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The epithelial tissues of the distal lung are continuously exposed to inhaled air, and are of research interest in studying respiratory exposure to both hazardous and therapeutic materials. Pharmaco-toxicological research depends on the development of sophisticated models of the alveolar epithelium, which better represent the different cell types present in the native lung and interactions between them. We developed an air-liquid interface (ALI) model of the alveolar epithelium which incorporates cell lines which bear features of type I (hAELVi) and type II (NCI-H441) epithelial cells. We compared morphology of single cells and the structure of cell layers of the two lines using light and electron microscopy. Working both in monotypic cultures and cocultures, we measured barrier function by trans-epithelial electrical resistance (TEER), and demonstrated that barrier properties can be maintained for 30 days. We created a mathematical model of TEER development over time based on these data in order to make inferences about the interactions occurring in these culture systems. We assessed expression of a panel of relevant genes that play important roles in barrier function and differentiation. The coculture model was observed to form a stable barrier akin to that seen in hAELVi, while expressing surfactant protein C, and having a profile of expression of claudins and aquaporins appropriate for the distal lung. We described cavities which arise within stratified cell layers in NCI-H441 and cocultured cells, and present evidence that these cavities represent an aberrant apical surface. In summary, our results support the coculture of these two cell lines to produce a model which better represents the breadth of functions seen in native alveolar epithelium.

As the use of nanoparticles (NPs) is increasing, the potential toxicity and behavior of NPs in living systems need to be better understood. Our goal was to evaluate the developmental toxicity and bio-distribution of two different sizes of fluorescently-labeled SiO2 NPs, 25 and 115 nm, with neutral surface charge or with different surface functionalization, rendering them positively or negatively charged, in order to predict the effect of NPs in humans. We performed a zebrafish embryo toxicity test (ZFET) by exposing the embryos to SiO2 NPs starting from six hours post fertilization (hpf). Survival rate, hatching time, and gross morphological changes were assessed at 12, 24, 36, 48, 60, and 72 hpf. We evaluated the effect of NPs on angiogenesis by counting the number of sub-intestinal vessels between the second and seventh intersegmental vessels and gene expression analysis of vascular endothelial growth factor (VEGF) and VEGF receptors at 72 hpf. SiO2 NPs did not show any adverse effects on survival rate, hatching time, gross morphology, or physiological angiogenesis. We found that SiO2 NPs were trapped by the chorion up until to the hatching stage. After chemical removal of the chorion (dechorionation), positively surface-charged SiO2 NPs (25 nm) significantly reduced the survival rate of the fish compared to the control group. These results indicate that zebrafish chorion acts as a physical barrier against SiO2 NPs, and removing the chorions in ZFET might be necessary for evaluation of toxicity of NPs.

The human bronchial epithelium is the first line of defense against atmospheric particles, pollutants, and respiratory pathogens such as the novel SARS-CoV-2. The epithelial cells form a tight barrier and secrete proteins that are major components of the mucosal immune response. Functional in vitro models of the human lung are essential for screening the epithelial response and assessing the toxicity and barrier crossing of drugs, inhaled particles, and pollutants. However, there is a lack of models to investigate the effect of chronic exposure without resorting to animal testing. Here, we developed a 3D model of the human bronchial epithelium using Calu-3 cell line and demonstrated its viability and functionality for 21 days without subculturing. We investigated the effect of reduced Fetal Bovine Serum supplementation in the basal medium and defined the minimal supplementation needed to maintain a functional epithelium, so that the amount of exogenous serum proteins could be reduced during drug testing. The long-term evolution of the epithelial cell secretome was fully characterized by quantitative mass spectrometry in two preclinical models using Calu-3 or primary NHBE cells. 408 common secreted proteins were identified while significant differences in protein abundance were observed with time, suggesting that 7–10 days are necessary to establish a mature secretome in the Calu-3 model. The associated Reactome pathways highlight the role of the secreted proteins in the immune response of the bronchial epithelium. We suggest this preclinical 3D model can be used to evaluate the long-term toxicity of drugs or particles on the human bronchial epithelium, and subsequently to investigate their effect on the epithelial cell secretions.

