Biology subjects

Estes, M. K.

Publications and source records attributed to Estes, M. K..

31 records · Page 2Linked to original sources

bioRxiv2021

Dasabuvir inhibits human norovirus infection in human intestinal enteroids

Human noroviruses (HuNoVs) are acute viral gastroenteritis pathogens that affect all age groups, yet no approved vaccines and drugs to treat HuNoV infection are available. In this study, with a human intestinal enteroid (HIE) culture system where HuNoVs are able to replicate reproducibly, we screened an antiviral compound library to identify compound(s) showing anti-HuNoV activity. Dasabuvir, which has been developed as an anti-hepatitis C virus agent, was found to inhibit HuNoV infection in HIEs at micromolar concentrations. Dasabuvir also inhibited severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human A rotavirus (RVA) infection in HIEs. To our knowledge, this is the first study to screen an antiviral compound library for HuNoV using HIEs and we successfully identified dasabuvir as a novel anti-HuNoV inhibitor that warrants further investigation.

microbiology↗

bioRxiv2021

Drivers of Transcriptional Variance in Human Intestinal Epithelial Organoids

Background & AimsHuman intestinal epithelial organoids (enteroids and colonoids) are tissue cultures used for understanding the physiology of the intestinal epithelium. Here, we explored the effect on the transcriptome of common variations in culture methods, including extracellular matrix substrate, format, tissue segment, differentiation status, and patient heterogeneity. MethodsRNA-sequencing datasets from 251 experiments performed on 35 human enteroid and colonoid lines from 28 patients were aggregated from several groups in the Texas Medical Center. DESeq2 and Gene Set Enrichment Analysis (GSEA) was used to identify differentially expressed genes and enriched of pathways. ResultsPERMANOVA, Pearson correlations, and dendrogram analysis of all data indicated three tiers of influence of culture methods on transcriptomic variation: substrate (collagen vs. Matrigel) and format (3D, transwell, and monolayer) had the largest effect (7,271-1,305 differentially expressed genes-DEGs); segment of origin (duodenum, jejunum, ileum, colon) and differentiation status had a moderate effect (5,977-420 DEGs), and patient heterogeneity and specific experimental manipulations (e.g., pathogen infection) had the smallest effect. GSEA identified hundreds of pathways that varied between culture methods, such as IL1 cytokine signaling enriched in transwell vs. monolayer cultures, and cholesterol biosynthesis genes enriched in Matrigel vs. collagen cultures. ConclusionsSurprisingly large differences in organoid transcriptome were driven by variations in culture methods such as format and substrate, whereas experimental manipulations such as infection had modest effects. These results show that common variations in culture conditions can have large effects on intestinal organoids and should be accounted for when designing experiments and comparing results between laboratories. Our data constitute the largest RNA-seq dataset interrogating human intestinal organoids.

molecular biology↗

bioRxiv2020

New Insights and Enhanced Human Norovirus Cultivation in Human Intestinal Enteroids

Human noroviruses (HuNoVs) are the leading cause of epidemic and sporadic acute gastroenteritis worldwide. We previously demonstrated human intestinal stem cell-derived enteroids (HIEs) support cultivation of several HuNoV strains. However, HIEs did not support virus replication from every HuNoV-positive stool sample, which led us to test and optimize new media conditions, identify characteristics of stool samples that allow replication, and evaluate consistency of replication over time. Optimization of our HIE-HuNoV culture system has shown that: 1) A new HIE culture media made with conditioned medium from a single cell line and commercial media promote robust replication of HuNoV strains that replicated poorly in HIEs grown in our original culture media made with conditioned media from 3 separate cell lines; 2) GI.1, eleven GII genotypes (GII.1, GII.2, GII.3, GII.4, GII.6, GII.7, GII.8, GII.12, GII.13, GII.14 and GII.17) and six GII.4 variants, can be cultivated in HIEs; 3) successful replication is more likely with virus in stools with higher virus titers; 4) GII.4_Sydney_2012 virus replication was reproducible over three years; and 5) HuNoV infection is restricted to the small intestine, based on replication in duodenal and ileal HIEs but not colonoids from the same donors. These results improve the HIE culture system for HuNoV replication. Use of HIEs by several laboratories worldwide to study the molecular mechanisms that regulate HuNoV replication confirms the usefulness of this culture system and our optimized methods for virus replication will advance the development of effective therapies and methods for virus control. ImportanceHuman noroviruses (HuNoVs) are highly contagious and cause acute and sporadic diarrheal illness in all age groups. In addition, chronic infections occur in immunocompromised cancer and transplant patients. These viruses are antigenically and genetically diverse and there are strain-specific differences in binding to cellular attachment factors. In addition, new discoveries are being made on strain-specific differences in virus entry and replication and the epithelial cell response to infection in human intestinal enteroids. Human intestinal enteroids are a biologically-relevant model to study HuNoVs; however, not all strains can be cultivated at this time. A complete understanding of HuNoV biology thus requires cultivation conditions that will allow the replication of multiple strains. We report optimization of HuNoV cultivation in human intestinal enteroid cultures to increase the numbers of cultivatable strains and the magnitude of replication, which is critical for testing antivirals, neutralizing antibodies and methods of virus inactivation.

