to infection by commensal and Shiga toxin
Funding : This work was supported by National Institutes of Health ( https://projectreporter.nih.gov/ reporter.cfm ) Grants R21AI115003 , R01-AI064893 , U01-AI075498 , U19-AI116491 , to AAW . 
Core support was obtained from the CCTST ( Center for Clinical and Translational Science and Training , for Advancing Translational Sciences Award Number 1UL1TR001425-01 ) and by NIDDK 
Abstract 
Intestinal organoids model human responses 
Sayali S. Karve , Suman Pradhan , Doyle V. Ward , Alison A. Weiss 1☯¤ 1☯ 2 1*
1 Department of Molecular Genetics , Biochemistry , and Microbiology , University of Cincinnati , Cincinnati , Ohio , United States of America , 2 Center for Microbiome Research and Department of Microbiology and 
Physiological Systems , University of Massachusetts Medical School , Worcester , Massachusetts , United States of America 
Infection with Shiga toxin ( Stx ) producing Escherichia coli O157 : H7 can cause the potentially fatal complication hemolytic uremic syndrome , and currently only supportive therapy is available . 
Lack of suitable animal models has hindered study of this disease . 
Induced human intestinal organoids ( iHIOs ) , generated by in vitro differentiation of pluripotent stem cells , represent differentiated human intestinal tissue . 
We show that iHIOs with addition of human neutrophils can model E. coli intestinal infection and innate cellular responses . 
Commensal and O157 : H7 introduced into the iHIO lumen replicated rapidly achieving high num-integrity was observed after 4 hours . 
O157 : H7 grew as filaments , consistent with activation of the bacterial SOS stress response . 
SOS is induced by reactive oxygen species ( ROS ) , and O157 : H7 infection increased ROS production . 
Transcriptional profiling ( RNAseq ) dem-onstrated that both commensal and O157 : H7 upregulated genes associated with gastrointestinal maturation , while infection with O157 : H7 upregulated inflammatory responses , including interleukin 8 ( IL-8 ) . 
IL-8 is associated with neutrophil recruitment , and infection 
Introduction
Shiga toxin producing E. coli ( STEC ) , including O157 : H7 are an important cause of diarrheal disease , causing about 265,000 illnesses yearly in the US [ 1 ] . 
Shiga toxin ( Stx ) is responsible for the life-threatening systemic complication , hemolytic uremic syndrome ( HUS ) . 
Currently , supportive therapy is the only treatment , and importantly , patients treated with antibiotics are more are more likely to develop severe disease , including HUS [ 2 ] . 
Stx is an AB5 toxin ; the B-pentamer promotes entry of A-subunit into the mammalian cytoplasm , and the enzymatic Asubunit damages ribosomes , inhibiting protein synthesis [ 3 ] . 
Recent studies have shown that the A - and B-subunits can circulate independently , and active toxin is formed by subunit association on the target cell surface [ 4 ] . 
The genes for Stx are encoded in the late-gene region of lysogenic bacteriophages , and are silent until viral lytic replication is triggered by the bacterial SOS stress response [ 5 ] . 
Some antibiotics upregulate Stx expression , and this is likely responsible for the association of antibiotic treatment with increased risk for severe disease [ 6 -- 8 ] . 
O157 : H7 do not naturally infect mice , and there is a need for human model systems . 
Remarkable progress has been made using human tissue specific stem-cell propagated enter-oids [ 9 ] . 
Especially important has been the demonstration that the previously noncultivatable pathogen , human norovirus , can replicate in human intestinal enteroids [ 10 ] . 
Enteroids have been used to model bacterial infection , including studies with E. coli O157 : H7 [ 11 ] . 
Some studies have been performed using enteroid monolayers in transwells , with tissue culture medium on both the apical and basolateral surfaces . 
The presence of glucose-rich medium on the apical surface does not replicate the nutrient environment in the intestinal lumen , and presents a technical problem for studying bacteria , such as E. coli , which replicate rapidly and produce bi-products that can be toxic to cells . 
Furthermore , the smaller size of enteroids makes micro-injection challenging . 
Pluripotent stem-cell `` induced human intestinal organoids '' ( iHIOs ) represent a new experimental model to study enteric pathogens [ 12,13 ] . 
iHIOs are generated entirely in vitro from pluripotent embryonic stem cells by a process that mimics normal differentiation [ 12 ] , and represent a potentially infinite source of identical tissue samples . 
iHIOs represent human tissue from the distal portion of the small intestine [ 12 ] , the tissues favored for initial attachment of E. coli O157 : H7 [ 14 ] . 
iHIOs adopt the three-dimensional architecture of the human intestine 
[ 12 ] . 
The epithelium contains absorptive enterocytes and the major secretory lineages ( paneth cells , enteroendocrine cells , and goblet cells ) , and intestinal functions such as peptide transport and mucus secretion by goblet cells are maintained [ 12 ] . 
The epithelium is surrounded by a stratified mesenchyme which contains smooth muscle cells and sub-epithelial fibroblasts [ 12 ] . 
iHIOs have been successfully used to model features of embryonic development [ 16 ] and inflammatory bowel disease [ 17 ] . 
