L’interleukine (IL)-17 et le TNFa sont deux cytokines pro-inflammatoires jouant un rôle important dans diverses maladies inflammatoires systémiques et auto-immunes affectant différents organes et tissus comme le foie et les muscles. Cependant, les rôles de l’IL-17 et du TNFa restent encore mal compris dans les muscles et le foie, qui est impliqué dans la réponse en phase aiguë. En utilisant des cultures de myoblastes, d’hépatocytes et de cellules stellaires hépatiques humaines, nous avons trouvé que l’IL-17 et le TNFa augmentent en synergie la sécrétion de la cytokine pro-inflammatoire IL-6 et de plusieurs chimiokines. Dans les myoblastes, l’IL-17 et le TNFa induisent un stress oxydatif et une dérégulation de calcium montrant ainsi que les processus pathologiques immuns et non-immuns interagissent. Dans les hépatocytes, en augmentant en synergie les niveaux de la CRP et des transaminases, l’IL-17 et le TNFa participent à l’inflammation systémique et aux dommages cellulaires. Etant donné que des infiltrats de cellules immunitaires sont retrouvés lors d’atteintes inflammatoires, les interactions cellulaires contribuent certainement à la chronicité de l’inflammation. Des cellules mononuclées du sang périphérique activées ou non ont ainsi été placées en co-cultures avec les myoblastes, les hépatocytes et les cellules stellaires. Par comparaison aux monocultures, les productions de l’IL-6 et de la chimiokine IL-8 étaient augmentées dans les co-cultures. L’IL-17 et le TNFa contribuaient partiellement à ces effets. Les effets systémiques de l’IL-17 et du TNFa en font donc des cibles thérapeutiques attrayantes pour le traitement des nombreuses maladies inflammatoires systémiques / Interleukin-17A (IL-17) and tumor necrosis factor-a (TNFa) are two pro-inflammatory cytokines playing an important role in various systemic inflammatory and autoimmune disorders affecting different organs and tissues including the liver and the muscles. However, the roles of IL-17 and TNFa are not fully understood in the muscles and also in liver, which is crucial in the acute phase response. By using cultures of human myoblasts, primary human hepatocytes, human HepaRG cells and LX-2 hepatic stellate cells, we found that IL-17 and TNFa increase in synergy the production of the pro-inflammatory cytokine IL-6 and chemokines (IL-8, CCL20, MCP-1). In myoblasts, the IL-17 and TNFa stimulation induces endoplasmic reticulum stress and calcium dysregulation showing that immune and non-immune pathogenic mechanisms interplay. In hepatocytes, IL-17 and TNFa mediate systemic inflammation and cell damage by increasing in synergy the CRP acute-phase protein and transaminase levels through the induction of IL-6. Since active liver and muscle disorders are characterized by inflammatory infiltrates of immune cells, the cell interactions play certainly an important role in the chronicity of the inflammation. Peripheral blood mononuclear cells activated or not were therefore co-cultured with myoblasts, hepatocytes and/or hepatic stellate cells to assess the inflammatory role of the cell-cell interactions. Co-cultures enhance the production of IL-6 and IL-8 compared to monocultures. IL-17 and TNFa contribute partially to these inductions. The systemic effects of IL-17 and/or TNFa make them attractive therapeutic targets for the treatment of various systemic inflammatory disorders

Myofibroblast expansion is a critical event in the pathogenesis of liver fibrosis. The activation of hepatic stellate cells (HSC) to myofibroblast (MFB) results in the enhanced production of extracellular matrix (ECM). We have studied the effect of fibroblast growth factor 1 (FGF-1) on liver MFB. In the second part we investigated effect of transforming growth factor β1 (TGF-β1) and FGF-1 on cell line HSC-T6. Cells were cultured on plastic dishes and in 3D collagen gel mimicking fibrotic tissue. MFB were isolated by repeated passaging of nonparenchymal liver cell fraction. The transfer of MFB from plastic dishes to collagen gel resulted in the change in their shape and phenotype. The expression of cytokine TGF-β1 and of MFB markers, α-smooth muscle actin (α-SMA) and cellular fibronectin (EDA-FN) on protein level was significantly decreased in collagen gel. The experiments with SB 431542, the inhibitor of TGF-β receptor type I, showed that EDA-FN and α-SMA are differently regulated. EDA-FN expression is dependent on TGF-β1, while the expression of α-SMA is primarily determined by the environment and modified by TGF-β1. EDA-FN is more sensitive to the U0126, the inhibitor of protein kinases MEK 1 and 2. Collagen gel does not change the expression of metalloproteinase MMP-2 but activates the proenzyme....

