Human Ileum Research Articles

P1138 Curcumin-QingDai combination for patients with active Crohn’s disease: Initial real-world experience from a retrospective multi-center cohort study

Abstract Background A combination of Curcumin and QingDai (CurQD) was shown to exert aryl-hydrocarbon-receptor (AhR) pathway activation and to be effective in ulcerative colitis. However, its benefit in Crohn’s disease (CD) has not been studied Methods A retrospective cohort study of patients with active CD treated with CurQD at three academic centers. Active disease was defined by PRO-2 SF≥2 and AP ≥1. The primary endpoint was the rate of clinical remission at the end-of-induction (weeks 8-12), PRO2 SF≤1 and AP=0. Secondary endpoints included biomarker response (FCP drop ≥50% in a patient with FCP >250mcg/gr at baseline) and remission (FCP drop ≥50% and FCP<250mcg/gr at end-of-induction) and CurQD retention. Public-domain GTEx dataset was explored for mRNA expression of the target AhR in colon versus small intestinal mucosa Results Thirty patients were identified for the safety dataset, of whom 13/30 (43%) were bio-experienced, and 25 were eligible for inclusion in the efficacy analysis,. Clinical PRO2 remission at the end of induction was achieved in 12/25 (48%) of patients and clinical response in 19/25 (76%), with an overall reduction in median PRO2 score from 15 (95%CI 13-19.7) to 4 (95%CI 2-8.7, p<0.001). Biomarker remission and response were observed in 11/20 (55%) and 15/20 (75%), respectively, of patients with baseline FCP>250mcg/gr, with an overall reduction in median FCP from a baseline 639 mcg/gr (95%CI 128-5480) to 138mcg/gr (95%CI 5-1470, p=0.001). Biomarker remission was observed in 8/11 (73%) of patients with colonic-involving L2/L3 disease versus 3/9 (33%) L1 extent (OR 4.5, 95%CI 0.8- 24, p=0.08). After 8-months’ median follow-up (range 2-26 months), 16/19 (84%) of responding patients were still taking CurQD. Three patients experienced headaches and three had abdominal pain/diarrhea. Two of the six stopped CurQD due to these symptoms. One patient had elevated liver transaminases X3/normal, which resolved upon halving CurQD dose. To explore if the signal for a difference in efficacy in patients with or without colonic involvment relates to different expression of the target AhR in colon versus small bowel, an analysis of GTEx dataset was performed, which revealed comparable AhR mRNA expression levels in human colonic and terminal ileum segments. Conclusion In this first-reported real-world experience, the AhR-agonist CurQD was effective in inducing and maintaining clinical and biomarker remission in CD patients, including in biologic-experienced patients

Single-cell transcriptomics of human organoid-derived enteroendocrine cell populations from the small intestine.

Gut hormones control intestinal function, metabolism and appetite, and have been harnessed therapeutically to treat type 2 diabetes and obesity. Our understanding of the enteroendocrine axis arises largely from animal studies, but intestinal organoid models make it possible to identify, genetically modify and purify human enteroendocrine cells (EECs). This study aimed to map human EECs using single-cell RNA sequencing. Organoids derived from human duodenum and ileum were genetically modified using CRISPR-Cas9 to express the fluorescent protein Venus driven by the chromogranin-A promoter. Fluorescent cells from CHGA-Venus organoids were purified by flow cytometry and analysed by 10X single-cell RNA sequencing. Cluster analysis separated EEC populations, allowing an examination of differentially expressed hormones, nutrient-sensing machinery, transcription factors and exocytotic machinery. Bile acid receptor GPBAR1 was most highly expressed in L-cells (producing glucagon-like peptide 1 and peptide YY), long-chain fatty acid receptor FFAR1 was highest in I-cells (cholecystokinin), K-cells (glucose-dependent insulinotropic polypeptide) and L-cells, short-chain fatty acid receptor FFAR2 was highest in ileal L-cells and enterochromaffin cells, olfactory receptor OR51E1 was notably expressed in ileal enterochromaffin cells, and the glucose-sensing sodium glucose cotransporter SLC5A1 was highly and differentially expressed in K- and L-cells, reflecting their known responsiveness to ingested glucose. The organoid EEC atlas was merged with published data from human intestine and organoids, with good overlap between enteroendocrine datasets. Understanding the similarities and differences between human EEC types will facilitate the development of drugs targeting the enteroendocrine axis for the treatment of conditions such as diabetes, obesity and intestinal disorders. KEY POINTS: Gut hormones regulate intestinal function, nutrient homeostasis and metabolism and form the basis of the new classes of drugs for obesity and diabetes. As enteroendocrine cells (EECs) comprise only ∼1% of the intestinal epithelium, they are under-represented in current single-cell atlases. To identify, compare and characterise human EECs we generated chromogranin-A labelled organoids from duodenal and ileal biopsies by CRISPR-Cas9. Fluorescent chromogranin-A positive EECs were purified and analysed by single-cell RNA sequencing, revealing predominant cell clusters producing different gut hormones. Cell clusters exhibited differential expression of nutrient-sensing machinery including bile acid receptors, long- and short-chain fatty acid receptors and glucose transporters. Organoid-derived EECs mapped well onto data from native intestinal cell populations, extending coverage of EECs.

