Bacteroides ovatus Promotes IL-22 Production and Reduces Trinitrobenzene Sulfonic Acid-Driven Colonic Inflammation.

The intestinal microbiota influences the development and function of the mucosal immune system. However, the exact mechanisms by which commensal microbes modulate immunity is not clear. We previously demonstrated that commensal Bacteroides ovatus ATCC 8384 reduces mucosal inflammation. Herein, we aimed to identify immunomodulatory pathways employed by B. ovatus. In germ-free mice, mono-association with B. ovatus shifted the CD11b+/CD11c+ and CD103+/CD11c+ dendritic cell populations. Because indole compounds are known to modulate dendritic cells, B. ovatus cell-free supernatant was screened for tryptophan metabolites by liquid chromatography-tandem mass spectrometry and larger quantities of indole-3-acetic acid were detected. Analysis of cecal and fecal samples from germ-free and B. ovatus mono-associated mice confirmed that B. ovatus could elevate indole-3-acetic acid concentrations in vivo. Indole metabolites have previously been shown to stimulate immune cells to secrete the reparative cytokine IL-22. Addition of B. ovatus cell-free supernatant to immature bone marrow-derived dendritic cells stimulated IL-22 secretion. The ability of IL-22 to drive repair in the intestinal epithelium was confirmed using a physiologically relevant human intestinal enteroid model. Finally, B. ovatus shifted the immune cell populations in trinitrobenzene sulfonic acid-treated mice and up-regulated colonic IL-22 expression, effects that correlated with decreased inflammation. Our data suggest that B. ovatus-produced indole-3-acetic acid promotes IL-22 production by immune cells, yielding beneficial effects on colitis.

Enhancing responsiveness of human jejunal enteroids to host and microbial stimuli.

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. ABSTRACT: 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.

Human intestinal enteroids as a model of Clostridioides difficile-induced enteritis.

Clostridioides difficile is an important nosocomial pathogen that produces toxins to cause life-threatening diarrhea and colitis. Toxins bind to epithelial receptors and promote the collapse of the actin cytoskeleton. C. difficile toxin activity is commonly studied in cancer-derived and immortalized cell lines. However, the biological relevance of these models is limited. Moreover, no model is available for examining C. difficile-induced enteritis, an understudied health problem. We hypothesized that human intestinal enteroids (HIEs) express toxin receptors and provide a new model to dissect C. difficile cytotoxicity in the small intestine. We generated biopsy-derived jejunal HIE and Vero cells, which stably express LifeAct-Ruby, a fluorescent label of F-actin, to monitor actin cytoskeleton rearrangement by live-cell microscopy. Imaging analysis revealed that toxins from pathogenic C. difficile strains elicited cell rounding in a strain-dependent manner, and HIEs were tenfold more sensitive to toxin A (TcdA) than toxin B (TcdB). By quantitative PCR, we paradoxically found that HIEs expressed greater quantities of toxin receptor mRNA and yet exhibited decreased sensitivity to toxins when compared with traditionally used cell lines. We reasoned that these differences may be explained by components, such as mucins, that are present in HIEs cultures, that are absent in immortalized cell lines. Addition of human-derived mucin 2 (MUC2) to Vero cells delayed cell rounding, indicating that mucus serves as a barrier to toxin-receptor binding. This work highlights that investigation of C. difficile infection in that HIEs can provide important insights into the intricate interactions between toxins and the human intestinal epithelium.NEW & NOTEWORTHY In this article, we developed a novel model of Clostridioides difficile-induced enteritis using jejunal-derived human intestinal enteroids (HIEs) transduced with fluorescently tagged F-actin. Using live-imaging, we identified that jejunal HIEs express high levels of TcdA and CDT receptors, are more sensitive to TcdA than TcdB, and secrete mucus, which delays toxin-epithelial interactions. This work also optimizes optically clear C. difficile-conditioned media suitable for live-cell imaging.

A pathologist-AI collaboration framework for enhancing diagnostic accuracies and efficiencies.

