The airways of the lung are the primary sites of disease in asthma and cystic fibrosis. Here we study the cellular composition and hierarchy of the mouse tracheal epithelium by single-cell ...RNA-sequencing (scRNA-seq) and in vivo lineage tracing. We identify a rare cell type, the Foxi1
pulmonary ionocyte; functional variations in club cells based on their location; a distinct cell type in high turnover squamous epithelial structures that we term 'hillocks'; and disease-relevant subsets of tuft and goblet cells. We developed 'pulse-seq', combining scRNA-seq and lineage tracing, to show that tuft, neuroendocrine and ionocyte cells are continually and directly replenished by basal progenitor cells. Ionocytes are the major source of transcripts of the cystic fibrosis transmembrane conductance regulator in both mouse (Cftr) and human (CFTR). Knockout of Foxi1 in mouse ionocytes causes loss of Cftr expression and disrupts airway fluid and mucus physiology, phenotypes that are characteristic of cystic fibrosis. By associating cell-type-specific expression programs with key disease genes, we establish a new cellular narrative for airways disease.
Specialized RNA-seq methods are required to identify the 5' ends of transcripts, which are critical for studies of gene regulation, but these methods have not been systematically benchmarked. We ...directly compared six such methods, including the performance of five methods on a single human cellular RNA sample and a new spike-in RNA assay that helps circumvent challenges resulting from uncertainties in annotation and RNA processing. We found that the 'cap analysis of gene expression' (CAGE) method performed best for mRNA and that most of its unannotated peaks were supported by evidence from other genomic methods. We applied CAGE to eight brain-related samples and determined sample-specific transcription start site (TSS) usage, as well as a transcriptome-wide shift in TSS usage between fetal and adult brain.
Intestinal epithelial cells absorb nutrients, respond to microbes, function as a barrier and help to coordinate immune responses. Here we report profiling of 53,193 individual epithelial cells from ...the small intestine and organoids of mice, which enabled the identification and characterization of previously unknown subtypes of intestinal epithelial cell and their gene signatures. We found unexpected diversity in hormone-secreting enteroendocrine cells and constructed the taxonomy of newly identified subtypes, and distinguished between two subtypes of tuft cell, one of which expresses the epithelial cytokine Tslp and the pan-immune marker CD45, which was not previously associated with non-haematopoietic cells. We also characterized the ways in which cell-intrinsic states and the proportions of different cell types respond to bacterial and helminth infections: Salmonella infection caused an increase in the abundance of Paneth cells and enterocytes, and broad activation of an antimicrobial program; Heligmosomoides polygyrus caused an increase in the abundance of goblet and tuft cells. Our survey highlights previously unidentified markers and programs, associates sensory molecules with cell types, and uncovers principles of gut homeostasis and response to pathogens.
In the small intestine, a niche of accessory cell types supports the generation of mature epithelial cell types from intestinal stem cells (ISCs). It is unclear, however, if and how immune cells in ...the niche affect ISC fate or the balance between self-renewal and differentiation. Here, we use single-cell RNA sequencing (scRNA-seq) to identify MHC class II (MHCII) machinery enrichment in two subsets of Lgr5+ ISCs. We show that MHCII+ Lgr5+ ISCs are non-conventional antigen-presenting cells in co-cultures with CD4+ T helper (Th) cells. Stimulation of intestinal organoids with key Th cytokines affects Lgr5+ ISC renewal and differentiation in opposing ways: pro-inflammatory signals promote differentiation, while regulatory cells and cytokines reduce it. In vivo genetic perturbation of Th cells or MHCII expression on Lgr5+ ISCs impacts epithelial cell differentiation and IEC fate during infection. These interactions between Th cells and Lgr5+ ISCs, thus, orchestrate tissue-wide responses to external signals.
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•Intestinal stem cells (ISCs) are non-conventional antigen-presenting cells by MHCII•Interactions between ISCs and T helper subsets modulate distinct ISC fates•Epithelial MHCII is important for epithelial-cell remodeling following infection•Regulatory T cells and their key cytokine IL-10 support ISC renewal
Intestinal stem cells act as non-conventional antigen presenting cells, and these interactions with T helper cells modulate ISC renewal and differentiation to shape the intestine.