PPARα affects hepatic lipid homeostasis by perturbing necroptosis signals in the intestinal epithelium

Rapid turnover of the intestinal epithelium is a critical strategy to balance the uptake of nutrients and defend against environmental insults, whereas inappropriate death promotes the spread of inflammation. PPARα is highly expressed in the small intestine and regulates the absorption of dietary lipids. However, as a key mediator of inflammation, the impact of intestinal PPARα signaling on cell death pathways is unknown. Here, we show that Pparα deficiency of intestinal epithelium up-regulates necroptosis signals, disrupts the gut vascular barrier, and promotes LPS translocation into the liver. Intestinal Pparα deficiency drives age-related hepatic steatosis and aggravates hepatic fibrosis induced by a high-fat plus high-sucrose diet (HFHS). PPARα levels correlate with TRIM38 and MLKL in the human ileum. Inhibition of PPARα up-regulates necroptosis signals in the intestinal organoids triggered by TNF-α and LPS stimuli via TRIM38/TRIF and CREB3L3/MLKL pathways. Butyric acid ameliorates hepatic steatosis induced by intestinal Pparα deficiency through the inhibition of necroptosis. Our data suggest that intestinal PPARα is essential for the maintenance of microenvironmental homeostasis and the spread of inflammation via the gut–liver axis.

Diet shapes the metabolite profile in the intact human ileum, which affects PYY release.

The human ileum contains a high density of enteroendocrine L-cells, which release the appetite-suppressing hormones glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY) in response to food intake. Recent evidence highlighted the potential role of food structures in PYY release, but the link between food structures, ileal metabolites, and appetite hormone release remains unclear owing to limited access to intact human ileum. In a randomized crossover trial (ISRCTN11327221; isrctn.com), we investigated the role of human ileum in GLP-1 and PYY release by giving healthy volunteers diets differing in fiber and food structure: high-fiber (intact or disrupted food structures) or low-fiber disrupted food structures. We used nasoenteric tubes to sample chyme from the intact distal ileum lumina of humans in the fasted state and every 60 min for 480 min postprandially. We demonstrate the highly dynamic, wide-ranging molecular environment of the ileum over time, with a substantial decrease in ileum bacterial numbers and bacterial metabolites after food intake. We also show that high-fiber diets, independent of food structure, increased PYY release compared with a low-fiber diet during 0 to 240 min postprandially. High-fiber diets also increased ileal stachyose, and a disrupted high-fiber diet increased certain ileal amino acids. Treatment of human ileal organoids with ileal fluids or an amino acid and stachyose mixture stimulated PYY expression in a similar profile to blood PYY concentrations, confirming the role of ileal metabolites in PYY release. Our study demonstrates the diet-induced changes over time in the metabolite environment of intact human ileum, which play a role in PYY release.

