Saturated fatty acids differently affect mitochondrial function and the intestinal epithelial barrier depending on their chain length in the in vitro model of IPEC-J2 enterocytes

Introduction: Maintenance of the intestinal barrier mainly relies on the mitochondrial function of intestinal epithelial cells that provide ATP through oxidative phosphorylation (OXPHOS). Dietary fatty acid overload might induce mitochondrial dysfunction of enterocytes and may increase intestinal permeability as indicated by previous in vitro studies with palmitic acid (C16:0). Yet the impact of other dietary saturated fatty acids remains poorly described.Methods: To address this question, the in vitro model of porcine enterocytes IPEC-J2 was treated for 3 days with 250 µM of lauric (C12:0), myristic (C14:0), palmitic (C16:0) or stearic (C18:0) acids.Results and discussion: Measurement of the transepithelial electrical resistance, reflecting tight junction integrity, revealed that only C16:0 and C18:0 increased epithelial permeability, without modifying the expression of genes encoding tight junction proteins. Bioenergetic measurements indicated that C16:0 and C18:0 were barely β-oxidized by IPEC-J2. However, they rather induced significant OXPHOS uncoupling and reduced ATP production compared to C12:0 and C14:0. These bioenergetic alterations were associated with elevated mitochondrial reactive oxygen species production and mitochondrial fission. Although C12:0 and C14:0 treatment induced significant lipid storage and enhanced fusion of the mitochondrial network, it only mildly decreased ATP production without altering epithelial barrier. These results point out that the longer chain fatty acids C16:0 and C18:0 increased intestinal permeability, contrary to C12:0 and C14:0. In addition, C16:0 and C18:0 induced an important energy deprivation, notably via increased proton leaks, mitochondrial remodeling, and elevated ROS production in enterocytes compared to C12:0 and C14:0

Rôle des métabolites du lait maternel (Acides Gras à Chaîne Courte et Polyamines) sur la physiologie intestinale : étude dans un modèle in vitro d’épithélium intestinal

International audienceLe lait maternel (LM) est associé à des bénéfices majeurs à court et long termes sur lasanté et le développement des nourrissons. Cependant à travers le monde, les préparationspour nourrissons (PPN), substituts du LM, sont largement utilisées. Bien que ces PPN répondentaux besoins nutritionnels des nouveau-nés, elles sont dépourvues de nombreux composantsprésents dans le LM tels que les oligosaccharides du LM, des immunoglobulines, une multitudede métabolites ainsi qu’une fraction microbienne complexe. Parmi les métabolites présentsdans le LM, on retrouve des acides gras à chaîne courte (butyrate, acétate, propionate) ainsique des polyamines (spermine, spermidine, putrescine). Ces métabolites sont connus pour êtreproduits au sein du tractus gastro-intestinal avec des effets sur la physiologie de l’hôte.Cependant, leurs apports par le LM sur le développement du nourrisson sont peu connus.L’objectif de mon travail a donc été d’étudier leurs effets sur les différentes fonctionsintestinales via un modèle in vitro d’épithélium intestinal composé de quatre types cellulaires :des Caco-2 (modèle d’entérocyte), des HT29-MTX (modèle de cellule à mucus), des NCI-H716(modèle de cellule entéro-endocrine) et des cellules M. Nous avons exposé ce modèle auxmétabolites aux concentrations du LM et mesuré par biologie moléculaire l’expression de gènesmarqueurs des différentes fonctions intestinales. Ainsi nous avons mis en évidence que lebutyrate et les polyamines régulaient à la hausse l’expression certains gènes des jonctionsserrées tels que les claudines 1, 3, 4, 7 et l’occludine. Ces métabolites ont également étécapables d’agir sur l’expression de gènes de l’immunité tels que le récepteur à l’IL-22R, MyD88et TGFB1. Les métabolites du LM semblent donc impacter la fonction barrière et immunitairede l’intestin et la poursuite de leur étude est primordiale pour comprendre leur rôle sur ledéveloppement du nourrisson afin d’envisager leur incorporation dans les PPN

Human milk metabolites are bioactive and can modulate gut physiology : in vitro study in a pluricellular model of intestinal epithelium

