Assessment of transporter-mediated efflux of nintedanib using in vitro cell line models of idiopathic pulmonary fibrosis.

Objective: We aimed to investigate the involvement of efflux transporters, including multidrug resistant protein 1 (MDR1), multidrug resistance-associated protein 1 (MRP1), MRP2, and breast cancer resistance protein (BCRP), in the intracellular accumulation of the antifibrotic agent nintedanib in fibrotic lung cells. Methods: We used transforming growth factor-ß1 (TGF-ß1)-treated human lung fibroblasts (WI-38) and alveolar epithelial cells (A549) as in vitro models. The expression and activities of efflux transporters in TGF-ß1-treated WI-38 and A549 cells were evaluated using immunoblotting and flow cytometry. Cells were treated with nintedanib and then incubated with inhibitors of these transporters. The intracellular concentration of nintedanib was determined. Results: MDR1, MRP1, MRP2, and BCRP were found to be expressed in WI-38 and A549 cells with or without TGF-ß1 stimulation, with the exception of MRP2 in WI-38 cells. The efflux activities of these transporters were observed in these cells. MDR1 inhibitors significantly increased the intracellular accumulation of nintedanib, whereas MRP inhibitors did not show an effect. The BCRP inhibitor significantly increased the transporter activity in A549 cells but not in WI-38 cells. Conclusion: This study suggests that the efflux via MDR1 and BCRP is involved in the intracellular accumulation of nintedanib in fibrotic lung cells.

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment.

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor α) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

From organ to cell: Multi-level telomere length assessment in patients with idiopathic pulmonary fibrosis.

RATIONALE: A subset of patients with idiopathic pulmonary fibrosis (IPF) contains short leukocyte telomeres or telomere related mutations. We previously showed that alveolar type 2 cells have short telomeres in fibrotic lesions. Our objectives were to better understand how telomere shortening associates with fibrosis in IPF lung and identify a subset of patients with telomere-related disease. METHODS: Average telomere length was determined in multiple organs, basal and apical lung, and diagnostic and end-stage fibrotic lung biopsies. Alveolar type 2 cells telomere length was determined in different areas of IPF lungs. RESULTS: In IPF but not in controls, telomere length in lung was shorter than in other organs, providing rationale to focus on telomere length in lung. Telomere length did not correlate with age and no difference in telomere length was found between diagnostic and explant lung or between basal and apical lung, irrespective of the presence of a radiological apicobasal gradient or fibrosis. Fifteen out of 28 IPF patients had average lung telomere length in the range of patients with a telomerase (TERT) mutation, and formed the IPFshort group. Only in this IPFshort and TERT group telomeres of alveolar type 2 cells were extremely short in fibrotic areas. Additionally, whole exome sequencing of IPF patients revealed two genetic variations in RTEL1 and one in PARN in the IPFshort group. CONCLUSIONS: Average lung tissue telomere shortening does not associated with fibrotic patterns in IPF, however, approximately half of IPF patients show excessive lung telomere shortening that is associated with pulmonary fibrosis driven by telomere attrition.

Human pluripotent stem cell-derived alveolar epithelial cells are alternatives for in vitro pulmotoxicity assessment.

Human pluripotent stem cell (hPSC)-derived alveolar epithelial cells (AECs) provide new opportunities for understanding lung development and the treatment of pulmonary diseases. However, toxicity assessments using hPSC-AECs have not been undertaken. In this study, we generated functional AECs from hPSCs and evaluated their inflammatory and apoptotic responses to cadmium (Cd) exposure (1, 5, and 10 µM) for 24 h compared with the human bronchial epithelial cell line (BEAS-2B) and primary AECs as controls. Our data showed that Cd (10 µM) treatment induced substantial inflammatory responses and apoptosis in BEAS-2B cells, but not in both hPSC-AECs and primary AECs. Interestingly, conditioned medium from AEC cultures significantly alleviated apoptotic and inflammatory responses to Cd exposure in BEAS-2B cells. Using cytokine arrays, several potential factors secreted from hPSC-AECs and primary AECs were detected and may be involved in reducing Cd-induced cytotoxicity. We also observed higher expression of surfactant proteins B and C in both hPSC-AECs and primary AECs, which may contribute to protection against Cd-induced cytotoxicity. These results suggested that hPSC-AECs phenotypically and functionally resemble primary AECs and could be more biologically relevant alternatives for evaluating the pathological contribution of confirmed or potential pulmotoxic materials included in smoking and microdust.