Oxidative stress and inflammation response after nanoparticle exposure: differences between human lung cell monocultures and an advanced three-dimensional model of the human epithelial airways

Combustion-derived and manufactured nanoparticles (NPs) are known to provoke oxidative stress and inflammatory responses in human lung cells; therefore, they play an important role during the development of adverse health effects. As the lungs are composed of more than 40 different cell types, it is of particular interest to perform toxicological studies with co-cultures systems, rather than with monocultures of only one cell type, to gain a better understanding of complex cellular reactions upon exposure to toxic substances. Monocultures of A549 human epithelial lung cells, human monocyte-derived macrophages and monocyte-derived dendritic cells (MDDCs) as well as triple cell co-cultures consisting of all three cell types were exposed to combustion-derived NPs (diesel exhaust particles) and to manufactured NPs (titanium dioxide and single-walled carbon nanotubes). The penetration of particles into cells was analysed by transmission electron microscopy. The amount of intracellular reactive oxygen species (ROS), the total antioxidant capacity (TAC) and the production of tumour necrosis factor (TNF)-alpha and interleukin (IL)-8 were quantified. The results of the monocultures were summed with an adjustment for the number of each single cell type in the triple cell co-culture. All three particle types were found in all cell and culture types. The production of ROS was induced by all particle types in all cell cultures except in monocultures of MDDCs. The TAC and the (pro-)inflammatory reactions were not statistically significantly increased by particle exposure in any of the cell cultures. Interestingly, in the triple cell co-cultures, the TAC and IL-8 concentrations were lower and the TNF-alpha concentrations were higher than the expected values calculated from the monocultures. The interplay of different lung cell types seems to substantially modulate the oxidative stress and the inflammatory responses after NP exposure.

Human prostate fibroblasts induce growth and confer castration resistance and metastatic potential in LNCaP Cells

The tumor microenvironment is important for progressive and metastatic disease.

Atypical expression and distribution of embryonic stem cell marker, OCT4, in human lung adenocarcinoma

Lung cancer is one of the leading causes of cancer-related deaths in the world. Although the origin still remains to be resolved, a prevailing hypothesis implies the involvement of cancer stem cells (CSCs) responsible for tumor initiation, maintenance, and progression. Embryonic stem cell marker, OCT4, encoding the spliced variants OCT4A and OCT4B, has recently been shown to have a dual role; as a potential adult stem cell marker and as a CSC marker in germline and somatic tumors.

A comparison of acute and long-term effects of industrial multiwalled carbon nanotubes on human lung and immune cells in vitro

The close resemblance of carbon nanotubes to asbestos fibers regarding their high aspect ratio, biopersistence and reactivity increases public concerns on the widespread use of these materials. The purpose of this study was not only to address the acute adverse effects of industrially produced multiwalled carbon nanotubes (MWCNTs) on human lung and immune cells in vitro but also to further understand if their accumulation and biopersistence leads to long-term consequences or induces adaptive changes in these cells. In contrast to asbestos fibers, pristine MWCNTs did not induce overt cell death in A549 lung epithelial cells and Jurkat T lymphocytes after acute exposure to high doses of this material (up to 30 g/ml). Nevertheless, very high levels of reactive oxygen species (ROS) and decreased metabolic activity were observed which might affect long-term viability of these cells. However, the continuous presence of low amounts of MWCNTs (0.5 g/ml) for 6 months did not have major adverse long-term effects although large amounts of nanotubes accumulated at least in A549 cells. Moreover, MWCNTs did not appear to induce adaptive mechanisms against particle stress in long-term treated A549 cells. Our study demonstrates that despite the high potential for ROS formation, pristine MWCNTs can accumulate and persist within cells without having major long-term consequences or inducing adaptive mechanisms.