Unprofessional behaviour undermines organizational trust and negatively affects patient safety, the clinical learning environment, and clinician well-being. Improving professionalism in healthcare ...organizations requires insight into the frequency, types, sources, and targets of unprofessional behaviour in order to refine organizational programs and strategies to prevent and address unprofessional behaviours.
To investigate the types and frequency of perceived unprofessional behaviours among health care professionals and to identify the sources and targets of these behaviours.
Data was collected from 2017-2019 based on a convenience sample survey administered to all participants at the start of a mandatory professionalism course for health care professionals including attending physicians, residents and advanced practice providers (APPs) working at one academic hospital in the United States.
Out of the 388 participants in this study, 63% experienced unprofessional behaviour at least once a month, including failing to respond to calls/pages/requests (44.3%), exclusion from decision-making (43.0%) and blaming behaviour (39.9%). Other monthly experienced subtypes ranged from 31.7% for dismissive behaviour to 4.6% for sexual harassment. Residents were more than twice as likely (OR 2.25, p<0.001)) the targets of unprofessional behaviour compared to attending physicians. Female respondents experienced more discriminating behaviours (OR 2.52, p<0.01). Nurses were identified as the most common source of unprofessional behaviours (28.1%), followed by residents from other departments (21%).
Unprofessional behaviour was experienced frequently by all groups, mostly inflicted on these groups by those outside of the own discipline or department. Residents were most frequently identified to be the target and nurses the source of the behaviours. This study highlights that unprofessional behaviour is varied, both regarding types of behaviours as well as targets and sources of such behaviours. This data is instrumental in developing training and remediation initiatives attuned to specific professional roles and specific types of professionalism lapses.
A streamlined and readily accessible sample preparation protocol has been developed to enable TMT-based proteomic profiling of relatively low numbers of cells directly from a flow cytometer. These ...methods were applied to 12 freshly isolated immune cell types from mice to a depth of over 7000 quantified proteins. These data recapitulate many aspects of known immunology, nominate new cell type specific protein markers, and provide evidence for post-transcriptional regulation of gene expression across the immune system.
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Highlights
•A low input proteomic profiling sample preparation workflow has been developed.•The protocol components are accessible and are widely applicable.•Over 7000 proteins across 12 immune cell types were quantified from 3e5 cells.•The data provide evidence for global post-transcriptional regulation.
Proteomic profiling describes the molecular landscape of proteins in cells immediately available to sense, transduce, and enact the appropriate responses to extracellular queues. Transcriptional profiling has proven invaluable to our understanding of cellular responses; however, insights may be lost as mounting evidence suggests transcript levels only moderately correlate with protein levels in steady state cells. Mass spectrometry-based quantitative proteomics is a well-suited and widely used analytical tool for studying global protein abundances. Typical proteomic workflows are often limited by the amount of sample input that is required for deep and quantitative proteome profiling. This is especially true if the cells of interest need to be purified by fluorescence-activated cell sorting (FACS) and one wants to avoid ex vivo culturing. To address this need, we developed an easy to implement, streamlined workflow that enables quantitative proteome profiling from roughly 2 μg of protein input per experimental condition. Utilizing a combination of facile cell collection from cell sorting, solid-state isobaric labeling and multiplexing of peptides, and small-scale fractionation, we profiled the proteomes of 12 freshly isolated, primary murine immune cell types. Analyzing half of the 3e5 cells collected per cell type, we quantified over 7000 proteins across 12 key immune cell populations directly from their resident tissues. We show that low input proteomics is precise, and the data generated accurately reflects many aspects of known immunology, while expanding the list of cell-type specific proteins across the cell types profiled. The low input proteomics methods we developed are readily adaptable and broadly applicable to any cell or sample types and should enable proteome profiling in systems previously unattainable.
