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- Fresard, Laure, et al.
(författare)
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Identification of rare-disease genes using blood transcriptome sequencing and large control cohorts
- 2019
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Ingår i: Nature Medicine. - : NATURE PUBLISHING GROUP. - 1078-8956 .- 1546-170X. ; 25:6, s. 911-919
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Tidskriftsartikel (refereegranskat)abstract
- It is estimated that 350 million individuals worldwide suffer from rare diseases, which are predominantly caused by mutation in a single gene(1). The current molecular diagnostic rate is estimated at 50%, with whole-exome sequencing (WES) among the most successful approaches(2-5). For patients in whom WES is uninformative, RNA sequencing (RNA-seq) has shown diagnostic utility in specific tissues and diseases(6-8). This includes muscle biopsies from patients with undiagnosed rare muscle disorders(6,9), and cultured fibroblasts from patients with mitochondrial disorders(7). However, for many individuals, biopsies are not performed for clinical care, and tissues are difficult to access. We sought to assess the utility of RNA-seq from blood as a diagnostic tool for rare diseases of different pathophysiologies. We generated whole-blood RNA-seq from 94 individuals with undiagnosed rare diseases spanning 16 diverse disease categories. We developed a robust approach to compare data from these individuals with large sets of RNA-seq data for controls (n = 1,594 unrelated controls and n = 49 family members) and demonstrated the impacts of expression, splicing, gene and variant filtering strategies on disease gene identification. Across our cohort, we observed that RNA-seq yields a 7.5% diagnostic rate, and an additional 16.7% with improved candidate gene resolution.
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- Gustafsson, Jenny K, 1981, et al.
(författare)
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Intestinal goblet cells sample and deliver lumenal antigens by regulated endocytic uptake and transcytosis
- 2021
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Ingår i: eLife. - 2050-084X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Intestinal goblet cells maintain the protective epithelial barrier through mucus secretion and yet sample lumenal substances for immune processing through formation of goblet cell associated antigen passages (GAPs). The cellular biology of GAPs and how these divergent processes are balanced and regulated by goblet cells remains unknown. Using high-resolution light and electron microscopy, we found that in mice, GAPs were formed by an acetylcholine (ACh)-dependent endocytic event remarkable for delivery of fluid-phase cargo retrograde into the trans-golgi network and across the cell by transcytosis - in addition to the expected transport of fluid-phase cargo by endosomes to multi-vesicular bodies and lysosomes. While ACh also induced goblet cells to secrete mucins, ACh-induced GAP formation and mucin secretion were functionally independent and mediated by different receptors and signaling pathways, enabling goblet cells to differentially regulate these processes to accommodate the dynamically changing demands of the mucosal environment for barrier maintenance and sampling of lumenal substances. eLife digest Cells in the gut need to be protected against the many harmful microbes which inhabit this environment. Yet the immune system also needs to 'keep an eye' on intestinal contents to maintain tolerance to innocuous substances, such as those from the diet. The 'goblet cells' that are part of the gut lining do both: they create a mucus barrier that stops germs from invading the body, but they also can pass on molecules from the intestine to immune cells deep in the tissue to promote tolerance. This is achieved through a 'GAP' mechanism. A chemical messenger called acetylcholine can trigger both mucus release and the GAP process in goblet cells. Gustafsson et al. investigated how the cells could take on these two seemingly opposing roles in response to the same signal. A fluorescent molecule was introduced into the intestines of mice, and monitored as it pass through the goblet cells. This revealed how the GAP process took place: the cells were able to capture molecules from the intestines, wrap them in internal sack-like vesicles and then transport them across the entire cell. To explore the role of acetylcholine, Gustafsson et al. blocked the receptors that detect the messenger at the surface of goblet cells. Different receptors and therefore different cascades of molecular events were found to control mucus secretion and GAP formation; this explains how the two processes can be performed in parallel and independently from each other. Understanding how cells relay molecules to the immune system is relevant to other tissues in contact with the environment, such as the eyes, the airways, or the inside of the genital and urinary tracts. Understanding, and then ultimately harnessing this mechanism could help design of new ways to deliver drugs to the immune system and alter immune outcomes.
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- Knoop, K. A., et al.
(författare)
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In vivo labeling of epithelial cell-associated antigen passages in the murine intestine
- 2020
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Ingår i: Lab Animal. - : Springer Science and Business Media LLC. - 0093-7355 .- 1548-4475. ; 49, s. 79-88
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Tidskriftsartikel (refereegranskat)abstract
- Goblet cell-associated antigen passages can deliver luminal substances to antigen-presenting cells to induce antigen-specific T cell responses. This protocol describes how to identify and quantify intestinal epithelial cells that have the capacity to take up luminal substances, by intraluminal injection of fluorescent dextran, tissue sectioning for slide preparation and imaging with fluorescence microscopy. The intestinal immune system samples luminal contents to induce adaptive immune responses that include tolerance in the steady state and protective immunity during infection. How luminal substances are delivered to the immune system has not been fully investigated. Goblet cells have an important role in this process by delivering luminal substances to the immune system through the formation of goblet cell-associated antigen passages (GAPs). Soluble antigens in the intestinal lumen are transported across the epithelium transcellularly through GAPs and delivered to dendritic cells for presentation to T cells and induction of immune responses. GAPs can be identified and quantified by using the ability of GAP-forming goblet cells to take up fluorescently labeled dextran. Here, we describe a method to visualize GAPs and other cells that have the capacity to take up luminal substances by intraluminal injection of fluorescent dextran in mice under anesthesia, tissue sectioning for slide preparation and imaging with fluorescence microscopy. In contrast to in vivo two-photon imaging previously used to identify GAPs, this technique is not limited by anatomical constraints and can be used to visualize GAP formation throughout the length of the intestine. In addition, this method can be combined with common immunohistochemistry protocols to visualize other cell types. This approach can be used to compare GAP formation following different treatments or changes to the luminal environment and to uncover how sampling of luminal substances is altered in pathophysiological conditions. This protocol requires 8 working hours over 2-3 d to be completed.
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- Knoop, K. A., et al.
(författare)
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Synchronization of mothers and offspring promotes tolerance and limits allergy
- 2020
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Ingår i: JCI insight. - : American Society for Clinical Investigation. - 2379-3708. ; 5:15
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Tidskriftsartikel (refereegranskat)abstract
- Allergic disorders, characterized by Th2 immune responses to environmental substances, are increasingly common in children in Western societies. Multiple studies indicate that breastfeeding, early complementary introduction of food allergens, and antibiotic avoidance in the first year of life reduces allergic outcomes in at-risk children. Why the benefit of these practices is restricted to early life is largely unknown. We identified a preweaning interval during which dietary antigens are assimilated by the colonic immune system. This interval is under maternal control via temporal changes in breast milk, coincides with an influx of naive T cells into the colon, and is followed by the development of a long-lived population of colonic peripherally derived Tregs (pTregs) that can be specific for dietary antigens encountered during this interval. Desynchronization of mothers and offspring produced durable deficits in these pTregs, impaired tolerance to dietary antigens introduced during and after this preweaning interval, and resulted in spontaneous Th2 responses. These effects could be rescued by pTregs from the periweaning colon or by Tregs generated in vitro using periweaning colonic antigen-presenting cells. These findings demonstrate that mothers and their offspring are synchronized for the development of a balanced immune system.
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