INVESTIGATING MACROPHAGES AND MONOCYTES AS PROFIBROTIC IMMUNOPATHOGENIC CONTRIBUTORS TO PULMONARY FIBROSIS

Abstract

Idiopathic pulmonary fibrosis (IPF) is a fatal and relentless form of interstitial lung disease, characterized by excessive deposition of extracellular matrix in the lung tissue, declining lung function, and ultimately, respiratory failure. The prognosis of IPF is relatively poor and comparable to some aggressive forms of cancer, with a median survival of just 3 to 5 years after diagnosis. Etiology of IPF remains widely unknown and anti-fibrotic interventions options are limited, with just two drugs, nintedanib and pirfenidone, being approved for treatment of the disease. Although these medications slow disease progression and may extend survival, they are not curative and cannot halt or reverse fibrogenesis. Thus, there is a critical need to investigate the mechanisms that drive disease and strategies to target them. It is believed that macrophages are vital contributors implicated in the pathogenesis of IPF. Through secretion of profibrotic mediators, interaction with various cell types, and mediation of wound healing responses, multiple studies have shown that macrophages drive profibrotic processes. The quantities of both alternatively activated macrophages in the lung and circulating monocytes in the blood have been found to be increased in IPF patients. Additionally, depletion of these cells in animal models of pulmonary fibrosis have shown that they are fundamental for development of the fibrotic response. However, the detailed attributes and mechanisms of these cells remain to be elucidated. Recently, there has been growing interest in the mechanism of macrophage-myofibroblast transition (MMT), where macrophages transform into myofibroblast-like cells that are key effectors in fibrosis. We begin by exploring the evidence for MMT in lung tissue from IPF patients, to gain further insight into the profibrotic mechanisms of macrophages present in the disease. Through mining of a single cell RNAseq dataset of lung tissue explants from IPF patients and controls, and using our curated biobank of IPF surgical lung biopsies for various tissue-based assessments, we demonstrate findings supporting myeloid origin of ACTA2/α-SMA positive cells in IPF. Next, we establish and validate a novel, translational approach for investigation of macrophage profibrotic programming in the lung. Given the interactive and dynamic nature of macrophages, as well as their high degree of phenotypic plasticity, traditional in vitro systems present major limitations in the translation of research findings to true macrophage behaviour in disease. Precision-cut lung slices (PCLS) are living tissue slices derived from the full organ which bypass the limitation of artificially recreating the lung architecture and recapitulating the sophisticated microenvironment. Using our polarization cocktail and PCLS, we develop a moderate-throughput, biologically-relevant system for profibrotic macrophage programming in the lung. We also demonstrate induction of overall features of fibrosis in our system, which we show may be attributable to MMT, as described previously. Complementing our novel platform, we also describe the implementation of high-content imaging using Iterative Bleaching Extends Multiplexity (IBEX) to explore cellular phenotype and spatial characteristics in PCLS, which has potential for expansion to other cultured organ slice systems. Lastly, we investigate the attributes and mechanisms of circulating monocytes isolated from the blood of IPF patients. We confirm their increased quantity in IPF and uncover an aberrant metabolic phenotype. We show that gatekeeper enzyme PDK4 may function as a potential associated target that is also implicated in macrophage polarization, and further explore the mechanistic involvement of aberrant metabolism using our developed PCLS system. Overall, the findings presented in this thesis support the pursuit of knowledge to better understand the profibrotic contribution of macrophages and monocytes in IPF, and offer insights for the development of novel therapeutic interventions in fibrosis.