A fibrose hepática é uma patologia que acompanha outras doenças crônicas do fígado como a cirrose e o hepatocarcinoma. As células estreladas hepáticas (HSC, do inglês hepatic stellate cells) compõem uma população celular heterogênea que se caracteriza por transitar entre dois fenótipos. As células com fenótipo quiescente possuem a capacidade de armazenar vitamina A em gotas lipídicas. Os insultos ao fígado desencadeiam uma resposta inflamatória que gera estímulos parácrinos e autócrinos mediados por citocinas e espécies reativas. Neste contexto, as HSC assumem um fenótipo ativado fibrogênico e tornam-se responsáveis pela cicatrização hepática. Danos crônicos ao fígado levam a uma deposição de matriz extracelular exagerada que configura o estado patológico da fibrose. O resveratrol (RSV – 3,4’,5-tri-hidroxi-trans-estilbeno) é uma fitoalexina produzida por algumas espécies de plantas. Inúmeros efeitos benéficos à saúde são atribuídos ao RSV por causa do seu potencial antioxidante, antiinflamatório e pró-apoptótico. Estudos anteriores mostraram que tratamento da GRX, uma linhagem murina de HSC ativadas, com concentrações de RSV próximas as biodisponíveis (0,1 a 1 μM) resultou em parada do ciclo na fase S com consequente inibição de proliferação celular, um efeito associado à citotoxicidade e que pode favorecer a resolução da fibrose hepática. Neste estudo, por técnicas espectrofotométricas, foi demonstrado que tratamento da GRX por 24 horas com concentrações entre 0,1 a 50 μM de RSV promoveu um efeito pró-oxidante que causa uma citotoxicidade dependente da dose, bastante aumentada no grupo tratado com a concentração mais alta. Os efeitos citotóxicos atenuados encontrados nas células tratadas por 120 horas sugerem que a GRX pode se tornar resistente a estes efeitos. O potencial pró-oxidante do RSV foi o ponto de partida para investigar a possibilidade de que esta fitoalexina provocasse uma alteração no metabolismo mitocondrial da GRX. Para isso, os efeitos do RSV (1 a 50 μM) na função mitocondrial, na indução de morte mediada por estas organelas e na autofagia/mitofagia foram investigados por técnicas de espectrofotometria, de imunocitoquímica, de citometria de fluxo, de microscopia confocal e de microscopia eletrônica de transmissão em GRX tratadas por 24 e 120 horas. Foi demonstrado que todas as concentrações de RSV promovem apoptose por meio da ativação de caspases, alteram a dinâmica/função mitocondrial e induzem o aumento de autofagia/mitofagia na GRX. No entanto, o RSV provocou biogênese de mitocôndrias nos grupos tratados com 1 e 10 μM, enquanto que o tratamento com 50 μM causou dano celular evidente na GRX, sem induzir biogênese de mitocôndrias. Desta forma, é possível que a citotoxicidade “dose-dependente” do RSV, que causa a morte celular e dano oxidativo em 24 horas de tratamento, esteja relacionada com o desequilíbrio entre a indução concomitante de apoptose mediada por dano mitocondrial, autofagia/mitofagia e biogênese de mitocôndrias. Por fim, foi investigada a liberação de TNF-α, Interleucina-6 e Interleucina-10 pela GRX tratada por 24 e 120 horas com RSV (0,1 a 50 μM), considerando o papel antiinflamatório do RSV e o papel das HSC ativadas na sinalização autócrina que contribui para a modulação fenotípica destas células. Foi demonstrado que o tratamento da GRX com RSV por 24 e 120 horas induziu a redução da liberação de Interleucina-6; enquanto que a liberação de TNF-α e Interleucina-10 foi aumentada. Estes resultados confirmam um efeito antiinflamatório do RSV que deve contribuir na prevenção da ativação ou da perpetuação do estado ativado das HSC por meio de sinalização autócrina. Ainda que a concentração do RSV seja importante para efetivamente induzir a morte das HSC ativadas, o tratamento com esta fitoalexina pode ser promissor para a resolução da fibrose hepática por diminuir a população de células ativadas e, possivelmente, prevenir a perpetuação do estado fenotípico ativado. Estudos avaliando indicadores de quiescência em células tratadas são ainda necessários para desvendar completamente os efeitos do RSV quanto às possibilidades de inibição da perpetuação ou reversão fenotípica das HSC ativadas. / Liver fibrosis is a disease that accompanies other hepatic chronic diseases such as cirrhosis and hepatocellular carcinoma. Hepatic stellate cells (HSC) are a heterogeneous cell population characterized by transiting between two phenotypes. Cells with a quiescent phenotype are able to store vitamin A into lipid droplets. Damage to the liver trigger an inflammatory response that generates paracrine and autocrine stimulation mediated by cytokines and reactive species. In this context, HSC assume an activated and fibrogenic phenotype responsive for hepatic wound-healing. Chronic insults to the liver lead to an excessive deposition of extracellular matrix that configures the pathological state of fibrosis. Resveratrol (RSV – 3,4’,5-tri-hidroxi-trans-stilbeno) is a phytoalexin produced by some species of plants. Several beneficial effects are attributed to this molecule due to its antioxidant, antiproliferative and pro-apoptotic potential. Previous studies showed that treatment with bioavailable concentrations of RSV (0.1 to 1 μM) promoted an arrest cycle at the S phase in GRX, a murine activated HSC model, leading to cell proliferation inhibition, a cytotoxic effect that contributes to the liver fibrosis resolution. In this study, it was shown by spectrophotometric techniques that GRX treatment for 24 hours at concentrations between 0.1 to 50 μM of RSV promoted a fairly clear pro-oxidant effect that causes a dose-dependent cytotoxicity that was higher in the group treated with 50 μM. The attenuated cytotoxicity found after 120 hours of GRX treatment suggest that these cells became resistant to this effect. The pro-oxidant potential of RSV was the starting point for investigating the possibility that this phytoalexin would cause a change in the GRX mitochondrial metabolism. Thus, the effects of RSV (1 to 50 μM) on altering the mitochondrial function, on inducing mitochondrial-mediated cell death, and autophagy/mitofagia were investigated in GRX treated for 24 and 120 hours by spectrophotometric techniques, immunocytochemistry, flow cytometry, confocal microscopy, and transmission electron microscopy. All the RSV concentrations promote cell apoptosis through caspases activation, alter the mitochondrial dynamics and function, and induce an increase of autophagy/mitofagia. Curiously, only 1 and 10 μM of RSV induced mitochondrial biogenesis in GRX, while the highest concentration caused an evident cell damage without inducing mitochondrial biogenesis. Thus, it is possible that the "dose-dependent" cytotoxicity of RSV, which causes cell death and oxidative damage in 24 hours of treatment, is related to an imbalance between the concomitant induction of mitochondrial-mediated apoptosis, autophagy/mitofagia, and mitochondrial biogenesis. Finally, it was investigated the release of TNF-α, Interleukin-6 and Interleukin-10 by GRX treated for 24 and 120 hours with RSV (0.1 to 50 μM), considering the anti-inflammatory role of RSV and the autocrine signalling role of HSC that contributes to the perpetuation of its activated phenotype. It was demonstrated that GRX treatment with RSV for 24 and 120 hours reduced the release of Interleukin-6 in the culture medium; whereas the release of TNF-α and Interleukin-10 was increased. These results confirm the anti-inflammatory properties of RSV and may contribute to the prevention of HSC activation through autocrine signalling. Although RSV concentration is important to effectively induce activated HSC death, cells treatment with this phytoalexin may be promising for liver fibrosis resolution through decreasing the population of activated cells or through preventing the perpetuation of activated state of HSC. Future studies evaluating the quiescence indicators of GRX under RSV treatment are still needed to fully unravel the effects of this phytoalexin on inhibiting the perpetuation of activated HSC or reversing its activated phenotype.

