Macrophage autophagy protects mice from cerium oxide nanoparticle-induced lung fibrosis

Abstract

Background: Cerium (Ce) is a rare earth element, rapidly oxidizing to form CeO₂, and currently used in numerous commercial applications, especially as nanoparticles (NP). The potential health effects of Ce remain uncertain, but literature indicates the development of rare earth pneumoconiosis accompanied with granuloma formation, interstitial fibrosis and inflammation. The exact underlying mechanisms are not yet completely understood, and we propose that autophagy could be an interesting target to study, particularly in macrophages. Therefore, the objective of our study was to investigate the role of macrophagic autophagy after pulmonary exposure to CeO₂ NP in mice. Mice lacking the early autophagy gene Atg5 in their myeloid lineage and their wildtype counterparts were exposed to CeO₂ NP by single oropharyngeal administration and sacrificed up to 1 month after. At that time, lung remodeling was thoroughly characterized (inflammatory cells infiltration, expression of fibrotic markers such as αSMA, TGFβ1, total and type I and III collagen deposition), as well as macrophage infiltration (quantification and M1/M2 phenotype).

Results: Such pulmonary exposure to CeO₂ NP induces a progressive and dose-dependent lung fibrosis in the bronchiolar and alveolar walls, together with the activation of autophagy. Blockage of macrophagic autophagy protects from alveolar but not bronchiolar fibrosis, via the modulation of macrophage polarization towards M2 phenotype.

Conclusion: In conclusion, our findings bring novel insight on the role of macrophagic autophagy in lung fibrogenesis, and add to the current awareness of pulmonary macrophages as important players in the disease.

Keywords: Alveolar fibrosis - autophagy - macrophage polarization; Nanoparticle.

Fig. 1

Exposure to CeO₂ NP induces a progressive lung remodeling. Representative lung tissue sections of C57Bl/6 mice exposed to Saline or CeO₂ NP and stained with Hematoxylin Eosin (HE) after 1 day (Panel a), 7 days (Panel b) or 28 days (Panel c). Original magnification × 200. Panel d: higher magnification of alveolar macrophages with internalized NP

Fig. 2

Exposure to CeO₂ NP induces an increase in BAL cellularity. Quantification of total cell count in BAL fluid (Panel a). Each individual circle represents the value obtained from one animal (empty circle: saline exposure – plain circle: CeO₂ NP-exposure). *p < 0.05. Typical images of BAL macrophages from Saline-exposed (Panel b) or CeO₂ NP-exposed (Panel c) mice sacrifice after 24 h, 1 week or 28 days

Fig. 3

Exposure to CeO₂ NP induces autophagy. Representative lung tissue sections of GFP-LC3 mice exposed to Saline or CeO₂ NP after 1 day (Panel a), 7 days (Panel b) or 28 days (Panel c). Original magnification × 200. Panel d: representative lung tissue sections of C57Bl/6 mice exposed to Saline or 50 μg CeO₂ NP and stained, after 28 days, for Atg5. Original magnification × 200

Fig. 4

Atg5^+/− mice are protected from alveolar remodeling. Representative lung tissue sections of WT and Atg5^+/− mice exposed to Saline or CeO₂ NP (5 or 50 μg, observation at 28 days), after HES (Panel a and b for quantification), Sirius red (Panel c and d for quantification), Collagen type I (Panel e and f for quantification), Collagen type III (Panel g and h for quantification), αSMA (Panel i and j for quantification) and TGF-ß (Panel k and l for quantification). Original magnification × 200. Each individual circle represents the mean value obtained from one animal. Empty circle: saline exposure. Grey circle: 5 μg CeO₂ NP exposure. Plain circle: 50 μg CeO₂ NP exposure. *p < 0.05

Fig. 5

Atg5^+/− mice are not protected from bronchiolar remodeling. Representative lung tissue sections of WT and Atg5^+/− mice exposed to Saline or CeO₂ NP (5 or 50 μg, observation at 28 days), after HES (Panel a and b for quantification), Sirius red (Panel c and d for quantification), Collagen type I (Panel e and f for quantification), Collagen type III (Panel g and h for quantification), αSMA (Panel i and j for quantification) and TGF-ß (Panel k and l for quantification). Original magnification × 200. Each individual circle represents the mean value obtained from one animal. Empty circle: saline exposure. Grey circle: 5 μg CeO₂ NP exposure. Plain circle: 50 μg CeO₂ NP exposure. *p < 0.05

Fig. 6

Characterization of CeO₂-induced macrophage recruitment and polarization in mice. Representative lung tissue sections of C57Bl/6 mice exposed to Saline or CeO₂ NP (5 or 50 μg, observation at 28 days), after immunostaining for CD107b, as a marker of total number of macrophages (Panel a). Original magnification × 200. Quantification of CD107b positive cells (Panel b). Representative lung tissue sections of mice exposed to Saline or CeO₂ NP (5 or 50 μg, observation at 28 days), after immunostaining for CD80 (Panel c and d for quantification), CD68 (Panel e and f for quantification), iNOS (Panel g and h for quantification), CD206 (Panel i and j for quantification), CD163 (Panel k and l for quantification) or Arginase 1 (Panel m and n for quantification). Original magnification × 200. Legends as in Fig. 4

Fig. 7

Fibroblast differentiation in vitro. Representative images of WT fibroblasts exposed to supernatant of Vehicle (Control) or CeO₂ NP-exposed macrophages obtained from WT or Atg5+/− mice, and immuno-stained for SMA, Collagen type I or type III