Commensal Bacterial Endocytosis in Epithelial Cells Is Dependent on Myosin Light Chain Kinase–Activated Brush Border Fanning by Interferon-γ

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The American Journal of Pathology, Vol. -, No. -, - 2014

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Commensal Bacterial Endocytosis in Epithelial Cells Is Dependent on Myosin Light Chain KinaseeActivated Brush Border Fanning by Interferon-g Q23

Li-Ling Wu,* Wei-Hao Peng,y Wei-Ting Kuo,* Ching-Ying Huang,* Yen-Hsuan Ni,z Kuo-Shyan Lu,y Jerrold R. Turner,x and Linda C.H. Yu* From the Graduate Institutes of Physiology* and Anatomy and Cell Biology,y and the Department of Pediatrics,z National Taiwan University College of Medicine and Hospital, Taipei, Taiwan; and the Department of Pathology,x University of Chicago, Chicago, Illinois Accepted for publication May 5, 2014. Address correspondence to Linda C.H. Yu, Ph.D., Graduate Institute of Physiology, National Taiwan University College of Medicine, Ste. 1020, #1 Jen-Ai Rd. Sec. I, Taipei 100, Taiwan. E-mail: lchyu@ ntu.edu.tw.

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Abnormal bacterial adherence and internalization in enterocytes have been documented in Crohn disease, celiac disease, surgical stress, and intestinal obstruction and are associated with low-level interferon (IFN)-g production. How commensals gain access to epithelial soma through densely packed microvilli rooted on the terminal web (TW) remains unclear. We investigated molecular and ultrastructural mechanisms of bacterial endocytosis, focusing on regulatory roles of IFN-g and myosin light chain kinase (MLCK) in TW myosin phosphorylation and brush border fanning. Mouse intestines were sham operated on or obstructed for 6 hours by loop ligation with intraluminally administered ML-7 (a MLCK inhibitor) or Y27632 (a Rho-associated kinase inhibitor). After intestinal obstruction, epithelial endocytosis and extraintestinal translocation of bacteria were observed in the absence of tight junctional damage. Enhanced TW myosin light chain phosphorylation, arc formation, and brush border fanning coincided with intermicrovillous bacterial penetration, which were inhibited by ML-7 and neutralizing antieIFN-g but not Y27632. The phenomena were not seen in mice genetically deficient for long MLCK-210 or IFN-g. Stimulation of human Caco-2BBe cells with IFN-g caused MLCK-dependent TW arc formation and brush border fanning, which preceded caveolin-mediated bacterial internalization through cholesterol-rich lipid rafts. In conclusion, epithelial MLCK-activated brush border fanning by IFN-g promotes adherence and internalization of normally noninvasive enteric bacteria. Transcytotic commensal penetration may contribute to initiation or relapse of chronic inflammation. (Am J Pathol 2014, -: 1e15; http://dx.doi.org/10.1016/j.ajpath.2014.05.003)

Commensal bacteria, estimated at 100 trillion (1014), are normally confined to the gut lumen and not in direct contact with epithelial cells. However, abnormal bacterial adherence and internalization in enterocytes have been documented in patients and experimental models with Crohn disease,1,2 celiac disease,3,4 chronic psychological stress,5 surgical manipulation,6 and intestinal obstruction (IO).7 Recent in vitro studies have found transcellular passage of nonpathogenic, noninvasive bacteria in epithelial monolayers after exposure to inflammatory and metabolic stress, such as interferon (IFN)-g,8,9 hypoxia,10 and mitochondrial damage.11 To date, the molecular mechanisms of bacterial endocytosis in enterocytes remain unclear. Polarized intestinal epithelial cells are endowed with densely packed apical microvilli or brush border (BB),

which act as an ultrastructural barrier that impedes physical contact between enteric microbes and cellular soma.12 However, recent findings have revealed that commensal bacteria are internalized into epithelial cells via cholesterolrich lipid rafts situated at invaginations of apical membrane between adjacent microvilli.8,13,14 It is still poorly understood how microbes of 0.5 to 1 mm gain access to the base of the intermicrovillous cleft. Actin-cored microvilli are rooted in the filamentous meshwork of the terminal web Supported by grants NSC99-2320-B-002-024-MY3, NSC102-2325-B002-035, and NSC102-2628-B-002-009-MY3 from the National Science Council Taiwan and grant 10R7807 from the National Taiwan University. Disclosures: None declared.

Copyright ª 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2014.05.003

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Wu et al (TW), which consists of multiple proteins, including actin, myosin, fodrin, and spectrin.15 Early reports revealed that the phosphorylation of myosin light chain (MLC) in the TW region, via unknown kinases, leads to BB fanning in enterocytes.16,17 Other studies have found that the phosphorylation of perijunctional MLC by myosin light chain kinase (MLCK) and rho-associated kinase (ROCK) is involved in epithelial tight junction (TJ) disruption.18,19 We hypothesized that TW MLC contraction and BB fanning may allow bacterial penetration through an enlarged intermicrovillous cleft to initiate apical endocytosis. The roles of MLCK and ROCK in mechanisms of bacterial endocytosis and their correlation with TJ changes have yet to be determined. Previous studies from our laboratory found increased bacterial translocation (BT) to extraintestinal organs after IO by loop ligation.20,21 The current aim was to investigate molecular and ultrastructural mechanisms of apical bacterial endocytosis in enterocytes of IO models, focusing on the role of MLCK-dependent TW myosin phosphorylation and BB fanning. The regulatory role of IFN-g was also examined using genetically deficient mice and epithelial cell cultures.

sign of cyanosis was seen during the experiment. After ligation, phosphate-buffered saline (PBS) vehicle, 10 mmol/L ML-7 (a specific MLCK inhibitor; Sigma, St. Louis, MO), Q5 or 50 mmol/L Y27632 (a specific ROCK inhibitor; Sigma) was administered intraluminally in a volume of 0.25 mL using a 26-gauge needle with polyethylene tubing 10. The hole in the gut wall was closed, and the abdominal layers were sutured after surgery. The mice were placed back into steel cages, fasted but with free access to water, and sacrificed at various time points.

Blockade Studies with antieIFN-g Hamster anti-mouse IFN-g clone H22 (BioLegend, San Diego, CA) was used to neutralize IFN-g, and hamster IgG1 isotype antibody was used as a control.23 After bowel ligation, mice were i.p. injected with 0, 10, 50, or 100 mg of antieIFN-g or 100 mg of IgG1 isotype in 0.1 mL of sterile PBS. All mice were sacrificed 6 hours after the surgical procedure. The neutralizing effect was verified in mucosal samples using an enzyme-linked immunosorbent assay (ELISA) kit for IFN-g (eBioscience, San Diego, CA).

Quantification of Luminal Bacteria in Small Intestines

Materials and Methods Animals Specific pathogen-free BALB/c and C57BL/6 male mice (6 to 8 weeks old) were obtained from the Animal Center of National Taiwan University. MLCK/ mice (B6 background) that lacked the long 210-kDa MLCK were obtained from Dr. J. R. Turner (University of Chicago, Chicago, IL).22 IFN-g/ mice (B6 background) were obtained from the Jackson Laboratory (Chicago, IL). All experimental procedures were approved by the Animal Care and Use Committee of National Taiwan University.

