Human Intestinal Enteroids: New Models to Study Gastrointestinal Virus Infections

Zou, Winnie Y.; Blutt, Sarah E.; Crawford, Sue E.; Ettayebi, Khalil; Zeng, Xi; Saxena, Kapil; Ramani, Sasirekha; Karandikar, Umesh C.; Zachos, Nicholas C.; Estes, Mary K.

doi:10.1007/7651_2017_1

Cited by 124 publications

(127 citation statements)

References 23 publications

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“…Lentiviral transduction of HIEs. A cell suspension was prepared from three-dimensional (3D) undifferentiated jejunal HIEs cultivated as previously described (2,33). After trypsinization and pelleting of the cells at 300 ϫ g, the resulting cell pellet was suspended at a concentration of 3 ϫ 10 5 cells per ml of concentrated lentivirus supplemented with 10 M Rho-associated protein kinase (ROCK) inhibitor Y-27632 (catalog no.…”

Section: Methodsmentioning

confidence: 99%

Genetic Manipulation of Human Intestinal Enteroids Demonstrates the Necessity of a Functional Fucosyltransferase 2 Gene for Secretor-Dependent Human Norovirus Infection

Haga

Ettayebi

Tenge

et al. 2020

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Human noroviruses (HuNoVs) are the leading cause of nonbacterial gastroenteritis worldwide. Histo-blood group antigen (HBGA) expression is an important susceptibility factor for HuNoV infection based on controlled human infection models and epidemiologic studies that show an association of secretor status with infection caused by several genotypes. The fucosyltransferase 2 gene (FUT2) affects HBGA expression in intestinal epithelial cells; secretors express a functional FUT2 enzyme, while nonsecretors lack this enzyme and are highly resistant to infection and gastroenteritis caused by many HuNoV strains. These epidemiologic associations are confirmed by infections in stem cell-derived human intestinal enteroid (HIE) cultures. GII.4 HuNoV does not replicate in HIE cultures derived from nonsecretor individuals, while HIEs from secretors are permissive to infection. However, whether FUT2 expression alone is critical for infection remains unproven, since routinely used secretor-positive transformed cell lines are resistant to HuNoV replication. To evaluate the role of FUT2 in HuNoV replication, we used CRISPR or overexpression to genetically manipulate FUT2 gene function to produce isogenic HIE lines with or without FUT2 expression. We show that FUT2 expression alone affects both HuNoV binding to the HIE cell surface and susceptibility to HuNoV infection. These findings indicate that initial binding to a molecule(s) glycosylated by FUT2 is critical for HuNoV infection and that the HuNoV receptor is present in nonsecretor HIEs. In addition to HuNoV studies, these isogenic HIE lines will be useful tools to study other enteric microbes where infection and/or disease outcome is associated with secretor status.IMPORTANCE Several studies have demonstrated that secretor status is associated with susceptibility to human norovirus (HuNoV) infection; however, previous reports found that FUT2 expression is not sufficient to allow infection with HuNoV in a variety of continuous laboratory cell lines. Which cellular factor(s) regulates susceptibility to HuNoV infection remains unknown. We used genetic manipulation of HIE cultures to show that secretor status determined by FUT2 gene expression is necessary and sufficient to support HuNoV replication based on analyses of isogenic lines that lack or express FUT2. Fucosylation of HBGAs is critical for initial binding and for modification of another putative receptor(s) in HIEs needed for virus uptake or uncoating and necessary for successful infection by GI.1 and several GII HuNoV strains.

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Section: Methodsmentioning

confidence: 99%

Genetic Manipulation of Human Intestinal Enteroids Demonstrates the Necessity of a Functional Fucosyltransferase 2 Gene for Secretor-Dependent Human Norovirus Infection

Haga

Ettayebi

Tenge

et al. 2020

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“…Colonoid cultures were maintained via passaging once per week. To generate 2D monolayers, colonoids grown as described above were spun out of matrigel and dissociated into a single cell suspension using Trypsin/EDTA as described previously . Following one day of culture in complete growth media, cells were switched to differentiation media (growth media minus Wnt3a, R‐Spondin and with a 50% reduction in Noggin) for 4‐5 days of differentiation into monolayers of differentiated colonic epithelium.…”

