Interactions of Inhaled Nanoparticles with Rat Alveolar Epithelial Cell Monolayers
Authors: Sipos Arnold , Kim Kwang‐Jin , Chow Robert H. , Borok Zea , Crandall Edward D. 
Published: June 2021

Rationale

To understand the contribution to lung pathogenesis of ambient ultrafine air pollution particles, knowledge of basic mechanisms underlying how inhaled nanoparticles interact with alveolar epithelial cells (AEC) is needed. We have reported that polystyrene nanoparticles (PNP) at the apical surface of primary rat alveolar AEC monolayers (RAECM) are taken up in a manner dependent on exposure time, dose and particle size via a non‐endocytic ‘diffusional’ process, induce autophagy, and exit via both fast intracellular calcium‐dependent lysosomal exocytosis and a slower (‘diffusional’) process. In this study, we further investigated intracellular fate of PNP.

Methods

RAECM were apically exposed to near‐infrared labeled PNP (80 μg/mL; carboxylated; 20 nm). AEC were subjected to confocal imaging throughout the volume of the cells. In each z plane, total intracellular fluorescence intensity as well as vesicular and cytosolic PNP were determined and normalized to total intracellular PNP (PNPic). Volumes of endosomal, autophagosomal and lysosomal compartments, and colocalization of PNP in cytosolic vs vesicular compartments, were assessed using an early endosome marker (Rab5) tagged with green fluorescent protein (GFP), quinacrine, microtubule‐associated protein 1 light chain 3 (LC3B)‐GFP, and Magic Red (a lysosomal marker). [PNPic] was then determined by an isotropic method (cuvette‐based microfluorometry) utilizing AEC lysates as a function of time. [PNP] in vesicles ([PNPv]) and cytosol ([PNPc]) were estimated from the respective fractions of compartmental volumes and co‐localized PNP in each compartment.

Results

After 12 hr, [PNPic] reached a plateau of 1.84 mg/mL. Distribution of intracellular PNP was ~80% in intracellular vesicles (primarily lysosomes) and ~20% in cytosol at steady state, equivalent to 8.86 mg/ml and 0.58 mg/mL for [PNPv] and [PNPc], respectively. About 3% of total intracellular PNP was observed in Rab5‐GFP positive early endosomes by 3 hr after PNP exposure, but inhibition of classical endocytosis pathways did not alter PNP uptake kinetics. In contrast, blockade of autophagosome formation by 3‐methyladenine (3MA) markedly reduced PNP uptake and decreased steady state [PNPic] by ~75% with no detectable vesicular PNP. Inhibition of autophagosome‐lysosome fusion by bafilomycin lowered steady state PNPic by ~20% compared to control, with ~81% in cytosol and ~19% in vesicles.

In AEC exposed to PNP, [PNPv] exceeds both [PNPc] and [PNPic], with [PNPc]>>apical [PNP]. Cytosolic presence of PNP triggers autophagosome formation, leading to PNP accumulation in lysosomes. Inhibition of autophagy leads to decreased PNP entry and [PNPic]. These data suggest that intracellular processing of PNP is regulable, offering possible approaches to amelioration of inhaled nanoparticle‐related cytotoxicity in AEC.

Support or Funding Information

Funding: NIH; Will Rogers Institute; Hastings and Whittier Foundations

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.