In this study , iHIOs were infected with commensal as well as pathogenic E. coli O157 : H7 . 
The iHIOs were not damaged by infection with commensal E. coli ; however , O157 : H7 produced a very severe and rapid loss of epithelial structural integrity . 
Materials and methods
E. coli strains
We used strains characterized in previous studies . 
Bacterial assemblies and sequence data for strains ECOR13 and PT29 are deposited under NCBI BioProject ID : PRJNA359210 . 
E. coli 
O157 : H7 , PT29S , is a spontaneous streptomycin resistant mutant of PT29S previously isolated from a patient [ 18 ] . 
PT29 is sequence type ST-11 ( https://cge.cbs.dtu.dk/services/MLST/ ) and only possesses the genes for the more potent from of Shiga toxin , Shiga toxin type 2 ( Stx2 ) , not Shiga toxin type 1 ( Stx1 ) . 
In addition to Stx , the Virulence Finder search program ( https://cge . 
cbs.dtu.dk / services/VirulenceFinder / ) , revealed PT29 possessed virulence factor genes typical for O157 : H7 including ; tir ( translocated intimin receptor protein ) , eae ( intimin ) , espA ( type III secretion system ) , espB ( secreted protein B ) , espF ( type III secretion system ) , ehxA ( enterohae-molysin ) , espP ( extracellular serine protease plasmid-encoded ) , iss ( increased serum survival ) , espJ ( prophage-encoded type III secretion system effector ) , etpD ( type II secretion protein ) , astA ( EAST-1 heat-stable toxin ) , nleA-C ( non-LEE encoded effectors A-C ) , katP ( plasmid-encoded catalase peroxidase ) , toxB ( toxin B ) , and iha ( adherence protein ) . 
Commensal strain SGUC183 , also known as 183ϕS [ 8 ] is streptomycin and gentamicin resistant derivative of ECOR13 , a non-pathogenic E. coli isolated from a healthy person in Sweden , and is part of the Michigan State University STEC Center ECOR collection [ 8 ] . 
ECOR13 is Group A , Sequence Type ST-44 . 
The only hit using the Virulence Finder program was glutamate decarboxylase ( GadB ) , which converts glutamate to gamma-aminobutyrate . 
This activity helps to maintain neutral intracellular pH following exposure to extremely acidic conditions , such as transit through the stomach [ 19 ] . 
Streptomycin resistance was selected as a spontane ¬ 
Reagents and equipment used in iHIO culture
Matrigel basement membrane matrix ( BD Biosciences , cat . 
356234 ) , and extracellular matrix gel ( Sigma , cat . 
E1270 ) were used to embed the iHIOs in order to support development of 
3-dimensional architecture . 
Gut media for iHIO culture was prepared using advanced Dulbecco 's Modified Eagle Medium/Ham 's F-12 ( DMEM/F12 ) ( Gibco , Invitrogen , cat . 
12634 -- 028 ) supplemented with B27 insulin ( Invitrogen , cat . 
17504044 ) , N2 supplement ( Invitrogen , cat . 
17502048 ) , 2 mM L-glutamine ( Fisher , cat . 
SH3003401 ) , 15 mM HEPES ( Invitrogen , cat . 
15630080 ) , 100 ng ml epidermal growth factor ( R&D Systems , cat . 
236-EG-200 ) , and either 2 -1 mM penicillin/streptomycin ( Invitrogen , cat . 
15140 -- 122 ) or penicillin alone ( Amresco , cat . 
E480-20ML ) . 
iHIOs were maintained in tissue culture treated Nucleon delta 4-well ( Nunc , cat . 
registry number 0043 ) [ 12 ] were obtained from Pluripotent Stem Cell Facility and Organoid Core at Cincinnati Children 's Hospital and Medical Center . 
iHIOs were maintained in reconwith a micropipette puller ( Sutter Instrument Company ) . 
The sealed tips of the capillaries were cut open using Cuterz glass scissors , and the capillaries were loaded onto Nanoject II auto-nanoliter injector ( Fisher , cat . 
13-681-455 ) . 
Microinjections were performed , and before and after injection images of iHIOs were obtained using a stereomicroscope ( Leica ) . 
In some studies iHIOs were co-injected with 2.5 mg ml of fluorescent dye fluorescein isothiocyanate -1 
( FITC ) to label the lumen and to assess maintenance of the epithelial barrier as reported in previous studies [ 21 ] . 
The iHIOs were incubated at 37 ˚C in a humidified chamber containing 5 % CO2 for 5 days . 
For bacterial infections , approximately 10 3 E. coli cells were microinjected into the iHIO lumen . 
The iHIOs infected with E. coli were incubated in reconstituted gut media containing penicillin ( final concentration 100 U ml ) at 37 ˚C in a humidified chamber -1 with 5 % CO2 for 1 day . 
Images of the injected iHIOs were collected using Zeiss LSM710 Live Duo Confocal Microscope . 
Cryosectioning and staining of iHIOs . 
iHIOs were fixed in 4 % paraformaldehyde ( 2 to 4 hours ) followed by 30 % sucrose ( overnight ) . 
Organoids were prepared for cryosectioning by freezing at -20 ˚C in Tissue Freezing Medium ( Fisher , cat . 