Surgical Procedure of IO In the first set of experiments, mice were randomly divided to 2 groups: sham operation (sham) and IO. Both groups were sacrificed 1, 3, 6, and 24 hours after the surgical procedure. In the next set of experiments, mice were randomized to four groups: sham, IO and vehicle, IO and ML-7, and IO and Y27632; all four groups were sacrificed 6 hours after the surgical procedure. Mice were fasted overnight but allowed to drink water ad libitum. After i.p. anesthetization with 40 mg/kg of sodium pentobarbital, mice underwent aseptic laparotomy. In the IO group, the distal small intestine was mechanically obstructed with 4-0 nylon at both ends for a 10-cm loop ligation ending 1-cm proximal to the ileocecal junction.21 Mice in the sham group received laparotomy and mock manipulation of the gut without ligation. In all animals, care was taken not to occlude or puncture mesenteric vessels, and no

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At the time of sacrifice, the distal small intestines of mice in the sham group were ligated to a 10-cm loop as in the IO mice. The intestinal loops of sham and IO mice were excised and luminally instilled with 0.25 mL of sterile PBS through the open end. The intestinal loop was then closed tightly and the lavage solution distributed by rocking the section back and forth 10 times before drainage. The lavage was plated onto fresh blood agars for culturing, and luminal bacteria counts were expressed as log10 of colony-forming units (CFUs) per centimeter of intestinal segment.21

Analysis of BT The liver and spleen tissues were homogenized and sonicated in sterile PBS at a ratio of 1 mg to 10 mL. Undiluted lysate (200 mL) was inoculated onto fresh blood agar plates. After incubation at 37 C for 24 hours, bacterial counts were expressed as log10 of CFUs per gram of tissue.21,24

Quantification and Classification of Epithelial Endocytosed Bacteria Intestinal epithelial cells were isolated using a previously described protocol.25,26 A total of 2  106 isolated epithelial cells were incubated with 300 mg/mL of gentamicin (Sigma) for 1 hour.26 After washing, cells were lyzed with 1% Triton X-100 in PBS for 10 minutes on ice. The cell lysate was plated onto agar plates overnight at 37 C. The numbers of bacterial colonies are presented as log10 CFU per 106 cells. The individual bacterial colonies were picked out for DNA extraction and sequencing (see below).

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Bacterial Internalization and Terminal Web Myosin Q1

Bacterial Profiling by Denaturing Gradient Gel Electrophoresis and 16S Ribosomal DNA Sequencing

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The DNA of luminal contents in mouse small intestine was extracted using a QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) and subjected to polymerase chain reaction (PCR) in a T3000 Thermocycler (Biometra, Goettingen, Germany).27 The amplification of the V2-3 variable region of the 16S ribosomal RNA gene was performed using a forward primer (50 -CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGTCCTACGGGAGGCAGCAG-30 ) and a reverse primer (50 -ATTACCGCGGCTGCTGG-30 ). The forward primer contains an additional 40-nucleotide GC-rich sequence (GC clamp; underlined characters) at the 50 end. The PCR mixture contained 200 ng of template DNA, 1.5 mL of forward primer, 1.0 mL of reverse primer, 0.5 mL of Taq polymerase, 0.5 mL dNTPs (25 mmol/L), and 5 mL of 10 PCR buffer in a total reaction volume of 50 mL. All PCR amplifications were initiated at 94 C for 5 minutes. The samples were then denatured at 94 C for 30 seconds, followed by annealing at 53 C for 30 seconds and elongation at 72 C for 30 seconds, repeated for 35 cycles. A final elongation step at 72 C for 10 minutes was included, and the PCR products were separated on a gradient gel with denaturants.27 The 8% (m/v) polyacrylamide solution was used to cast a gel with denaturant gradients ranging from 40% to 60% [100% corresponding to 7 mol/L urea and 40% (v/v) formamide]. Electrophoresis was performed at a constant voltage of 50 V at 60 C for 12 hours. The dominant bands were punched from the gels for DNA extraction and reamplified using the forward primer (without the GC clamp) and the reverse primer. The PCR products were subcloned into plasmids and purified. The PCR fragments were sequenced and then classified using the naive Bayesian classifier provided by the ribosomal database project.28 Sequences from 16S gene libraries were deposited to the DNA Databank of Japan (http://www.ddbj.nig.ac.jp; accession numbers AB771416 to AB771423).

Fluorescent in Situ Hybridization Intestinal tissues were fixed in Carnoy’s solution (Ricca Chemical Company, Arlington, TX), embedded in paraffin wax, and processed according to a standard protocol.25 Briefly, 5-mm-thick sections were mounted onto glass slides and deparaffinized. Slides were treated with 1% Triton X-100 for 90 seconds and then incubated in PBS that contained 5 mg/mL of lysozyme for 20 minutes at 37 C. Slides were rinsed thoroughly with water and air-dried. Sections were preincubated at 46 C for 60 minutes in a hybridization buffer that contained 900 mmol/L NaCl, 20 mmol/L TrisHCl, 15% formamide, and 0.01% SDS (pH 7.4). Prewarmed hybridization buffer that contained 0.1 mmol/L oligonucleotide probe was applied to the tissue sections. These included 50 -end fluorescein isothiocyanateelabeled universal bacterial probe (EUB338) (50 -GCTGCCTCCCGTAGGAGT-30 ) and negative control probe (non-EUB338)

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(50 -ACATCCTACGGGAGGC-30 ), as well as 50 -end Cy3labeled probes for Lactobacillus/Enterococcus (Lab158) (50 -GGTATTAGCACCTGTTTCCA-30 ), Escherichia coli (Ecol1513) (50 -CACCGTAGTGCCTCGTCATCA-30 ), Staphylococcus (STA) (50 -TCCTCCATATCTCTGCGC30 ), and Bacteroides (Bac 303) (50 -CCAATGTGGGGGACCTT-30 ) (Genomics BioSci and Tech, Taipei, Taiwan).29 After incubation overnight in a dark humid chamber at 46 C, slides were rinsed thoroughly with sterile doubledistilled water and then stained with Hoechst dye to visualize cell nuclei. Images were captured under a fluorescence microscope.

Preparation of Fluorescent E. coli The expression plasmid pRSET that contains a green fluorescent protein (GFP) was transformed into nonpathogenic competent E. coli BL21 (Novagen, Madison, WI) and grown on LB plates supplemented with 100 mg/mL of ampicillin Q7 (Sigma) at 37 C overnight. Single colonies were seeded into 5 mL of LB broth with 100 mg/mL of ampicillin and placed on an incubated shaker (200 rpm) at 37 C till the OD600 reached 0.6. The GFP expression in E. coli was induced by adding 0.1 mmol/L isopropyl b-D-1-thiogalactopyranoside for incubation of 1.5 hours. After centrifugation, the pellet was mixed with 0.5 mL glycerol and stored at 80 C until use.

Administration of Live Fluorescent Bacteria into Intestinal Loops We resuspended 5  108 GFP-expressing E. coli (E. colie GFP) in 0.25 mL of PBS and inoculated into the obstructed loop. After 1, 3, 6, and 24 hours, the intestinal segments were excised and fixed in 2% paraformaldehyde (PFA) for 30 minutes at 4 C. Tissues were snap frozen in OCT solution (Thermo, West Palm Beach, FL) and cut into 8-mm sections with a cryostat and mounted on glass slides.21 Tissues were blocked with fetal bovine serum for 1.5 hours and incubated with 5 U/mL of phalloidin conjugated to Alexa-633 (Invitrogen, Carlsbad, CA) for 30 minutes. The slides were stained with a Hoechst dye, and images were captured under a fluorescence microscope.