Little is known about how metabolites couple tissue-specific stem cell function with physiology. Here we show that, in the mammalian small intestine, the expression of Hmgcs2 ...(3-hydroxy-3-methylglutaryl-CoA synthetase 2), the gene encoding the rate-limiting enzyme in the production of ketone bodies, including beta-hydroxybutyrate (βOHB), distinguishes self-renewing Lgr5+ stem cells (ISCs) from differentiated cell types. Hmgcs2 loss depletes βOHB levels in Lgr5+ ISCs and skews their differentiation toward secretory cell fates, which can be rescued by exogenous βOHB and class I histone deacetylase (HDAC) inhibitor treatment. Mechanistically, βOHB acts by inhibiting HDACs to reinforce Notch signaling, instructing ISC self-renewal and lineage decisions. Notably, although a high-fat ketogenic diet elevates ISC function and post-injury regeneration through βOHB-mediated Notch signaling, a glucose-supplemented diet has the opposite effects. These findings reveal how control of βOHB-activated signaling in ISCs by diet helps to fine-tune stem cell adaptation in homeostasis and injury.
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•HMGCS2 enriches for Lgr5+ ISCs to generate the ketone body βOHB•βOHB depletion reduces stemness, alters differentiation, and hampers regeneration•βOHB, through class I HDAC inhibition, reinforces the NOTCH program in ISCs•Dietary fat and glucose counter-regulate ketone body signaling to instruct ISCs
Ketone body metabolites inform intestinal stem cell decisions in response to diverse diets.
The clear role of autophagy in human inflammatory diseases such as Crohn disease was first identified by genome-wide association studies and subsequently dissected in multiple mechanistic studies. ...ATG16L1 has been particularly well studied in knockout and hypomorph settings as well as models recapitulating the Crohn disease-associated T300A polymorphism. Interestingly, ATG16L1 has a single homolog, ATG16L2, which is independently implicated in diseases, including Crohn disease and systemic lupus erythematosus. However, the contribution of ATG16L2 to canonical autophagy pathways and other cellular functions is poorly understood. To better understand its role, we generated and analyzed the first, to our knowledge, ATG16L2 knockout mouse. Our results show that ATG16L1 and ATG16L2 contribute very distinctly to autophagy and cellular ontogeny in myeloid, lymphoid, and epithelial lineages. Dysregulation of any of these lineages could contribute to complex diseases like Crohn disease and systemic lupus erythematosus, highlighting the value of examining cell-specific effects. We also identify a novel genetic interaction between ATG16L2 and epithelial ATG16L1. These findings are discussed in the context of how these genes may contribute distinctly to human disease.
Pulmonary neuroendocrine cells (PNECs) have crucial roles in airway physiology and immunity by producing bioactive amines and neuropeptides (NPs). A variety of human diseases exhibit PNEC ...hyperplasia. Given accumulated evidence that PNECs represent a heterogenous population of cells, we investigate how PNECs differ, whether the heterogeneity is similarly present in mouse and human cells, and whether specific disease involves discrete PNECs. Herein, we identify three distinct types of PNECs in human and mouse airways based on single and double positivity for TUBB3 and the established NP markers. We show that the three PNEC types exhibit significant differences in NP expression, homeostatic turnover, and response to injury and disease. We provide evidence that these differences parallel their distinct cell of origin from basal stem cells (BSCs) or other airway epithelial progenitors.
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•Rare TUBB3+ solitary pulmonary neuroendocrine cells reside in the conducting airway•Airway basal stem cells generate TUBB3+ pulmonary neuroendocrine cells•TUBB3 is required for cellular protrusions of pulmonary neuroendocrine cells•TUBB3+ pulmonary neuroendocrine cells are amplified in infant diseases
Mou et al. find that the conducting airway harbors three distinct types of pulmonary neuroendocrine cells, distinguished by gene expression, cell of origin, and existence in solitary and clustered configurations. The expansion of PNEC types is selectively associated with NEHI and SIDS, suggesting their contribution to disease.
Despite advances in high-dimensional cellular analysis, the molecular profiling of dynamic behaviors of cells in their native environment remains a major challenge. We present a method that allows us ...to couple the physiological behaviors of cells in an intact murine tissue to deep molecular profiling of individual cells. This method enabled us to establish a novel molecular signature for a striking migratory cellular behavior following injury in murine airways.
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