15-183-13 ) . 
Cryosections ( 10 μm ) were prepared with BD Cryotome FSE Cryostat and the sections placed on a plus glass microscope slides . 
Histologic stains are listed in S1 Table . 
To stain , sections were fixed in cold acetone ( 10 minutes ) , rinsed with distilled water , and blocked in blocking buffer ( PBS containing 
10 % goat serum , 1 % bovine serum albumin ( BSA ) and 0.01 % Triton X-100 ) for 2 hours at room temperature in a humidified chamber . 
The slides were drained and stained with primary antibody ( 1:500 ) in blocking buffer in a humidified chamber at 4 ˚C overnight . 
Sections were rinsed twice with wash buffer ( PBS with 0.1 % BSA and 0.025 % Triton X-100 ) , and secondary antibody was diluted in PBS ( 1:1000 ) and was applied to the sections . 
The sections were allowed to incubate with the secondary antibody for 2 hours in dark at room temperature . 
The sections were washed with PBS and DNA was counterstained with Hoechst ( 1 μg / mL ) or DAPI ( 0.5 μg / mL ) dye for 2 minutes in dark . 
Stained sections were air-dried and mounted using VectaMount permanent mounting medium . 
Analysis was performed using Zeiss LSM710 Live Duo Confocal Microscope . 
Merged images were generated and the FITC fluores ¬ 
After indicated incubation times at 37 ˚C with 5 % CO2 in a humidified chamber , the organoids were removed from the 3-dimensional culture matrix , transferred to an eppendorf tube , and washed with ice cold PBS . 
The organoids were then transferred to a sterile 2-ml tissue homogenizer , disrupted , and suspended in 100 μl PBS . 
Subsequent dilutions were plated on L-agar plates , and incubated at 37 ˚C overnight . 
The total number of bacteria per organoid was calculated based on the colony forming units ( CFU ) observed on the agar plates on the next day . 
Antibiotics are needed to confine bacterial growth to the lumen . 
Except where indicated , bacterial challenge studies were performed with streptomycin-resistant strains using extracellular matrix without gentamicin , and tissue culture media supplemented with penicil-lin and streptomycin . 
Penicillin-sensitive strains were able to grow in the organoid lumen , but not in the tissue culture medium when penicillin was in the tissue culture media . 
Gentamicin and streptomycin inhibited growth of antibiotic sensitive E. coli C600 strain [ 7 ] injected into the lumen . 
Matrigel is not available without antibiotics , so we transferred the organoids to the antibiotic-free extracellular matrix gel ( Sigma Aldrich , cat . 
# E1270 ) when working with genta ¬ 
Western blots to quantify Stx production
The iHIOs were infected with 10 PT29S cells and incubated at 37 ˚C with 5 % CO2 in a hum 3 ified chamber . 
After the indicated incubation times , the iHIO suspension was obtained as described above . 
No signal was detected in the supernatants of the lysed organoids ( data not shown ) . 
To determine whether the toxin was bound to the cell membrane , the lysate was centrifuged at 8600 x g 5 minutes at 4 ˚C and the supernatant and pellet fractions were analyzed by Western blot . 
Proteins were resolved in Bio-Rad Mini PROTEAN Tetra Cell using the 4 -- 15 % precast Mini-PROTEAN TGX ™ gel . 
Samples were boiled for 7 minutes in sample buffer ( 1M Tris , pH 
6.8 , 50 % glycerol , 10 % SDS , 0.5 % bromophenol blue , 0.5 % beta-mercaptoethanol ) before being loaded in a 15 μL volume . 
Gels were run at a constant 30 milliamps until the bromophenol blue dye reached the bottom of the gel . 
Proteins were transferred to a PVDF membrane in a Hoefer TE series transphor electrophoresis unit at 100 V for 1 hour using chilled transfer buffer ( 10 % methanol , 24 mM Tris pH 8.3 , 194 mM glycine ) . 
After transfer , the PVDF membrane was wetted in 100 % methanol for 1 minute followed by PBS for two minutes . 
The membrane was incubated with primary antibody rabbit polyclonal recognizing Stx2 A - and Bsubunits ( 1:5000 ) in Odyssey blocking diluent with 2 % Tween 20 , overnight followed by three washes in PBS-T ( PBS with 0.1 % Tween 20 ) . 
IRDye 800CW Diluted Secondary antibody ( Goat anti-rabbit ) ( 1:10,000 ) in Odyssey blocking diluent with 0.2 % Tween 20 was added to the membrane and incubated in the dark for one hour at room temperature with gentle shaking . 
The membrane was rinsed with PBS-T with vigorous shaking for 5 minutes . 
The washing was repeated three times and finally rinsed with PBS to remove the residual Tween 20 before the membrane was imaged in the Odyssey Family Imaging System ( LI-COR ; Odyssey CLx 1 
Near-Infrared ( NIR ) imaging system ) for the presence of Stx2a . 
Purified Stx2a at 25 and 50 ng was used as the positive control . 
The respective protein bands were quantified compared to the 
Stx2a standards using LI-COR Image Studio 4.0 software.
