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

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  • Surg Today (2006) 36:287290DOI 10.1007/s00595-005-3144-y

    Short Communication

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

    Yuji Toiyama, Toshimitsu Araki, Shigeyuki Yoshiyama, Jun-ichiro Hiro, Chikao Miki, and Masato Kusunoki

    Second Department of Surgery, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan

    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

    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

    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.

    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.

    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.

    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.

    Key words Pouchitis Toll-like receptor Ulcerativecolitis

    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

    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

    Reprint requests to: M. KusunokiReceived: February 3, 2005 / Accepted: July 12, 2005

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  • 288 Y. Toiyama et al: Toll-Like Receptor Expression in the Ileal Pouch

    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

    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).

    TLR3 and TLR5 were weakly expressed on epithelialcells throughout the normal control. But in both histo-logically inflamed and noninf