Acute in vivo pulmonary toxicity assessment of occupationally relevant particulate matter from a cellulose nanofiber board.

Cellulose nanofibers (CNFs) are an emerging engineered nanomaterial that are utilized in a variety of applications, including as a replacement for urea-formaldehyde, and other adhesives, as the binding agent in manufactured fiber and particle boards. To ensure the health and well-being of those producing, installing, or otherwise using cellulose nanofiber boards (CNFBs) it is imperative that the particulate matter (PM) produced during CNFB manipulation be evaluated for toxicity. We developed and internally verified a generation system to examine the PM produced by sanding CNFB using aluminum oxide sandpaper. With 80-grit sandpaper our system produced a low dispersity aerosol, as determined by a scanning mobility particle sizer and an optical particle counter, with a geometric mean of 28 nm (GSD = 1.60). ICP-MS evaluation showed little difference in metal concentrations between CNFB PM and nonsanded CNFB stock. We then used the system to simultaneously generate and expose both male and female C57BL/6J mice acutely for 4 hours at a concentration of 7.9 mg/m3. Sham-exposed controls were treated similarly but without sanding the CNFB. Analysis of bronchoalveolar lavage (BAL) fluid biomarkers showed no signs of inflammatory response at either 4- or 24-hours post exposure. Further, BAL cell viability, number of total cells, and pulmonary cellular recruitment were not significantly changed between the sham-exposed controls and CNFB-exposed mice. Histology further confirmed no pulmonary toxicity as a result of CNFB PM inhalation. We conclude that inhalation of a high concentration of the PM from manipulation of a CNFB did not produce acute toxic responses within 24 hours of exposure.

Toxicity assessment of metal oxide nanomaterials using in vitro screening and murine acute inhalation studies.

Characterizations and in vitro toxicity screening were performed on metal oxide engineered nanomaterials (ENMs) independently comprising ZnO, CuO, CeO2, Fe2O3, WO3, V2O5, TiO2, Al2O3 and MgO. Nanomaterials that exhibited the highest toxicity responses in the in vitro screening assays (ZnO, CuO, and V2O5) and the lesser explored material WO3 were tested for acute pulmonary toxicity in vivo. Female and male mice (C57Bl/6J) were exposed to aerosolized metal oxide ENMs in a nose-only exposure system and toxicity outcomes (biomarkers of cytotoxicity, immunotoxicity, inflammation, and lung histopathology) at 4 and 24 h after the start of exposure were assessed. The studies were performed as part of the NIEHS Nanomaterials Health Implications Research consortium with the purpose of investigating the effects of ENMs on various biological systems. ENMs were supplied by the Engineered Nanomaterials Resource and Coordination Core. Among the ENMs studied, the highest toxicity was observed for CuO and ZnO NPs in both in vitro and in vivo acute models. Compared to sham-exposed controls, there was a significant increase in bronchoalveolar lavage neutrophils and proinflammatory cytokines and a loss of macrophage viability at both 4 h and 24 h for ZnO and CuO but not seen for V2O5 or WO3. These effects were observed in both female and male mice. The cell viability performed after in vitro exposure to ENMs and assessment of lung inflammation after acute inhalation exposure in vivo were shown to be sensitive endpoints to predict ENM acute toxicity.

Comparison of in vitro toxicity of aerosolized engineered nanomaterials using air-liquid interface mono-culture and co-culture models.

Airborne engineered nanomaterials (ENMs) can readily enter the human body through inhalation potentially leading to adverse health effects such as cardiovascular and pulmonary diseases. Our group has previously utilized and validated an integrated low flow system capable of generating and depositing airborne ENMs directly onto cells at an air-liquid interface (ALI). To further improve this ALI method for an even closer representation of the in vivo system, a co-culture model containing epithelial, endothelial and macrophage cell lines (A549, EA.hy 926, and THP-1 differentiated macrophages) was established and validated for testing ENMs toxicity. In the co-culture model, cells were exposed to citrate-capped gold (Au), 15% silver on silica (Ag-SiO2) and copper oxide (CuO) ENMs under the same protocol (4 h ALI exposure with a target concentration of 3.5 mg/m3) and compared to responses with A549 cells only or THP-1 differentiated cells only. The toxicological profile was assessed by measuring cell viability, reactive oxygen species (ROS) production, lactate dehydrogenase (LDH) release, and interleukin (IL)-8 concentration. Results showed that 15% Ag-SiO2 induced more oxidative stress-related toxicity in the co-culture than in A549 cells alone. Both 15% Ag-SiO2 and CuO exposure produced significantly higher levels of IL-8 in the co-culture compared with A549 cells alone. Citrate-capped Au was largely inert. Further exposures of CuO on macrophages alone provided evidence of cell-cell interaction in the co-culture model. In addition, the co-culture model exhibited a similar response to primary human bronchial epithelial cells in terms of ROS and IL-8 responses after CuO exposure, suggesting a more advanced refinement of the conventional model for in vitro inhalation study.