Expression of Concern: 'Ileibacterium massiliense' gen. nov., sp. nov., a new bacterial species isolated from human ileum of a patient with Crohn disease

Expression of Concern: 'Ileibacterium massiliense' gen. nov., sp. nov., a new bacterial species isolated from human ileum of a patient with Crohn disease

Single nuclear RNA sequencing of terminal ileum in patients with cirrhosis demonstrates multi-faceted alterations in the intestinal barrier

Patients with cirrhosis have intestinal barrier dysfunction but the role of the individual cell types in human small intestine is unclear. We performed single-nuclear RNA sequencing (snRNAseq) in the pinch biopsies of terminal ileum of four age-matched men [56 years, healthy control, compensated, early (ascites and lactulose use) and advanced decompensated cirrhosis (ascites and rifaximin use)]. Cell type proportions, differential gene expressions, cell-type specific pathway analysis using IPA, and cellular crosstalk dynamics were compared. Stem cells, enterocytes and Paneth cells were lowest in advanced decompensation. Immune cells like naive CD4 + T cells were lowest while ITGAE + cells were highest in advanced decompensation patients. MECOM had lowest expression in stem cells in advanced decompensation. Defensin and mucin sulfation gene (PAPSS2) which can stabilize the mucus barrier expression were lowest while IL1, IL6 and TNF-related genes were significantly upregulated in the enterocytes, goblet, and Paneth cells in decompensated subjects. IPA analysis showed higher inflammatory pathways in enterocytes, stem, goblet, and Paneth cells in decompensated patients. Cellular crosstalk analysis showed that desmosome, protease-activated receptors, and cadherin-catenin complex interactions were most perturbed in decompensated patients. In summary, the snRNAseq of the human terminal ileum in 4 subjects (1 control and three cirrhosis) identified multidimensional alteration in the intestinal barrier with lower stem cells and altered gene expression focused on inflammation, mucin sulfation and cell–cell interactions with cirrhosis decompensation.

Low-dose daily folic acid (400 μg) supplementation does not affect regulation of folate transporters found present throughout the terminal ileum and colon of humans: a randomized clinical trial

BackgroundFolic acid supplementation during the periconceptional period reduces the risk of neural tube defects in infants, but concern over chronic folic acid exposure remains. An improved understanding of folate absorption may clarify potential risks. Folate transporters have been characterized in the small intestine, but less so in the colon of healthy, free-living humans. The impact of folic acid fortification or supplementation on regulation of these transporters along the intestinal tract is unknown. ObjectiveThe objective was to characterize expression of folate transporters/receptor (FT/R) and folate hydrolase, glutamate carboxypeptidase II (GCPII), from the terminal ileum and throughout the colon of adults and assess the impact of supplemental folic acid. MethodsIn this 16-wk open-labeled randomized clinical trial, adults consumed a low folic acid-containing diet, a folate-free multivitamin, and either a 400 μg folic acid supplement or no folic acid supplement. Dietary intakes and blood were assessed at baseline, 8 wk, and 16 wk (time of colonoscopy). Messenger RNA (mRNA) expression and protein expression of FT/R and GCPII were assessed in the terminal ileum, cecum, and ascending and descending colon. ResultsAmong 24 randomly assigned subjects, no differences in dietary folate intake or blood folate were observed at baseline. Mean ± SD red blood cell folate at 16 wk was 1765 ± 426 and 911 ± 242 nmol/L in the 400 and 0 μg folic acid group, respectively (P < 0.0001). Reduced folate carrier, proton-coupled folate transporter, and folate-receptor alpha expression were detected in the terminal ileum and colon, as were efflux transporters of breast cancer resistance protein and multidrug resistance protein-3. Other than a higher mRNA expression of FR-alpha and GCPII in the 400 μg supplement group in the ascending colon, no treatment differences were observed (P < 0.02). ConclusionsFolate transporters are present throughout the terminal ileum and colon; there is little evidence that a low dose of folic acid supplementation affects colonic absorption.This trial was registered at clinicaltrials.gov as NCT03421483.

