RESUMO
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.
Assuntos
Cálcio , Intestinos , Humanos , Cálcio/análise , Mucosa Intestinal/química , Organoides/química , Microscopia de Fluorescência/métodosRESUMO
Rotavirus causes life-threatening diarrhea in children, resulting in â¼200,000 deaths/year. The current treatment during infection is Oral Rehydration Solution which successfully replenishes fluids but does not alleviate diarrhea volume or severity. As a result, there is an urgent need to better understand rotavirus pathophysiology and develop more effective pediatric therapeutics. Rotavirus primarily infects the tips of small intestinal villi, yet has far-reaching effects on cell types distant from infected cells. We recently identified that rotavirus infected cells release the purinergic signaling molecule ADP, which activates P2Y1 receptors on nearby uninfected cells in vitro . To elucidate the role of purinergic signaling via P2Y1 receptors during rotavirus infection in vivo , we used the mouse-like rotavirus strain D6/2 which generates a severe infection in mice. C57BL/6J mouse pups were given an oral gavage of D6/2 rotavirus and assessed over the course of 5-7 days. Beginning at day 1 post infection, infected pups were treated daily by oral gavage with saline or 4 mg/kg MRS2500, a selective P2Y1 antagonist. Mice were monitored for diarrhea severity, diarrhea incidence, and viral shedding. Neonatal mice were euthanized at days 3 and 5 post-infection and small intestine was collected to observe infection. MRS2500 treatment decreased the severity, prevalence, and incidence of rotavirus diarrhea. Viral stool shedding, assessed by qPCR for rotavirus gene levels, revealed that MRS2500 treated pups had significantly lower viral shedding starting at day 4 post infection compared to saline treated pups, which suggests P2Y1 signaling may enhance rotavirus replication. Finally, we found that inhibition of P2Y1 with MRS2500 limited transmitted rotavirus diarrhea to uninfected pups within a litter. Together, these results suggest that P2Y1 signaling is involved in the pathogenesis of a homologous murine rotavirus strain, making P2Y1 receptors a promising anti-diarrheal, anti-viral therapeutic target to reduce rotavirus disease burden.
RESUMO
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.
Assuntos
Interações entre Hospedeiro e Microrganismos , Imagem Óptica , Interações Hospedeiro-Patógeno , Linhagem Celular , Microscopia de FluorescênciaRESUMO
Acute gastroenteritis remains the second leading cause of death among children under the age of 5 worldwide. While enteric viruses are the most common etiology, the drivers of their virulence remain incompletely understood. We recently found that cells infected with rotavirus, the most prevalent enteric virus in infants and young children, initiate hundreds of intercellular calcium waves that enhance both fluid secretion and viral spread. Understanding how rotavirus triggers intercellular calcium waves may allow us to design safer, more effective vaccines and therapeutics, but we still lack a mechanistic understanding of this process. In this study, we used existing virulent and attenuated rotavirus strains, as well as reverse engineered recombinants, to investigate the role of rotavirus nonstructural protein 4 (NSP4) in intercellular calcium wave induction using in vitro , organoid, and in vivo model systems. We found that the capacity to induce purinergic intercellular calcium waves (ICWs) segregated with NSP4 in both simian and murine-like rotavirus backgrounds, and NSP4 expression alone was sufficient to induce ICWs. NSP4's ability to function as a viroporin, which conducts calcium out of the endoplasmic reticulum, was necessary for ICW induction. Furthermore, viroporin activity and the resulting ICWs drove transcriptional changes indicative of innate immune activation, which were lost upon attenuation of viroporin function. Multiple aspects of RV disease severity in vivo correlated with the generation of ICWs, identifying a critical link between viroporin function, intercellular calcium waves, and enteric viral virulence.
RESUMO
IMPORTANCE: Many 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.