Assessment of production of reactive oxygen species
iHIOs were infected with 10 commensal or 10 pathogenic O157 : H7 in a medium devoid of 3 3 antibiotics and incubated at 37 ˚C with 5 % CO2 in a humidified chamber for a period of 4 h. Saline alone injected organoids were used as controls . 
At the indicated time , the iHIOs were again injected with 230 nL at 830 nM concentration of ROS detection reagent from Enzo Life Sciences . 
The iHIOs were further incubated at 37 ˚C with 5 % CO2 in a humidified chamber for an hour and the fluorescent intensity observed under a fluorescent microscope ( Nikon Eclipse 
TE2000-U) and the fluorescence quantitated by image processing program ImageJ.
Isolation and labeling of PMNs
De-identified human peripheral blood was obtained from the Cell Processing Core at Cincinnati Children 's Hospital Medical Center . 
5.0 ml of blood in EDTA was carefully layered onto 5.0 ml of Polymorphprep ™ ( Axis-Shield , Cat # 2017 -- 11 ) , and centrifuged at 500 G for 35 min at room temperature . 
The upper band of plasma and mononuclear cells was removed , and the lower band of PMNs was harvested . 
An equal volume of half-strength HEPES-buffered saline 
( 0.425 % ( w/v ) NaCl , 5 mM HEPES-NaOH , pH 7.4 ) was added to the PMN suspension . 
The PMNs were harvested by centrifugation at 400 G for 10 min at room temperature and suspended in the modified gut medium . 
Cell counts were performed using the 40μm Scepter ™ 
Cell Counter Sensor ( Millipore , Cat # PHCC40050 ) . 
The purified PMNs were labeled with 5 μM CellTracker ™ Violet BMQC dye ( Cat # C10094 , Molecular Probes ) for 30 minutes , centri-fuged to remove excess dye and the washed PMNs were suspended at the required number in gut medium . 
CO2 in a humidified chamber . 
For experiments without antibiotics , after injection the orga-noids were washed 3 times with sterile PBS to remove extracellular bacteria . 
After 4 hours , 5 X 10 PMNs in 20 4 μL were added to the wells and incubated for the indicated times . 
Fluorescent intensity of the labeled PMNs was observed on intact organoids by confocal microscope ( Zeiss LSM710 LIVE Duo ) , and quantified by image processing using ImageJ . 
A standard plane of focus was used for all confocal images ; the presence of the green FITC fluorescence indicates the image included the luminal compartment . 
The outline of the bright field image was used to define the boundaries of the organoid , and violet fluorescence within the boundary was considered to be due to internalized PMNs . 
Values were normalized to account for difference in 
Bioinformatics RNA-seq data analysis
RNA-seq was performed by Genomics , Epigenomics and Sequencing Core ( GESC ) in the University of Cincinnati . 
For each treatment , the total RNA from three independent iHIOs was extracted by using mirVana miRNA Isolation Kit ( Lifetech , Grand Island , NY ) with total RNA extraction protocol . 
Briefly , iHIOs were lyzed with lysis/binding buffer , treated with homoge-nate additive , and extracted with acid-phenol : chloroform . 
The supernatant was mixed with ethanol and passed through the filter cartridge . 
Bound RNA was washed and eluted . 
RNA concentrations were determined by Nanodrop ( Thermo Scientific , Wilmington , DE ) , and integ-rity was determined by Bioanalyzer ( Agilent , Santa Clara , CA ) . 
The Apollo 324 system 
( WaferGen , Fremont , CA ) and PrepX PolyA script was used for automatic polyA RNA isolation . 
The library was prepared using PrepX mRNA Library kit ( WaferGen ) and Apollo 324 
NGS automatic library prep system . 
Isolated RNA was RNase III fragmented , adaptor-ligated and converted to cDNA with Superscript III reverse transcriptase ( Lifetech , Grand Island , 
NY ) , followed by automatic purification using Agencourt AMPure XP beads ( Beckman Coulter , Indianapolis IN ) . 
The targeted cDNA fragments were around 200 base pairs ( bp ) . 
Universal ( SR ) and index-specific primers were added to each adaptor-ligated cDNA sample and the amplified library was enriched by AMPure XP beads purification , and quality and yield of the library was assessed by Kapa Library Quantification kit ( Kapabiosystem , Woburn , MA ) using ABI 's 9700HT real-time PCR system ( Lifetech ) . 
Individually indexed libraries were proportionally pooled ( 20 -- 50 million reads per sample ) for clustering in cBot system ( Illumina , San Diego , CA ) . 
Libraries at the final concentration of 15.0 pM were clustered onto a single read 
( SR ) flow cell using Illumina 's TruSeq SR Cluster kit v3 , and sequenced for 50 bp using TruSeq SBS kit on Illumina HiSeq system . 
To analyze differential gene expression , sequence reads were aligned to the human genome using the TopHat aligner [ 22 ] , and reads aligning to each known transcript were counted using Bioconductor packages for next-generation sequencing data analysis [ 23 ] . 
The differential expression analysis between different sample types was performed for each gene separately using the edgeR Bioconductor package [ 24 ] . 
The statistical significance of differential expression is established based on the FDR ( False discovery rate ) - adjusted p-values and are indicated as the values in the padj columns in S2 and S3 Tables ) [ 25 ] . 