microbiology↗

bioRxiv2020

Coronin-1 is necessary for enteric pathogen-induced transcytosis across human ileal enteroid monolayers expressing M cells

In the intestine, luminal sampling by microfold (M) cells is crucial for inducing protective mucosal immune responses but can also serve as an entry pathway for pathogens, including bacteria and viruses. Enteric pathogens can influence intestinal M cell function; however, the molecular mechanisms involved in the regulation of uptake and transcytosis of gut cargo by human M cells remain to be determined. Understanding the mechanisms responsible for regulating human M cell function requires a relevant human model. In this study, human ileal enteroids established from healthy donors were grown as confluent monolayers on permeable supports and differentiated to express mature M cells. Enteric pathogens including enteropathogenic E. coli (EPEC), adherent invasive E. coli (AIEC), and human rotavirus were apically exposed to M cell enteroid monolayers. M cell-mediated uptake and transcytosis was compared in enteroids infected by pathogenic or commensal bacteria (HS strain). EPEC and AIEC, but not HS, stimulated M cell uptake and transcytosis. We discovered that this pathogenspecific effect was dependent on expression of coronin 1a, a cytoskeletal remodeling protein. Using stable coronin 1a knockdown (KD) enteroids, we observed that EPEC-stimulated transcytosis of fluorescent beads was lost and associated with a significant decrease in the number of glycoprotein-2 positive (Gp-2+ve) M cells. The results of these studies demonstrate that coronin 1a is required for uptake and transcytosis of luminal cargo across human M cells and that coronin 1a is necessary for differentiation of mature M cells that actively transcytose luminal gut antigens in response to pathogenic, but not commensal, microbes.

cell biology↗

bioRxiv2020

Isolation, culture and maintenance of rabbit intestinal organoids, and organoid-derived cell monolayers

Organoids emulate many aspects of their parental tissue and have been used to study pathogen-host interactions, tissue development and regeneration, metabolic diseases, and other complex biological processes. Here, we report a robust protocol for the isolation, maintenance and differentiation of rabbit small intestinal organoids and organoid-derived cell monolayers. We also report conditions that sustain an intestinal stem cell population in spheroid culture. Rabbit intestinal spheroids and monolayer cultures propagated and expanded most efficiently in L-WRN-conditioned medium that contained the signalling factors Wnt, R-spondin and Noggin, and that had been supplemented with ROCK and TGF-{beta} inhibitors. Organoid and monolayer differentiation was initiated by switching to a medium that contained less of the L-WRN-conditioned medium and was supplemented with ROCK and Notch signalling inhibitors. Using immunofluorescence staining and RT-qPCR, we demonstrate that organoids contained enterocytes, enteroendocrine cells, goblet cells and Paneth cells. These findings demonstrate that our rabbit intestinal organoids have many of the multi-cellular characteristics of, and closely resemble, an intestinal epithelium. This newly established organoid culture system will provide a useful tool to study rabbit gastrointestinal physiology and disease. For example, organoids and organoid-derived cells may be used to propagate and study caliciviruses and other enterotropic pathogens that cannot be grown in conventional cell culture systems.

cell biology↗

bioRxiv2020

Obesity phenotypes are preserved in intestinal stem cell enteroids from morbidly obese patients

