Bacterial colonization stimulates a complex physiological response in the immature human intestinal epithelium
- University of Michigan, United States
- Montana State University, United States
Figures
Figure 1 with 3 supplements
HIOs can be stably associated with non-pathogenic E. coli.
(A) Mean fluorescent intensity of a human intestinal organoid (HIO) containing live GFPE. coli str. ECOR2. The lower panels show representative images from the time series. Representative of three …
Figure 1—figure supplement 1
Pluripotent stem-cell-derived intestinal epithelium transcriptionally resembles the immature human intestinal epithelium.
(A) Pearson’s correlation matrix with heirarchical clustering forwhole-transcriptome normalized RNA-seq gene counts from epithelium isolated from the tissuesindicated on the axes. (B) Pearson’s …
Figure 1—figure supplement 2
Phylogenetic tree based on maximum liklihood genomic distance among E.coli str.
ECOR2 (Ochman and Selander, 1984), the strain used in the HIO colonization experiments, closely related E. coli isolates available on the PATRIC (Wattam et al., 2017) database, and pathogenic type …
Figure 1—figure supplement 3
HIO colonization protocol.
(A) Schematic representation of the microinjection of HIOs with live E. coli. See Materials and Methods for additional details. (B) A comparison of CFU/HIO at 24 hpost-microinjection of HIOs …
Figure 2
(A) Log2-transformed fold change in normalized RNA-seq gene counts in E. coli colonized HIOs at 24, 48, and 96 hr post-microinjection with 104 live E. coli relative to PBS-injected HIOs. …
Figure 3 with 1 supplement
Bacterial colonization results in a transient increase in epithelial proliferation and the maturation of enterocytes.
(A) Representative confocal micrographs of HIOs injected with PBS or 104 CFU E. coli str. ECOR2 at 24–96 hr post-microinjection and stained with fluorescent indicators for for EdUDNA, E-cadherin, …
Figure 3—figure supplement 1
Pathview (Luo and Brouwer, 2013) plot of the KEGG (Kanehisa and Goto, 2000) pathway showing the Cell mitotic cycle ('HSA 04110') superimposed with RNA-seq expression data corresponding to the log2 -transformed fold change in expression of cell cycle transcripts from HIOs microinjected with E. coli relative to PBS-injected HIOs at 48 h post-microinjection.
Mean of 4 biological replicates, each representing 5-6 pooled HIOs.
Figure 4
E. coli colonization is associated with a reduction in luminal oxygen concentration.
(A) Luminal oxygen concentration in human intestinal organoids at 0–48 hr post-microinjection with 104 CFU live E. coli. p Values reflect results of unpaired one-tailed Students t-tests for the …
Figure 5 with 2 supplements
NF-κB integrates complex microbial and hypoxic stimuli.
(A) Analysis scheme for identifying genes sets representing the components of the transcriptional response to live E. coli that could be recapitulated with heat-inactivated E. coli (contact induced) …
Figure 5—figure supplement 1
NF-κB signaling pathway acticvation in HIOs.
(A) Heatmap representation of time-dependent normalized RNA-seqgene expression for NF-κB pathway components in HIOs at 0-96 hr post-microinjection. (B) Western blot of phosphorylated p65 and total …
Figure 5—figure supplement 2
Secretion of AMPs (BD-1 & BD-2), cytokines (IL-6 & IL-8), and thepro-angiogenesis growth factor VEGF in HIOs microinjected with PBS, 10 4 CFU E.
coli, or anequivalent concentration of heat-inactivated E. coli and cultured under standard cell cultureconditions or hypoxic conditions (1% O2 , 5% CO2 , 94% N 2) for 24 hr as measured by ELISA. N …
Figure 6 with 2 supplements
Bacterial colonization promotes secretion of antimicrobial peptides.
(A) Normalized fold change in antimicrobial peptide (AMP) gene expression in E. coli-associated HIOs at 24 hr relative to PBS control treatment. (B) Normalized fold change in expression of DEFB4A, …
Figure 6—figure supplement 1
Normalized fold change in expression of DEFB4B, a duplicated gene encoding human β-defensin 2 (BD-2) peptide, in each of the conditions indicated relative to PBS control treatment.
Concentration of BD-2 peptide in culture supernatant at 24 hr as measured by ELISA in HIO cultures treated as indicated. p-Values represents the results of a two-tailed Student’s t-test for the …
Figure 6—figure supplement 2
BD-2 inhibits E. coli growth in vitro.
(A) Optical density (600 nm) of E. coli str. ECOR2 and E. coli str. K12suspension cultures supplemented with PBS, BD-2 (1 μg/ml), or heat-inactivated BD-2 at 10-min intervals over a 6-hr period at …
Figure 7 with 1 supplement
Bacterial colonization promotes expression of epithelial mucins and glycotransferases.
(A) Heatmap of normalized RNA-seq glycotransferase and mucin gene counts of HIOs associated with E.coli at 0–96 hr post-microinjection. N = 4 (0 hr), 5 (24 hr), 3 (48 hr), and 4 (96 hr) biological …
Figure 7—figure supplement 1
Representative confocal micrographs of HIOs treated as indicated.
Fluorescent immunostaining pseudocoloring applied as indicated in the figure legend. 40X optical magnification with 3X digital zoom.
Figure 8 with 1 supplement
Bacterial colonization promotes resilience of the epithelial barrier via NF-κB.
(A) Heatmap of RNA-seq data indicating the relative expression of genes associated with the Adherens junction or Cell-cell junction assembly based on annotation in the REACTOME database. Results …
Figure 8—figure supplement 1
Representative confocal micrographs of HIOs treated as indicated.
Fluorescent immunostaining pseudocoloring applied as indicated in the figure legends. 60X optical magnification with 2X digital zoom.
Videos
Video 1
Animation of individual epifluorescent microscopy images from a human intestinal organoid (HIO) containing live GFP E. coli str.
ECOR2. Images were captured at 10 min intervals over the course of 18 hr an coalated in sequential order. Representative of three independent experiments. See Figure 1A.
Additional files
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Supplementary file 1
List of differentially expressed genes and the Gene Set (I-IV) assignments used in the analyses presented in Figure 5.
- https://doi.org/10.7554/eLife.29132.022
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Supplementary file 2
Immunostaining conditions for all immunohistochemistry data presented in this manuscript.
NDS, Normal donkey serum; PBS, phosphate buffered saline.
- https://doi.org/10.7554/eLife.29132.023
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Transparent reporting form
- https://doi.org/10.7554/eLife.29132.024
