Human Intestinal Epithelium Research Articles

504. Latent HHV7 Infection Attenuates Beneficial Host-Microbe Interactions in the Human Gut

Abstract Background Human Herpes Virus 7 (HHV7) infection results in a lifelong latent infection with over 95% of adults seropositive for HHV7. Lymphocytes are the best understood host cell type for latent HHV7, with the gastric mucosa another site of latent infection. Herpesviridae latency affects host processes like interferon signaling, antigen presentation, and proliferation—the same host pathways are implicated in the beneficial responses to commensal microbes. Methods Adult human colonic organoids with or without latent HHV7 infection were co-cultured with B. longum in asymmetric oxygen conditions that allow for live interaction (F1). The transcriptional state after 24h of interactions was determined with RNA-sequencing. The transcriptional state was compared between axenic (no microbes) or after co-culture and with or without latent HHV7. F1: Establishing oxygen gradients across epithelial monolayers (A) Schematic of oxygen gradient apparatus (B) Closeup view of individual well (C) Growth of the anaerobe B. longum was identical in the apparatus with hypoxic epithelial compared to full anaerobic growth (D) Butyrate administration protects the epithelium from inflammatory effects of deoxycholate. Results Screening human intestinal organoids (that lack lymphocytes and are strictly of the endodermal lineage) we have generated evidence that intestinal stem cells (ISC) and IEC can be a host cell for latent HHV7 infection in some but not all people (T1). RNA-sequencing reveals that this is true latency, with ISC and IEC containing HHV7 without transcripts from any of the protein-coding genes in the HHV7 genome. Further we have observed that latent HHV7 is mosaic in the gut, not affecting all of the ISC in an individual (T1). In organoid-derived human IEC we have noted co-culture with the commensal bacteria B. longum suppresses key executors of apoptosis and pyroptosis (F2). These same effects are no longer observed with the B. longum is interacting with IEC latently infected with HHV7. A similar pattern was observed with NFkB regulators (F3). T1: Latent HHV7 is found in some intestinal organoids derived from healthy adults From the University of Michigan Translational Tissue Modeling Laboratory (TTML) organoid registry. Each donor is assigned a unique numerical ID. Viral panel is from a commercial multiplex PCR screen. Notably, donor 87 has colonic organoids with and without latent HHV7. F2: Co-culture with the commensal microbe B. longum suppresses most apoptosis and pyroptosis executors only in an organoid without latent HHV7 infection. Fragments per kilobase per millions of reads (FPKM) from RNA-sequencing of fully-differentiated organoid-derived human intestinal epithelium after 24 hours of co-culture with B.longum (Orange) or remaining axenic (without microbes; Blue). Results from cell without (left) or with (right) latent HHV7. F3: Stimulation of the NFkB inhibitor NFKBIA by co-culture with the commensal microbe B. longum is absent in organoids with latent HHV7 infection. Fragments per kilobase per millions of reads (FPKM) from RNA-sequencing of fully-differentiated organoid-derived human intestinal epithelium after 24 hours of co-culture with B.longum (Orange) or remaining axenic (without microbes; Blue). Results from cell without (left) or with (right) latent HHV7. Conclusion Latent HHV7 in the human gut epithelium may attenuate the effect of beneficial microbes like B. longum (F4). The evidence for mosaicism of latent HHV7 in the human gut adds a spatial aspect to these attenuating effects that could be relevant for specific disease processes (e.g. skip lesions in Crohn’s disease). Confirmation of these findings, estimating the true prevalence of latent HHV7 in the gut, and identification of the HHV7 latency mechanism will be required to understand the full implications of these findings. F4: A Model of Latent HHV7 Infection Attenuating Responses to Commensal Microbes. Based on our findings, latent HHV7 infection in the intestinal epithelium may leave the gut mucosa at increased risk for injury by impairing the beneficial response to commensal microbes in a spatially complex manner due to the apparent mosaic nature of latent HHV7 infection in the gut. Disclosures Jonathan L. Golob, MD/PhD, Loxo Oncology: Employment of Spouse.

