It is unclear why disease occurs in only a small proportion of persons carrying common risk alleles of disease susceptibility genes. Here we demonstrate that an interaction between a specific virus ...infection and a mutation in the Crohn's disease susceptibility gene Atg16L1 induces intestinal pathologies in mice. This virus-plus-susceptibility gene interaction generated abnormalities in granule packaging and unique patterns of gene expression in Paneth cells. Further, the response to injury induced by the toxic substance dextran sodium sulfate was fundamentally altered to include pathologies resembling aspects of Crohn's disease. These pathologies triggered by virus-plus-susceptibility gene interaction were dependent on TNFα and IFNγ and were prevented by treatment with broad spectrum antibiotics. Thus, we provide a specific example of how a virus-plus-susceptibility gene interaction can, in combination with additional environmental factors and commensal bacteria, determine the phenotype of hosts carrying common risk alleles for inflammatory disease.
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► Mice with mutations in the Crohn's disease gene Atg16L1 display intestinal disease ► Disease requires murine norovirus (MNV) infection together with Atg16L1 mutation ► Viral triggering of disease is dependent on virus strain and timing of infection ► Disease in response to mucosal injury involves cytokines and commensal bacteria
Cytokine modulation of autophagy is increasingly recognized in disease pathogenesis, and current concepts suggest that type 1 cytokines activate autophagy, whereas type 2 cytokines are inhibitory. ...However, this paradigm derives primarily from studies of immune cells and is poorly characterized in tissue cells, including sentinel epithelial cells that regulate the immune response. In particular, the type 2 cytokine IL13 (interleukin 13) drives the formation of airway goblet cells that secrete excess mucus as a characteristic feature of airway disease, but whether this process is influenced by autophagy was undefined. Here we use a mouse model of airway disease in which IL33 (interleukin 33) stimulation leads to IL13-dependent formation of airway goblet cells as tracked by levels of mucin MUC5AC (mucin 5AC, oligomeric mucus/gel forming), and we show that these cells manifest a block in mucus secretion in autophagy gene Atg16l1-deficient mice compared to wild-type control mice. Similarly, primary-culture human tracheal epithelial cells treated with IL13 to stimulate mucus formation also exhibit a block in MUC5AC secretion in cells depleted of autophagy gene ATG5 (autophagy-related 5) or ATG14 (autophagy-related 14) compared to nondepleted control cells. Our findings indicate that autophagy is essential for airway mucus secretion in a type 2, IL13-dependent immune disease process and thereby provide a novel therapeutic strategy for attenuating airway obstruction in hypersecretory inflammatory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis lung disease. Taken together, these observations suggest that the regulation of autophagy by Th2 cytokines is cell-context dependent.
Delivery of granule contents to epithelial surfaces by secretory cells is a critical physiologic process. In the intestine, goblet cells secrete mucus that is required for homeostasis. Autophagy ...proteins are required for secretion in some cases, though the mechanism and cell biological basis for this requirement remain unknown. We found that in colonic goblet cells, proteins involved in initiation and elongation of autophagosomes were required for efficient mucus secretion. The autophagy protein LC3 localized to intracellular multi‐vesicular vacuoles that were consistent with a fusion of autophagosomes and endosomes. Using cultured intestinal epithelial cells, we found that NADPH oxidases localized to and enhanced the formation of these LC3‐positive vacuoles. Both autophagy proteins and endosome formation were required for maximal production of reactive oxygen species (ROS) derived from NADPH oxidases. Importantly, generation of ROS was critical to control mucin granule accumulation in colonic goblet cells. Thus, autophagy proteins can control secretory function through ROS, which is in part generated by LC3‐positive vacuole‐associated NADPH oxidases. These findings provide a novel mechanism by which autophagy proteins can control secretion.
In vivo findings on mucus secretion in the colon illustrate how autophagy proteins can control secretion mechanisms via ROS generation.
Nutrient absorption is the basic function that drives mammalian intestinal biology. To facilitate nutrient uptake, the host's epithelial barrier is composed of a single layer of cells. This ...constraint is problematic, as a design of this type can be easily disrupted. The solution during the course of evolution was to add numerous host defense mechanisms that can help prevent local and systemic infection. These mechanisms include specialized epithelial cells that produce a physiochemical barrier overlying the cellular barrier, robust and organized adaptive and innate immune cells, and the ability to mount an inflammatory response that is commensurate with a specific threat level. The autophagy pathway is a critical cellular process that strongly influences all these functions. Therefore, a fundamental understanding of the components of this pathway and their influence on inflammation, immunity, and barrier function will facilitate our understanding of homeostasis in the gastrointestinal tract.
A coding polymorphism (Thr300Ala) in the essential autophagy gene, autophagy related 16-like 1 (ATG16L1), confers increased risk for the development of Crohn disease, although the mechanisms by which ...single disease-associated polymorphisms contribute to pathogenesis have been difficult to dissect given that environmental factors likely influence disease initiation in these patients. Here we introduce a knock-in mouse model expressing the Atg16L1 T300A variant. Consistent with the human polymorphism, T300A knock-in mice do not develop spontaneous intestinal inflammation, but exhibit morphological defects in Paneth and goblet cells. Selective autophagy is reduced in multiple cell types from T300A knock-in mice compared with WT mice. The T300A polymorphism significantly increases caspase 3- and caspase 7-mediated cleavage of Atg16L1, resulting in lower levels of full-length Atg16Ll T300A protein. Moreover, Atg16L1 T300A is associated with decreased antibacterial autophagy and increased IL-1β production in primary cells and in vivo. Quantitative proteomics for protein interactors of ATG16L1 identified previously unknown nonoverlapping sets of proteins involved in ATG16L1dependent antibacterial autophagy or IL-1β production. These findings demonstrate how the T300A polymorphism leads to cell typeand pathway-specific disruptions of selective autophagy and suggest a mechanism by which this polymorphism contributes to disease.
