Key points
Determining the signalling cascade of epithelial repair, using murine gastric organoids, allows definition of regulatory processes intrinsic to epithelial cells, at the same time as ...validating and dissecting the signalling cascade with more precision than is possible in vivo
Following single cell damage, intracellular calcium selectively increases within cells adjacent to the damage site and is essential for promoting repair.
Trefoil factor 2 (TFF2) acts via chemokine C‐X‐C receptor 4 and epidermal growth factor receptor signalling, including extracellular signal‐regulated kinase activation, to drive calcium mobilization and promote gastric repair.
Sodium hydrogen exchanger 2, although essential for repair, acts downstream of TFF2 and calcium mobilization.
The gastric mucosa of the stomach is continually exposed to environmental and physiological stress factors that can cause local epithelial damage. Although much is known about the complex nature of gastric wound repair, the stepwise process that characterizes epithelial restitution remains poorly defined. The present study aimed to determine the effectors that drive gastric epithelial repair using a reductionist culture model. To determine the role of trefoil factor 2 (TFF2) and intracellular calcium (Ca2+) mobilization in gastric restitution, gastric organoids were derived from TFF2 knockout (KO) mice and yellow Cameleon‐Nano15 (fluorescent calcium reporter) transgenic mice, respectively. Inhibitors and recombinant protein were used to determine the upstream and downstream effectors of gastric restitution following photodamage (PD) to single cells within the gastric organoids. Single cell PD resulted in parallel events of dead cell exfoliation and migration of intact neighbouring cells to restore a continuous epithelium in the damage site. Under normal conditions following PD, Ca2+ levels increased within neighbour migrating cells, peaking at ∼1 min, suggesting localized Ca2+ mobilization at the site of cell protrusion/migration. TFF2 KO organoids exhibit delayed repair; however, this delay can be rescued by the addition of exogenous TFF2. Inhibition of epidermal growth factor receptor (EGFR), extracellular signal‐regulated kinase (ERK)1/2 or a TFF2 receptor, chemokine C‐X‐C receptor 4 (CXCR4), resulted in significant delay and dampened Ca2+ mobilization. Inhibition of sodium hydrogen exchanger 2 (NHE2) caused significant delay but did not affect Ca2+ mobilization. A similar delay was observed in NHE2 KO organoids. In TFF2 KO gastric organoids, the addition of exogenous TFF2 in the presence of EGFR or CXCR4 inhibition was unable to rescue repair. The present study demonstrates that intracellular Ca2+ mobilization occurs within gastric epithelial cells adjacent to the damage site to promote repair by mechanisms that involve TFF2 signalling via CXCR4, as well as activation of EGFR and ERK1/2. Furthermore NHE2 is shown to be important for efficient repair and to operate via a mechanism either downstream or independent of calcium mobilization.
Key points
Determining the signalling cascade of epithelial repair, using murine gastric organoids, allows definition of regulatory processes intrinsic to epithelial cells, at the same time as validating and dissecting the signalling cascade with more precision than is possible in vivo
Following single cell damage, intracellular calcium selectively increases within cells adjacent to the damage site and is essential for promoting repair.
Trefoil factor 2 (TFF2) acts via chemokine C‐X‐C receptor 4 and epidermal growth factor receptor signalling, including extracellular signal‐regulated kinase activation, to drive calcium mobilization and promote gastric repair.
Sodium hydrogen exchanger 2, although essential for repair, acts downstream of TFF2 and calcium mobilization.
Key points
Enteroids are a physiologically relevant model to examine the human intestine and its functions.
Previously, the measurable cytokine response of human intestinal enteroids has been limited ...following exposure to host or microbial pro‐inflammatory stimuli.
Modifications to enteroid culture conditions facilitated robust human cytokine responses to pro‐inflammatory stimuli.
This new human enteroid culture methodology refines the ability to study microbiome:human intestinal epithelium interactions in the laboratory.