International audienceHuman milk (HM) is associated with major short- and long-term health benefits for infants. However, infant formulas (IF), substitutes for HM, are widely used for infant nutrition. Although these IFs meet the nutritional needs of newborns, they are devoid of many bioactive compounds present in HM, such as immunoglobulins, hormones and a multitude of metabolites. HM metabolites include short chain fatty acids (SCFAs) (butyric acid, acetic acid, propionic acid), polyamines (putrescine, spermine, spermidine), tryptophan derivatives (indole, indole-lactic acid, kynurenine) as well as GABA and lactate. These metabolites are known to be produced within the gastrointestinal tract by the intestinal microbiota and to have effects on the host physiology but their relevance in HM has not been studied so far. The objective of our study was therefore to decipher the effects of HM metabolites on intestinal physiology using an in vitro pluricellular and polarized model of intestinal epithelium, including Caco-2 (as enterocytes), HT-29 MTX (as goblet cells), NCI-H716 (as enteroendocrine cells) and M cells. HM metabolites were studied at concentrations close to those found in HM, that is quite low. Their effects on trans-epithelial electric resistance (TEER) and on the expression of genes involved in the intestinal barrier, immune, antioxidant, endocrine and digestive functions were analyzed. SCFAs strongly modulated different intestinal functions, particularly the immune one with a significant downregulation of genes coding for IL-8, MyD88 and TFF3. They also modulated genes encoding tight junctions, as did GABA and polyamines, upregulating CLDN3, TJP1 and CLDN4 respectively and downregulating CLDN1 for SCFA and polyamines and CLDN7 for GABA. In parallel SCFAs significantly increased TEER, highlighting a potential reinforcing effect on the epithelial barrier while polyamines and GABA had no effect on TEER. Finally, SCFAs, GABA and lactic acid modulated the expression of some transporters involved in nutrition such as MCT1, GLUT1 and SGLT1 respectively. HM metabolites, despite their low concentration, are able to impact the intestinal barrier physiology, inviting us to consider their implementation in IF for a better mimicry of HM health benefits

Alterations of mitochondrial function and epithelial homeostasis by saturated fatty acids according to their nature on the in vitro model of enterocyte IPEC-J2

International audienceObesity is characterized by a low-grade inflammation associated with disturbances of small intestine permeability. The latter notably relieson mitochondrial function of intestinal epithelial cells (IEC). Yet changes in lipid metabolism of IEC induced by high fat diet (HFD) mightalter mitochondrial function of IEC. We thus wondered whether saturated fats alter mitochondrial function of IEC and intestinal permeability.The in vitro model of enterocyte IPEC-J2 was treated for 3 days with lauric (C12:0), myristic (C14:0), palmitic (C16:0) and stearic (C18:0)acids, abundantly found in HFDs, alone at 250 μM or in mix with 250 μM each. Mitochondrial function was assessed by the Seahorsetechnology while epithelial permeability was evaluated by measuring the transepithelial electrical resistance.Treatment with the mix of fatty acids induced enterocyte steatosis and oxidative stress concomitant to antioxidant machinery activationbut decreased β-oxidation activity during the first hours of treatment, indicating enterocyte metabolic adaptation. Mitochondrial functionwas altered after 3 days of treatment with marked decreased respiration and lower mitochondrial ATP production rate linked with increasedepithelial permeability compared to control cells. Although 3 days of treatment with each fatty acid provoked enterocyte steatosis, C12:0and C14:0 induced greater lipid storage than C16:0 and C18:0. Only C16:0 decreased mitochondrial respiration of IPEC-J2 while C16:0 andC18:0 lowered the mitochondrial ATP production rate and increased epithelial permeability.In conclusion, chronic treatment with a mix of fatty acids simulating HFD, induced alterations of enterocyte metabolism and increasedpermeability likely due to the effect of C16:0 and/or C18:0

Les métabolites bactériens naturellement présents dans le lait maternel possèdent des propriétés bioactives : étude in vitro dans un modèle quadricellulaire d’épithélium intestinal et un modèle de neurones centraux.

