Effects of nanoparticles on sensitivity to injury and role of autophagy in alveolar epithelial cells

Abstract

Autophagy, a housekeeping mechanism, is crucial in the maintenance of normal cellular function. Although the involvement of autophagic processes is recognized in numerous diseases, it is unknown how cellular homeostasis might be affected by alterations in available autophagic activity and/or autophagic capacity. In this study, we measured autophagic flux and cellular damage in primary cultured monolayers of rat alveolar epithelial cells (AEC) exposed separately and sequentially to two different autophagy inducers. Rat AEC monolayers were exposed apically for 24 hrs to (1) polystyrene nanoparticles (PNP, 20 nm, carboxylated, near-infrared dye-labeled), (2) tunicamycin (TN, a disruptor of protein synthesis and inducer of the unfolded protein response, at 1-15 μg/mL), or (3) TN (1-15 μg/mL) after 12 hrs of PNP pre-incubation. Release of the cytoplasmic enzyme lactate dehydrogenase (LDH) was used as an indicator of cellular damage and quantified by an LDH assay from Dojindo (Rockville, MD). Autophagic flux was assessed by live cell imaging using confocal microscopy and 0.1 μM DAPRed (Dojindo; Rockville, MD) in the presence (for 1 hr) and absence of 40 μM chloroquine. Serial z sections were collected over the entire cell volume of live single cells to detect DAPRed (autophagosome) fluorescence. PNP were taken up into AEC, where their cytosolic presence induced a gradually increased autophagic flux, reaching a steady state at ~10-24 hrs. When AEC were exposed to TN alone for 24 hrs, autophagy was also activated. Cellular damage in the presence of TN, determined by increased LDH release, revealed dose-dependent injury with LD50 of 8.12 μg/mL over 24 hr TN exposure. When AEC were pre-exposed to PNP for 12 hrs and subsequently exposed to TN, increased LDH release was seen with LD50 of 3.95 μg/mL. In summary, the intracellular presence of PNP taken up from apical fluid of rat AEC monolayers activated autophagy. When cellular protein synthesis was disrupted with TN alone, autophagy was also activated. Furthermore, TN induced dose-dependent cellular damage in AEC. However, after pre-exposure to PNP, TN induced greater cellular damage observed as increased LDH release. These results suggest that autophagic capacity is limited and that pre-induction of autophagy by inhaled PNP makes AEC more susceptible to secondary injury by TN. These findings are consistent with the hypothesis that environmental stressors (e.g., ambient air nanoparticles) exert their harmful effects, at least in part, by reducing available autophagic activity/capacity, thereby causing nanoparticle-exposed AEC to be more susceptible to secondary injury. Funding: NIH; WRMPPF. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.