The Expression Patterns of Toll-Like Receptors in the Ileal Pouch Mucosa of Postoperative Ulcerative Colitis Patients

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<ul><li><p>Surg Today (2006) 36:287290DOI 10.1007/s00595-005-3144-y</p><p>Short Communication</p><p>The Expression Patterns of Toll-Like Receptors in the Ileal PouchMucosa of Postoperative Ulcerative Colitis Patients</p><p>Yuji Toiyama, Toshimitsu Araki, Shigeyuki Yoshiyama, Jun-ichiro Hiro, Chikao Miki, and Masato Kusunoki</p><p>Second Department of Surgery, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan</p><p>pouches of 100 : 1, compared to 4 : 1 in ileostomyeffluent, has been noted, and observations suggestingthat anaerobes play a role in this etiology thus requirefurther study.3</p><p>The Toll-like receptor (TLR) family comprises atleast eight human homologues of the Drosophila Tollprotein which appear to be key regulators in the differ-ential cellular recognition of conserved molecular pat-terns associated with microbial pathogens.4 Among thisfamily, TLR2 and TLR4 have been most extensivelystudied to date, and these studies have variably sug-gested that both TLRs may serve as potential mainmediators of lipopolysaccharide (LPS) signaling.5</p><p>Alexopoulou et al.6 demonstrated that viral dsRNArecognition relies on the TLR3, and Flagellin, a patho-gen-associated molecular pattern (PAMP), has beenidentified as the specific ligand for TLR5.</p><p>We speculate that an immune imbalance in pouchitismight result from an exaggerated activation of themucosal innate immune system in response to thebacterial products of the lumen initiated throughthe dysregulation of TLRs. However, it remains unclearas to whether TLRs are expressed in the ileum mucosain vivo and how such expression may be altered in thepouch mucosa either with or without inflammation. Theaim of this study was thus to evaluate the expression ofvarious PAMP receptors TLR2, -3, -4, and -5 in thepouch mucosa of postoperative UC patients in compari-son with those in normal controls.</p><p>Biopsy samples were obtained from three patientsdiagnosed to have pouchitis (3 months, 16 months, and24 months after undergoing ileoanal anastomosis).After a total proctocolectomy with a diverting ileo-stomy, noninflamed pouch mucosa specimens from fivepatients were also collected. Normal ileal mucosa speci-mens were obtained from five patients with ascendingcolon cancer who underwent surgery at Mie UniversityHospital. Informed consent was obtained from eachpatient.</p><p>AbstractThe aim of this study was to evaluate the expressionpattern of Toll-like receptors (TLRs) in the pouch mu-cosa of ulcerative colitis patients in comparison withthat in the ileum mucosa of noninflammatory boweldisease patients. Pouch mucosal biopsy specimens werecollected from postoperative patients who had under-gone surgery for ulcerative colitis. Normal ileum speci-mens were collected from colon cancer patients. Thespecimens were assessed by immunofluorescence his-tochemistry using TLR2, TLR3, TLR4, and TLR5polyclonal antibodies. The normal ileal mucosa consti-tutively expressed TLR3 and TLR5, whereas TLR2 andTLR4 were barely detectable. In the mucosa of activepouchitis, TLR2 and TLR4 was strongly upregulated,and TLR4 was upregulated even in a noninflamedpouch. No TLR3 or TLR5 expression was detectable.These data suggest that pouchitis may be associatedwith distinctive changes in selective TLR expression inthe pouch mucosa, and that TLR4 alterations in theinnate response system may contribute to the pathogen-esis of these disorders in particular.