Obesity and obesity-related comorbidities are significant health care challenges. Bariatric surgery (BS) is the most effective therapy for treating obesity and type 2 diabetes. A barrier in the development of therapeutic alternatives is incomplete mechanistic understanding of the benefits of BS and the lack of human intestinal models that recapitulate the pathophysiology of obesity. Using adult intestinal stem cell-derived enteroid cultures established from healthy lean subjects and morbidly obese patients, including post-BS cases, four phenotypes correlating patient BMI and intestinal glucose absorption were identified suggesting that enteroids retain patient phenotype heterogeneity associated with healthy and diseased state. In a sub-population of obese patients, increased dietary glucose absorption and gluconeogenesis was due to significantly higher expression of intestinal carbohydrate transporters (SGLT1, GLUT2 and GLUT5) and gluconeogenic enzymes (PEPCK1 and G6Pase) compared to enteroids from lean subjects that demonstrated low glucose absorption and lacked gluconeogenesis. Enteroids established from successful BS cases exhibited low glucose absorption similar to that observed in lean subjects. These data show that human enteroids preserve the patient phenotype in long-term cultures and represent a reliable preclinical model to study the heterogeneity of the obesity mechanisms, which is necessary to determine the efficacy of therapeutic interventions.

physiology↗

bioRxiv2020

ROTAVIRUS INDUCES INTERCELLULAR CALCIUM WAVES THROUGH ADP SIGNALING

Rotavirus causes severe diarrheal disease in children by broadly dysregulating intestinal homeostasis. However, the underlying mechanism(s) of rotavirus-induced dysregulation remains incompletely characterized. Here we show that rotavirus-infected cells produce paracrine signals that manifest as intercellular calcium waves (ICWs); which are observed in both cell lines and human intestinal enteroids. Rotavirus ICWs are caused by the release of extracellular adenosine diphosphate (ADP) that activates P2Y1 purinergic receptors on neighboring cells and are blocked by P2Y1 antagonists or CRISPR/Cas9 knockout of P2Y1. Blocking the paracrine ADP signal reduces rotavirus replication, inhibits rotavirus-induced serotonin release and fluid secretion, and reduces diarrhea severity in neonatal mice. This is the first evidence that viruses exploit ICWs to amplify diarrheal signaling; a finding which has broad implications for gastrointestinal physiology. SummaryRotavirus triggers the extracellular release of ADP from infected cells to dysregulate nearby uninfected cells and activate pro-disease pathways.

microbiology↗

bioRxiv2019

Human Rotavirus Diarrhea Is Associated with Altered Trafficking and Expression of Apical Membrane Transport Proteins

BackgroundRotavirus (RV) is the 5th leading cause of death in children <5 years old but the leading cause of diarrhea related deaths in this age group. The mechanism of RV diarrhea involves decreased activity of Na+-dependent solute transporters with increased luminal secretion of Cl- in the absence of significant histologic damage. While our understanding of RV diarrhea has come from studies in animal models and cancer cell lines, the mechanism of the diarrhea and the transport proteins affected in human RV disease remains only partially understood. This understanding is likely to impact drug development therapy for RV diarrhea.\n\nMethodsFormalin-fixed paraffin-embedded small intestinal specimens from patients diagnosed with RV diarrhea (confirmed by anti-RV antibodies) were analyzed by immunofluorescence for changes in apical/basolateral ion/nutrient transporters/channels as well as tight junctional and cytoskeletal proteins. Proximal small intestinal enteroids generated from biopsies obtained from healthy human subjects were grown as monolayers, differentiated to resemble villus epithelial cells, and infected with human RV.\n\nResultsRV diarrhea was associated with reduced expression and intracellular localization of transport proteins normally found in the brush border membranes, including SGLT1, NHE3, NHE2, the Na+-dependent amino acid transporter SLC6A19, and CFTR. In contrast, basolateral proteins, including Na+/K+-ATPase, NKCC1, and {beta}-catenin, the brush border marker ezrin, as well as the tight junction protein, ZO-1, were expressed and localized normally. RV-induced mislocalization of NHE3, SGLT1, SLC6A19 and CFTR was also seen when human small intestinal enteroids were infected with RV.\n\nConclusionsThese data demonstrate a new pathophysiologic mechanism of acute diarrhea in which expression of multiple apical transport proteins are reduced. This acute diarrhea is likely to be caused by an effect on a common apical trafficking pathway, as exemplified by RV diarrhea, and its contribution to other enteric pathogen-induced diarrheal diseases should be determined.