In pathology, the deployment of artificial intelligence (AI) in clinical settings is constrained by limitations in data collection and in model transparency and interpretability. Here we describe a digital pathology framework, nuclei.io, that incorporates active learning and human-in-the-loop real-time feedback for the rapid creation of diverse datasets and models. We validate the effectiveness of the framework via two crossover user studies that leveraged collaboration between the AI and the pathologist, including the identification of plasma cells in endometrial biopsies and the detection of colorectal cancer metastasis in lymph nodes. In both studies, nuclei.io yielded considerable diagnostic performance improvements. Collaboration between clinicians and AI will aid digital pathology by enhancing accuracies and efficiencies.

Comparison of nucleocapsid antigen with strand-specific reverse-transcription PCR for monitoring SARS-CoV-2 infection.

BACKGROUND: Tests that sensitively detect the presence of actively replicating SARS-CoV-2 may improve patient care by allowing the safe and timely discontinuation of isolation. Correlates of active replication include nucleocapsid antigen and virus minus-strand RNA. METHODS: Qualitative agreement of the DiaSorin LIAISON SARS-CoV-2 nucleocapsid antigen chemiluminescent immunoassay (CLIA) with minus-strand RNA was determined using 402 upper respiratory specimens from 323 patients previously tested using a laboratory-developed SARS-CoV-2 strand-specific RT-qPCR. Nucleocapsid antigen levels, minus-strand and plus-strand cycle threshold values, as well as virus culture, were used to evaluate discordant specimens. Receiver operating characteristic curves were also used to identify virus RNA thresholds for active replication, including values harmonized to the World Health Organization International Standard. RESULTS: Overall agreement was 92.0% [95% confidence interval (CI): 89.0 - 94.5], positive percent agreement was 90.6% (95% CI: 84.4 - 95.0), and negative percent agreement was 92.8% (95% CI: 89.0 - 95.6). The kappa coefficient was 0.83 (95% CI: 0.77 - 0.88). Discordant specimens contained low levels of nucleocapsid antigen and minus-strand RNA. 84.8% (28/33) were negative by culture. Sensitivity-optimized plus-strand RNA thresholds for active replication were 31.6 cycles or 3.64 log10 IU/mL; resulting in 100.0% sensitivity (95% CI: 97.6 to 100.0) and 55.9 specificity (95% CI: 49.7 to 62.0). CONCLUSIONS: Detection of nucleocapsid antigen by CLIA performs equivalently to minus-strand detection via strand-specific RT-qPCR, though these methods may overestimate replication-competent virus compared to culture. Careful implementation of biomarkers for actively replicating SARS-CoV-2 has the potential to inform infection control decision-making and patient management.

Case study of water-soluble metal containing organic constituents of biomass burning aerosol.

Natural and prescribed biomass fires are a major source of aerosols that may persist in the atmosphere for several weeks. Biomass burning aerosols (BBA) can be associated with long-range transport of water-soluble N-, S-, P-, and metal-containing species. In this study, BBA samples were collected using a particle-into-liquid sampler (PILS) from laboratory burns of vegetation collected on military bases in the southeastern and southwestern United States. The samples were then analyzed using high resolution electrospray ionization mass spectrometry (ESI/HR-MS) that enabled accurate mass measurements for hundreds of species with m/z values between 70 and 1000 and assignment of elemental formulas. Mg, Al, Ca, Cr, Mn, Fe, Ni, Cu, Zn, and Ba-containing organometallic species were identified. The results suggest that the biomass may have accumulated metal-containing species that were re-emitted during biomass burning. Further research into the sources, dispersion, and persistence of metal-containing aerosols, as well as their environmental effects, is needed.

Mucin-Degrading Microbes Release Monosaccharides That Chemoattract Clostridioides difficile and Facilitate Colonization of the Human Intestinal Mucus Layer.

It is widely accepted that the pathogen Clostridioides difficile exploits an intestinal environment with an altered microbiota, but the details of these microbe-microbe interactions are unclear. Adherence and colonization of mucus has been demonstrated for several enteric pathogens and it is possible that mucin-associated microbes may be working in concert with C. difficile. We showed that C. difficile ribotype-027 adheres to MUC2 glycans and using fecal bioreactors, we identified that C. difficile associates with several mucin-degrading microbes. C. difficile was found to chemotax toward intestinal mucus and its glycan components, demonstrating that C. difficile senses the mucus layer. Although C. difficile lacks the glycosyl hydrolases required to degrade mucin glycans, coculturing C. difficile with the mucin-degrading Akkermansia muciniphila, Bacteroides thetaiotaomicron, and Ruminococcus torques allowed C. difficile to grow in media that lacked glucose but contained purified MUC2. Collectively, these studies expand our knowledge on how intestinal microbes support C. difficile.

