca. The expression of mRNA was quantified and evaluated by RT-PCR and subsequently, real-time PCR was performed. After infection of the THP-1 differentiated cells in macrophages, it was observed that there was an increase expression of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and TLRs (TLR2 and TLR4) compared to infection control. Statistical analyses allowed expression correlation of these cytokines and TLRs, and through those results, it was possible to see that TLR2 regulates pro-inflammatory cytokines (IL-1β , IL-6 and TNF-α) expression and those clinical isolates with four resistance have better signal of pro-inflammatory cytokines and TLRs expression, while sensitive clinical isolates, has a weak signal.

Les céréales et les produits issus de leur transformation sont susceptibles d’être contaminés par des espèces fongiques capables de produire des mycotoxines. L’Homme est ainsi exposé tout au long de sa vie à travers son alimentation à ces contaminants naturels, généralement à de faibles doses et en mélange. Cependant, l’incidence de la présence simultanée de ces toxines sur notre santé, à court terme comme à plus long terme, ainsi que les mécanismes responsables de leur toxicité sont encore peu ou mal caractérisés. L’utilisation de modèles cellulaires humains pertinents et adaptés est particulièrement importante pour de telles études. L’épithélium intestinal et le système immunitaire, qui constituent la première barrière de défense de l’hôte suite à l’ingestion de contaminants alimentaires, ainsi que le foie, de par son rôle majeur dans la biotransformation des xénobiotiques, représentent des modèles d’étude pertinents en toxicologie. Dans le cadre de cette étude, des modèles cellulaires humains d’origine intestinale (Caco-2), immunitaire (THP-1) et hépatique (HepaRG) ont été employés pour évaluer le risque associé à la co-exposition aux mycotoxines de Fusarium (appelées fusariotoxines) qui sont parmi les plus problématiques dans nos régions. Différents types d’interactions, tels que de l’antagonisme et du synergisme, ont pu être observés sur la viabilité cellulaire en fonction de la nature du mélange, des doses testées, de la lignée cellulaire étudiée et du modèle mathématique utilisé pour prédire les effets combinés. Des interactions ont également été observées à l’échelle moléculaire, les effets des mélanges étant très différents de ceux induits par les toxines individuellement sur le protéome des cellules. D’autres résultats obtenus interrogent sur la façon dont les mycotoxines déclenchent réellement la réponse cellulaire. De plus, les interactions entre cellules cocultivées semblent capables de modifier la réponse cellulaire suite à l’exposition à ces toxines. Ces résultats soulignent l’importance de développer des modèles in vitro de plus en plus sophistiqués et s’approchant des conditions in vivo pour permettre une meilleure caractérisation du risque « mycotoxine ». / Cereals and cereal-based products are susceptible to be contaminated by mycotoxin-producing fungi.Thus, through their diet, humans are exposed throughout their life to these natural food contaminants, mostly at low doses and in mixture. However, the health impact of the simultaneous exposure to these toxins, in acute and chronic conditions, as well as the mechanism related to their toxicity, are still poorly characterized. The use of relevant and suitable human cell models is therefore of particular importance for such studies. The intestinal epithelium and immune system, which constitute the first host defense barrier following the food contaminant uptake, as well as the liver, due to its major function in xenobiotic biotransformation, are relevant for toxicity studies. In the framework of study, the intestinal (Caco-2), immune (THP-1) and hepatic (HepaRG) human cell models were used for risk assessment associated with co-exposure to Fusarium mycotoxins (called fusariotoxins) which are the most problematic in our regions. Different type of interactions, such as antagonism and synergism, were observed on cell viability depending on the nature of the mixture, tested concentration, studied cell line and used mathematical model to predict the combined effects. Interactions were also highlighted at the molecular level, the effects of mixtures being very different from those induced by the toxins alone on the cell proteome. Other results raised the question about how mycotoxins actually trigger the cellular response. In addition, cell-cell interactions in co-cultured systems appeared to modify the cellular response following exposures to these toxins. Overall, the obtained results highlighted the relevance of developing in vitro models increasingly sophisticated and closer to in vivo conditions to allow for a better characterization of the "mycotoxin" risk.