CFTR gene transfer with AAV improves early cystic fibrosis pig phenotypes.

The physiological components that contribute to cystic fibrosis (CF) lung disease are steadily being elucidated. Gene therapy could potentially correct these defects. CFTR-null pigs provide a relevant model to test gene therapy vectors. Using an in vivo selection strategy that amplifies successful capsids by replicating their genomes with helper adenovirus coinfection, we selected an adeno-associated virus (AAV) with tropism for pig airway epithelia. The evolved capsid, termed AAV2H22, is based on AAV2 with 5 point mutations that result in a 240-fold increased infection efficiency. In contrast to AAV2, AAV2H22 binds specifically to pig airway epithelia and is less reliant on heparan sulfate for transduction. We administer AAV2H22-CFTR expressing the CF transmembrane conductance regulator (CFTR) cDNA to the airways of CF pigs. The transduced airways expressed CFTR on ciliated and nonciliated cells, induced anion transport, and improved the airway surface liquid pH and bacterial killing. Most gene therapy studies to date focus solely on Cl- transport as the primary metric of phenotypic correction. Here, we describe a gene therapy experiment where we not only correct defective anion transport, but also restore bacterial killing in CFTR-null pig airways.

Integrin α6ß4 identifies human distal lung epithelial progenitor cells with potential as a cell-based therapy for cystic fibrosis lung disease.

To develop stem/progenitor cell-based therapy for cystic fibrosis (CF) lung disease, it is first necessary to identify markers of human lung epithelial progenitor/stem cells and to better understand the potential for differentiation into distinct lineages. Here we investigated integrin α6ß4 as an epithelial progenitor cell marker in the human distal lung. We identified a subpopulation of α6ß4(+) cells that localized in distal small airways and alveolar walls and were devoid of pro-surfactant protein C expression. The α6ß4(+) epithelial cells demonstrated key properties of stem cells ex vivo as compared to α6ß4(-) epithelial cells, including higher colony forming efficiency, expression of stem cell-specific transcription factor Nanog, and the potential to differentiate into multiple distinct lineages including basal and Clara cells. Co-culture of α6ß4(+) epithelial cells with endothelial cells enhanced proliferation. We identified a subset of adeno-associated virus (AAVs) serotypes, AAV2 and AAV8, capable of transducing α6ß4(+) cells. In addition, reconstitution of bronchi epithelial cells from CF patients with only 5% normal α6ß4(+) epithelial cells significantly rescued defects in Cl(-) transport. Therefore, targeting the α6ß4(+) epithelial population via either gene delivery or progenitor cell-based reconstitution represents a potential new strategy to treat CF lung disease.

Enhanced sialic acid-dependent endocytosis explains the increased efficiency of infection of airway epithelia by a novel adeno-associated virus.

We previously used directed evolution in human airway epithelia to create adeno-associated virus 2.5T (AAV2.5T), a highly infectious chimera of AAV2 and AAV5 with one point mutation (A581T). We hypothesized that the mechanism for its increased infection may be a higher binding affinity to the surface of airway epithelia than its parent AAV5. Here, we show that, like AAV5, AAV2.5T, uses 2,3N-linked sialic acid as its primary receptor; however, AAV2.5T binds to the apical surface of human airway epithelia at higher levels and has more receptors than AAV5. Furthermore, its binding affinity is similar to that of AAV5. An alternative hypothesis is that AAV2.5T interaction with 2,3N-linked sialic acid may instead be required for cellular internalization. Consistent with this, AAV2.5T binds but fails to be internalized by CHO cells that lack surface expression of sialic acid. Moreover, whereas AAV2.5T binds similarly to human (rich in 2,3N-linked sialic acid) and pig airway epithelia (2,6N-linked sialic acid), significantly more virus was internalized by human airway. Subsequent transduction correlated with the level of internalized rather than surface-bound virus. We also found that human airway epithelia internalized significantly more AAV2.5T than AAV5. These data suggest that AAV2.5T has evolved to utilize specific 2,3N-linked sialic acid residues on the surface of airway epithelia that mediate rapid internalization and subsequent infection. Thus, sialic acid serves as not just an attachment factor but is also required for AAV2.5T internalization, possibly representing an important rate-limiting step for other viruses that use sialic acids.