A fibrose hepática é uma patologia que acompanha outras doenças crônicas do fígado como a cirrose e o hepatocarcinoma. As células estreladas hepáticas (HSC, do inglês hepatic stellate cells) compõem uma população celular heterogênea que se caracteriza por transitar entre dois fenótipos. As células com fenótipo quiescente possuem a capacidade de armazenar vitamina A em gotas lipídicas. Os insultos ao fígado desencadeiam uma resposta inflamatória que gera estímulos parácrinos e autócrinos mediados por citocinas e espécies reativas. Neste contexto, as HSC assumem um fenótipo ativado fibrogênico e tornam-se responsáveis pela cicatrização hepática. Danos crônicos ao fígado levam a uma deposição de matriz extracelular exagerada que configura o estado patológico da fibrose. O resveratrol (RSV – 3,4’,5-tri-hidroxi-trans-estilbeno) é uma fitoalexina produzida por algumas espécies de plantas. Inúmeros efeitos benéficos à saúde são atribuídos ao RSV por causa do seu potencial antioxidante, antiinflamatório e pró-apoptótico. Estudos anteriores mostraram que tratamento da GRX, uma linhagem murina de HSC ativadas, com concentrações de RSV próximas as biodisponíveis (0,1 a 1 μM) resultou em parada do ciclo na fase S com consequente inibição de proliferação celular, um efeito associado à citotoxicidade e que pode favorecer a resolução da fibrose hepática. Neste estudo, por técnicas espectrofotométricas, foi demonstrado que tratamento da GRX por 24 horas com concentrações entre 0,1 a 50 μM de RSV promoveu um efeito pró-oxidante que causa uma citotoxicidade dependente da dose, bastante aumentada no grupo tratado com a concentração mais alta. Os efeitos citotóxicos atenuados encontrados nas células tratadas por 120 horas sugerem que a GRX pode se tornar resistente a estes efeitos. O potencial pró-oxidante do RSV foi o ponto de partida para investigar a possibilidade de que esta fitoalexina provocasse uma alteração no metabolismo mitocondrial da GRX. Para isso, os efeitos do RSV (1 a 50 μM) na função mitocondrial, na indução de morte mediada por estas organelas e na autofagia/mitofagia foram investigados por técnicas de espectrofotometria, de imunocitoquímica, de citometria de fluxo, de microscopia confocal e de microscopia eletrônica de transmissão em GRX tratadas por 24 e 120 horas. Foi demonstrado que todas as concentrações de RSV promovem apoptose por meio da ativação de caspases, alteram a dinâmica/função mitocondrial e induzem o aumento de autofagia/mitofagia na GRX. No entanto, o RSV provocou biogênese de mitocôndrias nos grupos tratados com 1 e 10 μM, enquanto que o tratamento com 50 μM causou dano celular evidente na GRX, sem induzir biogênese de mitocôndrias. Desta forma, é possível que a citotoxicidade “dose-dependente” do RSV, que causa a morte celular e dano oxidativo em 24 horas de tratamento, esteja relacionada com o desequilíbrio entre a indução concomitante de apoptose mediada por dano mitocondrial, autofagia/mitofagia e biogênese de mitocôndrias. Por fim, foi investigada a liberação de TNF-α, Interleucina-6 e Interleucina-10 pela GRX tratada por 24 e 120 horas com RSV (0,1 a 50 μM), considerando o papel antiinflamatório do RSV e o papel das HSC ativadas na sinalização autócrina que contribui para a modulação fenotípica destas células. Foi demonstrado que o tratamento da GRX com RSV por 24 e 120 horas induziu a redução da liberação de Interleucina-6; enquanto que a liberação de TNF-α e Interleucina-10 foi aumentada. Estes resultados confirmam um efeito antiinflamatório do RSV que deve contribuir na prevenção da ativação ou da perpetuação do estado ativado das HSC por meio de sinalização autócrina. Ainda que a concentração do RSV seja importante para efetivamente induzir a morte das HSC ativadas, o tratamento com esta fitoalexina pode ser promissor para a resolução da fibrose hepática por diminuir a população de células ativadas e, possivelmente, prevenir a perpetuação do estado fenotípico ativado. Estudos avaliando indicadores de quiescência em células tratadas são ainda necessários para desvendar completamente os efeitos do RSV quanto às possibilidades de inibição da perpetuação ou reversão fenotípica das HSC ativadas. / Liver fibrosis is a disease that accompanies other hepatic chronic diseases such as cirrhosis and hepatocellular carcinoma. Hepatic stellate cells (HSC) are a heterogeneous cell population characterized by transiting between two phenotypes. Cells with a quiescent phenotype are able to store vitamin A into lipid droplets. Damage to the liver trigger an inflammatory response that generates paracrine and autocrine stimulation mediated by cytokines and reactive species. In this context, HSC assume an activated and fibrogenic phenotype responsive for hepatic wound-healing. Chronic insults to the liver lead to an excessive deposition of extracellular matrix that configures the pathological state of fibrosis. Resveratrol (RSV – 3,4’,5-tri-hidroxi-trans-stilbeno) is a phytoalexin produced by some species of plants. Several beneficial effects are attributed to this molecule due to its antioxidant, antiproliferative and pro-apoptotic potential. Previous studies showed that treatment with bioavailable concentrations of RSV (0.1 to 1 μM) promoted an arrest cycle at the S phase in GRX, a murine activated HSC model, leading to cell proliferation inhibition, a cytotoxic effect that contributes to the liver fibrosis resolution. In this study, it was shown by spectrophotometric techniques that GRX treatment for 24 hours at concentrations between 0.1 to 50 μM of RSV promoted a fairly clear pro-oxidant effect that causes a dose-dependent cytotoxicity that was higher in the group treated with 50 μM. The attenuated cytotoxicity found after 120 hours of GRX treatment suggest that these cells became resistant to this effect. The pro-oxidant potential of RSV was the starting point for investigating the possibility that this phytoalexin would cause a change in the GRX mitochondrial metabolism. Thus, the effects of RSV (1 to 50 μM) on altering the mitochondrial function, on inducing mitochondrial-mediated cell death, and autophagy/mitofagia were investigated in GRX treated for 24 and 120 hours by spectrophotometric techniques, immunocytochemistry, flow cytometry, confocal microscopy, and transmission electron microscopy. All the RSV concentrations promote cell apoptosis through caspases activation, alter the mitochondrial dynamics and function, and induce an increase of autophagy/mitofagia. Curiously, only 1 and 10 μM of RSV induced mitochondrial biogenesis in GRX, while the highest concentration caused an evident cell damage without inducing mitochondrial biogenesis. Thus, it is possible that the "dose-dependent" cytotoxicity of RSV, which causes cell death and oxidative damage in 24 hours of treatment, is related to an imbalance between the concomitant induction of mitochondrial-mediated apoptosis, autophagy/mitofagia, and mitochondrial biogenesis. Finally, it was investigated the release of TNF-α, Interleukin-6 and Interleukin-10 by GRX treated for 24 and 120 hours with RSV (0.1 to 50 μM), considering the anti-inflammatory role of RSV and the autocrine signalling role of HSC that contributes to the perpetuation of its activated phenotype. It was demonstrated that GRX treatment with RSV for 24 and 120 hours reduced the release of Interleukin-6 in the culture medium; whereas the release of TNF-α and Interleukin-10 was increased. These results confirm the anti-inflammatory properties of RSV and may contribute to the prevention of HSC activation through autocrine signalling. Although RSV concentration is important to effectively induce activated HSC death, cells treatment with this phytoalexin may be promising for liver fibrosis resolution through decreasing the population of activated cells or through preventing the perpetuation of activated state of HSC. Future studies evaluating the quiescence indicators of GRX under RSV treatment are still needed to fully unravel the effects of this phytoalexin on inhibiting the perpetuation of activated HSC or reversing its activated phenotype.

