Human Intestinal Enteroids: a New Model To Study Human Rotavirus Infection, Host Restriction, and Pathophysiology - PubMed (original) (raw)

. 2015 Oct 7;90(1):43-56.

doi: 10.1128/JVI.01930-15. Print 2016 Jan 1.

Sarah E Blutt 1, Khalil Ettayebi 1, Xi-Lei Zeng 1, James R Broughman 1, Sue E Crawford 1, Umesh C Karandikar 1, Narayan P Sastri 1, Margaret E Conner 1, Antone R Opekun 2, David Y Graham 3, Waqar Qureshi 2, Vadim Sherman 4, Jennifer Foulke-Abel 5, Julie In 5, Olga Kovbasnjuk 5, Nicholas C Zachos 5, Mark Donowitz 5, Mary K Estes 6

Affiliations

Human Intestinal Enteroids: a New Model To Study Human Rotavirus Infection, Host Restriction, and Pathophysiology

Kapil Saxena et al. J Virol. 2015.

Abstract

Human gastrointestinal tract research is limited by the paucity of in vitro intestinal cell models that recapitulate the cellular diversity and complex functions of human physiology and disease pathology. Human intestinal enteroid (HIE) cultures contain multiple intestinal epithelial cell types that comprise the intestinal epithelium (enterocytes and goblet, enteroendocrine, and Paneth cells) and are physiologically active based on responses to agonists. We evaluated these nontransformed, three-dimensional HIE cultures as models for pathogenic infections in the small intestine by examining whether HIEs from different regions of the small intestine from different patients are susceptible to human rotavirus (HRV) infection. Little is known about HRVs, as they generally replicate poorly in transformed cell lines, and host range restriction prevents their replication in many animal models, whereas many animal rotaviruses (ARVs) exhibit a broader host range and replicate in mice. Using HRVs, including the Rotarix RV1 vaccine strain, and ARVs, we evaluated host susceptibility, virus production, and cellular responses of HIEs. HRVs infect at higher rates and grow to higher titers than do ARVs. HRVs infect differentiated enterocytes and enteroendocrine cells, and viroplasms and lipid droplets are induced. Heterogeneity in replication was seen in HIEs from different patients. HRV infection and RV enterotoxin treatment of HIEs caused physiological lumenal expansion detected by time-lapse microscopy, recapitulating one of the hallmarks of rotavirus-induced diarrhea. These results demonstrate that HIEs are a novel pathophysiological model that will allow the study of HRV biology, including host restriction, cell type restriction, and virus-induced fluid secretion.

Importance: Our research establishes HIEs as nontransformed cell culture models to understand human intestinal physiology and pathophysiology and the epithelial response, including host restriction of gastrointestinal infections such as HRV infection. HRVs remain a major worldwide cause of diarrhea-associated morbidity and mortality in children ≤5 years of age. Current in vitro models of rotavirus infection rely primarily on the use of animal rotaviruses because HRV growth is limited in most transformed cell lines and animal models. We demonstrate that HIEs are novel, cellularly diverse, and physiologically relevant epithelial cell cultures that recapitulate in vivo properties of HRV infection. HIEs will allow the study of HRV biology, including human host-pathogen and live, attenuated vaccine interactions; host and cell type restriction; virus-induced fluid secretion; cell-cell communication within the epithelium; and the epithelial response to infection in cultures from genetically diverse individuals. Finally, drug therapies to prevent/treat diarrheal disease can be tested in these physiologically active cultures.

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Figures

FIG 1

FIG 1

Characterization of differentiated human jejunal enteroids. (A) Representative images of jejunal enteroids grown over 5 days from intestinal crypts (bar = 50 μm). (B) After 5 days of growth in complete medium with growth factors (CMGF+ medium), enteroids typically result in two major morphologies, multilobular (left) (bar = 150 μm) and cystic (right) (bar = 100 μm). (C) Upon differentiation, enteroids contain the four major mature cell types of the small intestinal epithelium. (Left) Chromogranin A-containing enteroendocrine cells (green) and sucrase-isomaltase-expressing enterocytes (arrowheads) (bar = 10 μm). (Middle) Periodic acid-Schiff stain-reacting goblet cells (purple) (bar = 20 μm). (Right) Lysozyme-containing Paneth cells (green) (bar = 10 μm). E-cadherin (red) and DAPI (4′,6-diamidino-2-phenylindole) nuclear staining (blue) are shown in the left and right panels. (D) qRT-PCR results showing the fold change in levels of transcripts in differentiated enteroids relative to the transcript levels in undifferentiated enteroids. Transcript levels were first normalized to GAPDH levels prior to obtaining the relative fold change by using the 2−ΔΔ_CT_ method. Shown are markers for enterocytes (EC), enteroendocrine cells (EE), goblet cells (GC), Paneth cells (PC), and stem cells (SC). Gene symbols represent lactase (LCT), sucrase-isomaltase (SI), alkaline phosphatase (ALPI), chromogranin A (CHGA), synaptophysin (SYP), mucin 2 (MUC2), trefoil factor 3 (TFF3), lysozyme (LYZ), defensin alpha 5 (DEFA5), antigen identified by monoclonal antibody Ki-67 (MKI67), and leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5) genes. Error bars indicate standard errors of the means (n = 3).