cell biology↗

bioRxiv2019

Rotavirus Calcium Dysregulation Manifests as Dynamic Calcium Signaling in the Cytoplasm and Endoplasmic Reticulum

Like many viruses, rotavirus (RV) dysregulates calcium homeostasis by elevating cytosolic calcium ([Ca2+]cyt) and decreasing endoplasmic reticulum (ER) stores. While an overall, monophasic increase in [Ca2+]cyt during RV infection has been shown, the nature of the RV-induced aberrant calcium signals and how they manifest over time at the single-cell level have not been characterized. Thus, we generated cell lines and human intestinal enteroids (HIEs) stably expressing cytosolic and/or ER-targeted genetically-encoded calcium indicators to characterize calcium signaling throughout RV infection by time-lapse imaging. We found that RV induces highly dynamic [Ca2+]cyt signaling that manifest as hundreds of discrete [Ca2+]cyt spikes, which increase during peak infection. Knockdown of nonstructural protein 4 (NSP4) attenuates the [Ca2+]cyt spikes, consistent with its role in dysregulating calcium homeostasis. RV-induced [Ca2+]cyt spikes were primarily from ER calcium release and were attenuated by inhibiting the store-operated calcium entry (SOCE) channel Orai1. RV-infected HIEs also exhibited prominent [Ca2+]cyt spikes that were attenuated by inhibiting SOCE, underlining the relevance of these [Ca2+]cyt spikes to gastrointestinal physiology and role of SOCE in RV pathophysiology. Thus, our discovery that RV increases [Ca2+]cyt by dynamic Ca2+ signaling, establishes a new, paradigm-shifting understanding of the spatial and temporal complexity of virus-induced Ca2+ signaling.

microbiology↗

bioRxiv2019

Human intestinal enteroids with inducible neurogenin-3 expression as a novel model of gut hormone secretion

BackgroundEnteroendocrine cells (EECs) are specialized epithelial cells that produce molecules vital for intestinal homeostasis, but due to their limited numbers, in-depth functional studies have remained challenging. Human intestinal enteroids (HIEs) that are derived from intestinal crypt stem cells are a biologically relevant in vitro model of the intestinal epithelium. HIEs contain all intestinal epithelial cell types; however, like the intestine, HIEs spontaneously produce few EECs, which limits their study.\n\nMethodsTo increase the number of EECs in HIEs, we used lentivirus transduction to stably engineer jejunal HIEs with doxycycline-inducible expression of neurogenin-3 (NGN3), a transcription factor that drives EEC differentiation (tetNGN3-HIEs). We examined the impact of NGN3 induction on EECs by quantifying the increase in the enterochromaffin cells and other EEC subtypes. We functionally assessed secretion of serotonin and EEC hormones in response to norepinephrine and rotavirus infection.\n\nResultsTreating tetNGN3-HIEs with doxycycline induced a dose-dependent increase of chromogranin A (ChgA)-positive and serotonin-positive cells, demonstrating increased enterochromaffin cell differentiation. Despite increased ChgA-positive cells, other differentiated cell types of the epithelium remained largely unchanged by gene expression and immunostaining. RNA sequencing of doxycycline-induced tetNGN3- HIEs identified increased expression of key hormones and enzymes associated with several other EEC subtypes. Doxycycline-induced tetNGN3-HIEs secreted serotonin, monocyte chemoattractant protein-1, glucose-dependent insulinotropic peptide, peptide YY, and ghrelin in response to norepinephrine and rotavirus infection, further supporting the presence of multiple EEC types.\n\nConclusionsWe have combined HIEs and inducible-NGN3 expression to establish a flexible in vitro model system for functional studies of EECs in enteroids and advance the molecular and physiological investigation of EECs.\n\nSynopsisEnteroendocrine cells have low abundance but exert widespread effects on gastrointestinal physiology. We engineered human intestinal enteroids with inducible expression of neurogenin-3, resulting in increased enteroendocrine cells and facilitating investigations of host responses to the dynamic intestinal environment.