19,076 transcripts were characterized ; 18,543 were identified as genes and 15,448 were associated with a gene ontology ( GO ) term using the gene ontology analysis program GOrilla [ 26 ] . 
As expected transcripts ( e.g. IL-13 , IL-25 , IL-22 , INF-γ , TNF , and IL-12 ) restricted to hematopoietic lineages were not detected . 
Venn diagrams were prepared using the online tool from Bioinformatics & Evolutionary Genomics ( http://bioinformatics.psb.ugent.be/webtools/Venn/ ) . 
Results and discussion
Sensitivity to LPS depends on route of exposure
iHIOs resemble sterile neonatal tissue [ 15 ] . 
Microbial colonization promotes maturation of the neonatal intestine , and Gram negative lipopolysaccharide ( LPS ) elicits strong responses , which are dependent on the cell-surface that is exposed . 
For iHIOs , introduction of LPS into the lumen mimics natural intestinal colonization , while addition of LPS to the tissue culture medium mimics life-threatening septicemia . 
To assess LPS toxicity , the lumen was labeled with the fluorescent dye , fluorescein isothiocyanate ( FITC ) , and the fluorescence was monitored to indicate maintenance of barrier function ( Fig 2 ) . 
Luminal addition of up to 10 ng of LPS did not compromise barrier function ( Fig 2A ) . 
This intraluminal concentration is about 
20,000 ng/ml , assuming a spherical organoid with a diameter of 1 mm has a volume of about 0.5 μL . 
In contrast , iHIOs were extremely sensitive to LPS added to the surrounding medium 
To assess whether E. coli can replicate and persist in the iHIO lumen , approximately 103 nonpathogenic commensal strain of E. coli ( SGUC183 ) or clinical isolate ( PT29S ) of O157 : H7 , which only expressed Stx2a were microinjected into the iHIO lumen under conditions that prevented bacterial growth in the tissue culture medium . 
The iHIOs were able to support the growth of both E. coli strains with virtually identical growth rates , although the O157 : H7 strain had a slightly longer lag phase ( Fig 3A ) . 
Biphasic growth rates were observed . 
During the first 4 hours , both strains had a doubling time of about 30 minutes , similar to in vitro growth rates with aeration in nutrient rich medium . 
After about 4 hours , much slower doubling times of about 3 hours were observed , suggesting changes in the lumen environment , such as nutrient or oxygen depletion . 
At 24 hours about 10 commensal bacteria were recovered , while after 72 6 hour over 10 commensal bacteria were recovered ( data not shown ) . 
Assuming iHIOs are hol-7 low spheres about 0.1 cm in diameter ( radius = 0.05 cm ) , the internal volume is equal to 4/3 πR3 ( or 5.24 x 10 − 4 ml ) , for an estimated density of about 1.9 x 1010 bacteria per ml , within the range of bacterial density in the human ileum ( about 10 per ml ) and colon ( about 10 per 8 12 
At 24 hours, commensal E. coli but not pathogenic O157:H7 were recovered from the
iHIOs . 
Furthermore , after 24 hours , organoids challenged with O157 : H7 were fragile and often broke apart when removed from the extracellular matrix support . 
To determine if the inability to recover O157 : H7 at 24 hours was due to loss of the epithelial barrier and subsequent exposure to the antibiotics from the tissue culture medium , at 18 hours the medium standard deviation were determined at the indicated times from three different iHIOs for each strain . 
Closed symbols , in a separate experiment iHIOs were injected with 10 commensal E. coli ( SGUC183 , squares ) , or 3 pathogenic O157 : H7 ( PT29S , triangles ) as above , but at 18 hours , the medium was replaced with medium lacking penicillin , and bacterial counts were assessed at 27 hours post inoculation . 
B-C , Commensal E. coli 
( B ) replicates in the lumen without damaging the iHIO , while O157 : H7 ( C ) damages the actin layer . 
Cryosections of iHIOs 18 hours after injection were stained for DNA ( blue ) , bacteria ( green , anti-E . 
coli for commensal , anti-O157 for O157 : H7 ) , and F-actin ( red ) . 
Bar indicates 20 μm . 
D-E . 
Cryosection 1 hour after infection with O157 : H7 , stained for nuclear and bacterial DNA ( DAPI , blue ) , E-cadherin ( green ) , and F-actin ( red ) . 
White arrowheads represent bacterial nucleoids co-localized with actin . 
( D ) , Bar indicates 10 μm . 
( E ) , Magnified image of D , bar indicates 2 μm . 
F-G . 
Cryosections 4 hours after infection with O157 : H7 . 
White arrowheads represent bacterial co-localization with actin . 
( F ) , stained for nuclear and bacterial DNA ( DAPI , blue ) , E-cadherin ( green ) , and F-actin ( red ) , bar indicates 10 μm . 
( G ) , stained for nuclear and bacterial DNA ( DAPI , blue ) , F-actin ( red ) , and anti-O157 ( green ) , bar indicates 5 μm . 
H-I . 
Cryosections 18 hours after infection with O157 : H7 . 
Yellow arrows indicate filamentous E. coli . 
( H ) , stained for nuclear and bacterial DNA ( DAPI , blue ) , E-cadherin ( green ) , and F-actin ( red ) , bar indicates 10 μm . 