Impact of selected novel plant bioactives on improvement of impaired gut barrier function using human primary cell intestinal epithelium

Gut barrier function is compromised in the obese state. The N-trans caffeoyltyramine (NCT) and N-trans feruloyltyramine (NFT), two naturally occurring bioactive compounds in hemp hulls, identified using in silico approaches, have the potential to improve gut barrier function and their effects were studied here in vitro. Proliferative human transverse colon epithelial cells were plated and co-cultured with tumor necrosis factor (TNF) along with NCT, NFT or NCT/NFT (2.2 ratio) post-differentiation, over a 48-hour period to induce inflammation and to observe the effects of NCT and NFT. A decrease in transepithelial electrical resistance (TEER) and increase in the intestinal permeability were observed with increased addition of TNF. Co-administration of NCT and NFT demonstrated a dose-dependent and statistically significant reversal of impaired TEER and intestinal permeability. NCT and NFT demonstrated a physiologically relevant reversal of impaired gut barrier function in the setting of inflammation via significant improvement in TEER and percent permeability.

Effect of oxidation and in vitro intestinal hydrolysis on phospholipid toxicity towards HT29 cell line serving as a model of human intestinal epithelium

Oxidation of food-derived phospholipids (PLs) can influence nutrient digestion and induce oxidative stress in gastrointestinal epithelium. In this study, hen egg yolk PL fraction was used to evaluate the effect of lipoxygenase (LOX)-induced PL oxidation on the rate of PL hydrolysis catalyzed by pancreatic phospholipase A2 (PLA2) in the presence of bile salts (BSs). Then, PL/BS solutions containing native or oxidized PLs were used in in vitro intestinal digestion to assess the effect of PL oxidation and hydrolysis on the toxicity towards HT29 cell line. Based on the obtained results, we suggest that hexanal and (E)-2-nonenal, formed by the decomposition of PL hydroperoxides, inhibited PLA2 activity. The cell exposure to simulated intestinal fluid (SIF) containing BSs decreased HT29 cell viability and significantly damaged cellular DNA. However, the genotoxic effect was reversed in the presence of all tested PL samples, while the protective effect against the BS-induced cytotoxicity was observed for native non-hydrolyzed PLs, but was not clearly visible for other samples. This can result from an overlap of other toxic effects such as lipotoxicity or disturbance of cellular redox homeostasis. Taking into account the data obtained, it was proposed that the PLA2 activity decline in the presence of PL oxidation products may be a kind of protective mechanism against rapid release of oxidized FAs characterized by high cytotoxic effect towards intestinal epithelium cells.

Newly Designed Primers for the Sequencing of the inlA Gene of Lineage I and II Listeria monocytogenes Isolates.

Listeria monocytogenes is a major human foodborne pathogen responsible for listeriosis. The virulence factor Internalin A (inlA) has a key role in the invasion of L. monocytogenes into the human intestinal epithelium, and the presence of premature stop-codons (PMSC) mutations in the inlA gene sequence is correlated with attenuated virulence. The inlA sequencing process is carried out by dividing the gene into three sections which are then reassembled to obtain the full gene. The primers available however were only able to entirely amplify the lineage II isolates. In this study, we present a set of new primers which allow inlA sequencing of isolates belonging to both lineages, since lineage I isolates are the ones most frequently associated to clinical cases. Using newly designed primers, we assessed the presence of inlA PMSCs in food, food processing environments and clinical isolates.

OR08-4 Endocrine Mechanisms of an Orphan G Protein-Coupled Receptor Regulating Metabolic Homeostasis