Fusobacterium nucleatum Secretes Outer Membrane Vesicles and Promotes Intestinal Inflammation.

Multiple studies have implicated microbes in the development of inflammation, but the mechanisms remain unknown. Bacteria in the genus Fusobacterium have been identified in the intestinal mucosa of patients with digestive diseases; thus, we hypothesized that Fusobacterium nucleatum promotes intestinal inflammation. The addition of >50 kDa F. nucleatum conditioned media, which contain outer membrane vesicles (OMVs), to colonic epithelial cells stimulated secretion of the proinflammatory cytokines interleukin-8 (IL-8) and tumor necrosis factor (TNF). In addition, purified F. nucleatum OMVs, but not compounds <50 kDa, stimulated IL-8 and TNF production; which was decreased by pharmacological inhibition of Toll-like receptor 4 (TLR4). These effects were linked to downstream effectors p-ERK, p-CREB, and NF-κB. F. nucleatum >50-kDa compounds also stimulated TNF secretion, p-ERK, p-CREB, and NF-κB activation in human colonoid monolayers. In mice harboring a human microbiota, pretreatment with antibiotics and a single oral gavage of F. nucleatum resulted in inflammation. Compared to mice receiving vehicle control, mice treated with F. nucleatum showed disruption of the colonic architecture, with increased immune cell infiltration and depleted mucus layers. Analysis of mucosal gene expression revealed increased levels of proinflammatory cytokines (KC, TNF, IL-6, IFN-γ, and MCP-1) at day 3 and day 5 in F. nucleatum-treated mice compared to controls. These proinflammatory effects were absent in mice who received F. nucleatum without pretreatment with antibiotics, suggesting that an intact microbiome is protective against F. nucleatum-mediated immune responses. These data provide evidence that F. nucleatum promotes proinflammatory signaling cascades in the context of a depleted intestinal microbiome.IMPORTANCE Several studies have identified an increased abundance of Fusobacterium in the intestinal tracts of patients with colon cancer, liver cirrhosis, primary sclerosing cholangitis, gastroesophageal reflux disease, HIV infection, and alcoholism. However, the direct mechanism(s) of action of Fusobacterium on pathophysiological within the gastrointestinal tract is unclear. These studies have identified that F. nucleatum subsp. polymorphum releases outer membrane vesicles which activate TLR4 and NF-κB to stimulate proinflammatory signals in vitro Using mice harboring a human microbiome, we demonstrate that F. nucleatum can promote inflammation, an effect which required antibiotic-mediated alterations in the gut microbiome. Collectively, these results suggest a mechanism by which F. nucleatum may contribute to intestinal inflammation.

Bifidobacterium dentium-derived y-glutamylcysteine suppresses ER-mediated goblet cell stress and reduces TNBS-driven colonic inflammation.

Endoplasmic reticulum (ER) stress compromises the secretion of MUC2 from goblet cells and has been linked with inflammatory bowel disease (IBD). Although Bifidobacterium can beneficially modulate mucin production, little work has been done investigating the effects of Bifidobacterium on goblet cell ER stress. We hypothesized that secreted factors from Bifidobacterium dentium downregulate ER stress genes and modulates the unfolded protein response (UPR) to promote MUC2 secretion. We identified by mass spectrometry that B. dentium secretes the antioxidant γ-glutamylcysteine, which we speculate dampens ER stress-mediated ROS and minimizes ER stress phenotypes. B. dentium cell-free supernatant and γ-glutamylcysteine were taken up by human colonic T84 cells, increased glutathione levels, and reduced ROS generated by the ER-stressors thapsigargin and tunicamycin. Moreover, B. dentium supernatant and γ-glutamylcysteine were able to suppress NF-kB activation and IL-8 secretion. We found that B. dentium supernatant, γ-glutamylcysteine, and the positive control IL-10 attenuated the induction of UPR genes GRP78, CHOP, and sXBP1. To examine ER stress in vivo, we first examined mono-association of B. dentium in germ-free mice which increased MUC2 and IL-10 levels compared to germ-free controls. However, no changes were observed in ER stress-related genes, indicating that B. dentium can promote mucus secretion without inducing ER stress. In a TNBS-mediated ER stress model, we observed increased levels of UPR genes and pro-inflammatory cytokines in TNBS treated mice, which were reduced with addition of live B. dentium or γ-glutamylcysteine. We also observed increased colonic and serum levels of IL-10 in B. dentium- and γ-glutamylcysteine-treated mice compared to vehicle control. Immunostaining revealed retention of goblet cells and mucus secretion in both B. dentium- and γ-glutamylcysteine-treated animals. Collectively, these data demonstrate positive modulation of the UPR and MUC2 production by B. dentium-secreted compounds.