Not Only Length Matters! Impact of the Ileal Width on the Capacity of the Orthotopic Neobladder: The AADAPT Formula Tested on the Animal Model

BackgroundThe capacity of a given shape of an orthotopic ileal neobladder (ONB) varies significantly, although the same length of preterminal ileum is utilised. ObjectiveTo investigate the variability of the human ileal width and to create a mathematical formula that calculates its impact on the neobladder capacity. Design, setting, and participantsDuring 50 consecutive cases of robotic pelvic surgery, a segment of preterminal ileum was identified and the width was measured. A mathematical formula was created to calculate, for a given ileal length and width, the neobladder capacity and, for a given ileal width and neobladder capacity, the length of the (pre)terminal ileum to harvest. The accuracy of our model was tested on 28 pouches created by swine ileum. Outcome measurements and statistical analysisThe interindividual variability of the ileal width and its impact on the ileal neobladder capacity was investigated. Results and limitationsThe mean hemicircumference of the human distal ileum is 2.43 ± 0.39 cm (range 2–3.5 cm). According to our geometric model and as confirmed in the swine model, an increase of 1 cm in ileal width increases the neobladder capacity by 85%. The Pearson correlation coefficient reported a strong positive relationship between the formula-calculated and effective volumes of the pouch (r = 0.97). Moreover, for the same target capacity, 1 cm of difference in the ileal width implies harvesting 20 cm less ileum. A lack of testing on humans and application only to spheroidal neobladders are the main limits. ConclusionsThe ileal width impacts the capacity of the ONB. For a given type of ONB, no standard length of ileum should be harvested; instead, the length should be tailored to the width of the ileum for a given patient. Clinical studies are required to confirm our model. Patient summaryWe demonstrated the variability of the ileal width among humans, and we provided a mathematical formula tested on swine that evaluates the impact of the ileal width on the capacity of the orthotopic ileal neobladder.

Aryl hydrocarbon receptor utilises cellular zinc signals to maintain the gut epithelial barrier

Zinc and plant-derived ligands of the aryl hydrocarbon receptor (AHR) are dietary components affecting intestinal epithelial barrier function. Here, we explore whether zinc and the AHR pathway are linked. We show that dietary supplementation with an AHR pre-ligand offers protection against inflammatory bowel disease in a mouse model while protection fails in mice lacking AHR in the intestinal epithelium. AHR agonist treatment is also ineffective in mice fed zinc depleted diet. In human ileum organoids and Caco-2 cells, AHR activation increases total cellular zinc and cytosolic free Zn2+ concentrations through transcription of genes for zinc importers. Tight junction proteins are upregulated through zinc inhibition of nuclear factor kappa-light-chain-enhancer and calpain activity. Our data show that AHR activation by plant-derived dietary ligands improves gut barrier function at least partly via zinc-dependent cellular pathways, suggesting that combined dietary supplementation with AHR ligands and zinc might be effective in preventing inflammatory gut disorders.

NAMPT inhibition relieves intestinal inflammation by regulating macrophage activation in experimental necrotizing enterocolitis

Nicotinamide phosphoribosyl transferase (NAMPT) is associated with various NAD+ -consuming enzymatic reactions. The precise role in intestinal mucosal immunity in necrotizing enterocolitis (NEC) is not well defined. Here, we examined whether NAMPT inhibition by the highly specific inhibitor FK866 could alleviate intestinal inflammation during the pathogenesis of NEC. In the present study, we showed that NAMPT expression was upregulated in the human terminal ileum of human infants with NEC. FK866 administration attenuated M1 macrophage polarization and relieved the symptoms of experimental NEC pups. FK866 inhibited intercellular NAD+ levels, macrophage M1 polarization, and the expression of NAD+ -dependent enzymes, such as poly (ADP ribose) polymerase 1 (PARP1) and Sirt6. Consistently, the capacity of macrophages to phagocytose zymosan particles, as well as antibacterial activity, were impaired by FK866, whereas NMN supplementation to restore NAD+ levels reversed the changes in phagocytosis and antibacterial activity. In conclusion, FK866 reduced intestinal macrophage infiltration and skewed macrophage polarization, which is implicated in intestinal mucosal immunity, thereby promoting the survival of NEC pups.

