Effects of preconditioning with TNFα and IFNγ in angiogenic potential of mesenchymal stromal cell-derived extracellular vesicles.

Introduction: Mesenchymal stromal cells (MSCs) have demonstrated therapeutic properties both in vitro and in vivo to treat various diseases, including anti-inflammatory, immunomodulatory and pro-angiogenic effects. These therapeutic effects are mediated by their secretome composed of soluble factors and extracellular vesicles (EVs). The composition of EVs reflects the molecular and functional characteristics of parental cells. MSC preconditioning can alter the composition of EVs, thereby influencing their therapeutic potential. Methods: MSCs were subjected to preconditioning with two cytokines, TNFα and IFNγ. Following 24 h of preconditioning, MSC-EVs secreted into the culture supernatant were isolated through tangential filtration. Particle concentration and size distribution were measured by nanoparticle tracking analysis, and the surface antigen expression of the EV-specific CD63 was quantified via Enzyme Linked ImmunoSorbent Assay. The angiogenic potential of MSCEVs obtained after preconditioning MSCs was assessed by the analysis of their protein composition and their influence on human umbilical vein endothelial cell (HUVECs) proliferation, migration, and tube-forming ability. Results: Preconditioning with TNFα and IFNγ did not influence the MSC-EV profile but did induce changes in their protein content. Indeed, the expression of pro-angiogenic proteins increased in EVs from preconditioned MSCs compared to EVs from no-preconditioned MSCs. EVs from preconditioned MSCs tend to stimulate HUVEC migration, proliferation and tubeforming ability. These observations imply the presence of a pro-angiogenic potential in EVs obtained after preconditioning of MSCs with TNFα and IFNγ. Discussion: In conclusion, it appears that the pro-angiogenic potential of EVs is enhanced through preconditioning of MSCs with TNFα and IFNγ. The use of these MSCs-EVs in therapy would circumvent the limitations of current cell-based therapies. Indeed, the therapeutic potential of MSC-EVs presents an attractive strategy for exploiting the clinical benefits of MSC therapy. For example, in the field of regenerative medicine, the exploitation of cell-free therapy using highly pro-angiogenic MSC-EVs is of great interest.

Critical Review on Toxicological Mechanisms Triggered by Inhalation of Alumina Nanoparticles on to the Lungs.

Alumina nanoparticles (Al2O3 NPs) can be released in occupational environments in different contexts such as industry, defense, and aerospace. Workers can be exposed by inhalation to these NPs, for instance, through welding fumes or aerosolized propellant combustion residues. Several clinical and epidemiological studies have reported that inhalation of Al2O3 NPs could trigger aluminosis, inflammation in the lung parenchyma, respiratory symptoms such as cough or shortness of breath, and probably long-term pulmonary fibrosis. The present review is a critical update of the current knowledge on underlying toxicological, molecular, and cellular mechanisms induced by exposure to Al2O3 NPs in the lungs. A major part of animal studies also points out inflammatory cells and secreted biomarkers in broncho-alveolar lavage fluid (BALF) and blood serum, while in vitro studies on lung cells indicate contradictory results regarding the toxicity of these NPs.

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-1ß, 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.

Alumina nanoparticles size and crystalline phase impact on cytotoxic effect on alveolar epithelial cells after simple or HCl combined exposures.

Applications using alumina nanoparticles (Al2O3 NPs) have incredibly increased in different fields of activity. In defense and aerospace fields, solid composite propellants use leads to complex combustion aerosols emissions containing high concentrations of Al2O3 NPs and hydrogen chloride gas (HCl). To better characterize potential hazard resulting from exposure to these aerosols, this study assesses cytotoxic effects of mixtures containing both compounds on human pulmonary alveolar epithelial cells (A549 cell line) after 24 h exposures. After all co-exposures cell viability was >80%. However co-exposures decrease normalized real-time cell index. Significant decreases of intracellular reduced glutathione pool were also observed after co-exposures to γ-10 nm or γ/δ-13 nm Al2O3 NPs and HCl. Co-incubations with γ/δ-13 nm or γ-500 nm Al2O3 particles and HCl induced significant DNA double-strand breaks increases. Moreover all co-exposures and HCl alone disrupted cell cycle (increased G1 phase cells). Transmission Electron Microscopy (TEM) observations revealed γ/δ-13 nm Al2O3NPs adsorption and internalization in cell cytoplasm only, suggesting indirect genotoxic effects. According to our results Al2O3 particles/HCl mixtures can induce cytotoxic effects and Al2O3 size and crystallinity are two main parameters influencing cytotoxic mechanisms.

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 6h, 24h, 48h and 72h of incubation with THP-1 monocytic cells (concentrations tested from 10 to 50µg/mL). From 6h to 72h 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 24h 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-1ß, IL-6, IL-8, TNF-α and IL-10) was also measured in supernatants after 6h or 24h 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.

Specific uptake and genotoxicity induced by polystyrene nanobeads with distinct surface chemistry on human lung epithelial cells and macrophages.

Nanoparticle surface chemistry is known to play a crucial role in interactions with cells and their related cytotoxic effects. As inhalation is a major route of exposure to nanoparticles, we studied specific uptake and damages of well-characterized fluorescent 50 nm polystyrene (PS) nanobeads harboring different functionalized surfaces (non-functionalized, carboxylated and aminated) on pulmonary epithelial cells and macrophages (Calu-3 and THP-1 cell lines respectively). Cytotoxicity of in mass dye-labeled functionalized PS nanobeads was assessed by xCELLigence system and alamarBlue viability assay. Nanobeads-cells interactions were studied by video-microscopy, flow cytometry and also confocal microscopy. Finally ROS generation was assessed by glutathione depletion dosages and genotoxicity was assessed by γ-H2Ax foci detection, which is considered as the most sensitive technique for studying DNA double strand breaks. The uptake kinetic was different for each cell line. All nanobeads were partly adsorbed and internalized, then released by Calu-3 cells, while THP-1 macrophages quickly incorporated all nanobeads which were located in the cytoplasm rather than in the nuclei. In parallel, the genotoxicity study reported that only aminated nanobeads significantly increased DNA damages in association with a strong depletion of reduced glutathione in both cell lines. We showed that for similar nanoparticle concentrations and sizes, aminated polystyrene nanobeads were more cytotoxic and genotoxic than unmodified and carboxylated ones on both cell lines. Interestingly, aminated polystyrene nanobeads induced similar cytotoxic and genotoxic effects on Calu-3 epithelial cells and THP-1 macrophages, for all levels of intracellular nanoparticles tested. Our results strongly support the primordial role of nanoparticles surface chemistry on cellular uptake and related biological effects. Moreover our data clearly show that nanoparticle internalization and observed adverse effects are not necessarily associated.

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

As 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 Ω 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.

Cell cooperation and role of the P2X7 receptor in pulmonary inflammation induced by nanoparticles.

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-1ß, IL-6 and IL-8) in response to NPs exposure was higher in co-cultures than in mono-cultures. A functional P2X7 receptor was found in all the cell lines studied. Its involvement in IL-1ß 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 NPs 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-1ß secretion, and likely through 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.