Biological effects of double-walled carbon nanotubes on the innate immune system: An in vitro study on THP-1 human monocytes

DWCNTs have numerous industrial and biomedical applications and several studies reported that they could act as immunomodulator systems. The immune system is the first line of defence of the human body when exposed to particulate matter. In order to investigate DWCNTs’ role on innate immunity, we used THP-1 monocytic cells for the purpose of this study. We showed that DWCNTs were not cytotoxic until 6 h, 24 h, 48 h and 72 h of incubation with THP-1 monocytic cells (concentrations tested from 10 to 50 μg/mL). From 6 h to 72 h of incubation of THP-1 cells with DWCNTs, we measured a significant increase of the baseline cell index using xCELLigence® technology showing cell adhesion. After 24 h of exposure, DWCNTs agglomerates were localized in THP-1 monocyte cytoplasm and cell adhesion was observed simultaneously with a significant increase in the expression of CD11b and CD14 cell surface proteins. Pro-inflammatory cytokine secretion (IL-1β, IL-6, IL-8, TNF-α and IL-10) was also measured in supernatants after 6 h or 24 h of exposure to DWCNTs. This pro-inflammatory response was increased in THP-1 monocytic cells pre-treated with LPS. Altogether, our data indicate that DWCNTs induce an increased pro-inflammatory response of THP-1 monocytes and seem to modulate cell surface protein expression confirming that DWCNTs could act as stimulators of innate immunity

Nose-only inhalations of high-dose alumina nanoparticles/hydrogen chloride gas mixtures induce strong pulmonary pro-inflammatory response: a pilot study

Objective Solid composite propellants combustion, in aerospace and defense fields, can lead to complex aerosols emission containing high concentrations of alumina nanoparticles (Al2O3 NPs) and hydrogen chloride gas (HClg). Exposure to these mixtures by inhalation is thus possible but literature data toward their pulmonary toxicity are missing. To specify hazards resulting from these combustion aerosols, a pilot study was implemented. Materials and methods Male Wistar rats were nose-only exposed to Al2O3 NPs (primary size 13 nm, 10 g/L suspension leading to 20.0–22.1 mg/m3 aerosol) and/or to HClg aerosols (5 ppm target concentration) following two exposure scenarios (single exposures (SE) or repeated exposures (RE)). Bronchoalveolar lavage fluids (BALF) content and lungs histopathology were analyzed 24 h after exposures. Results Repeated co-exposures increased total proteins and LDH concentrations in BALF indicating alveolar–capillary barrier permeabilization and cytolysis. Early pulmonary inflammation was induced after RE to Al2O3 NPs ± HClg resulting in PMN, TNF-α, IL-1β, and GRO/KC increases in BALF. Both exposure scenarios resulted in pulmonary histopathological lesions (vascular congestions, bronchial pre-exfoliations, vascular and interalveolar septum edemas). Lung oxidative damages were observed in situ following SE. Conclusion Observed biological effects are dependent on both aerosol content and exposure scenario. Results showed an important pro-inflammatory effect of Al2O3 NPs/HClg mixtures on the lungs of rat 24 h after exposure. This pilot study raises concerns toward potential long-term pulmonary toxicity of combustion aerosols and highlights the importance for further studies to be led in order to define dose limitations and exposure thresholds for risk management at the work place

Development of in vitro models of the alveolo-capillary barrier to study the toxicity and the passage of nanoparticles

Après exposition par inhalation, les nanoparticules (NPs) peuvent atteindre les alvéoles pulmonaires, se retrouver au niveau de la barrière alvéolo-capillaire (BAC), et induire une toxicité locale et / ou franchir cette barrière pour se retrouver dans la circulation sanguine. Dans ce contexte, l’objectif de ce travail a été de développer des modèles de co-cultures in vitro simples à mettre en œuvre (utilisation de lignées cellulaires humaines), pour étudier les effets des NPs au niveau de la BAC. Dans un premier temps, des co-cultures de cellules épithéliales alvéolaires ou de phénotype proche (lignées A549 ou NCI-H441), et de macrophages (lignée THP-1), ont permis l’étude des effets pro-inflammatoires des NPs de SiO2 et de TiO2. Avec ces modèles nous avons montré l’importance de la coopération cellulaire mise en jeu lors des processus inflammatoires liés aux NPs, mais aussi le rôle du ratio cellulaire employé dans ces réponses. Dans un second temps, des co-cultures tridimensionnelles en chambres bicamérales associant des macrophages (lignée THP-1), des cellules épithéliales bronchiques (lignée Calu-3), et des cellules endothéliales pulmonaires microvasculaires (lignée HPMEC-ST1.6R), ont permis l’étude de l’impact de NPs fluorescentes de polystyrène sur l’intégrité de la BAC, et leur passage à travers cette barrière. Les cellules épithéliales Calu-3 permettent d’établir une barrière de qualité mais la membrane microporeuse servant de support aux cellules doit être optimisée pour ne pas être un frein au passage des NPs. Ce travail montre qu’un seul modèle ne permet pas d’étudier de façon optimale à la fois la toxicité et la translocation des NPs, et qu’une approche adaptée doit être envisagée en fonction du paramètre que l’on souhaite étudier.After inhalation, nanoparticles (NPs) can reach the alveoli and the alveolo-capillary barrier (ACB), and consequently induce local toxicity and / or cross this barrier to reach the bloodstream. In this context, the aim of this work was to develop co-culture in vitro models simple to implement (using human cell lines), to study effects of NPs on the ACB. In a first time, pro-inflammatory effects of SiO2 and TiO2 NPs were studied on co-cultures of alveolar epithelial cells (A549 and NCI-H441 cell lines), and macrophages (THP-1 cell line). We demonstrated the importance of cell cooperation during inflammatory processes caused by these NPs, and the role of the cellular ratio in these inflammatory responses. In a second time, effects of fluorescent polystyrene NPs on the ACB integrity, and their translocation were studied on three-dimensional co-cultures in bicameral chambers involving macrophages (THP-1 cell line), bronchial epithelial cells (Calu-3 cell line), and micro-vascular pulmonary endothelial cells (HPMEC ST1.6R cell line). The use of Calu-3 has provided a good barrier, but further investigations on microporous membranes are still needed to not interfere with NPs translocation. Altogether, these results show that a tailored approach should be considered in order to study toxicity or translocation of NPs