The orphan MRGPRF receptor is expressed in entero-endocrine cells of the human gut mucosa.

In the past years, it has become clear that the family of Mas-related G protein-coupled receptors plays a central role in neuro-immune communication at mucosal barrier surfaces, in particular in the skin. Remarkably, MRGPR expression at other mucosal surfaces remains poorly characterized. To fill this gap in our understanding, the present study was undertaken to screen and verify the expression of the human MRGPR family members in the mucosal biopsies of the human gastrointestinal (GI) tract. Our findings revealed that, of all human MRGPRs family members, only MRGPRF mRNA is expressed at detectable levels in human mucosal biopsies of both terminal ileum and sigmoid colon. Furthermore, immunohistochemical stainings revealed that MRGPRF is specifically expressed by mucosal entero-endocrine cells (EECs). Overall, this study showed for the first time that the human ileum and colonic mucosa represent a novel expression site for the orphan MRGPRF, more specifically in EECs.

Prokinetic Activity of Mulberry Fruit, Morus alba L.

The fruit of Morus alba L. (MAF) has been consumed as a food worldwide. MAF has also been widely used in traditional medicine for thousands of years in East Asia, and its diverse bioactivities have been reported in numerous publications. However, no prokinetic activity has been reported for MAF or its components. In the present study, therefore, we investigated the effects of MAF on gastrointestinal motor function by measuring the intestinal transit rate (ITR) of Evans blue in mice in vivo. The ITR values accelerated by MAF were significantly higher than those accelerated by cisapride or metoclopramide, suggesting that MAF has potential as a new prokinetic agent to replace cisapride and metoclopramide. We also investigated the effects of MAF on myogenic and neurogenic contractions in human intestinal smooth muscles by measuring spontaneous contractions of smooth muscle strips, smooth muscle contractions induced by neural stimulation, and migrating motor complexes from intestinal segments in the human ileum and sigmoid colon in situ. MAF increased both myogenic and neurogenic contractions to enhance ileal and colonic motility in the human intestine. Taken together, these results indicate that MAF enhanced intestinal motility by increasing both myogenic and neurogenic contractions, thereby accelerating the ITR.

Intestinal single-cell atlas reveals novel lymphocytes in pigs with similarities to human cells.

Lymphocytes can heavily influence intestinal health, but resolving intestinal lymphocyte function is challenging as the intestine contains a vastly heterogeneous mixture of cells. Pigs are an advantageous biomedical model, but deeper understanding of intestinal lymphocytes is warranted to improve model utility. Twenty-six cell types were identified in the porcine ileum by single-cell RNA sequencing and further compared with cells in human and murine ileum. Though general consensus of cell subsets across species was revealed, some porcine-specific lymphocyte subsets were identified. Differential tissue dissection and in situ analyses conferred spatial context, revealing similar locations of lymphocyte subsets in Peyer's patches and epithelium in pig-to-human comparisons. Like humans, activated and effector lymphocytes were abundant in the ileum but not periphery of pigs, suggesting tissue-specific and/or activation-associated gene expression. Gene signatures for peripheral and ileal innate lymphoid cells newly discovered in pigs were defined and highlighted similarities to human innate lymphoid cells. Overall, we reveal novel lymphocyte subsets in pigs and highlight utility of pigs for intestinal research applications.

Structure of the myenteric plexus in normal and diseased human ileum analyzed by X-ray virtual histology slices.