</p><p>Key words Pouchitis Toll-like receptor Ulcerativecolitis</p><p>The current treatment of choice for many patients re-quiring a colectomy for ulcerative colitis (UC) or famil-ial adenomatous polyposis is ileoanal anastomosis withpouch creation. The symptomatic inflammation of thepouch, a condition known as pouchitis, develops in from7% to 40% of all patients who undergo this surgery.1</p><p>At the present time, no clear microbiologic factor hasbeen shown to play a part in the etiology.2 However,a marked increase in the anaerobe/aerobe ratio in</p><p>Reprint requests to: M. KusunokiReceived: February 3, 2005 / Accepted: July 12, 2005</p><p>Used Mac Distiller 5.0.x Job OptionsThis report was created automatically with help of the Adobe Acrobat Distiller addition "Distiller Secrets v1.0.5" from IMPRESSED GmbH.You can download this startup file for Distiller versions 4.0.5 and 5.0.x for free from</p><p>GENERAL ----------------------------------------File Options: Compatibility: PDF 1.2 Optimize For Fast Web View: Yes Embed Thumbnails: Yes Auto-Rotate Pages: No Distill From Page: 1 Distill To Page: All Pages Binding: Left Resolution: [ 600 600 ] dpi Paper Size: [ 595.3 785.2 ] Point</p><p>COMPRESSION ----------------------------------------Color Images: Downsampling: Yes Downsample Type: Bicubic Downsampling Downsample Resolution: 150 dpi Downsampling For Images Above: 225 dpi Compression: Yes Automatic Selection of Compression Type: Yes JPEG Quality: Medium Bits Per Pixel: As Original BitGrayscale Images: Downsampling: Yes Downsample Type: Bicubic Downsampling Downsample Resolution: 150 dpi Downsampling For Images Above: 225 dpi Compression: Yes Automatic Selection of Compression Type: Yes JPEG Quality: Medium Bits Per Pixel: As Original BitMonochrome Images: Downsampling: Yes Downsample Type: Bicubic Downsampling Downsample Resolution: 600 dpi Downsampling For Images Above: 900 dpi Compression: Yes Compression Type: CCITT CCITT Group: 4 Anti-Alias To Gray: No</p><p> Compress Text and Line Art: Yes</p><p>FONTS ---------------------------------------- Embed All Fonts: Yes Subset Embedded Fonts: No When Embedding Fails: Warn and ContinueEmbedding: Always Embed: [ ] Never Embed: [ ]</p><p>COLOR ----------------------------------------Color Management Policies: Color Conversion Strategy: Convert All Colors to sRGB Intent: DefaultWorking Spaces: Grayscale ICC Profile: RGB ICC Profile: sRGB IEC61966-2.1 CMYK ICC Profile: U.S. Web Coated (SWOP) v2Device-Dependent Data: Preserve Overprint Settings: Yes Preserve Under Color Removal and Black Generation: Yes Transfer Functions: Apply Preserve Halftone Information: Yes</p><p>ADVANCED ----------------------------------------Options: Use and No Allow PostScript File To Override Job Options: Yes Preserve Level 2 copypage Semantics: Yes Save Portable Job Ticket Inside PDF File: No Illustrator Overprint Mode: Yes Convert Gradients To Smooth Shades: No ASCII Format: NoDocument Structuring Conventions (DSC): Process DSC Comments: No</p><p>OTHERS ---------------------------------------- Distiller Core Version: 5000 Use ZIP Compression: Yes Deactivate Optimization: No Image Memory: 524288 Byte Anti-Alias Color Images: No Anti-Alias Grayscale Images: No Convert Images (&lt; 257 Colors) To Indexed Color Space: Yes sRGB ICC Profile: sRGB IEC61966-2.1</p><p>END OF REPORT ----------------------------------------</p><p>IMPRESSED GmbHBahrenfelder Chaussee 4922761 Hamburg, GermanyTel. +49 40 897189-0Fax +49 40 897189-71Email: info@impressed.deWeb:</p><p>Adobe Acrobat Distiller 5.0.x Job Option File</p><p> /ColorImageDownsampleType /Bicubic /GrayImageDict &gt; /CalCMYKProfile (U.S. Web Coated (SWOP) v2) /ParseDSCComments false /PreserveEPSInfo false /MonoImageDepth -1 /AutoFilterGrayImages true /SubsetFonts false /GrayACSImageDict &gt; /ColorImageFilter /DCTEncode /AutoRotatePages /None /PreserveCopyPage