Transmission Electron Microscopy and Immunoelectron Microscopy Intestinal tissues were fixed in 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer (pH 7.4) for 2 hours at 22 C and rinsed with 0.05 mol/L Tris buffer (pH 7.4) for 4 hours at 4 C. Tissues were osmicated, dehydrated in a graded ethanol series, and embedded in epoxy resin. Vibratome sections (100 mm thick) were cut and washed in Tris buffer three times. Sections (70 nm thin) were then cut and examined with a Hitachi-7100 electron microscope (Hitachi Co., Tokyo, Japan). Electron micrographs of

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Wu et al epithelial cells were taken at magnifications of 6000, 15,000, and 30,000.21 For immunoelectron microscopy, 50-mm-thick vibratome sections were placed in 5% normal goat serum in 0.1 mol/L PBS for 1 hour at room temperature. Sections were incubated with a rabbit antiecaveolin-1 monoclonal antibody (1:500; Cell Signaling, Danvers, MA) for 48 hours at 4 C. After rinsing, the sections were incubated with affinity-purified biotinylated goat anti-rabbit IgG (1:200; Jackson Laboratory) for 2 hours. The sections were treated with avidin-biotin complex for 1 hour. After rinsing, sections were soaked for 10 minutes in 0.05 mol/L Tris buffer (pH 7.2), 0.0125% diaminobenzidine, and 0.35% imidazole. This was followed by 2 to 4 minutes in a fresh aliquot of diaminobenzidine mixture that had been activated with 0.003% hydrogen peroxide. Sections were washed in 0.1 mol/L sodium phosphate buffer and then dehydrated in an alcohol series followed by infiltration and embedding with propylene oxide and Epon. Sections 70 to 90 nm thin were collected onto copper mesh grids and examined with a Hitachi-7100 electron microscope at 90 kV. Controls consisted of omitting the primary antiserum, secondary antibody, or biotin-avidin complex.

Scanning Electron Microscopy

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Intestinal tissue were plated on 13 mm coverslips and treated as in the static adhesion assay. Tissues on the coverslip were fixed in 2.5% glutaraldehyde in PBS for 1 hour and then postfixed in aqueous 1% osmium tetroxide/ 1% tannic acid for 1 hour. After washing, samples were dehydrated in a graded ethanol series and critical point dried in a HCP-2 critical point drying device (Hitachi Co.). The specimens were mounted on metal stubs, coated with gold in a JEOL JFC-1100 Fine Coat Ion Sputter, and photographed in a scanning electron microscope (JEOL S330; JEOL, Tokyo, Japan).

Analysis of TW Contraction and BB Fanning Intestinal epithelial sheets were isolated for measurement of TW contraction and BB fanning according to the methods of Keller and Mooseker.16 Scraped mucosal samples were gently homogenized to obtain sheets of 5 to 10 epithelial cells connected by their intercellular junctions. Contraction of TW was assayed by placing the epithelial sheets in icecold contraction buffer with or without Ca2þ/ATP and immediately transferred to 37 C for 15 minutes before fixation. The epithelial sheets were photographed in a light microscope with differential interference contrast, and the width of the TW and the height of the arc were measured to determine the extent of TW contraction. For ultrastructural analysis of BB fanning, epithelial sheets were processed for transmission electron microscopy to obtain longitudinal and cross-sectional views of the BB. The extent of BB disorganization is determined according to the method of Tyska et al.30

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Immunofluorescent Staining of p-MLC and Fodrin in Intestinal Tissues Scraped mucosal tissues were fixed with 4% PFA for 1 hour at 4 C. The tissues were quenched with 50 mmol/L NH4Cl (Sigma) in PBS for 10 minutes and blocked with fetal bovine serum for 1 hour at room temperature. After washing, tissues were incubated with rabbit antiephosphorylated MLC2 (antiep-MLC2) (Ser19) (1:100; Cell Signaling) and mouse antiea-fodrin (1:100; Abcam, Cambridge, UK) overnight at 4 C. The tissues were washed in PBS thrice and incubated with goat anti-rabbit IgG conjugated to Alexa-488 and antimouse IgG conjugated to Alexa-594 for 1 hour at room temperature. After incubating with Hoechst dye for 30 minutes, the samples were plated onto slides with an ultrathin high-performance cover glass (Zeiss, Göttingen, Germany). The slides were observed under a fluorescent microscope.

Histopathologic Examination Intestinal tissues were fixed in 4% PFA and embedded in paraffin wax with proper orientation of the crypt to villus axis before sectioning. Sections (5 mm thick) were deparaffinized with xylene and graded ethanol, stained with hematoxylineosin, and observed under a light microscope.21

Western Blotting Intestinal mucosal proteins were extracted with complete radioimmunoprecipitation assay buffer and subjected to SDSPAGE (4% to 13% polyacrylamide) as described.21 The resolved proteins were then electrotransferred onto polyvinylidene difluoride or nitrocellulose membranes in a semidry blotter. Blots were blocked with 5% (w/v) nonfat dry milk in Tris-buffered saline (TBS) or 5% (w/v) bovine serum albumin in TBS with Tween 20 [0.1% (v/v) Tween 20 in TBS] for 1 hour, washed with TBS with Tween 20, and incubated with a primary antibody at 4 C overnight. The membrane was washed and incubated with a secondary antibody for 1 hour. After washing, the membranes were incubated with chemiluminescent solution and signals detected. The primary antibodies used included anti-MLC (1:500; Cell Signaling), antiep-MLC (1:500; Cell Signaling), antie b-actin (1:10,000; Sigma), and anti-MLCK (1:500; Santa Cruz, Dallas, TX). The secondary antibodies used were horseradish peroxidase (HRP)econjugated goat anti-mouse IgG (1:2000; Santa Cruz) and anti-rabbit IgG (1:1000; Cell Signaling).

Staining of TJs on Intestinal Tissues A 1-cm intestinal segment was fixed with 4% PFA and then snap frozen in OCT solution. The tissues were cut into 6 mm sections with a cryostat and mounted on precoated glass slides as previously described.21 Briefly, sections were prefixed in cold acetone, air-dried, and fixed again with 4% PFA. The

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tissues were permeabilized with 1% Triton X-100 for 1 minute, blocked with fetal bovine serum for 1.5 hours, and then incubated with rabbit antiezonula occludens protein 1 (1:100; Invitrogen) at 4 C overnight. After washing, tissues were incubated with goat anti-rabbit IgG conjugated to Alexa-488 for 1 hour. The slides were washed and stained with Hoechst dye for imaging under a fluorescence microscope.

flux rate of HRP (mol. wt., 44 kDa; Sigma). Briefly, HRP was added to the luminal buffer at a final concentration of 5  105 mol/L. Then 200-mL serosal samples were collected after 0, 30, 60, and 90 minutes, and the enzymatic activity of HRP was measured. The HRP fluxes were calculated according to a standard formula and were expressed as picomoles per square centimeter per hour.21,31