BACKGROUNDThe enteric nervous system (ENS) is situated along the entire gastrointestinal tract and is divided into myenteric and submucosal plexuses in the small and large intestines. The ENS consists of neurons, glial cells, and nerves assembled into ganglia, surrounded by telocytes, interstitial cells of Cajal, and connective tissue. Owing to the complex spatial organization of several interconnections with nerve fascicles, the ENS is difficult to examine in conventional histological sections of 3-5 μm.AIMTo examine human ileum full-thickness biopsies using X-ray phase-contrast nanotomography without prior staining to visualize the ENS.METHODSSix patients were diagnosed with gastrointestinal dysmotility and neuropathy based on routine clinical and histopathological examinations. As controls, full-thickness biopsies were collected from healthy resection ileal regions after hemicolectomy for right colon malignancy. From the paraffin blocks, 4-µm thick sections were prepared and stained with hematoxylin and eosin for localization of the myenteric ganglia under a light microscope. A 1-mm punch biopsy (up to 1 cm in length) centered on the myenteric plexus was taken and placed into a Kapton® tube for mounting in the subsequent investigation. X-ray phase-contrast tomography was performed using two custom-designed laboratory setups with micrometer resolution for overview scanning. Subsequently, selected regions of interest were scanned at a synchrotron-based end-station, and high-resolution slices were reported. In total, more than 6000 virtual slices were analyzed from nine samples.RESULTSIn the overview scans, the general architecture and quality of the samples were studied, and the myenteric plexus was localized. High-resolution scans revealed details, including the ganglia, interganglional nerve fascicles, and surrounding tissue. The ganglia were irregular in shape and contained neurons and glial cells. Spindle-shaped cells with very thin cellular projections could be observed on the surface of the ganglia, which appeared to build a network. In the patients, there were no alterations in the general architecture of the myenteric ganglia. Nevertheless, several pathological changes were observed, including vacuolar degeneration, autophagic activity, the appearance of sequestosomes, chromatolysis, and apoptosis. Furthermore, possible expulsion of pyknotic neurons and defects in the covering cellular network could be observed in serial slices. These changes partly corresponded to previous light microscopy findings.CONCLUSIONThe analysis of serial virtual slices could provide new information that cannot be obtained by classical light microscopy. The advantages, disadvantages, and future possibilities of this method are also discussed.

M cells of mouse and human Peyer's patches mediate the lymphatic absorption of an Astragalus hyperbranched heteroglycan

The gut cell wall is considered an impenetrable barrier to orally administrated polysaccharides. We recently reported a selective lymphatic route for Radix Astragali polysaccharide RAP to enter Peyer's patches (PPs) to trigger immune responses. However, how RAP enters PPs is unclear. Herein, we screened the intestinal epithelial cells of mice and found that the follicle-associated epithelium cells were specifically bound with FITC-RAP. Further studies in vitro and in vivo revealed that RAP was efficiently transported by microfold (M) cells. We also confirmed that M cell-transported RAP directly contacted dendritic cells. More importantly, for the first time, we verified this interesting M cell-mediated transcytosis of RAP in the human distal ileum. Mechanistically, we identified M cells to be the transporter cells that independently deliver RAP into the lymphatic system to trigger immune responses. This interesting transcytosis mechanism might apply to many other immunomodulatory polysaccharides orally dosed to human body.

Cross-species single-cell transcriptomic analysis reveals divergence of cell composition and functions in mammalian ileum epithelium.

Animal models are widely used for biomedical studies and drug evaluation. The small intestine plays key roles in nutrient absorption, hormone secretion, microbiota defense and drug absorption and metabolism. Although the intestinal structure of mammals is conserved, the differences on epithelial cell composition, functional assignments and drug absorption among mammals are largely unknown. Here, cross-species analysis of single-cell transcriptomic atlas of the ileum epithelium from mouse, rat, pig, macaque and human reveals the conserved and differential cell types and functions among species, identifies a new CA7+ cell type in pig, macaque and human ileum, uncovers the distinct expression pattern in enterocytes, enteroendocrine cells and Paneth cells, and defines the conserved and species-specific intestinal stem cell signature genes. The examination of drug absorption across species suggests that drug metabolism in mouse ileum is closer to human while drug transport in macaque ileum is more similar to human. Together, our data provide the comprehensive information about cell composition and functional assignments in five species, and offer the valuable guidance for animal model selection and drug testing.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13619-022-00118-7.

Elevated 5‐hydroxytryptamine in COVID‐19 Stimulates ANO1 Mediated Cl Secretion in Lung &amp; 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.