Investigating the interaction of cellulose nanofibers derived from cotton with a sophisticated 3D human lung cell coculture

Cellulose nanofibers are an attractive component of a broad range of nanomaterials. Their intriguing mechanical properties and low cost, as well as the renewable nature of cellulose make them an appealing alternative to carbon nanotubes (CNTs), which may pose a considerable health risk when inhaled. Little is known, however, concerning the potential toxicity of aerosolized cellulose nanofibers. Using a 3D in vitro triple cell coculture model of the human epithelial airway barrier, it was observed that cellulose nanofibers isolated from cotton (CCN) elicited a significantly (p < 0.05) lower cytotoxicity and (pro-)inflammatory response than multiwalled CNTs (MWCNTs) and crocidolite asbestos fibers (CAFs). Electron tomography analysis also revealed that the intracellular localization of CCNs is different from that of both MWCNTs and CAFs, indicating fundamental differences between each different nanofibre type in their interaction with the human lung cell coculture. Thus, the data shown in the present study highlights that not only the length and stiffness determine the potential detrimental (biological) effects of any nanofiber, but that the material used can significantly affect nanofiber-cell interactions.

MicroRNA-29b is involved in the Src-ID1 signaling pathway and is dysregulated in human lung adenocarcinoma

The c-Src kinase regulates cancer cell invasion through inhibitor of DNA binding/differentiation 1 (ID1). Src and ID1 are frequently overexpressed in human lung adenocarcinoma. The current study aimed at identifying microRNAs (miRNAs) involved in the Src-ID1 signaling in lung cancer. Incubation of lung cancer cells with the Src inhibitor saracatinib led to the upregulation of several miRNAs including miR-29b, which was the most highly upregulated miRNA with predicted binding to the ID1 3'-untranslated region (UTR). Luciferase reporter assays confirmed direct binding of miR-29b to the ID1 3'-UTR. Expression of miR-29b suppressed ID1 levels and significantly reduced migration and invasion. Expression of antisense-miR-29b (anti-miR-29b), on the other hand, enhanced ID1 mRNA and protein levels, and significantly increased lung cancer cell migration and invasion, a hallmark of the Src-ID1 pathway. The ectopic expression of ID1 in miR-29b-overexpressing cells was able to rescue the migratory potential of these cells. Both, anti-miR-29b and ID1 overexpression diminished the effects of the Src inhibitors saracatinib and dasatinib on migration and invasion. Saracatinib and dasatinib decreased c-Myc transcriptional repression on miR-29b and led to increased ID1 protein levels, whereas forced expression of c-Myc repressed miR-29b and induced ID1. In agreement, we showed direct recruitment of c-Myc to the miR-29b promoter. miR-29b was significantly downregulated in primary lung adenocarcinoma samples compared with matched alveolar lung tissue, and miR-29b expression was a significant prognostic factor for patient outcome. These results suggest that miR-29b is involved in the Src-ID1 signaling pathway, is dysregulated in lung adenocarcinoma and is a potential predictive marker for Src kinase inhibitors.

Effects of flame made zinc oxide particles in human lung cells - a comparison of aerosol and suspension exposures