Hepatocellular carcinoma (HCC) is the most common liver malignancy that usually develops in patients suffering from chronic liver diseases. One of the major problems faced in the treatment of HCC is severe chemoresistance. Endoplasmic reticulum (ER) stress and hepatic stellate cells play an important role in tumour survival and growth as well as fibrosis. This study further investigates the crosstalk between ER-stress and hepatic stellate cells in HCC resistant cells as well as their relation to chemoresistance markers expression. Mice with chemically induced HCC were divided in 3 different treatment group; one was only treated with doxorubicin, one only with pharmacological ER-stress inhibitor 4μ8C, and one was treated with a combination of doxorubicin and 4μ8C. Tumour burden, fibrosis and cell proliferation were assessed through histological analysis and ImageJ processing. Chemoresistance markers expression was evaluated through mRNAs determination using real-time qPCR. While the combined treatment consisting of doxorubicin and pharmacological ER-stress inhibitor (4μ8C) has shown to positively reduce tumour progression, ferroptosis and collagen deposition, consequently decreasing fibrosis, drug resistance markers’ expression, on the other hand, seems to be more intricate, thus indicating that further investigations are probably needed. / Hepatocellulärt karcinom (HCC) är den vanligaste maligniteten i levern som vanligtvis utvecklas hos patienter som lider av kroniska leversjukdomar. Ett av de största problemen vid behandling av HCC är svår kemoresistens. Stress i endoplasmatiska retikulum (ER) och hepatiska stellatceller spelar en viktig roll för tumörernas överlevnad och tillväxt samt för fibros. I denna studie undersöks vidare samspelet mellan ER-stress och hepatiska stellatceller i HCC-resistenta celler samt deras relation till uttryck av kemoresistensmarkörer. Möss med kemiskt inducerad HCC delades in i tre olika behandlingsgrupper; en behandlades enbart med doxorubicin, en enbart med den farmakologiska ER-stresshämmaren 4μ8C och en behandlades med en kombination av doxorubicin och 4μ8C. Tumörbörda, fibros och cellproliferation bedömdes genom histologisk analys och ImageJ-bearbetning. Kemoresistensmarkörernas uttryck utvärderades genom bestämning av mRNA med hjälp av qPCR i realtid. Medan kombinationsbehandlingen bestående av doxorubicin och farmakologisk ER-stresshämmare (4μ8C) har visat sig minska tumörprogressionen, ferroptos och kollagenavlagring och därmed minska fibros, verkar uttrycket av läkemedelsresistensmarkörer å andra sidan vara mer invecklat, vilket tyder på att det troligen behövs ytterligare undersökningar.