Abstract G protein-coupled receptors (GPCRs) are the largest protein family and amenable for pharmacological manipulation. GPCRs in the neuroendocrine and enteroendocrine systems respond to various biological cues and in turn exert crucial roles in maintaining whole body metabolic homeostasis. For example, GPCRs in the gastrointestinal tract are involved in maintaining glucose and energy homeostasis by regulating the release of gut hormones in response to luminal dietary nutrients as well as microbial metabolites. We recently identified that an orphan GPCR, Gpr17, was co-expressed in glucagon-like peptide-1 (GLP-1)-expressing EECs in human and rodent intestinal epithelium. We reported that acute genetic ablation of Gpr17 in intestinal epithelium (iKO) improved oral glucose tolerance and glucose-stimulated insulin secretion (GSIS) in female and male mice through increased secretion of incretin hormone in response to nutrient ingestion (Cell Reports, 2022). In the neuroendocrine system, we previously identified Gpr17 as the transcription target of Forkhead box protein O1 (FoxO1) in the hypothalamus and generated hypothalamic neuron specific Gpr17 knockout animals (Cell, 2012). Our results demonstrated that agouti-related peptide (AgRP) neuron Gpr17 knockout mice had increased satiety during fasting-refeeding challenge (Diabetes, 2015) and that proopiomelanocortin (POMC) neuron Gpr17 knockout mice had increased alpha-melanocyte stimulating hormone (aMSH) processing and increased firing of POMC neurons (Nutrition & Diabetes, 2019). However, how Gpr17 may regulate energy balance especially regarding leptin hormone signaling is unclear. To address this question, we used genetic knockout approach to generate Gpr17 conditional knockout in leptin receptor expressing neurons in the brain. We found Gpr17 deficiency in leptin receptor expressing neurons increased leptin sensitivity in animals and resulted in reduced food intake, which corresponded with reduced inflammatory response in the hypothalamus. Moreover, male Gpr17 whole body knockout mice have improved intraperitoneal glucose tolerance, and increased insulin sensitivity in hyperinsulinemic-euglycemic clamp studies. In conclusion, our collective body of work showed that ablation of Gpr17 signaling in the neuroendocrine and enteroendocrine systems led to improved neurohormonal regulation to maintain metabolic homeostasis, indicating functionally targeting this GPCR may lead to better therapeutic outcome of diabetes and obesity. Presentation: Saturday, June 11, 2022 12:15 p.m. - 12:30 p.m.

Bioengineering human intestinal mucosal grafts using patient-derived organoids, fibroblasts and scaffolds.

Tissue engineering is an interdisciplinary field that combines stem cells and matrices to form functional constructs that can be used to repair damaged tissues or regenerate whole organs. Tissue stem cells can be expanded and functionally differentiated to form 'mini-organs' resembling native tissue architecture and function. The choice of the scaffold is also pivotal to successful tissue reconstruction. Scaffolds may be broadly classified into synthetic or biological depending upon the purpose of the engineered organ. Bioengineered intestinal grafts represent a potential source of transplantable tissue for patients with intestinal failure, a condition resulting from extensive anatomical and functional loss of small intestine and therefore digestive and absorptive capacity. Prior strategies in intestinal bioengineering have predominantly used either murine or pluripotent cells and synthetic or decellularized rodent scaffolds, thus limiting their translation. Microscale models of human intestinal epithelium on shaped hydrogels and synthetic scaffolds are more physiological, but their regenerative potential is limited by scale. Here we present a protocol for bioengineering human intestinal grafts using patient-derived materials in a bioreactor culture system. This includes the isolation, expansion and biobanking of patient-derived intestinal organoids and fibroblasts, the generation of decellularized human intestinal scaffolds from native human tissue and providing a system for recellularization to form transplantable grafts. The duration of this protocol is 12 weeks, and it can be completed by scientists with prior experience of organoid culture. The resulting engineered mucosal grafts comprise physiological intestinal epithelium, matrix and surrounding niche, offering a valuable tool for both regenerative medicine and the study of human gastrointestinal diseases.

Human neutrophil IL1β directs intestinal epithelial cell extrusion during Salmonella infection.