International audienceLe lait maternel (LM) est un aliment complexe contenant de nombreux éléments dont des métabolites. Ceux-ci sont d’origine maternelle ou peuvent être synthétisés par les bactéries naturellement présentes dans le LM. C’est le cas des acides gras à chaîne courte (AGCC) (butyrate, acétate, propionate), des polyamines (putrescine, spermine, spermidine), du GABA et du lactate, qui sont retrouvés à faible concentration dans le LM. Ces métabolites sont connus pour être produits par le microbiote intestinal et être des acteurs de l’axe microbiote-intestin-cerveau, mais leur rôle au sein du LM a été peu étudié. L’objectif de cette étude a été d’évaluer les propriétés bioactives de ces métabolites bactériens sur les fonctions intestinales et cérébrales du nouveau-né. Ces métabolites ont été étudiés in vitro, à des concentrations proches de celles du LM, dans un modèle pluricellulaire et polarisé d’épithélium intestinal composé de Caco-2 (entérocytes), de HT-29-MTX (cellules à Gobelet), de NCI-H716 (cellules entéroendocrines) et de cellules M, ainsi que dans un modèle de neurones centraux. Les effets des métabolites sur la résistance électrique transépithéliale (TEER) et l'expression de marqueurs géniques ou protéiques des fonctions intestinales et cérébrales ont été analysés. Les AGCC ont agi sur des marqueurs de l’immunité intestinale en régulant négativement les gènes codant pour l'IL-8, MyD88 et TFF3. Ils ont également modulé l’expression de certaines jonctions serrées intestinales, tout comme le GABA et les polyamines, en augmentant l’expression de CLDN3, ZO-1 et CLDN4 respectivement et en diminuant celle de CLDN1 pour les AGCC et les polyamines et CLDN7 pour le GABA. En parallèle, les AGCC ont significativement augmenté la TEER, mettant en évidence un potentiel renforcement sur la barrière épithéliale. Les AGCC et le GABA ont également modulé certains marqueurs de connectivité et de maturation neuronales (synaptophysine, synapsine, PSD95 et doublecortine). En conclusion, notre étude montre que les métabolites bactériens du LM, malgré leur faible concentration, sont capables de moduler les fonctions intestinales et cérébrales in vitro, suggérant un impact potentiel sur la physiologie et développement du nourrisson

Saturated fatty acids differently affect mitochondrial function and the intestinal epithelial barrier depending on their chain length in the in vitro model of IPEC-J2 enterocytes

International audienceIntroduction: Maintenance of the intestinal barrier mainly relies on the mitochondrial function of intestinal epithelial cells that provide ATP through oxidative phosphorylation (OXPHOS). Dietary fatty acid overload might induce mitochondrial dysfunction of enterocytes and may increase intestinal permeability as indicated by previous in vitro studies with palmitic acid (C16:0). Yet the impact of other dietary saturated fatty acids remains poorly described. Methods: To address this question, the in vitro model of porcine enterocytes IPEC-J2 was treated for 3 days with 250 μM of lauric (C12:0), myristic (C14:0), palmitic (C16:0) or stearic (C18:0) acids.Results and discussion: Measurement of the transepithelial electrical resistance, reflecting tight junction integrity, revealed that only C16:0 and C18:0 increased epithelial permeability, without modifying the expression of genes encoding tight junction proteins. Bioenergetic measurements indicated that C16:0 and C18:0 were barely β-oxidized by IPEC-J2. However, they rather induced significant OXPHOS uncoupling and reduced ATP production compared to C12:0 and C14:0. These bioenergetic alterations were associated with elevated mitochondrial reactive oxygen species production and mitochondrial fission. Although C12:0 and C14:0 treatment induced significant lipid storage and enhanced fusion of the mitochondrial network, it only mildly decreased ATP production without altering epithelial barrier. These results point out that the longer chain fatty acids C16:0 and C18:0 increased intestinal permeability, contrary to C12:0 and C14:0. In addition, C16:0 and C18:0 induced an important energy deprivation, notably via increased proton leaks, mitochondrial remodeling, and elevated ROS production in enterocytes compared to C12:0 and C14:0

Radiosensitizing Fe-Au nanocapsules (hybridosomes®) increase survival of GL261 brain tumor-bearing mice treated by radiotherapy

International audienceGlioblastoma remains a cancer for which the effectiveness of treatments has shown little improvement over the last decades. For this pathology, multiple therapies combining resection, chemotherapy and radiotherapy remain the norm. In this context, the use of high-Z nanoparticles such as gold or hafnium to potentiate radiotherapy is attracting more and more attention. Here, we evaluate the potentiating effect of hollow shells made of gold and iron oxide nanoparticles (hybridosomes®) on the radiotherapy of glioblastoma, using murine GL261-Luc+ brain tumor model. While iron oxide seems to have no beneficial effect for radiotherapy, we observe a real effect of gold nanoparticles —despite their low amount— with a median survival increase of almost 20% compared to radiotherapy only and even 33% compared to the control group. Cellular and in vivo studies show that a molecule of interest nano-precipitated in the core of the hybridosomes® is released and internalized by the surrounding brain cells. Finally, in vivo studies show that hybridosomes® injected intra-tumorally are still present in the vicinity of the brain tumor more than 5 days after injection (duration of the Stupp protocol’s radiation treatment). Interestingly, one mouse treated with radiotherapy in the presence of gold-containing hybridosomes® survived 78 days. Monitoring of the tumoral growth of this long-term survivor using both MRI and bioluminescence revealed a decrease of the tumor size after treatment. These very encouraging results are a proof-of-concept that hybridosomes®, are really effective tools for combined therapies development (chemo-radiotherapy)