Background Predominantly, studies of nanoparticle (NPs) toxicology in vitro are based upon the exposure of submerged cell cultures to particle suspensions. Such an approach however, does not reflect particle inhalation. As a more realistic simulation of such a scenario, efforts were made towards direct delivery of aerosols to air-liquid-interface cultivated cell cultures by the use of aerosol exposure systems. This study aims to provide a direct comparison of the effects of zinc oxide (ZnO) NPs when delivered as either an aerosol, or in suspension to a triple cell co-culture model of the epithelial airway barrier. To ensure dose–equivalence, ZnO-deposition was determined in each exposure scenario by atomic absorption spectroscopy. Biological endpoints being investigated after 4 or 24h incubation include cytotoxicity, total reduced glutathione, induction of antioxidative genes such as heme-oxygenase 1 (HO–1) as well as the release of the (pro)-inflammatory cytokine TNFα. Results Off-gases released as by-product of flame ZnO synthesis caused a significant decrease of total reduced GSH and induced further the release of the cytokine TNFα, demonstrating the influence of the gas phase on aerosol toxicology. No direct effects could be attributed to ZnO particles. By performing suspension exposure to avoid the factor “flame-gases”, particle specific effects become apparent. Other parameters such as LDH and HO–1 were not influenced by gaseous compounds: Following aerosol exposure, LDH levels appeared elevated at both timepoints and the HO–1 transcript correlated positively with deposited ZnO-dose. Under submerged conditions, the HO–1 induction scheme deviated for 4 and 24h and increased extracellular LDH was found following 24h exposure. Conclusion In the current study, aerosol and suspension-exposure has been compared by exposing cell cultures to equivalent amounts of ZnO. Both exposure strategies differ fundamentally in their dose–response pattern. Additional differences can be found for the factor time: In the aerosol scenario, parameters tend to their maximum already after 4h of exposure, whereas under submerged conditions, effects appear most pronounced mainly after 24h. Aerosol exposure provides information about the synergistic interplay of gaseous and particulate phase of an aerosol in the context of inhalation toxicology. Exposure to suspensions represents a valuable complementary method and allows investigations on particle-associated toxicity by excluding all gas–derived effects.

In contrast to lung fibroblasts--no signs of senescence in skin fibroblasts of patients with emphysema

Smoking is known to be linked to skin ageing and there is evidence for premature senescence of parenchymal lung fibroblasts in emphysema. To reveal whether the emphysema-related changes in cellular phenotype extend beyond the lung, we compared the proliferation characteristics of lung and skin fibroblasts between patients with and without emphysema. Parenchymal lung fibroblasts and skin fibroblasts from the upper torso (thus limiting sun exposure bias) were obtained from patients without, or with mild, or with moderate to severe emphysema undergoing lung surgery. We analysed proliferation rate, population doublings (PD), staining for senescence-associated beta-galactosidase (beta-gal) and gene expression of IGFBP-3 and IGFBP-rP1. Population doubling time of lung fibroblasts differed between control, mild, and moderate to severe emphysema (median (IQR) 29.7(10.0), 33.4(6.1), 44.4(21.2) h; p=0.012) and staining for beta-gal was elevated in moderate to severe emphysema. Compared to control subjects, skin fibroblasts from patients with emphysema did not differ with respect to proliferation rate, PD and beta-gal staining, and showed a lower abundance of mRNA for IGFBP-3 and -rP1 (p<0.05, each). These results suggest that the induction of a senescent fibroblast phenotype by cigarette smoke, as observed in emphysema, primarily occurs in the lung.

Identification of mesenchymal stromal cells in human lung parenchyma capable of differentiating into aquaporin 5-expressing cells

The lack of effective therapies for end-stage lung disease validates the need for stem cell-based therapeutic approaches as alternative treatment options. In contrast with exogenous stem cell sources, the use of resident progenitor cells is advantageous considering the fact that the lung milieu is an ideal and familiar environment, thereby promoting the engraftment and differentiation of transplanted cells. Recent studies have shown the presence of multipotent 'mesenchymal stem cells' in the adult lung. The majority of these reports are, however, limited to animal models, and to date, there has been no report of a similar cell population in adult human lung parenchyma. Here, we show the identification of a population of primary human lung parenchyma (pHLP) mesenchymal stromal cells (MSCs) derived from intraoperative normal lung parenchyma biopsies. Surface and intracellular immunophenotyping by flow cytometry revealed that cultures do not contain alveolar type I epithelial cells or Clara cells, and are devoid of the following hematopoietic markers: CD34, CD45 and CXCR4. Cells show an expression pattern of surface antigens characteristic of MSCs, including CD73, CD166, CD105, CD90 and STRO-1. As per bone marrow MSCs, our pHLP cells have the ability to differentiate along the adipogenic, osteogenic and chondrogenic mesodermal lineages when cultured in the appropriate conditions. In addition, when placed in small airway growth media, pHLP cell cultures depict the expression of aquaporin 5 and Clara cell secretory protein, which is identified with that of alveolar type I epithelial cells and Clara cells, respectively, thereby exhibiting the capacity to potentially differentiate into airway epithelial cells. Further investigation of these resident cells may elucidate a therapeutic cell population capable of lung repair and/or regeneration.