( I ) , stained for nuclear and bacterial DNA ( DAPI , blue ) , F-actin ( red ) , anti-O157 ( green ) , bar indicates 10 μm . 
Representative images of experiments performed at least four times are shown . 
containing penicillin and streptomycin was replaced with antibiotic-free medium . 
The orga-noids were harvested at 27 hours post-infection ( 9 hours without antibiotics in the medium ) , and colony counts were assessed for both the tissue culture medium and the organoids . 
For the commensal , 2 x 10 CFU were recovered from the organoid at 27 hours ( Fig 3A , solid 6 square ) , but no viable bacteria were recovered from the tissue culture medium , suggesting the commensal bacteria continued to replicate within the confines of the lumen . 
In contrast , nine hours after antibiotic removal , 6 x 10 viable O157 : H7 were recovered from the organoid ( Fig 4 3A , solid triangle ) , and 3 x 10 were recovered from the tissue culture medium . 
These results 5 suggest that about 18 hours post-infection the O157 : H7 destroy the luminal barrier , and are killed if antibiotics are present in the medium . 
However , if antibiotics are not present in the 
Histologic characterization of iHIOs
Cryosections were examined to determine the effect of bacterial infection on iHIO morphology and the luminal epithelial layer . 
In sections taken at 18 hours , the epithelial layer of iHIOs injected with non-pathogenic SGUC183 was clearly defined by F-actin ( red ) and similar to PBS-injected organoids ( Fig 1B ) , with numerous bacteria ( green ) within the lumen ( Fig 3B ) . 
In contrast , the actin layer of iHIOs injected with O157 : H7 ( Fig 3C ) was clearly disrupted , there was no evidence for a luminal compartment and filamentous bacteria ( green ) were pres ¬ 
3D -- 3I ) . 
At one hour ( Fig 3D and 3E ) , while the lumen was clear , breaks in F-actin ( red ) were seen . 
At 4-hours post-infection ( Fig 3F and 3G ) , disrupted F-actin and loss of E-cadherin expression was apparent . 
At 18 hours post infection ( Fig 3H and 3I ) , the luminal border was gone , F-actin staining was sparse and randomly distributed , and the green E-cadherin staining iHIOs resemble the distal portion of the small intestine [ 12 ] , the tissues that are favored for initial attachment of E. coli O157 : H7 [ 14,30 ] . 
Adherence to human intestinal epithelium is a key determinant of pathogenicity . 
Strains possessing the locus of enterocyte effacement ( LEE ) display F-actin mediated intimate attachment to epithelial cells [ 31 -- 34 ] . 
Individual bacteria could be seen in the expanded images . 
O157 : H7 and other enteropathogenic E. coli display intimate attachment to intestinal epithelial cells mediated by the cytoskeletal protein F-actin [ 31 ] . 
Intimate contact can activate host antibacterial responses , such as production of reactive oxygen species ( ROS ) , which in turn can induce expression of Stx through activation of SOS response in STEC [ 35,36 ] . 
At 1-hour post infection ( Fig 3E , white arrowheads ) , DNA the size of a bacterial nucleoid ( blue staining ) and F-actin ( red ) were co-localized as evidenced by the purple in expanded merged confocal image . 
Pedestal formation was not observed , although such structures are typically visualized by electron microscopy . 
At 4-hours post infection ( Fig 3F ) numerous bacteria were seen in the lumen , growing primarily as coccobacilli . 
Co-localiza-tion of DNA and F-actin was observed ( Fig 3F , white arrowheads ) , and staining with the anti-E . 
coli antibody demonstrated that bacteria ( green ) were co-localized with the actin ( Fig 3G , white arrowhead ) . 
At 18 hours post infection ( Fig 3H ) , numerous filamentous DNA structures were seen . 
To verify that the filamentous structures were E. coli O157 : H7 , the organoids were stained with antibody to O157 LPS ( Fig 3I ) . 
Numerous green coccobacilli as well as long green filaments were seen , demonstrating that the small , sub-nuclear DNA structures were E. coli . 
Production of reactive oxygen species (ROS)
Filaments form when bacteria continue to replicate , but the daughter cells fail to separate , and occurs following exposure to DNA damaging agents , including ROS . 
The delay in septation is induced by the bacterial SOS system . 
It allows time for DNA damage repair , minimizing transfer of damaged chromosomes . 
ROS production was assessed in iHIOs were injected with saline , or 10 commensal or 10 pathogenic O157 : H7 . 
After 4 hours , bacterial recovery was 3 3 similar for both strains ( Fig 4A ) . 
Injection of O157 : H7 resulted in significantly increased ROS compared to the saline control or injection with the commensal strain ( Fig 4B and 4C ) . 
Stx production
While the SOS response is designed to protect chromosomal integrity , lysogenic bacteriophage use activation of the SOS response as a signal to initiate lytic replication and escape from a damaged host . 
Stx is phage-encoded , and activation of the SOS response initiates Stx expression [ 5 ] . 
Stx expression was observed in iHIOs infected with O157 : H7 . 
Stx2a was not detected by western blots at 1 , 2 , 4 and 6 hours post-infection ( Fig 5 ) . 