Infection of the human gut by Salmonella enterica Typhimurium (STM) results in a localized inflammatory disease that is not mimicked in murine infections. To determine mechanisms by which neutrophils, as early responders to bacterial challenge, direct inflammatory programming of human intestinal epithelium, we established a multi-component human intestinal organoid (HIO) model of STM infection. HIOs were micro-injected with STM and seeded with primary human polymorphonuclear leukocytes (PMN-HIOs). PMNs did not significantly alter luminal colonization of Salmonella, but their presence reduced intraepithelial bacterial burden. Adding PMNs to infected HIOs resulted in substantial accumulation of shed TUNEL+ epithelial cells that was driven by PMN Caspase-1 activity. Inhibition of Caspases-1, -3 or -4 abrogated epithelial cell death and extrusion in the infected PMN-HIOs but only Caspase-1 inhibition significantly increased bacterial burden in the PMN-HIO epithelium. Thus, PMNs promote cell death in human intestinal epithelial cells through multiple caspases as a protective response to infection. IL-1β was necessary and sufficient to induce cell shedding in the infected HIOs. These data support a critical innate immune function for human neutrophils in amplifying cell death and extrusion of human epithelial cells from the Salmonella-infected intestinal monolayer.

Development of a Primary Human Intestinal Epithelium Enriched in L-Cells for Assay of GLP-1 Secretion.

Type 2 diabetes mellitus is a chronic disease associated with obesity and dysregulated human feeding behavior. The hormone glucagon-like peptide 1 (GLP-1), a critical regulator of body weight, food intake, and blood glucose levels, is secreted by enteroendocrine L-cells. The paucity of L-cells in primary intestinal cell cultures including organoids and monolayers has made assays of GLP-1 secretion from primary human cells challenging. In the current paper, an analytical assay pipeline consisting of an optimized human intestinal tissue construct enriched in L-cells paired with standard antibody-based GLP-1 assays was developed to screen compounds for the development of pharmaceuticals to modulate L-cell signaling. The addition of the serotonin receptor agonist Bimu 8, optimization of R-spondin and Noggin concentrations, and utilization of vasoactive intestinal peptide (VIP) increased the density of L-cells in a primary human colonic epithelial monolayer. Additionally, the incorporation of an air-liquid interface culture format increased the L-cell number so that the signal-to-noise ratio of conventional enzyme-linked immunoassays could be used to monitor GLP-1 secretion in compound screens. To demonstrate the utility of the optimized analytical method, 21 types of beverage sweeteners were screened for their ability to stimulate GLP-1 secretion. Stevioside and cyclamate were found to be the most potent inducers of GLP-1 secretion. This platform enables the quantification of GLP-1 secretion from human primary L-cells and will have broad application in understanding L-cell formation and physiology and will improve the identification of modulators of human feeding behavior.

Bioengineered 3D Tissue Model of Intestine Epithelium with Oxygen Gradients to Sustain Human Gut Microbiome.

The human gut microbiome is crucial to hosting physiology and health. Therefore, stable in vitro coculture of primary human intestinal cells with a microbiome community is essential for understanding intestinal disease progression and revealing novel therapeutic targets. Here, a three-dimensional scaffold system is presented to regenerate an in vitro human intestinal epithelium that recapitulates many functional characteristics of the native small intestines. The epithelium, derived from human intestinal enteroids, contains mature intestinal epithelial cells and possesses selectively permeable barrier functions. Importantly, by properly positioning the scaffolds cultured under normal atmospheric conditions, two physiologically relevant oxygen gradients, a proximal-to-distal oxygen gradient along the gastrointestinal (GI) tract, and a radial oxygen gradient across the epithelium, are distinguished in the tissues when the lumens are faced up and down in cultures, respectively. Furthermore, the presence of the low oxygen gradients supported the coculture of intestinal epithelium along with a complex living commensal gut microbiome (including obligate anaerobes) to simulate temporal microbiome dynamics in the native human gut. This unique silk scaffold platform may enable the exploration of microbiota-related mechanisms of disease pathogenesis and host-pathogen dynamics in infectious diseases including the potential to explore the human microbiome-gut-brain axis and potential novel microbiome-based therapeutics.

