Sarcoidosis Extracellular Matrix Regulation of Immune Cell Phenotype

Abstract

Fibrotic lung involvement contributes to sarcoidosis fatality in western countries, afflicting up to 20% of patients with this condition. To date, development of fibrosis in sarcoidosis is poorly understood and often unpredictable, with persistent progression in some patients and spontaneous remission in others. Previous studies suggest that fibrotic sarcoidosis is driven by immune cell activity; however, there have been no studies exploring the influence of sarcoidosis lung extracellular matrix (ECM) on immune cell phenotype. The use of decellularization techniques produces scaffolds that preserve the architecture, biochemical components, and mechanical properties of the lung and can be utilized to model the microenvironment of lungs in diseases such as IPF, scleroderma, and COPD. However, this approach has not yet been applied to the study of sarcoidosis lungs. Here we characterized the architectural and biochemical aspects of decellularized stage IV sarcoidosis ECM and developed a culture system utilizing these decellularized scaffolds to investigate the fibrotic progression of immune cells in response to the diseased lung microenvironment. Methods: Explanted lung tissue obtained at autopsy was used to generate control lung scaffolds (CLS) and stage IV sarcoidosis lung scaffolds (SLS) through well characterized detergent-based decellularization methods. Histologic evaluation, LC/MS-based proteomics, and immunofluorescence were used to assess the anatomic and biochemical composition of the scaffolds. Peripheral blood mononuclear cells (PBMCs) isolated from subjects with Stage IV sarcoidosis were cultured in the CLS and SLS for 24 hours, 7 days, and 14 days of culture at which point their ability to adopt a mesenchymal phenotype was assessed through CD45 and pro-collagen Iα1 (pro-coll Iα1) staining. Results: Relative to CLS, SLS showed altered tissue architecture and changes in biochemical composition consistent with accelerated ECM turnover including increased detection and activity of metalloproteinases, decreased detection of basement membrane components such as collagen IV and laminin, and increased expression of proteins related to adhesion such as fibronectin and periostin. Functionally, these attributes combined to augment detection of PBMCs-derived CD45+ pro-coll Iα1+ cells after 7 days of culture. Conclusions: SLS demonstrate differences in biochemical composition reflecting ongoing ECM remodeling and enhanced cellular adhesion that are associated with a mesenchymal phenotype in cultured leukocytes. These factors may contribute to the development of pulmonary fibrosis in stage IV sarcoidosis lungs.