Comparison of the toxicity of diesel exhaust produced by bio- and fossil diesel combustion in human lung cells in vitro

Alternative fuels are increasingly combusted in diesel- and gasoline engines and the contribution of such exhausts to the overall air pollution is on the rise. Recent findings on the possible adverse effects of biodiesel exhaust are contradictive, at least partly resulting from the various fuel qualities, engine types and different operation conditions that were tested. However, most of the studies are biased by undesired interactions between the exhaust samples and biological culture media. We here report how complete, freshly produced exhausts from fossil diesel (B0), from a blend of 20% rapeseed-methyl ester (RME) and 80% fossil diesel (B20) and from pure rapeseed methyl ester (B100) affect a complex 3D cellular model of the human airway epithelium in vitro by exposing the cells at the air–liquid interface. The induction of pro-apoptotic and necrotic cell death, cellular morphology, oxidative stress, and pro-inflammatory responses were assessed. Compared to B0 exhaust, B20 exhaust decreased oxidative stress and pro-inflammatory responses, whereas B100 exhaust, depending on exposure duration, decreased oxidative stress but increased pro-inflammatory responses. The effects are only very weak and given the compared to fossil diesel higher ecological sustainability of biodiesel, it appears that – at least RME – can be considered a valuable alternative to pure fossil diesel.

Nanomaterials and the human lung: what is known and what must be deciphered to realise their potential advantages?

Due to the constant expansion within the nanotechnology industry in the last decade, nanomaterials are omnipresent in society today. Nanotechnology-based products have numerous different applications ranging from electronic (e.g., advanced memory chips) to industrial (e.g., coatings or composites) to biomedical (e.g., drug delivery systems, diagnostics). Although these new nanomaterials can be found in many "everyday" products, their effects on the human body have still to be investigated in order to identify not only their risk, but also their potential benefits towards human health. Since the lung is commonly thought to be the main portal of entry into the human body for nanomaterials released within the environment, this review will attempt to summarise the current knowledge and understanding of how nanomaterials interact with the respiratory tract. Furthermore, the advantages and disadvantages of different experimental model systems that are commonly used to study this exposure route to the human body will be discussed.

Regulation of NAMPT in human gingival fibroblasts and biopsies

Adipokines, such as nicotinamide phosphoribosyltransferase (NAMPT), are molecules, which are produced in adipose tissue. Recent studies suggest that NAMPT might also be produced in the tooth-supporting tissues, that is, periodontium, which also includes the gingiva. The aim of this study was to examine if and under what conditions NAMPT is produced in gingival fibroblasts and biopsies from healthy and inflamed gingiva. Gingival fibroblasts produced constitutively NAMPT, and this synthesis was significantly increased by interleukin-1β and the oral bacteria P. gingivalis and F. nucleatum. Inhibition of the MEK1/2 and NFκB pathways abrogated the stimulatory effects of F. nucleatum on NAMPT. Furthermore, the expression and protein levels of NAMPT were significantly enhanced in gingival biopsies from patients with periodontitis, a chronic inflammatory infectious disease of the periodontium, as compared to gingiva from periodontally healthy individuals. In summary, the present study provides original evidence that gingival fibroblasts produce NAMPT and that this synthesis is increased under inflammatory and infectious conditions. Local synthesis of NAMPT in the inflamed gingiva may contribute to the enhanced gingival and serum levels of NAMPT, as observed in periodontitis patients. Moreover, local production of NAMPT by gingival fibroblasts may represent a possible mechanism whereby periodontitis may impact on systemic diseases.