La leishmaniose viscérale est une maladie systémique mortelle en l’absence de traitement. Elle est due aux protozoaires Leishmania donovani et L. infantum, parasites des phagocytes mononucléés, capables d’envahir les organes lymphoïdes et le foie. Le contrôle de l’infection hépatique repose sur la mise en place d’une réponse granulomateuse efficace, promue par une réponse immunitaire Th1, dans un environnement tissulaire Th2. L’objectif de ce travail était l'étude du rôle d’une cytokine Th2 récemment décrite, l’IL-33, dans cette réponse hépatique complexe encore partiellement incomprise. Des dosages d’IL-33 sur des sérums de patients et la détection de cellules IL-33+ dans le foie d’un patient rennais ont placé l’IL-33 comme un biomarqueur possible de la maladie active. L’IL-33 étant exprimée dans les cellules étoilées du foie au cours d’hépatites chroniques, ces cellules ont été exposées à L. donovani. Leur permissivité aux leishmanies sans toxicité apparente ni perturbation de leurs propriétés fonctionnelles, ainsi que la persistance des leishmanies sur une culture de plusieurs semaines, nous ont conduit à proposer les cellules étoilées comme cellules sanctuaires possibles pour les leishmanies viscérotropes, contribuant donc potentiellement au portage asymptomatique. En revanche, elles ne sont pas apparues comme une source majeure d'IL-33 au cours de la leishmaniose viscérale. Chez des souris C57BL/6 et BALB/c infectées par L. donovani, l'IL-33 a été observée dans des cellules ne s'apparentant pas à des cellules étoilées, et principalement localisées dans les granulomes. Des cellules exprimant son récepteur ST2 ayant été également observées dans le foie, un rôle de l’axe IL-33/ST2 a été recherché. Les résultats obtenus chez des souris BALB/c déficientes en ST2 ou traitées par de l'IL-33 recombinante suggèrent que l'IL-33 régule négativement l'expression de cytokines Th1 (IL-12, IFN-γ) et l'infiltrat de neutrophiles et monocytes dans le foie, limitant ainsi le contrôle de la charge parasitaire. Ainsi, l'IL-33 semble être un facteur de susceptibilité pour la leishmaniose viscérale. En parallèle, des travaux entrepris sur des souris C57BL/6 infectées par L. donovani suggèrent de possibles rôles différentiels de l'IL-33 en fonction de l'environnement immunitaire inhérent au fond génétique de l'hôte. / Visceral leishmaniasis is a life-threatening systemic disease caused by Leishmania protozoans, L. donovani and L. infantum, which invade mononuclear phagocytes in the lymphoid organs and the liver. The control of the hepatic parasite burden depends on the granuloma formation, which is favored by a Th1 immune response in a Th2 tissue microenvironment. The aim of this work was to study the role of the recently described Th2 cytokine IL-33 in this complex immune response, which remains partially misunderstood. IL-33 dosages in different patient sera and IL-33+ cells detected in the liver of a patient from Rennes suggested that IL-33 could be a biomarker for active visceral leishmaniasis. As IL-33 was described in hepatic stellate cells during chronic hepatitis, these cells were exposed to L. donovani in primary culture. The cell permissivity to L. donovani and the parasite persistence during a long term culture led us to propose hepatic stellate cells as a new type of sanctuary cells, which could partially explain asymptomatic carriage. However, these cells were apparently not the main source of IL-33 during visceral leishmaniasis. In infected BALB/c and C57BL/6 mice, IL-33 was detected in the liver in non stellate cells preferentially localized in granulomas. The presence of cells expressing its specific receptor ST2 in the liver led us to explore the role of the IL-33/ST2 axis. BALB/c mice deficient in ST2 or treated with recombinant IL-33 and infected with L. donovani revealed that IL-33 downregulates the expression of Th1 key cytokines (IL-12, IFN-γ) and the recruitment of neutrophils and monocytes. Finally, IL-33 acts as a susceptibility factor during visceral leishmaniasis. Besides, the model of L. donovani infected C57BL/6 mice deficient in IL-33 or treated with recombinant IL-33 suggests possible differential roles of IL-33 depending on the immune environment related to the host genetic background.

Introdução: Já foi mostrado que o óxido nítrico (NO) age como um potente inibidor da peroxidação lipídica, um dos principais contribuintes para a fibrogênese na esteatohepatite não-alcoólica (EHNA). O S-Nitroso-N-acetilcisteína (SNAC), um doador de NO, modula a ativação de células estreladas hepáticas e tem mostrado efeitos benéficos na EHNA experimental. O objetivo deste estudo foi avaliar a eficácia do SNAC como um agente anti-fibrogênico na EHNA experimental. Métodos: Ratos Sprague-Dawley adultos foram alimentados com dieta hiperlipídica deficiente em colina e expostos a dietilnitrosamina (DEN) durante 8 semanas. Dez animais receberam SNAC (1,4 mg/ kg) por gavagem diariamente (grupo SNAC) e 10 receberam veículo (grupo EHNA). Três animais receberam somente dieta padrão e veículo (grupo Controle). Após este período, os animais foram sacrificados e o tecido hepático retirado para avaliação histológica, quantificação do colágeno e análises de expressão gênica. Genes relacionados à fibrose [metaloproteinase de matriz (MMP)- 2, 9 e 13, e, TGF b -1, colágeno-1a, inibidor tecidual de metaloproteinase (TIMP)-1 e 2] e genes relacionados ao estresse oxidativo [proteínas de choque térmico (HSP)-60 e 90] foram avaliados. Resultados: O SNAC levou a atenuação da fibrose hepática verificada pela quantificação de colágeno, e este efeito foi associado com supra-regulação da MMP- 13, MMP-9 e infra-regulação da HSP-60, TIMP-2, TGF b -1 e colágeno-1a. Conclusões: O SNAC apresenta propriedades anti-fibrogênicas no modelo de EHNA empregado, infra-regula moléculas pró-fibrogênicas e supra-regula a MMP-13, um fator determinante da degradação do colágeno intersticial. Nossa hipótese é que o SNAC, pela redução do estresse oxidativo, leva a um padrão de expressão gênica que favorece uma maior remoção de colágeno, com conseqüente atenuação da fibrose hepática na EHNA, sugerindo que SNAC é um potencial agente anti-fibrogênico neste contexto / Introduction: Nitric oxide (NO) has already been shown to act as a potent inhibitor of lipid peroxidation, a major contributor for fibrogenesis in nonalcoholic steatohepatitis (NASH). S-Nitroso-N-acetylcysteine (SNAC), an NO donor, modulates hepatic stellate cells activation and has shown beneficial effects on experimental NASH. The aim of this study was to evaluate the effectiveness of SNAC as an antifibrogenic agent in experimental NASH. Methods: Adult Sprague-Dawley rats were fed choline-deficient high fat diet and exposed to diethylnitrosamine during 8 