Transcriptomic profiling of macrophage polarization

Abstract

Macrophages perform a wide variety of crucial, and sometimes contradictory, functions. While “pro-inflammatory” activities like fighting off infections and “anti-inflammatory” activities like wound-healing traditionally have been attributed to M1 and M2 macrophage subsets, studies of macrophages in both in vitro and in vivo contexts suggest that macrophages are phenotypically plastic and may shift states in response to environmental changes. However, it remains unclear whether macrophages retain any persistent memory of past polarization states which may then impact their future repolarization to new states. In this dissertation, I first describe the evolving understanding of macrophage polarization and phenotypic plasticity and introduce commonly used models for macrophage polarization in humans and mice. I also outline recent advances in single-cell RNA-sequencing and some of the most popularly used platforms for single-cell transcriptomics. I then focus on my work characterizing macrophage polarization and repolarization in vitro, where I performed deep transcriptomic profiling at high temporal resolution as macrophages were polarized with cytokines that drive them into “M1” and “M2” molecular states. I find through trajectory analysis of their global transcriptomic profiles that macrophages which are first polarized to M1 or M2 and then subsequently repolarized demonstrate little to no memory of their polarization history. I observe complete repolarization both from M1 to M2 and vice versa, and I find that macrophage transcriptional phenotypes are defined by the current cell microenvironment, rather than an amalgamation of past and present states. In the following chapters, I present preliminary work aimed at characterizing alveolar macrophages, the tissue-resident macrophages of the lung: I first describe my attempts to identify key stimuli for triggering specification of alveolar macrophage fate. I then discuss preliminary results from single-cell RNA-seq profiling of alveolar macrophages in the context of pulmonary alveolar proteinosis. In the final chapter, I reflect on challenges I faced in adapting single-cell RNA-sequencing methods to work with primary tissue cells and summarize the main findings from my work.