Ussing Chamber Studies and Permeability Assay

Cell Culture Models and Bacterial Internalization Assays

Intestinal segments (1 cm) were opened along the mesenteric border and mounted in Ussing chamber systems (World Precision Instruments, Sarasota, FL) to measure the tissue conductance as previously described.21 The intestinal epithelial permeability was determined by the mucosal-to-serosal

Human Caco-2 epithelial cells (clone C2BBe) were seeded on 12-well transwells with filter membranes of with a pore size of 3.0 mm (2  105 cells/mL) and grown for 14 days until postconfluency to develop TJs and long BB.15,32 The

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Bacterial Internalization and Terminal Web Myosin

Figure 1 Mucosal adherence and penetration of enteric bacteria increases after obstruction. A: Analysis of bacterial strains in distal small intestines by denaturing gradient gel electrophoresis and 16S ribosomal DNA sequencing. Representative photoimage showing bands (indicated by numbers) with density change in IO mice compared with sham groups. The bacterial genus corresponding to each band number is shown. B: Histologic staining and bacterial hybridization in intestinal tissues of sham, IO at 6 hours, and IO at 24 hours mice. Intestinal sections were stained with H&E, a universal bacterial probe EUB338, or Hoechst dye. C: Higher magnification showing bacteria signals in epithelial cells (arrowheads) and lamina propia (arrows) in intestinal tissues of IO at 6 hours and IO at 24 hours mice. D: The penetrated bacterial strains were characterized in IO at 6 hours mice using specific probes targeting Escherichia coli, Staphylococcus, Lactobacillus, and Bacteroides. E: Temporal-spatial relationship between luminal bacteria and gut epithelium was examined by injecting 5  108 colony-forming units of E. colieGFP into obstructed loops immediately after ligation. Superimposed images of E. colieGFP (green), Hoechst (blue), and phalloidin-labeled F-actin (red) revealed bacterial attachment by 3 hours and penetration by 6 and 24 hours. Representative photomicrographs were obtained from at least four mice in each group. Scale bar Z 10 mm. GFP, Green fluorescent protein; H&E, hematoxylin-eosin; IO, intestinal obstruction.

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cells were basolaterally treated with recombinant IFN-g (100 IU/mL) for 48 hours and processed for bacterial assays. In some experiments, 20 mmol/L ML-7 and 50 mmol/L Y27632 were added before IFN-g treatment. In another setting, at the end of IFN-g treatment, cell monolayers were apically administered with 2 mmol/L filipin (Sigma) or 50 mmol/L methyl-b-cyclodextrin (Sigma) 30 minutes before the bacterial assays. The amount of BT and internalization in cells was determined using the method described by Clark et al.8 Briefly, 2  108 CFU/mL of fluorescent E. colieGFP was inoculated into the apical chamber of transwells and incubated with cells for 1 hour, and the bacterial numbers in basolateral solution and epithelial cells were determined.8

Transepithelial Electrical Resistance and Paracellular Permeability The transepithelial electrical resistance of cells was measured using an electrovoltohmeter before and after IFN-g treatment. Q9 The paracellular permeability was determined by apicalto-basal transport of a dextranefluorescein isothiocyanate probe (mol. wt., 3000) (Invitrogen) as previously described.21,33

ELISA-Based MLCK in Vitro Kinase Activity MLCK activity was determined using the method described by Al-Sadi et al,34 with slight modification. Cells were rinsed once with PBS and lyzed with radioimmunoprecipitation

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Figure 2

Bacterial endocytosis and myosin phosphorylation in epithelial cells are uncoupled with TJ damage at early time points of IO. A: Increased intracellular bacterial counts in enterocytes were seen as early as 3 hours and continued to increase after 6 and 24 hours of IO. The line indicates the median of each group. B: Intestinal tissue conductance after 3 and 6 hours of IO was comparable to those of the sham groups. The conductance value at IO at 24 hours was higher than that of sham controls. C: Luminal-to-serosal macromolecular flux did not increase until 24 hours after IO. D: Photoimages showing ZO-1 staining (green) in intestinal tissues. Higher magnification in insets revealed ZO-1 disruption at IO at 24 hours but not early time points. Staining of Factin (red) and cell nucleus (blue). E: Western blots reveal increased p-MLC in mucosal samples of IO groups after 3 to 24 hours compared with sham controls. F: Staining of p-MLC localizes to epithelial layers in IO mice. n Z 6 to 8 per group. *P < 0.05 versus respective sham. Scale bar Z 10 mm. CFU, colony-forming unit; HRP, horseradish peroxidase; IO, intestinal obstruction; p-MLC, phosphorylated myosin light chain; TJ, tight junction; ZO-1, zonula occludens protein 1.

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Bacterial Internalization and Terminal Web Myosin assay buffer. The supernatant of the cell lysates was adjusted to 5 mg/mL for the measurement of MLCK activity. Biotinylated MLC was diluted in PBS and used to coat streptavidin 96-well plates at 37 C for 1 hour. After washing with PBS, the plates were incubated with blocking solution (1 mg/mL of BSA in PBS) at 37 C for 1 hour and then washed with PBS. Kinase reaction buffer (90

mL), provided by the manufacturer (MBL International, Woburn, MA), and 10 mL of treated samples were added to the wells, and the kinase reaction was performed at 37 C for 30 to 60 minutes. The reaction was stopped by removing the reaction mixtures. The plates were rinsed three times with washing buffer and incubated with 5 ng/ mL of antie pMLC-S19 antibody at room temperature for

Figure 3 Epithelial endocytosis of bacteria is dependent on MLCK but not ROCK activation. Mice were intraluminally administered ML-7, Y27, or veh immediately after loop ligation, and tissues were collected 6 hours after IO. A: Intracellular bacterial counts decrease in enterocytes in IO mice administered ML-7 but not Y27. B: Liver bacterial counts reduce in IO plus ML-7 but not IO plus Y27 mice. C: Increased mucosal inflammation caused by IO is reduced by ML7 but not Y27. D: Representative transmission electron micrographs showing bacterial internalization in epithelial cells of IO plus veh and IO plus Y27 mice but not sham and IO plus ML-7 groups. E: Normal TJs were seen in all groups. E: Higher magnification of apical membrane and BB of enterocytes showing no sign of bacteria in sham mice, presence of bacteria in close proximity or adherent to BB, and entry into cells in IO plus veh mice. F: Representative scanning electron micrographs showing bacterial penetration of BB of IO plus veh mice but not sham controls (upper panel). Higher magnification of bacteria is shown in lower panels. In contrast to the flat BB carpets in sham mice, multiple dome structures with enlarged crevices (arrows) are observed in IO plus veh groups. The ultrastructural change was inhibited by ML-7 but not Y27. All representative images were obtained from at least four mice per group. n Z 6 per group. *P < 0.05 versus sham; yP < 0.05 versus IO plus veh. Scale bar Z 1 mm. BB, brush border; CFU, colony-forming unit; IO, intestinal obstruction; MLCK, myosin light chain kinase; MPO, ---; ROCK, rho-associated kinase; TJ, tight junction; veh, vehicle; Y27, Y27632.