Développement de modèles in vitro de la barrière alvéolo-capillaire pour l'étude de la toxicité et du passage des nanoparticules

Après exposition par inhalation, les nanoparticules (NPs) peuvent atteindre les alvéoles pulmonaires, se retrouver au niveau de la barrière alvéolo-capillaire (BAC), et induire une toxicité locale et / ou franchir cette barrière pour se retrouver dans la circulation sanguine. Dans ce contexte, l objectif de ce travail a été de développer des modèles de co-cultures in vitro simples à mettre en œuvre (utilisation de lignées cellulaires humaines), pour étudier les effets des NPs au niveau de la BAC. Dans un premier temps, des co-cultures de cellules épithéliales alvéolaires ou de phénotype proche (lignées A549 ou NCI-H441), et de macrophages (lignée THP-1), ont permis l étude des effets pro-inflammatoires des NPs de SiO2 et de TiO2. Avec ces modèles nous avons montré l importance de la coopération cellulaire mise en jeu lors des processus inflammatoires liés aux NPs, mais aussi le rôle du ratio cellulaire employé dans ces réponses. Dans un second temps, des co-cultures tridimensionnelles en chambres bicamérales associant des macrophages (lignée THP-1), des cellules épithéliales bronchiques (lignée Calu-3), et des cellules endothéliales pulmonaires microvasculaires (lignée HPMEC-ST1.6R), ont permis l étude de l impact de NPs fluorescentes de polystyrène sur l intégrité de la BAC, et leur passage à travers cette barrière. Les cellules épithéliales Calu-3 permettent d établir une barrière de qualité mais la membrane microporeuse servant de support aux cellules doit être optimisée pour ne pas être un frein au passage des NPs. Ce travail montre qu un seul modèle ne permet pas d étudier de façon optimale à la fois la toxicité et la translocation des NPs, et qu une approche adaptée doit être envisagée en fonction du paramètre que l on souhaite étudier.After inhalation, nanoparticles (NPs) can reach the alveoli and the alveolo-capillary barrier (ACB), and consequently induce local toxicity and / or cross this barrier to reach the bloodstream. In this context, the aim of this work was to develop co-culture in vitro models simple to implement (using human cell lines), to study effects of NPs on the ACB. In a first time, pro-inflammatory effects of SiO2 and TiO2 NPs were studied on co-cultures of alveolar epithelial cells (A549 and NCI-H441 cell lines), and macrophages (THP-1 cell line). We demonstrated the importance of cell cooperation during inflammatory processes caused by these NPs, and the role of the cellular ratio in these inflammatory responses. In a second time, effects of fluorescent polystyrene NPs on the ACB integrity, and their translocation were studied on three-dimensional co-cultures in bicameral chambers involving macrophages (THP-1 cell line), bronchial epithelial cells (Calu-3 cell line), and micro-vascular pulmonary endothelial cells (HPMEC ST1.6R cell line). The use of Calu-3 has provided a good barrier, but further investigations on microporous membranes are still needed to not interfere with NPs translocation. Altogether, these results show that a tailored approach should be considered in order to study toxicity or translocation of NPs.PARIS5-Bibliotheque electronique (751069902) / SudocPARIS-BIUM-Bib. électronique (751069903) / SudocSudocFranceF

Toxicité des perturbateurs endocriniens au cours du développement de la barrière hémato-testiculaire chez le rat

PARIS-BIUP (751062107) / SudocSudocFranceF

Coopération cellulaire et rôle du récepteur P2X7 dans l’inflammation pulmonaire induite par les nanoparticules

