Date of Award
Spring 2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Biomedical Engineering (ENAS)
First Advisor
Gonzalez, Anjelica
Abstract
Neutrophils are crucial innate immune cells that comprise about 50-70% of the white blood cell population in the human peripheral circulation. These polymorphonuclear leukocytes play a pivotal role in the innate immune system, acting as the primary line of defense against pathogens and facilitating tissue repair. Neutrophils exhibit a diverse repertoire of cytotoxic mechanisms to eliminate offending pathogenic agents and promote the resolution of inflammation. These include phagocytosis, degranulation, generating reactive oxygen species, and forming neutrophil extracellular traps, underscoring their versatility in host defense. A critical aspect of neutrophil functionality is their capacity to infiltrate sites of injury or infection, facilitated by transmigration across the venular wall. While the interactions between neutrophils and the vascular endothelium (EC) have been studied in detail, the roles of pericytes (PCs) and the basement membrane (BM) in regulating neutrophil trafficking, phenotype, and function remain insufficiently explored. Despite their pivotal role in the immune response, aberrant neutrophil behavior and recruitment significantly contribute to the initiation and progression of various inflammatory conditions. These include, but are not limited to, cancer, autoimmune disorders, diabetes mellitus, atherosclerosis, and sepsis. Consequently, elucidating the complex regulatory mechanisms governing the process of neutrophil trafficking and activity in the extravascular space emerges as a question of profound biological and clinical importance. Expanding upon the complex roles of neutrophils in host defense and inflammatory disorders, the notion of neutrophil heterogeneity introduces an additional dimension of complexity to their roles in homeostasis and disease. Traditionally regarded as terminally-differentiated homogeneous effector cells with discrete functions, accumulating evidence challenges this notion, revealing high complexity and diversity within the neutrophil population. Despite ongoing research, a consensus on the molecular signatures that definitively and reproducibly distinguish distinct neutrophil subsets in human peripheral circulation has yet to be established. The debate extends to whether these subsets represent true, distinct lineages or reflect varying states of maturation or differentiation. The classification of neutrophil subsets has historically depended on characteristics such as morphology, localization, surface marker expression, and density. These criteria, however, have proven insufficient for a comprehensive delineation of the neutrophil compartment, leading to the identification of overlapping subpopulations. This has further complicated the nomenclature and fueled controversies surrounding associated effector functions and ontogeny. Moreover, the bulk of the research has mainly concentrated on neutrophils in pathological contexts, with a relatively limited focus on their regulation and function in non-inflammatory baseline conditions, underscoring the need to properly characterize these leukocytes to understand their activity in physiological and pathological states. Therefore, the work presented here seeks to provide a comprehensive characterization of the transcriptional and proteomic diversity of human neutrophils within healthy peripheral circulation. To achieve this, we employed a combination of single-cell RNA sequencing (scRNA-seq) and advanced multiplex flow cytometry analyses to examine the transcriptional and proteomic landscapes of human neutrophils in healthy peripheral blood, alongside assessing their migratory capabilities and phenotypic alterations post-transmigration. Our findings identify three transcriptionally and proteomically distinct neutrophil subsets, each characterized by diverse effector functions and migratory patterns, uniquely altered upon crossing the vascular wall. As we come to identify inherent differences in neutrophil profiles that contribute to vascular transmigration, characteristics of the venular wall itself can lead to uncontrolled leukocyte extravasation, further exacerbating the inflammatory response. Identifying cellular and molecular determinants that contribute to neutrophil recruitment is challenging to accomplish in vivo and requires the development and application of experimental in vitro models. Such models are essential for validating hypotheses and comprehensively understanding the fundamental mechanisms and variations involved in neutrophil extravasation. These should not only mimic physiologically relevant conditions but also enable the real-time observation of rapid intercellular dynamics, thereby facilitating a detailed understanding of the events involved in neutrophil trafficking. Accordingly, this work introduces a novel approach toward addressing the existing limitations of current in vitro models of neutrophil recruitment. We report the development of a highly tunable submicron-level mesh scaffold that resembles the biochemical and biophysical properties of the vascular basement membrane (vBM). This bioengineered model was fabricated using electrospinning to create polyethylene glycol (PEG) fibrillar networks with adjustable parameters such as nanofiber diameter, density, and alignment. Recognizing the critical need for further exploration of the biophysical properties of the vBM, we employed the use of second harmonic imaging and atomic force microscopy to characterize the human microvascular BM's morphology and mechanical properties, which served as a guide for the design constraints of our in vitro system. Rendering the scaffold bioactive via the conjugation of adhesive moieties at the surface of the PEG fibers enabled the culture of microvascular EC and PC to create a composite construct of the microvascular wall. Moreover, we integrated our vBM mimetic with an in-house, customized imaging chamber to evaluate the interactions between migrating leukocytes and the vascular wall components using 4D microscopy. This allowed us to observe real-time interactions between neutrophils and each component of the microvascular wall, advancing our understanding of neutrophil extravasation. Overall, the research delineated herein addresses a critical gap in the current understanding of neutrophil phenotypes and their functional roles during vascular transmigration. It enriches our knowledge of neutrophils in healthy peripheral circulation, providing a more detailed perspective of their contribution to homeostasis and immune function. The introduction of a versatile and highly tunable vBM-mimetic system offers significant potential for further insights into cellular dynamics and interactions within the microvascular compartment in real time. By integrating this system with transcriptomic and proteomic analyses, this tool can uncover the biological functions and behaviors unique to transmigrating neutrophils, thereby elucidating their implications in both health and disease.
Recommended Citation
Morales, Laura C., "Neutrophil Heterogeneity and Transmigration: Decoding Molecular Profiles Through Multi-Omic Analysis and In Vitro Mimicry of the Venular Wall" (2024). Yale Graduate School of Arts and Sciences Dissertations. 1421.
https://elischolar.library.yale.edu/gsas_dissertations/1421