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1 hour. After rinsing the plates, goat anti-rabbit IgG antibody (1:2000) was added to the wells for incubation at 37 C for 1 hour. After rinsing, the plates were incubated with substrate solution (tetramethylbenzidine) at 37 C for 5 to 15 minutes. A stop solution that contained 0.5 N H2SO4 was added, and the absorbance was determined at 450 nm using a spectrometer.

RNA InterferenceeMediated Knockdown of MLCK or Caveolin-1 Cells were seeded at 2  105 cell/mL and transfected at 60% to 80% confluency the next day. Fifty-nanomolar siRNA in Opti-MEM RNAiMAx reagent (Santa Cruz) was added to cells in serum-free medium for overnight incubation. Cells

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Bacterial Internalization and Terminal Web Myosin were trypsinized and reseeded at 1  106 cells/mL on inserts of 12-well transwells. After 2 days, IFN-g was added basolaterally for 48 hours of incubation followed by bacterial internalization assays.

Immunofluorescent Staining and Confocal Microscopy The cell monolayer after exposure to E. colieGFP was processed for immunofluorescent staining. The cells on filter supports were fixed with 4% PFA, permeabilized with 0.5% Triton X-100, and quenched with 50 mmol/L NH4Cl. After blocking with 1% BSA, cells were stained with mouse monoclonal antie zonula occludens protein 1 conjugated with Alexa-594 (1:100; Invitrogen) or with phalloidin conjugated to Alexa-594 (5 U/mL; Invitrogen) to visualize F-actin. The cell nuclei were stained with Hoechst dye. The filter support was cut off and mounted on slides, and images were captured using a laser scanning confocal microscope (Zeiss LSM780).

Statistical Analysis

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All data are expressed as means  SEM, except that bacterial CFU values were presented as medians. When normality assumption was appropriate, data were analyzed by one-way analysis of variance, followed by the StudentNewman-Keuls method for multiple comparisons. When comparing the difference between two groups of bacterial CFU values that failed to satisfy the normality assumption, the nonparametric U-test was used (Sigma Plot version 10.0). P < 0.05 was considered statistically significant.

Results Enteric Bacterial Overgrowth Coincides with Extraintestinal Translocation After Obstruction A mouse model of IO was used to investigate the mechanisms of transepithelial bacterial penetration by loop ligation in the distal small intestine for 1, 3, 6, and 24 hours. The bacterial counts in the gut lumen, and liver and spleen tissues increased with time in IO mice compared with sham controls (Supplemental Figures S1 and S2). Bacterial DNA was extracted from luminal aspirate of the obstructed loops after

1055 6 hours and processed for 16S ribosomal DNA sequencing, 1056 where the strains included Staphylococcus, Enterococcus, Lactobacillus, Escherichia, and Bacteroides (Figure 1A). ½F1 1057 1058 Mucosal penetration of microbes was evident after 6 and 24 1059 hours of IO by hybridization using primers that targeted spe1060 cific bacterial strains (Figure 1, BeD). Although mucosal 1061 morphologic findings were histologically normal 6 hours after 1062 IO, villous blunting was found after 24 hours (Figure 1B). 1063 Additional experiments by inoculating live fluorescent 1064 1065 bacteria into ligated loops allowed us to visualize the temporal1066 spatial association of enteric bacteria and gut mucosa after 1067 Q11 obstruction. The GFP-tagged E. coli remained in the lumen 1068 separated from epithelial layers after 1 hour, formed a close 1069 contact with epithelial surfaces after 3 hours, and penetrated 1070 into the lamina propria after 6 and 24 hours (Figure 1E).

Epithelial Adherence and Endocytosis of Commensal Bacteria Is Uncoupled with TJ Damage The transepithelial routes of bacterial penetration were next investigated. The intracellular bacterial number in epithelial cells was significantly increased after 3, 6, and 24 hours of IO compared with respective sham controls (Figure 2A). Although no sign of paracellular permeability change was found at 6 hours of IO, elevated tissue conductance and macromolecular flux associated with TJ disruption were noted at 24 hours of IO (Figure 2, BeD, and Supplemental Figure S3). Furthermore, epithelial MLC phosphorylation levels were significantly augmented at both 6 hours and 24 hours of IO (Figure 2, E and F). Our results suggest that the onset of bacterial internalization and MLC phosphorylation in enterocytes were earlier than the increase in paracellular permeability. Therefore, all of the following experiments were conducted at 6 hours of IO. To assess the strains and diversity of endocytosed microbes, bacterial colonies derived from lyzed epithelial cells of IO mice were either subjected to Gram staining or DNA extraction. On the basis of the results of Gram staining, the percentages of colonies classified as Gram-positive rods and cocci and Gram-negative rods and cocci were 19%, 12%, 35%, and 31%, respectively. In addition, 3% of the bacterial clones were unclassified and unidentified. According to the

Figure 4 MLCK-dependent TW myosin phosphorylation and arc formation correlate with BB disarray and fanning. A: p-MLC colocalizes with fodrin in TW of epithelial cells in IO plus veh mice, which is inhibited by ML-7 but not Y27. B: Staining of p-MLC (green), fodrin (red), and cell nucleus (blue) is merged with DIC images to show orientation. The ultrastructures of BB, TW, and Nu are labeled. DIC images show TW arc formation on BB of IO plus veh mice but not sham controls, in which the phenomenon is inhibited by ML-7 but not Y27. C: The arc formation in epithelial sheets was potentiated by addition of Ca2þ/ATP in the contraction buffer. Electron micrographs showing BB fanning in IO plus veh mice, which was potentiated by addition of Ca2þ/ATP. D: The fanning is reduced in IO plus ML-7 but not IO plus Y27 mice. Scheme of the width (X ) and the height (Y ) of the TW arc in uncontracted and contracted conditions. E: The Y:X ratio was quantified to determine TW contraction. The extent of TW contraction was augmented in IO plus veh mice compared with sham controls, in which the addition of Ca2þ/ATP caused a further increase. F: The contraction level is decreased in IO plus ML-7 but not IO plus Y27 mice. Quantification of TW contraction in epithelial sheets obtained from sham and IO plus veh mice with ex vivo treatment with inhibitors. G: Representative electron micrographs showing crosssectional view of microvilli, in which the packing angles were quantified. H: Disarray of BB was seen in IO plus veh and IO plus Y27 mice but not sham and IO plus ML-7 mice. The microvillus number per area of intestinal tissues is given. I: The intermicrovillous space in windows of electron micrographs. n Z 5 per group. *P < 0.05 verus sham; yP < 0.05 versus IO plus veh; zP < 0.05 after addition of Ca2þ/ATP. Scale bar Z 1 mm. BB, brush border; DIC, differential interference contrast; IO, intestinal obstruction; MLCK, myosin light chain kinase; Nu, nucleus; p-MLC, phosphorylated myosin light chain; TW terminal web; veh, vehicle; w/o, without; Y27, Y27632.

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data of 16S ribosomal DNA sequencing, multiple strains of commensal bacteria (ie, Bacillus, Enterococcus, Staphylococcus, Escherichia, and Acinetobacter) were present within enterocytes. By using real-time imaging, we observed live motile bacteria in the gut lumen separated from epithelial surfaces by a thick static mucous layer in sham controls (Supplemental Video S1 and Supplemental Figure S4). In contrast, dynamic bacterial penetration through a turbulent, fluid mucous layer was seen in intestinal samples of IO mice (Supplemental Video S2 and Supplemental Figure S4). The

presence of the glycoprotein-rich mucous layer covering epithelial surfaces in both groups was confirmed by Alcian blue periodic acid-Schiff staining (Supplemental Figure S4).