Human-Derived Bifidobacterium dentium Modulates the Mammalian Serotonergic System and Gut-Brain Axis.

BACKGROUND & AIMS: The human gut microbiota can regulate production of serotonin (5-hydroxytryptamine [5-HT]) from enterochromaffin cells. However, the mechanisms underlying microbial-induced serotonin signaling are not well understood. METHODS: Adult germ-free mice were treated with sterile media, live Bifidobacterium dentium, heat-killed B dentium, or live Bacteroides ovatus. Mouse and human enteroids were used to assess the effects of B dentium metabolites on 5-HT release from enterochromaffin cells. In vitro and in vivo short-chain fatty acids and 5-HT levels were assessed by mass spectrometry. Expression of tryptophan hydroxylase, short-chain fatty acid receptor free fatty acid receptor 2, 5-HT receptors, and the 5-HT re-uptake transporter (serotonin transporter) were assessed by quantitative polymerase chain reaction and immunostaining. RNA in situ hybridization assessed 5-HT-receptor expression in the brain, and 5-HT-receptor-dependent behavior was evaluated using the marble burying test. RESULTS: B dentium mono-associated mice showed increased fecal acetate. This finding corresponded with increased intestinal 5-HT concentrations and increased expression of 5-HT receptors 2a, 4, and serotonin transporter. These effects were absent in B ovatus-treated mice. Application of acetate and B dentium-secreted products stimulated 5-HT release in mouse and human enteroids. In situ hybridization of brain tissue also showed significantly increased hippocampal expression of 5-HT-receptor 2a in B dentium-treated mice relative to germ-free controls. Functionally, B dentium colonization normalized species-typical repetitive and anxiety-like behaviors previously shown to be linked to 5-HT-receptor 2a. CONCLUSIONS: These data suggest that B dentium, and the bacterial metabolite acetate, are capable of regulating key components of the serotonergic system in multiple host tissues, and are associated with a functional change in adult behavior.

Reuterin disrupts Clostridioides difficile metabolism and pathogenicity through reactive oxygen species generation.

Antibiotic resistance is one of the world's greatest public health challenges and adjunct probiotic therapies are strategies that could lessen this burden. Clostridioides difficile infection (CDI) is a prime example where adjunct probiotic therapies could decrease disease incidence through prevention. Human-derived Lactobacillus reuteri is a probiotic that produces the antimicrobial compound reuterin known to prevent C. difficile colonization of antibiotic-treated fecal microbial communities. However, the mechanism of inhibition is unclear. We show that reuterin inhibits C. difficile outgrowth from spores and vegetative cell growth, however, no effect on C. difficile germination or sporulation was observed. Consistent with published studies, we found that exposure to reuterin stimulated reactive oxygen species (ROS) in C. difficile, resulting in a concentration-dependent reduction in cell viability that was rescued by the antioxidant glutathione. Sublethal concentrations of reuterin enhanced the susceptibility of vegetative C. difficile to vancomycin and metronidazole treatment and reduced toxin synthesis by C. difficile. We also demonstrate that reuterin is protective against C. difficile toxin-mediated cellular damage in the human intestinal enteroid model. Overall, our results indicate that ROS are essential mediators of reuterin activity and show that reuterin production by L. reuteri is compatible as a therapeutic in a clinically relevant model.

Rotavirus infection induces glycan availability to promote ileum-specific changes in the microbiome aiding rotavirus virulence.