Coronin 1A is Uniquely Expressed in the Human Follicle Associated Epithelium and is Required For Human M Cell Maturation and Function

IntroductionMicrofold (M) cells are specialized epithelial cells that play an integral role in immune surveillance of the gut luminal environment. They functionally act as gatekeepers within the follicle‐associated epithelium (FAE) of Peyer’s patches to regulate antigen delivery to immune cells of gut‐associated lymphoid tissue. Studies have associated damage in the FAE with chronic inflammatory conditions, such as IBD; however, studies designed to understand how human M cells contribute to disease onset and/or pathogenesis under pathogenic conditions remain incomplete. OBJECTIVE: Since M cells are differentiated from Lgr5+ intestinal stem cells, we proposed to utilize human enteroids to determine the factors that regulate human M cell differentiation and function. Quantitative proteomics analysis of human enteroids differentiated to express M cells revealed enriched expression of coronin 1A, an actin‐remodeling protein involved in phagocytosis and expressed immune cells. Since M cells engulf luminal gut antigens via phagocytosis, we hypothesized that coronin 1A is uniquely expressed in the human FAE and plays a role in gut antigen uptake and transcytosis in human M cells.MethodsHuman ileal enteroids derived from biopsies obtained from healthy donors were grown as monolayers and differentiated (no Wnt3a, R‐spondin‐1) for at least 5 days. To express M cells, cultures were exposed to RANKL and TNF during differentiation. Human expression of coronin 1A was validated by immunoblot and confocal microscopy of ileal biopsies obtained from healthy donors. Stable knockdown (KD) of coronin 1A in human enteroids was achieved by shRNA lentivirus and puromycin selection. M cell monolayers, expressing or lacking coronin 1A, were exposed apically to commensal (HS) and pathogenic strains of E. coli (EPEC, AIEC) to determine the contribution of gut microbes to stimulate M cell transcytosis, as measured by uptake of fluorescent polystyrene latex beads and immunoglobulins.ResultsCoronin 1A was expressed in human enteroid monolayers differentiated to express M cells, but not in monolayers differentiated to mimic the villus epithelium. Similar results were obtained in biopsy sections of the human ileum with coronin 1A detected in the FAE, but not in crypt or villus epithelia. This staining pattern was unique for human, as coronin 1A expression in the mouse ileum was confined to lamina propria and lymphoid follicle immune cells. Coronin 1A KD resulted in decreased expression of glycoprotein 2, a marker of mature M cells, while other markers of the FAE and M cells, including CD14, ICAM1, Spi‐B, SOX8, and FcRN remained unchanged. Coronin 1A KD also resulted in impaired uptake/transcytosis of IgG (via FcRN) as well as pathogen‐stimulated uptake/transcytosis of polystyrene beads by human M cells.ConclusionsThe studies show: (1) human enteroids can be differentiated to model coronin 1A expression in the intact human FAE; (2) coronin 1A expression is necessary for terminal differentiation and function of human M cells; and (3) coronin 1A is required for pathogen‐stimulated uptake and transcytosis of various types of luminal gut cargo. We conclude that M cell‐expressing enteroid monolayers can be used as a relevant human model to further interrogate tropism for host‐pathogen interactions, mechanisms of luminal surveillance, and response to oral vaccine therapies.

Eicosatetraynoic Acid and Butyrate Regulate Human Intestinal Organoid Mitochondrial and Extracellular Matrix Pathways Implicated in Crohn's Disease Strictures.