Bacterial Endocytosis is Dependent on MLCK Activation Associated with TW Contraction and BB Fanning Because epithelial MLC hyperphosphorylation paralleled bacterial endocytosis in the early phases of IO, we sought to determine the roles of MLCK and ROCK in mechanisms of bacterial internalization. Intraluminal administration of

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Figure 5 TW-associated BB fanning and bacterial endocytosis are absent in enterocytes of MLCK/ and IFN-g/ mice. A: p-MLC in TW of epithelial cells is seen after IO in WT but not mutant mice. B: Staining of p-MLC (green), fodrin (red), and cell nucleus (blue) is merged with DIC images to show orientation. TW arc formation caused by IO is seen in WT but not mutant mice and is potentiated by addition of Ca2þ/ATP. C: Electron micrographs of longitudinal and cross-sectional views of BB show IO-induced BB fanning in WT mice and attenuation of fanning in mutant mice. Bacterial counts decrease in intestinal epithelial cells of MLCK/ and IFN-g/ mice (D) and in liver and spleen tissues of MLCK/ and IFN-g/ mice (E). F: MLCK activity increases after IO in WT mice but not mutant mice. G: Mucosal IFN-g levels increase after IO in WT and MLCK/ mice but not IFN-g/ mice. n Z 6 per group. *P < 0.05 versus respective sham; yP < 0.05 versus IO in WT mice. Scale bar Z 1 mm. BB, brush border; CFU, colony-forming unit; DIC, differential interference contrast; IFN-g, interferon-g; IO, intestinal obstruction; MLCK, myosin light chain kinase; p-MLC, phosphorylated myosin light chain; TW terminal web; WT, wild type.

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1241 ML-7 prevented the increase in intracellular bacterial counts Electromicrographic images revealed that enteric bacteria 1242 in isolated enterocytes of IO mice, whereas treatment with were not in close contact with BB of epithelial cells in sham 1243 ½F3 Y27632 had no effect (Figure 3A). The extraintestinal controls (Figure 3, D and E). By contrast, the presence of 1244 bacterial counts and mucosal MPO activity were also adherent and internalized bacteria within enterocytes was 1245 decreased in mice treated with ML-7 but not Y27632 observed in IO mice (Figure 3, D and E). Using scanning 1246 (Figure 3, B and C). Comparable levels of gut luminal electron microscopy, we noted bacterial attachment on 1247 bacterial counts were seen in IO mice given vehicle, ML-7, multiple bouquet-like dome structures on villous surface of 1248 or Y27632, ruling out bactericidal effects of pharmacologic IO mice compared with the smooth villous exterior without 1249 inhibitors (Supplemental Figure S5A). bacteria in sham controls (Figure 3F). In addition, enlarged 1250 1251 1252 1253 1254 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267 1268 1269 1270 1271 1272 1273 1274 1275 1276 1277 1278 1279 1280 1281 1282 1283 1284 1285 1286 1287 1288 1289 1290 1291 1292 Figure 6 IFN-g triggers MLCK-dependent ROCK-independent TW myosin phosphorylation and bacterial endocytosis in human epithelial Caco-2 cells. A: 1293 Caco-2 cells serosally treated with IFN-g for 48 hours do not show changes in TER and dextran flux. B: Treatment with IFN-g increases MLCK activity. C: IFN-g 1294 increases the expression levels of MLCK in cells. D: IFN-g enhances MLCK-dependent MLC phosphorylation. E: TW myosin phosphorylation parallels arc formation after IFN-g treatment, which is inhibited by ML-7 but not Y27. Superimposed images of p-MLC (green) and fodrin (red) staining and DIC images of 1295 epithelial monolayer with cell nuclei (blue). F: Confocal images show adherence and internalization of E. colieGFP (green) in IFN-getreated cells with intact 1296 tight junctions, ZO-1 (red). G: The phenomenon is inhibited by ML-7 but not Y27. Bacterial endocytosis and transcytosis increases after exposure to IFN-g 1297 compared with medium, which is inhibited by ML-7 but not Y27. H: Transfection with siMLCK decreases IFN-geinduced bacterial endocytosis compared with 1298 siCON. I: Reduction of protein expression and activity level of MLCK confirms the efficiency of gene silencing. n Z 6 per group. *P < 0.05 versus medium; 1299 y P < 0.05 versus veh (G) or siCON (H). Scale bar Z 1 mm. CFU, colony-forming unit; DIC, differential interference contrast; IFN-g, interferon-g; MLCK, myosin 1300 light chain kinase; ROCK, ROCK, rho-associated kinase; siCON, siRNA to control; siMLCK, siRNA to MLCK; TER, transepithelial electrical resistance; TW, terminal 1301 web; veh, vehicle; Y27, Y27632; ZO-1, zonula occludens protein 1. 1302

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1427 1428 1429 1430 1431 1432 1433 1434 1435 1436 1437 IFN-g Is Involved in Mechanisms of Epithelial 1438 MLCK-Dependent TW Phosphorylation and Bacterial 1439 Endocytosis 1440 1441 A 2.5-times increase in mucosal IFN-g levels was observed 1442 in IO mice compared with sham controls (Supplemental 1443 Figure S6A). Administration of neutralizing antieIFN-g 1444 decreased the epithelial TW p-MLC levels (Supplemental 1445 Figure S6, B and C), and reduced BB fanning in IO mice 1446 (Supplemental Figure S6, DeG). Lower counts of endocy1447 tosed and translocated bacteria were found in mice admin1448 1449 istered antieIFN-g versus isotypes (Supplemental Figure S6, 1450 H and I). AntieIFN-g did not affect the luminal bacterial 1451 counts (Supplemental Figure S5B). 1452 Additional experiments were conducted in mice deficient 1453 in IFN-g and long MLCK (210 kDa) to confirm their roles 1454 in epithelial bacterial endocytosis. Significant decreases in 1455 the extent of TW p-MLC and arc formation (Figure 5, A and ½F5 1456 B) and BB fanning (Figure 5C) were seen in mutant mice 1457 compared with wild types. The IO-increased counts of 1458 endocytosed and translocated bacteria were also reduced in 1459 1460 1461 1462 1463 1464 1465 1466 1467 1468 1469 1470 1471 1472 1473 1474 1475 1476 1477 1478 1479 Figure 7 Myosin phosphorylation and arc formation of TW precedes Cav-1edependent bacterial internalization through cholesterol-rich lipid rafts. A: 1480 Administration of cholesterol-sequestering agents (filipin and MbCD) decreases IFN-geinduced bacterial endocytosis and translocation compared with veh 1481 controls. B: Filipin and MbCD have no effect on IFN-geinduced MLC phosphorylation and arc formation of TW. C: Staining of p-MLC (green), fodrin (red), and 1482 cell nucleus (blue) is merged with DIC images to show orientation. Transfection with siCav-1 decreases IFN-geinduced bacterial endocytosis and translocation 1483 compared with siCON. D: Western blots confirm the reduction of Cav-1 levels by gene silencing. E: IFN-geinduced MLC phosphorylation does not decrease by 1484 siCav-1. Electron microscopic images show colocalization of bacteria and caveolin-1 (arrowheads) in intestinal epithelial cells of IO mice, by immunostaining 1485 with antieCav-1 monoclonal antibody. n Z 6 per group. *P < 0.05 versus veh or siCON. Scale bar Z 1 mm. Cav-1, caveolin-1; CFU, colony-forming unit; DIC, 1486 differential interference contrast; IFN-g, interferon-g; IO, intestinal obstruction; MbCD, methyl-b-cyclodextrin; MLC, myosin light chain; p-MLC, phosphor1487 Q22 ylated myosin light chain; siCav-1, siRNA to caveolin-1; siCON, siRNA to control; TW, terminal web; veh, vehicle. 1488