Protective effect of bovine lactoferrin against Cronobacter sakazakii in human intestinal Caco-2/TC7 cells

Milk is a source of bioactive proteins with defensive properties of great value for protecting the newborn. The activity of bovine milk lactoferrin (LF) was investigated as an antibacterial agent in the internalisation of the emergent pathogen Cronobacter sakazakii into Caco-2/TC7 cells, a model of human intestinal epithelium. The effect of LF on oxidative stress and expression of Toll-like receptors (TLR) was also investigated. LF exerted a clear antibacterial activity against C. sakazakii, as well as an inhibitory effect on C. sakazakii adhesion and internalisation into Caco-2/TC7 cells. Incubation with C. sakazakii induced an increase in oxidative stress on the lipid fraction of Caco-2/TC7 cells, which was reversed by LF. Additionally, LF altered the expression of TLR, with a clear decrease in the expression levels of TLR4 at 24 h of incubation. These results suggest that LF has very interesting properties as a potential ingredient for functional foods.

TRPV6 channel mediates alcohol-induced gut barrier dysfunction and systemic response.

SUMMARYIntestinal epithelial tight junction disruption is a primary contributing factor in alcohol-associated endotoxemia, systemic inflammation, and multiple organ damage. Ethanol and acetaldehyde disrupt tight junctions by elevating intracellular Ca2+. Here we identify TRPV6, a Ca2+-permeable channel, as responsible for alcohol-induced elevation of intracellular Ca2+, intestinal barrier dysfunction, and systemic inflammation. Ethanol and acetaldehyde elicit TRPV6 ionic currents in Caco-2 cells. Studies in Caco-2 cell monolayers and mouse intestinal organoids show that TRPV6 deficiency or inhibition attenuates ethanol- and acetaldehyde-induced Ca2+ influx, tight junction disruption, and barrier dysfunction. Moreover, Trpv6−/− mice are resistant to alcohol-induced intestinal barrier dysfunction. Photoaffinity labeling of 3-azibutanol identifies a histidine as a potential alcohol-binding site in TRPV6. The substitution of this histidine, and a nearby arginine, reduces ethanol-activated currents. Our findings reveal that TRPV6 is required for alcohol-induced gut barrier dysfunction and inflammation. Molecules that decrease TRPV6 function have the potential to attenuate alcohol-associated tissue injury.

1356-P: Investigating the Metabolic Function of an Orphan G Protein–Coupled Receptor in the Intestinal Epithelium

G protein-coupled receptors (GPCRs) in the gastrointestinal tract are involved in maintaining glucose and energy homeostasis by regulating the release of gut hormones in response to luminal dietary nutrients as well as microbial metabolites. We recently identified that an orphan GPCR, Gpr17, was co-expressed in glucagon-like peptide-1 (GLP-1) -expressing EECs in human and rodent intestinal epithelium. However, it is unknown how Gpr17 ablation in the intestinal epithelium affects feeding behavior and satiety regulation. To address this question, we used genetic knockout approach to generate intestinal Gpr17-deficient mice and analyzed their glucose metabolism and feeding behavior. We showed that intestinal Gpr17-deficient mice had similar growth curve, body composition, and ad libitum food intake compared with littermate controls. We found that acute genetic ablation of Gpr17 in intestinal epithelium (iKO) improved oral glucose tolerance and glucose-stimulated insulin secretion (GSIS) in female and male mice. iKO mice responded to glucose or lipid ingestion with increased secretion of GLP-1, but not the other incretin glucose-dependent insulinotropic polypeptide (GIP) . Intestinal Gpr17-deficient mice responded to fasting-refeeding challenge with reduced fasting locomotor activity and less food intake after refeeding, suggesting increased satiety during the phase of rebound hyperphagia. Furthermore, comprehensive studies including gut morphology, gut epithelium heterogeneity by scRNA-seq, and microbiome analysis were examined but none of them was changed because of intestinal Gpr17 ablation. In addition, male Gpr17 whole body knockout mice have improved intraperitoneal glucose tolerance, and increased insulin sensitivity in hyperinsulinemic-euglycemic clamp studies. In conclusion, our work showed that ablation of intestinal Gpr17 signaling led to improved neurohormonal regulation to maintain metabolic homeostasis. Disclosure H.Ren: None. Funding National Institute of Health (R01DK120772)

Intestinal Absorption Study of a Granular Form of Ferric Pyrophosphate.

