Current Challenges to Align Inflammatory Key Events in Animals and Lung Cell Models In Vitro

Abstract

With numerous novel and innovative in vitro models emerging every year to reduce or replace animal testing, there is an urgent need to align the design, harmonization, and validation of such systems using in vitro-in vivo extrapolation (IVIVE) approaches. In particular, in inhalation toxicology, there is a lack of predictive and prevalidated in vitro lung models that can be considered a valid alternative for animal testing. The predictive power of such models can be enhanced by applying the Adverse Outcome Pathways (AOP) framework, which casually links key events (KE) relevant to IVIVE. However, one of the difficulties identified is that the endpoint analysis and readouts of specific assays in in vitro and animal models for specific toxicants are currently not harmonized, making the alignment challenging. We summarize the current state of the art in endpoint analysis in the two systems, focusing on inflammatory-induced effects and providing guidance for future research directions to improve the alignment.

Figure 1

Relevant dose metrics for IVIVE. From Loret et al. under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/).

Figure 2

Inflammation-related KEs - Simplified scheme of AOP173 with the highlight of three major events in the inflammatory response. This scheme was adapted from Villeneuve et al., 2018 published in open access that can be used under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/).

Figure 3

Key inflammatory endpoints identified by relevant assays to detect major changes in cell morphology, metabolism, and secretion. The assays to analyze cytotoxicity, cytokine and chemokine release, gene expression changes, and characterization of macrophage phenotype, can be measured in supernatant and collected cells from in vitro cell culture experiments to compare readouts with results acquired from bronchoalveolar lavage fluid (BALF) and tissue collected in vivo.

Figure 4

Hematoxylin and eosin (H&E) staining of healthy and bleomycin (BLM) treated mouse lungs. Mice were either instilled intratracheal with 50 μL of saline (control) or with BLM (1.52U/kg) to induce pulmonary fibrosis and sacrificed on day 14 following instillation. A. Control H&E-stained lung sections of mice treated as outlined above. B. H&E-stained lung sections of bleomycin mice.

Figure 5

Tumor necrosis factor α (TNF- α) release was measured by ELISA. TNF-α released into the basal supernatants as a marker of proinflammatory response in an EpiAlveolar tissue was measured over 21 days (D1-D21) upon exposure to Dörentrup Quartz (DQ₁₂) silica particles. Data are presented as relative to negative control. Data marked as with * were considered statistically significantly (p < 0.05) increased compared to negative control. Adapted with permission from ref (67), further permissions related to the material excerpted should be directed to the ACS.