crevices were seen on the BB carpet in IO groups (Figure 3F). These morphologic changes were inhibited by ML-7 but not Y27632 (Figure 3F), suggesting an MLCKdependent mechanism. We next examined whether MLCK-dependent phosphorylation is involved in TW arc formation and BB fanning in IO enterocytes. Compared with sham controls, heightened amounts of p-MLC colocalized with fodrin staining (an indicator of the TW region) in isolated epithelial sheets in IO mice (Figure 4A). The increase in the TW p-MLC level was inhibited in mice administered ML-7 but not Y27632 (Figure 4A). By high-resolution imaging and electron microscopy, a smooth lining of BB formed by highly organized microvilli was seen in epithelial sheets of sham controls, whereas TW arc formation and BB disarray were observed in those of IO mice (Figure 4, B and C). Ex vivo addition of calcium/ATP to epithelial sheets further potentiated the arc formation in IO groups (Figure 4, B and C). To quantify TW contraction, ratios of the height and width of the arc in each enterocyte were determined as shown in the scheme (Figure 4D). Totals of 140% and 210% were seen in IO groups compared with sham controls in calcium/ATP-free and -containing solutions, respectively (Figure 4E). In vivo and ex vivo administration with ML-7 blocked the epithelial TW contraction in IO mice (Figure 4, E and F). On the other hand, Y27632 had no effect. The organization of BB was further examined by quantifying the packing angles of microvilli and intermicrovillous space. Densely packed microvilli were seen in the crosssectional view of BB in sham controls, whereas a poor

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organization was observed in that of IO mice (Figure 4G). A wider range of packing angles of microvilli was seen in IO mice compared with sham controls (Figure 4G). A decrease in microvillus numbers and increase in intermicrovillous space per surface area were noted in IO mice compared with sham controls (Figure 4, H and I). The disorganized pattern of BB caused by IO was inhibited by ML-7 but not Y27632 (Figure 4, H and I).

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Bacterial Internalization and Terminal Web Myosin 1489 mutant mice (Figure 5, D and E). Comparable levels of gut 1490 luminal bacterial counts were seen between mutant mice and 1491 wild types (Supplemental Figure S5C). In contrast to the 1492 increases in mucosal MLCK activity and IFN-g levels by IO 1493 in wild-type mice, MLCK/ mice had increased IFN-g but 1494 no sign of MLCK activation, whereas IFN-g/ mice had 1495 the lack of both after IO (Figure 5, F and G). Collectively, 1496 our results confirmed that IFN-g activates MLCK1497 dependent myosin phosphorylation in TW and bacterial 1498 1499 transcytosis in enterocytes. 1500 1501 IFN-g Induces MLCK-Dependent TW Myosin 1502 Phosphorylation Preceding Caveolin-Mediated 1503 Bacterial Internalization through Lipid Rafts in 1504 1505 Caco-2BBe Cells 1506 1507 Caco-2BBe cells were used to further investigate the mo1508 lecular mechanisms of bacterial internalization. Cells treated 1509 with 100 U/mL of IFN-g had no change in paracellular 1510 ½F6 permeability (Figure 6A) but had heightened MLCK1511 dependent MLC phosphorylation associated with TW arc 1512 formation (Figure 6, BeE). After apical addition of E. colie 1513 GFP, bacterial adherence and internalization were observed 1514 without TJ changes in IFN-getreated cells (Figure 6F). 1515 Importantly, no sign of bacterial attachment was seen in 1516 medium control groups (Figure 6F). A significant increase in 1517 1518 endocytosed and translocated bacterial counts was noted in 1519 IFN-getreated cells (Figure 6G). All of the aforementioned 1520 phenomena were inhibited by ML-7 but not Y27632 1521 (Figure 6, EeG). Moreover, gene silencing of MLCK by 1522 siRNA also attenuated the IFN-geinduced increase in bac1523 terial endocytosis (Figure 6H). Knockdown efficiency was 1524 confirmed by decreased expression and activity of MLCK 1525 (Figure 6I). 1526 Lastly, cholesterol-sequestering agents (eg, filipin and 1527 methyl-b-cyclodextrin) and gene silencing of caveolin-1 1528 decreased IFN-geinduced bacterial endocytosis and trans1529 1530 ½F7 cytosis (Figure 7, A and C) but did not affect the level of 1531 MLC phosphorylation and arc formation of TW (Figure 7, B 1532 and D). These results suggest that BB fanning precedes the 1533 lipid raftemediated bacterial internalization. None of the 1534 inhibitors or chemical agents decreased the transepithelial 1535 electrical resistance values of cell monolayers (Supplemental 1536 Figure S7). 1537 1538 1539 Discussion 1540 1541 Abnormal commensal penetration plays a critical role in the 1542 pathogenesis of chronic intestinal inflammation and gut-derived 1543 septic complications. Recent data indicate that epithelial cells 1544 may apically internalize and transcytose bacteria, before TJ 1545 impairment, via unknown mechanisms.8,14 Our novel findings 1546 1547 indicate that MLCK-activated TW myosin phosphorylation and 1548 BB fanning by IFN-g stimulation allows bacterial penetration 1549 through intermicrovillous clefts for endocytosis. The bacterial 1550

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transcytotic route may contribute to the initiation or relapse of chronic inflammation. An intestinal obstructive loop model was used to induce bacterial overgrowth, accompanied by mucosal penetration and extraintestinal translocation. At early time points (6 hours) after ligation, MLCK-dependent TW MLC phosphorylation and BB fanning were associated with increased bacterial endocytosis. The possibility of paracellular or basolateral entry for microbes was ruled out because the TJ structures remained intact. Although bacterial sampling by M cells or dendritic cells may also contribute to microbial translocation,35,36 intracellular microbes were observed mainly in epithelial cells in our study. We found that IFN-geinduced TW contraction and arc formation preceded caveolin-mediated bacterial endocytosis through cholesterol-rich lipid rafts. Previous reports documented IFN-g overproduction by intraepithelial lymphocytes or lamina propria Th1 cells in association with the pathogenesis and disease severity of Crohn disease and celiac disease.3,4 Other than the proinflammatory role, IFN-g was linked with increased paracellular permeability by causing MLCKdependent perijunctional actinomyosin contraction and ROCK-dependent junctional protein endocytosis.18,37 The data from our group and others8 further emphasize a critical role of low-dose IFN-g in triggering transcellular bacterial passage before TJ damage, which may be responsible for relapse of chronic inflammation. Whether the internalized microbial strains simply reflect their dominance in the gut lumen as bystanders trapped by microvilli or are associated with unknown virulent invasive factors remains elusive. Previous reports documented high prevalence of adhesive and invasive E. coli in patients with Crohn disease, patients with irritable bowel syndrome, and those undergoing colectomy.38e40 These bacterial isolates are known to penetrate and survive intracellularly better than other nonpathogenic commensals when co-cultured with intestinal epithelial cells.40 Further experiments on characterizing the virulent factors of endocytosed bacteria are currently under way and will provide information on host-microbial interaction. The nonmuscle long MLCK (210 kDa) is the predominant isoform expressed in enterocytes with splice variants 1 and 2, which differed by the absence of a 69eamino acid region in the latter.19 We found that knockdown of long MLCK leads to ablation of bacterial endocytosis in epithelial cells. The long MLCK-1 is involved in perijunctional actinomyosin contraction,19 whereas the function of long MLCK-2 in enterocytes is currently unknown. Further research to verify specific roles of long MLCK-1 and -2 variants in response to IFN-g activation is needed.

