Alignment of in vitro and in vivo pulmonary inflammation models using crystalline quartz silica

Abstract

Background: Systematic in vitro-in vivo comparisons are increasingly used to assess the relevance and predictivity of in vitro lung models for inhalation toxicology and regulatory risk assessment. Here, we compared inflammatory endpoints across established in vitro and in vivo pulmonary models following exposure to crystalline quartz silica particles (DQ₁₂). To better align exposure timelines, in vitro responses assessed at 24 h were extended to 7 days, matching the post-exposure period recommended in OECD inhalation testing guidelines for animal testing. To test its potential and limitations, we utilized a harmonized in vitro co-culture model consisting of the human bronchial cell line Calu-3 and human monocyte-derived macrophages, which were exposed to DQ₁₂ particles.

Results: No increased cytotoxicity or impairment of barrier integrity, as assessed by transepithelial electrical resistance (TEER) and tight junction protein 1 (TJP1) gene expression, was observed 7 days after exposure in vitro, in contrast to clear tissue damage detected in vivo. However, we observed increased release of interleukin (IL)-6 and IL-8, measured at both protein and gene levels. Gene expressions of IL-1β, IL-6, and IL-8 showed positive correlations between the in vitro and in vivo models.

Conclusions: By extending exposure duration and aligning time points, this study identified inflammatory biomarkers that correlate between an in vitro lung model and in vivo data. These findings demonstrate the value of refined in vitro models for assessing particle-induced lung inflammation and support their relevance for hazard assessment.

Keywords: Animal testing; Crystalline quartz silica particles; In vitro toxicology; Inflammation; Inhalation toxicology; Lung cell model.

Fig. 1

Characterization of the Calu-3 and MDMs co-culture model. A Brightfield images with a scale bar of 100 μm show Calu-3 cells after seeding on top of the insert in a submerged condition until day 8. After 8 days, they were transferred to ALI. MDMs were added on day 15, as indicated with a yellow arrow, and exposure was performed on day 16. B Calu-3 monolayer nuclei DAPI staining (cyan) and F-actin (magenta). C TEER values from cell seeding until the exposure; data are presented as mean ± standard deviation, N = 4. D MDMs (yellow) live imaging with Vibrant DiD staining

Fig. 2

Characterization of DQ₁₂ particles. A SEM image of DQ₁₂ topographic features. B TEM image of DQ₁₂ deposition on TEM grids. C The size of DQ₁₂ particles was determined using TEM image analysis

Fig. 3

Effects on in vitro co-culture model upon 24 h post-exposure to DQ₁₂. A Cytotoxicity measured as LDH release from the apical side of the insert 24 h after exposure, normalized to 0.2% Triton X-100 control. Data are presented as mean ± standard deviation (N = 4); B Membrane integrity evaluated by TEER measurements, data are presented as mean ± standard deviation (N = 4); C IL-6 release measured by ELISA assay, positive control was LPS treated cells. Data are presented as mean ± standard deviation (N = 3), results marked as with ** were considered statistically significant (p < 0.01) according to ordinary one-way ANOVA with Dunnett’s multiple comparison test; D qPCR data of IL-1β, IL-6, and IL-8 gene expression profile as markers of pro-inflammatory response upon exposure to DQ₁₂, dose range from 10–300 µg/cm². Data for gene expression are presented as fold-change to the untreated control. The positive control was LPS-treated cells. Data are presented as mean ± standard deviation (N = 3)

Fig. 4

Experimental design for in vivo- in vitro comparison. Exposures were performed in both models, starting with the same batch of DQ₁₂ material. Cytotoxicity and inflammatory endpoints were measured after days 1, 3 and 7

Fig. 5

Effects on in vitro co-culture model upon 7 days post-exposure to DQ₁₂. A Cytotoxicity measured as LDH release after 7 days from the apical side of the insert, normalized to 0.2% Triton X-100 control, data are presented as mean ± standard deviation (N = 3); B Cytotoxicity was assessed in vivo by measuring LDH release in BALF. Rats were administered 8 mg/kg of DQ₁₂ via oropharyngeal aspiration in two doses of 200 µl of dH₂O, and BALF was collected at 24 h, 3 days, and 7 days post-administration. Control rats received 400 µl of dH₂O via oropharyngeal aspiration.; C Membrane integrity evaluated by TEER measurements. D H&E staining was performed in rat lung tissue following DQ₁₂ administration, following the same treatment protocol described in (B). Data are presented as mean ± standard deviation (N = 4 for in vitro, N = 6–8 for in vivo experiments). It should be noted that the in vitro doses (10–100 µg/cm²) are not intended to be dosimetrically equivalent to the calculated in vivo deposited dose (0.2–0.5 µg/cm²). The comparison in Fig. 5 is qualitative and endpoint-based rather than quantitative dosimetry

Fig. 6

In vitro-in vivo comparisons following exposure to DQ₁₂. A Increased IL-8 secretion in the in vitro model on days 3 and 7 following exposure. B Total number of cells counted in BALF at different time points post-DQ₁₂ administration in rats. Rats were administered 8 mg/kg of DQ₁₂ via oropharyngeal aspiration in two doses of 200 µl of dH₂O, and BALF was collected 24 h, 3 days, and 7 days post-administration. Control rats received 400 µl of dH₂O via oropharyngeal aspiration. C Diff Quick Staining of BALF cells following the exposure protocol as described in (B). D Interleukin 1β, interleukin-6, and interleukin-8 gene expression profile in vivo and in vitro on days 1,3 and 7 upon exposure to DQ₁₂ at 10 µg/cm² (low) or 100 µg/cm² (high). Rats were exposed as described in (B), receiving a calculated dose of 0.2-0.2.5ug/cm². Data are presented relative to the negative control (line); untreated cells served as a baseline; the positive control was LPS-treated cells. Data are presented as mean ± standard deviation (N = 5 for in vitro and N = 6–8 for in vivo)