Iron deficiency is one of the most prevalent nutritional disorders worldwide. The standard treatment involves iron supplementation, but this task is challenging because of poor solubility and organoleptic issues. Moreover, the need to increase iron bioavailability represents a challenge for treating iron-related disorders. In this study, gastroresistance and iron intestinal absorption of an innovative granular formulation composed of ferric pyrophosphate, modified starch and phospholipids branded as Ferro Fosfosoma® was investigated. Gastroresistant properties were studied using standard protocols, and a bioaccessible fraction was obtained by exposing a food supplement to in vitro digestion. This fraction was used for investigating iron absorption in Caco-2 and human follicle-associated intestinal epithelium (FAE) models. Ferro Fosfosoma® showed an improved resistance to gastric digestion and higher intestinal absorption than ferric pyrophosphate salt used as a control in both models. In the FAE model, Ferro Fosfosoma® induces larger iron absorption than in the Caco-2 monolayer, most likely due to the transcytosis ability of M cells. The larger iron absorption in the Ferro Fosfosoma®-treated FAE model corresponds to higher ferritin level, proving physiological iron handling that was once delivered by granular formulation. Finally, the formulation did not induce any alterations in viability and barrier integrity. To conclude, Ferro Fosfosoma® favors iron absorption and ferritin expression, while preserving any adverse effects.

A Protocol for the Cryopreservation of Human Intestinal Mucosal Biopsies Compatible With Single-Cell Transcriptomics and Ex Vivo Studies.

The heterogeneity of the human intestinal epithelium has hindered the understanding of the pathophysiology of distinct specialized cell types on a single-cell basis in disease states. Described here is a workflow for the cryopreservation of endoscopically obtained human intestinal mucosal biopsies, subsequent preparation of this tissue to yield highly viable fluorescence-activated cell sorting (FACS)isolated human intestinal epithelial cell (IEC) single-cell suspensions compatible with successful library preparation and deep single-cell RNA sequencing (scRNAseq). We validated this protocol in deep scRNAseq of 59,653 intestinal cells in 10 human participants. Furthermore, primary intestinal cultures were successfully generated from cryopreserved tissue, capable of surviving in short-term culture and suitable for physiological assays studying gut peptide secretion from rare hormone-producing enteroendocrine cells in humans. This study offers an accessible avenue for single-cell transcriptomics and ex vivo studies from cryopreserved intestinal mucosal biopsies. These techniques may be used in the future to dissect and define novel aberrations to the intestinal ecosystem that lead to the development and progression of disease states in humans, even in rare IEC populations.

SARS-CoV-2 induces barrier damage and inflammatory responses in the human iPSC-derived intestinal epithelium

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly spread and led to global health crises. COVID-19 causes well-known respiratory failure and gastrointestinal symptoms, such as diarrhea, nausea, and vomiting. Thus, human gastrointestinal cell models are urgently needed for COVID-19 research; however, it is difficult to obtain primary human intestinal cells. In this study, we examined whether human induced pluripotent stem cell (iPSC)-derived small intestinal epithelial cells (iPSC-SIECs) could be used as a SARS-CoV-2 infection model. We observed that iPSC-SIECs, such as absorptive and Paneth cells, were infected with SARS-CoV-2, and remdesivir treatment decreased intracellular SARS-CoV-2 replication in iPSC-SIECs. SARS-CoV-2 infection decreased expression levels of tight junction markers, ZO-3 and CLDN1, and transepithelial electrical resistance (TEER), which evaluates the integrity of tight junction dynamics. In addition, SARS-CoV-2 infection increased expression levels of proinflammatory genes, which are elevated in patients with COVID-19. These findings suggest iPSC-SIECs as a useful in vitro model for elucidating COVID-19 pathology and drug development.