Conclusion Our study revealed that apical bacterial internalization is regulated by IFN-geinduced MLCK-dependent BB

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Wu et al fanning. The understanding of intermicrovillous penetration and transcytotic pathways of commensal microbes may shed light on the development of therapeutic intervention to prevent relapse of chronic intestinal inflammation.

Acknowledgments We thank the staff of the imaging facility at the First Core Lab and the AM2 of Disease Animal Research Center for their technical assistance. Study concept and design: L.C.Y.; data acquisition: L.L.W., W.H.P., W.T.K., C.Y.H.; data analysis/interpretation: L.L.W., Y.H.N., K.S.L.; statistical analysis: L.L.W.; material and technical support: Y.H.N., K.S.L., J.R.T.; obtained funding: L.C.Y.; manuscript drafting and editing, literature research, and manuscript final version approval: all authors.

Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2014.05.003.

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Supplemental Figure S1 IO triggers enteric bacterial overgrowth and translocation to extraintestinal organs. Bacterial counts in the gut lumen and liver and spleen tissues were determined by aerobic culturing. Bacteria were grown under aerobic conditions. A: A time-dependent increase in luminal bacterial counts of distal small intestines was noted in IO mice compared with sham controls. Bacterial counts in liver (B) and spleen (C) tissues increased with time after IO. Each data point represents the value from one animal. Bars indicate median values. n Z 6 to 8 per group. *P < 0.05 versus respective sham. CFU, colony-forming unit; IO, intestinal obstruction. Supplemental Figure S2 IO for 6 hours triggers overgrowth of anaerobic bacteria and their translocation to extraintestinal organs. A: Increase in luminal anaerobic bacterial counts was noted in distal small intestine of IO mice compared with sham controls. The anaerobic bacterial counts in liver (B) and spleen (C) tissues increased after IO. Each data point represents one animal. Bars indicate median bacterial counts. n Z 6 per group. *P < 0.05 versus respective sham. CFU, colony-forming units; IO, intestinal obstruction. Supplemental Figure S3

Occludin cleavage is observed in intestinal tissue after 24 hours but not 6 hours of IO. Western blots showing the occludin levels in mice subjected to IO for 6 or 24 hours. Intraluminal administration of ML-7 and Y27632 in mice subjected to IO for 6 hours had no effect on occludin levels. Experiments were repeated twice. n Z 5 per group. IO, intestinal obstruction; veh, vehicle.

Supplemental Figure S4 Bacteria are separated from epithelium by a thick mucous layer in sham control mice, whereas bacterial penetration through mucous layer and adherence to epithelium are observed in IO mice. A: DIC images showing bacteria (arrows) and epithelial BB. B: Higher magnification of the surface mucous gel layer and epithelial cells after staining with AB-PAS. Staining is merged with DIC images. n Z 5 per group. AB-PAS, Alcian blue periodic acid-Schiff; BB, brush border; DIC, differential interference contrast; IO, intestinal obstruction. Supplemental Figure S5 Enteric bacterial counts in wild-type mice after administration of inhibitors or antibodies and in mutant mice. A: Increase in gut luminal bacterial counts was seen in IO mice administered veh, ML-7, or Y27632 compared with sham controls. B: Comparable luminal bacterial counts were seen in IO mice injected i.p. with antieIFN-g and isotype antibodies. C: Increase in gut luminal bacterial counts was noted after IO in WT, MLCK/, and IFN-g/ mice. Each data point represents one animal. Bars indicate the median bacterial counts. n Z 5 per group. *P < 0.05 versus respective sham. CFU, colony-forming unit; IFN-g, interferon-g; IO, intestinal obstruction; MLCK, myosin light chain kinase; veh, vehicle; WT, wild type. Supplemental Figure S6 IFN-g is involved in MLCK-dependent TW myosin phosphorylation and bacterial endocytosis in enterocytes. Mice were injected i.p. with antieIFN-g or isotype antibody before surgical procedures of IO. A: Increase in mucosal IFN-g levels caused by IO is inhibited by neutralizing antieIFN-g, whereas isotype antibody has no effect. B: Western blots reveal reduction of mucosal MLC phosphorylation by antieIFN-g compared with isotypes. C: Absence of p-MLC in the terminal web of epithelial cells in antieIFN-g groups compared with isotype controls. Immunostaining of p-MLC (green) and fodrin (red) are merged with DIC images. D: Representative scanning electron micrographs of intestinal tissues obtained from mice given isotype and antieIFN-g. E and F: Transmission electron micrographs of the longitudinal view of enterocytes show reduction of bacteria internalization and BB fanning in antieIFN-g groups compared with isotype controls. G: Cross-sectional view of enterocytes shows reduction of intermicrovillous space in antieIFN-g groups compared with isotype controls. H: AntieIFN-g decreases the intracellular bacterial counts in epithelial cells. I: Bacterial counts in liver and spleen tissues are reduced by antieIFN-g. The line indicates the median for each group. n Z 6 per group (A, C, and I). *P < 0.05 versus sham (A) or versus isotype (I). Scale bars: 1 mm (C, D, E, F, and G). CFU, colony-forming unit; IFN-g, interferon-g; IO, intestinal obstruction; MLC, myosin light chain, MLCK, myosin light chain kinase; p-MLC, phosphorylated myosin light chain. Supplemental Figure S7

Administration of inhibitors or sequestering agents and transfection of siRNA does not decrease the TER of cells. A: TER values in cells treated with IFN-g and inhibitors (ML-7 and Y27). B: IFN-getreated cells administered filipin and MbCD. C: IFN-getreated cells with transfection of siRNA. All experiments were repeated at least twice. n Z 6 per group. IFN-g, interferon-g; MbCD, methyl-b-cyclodextrin; siCav-1, small interfering caveolin-1; siCON, siRNA to control; siMLCK, siRNA to myosin light chain kinase; TER, transepithelial electrical resistance; veh, vehicle; Y27, Y27632.

Supplemental Video S1 mm. BB, brush border.

Live imaging of bacteria and epithelial layers in intestines of sham controls. The arrow points to the bacteria. Scale bar Z 5

Supplemental Video S2 Live imaging of bacteria and epithelial layers in intestines of IO mice. The arrow points to the bacteria. Scale bar Z 5 mm. BB, brush border; IO, intestinal obstruction.

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