The intestinal epithelium is the primary interface between the host, the gut microbiome and its external environment. Since the intestinal epithelium contributes to innate immunity as a first line of defence, understanding how the epithelium responds to microbial and host stimuli is an important consideration in promoting homeostasis. Human intestinal enteroids (HIEs) are primary epithelial cell cultures that can provide insights into the biology of the intestinal epithelium and innate immune responses. One potential limitation of using HIEs for innate immune studies is the relative lack of responsiveness to factors that stimulate epithelial cytokine production. We report technical refinements, including removal of extracellular antioxidants, to facilitate enhanced cytokine responses in HIEs. Using this new method, we demonstrate that HIEs have distinct cytokine profiles in response to pro‐inflammatory stimuli derived from host and microbial sources. Overall, we found that host‐derived cytokines tumour necrosis factor and interleukin‐1α stimulated reactive oxygen species and a large repertoire of cytokines. In contrast, microbial lipopolysaccharide, lipoteichoic acid and flagellin stimulated a limited number of cytokines and histamine did not stimulate the release of any cytokines. Importantly, HIE‐secreted cytokines were functionally active, as denoted by the ability of human blood‐derived neutrophil to migrate towards HIE supernatant containing interleukin‐8. These findings establish that the immune responsiveness of HIEs depends on medium composition and stimuli. By refining the experimental culture medium and creating an environment conducive to epithelial cytokine responses by human enteroids, HIEs can facilitate exploration of many experimental questions pertaining to the role of the intestinal epithelium in innate immunity.
Key points
Enteroids are a physiologically relevant model to examine the human intestine and its functions.
Previously, the measurable cytokine response of human intestinal enteroids has been limited following exposure to host or microbial pro‐inflammatory stimuli.
Modifications to enteroid culture conditions facilitated robust human cytokine responses to pro‐inflammatory stimuli.
This new human enteroid culture methodology refines the ability to study microbiome:human intestinal epithelium interactions in the laboratory.
Gastrointestinal organoids in the study of viral infections Gebert, J Thomas; Scribano, Francesca; Engevik, Kristen A ...
American journal of physiology: Gastrointestinal and liver physiology,
01/2023, Letnik:
324, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Viruses are among the most prevalent enteric pathogens. Although virologists historically relied on cell lines and animal models, human intestinal organoids (HIOs) continue to grow in popularity. ...HIOs are nontransformed, stem cell-derived, ex vivo cell cultures that maintain the cell type diversity of the intestinal epithelium. They offer higher throughput than standard animal models while more accurately mimicking the native tissue of infection than transformed cell lines. Here, we review recent literature that highlights virological advances facilitated by HIOs. We discuss the variations and limitations of HIOs, how HIOs have allowed for the cultivation of previously uncultivatable viruses, and how they have offered insight into tropism, entry, replication kinetics, and host-pathogen interactions. In each case, we discuss exemplary viruses and archetypal studies. We discuss how the speed and flexibility of HIO-based studies contributed to our knowledge of SARS-CoV-2 and antiviral therapeutics. Finally, we discuss the current limitations of HIOs and future directions to overcome these.
Recent technological advances in microscopy have facilitated novel approaches to investigate host-pathogen interactions. In particular, improvements in both microscope hardware and engineered ...biosensors have helped to overcome barriers to live-cell imaging with fluorescence microscopy. Live fluorescent microscopy allows for the detection of discrete signaling events and protein localization, improving our ability to assess the effects of pharmacologic agents, microbes, or infection with high temporal resolution. Here we describe a protocol for long-term live-cell fluorescence imaging of virus infected cell lines.