Elevated 5‐hydroxytryptamine in COVID‐19 Stimulates ANO1 Mediated Cl Secretion in Lung & Intestinal Epithelial Cells

Earlier studies demonstrated that blood 5‐hydroxytryptamine (5‐HT) is elevated in patients with COVID‐19‐associated diarrhea with higher severity of symptoms. We hypothesize that disruption of vectorial Cl transport by 5‐HT may be critical in determining the alveolar flooding and abnormalities in intestinal Cl secretion through stimulation of anoctamin 1(ANO1) Cl channel. Using western blot, immunostaining, and electrophysiology, we characterized the localization of human ANO1 and its stimulation by 5‐HT on Cl secretion in lung and intestinal epithelium. Because calcium‐activated chloride current in human intestinal epithelia remains controversial, we examined the localization of ANO1 in the human terminal ileum and colonic tissue using confocal microscopy. Our results indicated that ANO1 was localized predominantly at the brush‐border membrane and co‐localized with the brush‐border membrane marker villin. ANO1 is not present in goblet cells. The anti‐ANO1 antibody recognized a protein of appropriate size in human colonic tissue. The specificity of the ANO1 antibody was tested by immunoblot analysis of lysates from human colorectal cancer tissues, where it displayed amplified ANO1 protein expression. We next confirmed ANO1‐currents activated by 5‐HT in the Caco‐2 cells by patch‐clamp measurements of whole‐cell current. The application of 100 nM 5‐HT produced a typical outward rectification. CaCCinh‐A01, a specific ANO1 blocker, inhibited the currents. The half‐maximal effective concentration value for the effects of 5‐HT was estimated at 21.8 ± 13.7 nM with a Hill coefficient of 0.89 ± 0.16. These results indicated that 5‐HT evoked calcium‐activated Cl currents through ANO1 channels. ANO1 is expressed in Calu 3 cells. We next confirmed the presence of ANO1 currents activated by 5‐HT in Calu‐3 cells by the Ussing chamber experiments. Serosal addition of 5‐HT produced an immediate and significant increase in Isc in Calu‐3 cells that was inhibited by the ANO1 selective inhibitor T16Ainh‐A01. Finally, we demonstrate that SARS‐CoV2 infection led to enterochromaffin cell hyperplasia in the intestinal epithelium of Syrian Hamster with a possible elevation of 5‐HT, which could explain the severity of symptoms in COVID‐19 associated diarrheal patients.In conclusion, SARS‐CoV2 infection resulted in intestinal enterochromaffin cells hyperplasia that could elevate 5‐HT. Elevated 5‐HT activates luminal ANO1 CaCC in the intestinal and lung epithelium by a mechanism that appears to involve the rise of [Ca2+]i. Our data suggest that 5‐HT may be a critical determinant of the COVID‐19 associated diarrhea and flooding of alveoli that have considerable implications for COVID‐19 therapy.

Thiolated Chitosan Conjugated Liposomes for Oral Delivery of Selenium Nanoparticles.

This study aimed to design a hybrid oral liposomal delivery system for selenium nanoparticles (Lip-SeNPs) to improve the bioavailability of selenium. Thiolated chitosan, a multifunctional polymer with mucoadhesive properties, was used for surface functionalization of Lip-SeNPs. Selenium nanoparticle (SeNP)-loaded liposomes were manufactured by a single step microfluidics-assisted chemical reduction and assembling process. Subsequently, chitosan-N-acetylcysteine was covalently conjugated to the preformed Lip-SeNPs. The Lip-SeNPs were characterized in terms of composition, morphology, size, zeta potential, lipid organization, loading efficiency and radical scavenging activity. A co-culture system (Caco-2:HT29-MTX) that integrates mucus secreting and enterocyte-like cell types was used as a model of the human intestinal epithelium to determine adsorption, mucus penetration, release and transport properties of Lip-SeNPs in vitro. Thiolated Lip-SeNPs were positively charged with an average size of about 250 nm. Thiolated Lip-SeNPs tightly adhered to the mucus layer without penetrating the enterocytes. This finding was consistent with ex vivo adsorption studies using freshly excised porcine small intestinal tissues. Due to the improved mucoadhesion and retention in a simulated microenvironment of the small intestine, thiolated Lip-SeNPs might be a promising tool for oral selenium delivery.

3D in vitro morphogenesis of human intestinal epithelium in a gut-on-a-chip or a hybrid chip with a cell culture insert.