FIG 2

FIG 2

Human rotavirus infection and replication properties in human intestinal enteroids. (A) Jejunal enteroids from one patient (patient j11) were either mock infected or infected with RV at an MOI of 20 PFU/cell. At 20 hpi, enteroids were visualized by light microscopy (left) (bar = 50 μm) for cytopathic effect. Enteroids were also assessed for the percentage of infected cells by flow cytometry. Infected cells were defined as cells containing intracellular rotavirus antigen as detected by rabbit polyclonal antirotavirus serum. Examples of individual infection results (left) are accompanied by composite results from the experiment (right). (B) Enteroids generated from 11 different patients across the three sections of the small intestine were infected with HRV Ito at an MOI of 10 FFU/cell and assessed for the percentage of infected cells as described above for panel A. (C) A one-step growth curve for HRV Ito replication was performed over 30 h. Enteroids were infected at an MOI of 10 FFU/cell. At each of the 6 time points, enteroids and the surrounding supernatant were harvested, and the amount of infectious virus was quantified by a fluorescent-focus assay. Viral titer is displayed on the left y axis and is represented by black circles on the graph. Cytotoxicity was also measured at 2 hpi and 14 hpi and is represented as bars, with values displayed on the right y axis. (D) Jejunal enteroids from 5 different patients were infected with HRV Ito at an MOI of 0.5 FFU/cell, and the amount of infectious virus was quantified as described above for panel C at 2 hpi and 24 hpi. (E) Jejunal enteroids from one patient (patient j11) were infected with either RRV or HRV at an MOI of 0.5 FFU/cell, and the amount of infectious virus was quantified at 1.5 hpi and 24 hpi. (F) Jejunal enteroids from three patients were infected with either RV1 (Rotarix) or Wa, and the amount of infectious virus was quantified at 2 hpi and 24 hpi. In panels D to F, numbers above the bars show fold increases from 1.5 or 2 hpi (light gray bars) to 24 hpi (dark gray bars). (G) Secretor-negative enteroids from 3 different patients were infected with HRV strains Ito (left), Wa (middle), and RV1 (right) at an MOI of 0.5 FFU/cell. The amount of infectious virus at 1.5 hpi and 24 hpi was quantified as described above for panel C. Each bar represents the fold increase in viral growth from 1.5 hpi to 24 hpi. Statistical analysis was performed by using one-way ANOVA, followed by post hoc analysis with a Tukey HSD test. (H) Electron micrograph of an infected cell within an enteroid. Strain Ito particles (RV) adjacent to a lipid droplet (LD) and viroplasm (V) are shown (bar = 250 nm). Results are representative of data from duplicate (B, D, and F) or triplicate (A, C, and E) independent experiments. Each data bar represents means ± standard deviations for 3 samples within each independent experiment. Statistical analyses were performed by using Student's t test unless otherwise specified.