Section: Methodsmentioning

confidence: 99%

Regulation of neutrophil function by selective targeting of glycan epitopes expressed on the integrin CD11b/CD18

et al. 2019

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Polymorphonuclear neutrophils (PMNs) play a critical role in the innate immune response to invading pathogens. However, dysregulated mucosal trafficking of PMNs and associated epithelial tissue damage is a pathological hallmark of numerous inflammatory conditions including inflammatory bowel disease. The glycoprotein CD11b/CD18 plays a well‐described role in regulating PMN transepithelial migration and PMN inflammatory functions. Previous studies have demonstrated that targeting of the N‐linked glycan Lewis X on CD11b blocks PMN transepithelial migration (TEpM). Given evidence of glycosylation‐dependent regulation of CD11b/CD18 function, we performed MALDI TOF Mass Spectrometry (MS) analyses on CD11b/CD18 purified from human PMNs. Unusual glycan epitopes identified on CD11b/CD18 included high Mannose oligosaccharides recognized by the Galanthus Nivalis lectin and biantennary galactosylated N‐glycans recognized by the Phaseolus Vulgaris erythroagglutinin lectin. Importantly, we show that selective targeting of glycans on CD11b with such lectins results in altered intracellular signaling events that inhibit TEpM and differentially affect key PMN inflammatory functions including phagocytosis, superoxide release and apoptosis. Taken together, these data demonstrate that discrete glycan motifs expressed on CD11b/CD18 such as biantennary galactose could represent novel targets for selective manipulation of CD11b function and reduction of PMN‐associated tissue damage in chronic inflammatory diseases.

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“…The HIE system exhibits multiple advantages over traditional transformed intestinal epithelial cell lines (e.g., Caco-2 cells): 1. HIE only contain non-transformed human cells [10]; 2. they contain multiple cell types [13]; 3. they maintain the intestinal region-specific differences in culture [14]; 4. they can be differentiated into cell populations located in the villus or crypt regions [15]; and 5. HIE maintain the genetic identity of the host [10].…”

Section: Introductionmentioning

confidence: 99%

Astrovirus replication in human intestinal enteroids reveals multi-cellular tropism and an intricate host innate immune landscape

et al. 2019

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Human astroviruses (HAstV) are understudied positive-strand RNA viruses that cause gastroenteritis mostly in children and the elderly. Three clades of astroviruses, classic, MLBtype and VA-type have been reported in humans. One limitation towards a better understanding of these viruses has been the lack of a physiologically relevant cell culture model that supports growth of all clades of HAstV. Herein, we demonstrate infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts. A detailed investigation of infection of VA1, a member of the non-canonical HAstV-VA/HMO clade, showed robust replication in HIE derived from different patients and from different intestinal regions independent of the cellular differentiation status. Flow cytometry and immunofluorescence analysis revealed that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes, in HIE cultures. RNA profiling of VA1-infected HIE uncovered that the host response to infection is dominated by interferon (IFN)-mediated innate immune responses. A comparison of the antiviral host response in non-transformed HIE and transformed human colon carcinoma Caco-2 cells highlighted significant differences between these cells, including an increased magnitude of the response in HIE. Additional studies confirmed the sensitivity of VA1 to exogenous IFNs, and indicated that the endogenous IFN response of HIE to curtail the growth of strains from all three clades. Genotypic variation in the permissiveness of different HIE lines to HAstV could be overcome by pharmacologic inhibition of JAK/STAT PLOS Pathogens | https://doi.and ALSAC to SSC.; NIH R21 NS101371 to DW; Wellcome Trust: Ref 207498/Z/ signaling. Collectively, our data identify HIE as a universal infection model for HAstV and an improved model of the intestinal epithelium to investigate enteric virus-host interactions. Author summaryHuman astroviruses (HAstV) are understudied positive-strand RNA viruses that typically cause gastroenteritis mostly in children and the elderly, but more recent studies also implicate them in neurological disease in immunocompromised patients. To better understand these viruses, a physiologically relevant cell culture model that supports growth of all clades of HAstV would be highly beneficial. Herein, we demonstrated robust infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts from different patients and intestinal regions, making HIE a valuable model to study HAstV biology. Using this system, we identify for the first time that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes. Analysis of the antiviral host response to infection demonstrated that HIE respond to infection with a type I and III interferon response. This response reduced HAstV replication and when blocked resulted in increased infection. Establis...

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Human Intestinal Enteroids: New Models to Study Gastrointestinal Virus Infections

Cited by 124 publications

References 23 publications

Genetic Manipulation of Human Intestinal Enteroids Demonstrates the Necessity of a Functional Fucosyltransferase 2 Gene for Secretor-Dependent Human Norovirus Infection

Genetic Manipulation of Human Intestinal Enteroids Demonstrates the Necessity of a Functional Fucosyltransferase 2 Gene for Secretor-Dependent Human Norovirus Infection

Regulation of neutrophil function by selective targeting of glycan epitopes expressed on the integrin CD11b/CD18

Astrovirus replication in human intestinal enteroids reveals multi-cellular tropism and an intricate host innate immune landscape

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