Macrophages and alveolar epithelial cells are the first targets of inhaled nanoparticles (NPs) reaching the alveoli. Mono- or co-cultures of lung epithelial (A549 or NCI-H441) and macrophage (THP-1) cell lines were used to study the cell cooperation and the involvement of the P2X7 cell death receptor during the inflammation caused by SiO2 and TiO2 NPs. Here we show that secretion of pro-inflammatory cytokines (IL-1β, IL-6 and IL-8) in response to NP exposure was higher in co-cultures than in monocultures. A functional P2X7 receptor was found in all the cell lines studied. Its involvement in IL-1β secretion in co-cultures was demonstrated using a specific antagonist, the brilliant blue G. Furthermore, mono and co-cultures exhibited distinct secretion patterns of pro-inflammatory cytokines in response to NP exposure and we provide the first evidence that the P2X7 receptor is involved in the inflammation triggered by SiO2 and TiO2 NPs, by increasing IL-1β secretion, and likely via the inflammasome pathway. Altogether, our data indicate that cell co-cultures used in this study represent valid models to study the inflammatory mechanisms of NPs within the alveoli.Le développement très important des nanotechnologies, et donc la production à un niveau industriel de matériaux manufacturés à l’échelle nanométrique, impliquent des probabilités d’exposition de plus en plus élevées, avant tout chez les travailleurs concernés, mais également pour le grand public. De par leur petite taille (< 100 nm), le premier risque d’exposition aux nanoparticules (NPs) est lié à la pénétration par voie pulmonaire (inhalation) mais les données toxicologiques sur les NPs sont encore incomplètes à ce jour. Les études menées in vitro sont, pour des considérations éthiques et économiques, une alternative indispensable à l’approche in vivo. Bien que les modèles de toxicité in vitro, isolés de leur microenvironnement in vivo, ne puissent pas remplacer complètement les études in vivo, ils permettent, par leur simplicité, d’appréhender plus facilement les mécanismes d’action des toxiques. L’avantage des méthodes in vitro pour l’évaluation de la dangerosité des nanomatériaux est donc de fournir des outils de dépistage de toxicité plus simples, plus rapides et moins coûteux que l’approche in vivo

Characterization of a new coculture model of alveolo-capillary barrier developed to study the translocation and the toxicity of nanoparticles

One of the vital barriers in the human body is the air-blood barrier (ABB) in the lung. With an area of approximately 140 m2 the alveoli are functionally the most important element of the lung. Therefore, we developed an in vitro model that mimics the ABB in order to assess the translocation and the toxicity of nanoparticles (NP). In order to better mimic the reality, the model is composed of 3 cell types: THP-1 differentiated-macrophages and epithelial cells (A549 or NCI-H441) cultivated on the apical membrane of an insert, and HUVEC-CS endothelial cells on the basal side. Contrary to A549 cell line, NCI-H441 cells are not type II pneumocytes, but we showed that they are able to establish reliable junctional complexes. Consequently, they are good candidates to build a model of ABB. Coculture with THP-1 macrophages revealed to be essential to assess inflammatory process close to reality because 20 nm SiO2 and TiO2 NP-exposed macrophage's conditioned medium does not trigger the same inflammatory response compared to direct treatment of epithelial cells by NP. On the other hand, coculture of NCI-H441 cells with HUVEC-CS cells was tested with different coatings (collagen, elastin, fibronectin) to determine the best conditions in terms of cell differentiation. Moreover, different insert pore-sizes were tested (0.4 um; 1 um and 3 um) and optimal sizes (0.4 um and 1 um) were chosen according to NP translocation and cell growth. Therefore, the first results with our complete model underline the importance of using macrophages to assess inflammatory and translocation process on the ABB

Assessment of an in vitro model of pulmonary barrier to study the translocation of nanoparticles

International audienceAs the lung is one of the main routes of exposure to manufactured nanoparticles, we developed an in vitro model resembling the alveolo-capillary barrier for the study of nanoparticle translocation. In order to provide a relevant and ethical in vitro model, cost effective and easy-to-implement human cell lines were used. Pulmonary epithelial cells (Calu-3 cell line) and macrophages (THP-1 differentiated cells) were cultivated on the apical side and pulmonary endothelial cells (HPMEC-ST1.6R cell line) on the basal side of a microporous polyester membrane (Transwell). Translocation of non-functionalized (51 and 110 nm) and aminated (52 nm) fluorescent polystyrene (PS) nanobeads was studied in this system. The use of Calu-3 cells allowed high transepithelial electrical resistance (TEER) values (>1000 O cm2) in co-cultures with or without macrophages. After 24 h of exposure to non-cytotoxic concentrations of non-functionalized PS nanobeads, the relative TEER values (%/t0) were significantly decreased in co-cultures. Epithelial cells and macrophages were able to internalize PS nanobeads. Regarding translocation, Transwell membranes per se limit the passage of nanoparticles between apical and basal side. However, small non-functionalized PS nanobeads (51 nm) were able to translocate as they were detected in the basal side of co-cultures. Altogether, these results show that this co-culture model present good barrier properties allowing the study of nanoparticle translocation but research effort need to be done to improve the neutrality of the porous membrane delimitating apical and basal sides of the model