At 18-hours post infection , 4 ng and 10 ng Stx2a was detected in two separate experiments . 
Transcriptional profiling
Relative expression of linage specific genes . 
RNAseq was performed on iHIOs at 4 hours post-injection with PBS ( control ) or 10 commensal 3 E. coli or O157 : H7 . 
Expression of individual intestinal genes was examined ( Table 1 ) . 
Infection with the commensal and O157 : H7 strain resulted in slight ( approximately 2-fold ) , but significantly increased expression of epithelial transcription of proteins that participate in gastrointestinal defenses , such as alkaline phospha-tase , which is involved in detoxification of lipopolysaccharide ( 9 to 19-fold increase ) , the bacteriolytic enzyme lysozyme ( 6-fold increase ) , mucins involved in barrier function , including MUC2 ( 4 to 6 fold increase ) and MUC13 ( 4-fold increase ) , and a structural component of gas-signaling related to the innate immune defenses was also examined ( Table 1 ) . 
Bacterial infection constituted the first encounter of the sterile iHIOs with lipopolysaccharide ( LPS ) ; however , expression of the LPS receptor , TLR4 was not altered . 
Expression of IL-1β was highly upregulated by infection with either strain ; however , infection with O157 : H7 , but not the commensal strain , resulted in significant upregulation of the inflammatory mediators , IL-8 and IL ¬ 
18, and significant downregulation of NOD-like receptor, NLRC4.
Transcriptional enrichment analysis
Setting significance at P < 0.05 and using a 4-fold change compared to the PBS controls as the cutoff , infection with the commensal strain resulted in 317 differentially expressed genes ( 95 upregulated and 222 downregulated ) , while infection with the pathogenic O157 : H7 strain resulted in 429 differentially expressed genes ( 160 upregulated and 269 downregulated ) . 
The most significantly ( P < 3E-10 ) GO category uniquely upregulated by O157 : H7 infection ( S1A 
Fig ) was `` Chemokine-mediated signaling pathway '' ( GO :0070098 ) , with upregulation of the genes indicated in S1A Fig , box . 
Other categories uniquely upregulated by O157 : H7 included 
`` regulation of response to wounding '' , and the classical MAP kinase pathway , `` positive regulation of ERK1 and ERK2 cascade '' . 
Infection with either strain resulted in upregulation of the 
GO term , `` digestive system process '' . 
The most significant downregulated GO process category for both E. coli strains was the GO term , `` Multicellular organismal process '' ( GO :0032501 ) ; with 82 down-regulated genes for the commensal strain and 88 for O157 : H7 . 
The upregulated GO terms were compared ( Fig 6 ) . 
Both commensal and O157 : H7 infection up-regulated the GO terms `` Response to iron '' ( GO :0010039 ) and `` Regulation of vascular endothelial growth factor receptor signaling pathway '' ( GO :0030947 ) , and `` Maintenance of gastrointestinal epithelium '' ( GO :0030277 ) . 
O157 : H7 uniquely upregulated `` Chemokine mediated signaling pathways '' ( GO :0070098 ) . 
PMNs and infected iHIOs
The inflammatory mediator , IL-8 , is associated with neutrophil recruitment , alternatively breach of the intestinal barrier could promote recruitment by the presence of pathogen-associ-ated molecular patterns , such as LPS . 
We assessed whether pathogenic O157 : H7 promoted neutrophil recruitment . 
iHIOs were injected with saline , or 10 commensal or O157 : H7 , in the 3 presence of the fluorescent dye FITC to label the lumen , and incubated for four hours to allow for chemokine expression . 
Human PMNs ( polymorphonuclear leukocytes ) , a population comprise primarily of neutrophils , were labeled with fluorescent cell-tracker dye , and 5 x 10 were 4 added to the medium . 
Initial studies were done in the absence of antibiotics to allow for assessment of the potential of PMNs to reduce bacterial numbers ( Fig 7A ) . 
Bacterial recovery from the iHIOs was similar at 4 hours before addition of the PMNs . 
Both strains grew within the organoid after addition of the PMNs , and at 18 hours bacterial recovery from the iHIO in the presence of PMNs ( Fig 7A , full graph ) was not statistically different from growth in the absence of PMNs ( Fig 7A ) . 
The culture medium was also sampled . 
About 
1300 O157 : H7 were recovered from the media , but only 32 commensal bacteria were recovered from the media , suggesting O157 : H7 may have breached the epithelial barrier . 
As shown in Fig 3A , antibiotics can access and kill the bacteria if the epithelial barrier is breached . 
The influence of antibiotics in the tissue culture medium on bacterial recovery was assessed in the presence and absence of PMNs ( Fig 7B ) . 
At 6 and 8 hours , bacterial recovery was similar in the presence or absence of PMNs . 
However , at 23 hours post-infection , the commensal strain was recovered whether or not PMNs were present , but no viable O157 : H7 were recovered . 
This is consistent with O157 : H7 induced loss of the intestinal barrier , and further suggests that the presence of PMNs can not prevent the epithelial damage . 
Epithelial barrier function was further evaluated by quantifying fluorescence of FITC injected into the lumen fluorescence was recovered from the iHIOs injected with the commensal strain at 18 hours . 