Human intestinal morphogenesis establishes 3D epithelial microarchitecture and spatially organized crypt-villus characteristics. This unique structure is necessary to maintain intestinal homeostasis by protecting the stem cell niche in the basal crypt from exogenous microbial antigens and their metabolites. Also, intestinal villi and secretory mucus present functionally differentiated epithelial cells with a protective barrier at the intestinal mucosal surface. Thus, re-creating the 3D epithelial structure is critical to building in vitro intestine models. Notably, an organomimetic gut-on-a-chip can induce spontaneous 3D morphogenesis of an intestinal epithelium with enhanced physiological function and biomechanics. Here we provide a reproducible protocol to robustly induce intestinal morphogenesis in a microfluidic gut-on-a-chip as well as in a Transwell-embedded hybrid chip. We describe detailed methods for device fabrication, culture of Caco-2 or intestinal organoid epithelial cells in conventional setups as well as on microfluidic platforms, induction of 3D morphogenesis and characterization of established 3D epithelium using multiple imaging modalities. This protocol enables the regeneration of functional intestinal microarchitecture by controlling basolateral fluid flow within 5 d. Our in vitro morphogenesis method employs physiologically relevant shear stress and mechanical motions, and does not require complex cellular engineering or manipulation, which may be advantageous over other existing techniques. We envision that our proposed protocol may have a broad impact on biomedical research communities, providing a method to regenerate in vitro 3D intestinal epithelial layers for biomedical, clinical and pharmaceutical applications.

High-Definition DIC Imaging Uncovers Transient Stages of Pathogen Infection Cycles on the Surface of Human Adult Stem Cell-Derived Intestinal Epithelium.

ABSTRACTInteractions between individual pathogenic microbes and host tissues involve fast and dynamic processes that ultimately impact the outcome of infection. Using live-cell microscopy, these dynamics can be visualized to study, e.g., microbe motility, binding and invasion of host cells, and intrahost-cell survival. Such methodology typically employs confocal imaging of fluorescent tags in tumor-derived cell line infections on glass. This allows high-definition imaging but poorly reflects the host tissue’s physiological architecture and may result in artifacts. We developed a method for live-cell imaging of microbial infection dynamics on human adult stem cell-derived intestinal epithelial cell (IEC) layers. These IEC layers are grown in apical imaging chambers, optimized for physiological cell arrangement and fast, but gentle, differential interference contrast (DIC) imaging. This allows subsecond visualization of both microbial and epithelial surface ultrastructure at high resolution without using fluorescent reporters. We employed this technology to probe the behavior of two model pathogens, Salmonella enterica serovar Typhimurium and Giardia intestinalis, at the intestinal epithelial surface. Our results reveal pathogen-specific swimming patterns on the epithelium and show that Salmonella lingers on the IEC surface for prolonged periods before host cell invasion, while Giardia uses circular swimming with intermittent attachments to scout for stable adhesion sites. The method even permits tracking of individual Giardia flagella, demonstrating that active flagellar beating and attachment to the IEC surface are not mutually exclusive. This work describes a generalizable and relatively inexpensive approach to resolving dynamic pathogen-IEC layer interactions, applicable even to genetically nontractable microorganisms.

Current Perspectives on Gastrointestinal Models to Assess Probiotic-Pathogen Interactions.

There are different models available that mimic the human intestinal epithelium and are thus available for studying probiotic and pathogen interactions in the gastrointestinal tract. Although, in vivo models make it possible to study the overall effects of a probiotic on a living subject, they cannot always be conducted and there is a general commitment to reduce the use of animal models. Hence, in vitro methods provide a more rapid tool for studying the interaction between probiotics and pathogens; as well as being ethically superior, faster, and less expensive. The in vitro models are represented by less complex traditional models, standard 2D models compromised of culture plates as well as Transwell inserts, and newer 3D models like organoids, enteroids, as well as organ-on-a-chip. The optimal model selected depends on the research question. Properly designed in vitro and/or in vivo studies are needed to examine the mechanism(s) of action of probiotics on pathogens to obtain physiologically relevant results.