Developing advanced cell culture models to study COVID-19-associated pulmonary fibrosis

Abstract

Patients with severe COVID-19-associated pneumonia are at risk to develop pulmonary fibrosis. To study the underlying mechanisms, we aim to develop advanced cell culture models that reliably reflect COVID-19-related profibrotic microenvironment. To identify key cellular players, we performed pilot immunohistochemistry analysis on lung tissue from COVID-19 patients with fibrosis collected during autopsy. Results revealed diffuse alveolar damage with macrophage infiltration, and myofibroblast accumulation with enriched collagen deposition surrounding the damaged alveoli. To mimic SARS-CoV-2 infection in alveoli, we infected human primary type II alveolar epithelial cells (AEC2) and found enhanced signaling of profibrotic cytokine transforming growth factor β (TGFβ) in some donors. To recreate the early fibrotic niche, an alveolar-macrophage-fibroblast (AMF) tri-culture model was established. After infecting AEC2 with SARS-CoV-2 in this AMF model, gene expression analysis provided evidence for fibroblast-to-myofibroblast transition. Furthermore, we found that overexpression of SARS-CoV-2 papain-like protease (PLpro) can promote TGFβ signaling in HEK293T and A549 cells. After infecting AEC2 with SARS-CoV-2 PLpro lentivirus in the AMF model, we found signs of epithelial-to-mesenchymal transition and fibroblast-to-myofibroblast transition. In future studies, we will use a detailed analysis of COVID-19-associated lung fibrosis with other types of lung fibrosis, to further refine COVID-19-related fibrosis models, including lung-on-chip models. This study was supported by a COVID-19 MKMD grant from ZonMw and Proefdiervrij (grant #114025007).

Authors 11

1. S Liu
   Affiliation as printed

   Department of Pulmonology, Leiden, Netherlands

2. Y Wang
   Affiliation as printed

   Department of Pulmonology, Leiden, Netherlands

3. Melissa Thaler Aachen Department of Medical Microbiology

   Leiden University

   Affiliation as printed

   Department of Medical Microbiology, Leiden, Netherlands

4. Annemarie M. van Schadewijk
   Affiliation as printed

   Department of Pulmonology, Leiden, Netherlands

5. M Van Huizen Aachen Department of Medical Microbiology

   Leiden University

   Affiliation as printed

   Department of Medical Microbiology, Leiden, Netherlands

6. D Cohen
   Affiliation as printed

   Department of Pathology, Leiden, Netherlands

7. Marjolein Kikkert Aachen Department of Medical Microbiology

   Leiden University

   Affiliation as printed

   Department of Medical Microbiology, Leiden, Netherlands

8. Martijn J. van Hemert Aachen Department of Medical Microbiology

   Leiden University

   Affiliation as printed

   Department of Medical Microbiology, Leiden, Netherlands

9. P Ten Dijke Aachen Department of Cell and Chemical Biology

   Leiden University

   Affiliation as printed

   Department of Cell and Chemical Biology, Leiden, Netherlands

10. Pieter S. Hiemstra
    Affiliation as printed

    Department of Pulmonology, Leiden, Netherlands

11. Anne M. van der Does
    Affiliation as printed

    Department of Pulmonology, Leiden, Netherlands