Résumé : Depuis l'interdiction en 2013 des tests sur les animaux par le Règlement cosmétique n°1223/2009, différentes méthodes in vitro ont été développées. Toutefois, selon un consensus scientifique, aucune méthode ne peut couvrir à elle seule l’ensemble des événements clés (KE) définis pour la sensibilisation cutanée.Après un état de l’art des méthodes alternatives relatives à la sensibilisation cutanée, nous avons sélectionné et comparé 3 tests pour ensuite déterminer la meilleure stratégie à suivre. Dans le but de proposer un nouveau test intégré, nous avons adressé l’ensemble des KEs au sein d’un même type cellulaire. La cellule dendritique (DC) jouant un rôle clé dans le développement de la dermatite de contact allergique (DCA), notre choix s’est porté sur la lignée humaine pro-monocytaire THP-1. Nous avons étudié comme événements initiaux (KE1) les formes réactives à l’oxygène (FRO) et le glutathion (GSH), la voie Nrf2-Keap1 (voie centrale de détoxication) et l’expression génique pour le KE2, ainsi que les modifications phénotypiques pour le KE3.Nous avons montré que les allergisants forts induisent une production précoce des FRO associée à une réduction du GSH. Ils activent également la voie Nrf2-Keap1 et induisent l’expression des marqueurs de surface cellulaire CD54 et CD86, ainsi qu’une production de cytokines spécifiques (IL-8, IL-18,...).Pour conclure, ce travail a permis de proposer un test intégrant l’ensemble des mesures biologiques comme différents KE au sein d’un même type cellulaire. / Abstract : Since the animal testing ban in 2013 by Cosmetics Regulation n°1223/2009, various in vitro methods have been developed. However, according to a scientific consensus, no single method can stand-alone to cover the different key events (KE) defined for skin sensitization.After a state of the art of alternative methods relating to skin sensitization, we selected and compared 3 tests to determine the best strategy to follow. In order to propose a new integrated test, we wanted to address all KE within the same cell line. Because dendritic cell (DC) plays a key role in the development of allergic contact dermatitis (ACD), we have chosen the pro-monocytic human line THP-1. We have studied as initial events (KE1), reactive oxygen species (ROS) and glutathione (GSH), Nrf2-Keap1 pathway (central detoxification pathway) and gene expression for KE2 as well as phenotypic modifications for KE3.We have shown that strong allergens are correlated with early production of FRO associated with GSH reduction. They also activate the Nrf2-Keap1 pathway and induce the expression of CD54 and CD86 cell surface markers as well as production of specific cytokines (IL-8, IL-18, etc.).To conclude, this work propose a new assay integrating all the biological measures as different KEs within the same cell.

During colonization of the cystic fibrosis airway Pseudomonas aeruginosa converts from non-mucoid to a mucoid phenotype, characterized by the production of the exopolysaccharide alginate. Alginate production has been shown to enhance survival by promoting biofilm formation, evading complement killing, and resisting phagocytosis. The mechanism by which alginate protects P. aeruginosa from phagocytosis is unclear. To investigate the role of alginate in the inhibition of phagocytosis, a human monocytic cell line (THP-1) and a murine alveolar macrophage cell line (MH-S) were used to determine the effects of alginate on macrophage binding, signaling, and phagocytosis. Phagocytosis assays using the mucoid cystic fibrosis clinical isolate FRD1, and its non-mucoid isogenic algD mutant FRD1131, revealed that alginate inhibits opsonic and non-opsonic phagocytosis. The inhibitory effect of alginate production is intrinsic to the bacteria as exogenous alginate was unable to protect non-mucoid FRD1131 from phagocytosis. Decreased binding of FRD1 compared to FRD1131 was also demonstrated by using the actin polymerization inhibitor cytochalasin D to inhibit phagocytosis. Furthermore, studies using blocking antibodies to CD11b and CD14 found that both of these receptors were important for the phagocytosis of FRD, and it is likely that these receptors are blocked by alginate. Alginate production by P. aeruginosa may reduce lipid raft formation, however, it was not found to affect acid sphingomyelinase activity, which is important for ceramide formation within the lipid raft. Decreased binding led to decreased signaling in macrophages demonstrated by reduction in level and alteration in kinetics of phosphorylation of AKT and ERK1/2 kinases. Signaling pathway inhibitors revealed that PI3K, but not MEK, activation was critical for phagocytosis of P. aeruginosa. Despite altered intracellular signaling in murine macrophages, both mucoid and non-mucoid P. aeruginosa induced similar levels of IL-8 and MIP-2 from human and murine macrophages, respectively. By understanding the pathways involved in mediating efficient phagocytosis of clinical isolates, it may be possible to develop a treatment to promote clearance by the resident alveolar macrophages. These experiments may serve as a model to evaluate the effectiveness of such treatments. This approach also provides valuable insight into previously unknown mechanisms of phagocytosis of P. aeruginosa.