Background The uptake of nanoparticles (NPs) by cells remains to be better characterized in order to understand the mechanisms of potential NP toxicity as well as for a reliable risk assessment. Real NP uptake is still difficult to evaluate because of the adsorption of NPs on the cellular surface. Results Here we used two approaches to distinguish adsorbed fluorescently labeled NPs from the internalized ones. The extracellular fluorescence was either quenched by Trypan Blue or the uptake was analyzed using imaging flow cytometry. We used this novel technique to define the inside of the cell to accurately study the uptake of fluorescently labeled (SiO 2 ) and even non fluorescent but light diffracting NPs (TiO 2 ). Time course, dose-dependence as well as the influence of surface charges on the uptake were shown in the pulmonary epithelial cell line NCI-H292. By setting up an integrative approach combining these flow cytometric analyses with confocal microscopy we deciphered the endocytic pathway involved in SiO 2 NP uptake. Functional studies using energy depletion, pharmacological inhibitors, siRNA-clathrin heavy chain induced gene silencing and colocalization of NPs with proteins specific for different endocytic vesicles allowed us to determine macropinocytosis as the internalization pathway for SiO 2 NPs in NCI-H292 cells. Conclusion The integrative approach we propose here using the innovative imaging flow cytometry combined with confocal microscopy could be used to identify the physico-chemical characteristics of NPs involved in their uptake in view to redesign safe NPs.

Background Increasing environmental and occupational exposures to nanoparticles (NPs) warrant deeper insight into the toxicological mechanisms induced by these materials. The present study was designed to characterize the cell death induced by carbon black (CB) and titanium dioxide (TiO 2 ) NPs in bronchial epithelial cells (16HBE14o- cell line and primary cells) and to investigate the implicated molecular pathways. Results Detailed time course studies revealed that both CB (13 nm) and TiO 2 (15 nm) NP exposed cells exhibit typical morphological (decreased cell size, membrane blebbing, peripheral chromatin condensation, apoptotic body formation) and biochemical (caspase activation and DNA fragmentation) features of apoptotic cell death. A decrease in mitochondrial membrane potential, activation of Bax and release of cytochrome c from mitochondria were only observed in case of CB NPs whereas lipid peroxidation, lysosomal membrane destabilization and cathepsin B release were observed during the apoptotic process induced by TiO 2 NPs. Furthermore, ROS production was observed after exposure to CB and TiO 2 but hydrogen peroxide (H 2 O 2 ) production was only involved in apoptosis induction by CB NPs. Conclusions Both CB and TiO 2 NPs induce apoptotic cell death in bronchial epithelial cells. CB NPs induce apoptosis by a ROS dependent mitochondrial pathway whereas TiO 2 NPs induce cell death through lysosomal membrane destabilization and lipid peroxidation. Although the final outcome is similar (apoptosis), the molecular pathways activated by NPs differ depending upon the chemical nature of the NPs.

Background Nanoplastics (NPs) are released into the environment through the degradation of plastic objects, leading to human exposure. Due to their small size, concerns have been raised about the potential hazards to the respiratory tract, as ultrafine and nanoparticles are known to penetrate till the alveolar regions of the lungs, potentially impairing their functions. Thus, in the present study, we used model polystyrene nanoparticles doped with the fluorescent metal europium (PS-Eu) to enhance the understanding of NPs hazard and investigate adverse outcomes associated with exposure in human lungs using alveolar (A549) and bronchial (Calu-3) cell models grown in 2D and 3D submerged conditions or quasi air-liquid interface (ALI) conditions (3D). Results Briefly, after in-dept physicochemical characterization of the particles, we assessed their impact on ROS production, cell viability (AlamarBlue and lactate dehydrogenase assays) and barrier integrity (lucifer yellow assay and TEER measurement), finding no negative effects in either model. However, in alveolar cells, particles increased acidic organelle activity. Transmission electron microscopy and Raman microscopy showed, in both models, a dose- and cell-dependent particle uptake with PS-Eu accumulating in numerous and large endo-lysosomes, which, in transwells-grown A549 cells, often contained also lamellar bodies (LBs), organelles involved in surfactants storage and secretion. After extensively quantifying surfactant proteins (SP) in the pellet and supernatant fractions of treated A549 cells, we observed a significant reduction in several members of this family, including surfactant protein B, which is crucial for lamellar body formation and surface tension regulation in the lungs. In quasi-ALI Calu-3 cultures instead, PS-Eu significantly upregulated interleukin 6 (IL-6) and increased transforming growth factor beta β (TGF-β), zonula occludens 1 (ZO-1), and mucin (MUC) 5B mRNA expressions causing a moderate proinflammatory response. Conclusion Our results show that PS-Eu exposure does not induce acute cytotoxicity in these models, but affects cell-specific functions like surfactant, mucin, and cytokine production. This underscores the limitations of relying solely on standard cytotoxicity tests for particle hazard assessment and highlights the importance of investigating cell function-specific signaling pathways. To support researchers in hazard assessment, we propose specific classes of biomarkers to test in in vitro lung models.