weeks, during which 10 animals received SNAC (1.4 mg/kg) by daily gavage (SNAC group) and 10 animals received vehicle (NASH group). Another group (Control; n=3) received only standard diet and vehicle. After this period, liver tissue was obtained for histological study, collagen quantification and gene expression analyses. Genes related to fibrosis [matrix metalloproteinase (MMP)-13, 9 and 2, TGFb-1, collagen-1a, tissue inhibitor of metalloproteinase (TIMP)-1 and 2] and genes related to oxidative stress [heat-shock protein (HSP)-60 and 90] were evaluated. Results: SNAC led to attenuation of liver fibrosis, verified through collagen quantification, and this effect was associated with up-regulation of MMP-13, MMP-9 and down-regulation of HSP-60, TIMP-2, TGFb-1 and collagen-1a. Conclusions: SNAC shows antifibrogenic properties in the NASH model employed, down-regulates profibrogenic molecules and up-regulates MMP-13, a key determinant of interstitial collagen degradation. We hypothesize that SNAC, through oxidative stress lowering, leads to a gene expression pattern that favors greater collagen removal, with consequent attenuation of liver fibrosis in NASH. Our findings suggest that SNAC is a potential antifibrogenic agent in this setting

During embryonic development a variety of tissues and organs such as the lung, eye, and kidney are being formed. The generation of functional organs is regulated by reciprocal cell-cell interactions. Via the secretion of soluble molecules one type of cells affect the fate of their neighboring cells. A central issue in organogenesis is how a cell interprets such extrinsic signals and adopts a specific fate, and how the cell in response to this signal establishes reciprocal signaling. Transcription factors play a critical role in this process and my thesis focuses on the role of the LIM-homeodomain transcription factor, Lhx2, in the development of three different organ systems, the liver, the hematopoietic system and the olfactory system. The liver is formed from endoderm of the ventral foregut and mesenchyme of the septum transversum (st) and its development depends upon signaling interactions between these two tissues. As the liver becomes a distinct organ it is colonized by hematopoietic cells and serves as hematopoietic organ until birth. The fetal liver provides a microenvironment that supports the expansion of the entire hematopoietic system (HS) including the hematopoietic stem cells (HSCs). Liver development in Lhx2-/- embryos is disrupted leading to a lethal anemia due to insufficient support of hematopoiesis. To further investigate the role of Lhx2 in liver development I analyzed gene expression from the Lhx2 locus during liver development in wild-type and Lhx2-/- mice. Lhx2 is expressed in the liver associated st mesenchymal cells that become integrated in the liver and contribute to a subpopulation of hepatic stellate cells in adult liver. Lhx2 is not required for the formation of these mesenchymal cells, suggesting that the phenotype in Lhx2-/- livers is due to the presence of defective mesenchymal cells. The putative role of Lhx2 in the expansion of the HS was examined by introducing Lhx2 cDNA into embryonic stem cells differentiated in vitro. This approach allowed for the generation of immortalized multipotent hematopoietic progenitor cell (HPC) lines that share many characteristics with normal HSCs. The Lhx2-dependent generation of HSC-like cell lines suggests that Lhx2 plays a role in the maintenance and/or expansion of the HS. To isolate genes putatively linked to Lhx2 function, genes differentially expressed in the HPC lines were isolated using a cDNA subtraction approach. This allowed for the identification of a few genes putatively linked to Lhx2 function, as well as several stem cell-specific genes. The antagonist of Wnt signalling, Dickkopf-1 (Dkk-1), was identified in the former group of genes as it showed a similar expression pattern in the fetal liver, as that of Lhx2 and expression of Dkk-1 in fetal liver and in HPC lines appeared to be regulated by Lhx2. This suggests that Dkk-1 plays a role in liver development and/or HSC physiology during embryonic development. During development of the olfactory epithelium (OE) neuronal progenitors differentiate into mature olfactory sensory neurons (OSNs) that are individually specified into over a thousand different subpopulations, each expressing a unique odorant receptor (OR) gene. The expression of Lhx2 in olfactory neurons suggested a potential role for Lhx2 in the development of OSNs. To address this OE from Lhx2-/- and wild-type mice was compared. In the absence of functional Lhx2 neuronal differentiation was arrested prior to onset of OR expression. Lhx2 is thus required for the development of OSN progenitors into functional, individually specified OSNs. Thus, Lhx2 trigger a variety of cellular responses in different organ systems that play important roles in organ development in vivo and stem cell expansion in vitro.

Introdução: Já foi mostrado que o óxido nítrico (NO) age como um potente inibidor da peroxidação lipídica, um dos principais contribuintes para a fibrogênese na esteatohepatite não-alcoólica (EHNA). O S-Nitroso-N-acetilcisteína (SNAC), um doador de NO, modula a ativação de células estreladas hepáticas e tem mostrado efeitos benéficos na EHNA experimental. O objetivo deste estudo foi avaliar a eficácia do SNAC como um agente anti-fibrogênico na EHNA experimental. Métodos: Ratos Sprague-Dawley adultos foram alimentados com dieta hiperlipídica deficiente em colina e expostos a dietilnitrosamina (DEN) durante 8 semanas. Dez animais receberam SNAC (1,4 mg/ kg) por gavagem diariamente (grupo SNAC) e 10 receberam veículo (grupo EHNA). Três animais receberam somente dieta padrão e veículo (grupo Controle). Após este período, os animais foram sacrificados e o tecido hepático retirado para avaliação histológica, quantificação do colágeno e análises de expressão gênica. Genes relacionados à fibrose [metaloproteinase de matriz (MMP)- 2, 9 e 13, e, TGF b -1, colágeno-1a, inibidor tecidual de metaloproteinase (TIMP)-1 e 2] e genes relacionados ao estresse oxidativo [proteínas de choque térmico (HSP)-60 e 90] foram avaliados. Resultados: O SNAC levou a atenuação da fibrose hepática verificada pela quantificação de colágeno, e este efeito foi associado com supra-regulação da MMP- 13, MMP-9 e infra-regulação da HSP-60, TIMP-2, TGF b -1 e colágeno-1a. Conclusões: O SNAC apresenta propriedades anti-fibrogênicas no modelo de EHNA empregado, infra-regula moléculas pró-fibrogênicas e supra-regula a MMP-13, um fator determinante da degradação do colágeno intersticial. Nossa hipótese é que o SNAC, pela redução do estresse oxidativo, leva a um padrão de expressão gênica