Uncovering the cytotoxic effects of air pollution with multi-modal imaging of in vitro respiratory models

Abstract

Annually, an estimated seven million deaths are linked to exposure to airborne pollutants. Despite extensive epidemiological evidence supporting clear associations between poor air quality and a range of short- and long-term health effects, there are considerable gaps in our understanding of the specific mechanisms by which pollutant exposure induces adverse biological responses at the cellular and tissue levels. The development of more complex, predictive, in vitro respiratory models, including two- and three-dimensional cell cultures, spheroids, organoids and tissue cultures, along with more realistic aerosol exposure systems, offers new opportunities to investigate the cytotoxic effects of airborne particulates under controlled laboratory conditions. Parallel advances in high-resolution microscopy have resulted in a range of in vitro imaging tools capable of visualizing and analysing biological systems across unprecedented scales of length, time and complexity. This article considers state-of-the-art in vitro respiratory models and aerosol exposure systems and how they can be interrogated using high-resolution microscopy techniques to investigate cell-pollutant interactions, from the uptake and trafficking of particles to structural and functional modification of subcellular organelles and cells. These data can provide a mechanistic basis from which to advance our understanding of the health effects of airborne particulate pollution and develop improved mitigation measures.

Keywords: bioimaging; cell and tissue culture models; microscopy; respiratory toxicology.

Figure 1.

The human respiratory tract and PM inhalation. A mixture of solid particles in the air are inhaled through the URT (nose, nasopharynx and the oropharynx). Typically, the nasal apparatus removes a higher fraction of larger particles (greater than 2.5 µm); however, a certain fraction of these large particles reaches the alveolar section, through ciliary clearance and mucus production from goblet cells. These particles (2.5 µm and less than 1 µm) move down to the LRT (larynx, trachea, bronchi, bronchioles and alveoli), where inhaled air is conducted to the alveolar surface for gas exchange, which takes place between the thin lining of alveolar type I epithelial cells. PMs < 0.1 µm can flow into the blood stream, where they can initiate inflammatory responses and/or affect other organs in the body.

Figure 2.

Model systems for respiratory toxicology. (a) The commonly employed submerged cell culture system. (b) Cells cultured in scaffolds to mimic the native lung composition of ECM. (c) Native slice cultures of PCLS with intact lung compartments of airways, vascular and alveolar localizations with structural and inflammatory cells. (d) Co-culture systems, PCLS and repopulated lung scaffolds subjected to a dynamic load, which mimics breathing patterns in vivo. (e) In vivo lung parameters in healthy individuals and patients with pulmonary diseases.

Figure 3.

Practical and theoretical limits to in vitro imaging. Schematic diagram illustrating the spatial resolution and depth penetration of different in vitro imaging techniques and how these relate to the size of relevant subcellular and cellular structures, PM size ranges (top) and respiratory model systems (right).

Figure 4.

Example of high-resolution images of respiratory models. (a) Internalization of PM_2.5 within the inner mitochondrial membrane in HTR-8 cells (arrow) and mitochondrial vacuolization revealed by TEM. Scale bars are 1 µm. Adapted from Naav et al. ([154], fig. 6A–C); copyright 2020; https://creativecommons.org/licenses/by/4.0/. (b) Ring-shaped structures of CD81 in the membrane of IgG-activated lung macrophages revealed using STORM. Scale bars are 5 μm in main image and 0.5 μm in zoomed images. Adapted from Ambrose et al. ([155], fig. 7B); copyright 2020, Elsevier Group; https://creativecommons.org/licenses/by/4.0/. (c) AFM topography images reveal morphological changes in activated eosinophils in individuals with acute asthma (bottom) compared with those from a health control (top). Scale bars are 5 µm. Adapted from Eaton et al. ([156], fig. 1d,e,g,h); copyright 2019, Frontiers in Physiology; https://creativecommons.org/licenses/by/4.0/. (d) Visualization of RSV particles (green) in cultured A549 cells stained for F-actin (red) and tubulin (cyan) using confocal microscopy (left), STED (middle) and deconvolved STED microscopy (right). Bottom row shows zoomed in view of part of the cell shown in the top row (RSV channel only). Scale bars are 5 µm in main image and 1 µm in inset image. Adapted from Mehedi et al. ([157], fig. 1 Merge and RSV F); copyright 2017, Bio-Protocol via PMC. (e) Colour-coded depth projection of a human-derived lung tissue scaffold. Image computed from a focal plane image series captured using an Airy beam light sheet fluorescence microscope. Scale bar is 100 µm; previously unpublished. (f) Multi-wall carbon nanotubes (pink) visible in a murine lung section (blue) imaged using stimulated Raman scattering. Scale bar is 50 µm. Adapted from Migliaccio et al. ([158], fig. 1C,D; Copyright 2021, Springer Nature Group; https://creativecommons.org/licenses/by/4.0/.