Rotavirus is a leading cause of viral gastroenteritis. A hallmark of rotavirus infection is increased cytosolic Ca2+ caused by nonstructural protein 4 (NSP4). NSP4 is a viral ion channel that ...releases endoplasmic reticulum (ER) Ca2+, and the increased Ca2+ signaling is critical for rotavirus replication. In addition to NSP4, host inositol 1,4,5-trisphosphate receptor (IP3R) ER Ca2+ channels may contribute to rotavirus-induced Ca2+ signaling and by extension, virus replication. Thus, we set out to determine the role of IP3R Ca2+ signaling during rotavirus infection using CRISPR/Cas9 IP3R-knockout of MA104 cells stably expressing the GCaMP6s Ca2+ indicator (MA104-GCaMP6s-IP3R-KO). Live Ca2+ imaging showed that IP3R-KO did not reduce Ca2+ signaling in infected cells but eliminated rotavirus-induced intercellular Ca2+ waves (ICWs) and, therefore, the increased Ca2+ signaling in surrounding, uninfected cells. MA104-GCaMP6s-IP3R-TKO cells showed similar rotavirus susceptibility, single-cycle replication, and viral protein expression as parental MA104-GCaMP6s cells. However, MA104-GCaMP6s-IP3R-TKO cells exhibited significantly smaller rotavirus plaques, decreased multi-round replication kinetics, and delayed virus spread, suggesting that rotavirus-induced ICW Ca2+ signaling stimulates virus replication and spread. Inhibition of ICWs by blocking the purinergic receptor P2RY1 (P2Y1), which mediates the ICW Ca2+ signals, also decreased rotavirus plaque size. Conversely, exogenous expression of P2Y1 in LLC-MK2-GCaMP6s cells, which natively lack P2Y1 and rotavirus ICWs, rescued the generation of rotavirus-induced ICWs and enabled plaque formation. In conclusion, this study shows that NSP4 Ca2+ signals fully support rotavirus replication in individual cells; however, IP3R is critical for rotavirus-induced ICWs and virus spread by priming Ca2+-dependent pathways in surrounding cells.IMPORTANCEMany viruses exploit host Ca2+ signaling to facilitate their replication; however, little is known about how Ca2+ signals from different host and viral channels contribute to the overall dysregulation of Ca2+ signaling or promote virus replication. Using cells lacking IP3R, a host ER Ca2+ channel, we delineated intracellular Ca2+ signals within virus-infected cells and intercellular Ca2+ waves (ICWs), which increased Ca2+ signaling in neighboring, uninfected cells. In infected cells, IP3R was dispensable for rotavirus-induced Ca2+ signaling and replication, suggesting the rotavirus NSP4 viroporin supplies these signals. However, IP3R-mediated ICWs increase rotavirus replication kinetics and spread, indicating that the Ca2+ signals from the ICWs may prime nearby uninfected cells to better support virus replication upon eventual infection. This “pre-emptive priming” of uninfected cells by exploiting host intercellular pathways in the vicinity of virus-infected cells represents a novel mechanism for viral reprogramming of the host to gain a replication advantage.
Nucleotides are potent extracellular signaling molecules during homeostasis, infection, and injury due to their ability to activate purinergic receptors. The nucleotide ATP activates P2X receptors ...(P2RXs), whereas the nucleotides ADP, ATP, UTP, and UDP-glucose selectively activate different P2Y receptors (P2RYs). Several studies have established crucial roles for P2 receptors during intestinal inflammatory and infectious diseases, yet the most extensive characterization of purinergic signaling has focused on immune cells and the central and enteric nervous systems. As epithelial cells serve as the first barrier against irritants and infection, we hypothesized that the gut epithelium may express multiple purinergic receptors that respond to extracellular nucleotide signals. Using the Human Protein Atlas and Gut Cell Survey, we queried single-cell RNA sequencing (RNAseq) data for the P2 purinergic receptors in the small and large intestines. In silico analysis reveals robust mRNA expression of P2RY1, P2RY2, P2RY11, and P2RX4 throughout the gastrointestinal tract. Human intestinal organoids exhibited a similar expression pattern with a prominent expression of P2RY1, P2RY2, and P2RX4, but this purinergic receptor repertoire was not conserved in T84, Caco2, and HT29 intestinal epithelial cell lines. Finally, P2YR1 and P2YR2 agonists elicited robust calcium responses in human intestinal organoids, but calcium responses were weaker or absent in the cell lines. These findings suggest that the gastrointestinal epithelia respond to extracellular purinergic signaling via P2RY1, P2RY2, P2RY11, and P2RX4 receptors and highlight the benefit of using intestinal organoids as a model of intestinal purinergic signaling.