que favorece uma maior remoção de colágeno, com conseqüente atenuação da fibrose hepática na EHNA, sugerindo que SNAC é um potencial agente anti-fibrogênico neste contexto / Introduction: Nitric oxide (NO) has already been shown to act as a potent inhibitor of lipid peroxidation, a major contributor for fibrogenesis in nonalcoholic steatohepatitis (NASH). S-Nitroso-N-acetylcysteine (SNAC), an NO donor, modulates hepatic stellate cells activation and has shown beneficial effects on experimental NASH. The aim of this study was to evaluate the effectiveness of SNAC as an antifibrogenic agent in experimental NASH. Methods: Adult Sprague-Dawley rats were fed choline-deficient high fat diet and exposed to diethylnitrosamine during 8 weeks, during which 10 animals received SNAC (1.4 mg/kg) by daily gavage (SNAC group) and 10 animals received vehicle (NASH group). Another group (Control; n=3) received only standard diet and vehicle. After this period, liver tissue was obtained for histological study, collagen quantification and gene expression analyses. Genes related to fibrosis [matrix metalloproteinase (MMP)-13, 9 and 2, TGFb-1, collagen-1a, tissue inhibitor of metalloproteinase (TIMP)-1 and 2] and genes related to oxidative stress [heat-shock protein (HSP)-60 and 90] were evaluated. Results: SNAC led to attenuation of liver fibrosis, verified through collagen quantification, and this effect was associated with up-regulation of MMP-13, MMP-9 and down-regulation of HSP-60, TIMP-2, TGFb-1 and collagen-1a. Conclusions: SNAC shows antifibrogenic properties in the NASH model employed, down-regulates profibrogenic molecules and up-regulates MMP-13, a key determinant of interstitial collagen degradation. We hypothesize that SNAC, through oxidative stress lowering, leads to a gene expression pattern that favors greater collagen removal, with consequent attenuation of liver fibrosis in NASH. Our findings suggest that SNAC is a potential antifibrogenic agent in this setting

[ES] La resistencia a la insulina se define como un aumento en la cantidad de insulina necesaria para conseguir la homeostasis de glucosa. Una de las complicaciones más comunes de la resistencia a la insulina es el defecto en la reparación de herida. El sustrato 2 del receptor de la insulina (IRS2) es un mediador clave para la señalización de insulina en hígado que actúa de puente entre los receptores de la insulina y del factor de crecimiento insulínico (IGF-1) y sus cascadas de señalización. Tanto la resistencia a la insulina como cambios en los niveles de expresión de IRS2 han sido asociados con el desarrollo y la progresión de enfermedades hepáticas graves. El daño hepático crónico generado por algunos factores derivados de la resistencia a la insulina ha sido establecido como determinante en la patofisiología de las enfermedades hepáticas. Sin embargo, sigue siendo una incógnita cómo el daño hepático generado por la resistencia a la insulina escapa de la extraordinaria capacidad de regeneración del hígado. Durante el daño hepático crónico, la reparación epitelial está mediada por las células progenitoras hepáticas (CPH) que se expanden y diferencian hasta hepatocitos o células biliares rodeadas de un nicho estromal formado por un conjunto de células estrelladas hepáticas (CEH), células inflamatorias, componentes de la matriz extracelular (ECM) y factores de crecimiento. El factor de crecimiento de fibroblastos 7 (FGF7), expresado por las CEH, resulta crítico para la respuesta de las CPH y para la regeneración hepática. Durante el daño hepático crónico las CEH se activan transdiferenciandose desde células quiescentes a células fibrogénicas (CEHa) denominadas miofibroblastos que depositan ECM para reemplazar el tejido dañado. Para alcanzar una correcta regeneración hepática se requiere la resolución de la activación ("reversión fibrogénica") de las CEH. Empleando el modelo de ratón Irs2-/- durante el daño hepático crónico con la dieta DDC 0.1% y los modelos in vitro humanos de CPH (HepaRG) y de CEH (CEH primarias y la línea celular LX-2), los resultados de este trabajo demuestran que la señalización de insulina-IRS2 es necesaria para la epitelización dirigida por la comunicación paracrina a través de FGF7 en el nicho de CPH. Por un lado, IRS2 es necesario en la población de CEH para permitir su supervivencia durante la reversión fibrogénica, un proceso que según nuestros resultados induce un aumento en la expresión de FGF7. Nuestros datos descubren un potencial mecanismo de regulación mediante el que IRS2 induce la expresión de FGF7 en CEH a través de la remodelación en la ECM mediada por NRF2 y el integrante de la ECM SERPINE1 durante las etapas tempranas de la reversión fibrogénica. El eje NRF2-SERPINE1 ha sido descrito anteriormente en fibroblastos de piel como esencial para la reepitelización de herida. NRF2 es el principal factor de transcripción de respuesta frente al estrés oxidativo (ruta canónica). En hepatocitos, la activación de NRF2 también puede inducirse a través de una ruta no canónica mediada por la proteína cargo de la autofagia P62. A pesar de que nuestros datos descubren a P62 como capaz de inducir la actividad de NRF2 en CPH, también demuestran que IRS2 activa a NRF2 en CPH y en CEH de manera independiente a la ruta no canónica mediada por P62. Por otro lado, demostramos que la señalización de insulina-IRS2, por promover la producción de FGF7, permite un novedoso bucle de inducción positiva mediante el que la respuesta a FGF7 en CPH promueve la expresión de su receptor, FGFR2b, favoreciendo su propia sensibilidad y sosteniendo la reparación epitelial. Futuras estrategias para potenciar en hígado la actividad de NRF2 y la señalización de FGF7 podrían servir para mejorar el pronóstico de los pacientes con resistencia a la insulina, diabetes o enfermedad metabólica por promover la reparación epitelial en hígado y reducir su riesgo de desarrollar patologías hepáticas graves con elevada tasa de mortalidad. / [CAT] La resistència a la insulina es defineix com un augment en la quantitat d'insulina necessària per a aconseguir l'homeòstasi de glucosa. Una de les complicacions més freqüents de la resistència a la insulina és el defecte en la reparació de ferida. El substrat 2 del receptor de la insulina (IRS2) és un mediador clau per a la senyalització d'insulina en fetge que actua de pont entre els receptors de la insulina i del factor de creixement insulínic (IGF-1) i les seues cascades de senyalització. Tant la resistència a la insulina com els canvis en els nivells d'expressió d'IRS2 han sigut associats amb el desenvolupament i la progressió de malalties hepàtiques greus. El mal hepàtic crònic generat per alguns factors derivats de la resistència a la insulina s'ha establert com a determinant