microbiology↗

bioRxiv2019

A novel human enteroid-anaerobe co-culture system to study microbial-host interaction under physiological hypoxia

Mechanistic investigations of host-microbe interactions in the human gut are limited by current co-culture model systems. The intestinal epithelium requires oxygen for viability, while gut bacteria are facultative or obligate anaerobes. The ability to model host-commensal interactions under dynamic oxygen conditions is critical to understanding host-pathogen interactions in the human gut. Here, we demonstrate a simple, cost-effective method for co-culturing obligate anaerobic bacteria with human intestinal enteroid monolayers under variable oxygen conditions. The Enteroid-Anaerobe Co-Culture (EACC) system is able to recapitulate the steep oxygen gradient seen in vivo and induce expression of hypoxia-associated phenotypes such as increased barrier integrity and expression of antimicrobial peptide genes. Using clinical strains of the commensal anaerobes Bacteroides thetaiotaomicron and Blautia sp. on established patient-derived intestinal enteroid cell lines under physiological hypoxia, the EACC system can sustain host-anaerobe interactions for at least 24 hours. Following co-culture with anaerobic bacteria, we demonstrate patient-specific differences in epithelial response, reinforcing the potential to develop a personalized medicine approach to bacteriotherapy and host-microbe interaction investigations. Our innovative EACC system provides a robust model for investigating host-microbe interactions in complex, patient-derived intestinal tissues, that facilitates study of mechanisms underlying the role of the microbiome in health and disease.

bioengineering↗

bioRxiv2018

Epithelial WNT2B and Desert Hedgehog are necessary for human colonoid regeneration after bacterial cytotoxin injury

Intestinal regeneration and crypt hyperplasia after radiation or pathogen injury relies on Wnt signaling to stimulate stem cell proliferation. Mesenchymal Wnts are essential for homeostasis and regeneration in mice, but the role of epithelial Wnts remains largely uncharacterized. Using the enterohemorrhagic E. coli secreted cytotoxin, EspP to induce injury to human colonoids, we evaluated a simplified, epithelial regeneration model that lacks mesenchymal Wnts. Here, we demonstrate that epithelial-produced WNT2B is upregulated following injury and essential for regeneration. Hedgehog signaling, specifically activation via the ligand Desert Hedgehog (DHH), but not Indian or Sonic Hedgehog, is another driver of regeneration and modulates WNT2B expression. These findings highlight the importance of epithelial WNT2B and DHH in regulating human colonic regeneration after injury.

physiology↗

bioRxiv2018

Bile acids target proteolipid nano-assemblies of EGFR and phosphatidic acid in the plasma membrane for stimulation of MAPK signaling

Bile acids are critical biological detergents in the gastrointestinal tract and also act as messengers to regulate a multitude of intracellular signaling events, including mitogenic signaling, lipid metabolism and endo/exocytosis. In particular, bile acids stimulate many receptors and ion channels on the cell surface, the mechanisms of which are still poorly understood. Membrane-associating proteins depend on the local spatial distribution of lipids in the plasma membrane (PM) for their function. Here, we report that the highly amphipathic secondary bile acid deoxycholic acid (DCA), a major constituent in the human bile, at doses <1M enhances the nanoclustering of phosphatidic acid (PA) but disrupts the local segregation of phosphoinositol 4,5-bisphosphate (PIP2) in the basolateral PM of the human colorectal adenocarcinoma Caco-2 cells. PA has been shown to be a key structural component of the signaling nano-domains of epidermal growth factor receptor (EGFR) on the cell surface. We show that DCA promotes the co-localization between PA and EGFR, the PA-driven EGFR dimerization/oligomerization and EGFR signaling. Depletion of PA abolishes the stimulatory effects of DCA on the EGFR oligomerization and signaling. This effect occurs in the cultured Caco-2 cells and the ex vivo human intestinal enteroids. We propose a novel mechanism, where the amphiphilic DCA monomers alter the nano-assemblies of anionic phospholipids and in turn change the dynamic structural integrity of the lipid-driven oligomerization of cell surface receptors and their signal transduction.

biophysics↗