A novel ex vivo approach for investigating profibrotic macrophage polarization using murine precision-cut lung slices

Megan Vierhout; Anmar Ayoub; Pareesa Ali; Vaishnavi Kumaran; Safaa Naiel; Takuma Isshiki; Joshua F E Koenig; Martin R J Kolb; Kjetil Ask

doi:10.1016/j.bbrc.2024.151038

A novel ex vivo approach for investigating profibrotic macrophage polarization using murine precision-cut lung slices

Biochem Biophys Res Commun. 2024 Nov 20:741:151038. doi: 10.1016/j.bbrc.2024.151038. Online ahead of print.

Authors

Megan Vierhout¹, Anmar Ayoub¹, Pareesa Ali¹, Vaishnavi Kumaran¹, Safaa Naiel¹, Takuma Isshiki², Joshua F E Koenig³, Martin R J Kolb¹, Kjetil Ask⁴

Affiliations

¹ Firestone Institute for Respiratory Health, Department of Medicine, McMaster University and the Research Institute of St. Joe's Hamilton, 50 Charlton Avenue East, Hamilton, Ontario, L8N 4A6, Canada; McMaster Immunology Research Centre, Department of Medicine, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4L8, Canada.
² Firestone Institute for Respiratory Health, Department of Medicine, McMaster University and the Research Institute of St. Joe's Hamilton, 50 Charlton Avenue East, Hamilton, Ontario, L8N 4A6, Canada; McMaster Immunology Research Centre, Department of Medicine, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4L8, Canada; Department of Respiratory Medicine, Toho University School of Medicine, 6-11-1 Omorinishi, Ota-ku, Tokyo, 143-8540, Japan.
³ Department of Respiratory Medicine, Toho University School of Medicine, 6-11-1 Omorinishi, Ota-ku, Tokyo, 143-8540, Japan; Schroeder Allergy and Immunology Research Institute, Faculty of Health Sciences, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4L8, Canada.
⁴ Firestone Institute for Respiratory Health, Department of Medicine, McMaster University and the Research Institute of St. Joe's Hamilton, 50 Charlton Avenue East, Hamilton, Ontario, L8N 4A6, Canada; McMaster Immunology Research Centre, Department of Medicine, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4L8, Canada. Electronic address: askkj@mcmaster.ca.

PMID: 39603027
DOI: 10.1016/j.bbrc.2024.151038

Abstract

Idiopathic pulmonary fibrosis (IPF) is fatal interstitial lung disease characterized by excessive scarring of the lung tissue and declining respiratory function. Given its short prognosis and limited treatment options, novel strategies to investigate emerging experimental treatments are urgently needed. Macrophages, as the most abundant immune cell in the lung, have key implications in wound healing and lung fibrosis. However, they are highly plastic and adaptive to their surrounding microenvironment, and thus to maximize translation of research to lung disease, there is a need to study macrophages in multifaceted, complex systems that are representative of the lung. Precision-cut lung slices (PCLS) are living tissue preparations derived from the lung that are cultured ex vivo, which bypass the need for artificial recapitulation of the lung milieu and architecture. Macrophage programming studies are traditionally conducted using isolated cells in vitro, thus our objective was to establish and validate a moderate-throughput, biologically-translational, viable model to study profibrotic polarization of pulmonary-resident macrophages using murine PCLS. To achieve this, we used a polarization cocktail (PC), consisting of IL-4, IL-13, and IL-6, over a 72-h time course. We first demonstrated no adverse effects of the PC on PCLS viability and architecture. Next, we showed that multiple markers of macrophage profibrotic polarization, including Arginase-1, CD206, YM1, and CCL17 were induced in PCLS following PC treatment. Through tissue microarray-based histological assessments, we directly visualized and quantified Arginase-1 and CD206 staining in PCLS in a moderate-throughput manner. We further delineated phenotype of polarized macrophages, and using high-plex immunolabelling with the Iterative Bleaching Extends Multiplexity (IBEX) method, showed that the PC effects both interstitial and alveolar macrophages. Substantiating the profibrotic properties of the system, we also showed expression of extracellular matrix components and fibrotic markers in stimulated PCLS. Finally, we demonstrated that clodronate treatment diminishes the PC effects on profibrotic macrophage readouts. Overall, our findings support a suitable complex model for studying ex vivo profibrotic macrophage programming in the lung, with future capacity for investigating experimental therapeutic candidates and disease mechanisms in pulmonary fibrosis.

Keywords: Macrophage programming; Macrophages; Precision-cut lung slices; Profibrotic polarization; Pulmonary fibrosis; ex vivo.