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Supplementary experimental data and analyses. (PDF 10858 kb)

Autophagy is a fundamental biological process of every cell that aims to maintain cellular homeostasis by degrading unnecessary macromolecules and non-functional organelles. Dysfunctional autophagy was reported in idiopathic pulmonary fibrosis (IPF), a disease with a remarkable age-related onset which may be triggered by chronic lung alveolar epithelial cell type II (AECII) injury and apoptosis. The principle role of autophagy in cellular homeostasis has been well defined, but remains to be studied in the evolution of several forms of lung fibrosis. Amiodarone (AD) and bleomycin are two lung fibrosis inducing agents which trigger disturbed autophagy within the AECII. In this study, we aimed to identify the role of the autophagy marker protein, LC3B in the development of these two forms of lung fibrosis. First, we performed knock down of LC3B in mouse lung epithelial cells and treated them with AD or bleomycin. LC3B knockdown rescued AD induced apoptosis but on the contrary, increased susceptibility to bleomycin induced injury and apoptosis. A systematic analysis of the LC3B-/- mice lungs revealed that aged LC3B-/- mice showed lung collagen deposition, increased cellularity, decreased alveolar spaces, increased apoptosis of AECII paralleled with surfactant alterations, increased lysosomal and endoplasmic reticulum stress providing clues on the importance of this distal autophagy protein in lung fibrosis development. Further, we were able to identify novel LC3B interaction partners within the AECII by mass spectrometric analysis which are being further confirmed. In conclusion, our studies reveal that autophagy has a pathomechanistic but context-specific role in the development of lung fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by excessive deposition of extracellular matrix (ECM). Objectives: We investigated the regulation of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in lung fibrosis. Methods: MMP and TIMP expression, collagenolytic activity and collagen content was assessed in IPF (n=16) versus donor (n=6) lung homogenates and accomplished by in-situ-zymography for gelatinolytic and collagenolytic activities, combined with MMP antigen detection. Role of MMP13 was assessed employing the bleomycin model of lung fibrosis in MMP-13-/- versus wild-type mice. Measurements and Main Results: In IPF, MMPs-1, 2, 7, 9 and 13, but not MMP-8, were significantly upregulated, whereas none of the TIMPs (1–4) were significantly altered. Collagen content was slightly increased and collagenolytic activity was most prominent in the airways and co-localized with MMP-13. We observed an exaggerated early inflammatory response and an augmented lung fibrosis in bleomycin-challenged MMP-13-/- versus wild-type mice, with elevated lung collagen content 28d after bleomycin challenge in the MMP-13-/- mice. Conclusions: Our data suggest that i) collagen deposition in IPF lungs is not primarily due to excessive TIMP production, but rather due to overwhelming ECM production in face of an overall increased, but spatially imbalanced collagenolytic activity, ii) preferential distribution of collagenolytic activity, largely MMP-13, in the airways offers an explanation for the development of honeycomb cysts and iii) despite an overall increase in inflammatory cell content the presence of MMP-13 seems to limit the overall extent of ECM deposition in lung fibrosis.