Multiple studies have identified changes within the gut microbiome in response to diarrheal-inducing bacterial pathogens. However, examination of the microbiome in response to viral pathogens remains understudied. Compounding this, many studies use fecal samples to assess microbiome composition; which may not accurately mirror changes within the small intestine, the primary site for most enteric virus infections. As a result, the functional significance of small intestinal microbiome shifts during infection is not well defined. To address these gaps, rotavirus-infected neonatal mice were examined for changes in bacterial community dynamics, host gene expression, and tissue recovery during infection. Profiling bacterial communities using 16S rRNA sequencing suggested significant and distinct changes in ileal communities in response to rotavirus infection, with no significant changes for other gastrointestinal (GI) compartments. At 1-d post-infection, we observed a loss in Lactobacillus species from the ileum, but an increase in Bacteroides and Akkermansia, both of which exhibit mucin-digesting capabilities. Concomitant with the bacterial community shifts, we observed a loss of mucin-filled goblet cells in the small intestine at d 1, with recovery occurring by d 3. Rotavirus infection of mucin-producing cell lines and human intestinal enteroids (HIEs) stimulated release of stored mucin granules, similar to in vivo findings. In vitro, incubation of mucins with Bacteroides or Akkermansia members resulted in significant glycan degradation, which altered the binding capacity of rotavirus in silico and in vitro. Taken together, these data suggest that the response to and recovery from rotavirus-diarrhea is unique between sub-compartments of the GI tract and may be influenced by mucin-degrading microbes.

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 unclear. We found that rotavirus-infected cells produce paracrine signals that manifested as intercellular calcium waves (ICWs), observed in cell lines and human intestinal enteroids. Rotavirus ICWs were caused by the release of extracellular adenosine 5'-diphosphate (ADP) that activated P2Y1 purinergic receptors on neighboring cells. ICWs were blocked by P2Y1 antagonists or CRISPR-Cas9 knockout of the P2Y1 receptor. Blocking the ADP signal reduced rotavirus replication, inhibited rotavirus-induced serotonin release and fluid secretion, and reduced diarrhea severity in neonatal mice. Thus, rotavirus exploited paracrine purinergic signaling to generate ICWs that amplified the dysregulation of host cells and altered gastrointestinal physiology to cause diarrhea.

Recovirus NS1-2 Has Viroporin Activity That Induces Aberrant Cellular Calcium Signaling To Facilitate Virus Replication.

Enteric viruses in the Caliciviridae family cause acute gastroenteritis in humans and animals, but the cellular processes needed for virus replication and disease remain unknown. A common strategy among enteric viruses, including rotaviruses and enteroviruses, is to encode a viral ion channel (i.e., viroporin) that is targeted to the endoplasmic reticulum (ER) and disrupts host calcium (Ca2+) homeostasis. Previous reports have demonstrated genetic and functional similarities between the nonstructural proteins of caliciviruses and enteroviruses, including the calicivirus NS1-2 protein and the 2B viroporin of enteroviruses. However, it is unknown whether caliciviruses alter Ca2+ homeostasis for virus replication or whether the NS1-2 protein has viroporin activity like its enterovirus counterpart. To address these questions, we used Tulane virus (TV), a rhesus enteric calicivirus, to examine Ca2+ signaling during infection and determine whether NS1-2 has viroporin activity that disrupts Ca2+ homeostasis. We found that TV increases Ca2+ signaling during infection and that increased cytoplasmic Ca2+ levels are important for efficient replication. Further, TV NS1-2 localizes to the endoplasmic reticulum, the predominant intracellular Ca2+ store, and the NS2 region has characteristics of a viroporin domain (VPD). NS1-2 had viroporin activity in a classic bacterial functional assay and caused aberrant Ca2+ signaling when expressed in mammalian cells, but truncation of the VPD abrogated these activities. Together, our data provide new mechanistic insights into the function of the NS2 region of NS1-2 and support the premise that enteric viruses, including those within Caliciviridae, exploit host Ca2+ signaling to facilitate their replication.IMPORTANCE Tulane virus is one of many enteric caliciviruses that cause acute gastroenteritis and diarrheal disease. Globally, enteric caliciviruses affect both humans and animals and amass >65 billion dollars per year in treatment and health care-associated costs, thus imposing an enormous economic burden. Recent progress has resulted in several cultivation systems (B cells, enteroids, and zebrafish larvae) to study human noroviruses, but mechanistic insights into the viral factors and host pathways important for enteric calicivirus replication and infection are still largely lacking. Here, we used Tulane virus, a calicivirus that is biologically similar to human noroviruses and can be cultivated by conventional cell culture, to identify and functionally validate NS1-2 as an enteric calicivirus viroporin. Viroporin-mediated calcium signaling may be a broadly utilized pathway for enteric virus replication, and its existence within caliciviruses provides a novel approach to developing antivirals and comprehensive therapeutics for enteric calicivirus diarrheal disease outbreaks.

