Differential analysis of scRNA-Seq data to characterize epithelial cells in health and disease

Barrier tissue maintenance and function is crucial to human health. These tissue systems, such as airways, the gut, lungs, and mammary gland, are surfaces for exchange of essential inputs to sustain life that must also tolerate significant insults from physical and biological hazards. Many individual specialized cells within these tissues collaborate to support proper tissue function. Over the course of a person’s lifetime, these tissues sustain a diverse array of perturbations in accordance with their core function. In the mammary gland this includes hormonally-driven re-arrangement to support the production of breast milk by mammary epithelial cells during lactation. In the respiratory system, this can include response to disease, such as allergic inflammation or infection with SARS-CoV2. Studies of these tissues and the cells of which they are composed can allow us to better understand how epithelia respond to diverse perturbations, leading to broad insights in how to mitigate dysfunction and promote health. The recent advent of high-resolution methods like single-cell RNA-Seq (scRNASeq) have enabled the study of these tissues with unprecedented resolution, revealing substantial cell-to-cell heterogeneity and the role of diverse cell states in health and disease. Increased accessibility of these technologies led to a parallel explosion of data collected from diverse tissues and disease states along with available computational methods for their analysis. We describe the use of these methods to identify biological insights from scRNA-seq applied to barrier tissues under diverse perturbations. These include the nasal airway epithelium during allergic inflammation and alterations in mammary epithelial cells over the course of lactation. Both studies find that epithelial diversification aids in maintenance of these barrier tissues. Additionally, we describe a database of scRNA-Seq datasets and its application to an exploratory analysis of potential target cells for the SARS-CoV2 virus including types of epithelial cells in the gut and airway. This work underscores the need to create easy-to-use databases to enable rapid progress during global pandemics. Taken together, this work provides a perspective on the utility of scRNA-Seq data for the study of health and disease to empower both cross-disciplinary collaborations and the development of accessible computational tools and resources.Ph.D

Allergic inflammatory memory in human respiratory epithelial progenitor cells

Barrier tissue dysfunction is a fundamental feature of chronic human inflammatory diseases [superscript 1]. Specialized subsets of epithelial cells—including secretory and ciliated cells—differentiate from basal stem cells to collectively protect the upper airway [superscript 2–4]. Allergic inflammation can develop from persistent activation [superscript 5] of type 2 immunity [superscript 6] in the upper airway, resulting in chronic rhinosinusitis, which ranges in severity from rhinitis to severe nasal polyps [superscript 7]. Basal cell hyperplasia is a hallmark of severe disease [superscript 7–9], but it is not known how these progenitor cells [superscript 2,10,11] contribute to clinical presentation and barrier tissue dysfunction in humans. Here we profile primary human surgical chronic rhinosinusitis samples (18,036 cells, n = 12) that span the disease spectrum using Seq-Well for massively parallel single-cell RNA sequencing [superscript 12], report transcriptomes for human respiratory epithelial, immune and stromal cell types and subsets from a type 2 inflammatory disease, and map key mediators. By comparison with nasal scrapings (18,704 cells, n = 9), we define signatures of core, healthy, inflamed and polyp secretory cells. We reveal marked differences between the epithelial compartments of the non-polyp and polyp cellular ecosystems, identifying and validating a global reduction in cellular diversity of polyps characterized by basal cell hyperplasia, concomitant decreases in glandular cells, and phenotypic shifts in secretory cell antimicrobial expression. We detect an aberrant basal progenitor differentiation trajectory in polyps, and propose cell-intrinsic [superscript 13], epigenetic [superscript 14,15] and extrinsic factors [superscript 11,16,17] that lock polyp basal cells into this uncommitted state. Finally, we functionally demonstrate that ex vivo cultured basal cells retain intrinsic memory of IL-4/IL-13 exposure, and test the potential for clinical blockade of the IL-4 receptor α-subunit to modify basal and secretory cell states in vivo. Overall, we find that reduced epithelial diversity stemming from functional shifts in basal cells is a key characteristic of type 2 immune-mediated barrier tissue dysfunction. Our results demonstrate that epithelial stem cells may contribute to the persistence of human disease by serving as repositories for allergic memories. KNational Institutes of Health (U.S.) (Grant 1DP2OD020839)National Institutes of Health (U.S.) (Grant 2U19AI089992)National Institutes of Health (U.S.) (Grant 1U54CA217377)National Institutes of Health (U.S.) (Grant P01AI039671)National Institutes of Health (U.S.) (Grant 5U24AI118672)National Institutes of Health (U.S.) (Grant 2RM1HG006193)National Institutes of Health (U.S.) (Grant 1R33CA202820)National Institutes of Health (U.S.) (Grant 2R01HL095791)National Institutes of Health (U.S.) (Grant 1R01AI138546)National Institutes of Health (U.S.) (Grant 1R01HL126554)National Institutes of Health (U.S.) (Grant 1R01DA046277)National Institutes of Health (U.S.) (Grant 2R01HL095791)Bill & Melinda Gates Foundation (Grant OPP1139972)Bill & Melinda Gates Foundation (Grant OPP1116944)National Institutes of Health (U.S.) (Grant 2R01GM081871–09 )National Cancer Institute (U.S.) (Grant P30-CA14051)National Institutes of Health (U.S.). Center for AIDS Research (Award P30 AI060354