Single-Cell Microchips for Profiling Macrophage Activation Dynamics [electronic resource].

9781687910714

Ann Arbor : ProQuest Dissertations & Theses, 2019.

1 online resource (217 p.)

Access is available to the Yale community.

Access restricted by licensing agreement.

Macrophages often serve as the front line of innate immune defense against a wide range of pathogenic or environmental stimuli. They are highly heterogeneous and dynamic, and may exhibit distinct and/or interchangeable phenotypes upon activation by external signals. How to characterize macrophage activation states and long-term adaption is important but still challenging because of cellular heterogeneity and plasticity. In my PhD work, I developed single-cell microchip technologies to measure the time course of macrophage responses to pathogenic stimulation in order to dissect the dynamics of macrophage activation and adaptation at the transcriptomic and proteomic levels.First, I developed a microchip assay that enables the longitudinal measurement of a large panel of cytokines secreted from thousands of single macrophages in parallel. This technology was applied to the study of cytokine secretions from human monocyte-derived macrophages (hMDMs) over 4 time points upon LPS-induced Toll-like receptor 4(TLR4) activation. The dynamic protein responses classified the cells into two major activation states, which were found to be dependent on the basal state of each cell. Single-cell RNA sequencing (scRNA-Seq) was performed on the same samples at the matched time points and the results confirmed at the transcriptomic level the two distinct states revealed by single-cell cytokine secretion assay. These data showed a cell-intrinsic heterogeneous response in a phenotypically homogeneous population.Second, I applied this longitudinal secretion measurement system as well as a microwell-based scRNA-Seq to murine bone marrow derived macrophages (mBMDMs) to measure the activation dynamics upon LPS stimulation in a way similar as above. The results indicate the existence of two sub-states of mBMDMs consistent with the observation on hMDM cells. Single-cell transcriptomic data further uncovers the molecular circuits underlying this dichotomy, indicating the existence of intrinsic heterogeneity independent of the LPS-induced M1 activation axis. Pathway analysis reveals that innate immune function and translation/extracellular matrix-associated circuits are associated with these two intrinsic cellular states. These results challenge the conventional model of a linear immune activation upon TLR4 ligand, pointing to a multi-dimensional model of macrophage activation heterogeneity.Finally, I combined single-cell-level omics studies and bulk measurements to investigate the longer time response of mBMDMs and the process of adaptation or tolerance. Using an endotoxin-induced innate immune tolerance model, I conducted single-cell protein secretion assay and scRNA-Seq in order to identify the subset of macrophages primarily contributing to immune activation or tolerance by searching for high expression genes associated with innate immune functions. We observed that endotoxin tolerance surprisingly increases the percentage of cytokine secretors. Single-cell data generated in this work for the first time revealed that cellular plasticity is a heterogeneous process and the activation is mainly driven by a subset of macrophages, which has a distinct time course of gene expression as well as selective recovery of activation potential during pathogen re-challenge. This demonstrated previously unknown features of cellular adaptation and memory within macrophage compartment, elevating the comprehension of complex innate immune memory.In summary, the work in my dissertation led to two single-cell microchip-based technologies to measure the dynamics of macrophage activation. We observed unanticipated heterogeneity throughout the activation process, demonstrated in both human monocyte-derived and murine bone marrow-derived macrophages. These results suggest an intrinsically heterogeneous activation dynamics, and open up a door toward the discovery of clinical biomarkers and therapeutic targets associated with the innate immune defense.

Dissertations & Theses @ Yale University.

Books / Online / Dissertations & Theses

English

January 17, 2020

Thesis (Ph.D.)--Yale University, 2019.

Yale University. Biomedical Engineering.