Longitudinal micro-computed tomography-derived biomarkers quantify non-resolving lung fibrosis in a silicosis mouse model

Abstract

In spite of many compounds identified as antifibrotic in preclinical studies, pulmonary fibrosis remains a life-threatening condition for which highly effective treatment is still lacking. Towards improving the success-rate of bench-to-bedside translation, we investigated in vivo µCT-derived biomarkers to repeatedly quantify experimental silica-induced pulmonary fibrosis and assessed clinically relevant readouts up to several months after silicosis induction. Mice were oropharyngeally instilled with crystalline silica or saline and longitudinally monitored with respiratory-gated-high-resolution µCT to evaluate disease onset and progress using scan-derived biomarkers. At weeks 1, 5, 9 and 15, we assessed lung function, inflammation and fibrosis in subsets of mice in a cross-sectional manner. Silica-instillation increased the non-aerated lung volume, corresponding to onset and progression of inflammatory and fibrotic processes not resolving with time. Moreover, total lung volume progressively increased with silicosis. The volume of healthy, aerated lung first dropped then increased, corresponding to an acute inflammatory response followed by recovery into lower elevated aerated lung volume. Imaging results were confirmed by a significantly decreased Tiffeneau index, increased neutrophilic inflammation, increased IL-13, MCP-1, MIP-2 and TNF-α concentration in bronchoalveolar lavage fluid, increased collagen content and fibrotic nodules. µCT-derived biomarkers enable longitudinal evaluation of early onset inflammation and non-resolving pulmonary fibrosis as well as lung volumes in a sensitive and non-invasive manner. This approach and model of non-resolving lung fibrosis provides quantitative assessment of disease progression and stabilization over weeks and months, essential towards evaluation of fibrotic disease burden and antifibrotic therapy evaluation in preclinical studies.

Figure 1

Experimental design. This scheme summarizes the followed protocol. X indicates a µCT scan. At four endpoints (1, 5, 9 and 15 weeks post instillation), a cohort of animals was sacrificed to obtain lung function and ex vivo measurements. At each endpoint, the number of animals is indicated and represents the number of animals up until that endpoint.

Figure 2

µCT visualizes and quantifies onset and progression of the inflammatory and fibrotic response upon silica-instillation. (a) Representative images of one control and one silica-instilled animal at selected time points, from baseline up until 15 weeks, visualize the inflammatory and fibrotic response. µCT-derived biomarkers reflect disease onset and progression in silica-instilled animals: (b) mean lung density expressed in HU (c) aerated lung volume (mL) (d) non-aerated lung volume (mL) (e) total lung volume (mL). Data are presented as mean ± SD. Full line represents longitudinal data. Mixed-effects model with Geisser–Greenhouse correction was based on the following number of animals for every timepoint: BL-w1 24 control and 34 silica mice; w2–5 18 control and 26 silica mice; w7–9 12 control and 16 silica mice; w11–15 6 control and 8 silica mice. *p value < 0.05; ***p value < 0.001; BL baseline; *under the x-axes indicate endpoints.

Figure 3

Pulmonary function data reflect fibrotic disease and correlate with in vivo µCT-derived biomarkers. At every endpoint, lung function was assessed by performing (a) deep inflation, (b) forced oscillations and (c) negative pressure-driven forced expiration maneuvers. Data are presented as mean ± SD. Dashed line represents cross-sectional data. Two-way ANOVA with Sidak’s correction for multiple comparison was based on the following number of animals: w1 6 control and 6 silica mice; w5 6 control and 10 silica mice; w9 6 control and 8 silica mice; w15 6 control and 8 silica mice. *p value < 0.05; **p value < 0.01; ***p value < 0.001. Correlations between µCT-derived biomarkers and lung function measurements indicate the agreement between both methods (d) aerated lung volume and inspiratory capacity (e) total lung volume and tissue hysteresivity (f) non-aerated lung volume and Tiffeneau index. For every correlation the Pearson correlation coefficient and the p value are given. Correlations only include silica-instilled animals.

Figure 4

Silica-instillation changes inflammatory readouts of BAL fluid and serum and increases the collagen content. At every endpoint, several ex vivo readouts for inflammatory and fibrotic responses were obtained. Silica instillation changed the number of viable cells (a) and neutrophils (c) but did not alter the number of macrophages (b) in the BAL fluid. Moreover, silica increased the protein concentration in the BAL fluid (d), the collagen content measured by OH-proline assay reflecting a fibrotic response on the silica particles (e) and the surfactant protein D (SpD) concentration in serum (f). Data are presented as mean ± SD. Dashed line represents cross-sectional data. Two-way ANOVA with Sidak’s correction for multiple comparison was based on the following number of animals: w1 6 control and 6 silica mice; w5 6 control and 10 silica mice; w9 6 control and 8 silica mice; w15 6 control and 8 silica mice. *p value < 0.05; **p value < 0.01; ***p value < 0.001. Correlations between µCT-derived biomarkers and ex vivo measurements indicate the agreement between the inflammatory and fibrotic disease and µCT-derived biomarkers (g) non-aerated lung volume and protein concentration in bronchoalveolar lavage fluid, (h) non-aerated lung volume and OH-proline content, (i) non-aerated lung volume and protein concentration in serum. For every correlation the Pearson correlation coefficient is given and the p value. Correlations only include silica-instilled animals.

Figure 5

Silica instillation induces a pro-inflammatory response characterized by an increased TNF-α, IL-13 and MIP-2 content in BAL fluid. At every endpoint, cytokine concentration was determined. Silica-instillation altered the amount of (a) TNF-α, (b) IL-13 and (c) MIP-2 concentration in the BAL fluid. Data is presented as mean ± SD. Dashed line represents cross-sectional data. Two-way ANOVA with Sidak’s correction for multiple comparison was based on the following number of animals: w1 6 control and 6 silica mice; w5 6 control and 10 silica mice; w9 6 control and 8 silica mice; w15 6 control and 8 silica mice. *p value < 0.05; **p value < 0.01; ***p value < 0.001.

Figure 6

Histopathological analysis of silica-instilled animals at every timepoint. Representative H&E stained images are shown for every timepoint for control and silica-instilled animals (magnification of 12.5 × and 50 ×). Representative Sirius Red images are shown for every timepoint for control and silica-instilled animals (magnification of 200 ×). For control animals, only native collagen is shown in centrilobular area. For silica-instilled animals, centrilobular located nodules with increasing amount of newly formed collagen (indicated with white arrows) are shown.