Radiosensitizing Fe-Au nanocapsules (hybridosomes®) increase survival of GL261 brain tumor-bearing mice treated by radiotherapy

International audienceGlioblastoma remains a cancer for which the effectiveness of treatments has shown little improvement over the last decades. For this pathology, multiple therapies combining resection, chemotherapy and radiotherapy remain the norm. In this context, the use of high-Z nanoparticles such as gold or hafnium to potentiate radiotherapy is attracting more and more attention. Here, we evaluate the potentiating effect of hollow shells made of gold and iron oxide nanoparticles (hybridosomes®) on the radiotherapy of glioblastoma, using murine GL261-Luc+ brain tumor model. While iron oxide seems to have no beneficial effect for radiotherapy, we observe a real effect of gold nanoparticles —despite their low amount— with a median survival increase of almost 20% compared to radiotherapy only and even 33% compared to the control group. Cellular and in vivo studies show that a molecule of interest nano-precipitated in the core of the hybridosomes® is released and internalized by the surrounding brain cells. Finally, in vivo studies show that hybridosomes® injected intra-tumorally are still present in the vicinity of the brain tumor more than 5 days after injection (duration of the Stupp protocol’s radiation treatment). Interestingly, one mouse treated with radiotherapy in the presence of gold-containing hybridosomes® survived 78 days. Monitoring of the tumoral growth of this long-term survivor using both MRI and bioluminescence revealed a decrease of the tumor size after treatment. These very encouraging results are a proof-of-concept that hybridosomes®, are really effective tools for combined therapies development (chemo-radiotherapy)

Radiosensitizing Fe-Au Nanocapsules (Hybridosomes®) increase survival of GL261 brain tumor-bearing mice treated by radiotherapy

International audienceGlioblastoma remains a cancer for which the effectiveness of treatments has shown little improvement over the last decades. For this pathology, multiple therapies combining resection, chemotherapy and radiotherapy remain the norm. In this context, the use of high-Z nanoparticles such as gold or hafnium to potentiate radiotherapy is attracting more and more attention. Here, we evaluate the potentiating effect of hollow shells made of gold and iron oxide nanoparticles (hybridosomes®) on the radiotherapy of glioblastoma, using murine GL261-Luc+brain tumor model. While iron oxide seems to have no beneficial effect for radiotherapy, we observe a real effect of gold nanoparticles -despite their low amount- with a median survival increase of almost 20% compared to radiotherapy only and even 33% compared to the control group. Cellular and in vivo studies show that a molecule of interest nano-precipitated in the core of the hybridosomes® is released and internalized by the surrounding brain cells. Finally, in vivo studies show that hybridosomes® injected intra-tumorally are still present in the vicinity of the brain tumor more than 5days after injection (duration of the Stupp protocol’s radiation treatment). Interestingly, one mouse treated with radiotherapy in the presence of gold-containing hybridosomes® survived 78days. Monitoring of the tumoral growth of this long-term survivor using both MRI and bioluminescence revealed a decrease of the tumor size after treatment. These very encouraging results are a proof-of-concept that hybridosomes®, are really effective tools for combined therapies development (chemo-radiotherapy)

Orthotopic model of lung cancer: isolation of bone micro-metastases after tumor escape from Osimertinib treatment

International audienceBackground: Osimertinib is a third generation tyrosine kinase inhibitor (TKI) that targets the epidermal growth factor receptor (EGFR) in lung cancer. However, although this molecule is not subject to some of the resistance mechanisms observed in response to first generation TKIs, ultimately, patients relapse because of unknown resistance mechanisms. New relevant non-small cell lung cancer (NSCLC) mice models are therefore required to allow the analysis of these resistance mechanisms and to evaluate the efficacy of new therapeutic strategies. Methods: Briefly, PC-9 cells, previously modified for luciferase expression, were injected into the tail vein of mice. Tumor implantation and longitudinal growth, almost exclusively localized in the lung, were evaluated by bioluminescence. Once established, the tumor was treated with osimertinib until tumor escape and development of bone metastases. Results: Micro-metastases were detected by bioluminescence and collected for further analysis. Conclusion: We describe an orthotopic model of NSCLC protocol that led to lung primary tumor nesting and, after osimertinib treatment, by metastases dissemination, and that allow the isolation of these small osimertinib-resistant micro-metastases. This model provides new biological tools to study tumor progression from the establishment of a lung tumor to the generation of drug-resistant micro-metastases, mimicking the natural course of the disease in human NSCLC patients