Pediatric clinical laboratories commonly measure tricarboxylic acid cycle intermediates for screening, diagnosis, and monitoring of specific inborn errors of metabolism, such as organic acidurias. In ...the past decade, the same tricarboxylic acid cycle metabolites have been implicated and studied in cancer. The accumulation of these metabolites in certain cancers not only serves as a biomarker but also directly contributes to cellular transformation, therefore earning them the designation of oncometabolites.
-2-hydroxyglutarate,
-2-hydroxyglutarate, succinate, and fumarate are the currently recognized oncometabolites. They are structurally similar and share metabolic proximity in the tricarboxylic acid cycle. As a result, they promote tumorigenesis in cancer cells through similar mechanisms. This review summarizes the currently understood common and distinct biological features of these compounds. In addition, we will review the current laboratory methodologies that can be used to quantify these metabolites and their downstream targets.
Oncometabolites play an important role in cancer biology. The metabolic pathways that lead to the production of oncometabolites and the downstream signaling pathways that are activated by oncometabolites represent potential therapeutic targets. Clinical laboratories have a critical role to play in the management of oncometabolite-associated cancers through development and validation of sensitive and specific assays that measure oncometabolites and their downstream effectors. These assays can be used as screening tools and for follow-up to measure response to treatment, as well as to detect minimal residual disease and recurrence.
Virtual reality (VR) displays are an increasingly popular medium for experiments on visual perception. This presents the challenge of showing precisely controlled stimuli on devices that were not ...primarily designed for research. Here we describe methods for controlling stimulus luminance in VR experiments created in Unity using the Built-in Render Pipeline. We discuss the Gamma/Linear setting, measuring luminance in a VR headset, and using color grading in Unity's Post-Processing Stack to make stimulus luminance proportional to achromatic RGB value. We provide MATLAB code that uses luminance measurements from a VR headset to generate the lookup table that Unity requires for linearizing luminance. We emphasize that when creating experiments in this complex environment, it is important to experiment with the rendering process to confirm that stimuli are displayed as expected. We show results of several such tests and provide code as a starting point for readers who wish to run further tests related to their own research.
Background & Aims Intestinal epithelial cells aid in mucosal defense by providing a physical barrier against entry of pathogenic bacteria and secreting antimicrobial peptides (AMPs). Autophagy is an ...important component of immune homeostasis. However, little is known about its role in specific cell types during bacterial infection in vivo. We investigated the role of autophagy in the response of intestinal epithelial and antigen-presenting cells to Salmonella infection in mice. Methods We generated mice deficient in Atg16l1 in epithelial cells (Atg16l1f/f × Villin-cre) or CD11c+ cells (Atg16l1f/f × CD11c-cre); these mice were used to assess cell type–specific antibacterial autophagy. All responses were compared with Atg16l1f/f mice (controls). Mice were infected with Salmonella enterica serovar typhimurium; cecum and small-intestine tissues were collected for immunofluorescence, histology, and quantitative reverse-transcription polymerase chain reaction analyses of cytokines and AMPs. Modulators of autophagy were screened to evaluate their effects on antibacterial responses in human epithelial cells. Results Autophagy was induced in small intestine and cecum after infection with S typhimurium , and required Atg16l1. S typhimurium colocalized with microtubule-associated protein 1 light chain 3β (Map1lc3b or LC3) in the intestinal epithelium of control mice but not in Atg16l1f/f × Villin-cre mice. Atg16l1f/f × Villin-cre mice also had fewer Paneth cells and abnormal granule morphology, leading to reduced expression of AMPs. Consistent with these defective immune responses, Atg16l1f/f × Villin-cre mice had increased inflammation and systemic translocation of bacteria compared with control mice. In contrast, we observed few differences between Atg16l1f/f × CD11c-cre and control mice. Trifluoperazine promoted autophagy and bacterial clearance in HeLa cells; these effects were reduced upon knockdown of ATG16L1. Conclusions Atg16l1 regulates autophagy in intestinal epithelial cells and is required for bacterial clearance. It also is required to prevent systemic infection of mice with enteric bacteria.
The work employs aliphatic and aromatic isothiocyanates and utilizes electrochemical techniques for the reaction. The process involves a two-electrode system with a conductive carbon working ...electrode and a counter electrode. An appropriate electrolyte, such as an organic solvent, is used, and upon applying an electric potential, electrochemical oxidation and nucleophilic substitution reactions occur, leading to the formation of the desired 1,3,4-oxadiazole-2-amines. Key advantages of this electrochemical method include its high functional group tolerance, enabling the synthesis of a wide range of compounds with different substituents. The reaction conditions can be fine-tuned for high conversion and selectivity, ensuring good overall yields of the target products. Additionally, the electrochemical method offers sustainability and convenience, making it a promising alternative to traditional synthesis. The use of electricity as an energy source is more environmentally friendly compared to traditional heating methods, contributing to greener and eco-friendly chemical processes.
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