Nanoparticles (NP) have a tendency to agglomerate after dispersion in physiological media, which can be prevented by the addition of serum. This may however result in modification of the toxic potential of particles due to the formation of protein corona. Our study aimed to analyze the role of serum that is added to improve the dispersion of 10 nm TiO 2 NPs on in vitro and in vivo effects following the exposure via the respiratory route. We characterized NP size, surface charge, sedimentation rate, the presence of protein corona and the oxidant-generating capacity after NP dispersion in the presence/absence of serum. The effect of serum on NP internalization, cytotoxicity and pro-inflammatory responses was assessed in a human pulmonary cell line, NCI-H292. Serum in the dispersion medium led to a slower sedimentation, but an enhanced cellular uptake of TiO 2 NPs. Despite this greater uptake, the pro-inflammatory response in NCI-H292 cells was lower after serum supplementation (used either as a dispersant or as a cell culture additive), which may be due to a reduced intrinsic oxidative potential of TiO 2 NPs. Interestingly, serum could be added 2 h after the NP treatment without affecting the pro-inflammatory response. We also determined the acute pulmonary and hepatic toxicity in vivo 24 h after intratracheal instillation of TiO 2 NPs in C57BL/6N mice. The use of serum resulted in an underestimation of the local acute inflammatory response in the lung, while a systemic response on glutathione reduction remained unaffected. In conclusion, serum as a dispersion agent for TiO 2 NPs can lead to an underestimation of the acute pro-inflammatory response in vitro and in vivo. To avoid potential unwanted effects of dispersants and medium components, we recommend that the protocol of NM preparation should be thoroughly tested, and reflect as close as possible realistic exposure conditions.

Background Silica nanoparticles (SiO 2 NPs) are widely used in industrial products. Surface modification of SiO 2 NPs is one of the promising strategies to develop safer nanomaterials by design. The present study was designed to determine the effects of amino or carboxyl functionalization of rhodamine-labeled SiO 2 NPs on cellular uptake and cytotoxicity. Methods In the in vivo arm of the study, male mice were randomly divided into seven groups (n = 6, each) and exposed to either amino (NH 2 )- or carboxyl (COOH)-functionalized, or non-functionalized (OH)-rhodamine-labeled SiO 2 NPs at 2 or 10 mg/kg bw, or endotoxin-free water as a control, by pharyngeal aspiration. At 24 h after administration, the mice were euthanized and bronchoalveolar lavage fluid (BALF) was collected for differential cell count and assessment of silica nanoparticle uptake using confocal microscopy. In the in vitro arm of the study, murine RAW264.7 macrophages were exposed to NH 2 -or COOH-functionalized or OH- rhodamine-labeled SiO 2 NPs. Nonspecific caspase inhibitor, necroptosis inhibitor, pyroptosis inhibitor and autophagy inhibitor were used to determine the roles of cell death signaling in cytotoxicity. Results The in vivo studies demonstrated significant increase in lung weight at 2 and 10 mg/kg bw by OH-SiO 2 NPs but not the other two SiO 2 NPs. At 10 mg/kg bw, COOH-SiO2NPs induced a significant increase in BALF macrophages, whereas OH- SiO 2 NPs significantly decreased macrophages. OH-SiO 2 NPs at 2 mg/kg bw and NH 2 - and COOH-SiO 2 NPs at 10 mg/kg bw significantly increased BALF neutrophiles. The in vitro studies showed greater NH 2 -SiO 2 NPs internalization into RAW264.7 macrophages than OH-SiO 2 NPs, while OH-SiO 2 NPs induced cytotoxicity and upregulation of IL-1β and TNF-α to greater extent than the other two types. Co-treatment with pan-caspase inhibitor and necroptosis inhibitor attenuated (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) cytotoxicity of OH-SiO 2 NPs. Conclusion NH 2 - or COOH-functionalization reduced the harmful changes observed with OH- SiO 2 NPs, which included increase in lung weight and BALF neutrophils at low dose in mice as well as decrease in cell viability and upregulation of proinflammatory cytokines in RAW264.7 macrophages. The results suggested that OH-SiO 2 NPs-induced cytotoxicity against macrophages was mediated at least in part through apoptotic/necroptotic signaling but was not related to internalization of particles. The results imply possible development of safer silica nanoparticles by amino- or carboxyl-functionalization of their silanols.