Several studies have revealed crucial roles for P2 receptors during inflammatory and infectious diseases, however, these have largely been demonstrated in immune cells and the enteric nervous system. Although epithelial cells serve as the first barrier against infection and inflammation, the role of purinergic signaling within the gastrointestinal tract remains largely unknown. This work expands our knowledge of purinergic receptor distribution and relative expression along the intestine.
The advent of the gastric organoid culture system has provided a new model to emulate native epithelial tissue in vitro. Gastric organoids grow from isolated epithelial stem cells and develop into ...three dimensional structures that can be used to study host physiology. Here we describe current laboratory protocols for growing gastric organoids and the microinjection of pathogens such as Helicobacter pylori into the lumen of gastric organoids in order to study the cellular response following infection.
Biological processes are dynamic. As a result, temporal analyses are necessary to fully understand the complex interactions that occurs within these systems. One example of a multifaceted biological ...process is restitution: the initial step in complex wound repair. Restitution is a dynamic process that depends on an elegant orchestration between damaged cells and their intact neighbors. Such orchestration enables the quick repair of the damaged area, which is essential to preserve epithelial integrity and prevent further injury. High quality dynamic data of the cellular and molecular events that make up the gastric restitution process has been documented. However, comprehensive dynamic models that connect all relevant molecular interactions to cellular behaviors are challenging to construct and experimentally validate. In order to efficiently provide feedback to ongoing experimental work, we have integrated dynamical modeling and machine learning to efficiently extract data-driven insights without incorporating detailed mechanisms. Dynamical models convert time course data into a set of static features, which are then subjected to machine learning analysis. The integrated analysis provides data-driven insights into how repair might be regulated in individual gastric organoids. We have provided a "
" of how such an analysis pipeline can be used to analyze any temporal dataset and provide timely data-driven insights.
Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory activity, actin dynamics, inflammatory ...responses, stem cell proliferation, and many other uncharacterized cellular functions. As such, mapping calcium signaling dynamics within the intestinal epithelium can provide insight into homeostatic cellular processes and unveil unique responses to various stimuli. Human intestinal organoids (HIOs) are a high-throughput, human-derived model to study the intestinal epithelium and thus represent a useful system to investigate calcium dynamics. This paper describes a protocol to stably transduce HIOs with genetically encoded calcium indicators (GECIs), perform live fluorescence microscopy, and analyze imaging data to meaningfully characterize calcium signals. As a representative example, 3-dimensional HIOs were transduced with lentivirus to stably express GCaMP6s, a green fluorescent protein-based cytosolic GECI. The engineered HIOs were then dispersed into a single-cell suspension and seeded as monolayers. After differentiation, the HIO monolayers were infected with rotavirus and/or treated with drugs known to stimulate a calcium response. An epifluorescence microscope fitted with a temperature-controlled, humidified live-imaging chamber allowed for long-term imaging of infected or drug-treated monolayers. Following imaging, acquired images were analyzed using the freely available analysis software, ImageJ. Overall, this work establishes an adaptable pipeline for characterizing cellular signaling in HIOs.
Many viruses exploit host Ca
signaling to facilitate their replication; however, little is known about how Ca
signals from different host and viral channels contribute to the overall dysregulation of ...Ca
signaling or promote virus replication. Using cells lacking IP
R, a host ER Ca
channel, we delineated intracellular Ca
signals within virus-infected cells and intercellular Ca
waves (ICWs), which increased Ca
signaling in neighboring, uninfected cells. In infected cells, IP
R was dispensable for rotavirus-induced Ca
signaling and replication, suggesting the rotavirus NSP4 viroporin supplies these signals. However, IP
R-mediated ICWs increase rotavirus replication kinetics and spread, indicating that the Ca
signals from the ICWs may prime nearby uninfected cells to better support virus replication upon eventual infection. This "pre-emptive priming" of uninfected cells by exploiting host intercellular pathways in the vicinity of virus-infected cells represents a novel mechanism for viral reprogramming of the host to gain a replication advantage.
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