Perturbagen analysis of Crohn's disease (CD) ileal gene expression data identified small molecules including eicosatetraynoic acid (ETYA), which may exert an antifibrotic effect. We developed a patient-specific human intestinal organoid (HIO) model system to test small molecule regulation of mitochondrial and wound-healing functions implicated in stricturing behavior. HIOs were made from CD induced pluripotent stem cells with and without a loss-of-function haplotype in the DUOX2 gene implicated in ileal homeostasis and characterized under basal conditions and following exposure to butyrate and ETYA using RNA sequencing, flow cytometry, and immunofluorescent and polarized light microscopy. Mitochondrial activity was measured using high-resolution respirometry and tissue stiffness using atomic force microscopy. HIOs expressed core mitochondrial and extracellular matrix (ECM) genes and enriched biologic functions implicated in CD ileal strictures; ECM gene expression was suppressed by both butyrate and ETYA, with butyrate also suppressing genes regulating epithelial proliferation. Consistent with this, butyrate, but not ETYA, exerted a profound effect on HIO epithelial mitochondrial function, reactive oxygen species production, and cellular abundance. Butyrate and ETYA suppressed HIO expression of alpha smooth muscle actin expressed by myofibroblasts, type I collagen, and collagen protein abundance. HIOs exhibited tissue stiffness comparable to normal human ileum; this was reduced by chronic ETYA exposure in HIOs carrying the DUOX2 loss-of-function haplotype. ETYA regulates ECM genes implicated in strictures and suppresses collagen content and tissue stiffness in an HIO model. HIOs provide a platform to test personalized therapeutics, including small molecules prioritized by perturbagen analysis.

EICOSATETRAYNOIC ACID REGULATES MITOCHONDRIAL AND EXTRA-CELLULAR MATRIX GENE EXPRESSION AND TISSUE STIFFNESS IN A HUMAN INTESTINAL ORGANOID MODEL OF ILEAL CROHN’S DISEASE

Abstract BACKGROUND Mutations in the DUOX2 intestinal epithelial cell NADPH oxidase are associated with risk for Crohn’s Disease (CD), and may influence wound healing and the development of strictures. Patients who develop strictures exhibit reduced expression of genes encoding mitochondrial sub-units, and increased expression of genes encoding extra-cellular matrix proteins, in the ileum at diagnosis. We conducted a perturbagen bioinformatics analysis which predicted that the cyclooxygenase and lipoxygenase inhibitor eicosatetraynoic-acid (ETYA) would reverse the ileal gene signature associated with stricture development. In the current study we tested effects of ETYA in a human intestinal organoid model (HIO) system. METHODS HIO were derived from wild type (WT) and DUOX2 mutant (DUOX2var) induced pluripotent stem cells (iPSCs) prepared from CD patients. WT and DUOX2var HIO were examined under basal conditions, and following exposure to ETYA for 72 hours or 12 days. Superoxide production in EPCAM+ epithelial cells and CD90+ stromal cells was measured by flow cytometry. RNA was prepared and expression of mitochondrial and extra-cellular matrix genes linked to strictures in patients was determined using RNA sequencing and a TaqMan Low Density Array (TLDA) card. HIO tissue stiffness was measured using atomic force microscopy (AFM). RESULTS ETYA reduced superoxide production by HIO EPCAM+ epithelial cells only in WT organoids; no effect was observed in DUOX2var HIO. This was specific, as no effect of ETYA upon superoxide production was detected in CD90+ stromal cells. Under basal conditions, RNA sequencing demonstrated that WT HIO expressed core mitochondrial and extra-cellular matrix genes and enriched biologic functions implicated in CD strictures. Expression of extra-cellular matrix and wound healing genes was increased in DUOX2var HIO under basal conditions. ETYA up-regulated expression of the COX5B, POLG2, and SLC25A27 mitochondrial genes associated with lower rates of strictures, while reducing expression of the ACTA2, VIM, and COL1A1 extra-cellular matrix genes associated with higher rates of strictures, independent of DUOX2 genotype. WT and DUOX2var HIO exhibited tissue stiffness comparable to normal human ileum under basal conditions; this was significantly reduced by ETYA exposure only in DUOX2var HIO. CONCLUSIONS ETYA regulates mitochondrial and extra-cellular matrix genes implicated in stricture formation in an HIO model system. DUOX2var HIO exhibit increased extra-cellular matrix gene expression under basal conditions, which is reduced by ETYA exposure in conjunction with a reduction in tissue stiffness. Collectively, these data confirm that HIO provide a relevant model system to study mechanisms regulating stricture formation in CD, including screening of small molecules prioritized by perturbagen bioinformatics analysis.