true /EncodeMonoImages true /ASCII85EncodePages false /PreserveOPIComments false /NeverEmbed [ ] /ColorImageDict &gt; /AntiAliasGrayImages false /GrayImageDepth -1 /CannotEmbedFontPolicy /Warning /EndPage -1 /TransferFunctionInfo /Apply /CalRGBProfile (sRGB IEC61966-2.1) /EncodeColorImages true /EncodeGrayImages true /ColorACSImageDict &gt; /Optimize true /ParseDSCCommentsForDocInfo false /GrayImageDownsampleThreshold 1.5 /MonoImageDownsampleThreshold 1.5 /AutoPositionEPSFiles false /GrayImageResolution 150 /AutoFilterColorImages true /AlwaysEmbed [ ] /ImageMemory 524288 /OPM 1 /DefaultRenderingIntent /Default /EmbedAllFonts true /StartPage 1 /DownsampleGrayImages true /AntiAliasColorImages false /ConvertImagesToIndexed true /PreserveHalftoneInfo true /CompressPages true /Binding /Left&gt;&gt; setdistillerparams&gt; setpagedevice</p></li><li><p>288 Y. Toiyama et al: Toll-Like Receptor Expression in the Ileal Pouch</p><p>A histopathological examination was done on the10% formalin-fixed, paraffin-embedded specimensby the pathologist at the Pathology Division. Afterdewaxing and dehydration, the sections were washedwith deionized water and phosphate-buffered saline(PBS), and then incubated for 20min in 20% Blockace(Dainippon Pharmaceutical, Suita, Japan) in 0.1Mphosphate buffer containing 0.005% saponin (Merck,Darmstadt, Germany, referred to as buffer A). Theywere then washed three times with PBS and subse-quently blocked in 20% Blockace in buffer A. Theywere incubated with either anti-TLR2, -TLR3, -TLR4,or -TLR5 polyclonal antibody (1 :100 to 1 : 500) (SantaCruz, Santa Cruz, CA, USA) with 0.1 M phosphatebuffer containing 4% Blockace and 0.001% saponin at</p><p>4C overnight. Fluorescein-conjugated goat antirab-bit IgG (Vector Laboratories, Burlingame, CA, USA)were used as secondary antibodies (1 : 1000) with AlexaFluor 594 halloidin (Invitrogen, Tokyo, Japan). Thespecimens were examined using Radiance 2000 confo-cal laser scanning microscopy (Bio-Rad, Hercules, CA,USA).</p><p>TLR3 and TLR5 were weakly expressed on epithelialcells throughout the normal control. But in both histo-logically inflamed and noninflamed pouch mucosaspecimens, the expression of TLR3 and TLR5 proteinexpression was not detectable (Fig. 1af). TLR2 expres-sion was barely detectable in the normal ileum mucosa.The intensity of the staining for TLR2 was not increasedin the epithelium of uninflamed pouch mucosa in com-</p><p>Fig. 1af. The expressions of TLR3 and TLR5 were mini-mally dectectable in the normal ileum, but they were notdetectable in the pouch mucosa with or without inflammation.a Normal ileum: TLR3. b Pouch mucosa without inflamma-</p><p>tion: TLR3. c Pouchitis: TLR3. d Normal ileum: TLR5.e Pouch mucosa without inflammation: TLR5. f Pouchitis:TLR5 (40)</p></li><li><p>289Y. Toiyama et al: Toll-Like Receptor Expression in the Ileal Pouch</p><p>parison with that in the normal ileum mucosa. Theupregulation of TLR2 protein expression inflamed inileal pouch mucosa was observed in the apical surface ofepithelial cells (Fig. 2ac). In contrast, both the in-flamed pouch mucosa and noninflamed ileum mucosafrom UC patients showed an intense epithelial cell ex-pression of TLR4 and this result was reproducible in allintestinal samples examined. Intense staining wasmostly present at apical and basolateral surfaces in theileal mucosal sections of UC patients (Fig. 2df).