FIG 2

FIG 2

Human rotavirus infection and replication properties in human intestinal enteroids. (A) Jejunal enteroids from one patient (patient j11) were either mock infected or infected with RV at an MOI of 20 PFU/cell. At 20 hpi, enteroids were visualized by light microscopy (left) (bar = 50 μm) for cytopathic effect. Enteroids were also assessed for the percentage of infected cells by flow cytometry. Infected cells were defined as cells containing intracellular rotavirus antigen as detected by rabbit polyclonal antirotavirus serum. Examples of individual infection results (left) are accompanied by composite results from the experiment (right). (B) Enteroids generated from 11 different patients across the three sections of the small intestine were infected with HRV Ito at an MOI of 10 FFU/cell and assessed for the percentage of infected cells as described above for panel A. (C) A one-step growth curve for HRV Ito replication was performed over 30 h. Enteroids were infected at an MOI of 10 FFU/cell. At each of the 6 time points, enteroids and the surrounding supernatant were harvested, and the amount of infectious virus was quantified by a fluorescent-focus assay. Viral titer is displayed on the left y axis and is represented by black circles on the graph. Cytotoxicity was also measured at 2 hpi and 14 hpi and is represented as bars, with values displayed on the right y axis. (D) Jejunal enteroids from 5 different patients were infected with HRV Ito at an MOI of 0.5 FFU/cell, and the amount of infectious virus was quantified as described above for panel C at 2 hpi and 24 hpi. (E) Jejunal enteroids from one patient (patient j11) were infected with either RRV or HRV at an MOI of 0.5 FFU/cell, and the amount of infectious virus was quantified at 1.5 hpi and 24 hpi. (F) Jejunal enteroids from three patients were infected with either RV1 (Rotarix) or Wa, and the amount of infectious virus was quantified at 2 hpi and 24 hpi. In panels D to F, numbers above the bars show fold increases from 1.5 or 2 hpi (light gray bars) to 24 hpi (dark gray bars). (G) Secretor-negative enteroids from 3 different patients were infected with HRV strains Ito (left), Wa (middle), and RV1 (right) at an MOI of 0.5 FFU/cell. The amount of infectious virus at 1.5 hpi and 24 hpi was quantified as described above for panel C. Each bar represents the fold increase in viral growth from 1.5 hpi to 24 hpi. Statistical analysis was performed by using one-way ANOVA, followed by post hoc analysis with a Tukey HSD test. (H) Electron micrograph of an infected cell within an enteroid. Strain Ito particles (RV) adjacent to a lipid droplet (LD) and viroplasm (V) are shown (bar = 250 nm). Results are representative of data from duplicate (B, D, and F) or triplicate (A, C, and E) independent experiments. Each data bar represents means ± standard deviations for 3 samples within each independent experiment. Statistical analyses were performed by using Student's t test unless otherwise specified.

FIG 3

FIG 3

Effect of differentiation status on susceptibility to HRV infection. (A) Undifferentiated and differentiated jejunal enteroids from the same patient were either mock infected or infected with HRV Ito at an MOI of 10 FFU/cell. At 20 hpi, single-cell suspensions were assessed for the presence of intracellular rotavirus antigen by flow cytometry. Results are representative of data from triplicate independent experiments. Each data bar represents the mean ± standard deviation for 3 samples within each independent experiment. (B) Undifferentiated and differentiated jejunal enteroids from the same patient (patient j2) were infected with HRV Ito at an MOI of 0.5 FFU/cell, and the amount of infectious virus was quantified at 1.5 hpi (light gray bars) and 24 hpi (dark gray bars). Fold increases are displayed above the bars. Results are representative of data from replicate experiments performed with enteroids from two additional patients (patients j3 and j11). Each data bar represents the mean ± standard deviation for 4 samples within each independent experiment. Statistical analyses were performed by using Student's t test.

FIG 4

FIG 4

Assessment of differentiated cell types infected by HRV. Ileal enteroids were either mock infected or infected with HRV Ito at an MOI of 10, fixed at 10 hpi, processed for immunofluorescence staining, and visualized by using confocal microscopy. (A) Paraffin-embedded sections of mock-infected (left) and HRV-infected (right) ileal enteroids were assessed for intracellular rotavirus antigen (green), E-cadherin (red), and nuclei (blue). Infected enterocytes (arrows) are identified as E-cadherin-expressing cells containing rotavirus antigen (bar = 20 μm). (B) Enteroendocrine cell infection was assessed by visualizing rotavirus antigen-containing cells (green) (left), chromogranin A-containing cells (magenta) (middle), and the merged image (right). Staining of nuclei (blue) and E-cadherin (red) is present in all panels (bar = 10 μm).

FIG 5

FIG 5

Fluid dynamics of human intestinal enteroids in response to rotavirus. (A) Duodenal HIEs were treated with forskolin and imaged over 40 min. The white line marks the enteroid lumen diameter (bar = 50 μm). (B) Duodenal HIEs were infected with HRV Ito at an MOI of 20 PFU/cell. HIEs were imaged from 2 hpi to 12 hpi. Images from four representative time points are shown. White lines demarcate the lumen diameter (bar = 50 μm). (C) Duodenal HIEs were either mock infected or infected with HRV as described above for panel B. The lumenal radius and the total enteroid radius were measured at 2 hpi and 6 hpi for mock-infected (n = 10) and HRV-infected (n = 9) HIEs. The ratio of these two radii is shown as lumen radius/total enteroid radius at 2 hpi and at 6 hpi. Black bars represent median values. (D) Duodenal HIEs were treated with forskolin (20 μM), carbachol (200 μM), the NSP4 wild-type (WT) peptide spanning residues 95 to 146 (20 μM), or the E120A/Q123A mutant NSP4 peptide spanning residues 95 to 146 (20 μM). Forskolin-treated HIEs (n = 5) were imaged for 10 min. Carbachol (n = 7)-, NSP4 wild-type peptide (n = 10)-, and NSP4 mutant peptide (n = 5)-treated HIEs were imaged for 40 min, and the rate of increase in the cross-sectional area is shown in square micrometers per minute. Statistical analyses of data in panels C and D were performed by using Student's t test.

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