Recruitment of PMNs . 
Recruitment of PMNs was monitored by microscopy ( Fig 7D -- 
7I ) . 
In the merged bright field and fluorescent images , the dark iHIO with a green , FITC-labeled lumen can be seen . 
PMNs ( violet ) were seen at the periphery of all iHIOs ( Fig 7D -- 7F and 7G -- 7I ) . 
For injection with saline or commensal , violet cells were primarily localized to the periphery of the iHIO . 
In contrast , for injection with O157 : H7 , violet cells were seen at the periphery , as well as within the iHIO and in some cases co-localize with the green stain that defines the lumen ( Fig 7I , white arrows ) . 
The violet signal within region corresponding to the body of the iHIO was quantified . 
Significantly more fluorescent signal was detected in the iHIOs infected with E. coli O157 : H7 than the saline or commensal-infected iHIOs at both 8.5 impermeant dye that stains cellular nucleic acids if the membrane has been compromised ( Fig 7K -- 7M ) . 
Significantly more fluorescent signal was detected in iHIOs infected with E. coli 
O157 : H7 compared to saline or commensal-infected iHIOs ( Fig 7N ) . 
Luminal presence of the phagocyte marker , CD11b , was also monitored in cryosections were in the lumen of the commensal infected iHIOs , as evidenced by red fluorescence ( Fig 8M ) and more were observed in the iHIOs infected with O157 : H7 ( Fig 7N ) . 
Less bacterial staining was observed for the commensal E. coli compared to E. coli O157 : H7 , likely due to the use of different bacterial antibodies , since Fig 7A demonstrated recovery of the two strains was similar . 
Conclusions
Commensal E. coli grew to high numbers in the previously sterile iHIO lumen without causing damage , demonstrating that like the neonatal intestine , the innate defenses of iHIOs are sufficient to contain non-pathogenic bacteria [ 37 ] . 
Tolerance of commensal bacteria is also seen in wild type mice , as well as severely immunodepleted NOD scid gamma ( NSG ) mice , lacking mature T cells , B cells , and natural killer ( NK ) cells . 
In contrast , growth of pathogenic O157 : 
H7 resulted in loss of epithelial barrier function . 
Thus some property or properties expressed by pathogenic O157 : H7 , but not commensal E. coli , is responsible for the rapid loss of epithelial barrier function . 
Both strains have been sequenced , and a likely candidate is the O157 : H7 LEE pathogenicity island , which is known to alter the integrity of the actin cytoskeleton , a cellular component necessary to maintain epithelial cell contact . 
A second candidate is Shiga toxin , which is known to kill cells . 
Whether either , both , or neither traits mediate the phenotype observed with O157 : H7 infection could be resolved by experiments with defined mutants in O157 : H7 . 
Pathogenic O157 : H7 activated innate defenses , including ROS production ( Fig 4 ) and several inflammatory immune responses ( S1A Fig , Table 1 ) . 
The different bacterial morphologies are consistent with differential activation of the host defenses [ 35 ] . 
The commensal strain grew normally as cocco-bacilli , while O157 : H7 displayed filamentous growth ( Figs 3 and 8 ) . 
In human disease , elevated neutrophil counts have been associated with development of HUS and fatal outcome [ 38,39 ] . 
IL-8 induces neutrophil-chemotaxis , and was upregulated by 
O157 : H7 . 
PMNs accumulated at the iHIO margins , migrated through the tissue and localized within the lumen . 
However , recruitment of PMNs did not prevent loss of epithelial barrier function ( Fig 7B and 7C ) or reduce the O157 : H7 numbers ( Fig 7A ) . 
This could be since long filamentous chains , as seen for O157 : H7 , can protect bacteria from phagocytosis [ 40,41 ] . 
Recruitment and activation of PMNs could contribute to tissue damage without helping to resolve the infection . 
Lack of experimental models has hampered investigation of human-restricted pathogens such as E. coli O157 : H7 . 
Our studies comparing infection of commensal to pathogenic E. coli demonstrate iHIOs represent a valuable model to study human-restricted enteric pathogens . 
Supporting information
S1 Fig . 
A , Highly significant GO PROCESS pathways upregulated by ( A ) O157 : H7 , PT29S and ( B ) . 
Commensal SGUC183 . 
S3 Table . 
RNAseq 4 hours post infection with O157 : H7 versus PBS ( samples in triplicate ) . 
( XLSX ) 
Acknowledgments
We would like to thank James Wells , Christopher Mayhew and Amy Pitstick from the Pluripotent Stem Cell and Organoid Core , Cincinnati Children 's Hospital Medical Center , and Chet 
Closson from the University of Cincinnati Live Microscopy Core for their valuable input . 
We also acknowledge the support from CCTST ( Center for Clinical and Translational Science and 
Training , for Advancing Translational Sciences Award Number 1UL1TR001425-01 ) and by NIDDK P30 DK078392 ( Pluripotent Stem Cell and Organoid Core and Live Microscopy 
Core ) of the Digestive Disease Research Core Center in Cincinnati . 
We thank the Biodefense and Emerging Infectious Diseases Research Resources Repository for providing purified Stx2a 
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