en la patofisiologia de les malalties hepàtiques. No obstant això, els motius pels quals el mal hepàtic generat per la resistència a la insulina escapa a l'extraordinària capacitat de regeneració del fetge són encara una incògnita. Durant el mal crònic, la reparació epitelial està mediada per les cèl·lules progenitores hepàtiques (CPH) que s'expandeixen i diferencien fins a hepatòcits o cèl·lules biliars envoltades d'un nínxol estromal format per un conjunt de cèl·lules estavellades hepàtiques (CEH), cèl·lules inflamatòries, components de la matriu extracelul·lar (ECM) i factors de creixement. El factor de creixement de fibroblasts 7 (FGF7), expressat en fetge per les CEH, resulta crític per a la resposta de les CPH i per a la regeneració hepàtica. Durant el mal hepàtic crònic les CEH s'activen transdiferenciant-se des de cèl·lules quiescents a cèl·lules fibrogèniques denominades miofibroblasts (CEH activades) que depositen ECM per a reemplaçar el teixit danyat. Per a aconseguir una correcta regeneració hepàtica es requereix la resolució de l'activació ("reversió fibrogènica") de les CEH. A partir de l'ús del model de ratolí Irs2-/- durant el mal hepàtic crònic amb la dieta DDC 0.1% i dels models in vitro humans de CPH (HepaRG) i de CEH (CEH primàries i la línia cel·lular LX-2), els resultats d'aquest treball demostren que la senyalització d'insulina-IRS2 és necessària per a l'epitelització dirigida per la comunicació paracrina mitjançant FGF7 en el nínxol de CPH. D'una banda, IRS2 és necessari en la població de CEH per a permetre la seua supervivència durant la reversió fibrogènica, que comporta un augment en l'expressió de FGF7. Les nostres dades descobreixen un potencial mecanisme de regulació mitjançant el qual IRS2 indueix l'expressió de FGF7 en CEH a través de la remodelació en la ECM mediada per NRF2 i per l'integrant de la ECM SERPINE1, que ocorre en les etapes primerenques de la reversió fibrogènica. L'eix NRF2-SERPINE1 ha sigut identificat anteriorment en fibroblasts de pell com a essencial per a la reparació de ferida. NRF2 és el principal factor de transcripció de resposta davant de l'estrés oxidatiu (ruta canònica). En hepatòcits, l'activació de NRF2 també pot induir-se a través d'una ruta no canònica mediada per la proteïna de càrrega de l'autofàgia P62. A pesar que les nostres dades indiquen que P62 és capaç d'induir l'activitat de NRF2 en CPH, també demostren que IRS2 activa NRF2 en CPH i CEH de manera independent a la ruta no canònica mediada per P62. D'altra banda, demostrem que la senyalització d'insulina-IRS2, per promoure la producció de FGF7, permet un nou bucle d'inducció positiva mitjançant el qual la resposta a FGF7 en CPH promou l'expressió del seu receptor, FGFR2b, afavorint la seua pròpia sensibilitat i sostenint la reparació epitelial. Futures estratègies per a potenciar en fetge l'activitat de NRF2 i la senyalització de FGF7 podrien servir per a millorar el pronòstic dels pacients amb resistència a la insulina, diabetis o malaltia metabòlica, per promoure la reparació epitelial en fetge i reduir, per tant, el seu risc de desenvolupar patologies hepàtiques greus amb elevada taxa de mortalitat. / [EN] Insulin resistance is defined as an increase in the amount of insulin that is necessary to achieve glucose homeostasis. One of the most prevalent complications of insulin resistance is the wound healing defect. Insulin receptor factor 2 (IRS2) is a key mediator of the insulin signaling in liver, which acts as a bridge between insulin and insulin growth factor 1 (IGF-1) receptors and its downstream molecular pathways. Both, insulin resistance and changes in the expression levels of IRS2, have been associated with the development and progression of severe liver diseases. Chronic liver injury produced by insulin resistance has been stablished as crucial in the pathophysiology of liver disease. However, it remains unknown how the chronic liver injury produced by insulin resistance escapes from the extraordinary ability of the liver to regenerate. During chronic liver injury, epithelial repair is mediated by liver progenitor cells (LPC), that expand and differentiate into hepatocytes and cholangiocytes surrounded by a stromal niche that consist of hepatic stellate cells (HSC), inflammatory cells, extracellular matrix (ECM) components and growth factors. Fibroblast growth factor 7 (FGF7), expressed by HSC, is critic for LPC response and liver regeneration. During chronic liver injury, HSC transdifferentiate from quiescent to active fibrogenic HSC (aHSC) called myofibroblast. aHSC deposit ECM to replace damaged tissue. A successful regeneration requires the resolution of the HSC activation, i.e., the "fibrogenic reversion" of HSC. Using the Irs2-/- mice model during chronic liver damage induced by 0.1% DDC diet and the human in vitro models of LPC (HepaRG) and HSC (primary HSC and the cell line LX-2), our results reveal a new role of insulin-IRS2 in the modulation of the paracrine FGF7 crosstalk in the LPC niche that drives LPC epithelization. On the one hand, IRS2 is necessary in HSC to allow survival during fibrogenic reversion, a process that according to our data induces an increase in FGF7 expression. Our results reveal a potential mechanism by which IRS2 promotes FGF7 expression in HSC through an ECM remodeling process that is mediated by NRF2 and the ECM constituent SERPINE1 during the early stages of fibrogenic reversion. NRF2-SERPINE1-mediated ECM remodeling has been previously identified in skin fibroblast as essential to promote re-epithelialization of wounds. NRF2 is a transcription factor that is activated in response to oxidative stress (canonical pathway). NRF2 activation in hepatocytes can be also induced by a non-canonical pathway mediated by the autophagy cargo protein P62. Although our data discovers a new ability of P62 to induce NRF2 activity in LPC, we also demonstrate that IRS2 activates NRF2 in LPC and HSC in a P62-independent manner. On the other hand, we demonstrate that insulin-IRS2 signaling, by promoting FGF7 production, enables a novel positive induction loop whereby FGF7 response in LPC promotes the expression of its receptor, FGFR2b, favoring its own sensitivity and sustaining epithelial repair. Future strategies to enhance NRF2 activity or FGF7 signaling in liver might be useful to improve the prognosis of insulin resistance, diabetic, and metabolic disease patients because of its uncovered ability to promote epithelial repair, thus, preventing the development of severe liver pathologies with high mortality risk. / Arámbul Anthony, MJ. (2022). Papel de IRS2 en la regulación de la comunicación a través de FGFs en el nicho de células progenitoras hepáticas [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/188913