Bifidobacterium dentium Fortifies the Intestinal Mucus Layer via Autophagy and Calcium Signaling Pathways.

Much remains unknown about how the intestinal microbiome interfaces with the protective intestinal mucus layer. Bifidobacterium species colonize the intestinal mucus layer and can modulate mucus production by goblet cells. However, select Bifidobacterium strains can also degrade protective glycans on mucin proteins. We hypothesized that the human-derived species Bifidobacterium dentium would increase intestinal mucus synthesis and expulsion, without extensive degradation of mucin glycans. In silico data revealed that B. dentium lacked the enzymes necessary to extensively degrade mucin glycans. This finding was confirmed by demonstrating that B. dentium could not use naive mucin glycans as primary carbon sources in vitro To examine B. dentium mucus modulation in vivo, Swiss Webster germfree mice were monoassociated with live or heat-killed B. dentium Live B. dentium-monoassociated mice exhibited increased colonic expression of goblet cell markers Krüppel-like factor 4 (Klf4), Trefoil factor 3 (Tff3), Relm-ß, Muc2, and several glycosyltransferases compared to both heat-killed B. dentium and germfree counterparts. Likewise, live B. dentium-monoassociated colon had increased acidic mucin-filled goblet cells, as denoted by Periodic Acid-Schiff-Alcian Blue (PAS-AB) staining and MUC2 immunostaining. In vitro, B. dentium-secreted products, including acetate, were able to increase MUC2 levels in T84 cells. We also identified that B. dentium-secreted products, such as γ-aminobutyric acid (GABA), stimulated autophagy-mediated calcium signaling and MUC2 release. This work illustrates that B. dentium is capable of enhancing the intestinal mucus layer and goblet cell function via upregulation of gene expression and autophagy signaling pathways, with a net increase in mucin production.IMPORTANCE Microbe-host interactions in the intestine occur along the mucus-covered epithelium. In the gastrointestinal tract, mucus is composed of glycan-covered proteins, or mucins, which are secreted by goblet cells to form a protective gel-like structure above the epithelium. Low levels of mucin or alterations in mucin glycans are associated with inflammation and colitis in mice and humans. Although current literature links microbes to the modulation of goblet cells and mucins, the molecular pathways involved are not yet fully understood. Using a combination of gnotobiotic mice and mucus-secreting cell lines, we have identified a human-derived microbe, Bifidobacterium dentium, which adheres to intestinal mucus and secretes metabolites that upregulate the major mucin MUC2 and modulate goblet cell function. Unlike other Bifidobacterium species, B. dentium does not extensively degrade mucin glycans and cannot grow on mucin alone. This work points to the potential of using B. dentium and similar mucin-friendly microbes as therapeutic agents for intestinal disorders with disruptions in the mucus barrier.

Human Intestinal Enteroids With Inducible Neurogenin-3 Expression as a Novel Model of Gut Hormone Secretion.

BACKGROUND & AIMS: Enteroendocrine cells (EECs) are specialized epithelial cells that produce molecules vital for intestinal homeostasis, but because of their limited numbers, in-depth functional studies have remained challenging. Human intestinal enteroids (HIEs) that are derived from intestinal crypt stem cells are biologically relevant in an in vitro model of the intestinal epithelium. HIEs contain all intestinal epithelial cell types; however, similar to the intestine, HIEs spontaneously produce few EECs, which limits their study. METHODS: To 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. RESULTS: Treating tetNGN3-HIEs with doxycycline induced a dose-dependent increase of chromogranin A (ChgA)-positive and serotonin-positive cells, showing 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. CONCLUSIONS: We 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.

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 calcium signaling, establishes a new, paradigm-shifting understanding of the spatial and temporal complexity of virus-induced calcium signaling.