</p><p>Cario and Podolsky,7 using immunofluorescence his-tochemistry, determined that TLR3 was significantlydownregulated in intestinal epithelial cells in activeCrohns disease (CD) but not in UC. In contrast,TLR4 was strongly upregulated in both UC andCD. Hausmann et al.8 have previously shown that an</p><p>induction of mRNA for TLR2, TLR4, and TLR5 Ininflammation-induced macrophages is located in thecolonic mucosa of inflammatory bowel disease (IBD).On the other hand, TLR4 has not been reported to bepresent in the epithelium from children with IBD.9</p><p>Our present study showed that the ileum mucosa ofnon-IBD patients constitutively express TLR3 andTLR5. In contrast, TLR3 and TLR5 were not detectedin the pouch mucosa with or without inflammation.Otherwise, TLR2 and TLR4 are barely detectable inthe normal ileum. The TLR2 expression remainedunchanged in the noninflamed pouch mucosa, but itbecame upregulated as the inflammation progressed.Interestingly, the TLR4 expressions were continuouslyupregulated in the pouch mucosa regardless of the de-gree of inflammation.</p><p>Fig. 2af. The expressions of TLR2 and TLR4 were onlybarely detectable in the normal ileum. In pouchitis, TLR2 andTLR4 were strongly upregulated in active pouchitis mucosa. a</p><p>Normal ileum: TLR2. b Pouch mucosa without inflammation:TLR2. c Pouchitis: TLR2. d Normal ileum: TLR4. e Pouchmucosa without inflammation: TLR4. f Pouchitis: TLR4 (40)</p></li><li><p>290 Y. Toiyama et al: Toll-Like Receptor Expression in the Ileal Pouch</p><p>TLR3 and TLR5 expressed on the basolateral surfaceof intestinal epithelia ought to be able to detect theinvasion of a large variety of microbes such as virus andSalmonella typhimurium and they are positioned to doso at a very early step in the invasive process.10 Support-ing this notion, mutations in TLR5 result in an increasedsusceptibility of mice to Salmonella infection.11 In apouch mucosa with UC, the loss of expression of thesereceptors may result in epithelial barrier dysfunctionwith subsequent inflammation.</p><p>Wolfs et al.12 previously reported that the enhancedTLR2 mRNA expression during renal inflammationwas found to be mediated by both interferon-g and tu-mor necrosis factor-a. In the present study, the TLR2expression of pouch mucosa was upregulated in linewith the degree of inflammation, thus suggesting thatthe alternation of expression represents a secondaryphenomenon induced by the inflammatory mediator.</p><p>Lumenal LPS is usually well tolerated in large quan-tities within the healthy intestine. This tolerance couldresult from TLR4 downregulation minimizing LPS rec-ognition.13 However, in UC, host tolerance towards lu-menal bacterial toxin may be broken,14 which could thusreflect an increased LPS recognition as a result of TLR4upregulation. A constant upregulation of TLR4 inthe pouch mucosa may therefore represent a hyper-response to commensal intraluminal bacteria, thusresulting in mucosal inflammation.</p><p>The present study is the first report demonstratingTLR expression to be altered in the pouch mucosa.Since the TLR4 expression was consistently upre-gulated both in the inflamed and noninflamed pouchmucosa, the upregulation of TLR4 may be partly in-volved in the pathogenesis or initiator of pouchitis.Moreover, the consistent loss of TLR3 and TLR5 ex-pression, which detects invasive pathogens, resulted inan increased pouch infection. However, the conclusive-ness of this preliminary report is limited due to the smallnumber of operations performed. Further studies thatfocus on the direct pathogenetic relevance and immuneconsequences of TLR dysregulation in pouchitis arethus needed.</p><p>References</p><p>1. Macafee DA, Abercrombie JF, Maxwell-Armstrong C. Pouchitis.Colorectal Dis 2004;6:14252.</p><p>2. Rubinstein MC, Fisher RL. Pouchitis: pathogenesis, diagnosis,and management. Gastroenterologist 1996;4:12933.</p><p>3. Nasmyth DG, Godwin PG, Dixon MF, Williams NS, Johnston D.Ileal ecology after pouch-anal anastomosis or ileostomy. A studyof mucosal morphology, fecal bacteriology, fecal volatile fattyacids,...</p></li></ul>