A growing body of epidemiological evidence links maternal exposure to air pollution with an increased risk of adverse pregnancy outcomes, such as preterm birth and low birth weight. Cerium dioxide nanoparticles (CeO 2 NPs or nanoceria) are emerging pollutants, used as additives in diesel fuels and cigarettes for their catalytic properties, and released into the environment. Due to their high surface-to-volume ratio and reactivity, CeO 2 NPs develop a surface coating during combustion, which may incorporate other released fuel-borne chemicals, such as benzo[a]pyrene (BaP), a known carcinogen, mutagen and reprotoxicant, raising concerns about their combined impacts on human health. To better reflect environmental reality, we produced BaP-coated CeO 2 NPs and exposed primary human trophoblasts and chorionic villi. Our findings show that BaP-coated CeO 2 NPs activate the aryl hydrocarbon receptor (AhR) pathway, enhancing trophoblast differentiation and syncytium formation, with effects distinct from those of BaP or CeO₂ NPs alone, or their unbound mixture. Additionally, exposure to CeO 2 NPs alone altered homeostasis of mitochondria, affecting their phenotype and function. While individual exposures or BaP-coated CeO 2 NPs had no detectable impact, parallel co-exposure resulted in a slight but significant reduction in basal respiration. Finally, uncoated CeO 2 NPs altered placental steroidogenesis, increasing estrone level while decreasing dehydroepiandrosterone level, with sex-specific effects. These findings suggest that CeO 2 NPs can influence the biological effects of BaP in the human placenta, including modulating trophoblast differentiation, as well as disrupting mitochondria homeostasis and steroid production, with potential implications for pregnancy outcomes in polluted environments.

Background Epidemiological emerging evidence shows that human exposure to some nanosized materials present in the environment would contribute to the onset and/or progression of Alzheimer’s disease (AD). The cellular and molecular mechanisms whereby nanoparticles would exert some adverse effects towards neurons and take part in AD pathology are nevertheless unknown. Results Here, we provide the prime evidence that titanium dioxide (TiO 2 ) and carbon black (CB) nanoparticles (NPs) bind the cellular form of the prion protein (PrP C ), a plasma membrane protein well known for its implication in prion diseases and prion-like diseases, such as AD. The interaction between TiO 2 - or CB-NPs and PrP C at the surface of neuronal cells grown in culture corrupts PrP C signaling function. This triggers PrP C -dependent activation of NADPH oxidase and subsequent production of reactive oxygen species (ROS) that alters redox equilibrium. Through PrP C interaction, NPs also promote the activation of 3-phosphoinositide-dependent kinase 1 (PDK1), which in turn provokes the internalization of the neuroprotective TACE α-secretase. This diverts TACE cleavage activity away from (i) TNFα receptors (TNFR), whose accumulation at the plasma membrane augments the vulnerability of NP-exposed neuronal cells to TNFα -associated inflammation, and (ii) the amyloid precursor protein APP, leading to overproduction of neurotoxic amyloid Aβ40/42 peptides. The silencing of PrP C or the pharmacological inhibition of PDK1 protects neuronal cells from TiO 2 - and CB-NPs effects regarding ROS production, TNFα hypersensitivity, and Aβ rise. Finally, we show that dysregulation of the PrP C -PDK1-TACE pathway likely occurs in the brain of mice injected with TiO 2 -NPs by the intra-cerebro-ventricular route as we monitor a rise of TNFR at the cell surface of several groups of neurons located in distinct brain areas. Conclusion Our in vitro and in vivo study thus posits for the first time normal cellular prion protein PrP C as being a neuronal receptor of TiO 2 - and CB-NPs and identifies PrP C -coupled signaling pathways by which those nanoparticles alter redox equilibrium, augment the intrinsic sensitivity of neurons to neuroinflammation, and provoke a rise of Aβ peptides. By identifying signaling cascades dysregulated by TiO 2 - and CB-NPs in neurons, our data shed light on how human exposure to some NPs might be related to AD.